The Transcriptome of Rhabdomyosarcoma Cells Infected with Cytolytic and Non-Cytolytic Variants of Coxsackievirus B2 Ohio-1

PubMed Central

Sävneby, Anna; Luthman, Johannes; Nordenskjöld, Fabian; Andersson, Björn

2016-01-01

The transcriptomes of cells infected with lytic and non-lytic variants of coxsackievirus B2 Ohio-1 (CVB2O) were analyzed using next generation sequencing. This approach was selected with the purpose of elucidating the effects of lytic and non-lytic viruses on host cell transcription. Total RNA was extracted from infected cells and sequenced. The resulting reads were subsequently mapped against the human and CVB2O genomes. The amount of intracellular RNA was measured, indicating lower proportions of human RNA in the cells infected with the lytic virus compared to the non-lytic virus after 48 hours. This may be explained by reduced activity of the cellular transcription/translation machinery in lytic enteroviral replication due to activities of the enteroviral proteases 2A and/or 3C. Furthermore, differential expression in the cells infected with the two virus variants was identified and a number of transcripts were singled out as possible answers to the question of how the viruses interact with the host cells, resulting in lytic or non-lytic infections. PMID:27760161

Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts

PubMed Central

Nath, Rahul; Mant, Christine A; Kell, Barbara; Cason, John; Bible, Jon M

2006-01-01

Background Human papillomavirus type 16 (HPV-16) E5 protein co-operates with epidermal growth factor to stimulate mitogenesis of murine fibroblasts. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-16 E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-growth of murine fibroblasts. Results We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-16 abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-16 E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-growth; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-growth and repression of p21 expression, despite high transcription levels. Conclusion The effects of HPV-16 E5 variants upon mitosis help to explain why Leu44Val65 HPV-16 E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK. PMID:16899131

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

PubMed

Fonseca, Dora Janeth; Ortega-Recalde, Oscar; Esteban-Perez, Clara; Moreno-Ortiz, Harold; Patiño, Liliana Catherine; Bermúdez, Olga María; Ortiz, Angela María; Restrepo, Carlos M; Lucena, Elkin; Laissue, Paul

2014-11-01

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

PubMed

Baillie, J Kenneth; Bretherick, Andrew; Haley, Christopher S; Clohisey, Sara; Gray, Alan; Neyton, Lucile P A; Barrett, Jeffrey; Stahl, Eli A; Tenesa, Albert; Andersson, Robin; Brown, J Ben; Faulkner, Geoffrey J; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Itoh, Masayoshi; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Mole, Damian; Bajic, Vladimir B; Heutink, Peter; Rehli, Michael; Kawaji, Hideya; Sandelin, Albin; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A; Hacohen, Nir; Freeman, Thomas C; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Hume, David A

2018-03-01

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease

PubMed Central

Gray, Alan; Neyton, Lucile P. A.; Barrett, Jeffrey; Stahl, Eli A.; Tenesa, Albert; Andersson, Robin; Brown, J. Ben; Faulkner, Geoffrey J.; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Kawaji, Hideya; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A.; Hacohen, Nir; Freeman, Thomas C.; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Hume, David A.

2018-01-01

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn’s disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits. PMID:29494619

Common genetic variants of the human UMOD gene are functional on transcription and predict plasma uric acid in two distinct populations

PubMed Central

Han, Jia; Liu, Ying; Rao, Fangwen; Nievergelt, Caroline M.; O’Connor, Daniel T.; Wang, Xingyu; Liu, Lisheng; Bu, Dingfang; Liang, Yu; Wang, Fang; Zhang, Luxia; Zhang, Hong; Chen, Yuqing; Wang, Haiyan

2013-01-01

Uromodulin (UMOD) genetic variants cause familial juvenile hyperuricemic nephropathy, characterized by hyperuricemia, decreased renal excretion of UMOD and uric acid; such findings suggest a role for UMOD in the regulation of plasma uric acid. We screened common variants across the UMOD locus in two populations, one from a community-based Chinese population, the other from California twins and siblings. Transcriptional activity of promoter variants was estimated in luciferase reporter plasmids transfected into HEK293 cells and mlMCD3 cells. By variance components in twin pairs, uric acid concentration and excretion were heritable traits. In the primary population from Beijing, we identified that carriers of haplotype GCC displayed higher plasma uric acid, and 3 UMOD promoter variants associated with plasma uric acid. UMOD promoter variants displayed reciprocal effects on urine uric acid excretion and plasma uric acid concentration, suggesting a primary effect on renal tubular handling of urate. These UMOD genetic marker-on-trait associations for uric acid were replicated in an independent American population sample. Site-directed mutagenesis at trait-associated UMOD promoter variants altered promoter activity in transfected luciferase reporter plasmids. These results suggest that UMOD promoter variants seem to initiate a cascade of transcriptional and biochemical changes influencing UMOD secretion, eventuating in elevation of plasma uric acid. PMID:23344472

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

Allele frequencies of variants in ultra conserved elements identify selective pressure on transcription factor binding.

PubMed

Silla, Toomas; Kepp, Katrin; Tai, E Shyong; Goh, Liang; Davila, Sonia; Catela Ivkovic, Tina; Calin, George A; Voorhoeve, P Mathijs

2014-01-01

Ultra-conserved genes or elements (UCGs/UCEs) in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations; Singaporean Chinese (n = 280) and Italian (n = 501) by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF<0.5%) of which 75% is not present in dbSNP137. UCEs association studies for complex human traits can use this information to model expected background variation and thus necessary power for association studies. By combining our data with 1000 Genome Project data, we show in three independent datasets that prevalent UCE variants (MAF>5%) are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level.

Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation.

PubMed

Miller, Clint L; Haas, Ulrike; Diaz, Roxanne; Leeper, Nicholas J; Kundu, Ramendra K; Patlolla, Bhagat; Assimes, Themistocles L; Kaiser, Frank J; Perisic, Ljubica; Hedin, Ulf; Maegdefessel, Lars; Schunkert, Heribert; Erdmann, Jeanette; Quertermous, Thomas; Sczakiel, Georg

2014-03-01

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3' untranslated region (3'-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3' UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.

Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

PubMed

Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A

2016-10-14

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

PubMed

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

PubMed Central

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

Discovery of novel transcripts of the human tissue kallikrein (KLK1) and kallikrein-related peptidase 2 (KLK2) in human cancer cells, exploiting Next-Generation Sequencing technology.

PubMed

Adamopoulos, Panagiotis G; Kontos, Christos K; Scorilas, Andreas

2018-03-31

Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). In addition, the expression profiles of each novel transcript were investigated with nested PCR experiments using variant-specific primers. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer. Copyright © 2018. Published by Elsevier Inc.

High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE

PubMed Central

Majoros, William H.; Campbell, Michael S.; Holt, Carson; DeNardo, Erin K.; Ware, Doreen; Allen, Andrew S.; Yandell, Mark; Reddy, Timothy E.

2017-01-01

Abstract Motivation: The accurate interpretation of genetic variants is critical for characterizing genotype–phenotype associations. Because the effects of genetic variants can depend strongly on their local genomic context, accurate genome annotations are essential. Furthermore, as some variants have the potential to disrupt or alter gene structure, variant interpretation efforts stand to gain from the use of individualized annotations that account for differences in gene structure between individuals or strains. Results: We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE (‘Assessing Changes to Exons’) converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detects gene-structure changes and their possible repercussions, and identifies several classes of possible loss of function. Novel transcripts predicted by ACE are commonly supported by spliced RNA-seq reads, and can be used to improve read alignment and transcript quantification when an individual-specific genome sequence is available. Using publicly available RNA-seq data, we show that ACE predictions confirm earlier results regarding the quantitative effects of nonsense-mediated decay, and we show that predicted loss-of-function events are highly concordant with patterns of intolerance to mutations across the human population. ACE can be readily applied to diverse species including animals and plants, making it a broadly useful tool for use in eukaryotic population-based resequencing projects, particularly for assessing the joint impact of all variants at a locus. Availability and Implementation: ACE is written in open-source C ++ and Perl and is available from geneprediction.org/ACE Contact: myandell@genetics.utah.edu or tim.reddy@duke.edu Supplementary information: Supplementary information is available at Bioinformatics online. PMID:28011790

High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE.

PubMed

Majoros, William H; Campbell, Michael S; Holt, Carson; DeNardo, Erin K; Ware, Doreen; Allen, Andrew S; Yandell, Mark; Reddy, Timothy E

2017-05-15

The accurate interpretation of genetic variants is critical for characterizing genotype-phenotype associations. Because the effects of genetic variants can depend strongly on their local genomic context, accurate genome annotations are essential. Furthermore, as some variants have the potential to disrupt or alter gene structure, variant interpretation efforts stand to gain from the use of individualized annotations that account for differences in gene structure between individuals or strains. We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE ('Assessing Changes to Exons') converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detects gene-structure changes and their possible repercussions, and identifies several classes of possible loss of function. Novel transcripts predicted by ACE are commonly supported by spliced RNA-seq reads, and can be used to improve read alignment and transcript quantification when an individual-specific genome sequence is available. Using publicly available RNA-seq data, we show that ACE predictions confirm earlier results regarding the quantitative effects of nonsense-mediated decay, and we show that predicted loss-of-function events are highly concordant with patterns of intolerance to mutations across the human population. ACE can be readily applied to diverse species including animals and plants, making it a broadly useful tool for use in eukaryotic population-based resequencing projects, particularly for assessing the joint impact of all variants at a locus. ACE is written in open-source C ++ and Perl and is available from geneprediction.org/ACE. myandell@genetics.utah.edu or tim.reddy@duke.edu. Supplementary information is available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Human Sex Determination at the Edge of Ambiguity

PubMed Central

Racca, Joseph D.; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B.; Weiss, Michael A.

2016-01-01

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent (“default”) cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. PMID:27576690

A new paradigm for transcription factor TFIIB functionality

PubMed Central

Gelev, Vladimir; Zabolotny, Janice M.; Lange, Martin; Hiromura, Makoto; Yoo, Sang Wook; Orlando, Joseph S.; Kushnir, Anna; Horikoshi, Nobuo; Paquet, Eric; Bachvarov, Dimcho; Schaffer, Priscilla A.; Usheva, Anny

2014-01-01

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects. PMID:24441171

Cis-Regulatory Variants Affect CHRNA5 mRNA Expression in Populations of African and European Ancestry

PubMed Central

Wang, Jen-Chyong; Spiegel, Noah; Bertelsen, Sarah; Le, Nhung; McKenna, Nicholas; Budde, John P.; Harari, Oscar; Kapoor, Manav; Brooks, Andrew; Hancock, Dana; Tischfield, Jay; Foroud, Tatiana; Bierut, Laura J.; Steinbach, Joe Henry; Edenberg, Howard J.; Traynor, Bryan J.; Goate, Alison M.

2013-01-01

Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels. PMID:24303001

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

PubMed

Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

2016-12-27

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer ( PCGEM1 , PEG3 , EPHA3 , and EFNB2 ) or other types of human cancers ( TOX3 , ST8SIA4 , and SLITRK3 ), and genes that are diagnostic/prognostic biomarkers of prostate cancer ( GRINA3 , and BCHE ).

Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

PubMed Central

Bhatia, Shipra; Gordon, Christopher T.; Foster, Robert G.; Melin, Lucie; Abadie, Véronique; Baujat, Geneviève; Vazquez, Marie-Paule; Amiel, Jeanne; Lyonnet, Stanislas; van Heyningen, Veronica; Kleinjan, Dirk A.

2015-01-01

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function. PMID:26030420

Cumulative role of rare and common putative functional genetic variants at NPAS3 in schizophrenia susceptibility.

PubMed

González-Peñas, Javier; Arrojo, Manuel; Paz, Eduardo; Brenlla, Julio; Páramo, Mario; Costas, Javier

2015-10-01

Schizophrenia may be considered a human-specific disorder arisen as a maladaptive by-product of human-specific brain evolution. Therefore, genetic variants involved in susceptibility to schizophrenia may be identified among those genes related to acquisition of human-specific traits. NPAS3, a transcription factor involved in central nervous system development and neurogenesis, seems to be implicated in the evolution of human brain, as it is the human gene with most human-specific accelerated elements (HAEs), i.e., .mammalian conserved regulatory sequences with accelerated evolution in the lineage leading to humans after human-chimpanzee split. We hypothesize that any nucleotide variant at the NPAS3 HAEs may lead to altered susceptibility to schizophrenia. Twenty-one variants at these HAEs detected by the 1000 genomes Project, as well as five additional variants taken from psychiatric genome-wide association studies, were genotyped in 538 schizophrenic patients and 539 controls from Galicia. Analyses at the haplotype level or based on the cumulative role of the variants assuming different susceptibility models did not find any significant association in spite of enough power under several plausible scenarios regarding direction of effect and the specific role of rare and common variants. These results suggest that, contrary to our hypothesis, the special evolution of the NPAS3 HAEs in Homo relaxed the strong constraint on sequence that characterized these regions during mammalian evolution, allowing some sequence changes without any effect on schizophrenia risk. © 2015 Wiley Periodicals, Inc.

Functional characterization of four naturally occurring variants of human pregnane X receptor (PXR): one variant causes dramatic loss of both DNA binding activity and the transactivation of the CYP3A4 promoter/enhancer region.

PubMed

Koyano, Satoru; Kurose, Kouichi; Saito, Yoshiro; Ozawa, Shogo; Hasegawa, Ryuichi; Komamura, Kazuo; Ueno, Kazuyuki; Kamakura, Shiro; Kitakaze, Masafumi; Nakajima, Toshiharu; Matsumoto, Kenji; Akasawa, Akira; Saito, Hirohisa; Sawada, Jun-Ichi

2004-01-01

Metabolism of administered drugs is determined by expression and activity of many drug-metabolizing enzymes, such as the cytochrome P450 (P450s) family members. Pregnane X receptor (PXR) is a master transcriptional regulator of many drug/xenobiotic-metabolizing enzymes, including P450s and drug transporters. In this study, we describe the functional analysis of four naturally occurring human PXR (hPXR) variants (R98C, R148Q, R381W, and I403V) that we have recently identified. By a reporter gene assay using the CYP3A4 promoter/enhancer reporter in COS-7 or HepG2 cells, it was found that the R98C variant failed to transactivate the CYP3A4 reporter. The R381W and I403V variants also showed varying degrees of reduction in transactivation, depending on the dose of PXR activators, rifampicin, clotrimazole, and paclitaxel. The transcriptional activities of the R148Q variant were not significantly different from that of the wild-type hPXR. The electrophoretic mobility shift assay revealed that only the R98C variant lacked DNA binding. Furthermore, the cellular localization of the hPXR proteins was analyzed. All four variants as well as the wild-type hPXR localized exclusively to the nucleus, regardless of the presence or absence of rifampicin. These data suggest that the R98C, R381W, and I403V hPXR variants, especially R98C, may influence the expression of drug-metabolizing enzymes and transporters, which are transactivated by PXR.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium.

PubMed

Torrado, Mario; Iglesias, Raquel; Nespereira, Beatriz; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T

2009-07-01

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

Identification of the thiamin pyrophosphokinase gene in rainbow trout: Characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

USGS Publications Warehouse

Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

2012-01-01

Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

PubMed

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-02-05

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

A high-quality human reference panel reveals the complexity and distribution of genomic structural variants.

PubMed

Hehir-Kwa, Jayne Y; Marschall, Tobias; Kloosterman, Wigard P; Francioli, Laurent C; Baaijens, Jasmijn A; Dijkstra, Louis J; Abdellaoui, Abdel; Koval, Vyacheslav; Thung, Djie Tjwan; Wardenaar, René; Renkens, Ivo; Coe, Bradley P; Deelen, Patrick; de Ligt, Joep; Lameijer, Eric-Wubbo; van Dijk, Freerk; Hormozdiari, Fereydoun; Uitterlinden, André G; van Duijn, Cornelia M; Eichler, Evan E; de Bakker, Paul I W; Swertz, Morris A; Wijmenga, Cisca; van Ommen, Gert-Jan B; Slagboom, P Eline; Boomsma, Dorret I; Schönhuth, Alexander; Ye, Kai; Guryev, Victor

2016-10-06

Structural variation (SV) represents a major source of differences between individual human genomes and has been linked to disease phenotypes. However, the majority of studies provide neither a global view of the full spectrum of these variants nor integrate them into reference panels of genetic variation. Here, we analyse whole genome sequencing data of 769 individuals from 250 Dutch families, and provide a haplotype-resolved map of 1.9 million genome variants across 9 different variant classes, including novel forms of complex indels, and retrotransposition-mediated insertions of mobile elements and processed RNAs. A large proportion are previously under reported variants sized between 21 and 100 bp. We detect 4 megabases of novel sequence, encoding 11 new transcripts. Finally, we show 191 known, trait-associated SNPs to be in strong linkage disequilibrium with SVs and demonstrate that our panel facilitates accurate imputation of SVs in unrelated individuals.

Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria.

PubMed

Hu, Liyan; Pandey, Amit V; Eggimann, Sandra; Rüfenacht, Véronique; Möslinger, Dorothea; Nuoffer, Jean-Marc; Häberle, Johannes

2013-11-29

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Exon 11 skipping of SCN10A coding for voltage-gated sodium channels in dorsal root ganglia

PubMed Central

Schirmeyer, Jana; Szafranski, Karol; Leipold, Enrico; Mawrin, Christian; Platzer, Matthias; Heinemann, Stefan H

2014-01-01

The voltage-gated sodium channel NaV1.8 (encoded by SCN10A) is predominantly expressed in dorsal root ganglia (DRG) and plays a critical role in pain perception. We analyzed SCN10A transcripts isolated from human DRGs using deep sequencing and found a novel splice variant lacking exon 11, which codes for 98 amino acids of the domain I/II linker. Quantitative PCR analysis revealed an abundance of this variant of up to 5–10% in human, while no such variants were detected in mouse or rat. Since no obvious functional differences between channels with and without the exon-11 sequence were detected, it is suggested that SCN10A exon 11 skipping in humans is a tolerated event. PMID:24763188

Profiling the array of Ca(v)3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations.

PubMed

Emerick, Mark C; Stein, Rebecca; Kunze, Robin; McNulty, Megan M; Regan, Melissa R; Hanck, Dorothy A; Agnew, William S

2006-08-01

We describe the regulated transcriptome of CACNA1G, a human gene for T-type Ca(v)3.1 calcium channels that is subject to extensive alternative RNA splicing. Fifteen sites of transcript variation include 2 alternative 5'-UTR promoter sites, 2 alternative 3'-UTR polyadenylation sites, and 11 sites of alternative splicing within the open reading frame. A survey of 1580 fetal and adult human brain full-length complementary DNAs reveals a family of 30 distinct transcripts, including multiple functional forms that vary in expression with development. Statistical analyses of fetal and adult transcript populations reveal patterns of linkages among intramolecular splice site configurations that change dramatically with development. A shift from nearly independent, biased splicing in fetal transcripts to strongly concerted splicing in adult transcripts suggests progressive activation of multiple "programs" of splicing regulation that reorganize molecular structures in differentiating cells. Patch-clamp studies of nine selected variants help relate splicing regulation to permutations of the gating parameters most likely to modify T-channel physiology in expressing neurons. Gating behavior reflects combinatorial interactions between variable domains so that molecular phenotype depends on ensembles of coselected domains, consistent with the observed emergence of concerted splicing during development. We conclude that the structural gene and networks of splicing regulatory factors define an integrated system for the phenotypic variation of Ca(v)3.1 biophysics during nervous system development. Copyright 2006 Wiley-Liss, Inc.

viGEN: An Open Source Pipeline for the Detection and Quantification of Viral RNA in Human Tumors.

PubMed

Bhuvaneshwar, Krithika; Song, Lei; Madhavan, Subha; Gusev, Yuriy

2018-01-01

An estimated 17% of cancers worldwide are associated with infectious causes. The extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using human transcriptome (RNA-seq) data from tumor samples. We present an open source bioinformatics pipeline viGEN, which allows for not only the detection and quantification of viral RNA, but also variants in the viral transcripts. The pipeline includes 4 major modules: The first module aligns and filter out human RNA sequences; the second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module quantifies read counts at the individual viral-gene level thus allowing for downstream differential expression analysis of viral genes between case and controls groups. The fourth module calls variants in these viruses. To the best of our knowledge, there are no publicly available pipelines or packages that would provide this type of complete analysis in one open source package. In this paper, we applied the viGEN pipeline to two case studies. We first demonstrate the working of our pipeline on a large public dataset, the TCGA cervical cancer cohort. In the second case study, we performed an in-depth analysis on a small focused study of TCGA liver cancer patients. In the latter cohort, we performed viral-gene quantification, viral-variant extraction and survival analysis. This allowed us to find differentially expressed viral-transcripts and viral-variants between the groups of patients, and connect them to clinical outcome. From our analyses, we show that we were able to successfully detect the human papilloma virus among the TCGA cervical cancer patients. We compared the viGEN pipeline with two metagenomics tools and demonstrate similar sensitivity/specificity. We were also able to quantify viral-transcripts and extract viral-variants using the liver cancer dataset. The results presented corresponded with published literature in terms of rate of detection, and impact of several known variants of HBV genome. This pipeline is generalizable, and can be used to provide novel biological insights into microbial infections in complex diseases and tumorigeneses. Our viral pipeline could be used in conjunction with additional type of immuno-oncology analysis based on RNA-seq data of host RNA for cancer immunology applications. The source code, with example data and tutorial is available at: https://github.com/ICBI/viGEN/.

Homo sapiens-Specific Binding Site Variants within Brain Exclusive Enhancers Are Subject to Accelerated Divergence across Human Population.

PubMed

Zehra, Rabail; Abbasi, Amir Ali

2018-03-01

Empirical assessments of human accelerated noncoding DNA frgaments have delineated presence of many cis-regulatory elements. Enhancers make up an important category of such accelerated cis-regulatory elements that efficiently control the spatiotemporal expression of many developmental genes. Establishing plausible reasons for accelerated enhancer sequence divergence in Homo sapiens has been termed significant in various previously published studies. This acceleration by including closely related primates and archaic human data has the potential to open up evolutionary avenues for deducing present-day brain structure. This study relied on empirically confirmed brain exclusive enhancers to avoid any misjudgments about their regulatory status and categorized among them a subset of enhancers with an exceptionally accelerated rate of lineage specific divergence in humans. In this assorted set, 13 distinct transcription factor binding sites were located that possessed unique existence in humans. Three of 13 such sites belonging to transcription factors SOX2, RUNX1/3, and FOS/JUND possessed single nucleotide variants that made them unique to H. sapiens upon comparisons with Neandertal and Denisovan orthologous sequences. These variants modifying the binding sites in modern human lineage were further substantiated as single nucleotide polymorphisms via exploiting 1000 Genomes Project Phase3 data. Long range haplotype based tests laid out evidence of positive selection to be governing in African population on two of the modern human motif modifying alleles with strongest results for SOX2 binding site. In sum, our study acknowledges acceleration in noncoding regulatory landscape of the genome and highlights functional parts within it to have undergone accelerated divergence in present-day human population.

Jannovar: a java library for exome annotation.

PubMed

Jäger, Marten; Wang, Kai; Bauer, Sebastian; Smedley, Damian; Krawitz, Peter; Robinson, Peter N

2014-05-01

Transcript-based annotation and pedigree analysis are two basic steps in the computational analysis of whole-exome sequencing experiments in genetic diagnostics and disease-gene discovery projects. Here, we present Jannovar, a stand-alone Java application as well as a Java library designed to be used in larger software frameworks for exome and genome analysis. Jannovar uses an interval tree to identify all transcripts affected by a given variant, and provides Human Genome Variation Society-compliant annotations both for variants affecting coding sequences and splice junctions as well as untranslated regions and noncoding RNA transcripts. Jannovar can also perform family-based pedigree analysis with Variant Call Format (VCF) files with data from members of a family segregating a Mendelian disorder. Using a desktop computer, Jannovar requires a few seconds to annotate a typical VCF file with exome data. Jannovar is freely available under the BSD2 license. Source code as well as the Java application and library file can be downloaded from http://compbio.charite.de (with tutorial) and https://github.com/charite/jannovar. © 2014 WILEY PERIODICALS, INC.

P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

PubMed

Miller, Walter L

2012-10-23

Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

A Syndromic Neurodevelopmental Disorder Caused by De Novo Variants in EBF3.

PubMed

Chao, Hsiao-Tuan; Davids, Mariska; Burke, Elizabeth; Pappas, John G; Rosenfeld, Jill A; McCarty, Alexandra J; Davis, Taylor; Wolfe, Lynne; Toro, Camilo; Tifft, Cynthia; Xia, Fan; Stong, Nicholas; Johnson, Travis K; Warr, Coral G; Yamamoto, Shinya; Adams, David R; Markello, Thomas C; Gahl, William A; Bellen, Hugo J; Wangler, Michael F; Malicdan, May Christine V

2017-01-05

Early B cell factor 3 (EBF3) is a member of the highly evolutionarily conserved Collier/Olf/EBF (COE) family of transcription factors. Prior studies on invertebrate and vertebrate animals have shown that EBF3 homologs are essential for survival and that loss-of-function mutations are associated with a range of nervous system developmental defects, including perturbation of neuronal development and migration. Interestingly, aristaless-related homeobox (ARX), a homeobox-containing transcription factor critical for the regulation of nervous system development, transcriptionally represses EBF3 expression. However, human neurodevelopmental disorders related to EBF3 have not been reported. Here, we describe three individuals who are affected by global developmental delay, intellectual disability, and expressive speech disorder and carry de novo variants in EBF3. Associated features seen in these individuals include congenital hypotonia, structural CNS malformations, ataxia, and genitourinary abnormalities. The de novo variants affect a single conserved residue in a zinc finger motif crucial for DNA binding and are deleterious in a fly model. Our findings indicate that mutations in EBF3 cause a genetic neurodevelopmental syndrome and suggest that loss of EBF3 function might mediate a subset of neurologic phenotypes shared by ARX-related disorders, including intellectual disability, abnormal genitalia, and structural CNS malformations. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

PubMed Central

Uzbekova, Svetlana; Roy-Sabau, Monica; Dalbiès-Tran, Rozenn; Perreau, Christine; Papillier, Pascal; Mompart, Florence; Thelie, Aurore; Pennetier, Sophie; Cognie, Juliette; Cadoret, Veronique; Royere, Dominique; Monget, Philippe; Mermillod, Pascal

2006-01-01

Background Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. Methods Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. Results We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. Conclusion Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes. PMID:16551357

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

PubMed Central

Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A.; Koyanagi, Kanako O.; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

2006-01-01

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

PubMed

Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

2014-10-03

The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of human placental lactogen and variant growth hormone genes in placentas.

PubMed

Martinez-Rodriguez, H G; Guerra-Rodriguez, N E; Iturbe-Cantu, M A; Martinez-Torres, A; Barrera-Saldaña, H A

1997-01-01

Previous studies comparing the expression levels of human placental lactogen (hPL) genes have shown varying results, due to, perhaps, the fact that in all of them only one placenta was being analyzed. Here, the expression of hPL and growth hormone variant (hGH-V) genes in fifteen term placentas was comparatively analyzed at the RNA level, using reverse transcription coupled to polymerase chain reaction (RT-PCR). The abundance of the combined RNA transcripts derived from these genes varied from one placenta to another. The authors found that hPL-4 transcripts were more abundant than those of hPL-3 in most samples (ratios from 1:1 to 6:1), transcripts from the putative hPL-1 pseudogene were more abundant at the unprocessed stage while those of the hGH-V gene were mostly processed. Again, the authors of this study observed wide variation from placenta to placenta in the abundance of both of these types of transcripts. The same was observed when a group of six placentas from abortuses and nine from pregnancies complicated by preclampsia, diabetes and hypertension was studied. The authors conclude that the disagreeing results reported in the literature which are not in agreement concerning the expression levels of hPL genes could be explained by normal variations of their expression levels among the different placentas analyzed.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

PubMed

Wilson, Thomas E; Arlt, Martin F; Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W

2015-02-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. © 2015 Wilson et al.; Published by Cold Spring Harbor Laboratory Press.

Large transcription units unify copy number variants and common fragile sites arising under replication stress

PubMed Central

Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W.

2015-01-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. PMID:25373142

Novel Variants in ZNF34 and Other Brain-Expressed Transcription Factors are Shared Among Early-Onset MDD Relatives

PubMed Central

Subaran, Ryan L.; Odgerel, Zagaa; Swaminathan, Rajeswari; Glatt, Charles E.; Weissman, Myrna M.

2018-01-01

There are no known genetic variants with large effects on susceptibility to major depressive disorder (MDD). Although one proposed study approach is to increase sensitivity by increasing sample sizes, another is to focus on families with multiple affected individuals to identify genes with rare or novel variants with strong effects. Choosing the family-based approach, we performed whole-exome analysis on affected individuals (n = 12) across five MDD families, each with at least five affected individuals, early onset, and prepubertal diagnoses. We identified 67 genes where novel deleterious variants were shared among affected relatives. Gene ontology analysis shows that of these 67 genes, 18 encode transcriptional regulators, eight of which are expressed in the human brain, including four KRAB-A box-containing Zn2+ finger repressors. One of these, ZNF34, has been reported as being associated with bipolar disorder and as differentially expressed in bipolar disorder patients compared to healthy controls. We found a novel variant—encoding a non-conservative P17R substitution in the conserved repressor domain of ZNF34 protein—segregating completely with MDD in all available individuals in the family in which it was discovered. Further analysis showed a common ZNF34 coding indel segregating with MDD in a separate family, possibly indicating the presence of an unobserved, linked, rare variant in that particular family. Our results indicate that genes encoding transcription factors expressed in the brain might be an important group of MDD candidate genes and that rare variants in ZNF34 might contribute to susceptibility to MDD and perhaps other affective disorders. PMID:26823146

Spleen-dependent regulation of antigenic variation in malaria parasites: Plasmodium knowlesi SICAvar expression profiles in splenic and asplenic hosts.

PubMed

Lapp, Stacey A; Korir-Morrison, Cindy; Jiang, Jianlin; Bai, Yaohui; Corredor, Vladimir; Galinski, Mary R

2013-01-01

Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera. We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry. SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

PubMed Central

Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

2016-01-01

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

Association of levels of fasting glucose and insulin with rare variants at the chromosome 11p11.2-MADD locus: Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study.

PubMed

Cornes, Belinda K; Brody, Jennifer A; Nikpoor, Naghmeh; Morrison, Alanna C; Chu, Huan; Ahn, Byung Soo; Wang, Shuai; Dauriz, Marco; Barzilay, Joshua I; Dupuis, Josée; Florez, Jose C; Coresh, Josef; Gibbs, Richard A; Kao, W H Linda; Liu, Ching-Ti; McKnight, Barbara; Muzny, Donna; Pankow, James S; Reid, Jeffrey G; White, Charles C; Johnson, Andrew D; Wong, Tien Y; Psaty, Bruce M; Boerwinkle, Eric; Rotter, Jerome I; Siscovick, David S; Sladek, Robert; Meigs, James B

2014-06-01

Common variation at the 11p11.2 locus, encompassing MADD, ACP2, NR1H3, MYBPC3, and SPI1, has been associated in genome-wide association studies with fasting glucose and insulin (FI). In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study, we sequenced 5 gene regions at 11p11.2 to identify rare, potentially functional variants influencing fasting glucose or FI levels. Sequencing (mean depth, 38×) across 16.1 kb in 3566 individuals without diabetes mellitus identified 653 variants, 79.9% of which were rare (minor allele frequency <1%) and novel. We analyzed rare variants in 5 gene regions with FI or fasting glucose using the sequence kernel association test. At NR1H3, 53 rare variants were jointly associated with FI (P=2.73×10(-3)); of these, 7 were predicted to have regulatory function and showed association with FI (P=1.28×10(-3)). Conditioning on 2 previously associated variants at MADD (rs7944584, rs10838687) did not attenuate this association, suggesting that there are >2 independent signals at 11p11.2. One predicted regulatory variant, chr11:47227430 (hg18; minor allele frequency=0.00068), contributed 20.6% to the overall sequence kernel association test score at NR1H3, lies in intron 2 of NR1H3, and is a predicted binding site for forkhead box A1 (FOXA1), a transcription factor associated with insulin regulation. In human HepG2 hepatoma cells, the rare chr11:47227430 A allele disrupted FOXA1 binding and reduced FOXA1-dependent transcriptional activity. Sequencing at 11p11.2-NR1H3 identified rare variation associated with FI. One variant, chr11:47227430, seems to be functional, with the rare A allele reducing transcription factor FOXA1 binding and FOXA1-dependent transcriptional activity. © 2014 American Heart Association, Inc.

Mutations in EBF3 Disturb Transcriptional Profiles and Cause Intellectual Disability, Ataxia, and Facial Dysmorphism.

PubMed

Harms, Frederike Leonie; Girisha, Katta M; Hardigan, Andrew A; Kortüm, Fanny; Shukla, Anju; Alawi, Malik; Dalal, Ashwin; Brady, Lauren; Tarnopolsky, Mark; Bird, Lynne M; Ceulemans, Sophia; Bebin, Martina; Bowling, Kevin M; Hiatt, Susan M; Lose, Edward J; Primiano, Michelle; Chung, Wendy K; Juusola, Jane; Akdemir, Zeynep C; Bainbridge, Matthew; Charng, Wu-Lin; Drummond-Borg, Margaret; Eldomery, Mohammad K; El-Hattab, Ayman W; Saleh, Mohammed A M; Bézieau, Stéphane; Cogné, Benjamin; Isidor, Bertrand; Küry, Sébastien; Lupski, James R; Myers, Richard M; Cooper, Gregory M; Kutsche, Kerstin

2017-01-05

From a GeneMatcher-enabled international collaboration, we identified ten individuals affected by intellectual disability, speech delay, ataxia, and facial dysmorphism and carrying a deleterious EBF3 variant detected by whole-exome sequencing. One 9-bp duplication and one splice-site, five missense, and two nonsense variants in EBF3 were found; the mutations occurred de novo in eight individuals, and the missense variant c.625C>T (p.Arg209Trp) was inherited by two affected siblings from their healthy mother, who is mosaic. EBF3 belongs to the early B cell factor family (also known as Olf, COE, or O/E) and is a transcription factor involved in neuronal differentiation and maturation. Structural assessment predicted that the five amino acid substitutions have damaging effects on DNA binding of EBF3. Transient expression of EBF3 mutant proteins in HEK293T cells revealed mislocalization of all but one mutant in the cytoplasm, as well as nuclear localization. By transactivation assays, all EBF3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene CDKN1A, and in situ subcellular fractionation experiments demonstrated that EBF3 mutant proteins were less tightly associated with chromatin. Finally, in RNA-seq and ChIP-seq experiments, EBF3 acted as a transcriptional regulator, and mutant EBF3 had reduced genome-wide DNA binding and gene-regulatory activity. Our findings demonstrate that variants disrupting EBF3-mediated transcriptional regulation cause intellectual disability and developmental delay and are present in ∼0.1% of individuals with unexplained neurodevelopmental disorders. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

regSNPs: a strategy for prioritizing regulatory single nucleotide substitutions

PubMed Central

Teng, Mingxiang; Ichikawa, Shoji; Padgett, Leah R.; Wang, Yadong; Mort, Matthew; Cooper, David N.; Koller, Daniel L.; Foroud, Tatiana; Edenberg, Howard J.; Econs, Michael J.; Liu, Yunlong

2012-01-01

Motivation: One of the fundamental questions in genetics study is to identify functional DNA variants that are responsible to a disease or phenotype of interest. Results from large-scale genetics studies, such as genome-wide association studies (GWAS), and the availability of high-throughput sequencing technologies provide opportunities in identifying causal variants. Despite the technical advances, informatics methodologies need to be developed to prioritize thousands of variants for potential causative effects. Results: We present regSNPs, an informatics strategy that integrates several established bioinformatics tools, for prioritizing regulatory SNPs, i.e. the SNPs in the promoter regions that potentially affect phenotype through changing transcription of downstream genes. Comparing to existing tools, regSNPs has two distinct features. It considers degenerative features of binding motifs by calculating the differences on the binding affinity caused by the candidate variants and integrates potential phenotypic effects of various transcription factors. When tested by using the disease-causing variants documented in the Human Gene Mutation Database, regSNPs showed mixed performance on various diseases. regSNPs predicted three SNPs that can potentially affect bone density in a region detected in an earlier linkage study. Potential effects of one of the variants were validated using luciferase reporter assay. Contact: yunliu@iupui.edu Supplementary information: Supplementary data are available at Bioinformatics online PMID:22611130

Differential HFE Gene Expression Is Regulated by Alternative Splicing in Human Tissues

PubMed Central

Proença, Daniela; Faustino, Paula

2011-01-01

Background The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum. PMID:21407826

Differential HFE gene expression is regulated by alternative splicing in human tissues.

PubMed

Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, Paula

2011-03-03

The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.

Genetic Variants in Transcription Factors Are Associated With the Pharmacokinetics and Pharmacodynamics of Metformin

PubMed Central

Goswami, S; Yee, SW; Stocker, S; Mosley, JD; Kubo, M; Castro, R; Mefford, JA; Wen, C; Liang, X; Witte, J; Brett, C; Maeda, S; Simpson, MD; Hedderson, MM; Davis, RL; Roden, DM; Giacomini, KM; Savic, RM

2014-01-01

One-third of type 2 diabetes patients do not respond to metformin. Genetic variants in metformin transporters have been extensively studied as a likely contributor to this high failure rate. Here, we investigate, for the first time, the effect of genetic variants in transcription factors on metformin pharmacokinetics (PK) and response. Overall, 546 patients and healthy volunteers contributed their genome-wide, pharmacokinetic (235 subjects), and HbA1c data (440 patients) for this analysis. Five variants in specificity protein 1 (SP1), a transcription factor that modulates the expression of metformin transporters, were associated with changes in treatment HbA1c (P < 0.01) and metformin secretory clearance (P < 0.05). Population pharmacokinetic modeling further confirmed a 24% reduction in apparent clearance in homozygous carriers of one such variant, rs784888. Genetic variants in other transcription factors, peroxisome proliferator–activated receptor-α and hepatocyte nuclear factor 4-α, were significantly associated with HbA1c change only. Overall, our study highlights the importance of genetic variants in transcription factors as modulators of metformin PK and response. PMID:24853734

An environment-dependent transcriptional network specifies human microglia identity.

PubMed

Gosselin, David; Skola, Dylan; Coufal, Nicole G; Holtman, Inge R; Schlachetzki, Johannes C M; Sajti, Eniko; Jaeger, Baptiste N; O'Connor, Carolyn; Fitzpatrick, Conor; Pasillas, Martina P; Pena, Monique; Adair, Amy; Gonda, David D; Levy, Michael L; Ransohoff, Richard M; Gage, Fred H; Glass, Christopher K

2017-06-23

Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Two Novel Variants Affecting CDKL5 Transcript Associated with Epileptic Encephalopathy.

PubMed

Neupauerová, Jana; Štěrbová, Katalin; Vlčková, Markéta; Sebroňová, Věra; Maříková, Tat'ána; Krůtová, Marcela; David, Staněk; Kršek, Pavel; Žaliová, Markéta; Seeman, Pavel; Laššuthová, Petra

2017-10-01

Variants in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been reported as being etiologically associated with early infantile epileptic encephalopathy type 2 (EIEE2). We report on two patients, a boy and a girl, with EIEE2 that present with early onset epilepsy, hypotonia, severe intellectual disability, and poor eye contact. Massively parallel sequencing (MPS) of a custom-designed gene panel for epilepsy and epileptic encephalopathy containing 112 epilepsy-related genes was performed. Sanger sequencing was used to confirm the novel variants. For confirmation of the functional consequence of an intronic CDKL5 variant in patient 2, an RNA study was done. DNA sequencing revealed de novo variants in CDKL5, a c.2578C>T (p. Gln860*) present in a hemizygous state in a 3-year-old boy, and a potential splice site variant c.463+5G>A in heterozygous state in a 5-year-old girl. Multiple in silico splicing algorithms predicted a highly reduced splice site score for c.463+5G>A. A subsequent mRNA study confirmed an aberrant shorter transcript lacking exon 7. Our data confirmed that variants in the CDKL5 are associated with EIEE2. There is credible evidence that the novel identified variants are pathogenic and, therefore, are likely the cause of the disease in the presented patients. In one of the patients a stop codon variant is predicted to produce a truncated protein, and in the other patient an intronic variant results in aberrant splicing.

Network perturbation by recurrent regulatory variants in cancer

PubMed Central

Cho, Ara; Lee, Insuk; Choi, Jung Kyoon

2017-01-01

Cancer driving genes have been identified as recurrently affected by variants that alter protein-coding sequences. However, a majority of cancer variants arise in noncoding regions, and some of them are thought to play a critical role through transcriptional perturbation. Here we identified putative transcriptional driver genes based on combinatorial variant recurrence in cis-regulatory regions. The identified genes showed high connectivity in the cancer type-specific transcription regulatory network, with high outdegree and many downstream genes, highlighting their causative role during tumorigenesis. In the protein interactome, the identified transcriptional drivers were not as highly connected as coding driver genes but appeared to form a network module centered on the coding drivers. The coding and regulatory variants associated via these interactions between the coding and transcriptional drivers showed exclusive and complementary occurrence patterns across tumor samples. Transcriptional cancer drivers may act through an extensive perturbation of the regulatory network and by altering protein network modules through interactions with coding driver genes. PMID:28333928

Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas.

PubMed

Bjørklund, Sunniva Stordal; Kristensen, Vessela N; Seiler, Michael; Kumar, Surendra; Alnæs, Grethe I Grenaker; Ming, Yao; Kerrigan, John; Naume, Bjørn; Sachidanandam, Ravi; Bhanot, Gyan; Børresen-Dale, Anne-Lise; Ganesan, Shridar

2015-07-17

Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.

Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zacapala-Gómez, Ana Elvira, E-mail: zak_ana@yahoo.com.mx; Del Moral-Hernández, Oscar, E-mail: odelmoralh@gmail.com; Villegas-Sepúlveda, Nicolás, E-mail: nvillega@cinvestav.mx

We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such asmore » adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.« less

Co-expression networks reveal the tissue-specific regulation of transcription and splicing

PubMed Central

Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D.H.; Jo, Brian; Gao, Chuan; McDowell, Ian C.; Engelhardt, Barbara E.

2017-01-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. PMID:29021288

Divergent Ah Receptor Ligand Selectivity during Hominin Evolution

PubMed Central

Hubbard, Troy D.; Murray, Iain A.; Bisson, William H.; Sullivan, Alexis P.; Sebastian, Aswathy; Perry, George H.; Jablonski, Nina G.; Perdew, Gary H.

2016-01-01

We have identified a fixed nonsynonymous sequence difference between humans (Val381; derived variant) and Neandertals (Ala381; ancestral variant) in the ligand-binding domain of the aryl hydrocarbon receptor (AHR) gene. In an exome sequence analysis of four Neandertal and Denisovan individuals compared with nine modern humans, there are only 90 total nucleotide sites genome-wide for which archaic hominins are fixed for the ancestral nonsynonymous variant and the modern humans are fixed for the derived variant. Of those sites, only 27, including Val381 in the AHR, also have no reported variability in the human dbSNP database, further suggesting that this highly conserved functional variant is a rare event. Functional analysis of the amino acid variant Ala381 within the AHR carried by Neandertals and nonhuman primates indicate enhanced polycyclic aromatic hydrocarbon (PAH) binding, DNA binding capacity, and AHR mediated transcriptional activity compared with the human AHR. Also relative to human AHR, the Neandertal AHR exhibited 150–1000 times greater sensitivity to induction of Cyp1a1 and Cyp1b1 expression by PAHs (e.g., benzo(a)pyrene). The resulting CYP1A1/CYP1B1 enzymes are responsible for PAH first pass metabolism, which can result in the generation of toxic intermediates and perhaps AHR-associated toxicities. In contrast, the human AHR retains the ancestral sensitivity observed in primates to nontoxic endogenous AHR ligands (e.g., indole, indoxyl sulfate). Our findings reveal that a functionally significant change in the AHR occurred uniquely in humans, relative to other primates, that would attenuate the response to many environmental pollutants, including chemicals present in smoke from fire use during cooking. PMID:27486223

ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

PubMed Central

Harris, Rebecca Louise; van den Berg, Carmen Wilma; Bowen, Derrick John

2012-01-01

Background. The asialoglycoprotein receptor (ASGPR) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR), expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver. PMID:22919488

GWAS4D: multidimensional analysis of context-specific regulatory variant for human complex diseases and traits.

PubMed

Huang, Dandan; Yi, Xianfu; Zhang, Shijie; Zheng, Zhanye; Wang, Panwen; Xuan, Chenghao; Sham, Pak Chung; Wang, Junwen; Li, Mulin Jun

2018-05-16

Genome-wide association studies have generated over thousands of susceptibility loci for many human complex traits, and yet for most of these associations the true causal variants remain unknown. Tissue/cell type-specific prediction and prioritization of non-coding regulatory variants will facilitate the identification of causal variants and underlying pathogenic mechanisms for particular complex diseases and traits. By leveraging recent large-scale functional genomics/epigenomics data, we develop an intuitive web server, GWAS4D (http://mulinlab.tmu.edu.cn/gwas4d or http://mulinlab.org/gwas4d), that systematically evaluates GWAS signals and identifies context-specific regulatory variants. The updated web server includes six major features: (i) updates the regulatory variant prioritization method with our new algorithm; (ii) incorporates 127 tissue/cell type-specific epigenomes data; (iii) integrates motifs of 1480 transcriptional regulators from 13 public resources; (iv) uniformly processes Hi-C data and generates significant interactions at 5 kb resolution across 60 tissues/cell types; (v) adds comprehensive non-coding variant functional annotations; (vi) equips a highly interactive visualization function for SNP-target interaction. Using a GWAS fine-mapped set for 161 coronary artery disease risk loci, we demonstrate that GWAS4D is able to efficiently prioritize disease-causal regulatory variants.

Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants.

PubMed

Pasquali, Lorenzo; Gaulton, Kyle J; Rodríguez-Seguí, Santiago A; Mularoni, Loris; Miguel-Escalada, Irene; Akerman, İldem; Tena, Juan J; Morán, Ignasi; Gómez-Marín, Carlos; van de Bunt, Martijn; Ponsa-Cobas, Joan; Castro, Natalia; Nammo, Takao; Cebola, Inês; García-Hurtado, Javier; Maestro, Miguel Angel; Pattou, François; Piemonti, Lorenzo; Berney, Thierry; Gloyn, Anna L; Ravassard, Philippe; Skarmeta, José Luis Gómez; Müller, Ferenc; McCarthy, Mark I; Ferrer, Jorge

2014-02-01

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Genetic Differences in the Immediate Transcriptome Response to Stress Predict Risk-Related Brain Function and Psychiatric Disorders

PubMed Central

Arloth, Janine; Bogdan, Ryan; Weber, Peter; Frishman, Goar; Menke, Andreas; Wagner, Klaus V.; Balsevich, Georgia; Schmidt, Mathias V.; Karbalai, Nazanin; Czamara, Darina; Altmann, Andre; Trümbach, Dietrich; Wurst, Wolfgang; Mehta, Divya; Uhr, Manfred; Klengel, Torsten; Erhardt, Angelika; Carey, Caitlin E.; Conley, Emily Drabant; Ripke, Stephan; Wray, Naomi R.; Lewis, Cathryn M.; Hamilton, Steven P.; Weissman, Myrna M.; Breen, Gerome; Byrne, Enda M.; Blackwood, Douglas H.R.; Boomsma, Dorret I.; Cichon, Sven; Heath, Andrew C.; Holsboer, Florian; Lucae, Susanne; Madden, Pamela A.F.; Martin, Nicholas G.; McGuffin, Peter; Muglia, Pierandrea; Noethen, Markus M.; Penninx, Brenda P.; Pergadia, Michele L.; Potash, James B.; Rietschel, Marcella; Lin, Danyu; Müller-Myhsok, Bertram; Shi, Jianxin; Steinberg, Stacy; Grabe, Hans J.; Lichtenstein, Paul; Magnusson, Patrik; Perlis, Roy H.; Preisig, Martin; Smoller, Jordan W.; Stefansson, Kari; Uher, Rudolf; Kutalik, Zoltan; Tansey, Katherine E.; Teumer, Alexander; Viktorin, Alexander; Barnes, Michael R.; Bettecken, Thomas; Binder, Elisabeth B.; Breuer, René; Castro, Victor M.; Churchill, Susanne E.; Coryell, William H.; Craddock, Nick; Craig, Ian W.; Czamara, Darina; De Geus, Eco J.; Degenhardt, Franziska; Farmer, Anne E.; Fava, Maurizio; Frank, Josef; Gainer, Vivian S.; Gallagher, Patience J.; Gordon, Scott D.; Goryachev, Sergey; Gross, Magdalena; Guipponi, Michel; Henders, Anjali K.; Herms, Stefan; Hickie, Ian B.; Hoefels, Susanne; Hoogendijk, Witte; Hottenga, Jouke Jan; Iosifescu, Dan V.; Ising, Marcus; Jones, Ian; Jones, Lisa; Jung-Ying, Tzeng; Knowles, James A.; Kohane, Isaac S.; Kohli, Martin A.; Korszun, Ania; Landen, Mikael; Lawson, William B.; Lewis, Glyn; MacIntyre, Donald; Maier, Wolfgang; Mattheisen, Manuel; McGrath, Patrick J.; McIntosh, Andrew; McLean, Alan; Middeldorp, Christel M.; Middleton, Lefkos; Montgomery, Grant M.; Murphy, Shawn N.; Nauck, Matthias; Nolen, Willem A.; Nyholt, Dale R.; O’Donovan, Michael; Oskarsson, Högni; Pedersen, Nancy; Scheftner, William A.; Schulz, Andrea; Schulze, Thomas G.; Shyn, Stanley I.; Sigurdsson, Engilbert; Slager, Susan L.; Smit, Johannes H.; Stefansson, Hreinn; Steffens, Michael; Thorgeirsson, Thorgeir; Tozzi, Federica; Treutlein, Jens; Uhr, Manfred; van den Oord, Edwin J.C.G.; Van Grootheest, Gerard; Völzke, Henry; Weilburg, Jeffrey B.; Willemsen, Gonneke; Zitman, Frans G.; Neale, Benjamin; Daly, Mark; Levinson, Douglas F.; Sullivan, Patrick F.; Ruepp, Andreas; Müller-Myhsok, Bertram; Hariri, Ahmad R.; Binder, Elisabeth B.

2015-01-01

Summary Depression risk is exacerbated by genetic factors and stress exposure; however, the biological mechanisms through which these factors interact to confer depression risk are poorly understood. One putative biological mechanism implicates variability in the ability of cortisol, released in response to stress, to trigger a cascade of adaptive genomic and non-genomic processes through glucocorticoid receptor (GR) activation. Here, we demonstrate that common genetic variants in long-range enhancer elements modulate the immediate transcriptional response to GR activation in human blood cells. These functional genetic variants increase risk for depression and co-heritable psychiatric disorders. Moreover, these risk variants are associated with inappropriate amygdala reactivity, a transdiagnostic psychiatric endophenotype and an important stress hormone response trigger. Network modeling and animal experiments suggest that these genetic differences in GR-induced transcriptional activation may mediate the risk for depression and other psychiatric disorders by altering a network of functionally related stress-sensitive genes in blood and brain. Video Abstract PMID:26050039

Isolation of genes negatively or positively co-expressed with human recombination activating gene 1 (RAG1) by differential display PCR (DD RT-PCR).

PubMed

Verkoczy, L K; Berinstein, N L

1998-10-01

Differential display PCR (DD RT-PCR) has been extensively used for analysis of differential gene expression, but continues to be hampered by technical limitations that impair its effectiveness. In order to isolate novel genes co-expressing with human RAG1, we have developed an effective, multi-tiered screening/purification approach which effectively complements the standard DD RT-PCR methodology. In 'primary' screens, standard DD RT-PCR was used, detecting 22 reproducible differentially expressed amplicons between clonally related cell variants with differential constitutive expression of RAG mRNAs. 'Secondary' screens used differential display (DD) amplicons as probes in low and high stringency northern blotting. Eight of 22 independent DD amplicons detected nine independent differentially expressed transcripts. 'Tertiary' screens used reconfirmed amplicons as probes in northern analysis of multiple RAG-and RAG+sources. Reconfirmed DD amplicons detected six independent RAG co-expressing transcripts. All DD amplicons reconfirmed by northern blot were a heterogeneous mixture of cDNAs, necessitating further purification to isolate single cDNAs prior to subcloning and sequencing. To effectively select the appropriate cDNAs from DD amplicons, we excised and eluted the cDNA(s) directly from regions of prior northern blots in which differentially expressed transcripts were detected. Sequences of six purified cDNA clones specifically detecting RAG co-expressing transcripts included matches to portions of the human RAG2 and BSAP regions and to four novel partial cDNAs (three with homologies to human ESTs). Overall, our results also suggest that even when using clonally related variants from the same cell line in addition to all appropriate internal controls previously reported, further screening and purification steps are still required in order to efficiently and specifically isolate differentially expressed genes by DD RT-PCR.

Methylation of CIITA promoter IV causes loss of HLA-II inducibility by IFN-γ in promyelocytic cells

PubMed Central

De Ambrosis, Alessandro; Banelli, Barbara; Pira, Giuseppina Li; Aresu, Ottavia; Romani, Massimo; Ferrini, Silvano; Accolla, Roberto S.

2008-01-01

The human promyelocytic cell line THP-1 expresses high level of HLA class II (HLA-II) molecules after IFN-γ treatment. Here, we report a variant of THP-1 that does not express HLA-II after IFN-γ. The variant's HLA-II phenotype is constant over time in culture and it is not related to a defective IFN-γ-signalling pathway. Transfection of CIITA, the HLA-II transcriptional activator, under the control of a cytomegalovirus promoter rescues high level of HLA-DR surface expression in the variant indicating that the biosynthetic block resides in the expression of CIITA and not in the CIITA-dependent transactivation of the HLA-II promoters. Treatment of the variant with 5-azacytidine (5-aza), which inhibits CpG methylation, restores inducibility of HLA-II by IFN-γ both at transcriptional and phenotypic level and antigen presenting and processing function of the variant. DNA studies demonstrate that the molecular defect of the THP-1 variant originates from the methylation of the CIITA promoter IV. Furthermore, treatment with 5-aza produces a substantial demethylation of CIITA promoter IV and a significant increase of IFN-γ-dependent HLA-II expression in another myelomonocytic cell line, U937. Therefore hyper-methylation of CIITA promoter IV may be a relevant mechanism of epigenetic control preventing HLA-II IFN-γ inducibility in the myelomonocytic cell lineage. PMID:18829986

The Evolution and Functional Impact of Human Deletion Variants Shared with Archaic Hominin Genomes

PubMed Central

Lin, Yen-Lung; Pavlidis, Pavlos; Karakoc, Emre; Ajay, Jerry; Gokcumen, Omer

2015-01-01

Allele sharing between modern and archaic hominin genomes has been variously interpreted to have originated from ancestral genetic structure or through non-African introgression from archaic hominins. However, evolution of polymorphic human deletions that are shared with archaic hominin genomes has yet to be studied. We identified 427 polymorphic human deletions that are shared with archaic hominin genomes, approximately 87% of which originated before the Human–Neandertal divergence (ancient) and only approximately 9% of which have been introgressed from Neandertals (introgressed). Recurrence, incomplete lineage sorting between human and chimp lineages, and hominid-specific insertions constitute the remaining approximately 4% of allele sharing between humans and archaic hominins. We observed that ancient deletions correspond to more than 13% of all common (>5% allele frequency) deletion variation among modern humans. Our analyses indicate that the genomic landscapes of both ancient and introgressed deletion variants were primarily shaped by purifying selection, eliminating large and exonic variants. We found 17 exonic deletions that are shared with archaic hominin genomes, including those leading to three fusion transcripts. The affected genes are involved in metabolism of external and internal compounds, growth and sperm formation, as well as susceptibility to psoriasis and Crohn’s disease. Our analyses suggest that these “exonic” deletion variants have evolved through different adaptive forces, including balancing and population-specific positive selection. Our findings reveal that genomic structural variants that are shared between humans and archaic hominin genomes are common among modern humans and can influence biomedically and evolutionarily important phenotypes. PMID:25556237

An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

PubMed

Saraco, Nora; Nesi-Franca, Suzana; Sainz, Romina; Marino, Roxana; Marques-Pereira, Rosana; La Pastina, Julia; Perez Garrido, Natalia; Sandrini, Romolo; Rivarola, Marco Aurelio; de Lacerda, Luiz; Belgorosky, Alicia

2015-01-01

Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied. A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient. © 2015 S. Karger AG, Basel.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants

PubMed Central

De Rosa, Veronica; Galgani, Mario; Porcellini, Antonio; Colamatteo, Alessandra; Santopaolo, Marianna; Zuchegna, Candida; Romano, Antonella; De Simone, Salvatore; Procaccini, Claudio; La Rocca, Claudia; Carrieri, Pietro Biagio; Maniscalco, Giorgia Teresa; Salvetti, Marco; Buscarinu, Maria Chiara; Franzese, Adriana; Mozzillo, Enza; La Cava, Antonio; Matarese, Giuseppe

2016-01-01

Human regulatory T cells (Treg cells) that develop from conventional T cells (Tconv cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced Treg cells (iTreg cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iTreg cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2–related suppressive activity of iTreg cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of Treg cells in health and in autoimmunity. PMID:26414764

The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.

PubMed

Bruschi, Francesca Virginia; Claudel, Thierry; Tardelli, Matteo; Caligiuri, Alessandra; Stulnig, Thomas M; Marra, Fabio; Trauner, Michael

2017-06-01

The genetic polymorphism I148M of patatin-like phospholipase domain-containing 3 (PNPLA3) is robustly associated with hepatic steatosis and its progression to steatohepatitis, fibrosis, and cancer. Hepatic stellate cells (HSCs) are key players in the development of liver fibrosis, but the role of PNPLA3 and its variant I148M in this process is poorly understood. Here we analyzed the expression of PNPLA3 during human HSC activation and thereby explored how a PNPLA3 variant impacts hepatic fibrogenesis. We show that expression of PNPLA3 gene and protein increases during the early phases of activation and remains elevated in fully activated HSCs (P < 0.01). Knockdown of PNPLA3 significantly decreases the profibrogenic protein alpha-smooth muscle actin (P < 0.05). Primary human I148M HSCs displayed significantly higher expression and release of proinflammatory cytokines, such as chemokine (C-C motif) ligand 5 (P < 0.01) and granulocyte-macrophage colony-stimulating factor (P < 0.001), thus contributing to migration of immune cells (P < 0.05). Primary I148M HSCs showed reduced retinol (P < 0.001) but higher lipid droplet content (P < 0.001). In line with this, LX-2 cells stably overexpressing I148M showed augmented proliferation and migration, lower retinol, and abolished retinoid X receptor/retinoid A receptor transcriptional activities but more lipid droplets. Knockdown of I148M PNPLA3 (P < 0.001) also reduces chemokine (C-C motif) ligand 5 and collagen1α1 expression (P < 0.05). Notably, I148M cells display reduced peroxisome proliferator-activated receptor gamma transcriptional activity, and this effect was attributed to increased c-Jun N-terminal kinase, thereby inhibiting peroxisome proliferator-activated receptor gamma through serine 84 phosphorylation and promoting activator protein 1 transcription. Conversely, the c-Jun N-terminal kinase inhibitor SP600125 and the peroxisome proliferator-activated receptor gamma agonist rosiglitazone decreased activator protein 1 promoter activity. These data indicate that PNPLA3 is required for HSC activation and that its genetic variant I148M potentiates the profibrogenic features of HSCs, providing a molecular mechanism for the higher risk of progression and severity of liver diseases conferred to patients carrying the I148M variant. (Hepatology 2017;65:1875-1890). © 2017 by the American Association for the Study of Liver Diseases.

Co-expression networks reveal the tissue-specific regulation of transcription and splicing.

PubMed

Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D H; Jo, Brian; Gao, Chuan; McDowell, Ian C; Engelhardt, Barbara E; Battle, Alexis

2017-11-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. © 2017 Saha et al.; Published by Cold Spring Harbor Laboratory Press.

High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE

USDA-ARS?s Scientific Manuscript database

We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE (“Assessing Changes to Exons”) converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detect...

Signatures of positive selection in the cis-regulatory sequences of the human oxytocin receptor (OXTR) and arginine vasopressin receptor 1a (AVPR1A) genes.

PubMed

Schaschl, Helmut; Huber, Susanne; Schaefer, Katrin; Windhager, Sonja; Wallner, Bernard; Fieder, Martin

2015-05-13

The evolutionary highly conserved neurohypophyseal hormones oxytocin and arginine vasopressin play key roles in regulating social cognition and behaviours. The effects of these two peptides are meditated by their specific receptors, which are encoded by the oxytocin receptor (OXTR) and arginine vasopressin receptor 1a genes (AVPR1A), respectively. In several species, polymorphisms in these genes have been linked to various behavioural traits. Little, however, is known about whether positive selection acts on sequence variants in genes influencing variation in human behaviours. We identified, in both neuroreceptor genes, signatures of balancing selection in the cis-regulative acting sequences such as transcription factor binding and enhancer sequences, as well as in a transcriptional repressor sequence motif. Additionally, in the intron 3 of the OXTR gene, the SNP rs59190448 appears to be under positive directional selection. For rs59190448, only one phenotypical association is known so far, but it is in high LD' (>0.8) with loci of known association; i.e., variants associated with key pro-social behaviours and mental disorders in humans. Only for one SNP on the OXTR gene (rs59190448) was a sign of positive directional selection detected with all three methods of selection detection. For rs59190448, however, only one phenotypical association is known, but rs59190448 is in high LD' (>0.8), with variants associated with important pro-social behaviours and mental disorders in humans. We also detected various signatures of balancing selection on both neuroreceptor genes.

Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms

PubMed Central

de la Hoya, Miguel; Soukarieh, Omar; López-Perolio, Irene; Vega, Ana; Walker, Logan C.; van Ierland, Yvette; Baralle, Diana; Santamariña, Marta; Lattimore, Vanessa; Wijnen, Juul; Whiley, Philip; Blanco, Ana; Raponi, Michela; Hauke, Jan; Wappenschmidt, Barbara; Becker, Alexandra; Hansen, Thomas V. O.; Behar, Raquel; Investigators, KConFaB; Niederacher, Diether; Arnold, Norbert; Dworniczak, Bernd; Steinemann, Doris; Faust, Ulrike; Rubinstein, Wendy; Hulick, Peter J.; Houdayer, Claude; Caputo, Sandrine M.; Castera, Laurent; Pesaran, Tina; Chao, Elizabeth; Brewer, Carole; Southey, Melissa C.; van Asperen, Christi J.; Singer, Christian F.; Sullivan, Jan; Poplawski, Nicola; Mai, Phuong; Peto, Julian; Johnson, Nichola; Burwinkel, Barbara; Surowy, Harald; Bojesen, Stig E.; Flyger, Henrik; Lindblom, Annika; Margolin, Sara; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Galastri, Laura; Olson, Janet E.; Hallberg, Emily; Giles, Graham G.; Milne, Roger L.; Andrulis, Irene L.; Glendon, Gord; Hall, Per; Czene, Kamila; Blows, Fiona; Shah, Mitul; Wang, Qin; Dennis, Joe; Michailidou, Kyriaki; McGuffog, Lesley; Bolla, Manjeet K.; Antoniou, Antonis C.; Easton, Douglas F.; Couch, Fergus J.; Tavtigian, Sean; Vreeswijk, Maaike P.; Parsons, Michael; Meeks, Huong D.; Martins, Alexandra; Goldgar, David E.; Spurdle, Amanda B.

2016-01-01

A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10−8. Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70–80% truncating transcripts, and ≈20–30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function. We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20–30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes. PMID:27008870

Cancer-specific SNPs originate from low-level heteroplasmic variants in human mitochondrial genomes of a matched cell line pair.

PubMed

Hedberg, Annica; Knutsen, Erik; Løvhaugen, Anne Silje; Jørgensen, Tor Erik; Perander, Maria; Johansen, Steinar D

2018-04-19

Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

Differential expression of Oct4 variants and pseudogenes in normal urothelium and urothelial cancer.

PubMed

Wezel, Felix; Pearson, Joanna; Kirkwood, Lisa A; Southgate, Jennifer

2013-10-01

The transcription factor octamer-binding protein 4 (Oct4; encoded by POU5F1) has a key role in maintaining embryonic stem cell pluripotency during early embryonic development and it is required for generation of induced pluripotent stem cells. Controversy exists concerning Oct4 expression in somatic tissues, with reports that Oct4 is expressed in normal and in neoplastic urothelium carrying implications for a bladder cancer stem cell phenotype. Here, we show that the pluripotency-associated Oct4A transcript was absent from cultures of highly regenerative normal human urothelial cells and from low-grade to high-grade urothelial carcinoma cell lines, whereas alternatively spliced variants and transcribed pseudogenes were expressed in abundance. Immunolabeling and immunoblotting studies confirmed the absence of Oct4A in normal and neoplastic urothelial cells and tissues, but indicated the presence of alternative isoforms or potentially translated pseudogenes. The stable forced expression of Oct4A in normal human urothelial cells in vitro profoundly inhibited growth and affected morphology, but protein expression was rapidly down-regulated. Our findings demonstrate that pluripotency-associated isoform Oct4A is not expressed by normal or malignant human urothelium and therefore is unlikely to play a role in a cancer stem cell phenotype. However, our findings also indicate that urothelium expresses a variety of other Oct4 splice-variant isoforms and transcribed pseudogenes that warrant further study. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Mutations in HIVEP2 are associated with developmental delay, intellectual disability, and dysmorphic features.

PubMed

Steinfeld, Hallie; Cho, Megan T; Retterer, Kyle; Person, Rick; Schaefer, G Bradley; Danylchuk, Noelle; Malik, Saleem; Wechsler, Stephanie Burns; Wheeler, Patricia G; van Gassen, Koen L I; Terhal, P A; Verhoeven, Virginie J M; van Slegtenhorst, Marjon A; Monaghan, Kristin G; Henderson, Lindsay B; Chung, Wendy K

2016-07-01

Human immunodeficiency virus type I enhancer binding protein 2 (HIVEP2) has been previously associated with intellectual disability and developmental delay in three patients. Here, we describe six patients with developmental delay, intellectual disability, and dysmorphic features with de novo likely gene-damaging variants in HIVEP2 identified by whole-exome sequencing (WES). HIVEP2 encodes a large transcription factor that regulates various neurodevelopmental pathways. Our findings provide further evidence that pathogenic variants in HIVEP2 lead to intellectual disabilities and developmental delay.

Biallelic germline and somatic mutations in malignant mesothelioma: multiple mutations in transcription regulators including mSWI/SNF genes.

PubMed

Yoshikawa, Yoshie; Sato, Ayuko; Tsujimura, Tohru; Otsuki, Taiichiro; Fukuoka, Kazuya; Hasegawa, Seiki; Nakano, Takashi; Hashimoto-Tamaoki, Tomoko

2015-02-01

We detected low levels of acetylation for histone H3 tail lysines in malignant mesothelioma (MM) cell lines resistant to histone deacetylase inhibitors. To identify the possible genetic causes related to the low histone acetylation levels, whole-exome sequencing was conducted with MM cell lines established from eight patients. A mono-allelic variant of BRD1 was common to two MM cell lines with very low acetylation levels. We identified 318 homozygous protein-damaging variants/mutations (18-78 variants/mutations per patient); annotation analysis showed enrichment of the molecules associated with mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complexes and co-activators that facilitate initiation of transcription. In seven of the patients, we detected a combination of variants in histone modifiers or transcription factors/co-factors, in addition to variants in mSWI/SNF. Direct sequencing showed that homozygous mutations in SMARCA4, PBRM1 and ARID2 were somatic. In one patient, homozygous germline variants were observed for SMARCC1 and SETD2 in chr3p22.1-3p14.2. These exhibited extended germline homozygosity and were in regions containing somatic mutations, leading to a loss of BAP1 and PBRM1 expression in MM cell line. Most protein-damaging variants were heterozygous in normal tissues. Heterozygous germline variants were often converted into hemizygous variants by mono-allelic deletion, and were rarely homozygous because of acquired uniparental disomy. Our findings imply that MM might develop through the somatic inactivation of mSWI/SNF complex subunits and/or histone modifiers, including BAP1, in subjects that have rare germline variants of these transcription regulators and/or transcription factors/co-factors, and in regions prone to mono-allelic deletion during oncogenesis. © 2014 UICC.

Functional characterization of rare FOXP2 variants in neurodevelopmental disorder.

PubMed

Estruch, Sara B; Graham, Sarah A; Chinnappa, Swathi M; Deriziotis, Pelagia; Fisher, Simon E

2016-01-01

Heterozygous disruption of FOXP2 causes a rare form of speech and language impairment. Screens of the FOXP2 sequence in individuals with speech/language-related disorders have identified several rare protein-altering variants, but their phenotypic relevance is often unclear. FOXP2 encodes a transcription factor with a forkhead box DNA-binding domain, but little is known about the functions of protein regions outside this domain. We performed detailed functional analyses of seven rare FOXP2 variants found in affected cases, including three which have not been previously characterized, testing intracellular localization, transcriptional regulation, dimerization, and interaction with other proteins. To shed further light on molecular functions of FOXP2, we characterized the interaction between this transcription factor and co-repressor proteins of the C-terminal binding protein (CTBP) family. Finally, we analysed the functional significance of the polyglutamine tracts in FOXP2, since tract length variations have been reported in cases of neurodevelopmental disorder. We confirmed etiological roles of multiple FOXP2 variants. Of three variants that have been suggested to cause speech/language disorder, but never before been characterized, only one showed functional effects. For the other two, we found no effects on protein function in any assays, suggesting that they are incidental to the phenotype. We identified a CTBP-binding region within the N-terminal portion of FOXP2. This region includes two amino acid substitutions that occurred on the human lineage following the split from chimpanzees. However, we did not observe any effects of these amino acid changes on CTBP binding or other core aspects of FOXP2 function. Finally, we found that FOXP2 variants with reduced polyglutamine tracts did not exhibit altered behaviour in cellular assays, indicating that such tracts are non-essential for core aspects of FOXP2 function, and that tract variation is unlikely to be a highly penetrant cause of speech/language disorder. Our findings highlight the importance of functional characterization of novel rare variants in FOXP2 in assessing the contribution of such variants to speech/language disorder and provide further insights into the molecular function of the FOXP2 protein.

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adamopoulos, Panagiotis G.; Kontos, Christos K.; Scorilas, Andreas

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′more » rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.« less

Truncating Variants in NAA15 Are Associated with Variable Levels of Intellectual Disability, Autism Spectrum Disorder, and Congenital Anomalies.

PubMed

Cheng, Hanyin; Dharmadhikari, Avinash V; Varland, Sylvia; Ma, Ning; Domingo, Deepti; Kleyner, Robert; Rope, Alan F; Yoon, Margaret; Stray-Pedersen, Asbjørg; Posey, Jennifer E; Crews, Sarah R; Eldomery, Mohammad K; Akdemir, Zeynep Coban; Lewis, Andrea M; Sutton, Vernon R; Rosenfeld, Jill A; Conboy, Erin; Agre, Katherine; Xia, Fan; Walkiewicz, Magdalena; Longoni, Mauro; High, Frances A; van Slegtenhorst, Marjon A; Mancini, Grazia M S; Finnila, Candice R; van Haeringen, Arie; den Hollander, Nicolette; Ruivenkamp, Claudia; Naidu, Sakkubai; Mahida, Sonal; Palmer, Elizabeth E; Murray, Lucinda; Lim, Derek; Jayakar, Parul; Parker, Michael J; Giusto, Stefania; Stracuzzi, Emanuela; Romano, Corrado; Beighley, Jennifer S; Bernier, Raphael A; Küry, Sébastien; Nizon, Mathilde; Corbett, Mark A; Shaw, Marie; Gardner, Alison; Barnett, Christopher; Armstrong, Ruth; Kassahn, Karin S; Van Dijck, Anke; Vandeweyer, Geert; Kleefstra, Tjitske; Schieving, Jolanda; Jongmans, Marjolijn J; de Vries, Bert B A; Pfundt, Rolph; Kerr, Bronwyn; Rojas, Samantha K; Boycott, Kym M; Person, Richard; Willaert, Rebecca; Eichler, Evan E; Kooy, R Frank; Yang, Yaping; Wu, Joseph C; Lupski, James R; Arnesen, Thomas; Cooper, Gregory M; Chung, Wendy K; Gecz, Jozef; Stessman, Holly A F; Meng, Linyan; Lyon, Gholson J

2018-05-03

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development. Copyright © 2018 American Society of Human Genetics. All rights reserved.

High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner*

PubMed Central

Kugler, Jamie E.; Horsch, Marion; Huang, Di; Furusawa, Takashi; Rochman, Mark; Garrett, Lillian; Becker, Lore; Bohla, Alexander; Hölter, Sabine M.; Prehn, Cornelia; Rathkolb, Birgit; Racz, Ildikó; Aguilar-Pimentel, Juan Antonio; Adler, Thure; Adamski, Jerzy; Beckers, Johannes; Busch, Dirk H.; Eickelberg, Oliver; Klopstock, Thomas; Ollert, Markus; Stöger, Tobias; Wolf, Eckhard; Wurst, Wolfgang; Yildirim, Ali Önder; Zimmer, Andreas; Gailus-Durner, Valérie; Fuchs, Helmut; Hrabě de Angelis, Martin; Garfinkel, Benny; Orly, Joseph; Ovcharenko, Ivan; Bustin, Michael

2013-01-01

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1tm1/tm1, Hmgn3tm1/tm1, and Hmgn5tm1/tm1 mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgntm1/tm1 lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner. PMID:23620591

An integrated map of genetic variation from 1,092 human genomes

PubMed Central

2012-01-01

Summary Through characterising the geographic and functional spectrum of human genetic variation, the 1000 Genomes Project aims to build a resource to help understand the genetic contribution to disease. We describe the genomes of 1,092 individuals from 14 populations, constructed using a combination of low-coverage whole-genome and exome sequencing. By developing methodologies to integrate information across multiple algorithms and diverse data sources we provide a validated haplotype map of 38 million SNPs, 1.4 million indels and over 14 thousand larger deletions. We show that individuals from different populations carry different profiles of rare and common variants and that low-frequency variants show substantial geographic differentiation, which is further increased by the action of purifying selection. We show that evolutionary conservation and coding consequence are key determinants of the strength of purifying selection, that rare-variant load varies substantially across biological pathways and that each individual harbours hundreds of rare non-coding variants at conserved sites, such as transcription-factor-motif disrupting changes. This resource, which captures up to 98% of accessible SNPs at a frequency of 1% in populations of medical genetics focus, enables analysis of common and low-frequency variants in individuals from diverse, including admixed, populations. PMID:23128226

Temporal Expression Profiling Identifies Pathways Mediating Effect of Causal Variant on Phenotype

PubMed Central

Gupta, Saumya; Radhakrishnan, Aparna; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M.; Gagneur, Julien; Sinha, Himanshu

2015-01-01

Even with identification of multiple causal genetic variants for common human diseases, understanding the molecular processes mediating the causal variants’ effect on the disease remains a challenge. This understanding is crucial for the development of therapeutic strategies to prevent and treat disease. While static profiling of gene expression is primarily used to get insights into the biological bases of diseases, it makes differentiating the causative from the correlative effects difficult, as the dynamics of the underlying biological processes are not monitored. Using yeast as a model, we studied genome-wide gene expression dynamics in the presence of a causal variant as the sole genetic determinant, and performed allele-specific functional validation to delineate the causal effects of the genetic variant on the phenotype. Here, we characterized the precise genetic effects of a functional MKT1 allelic variant in sporulation efficiency variation. A mathematical model describing meiotic landmark events and conditional activation of MKT1 expression during sporulation specified an early meiotic role of this variant. By analyzing the early meiotic genome-wide transcriptional response, we demonstrate an MKT1-dependent role of novel modulators, namely, RTG1/3, regulators of mitochondrial retrograde signaling, and DAL82, regulator of nitrogen starvation, in additively effecting sporulation efficiency. In the presence of functional MKT1 allele, better respiration during early sporulation was observed, which was dependent on the mitochondrial retrograde regulator, RTG3. Furthermore, our approach showed that MKT1 contributes to sporulation independent of Puf3, an RNA-binding protein that steady-state transcription profiling studies have suggested to mediate MKT1-pleiotropic effects during mitotic growth. These results uncover interesting regulatory links between meiosis and mitochondrial retrograde signaling. In this study, we highlight the advantage of analyzing allele-specific transcriptional dynamics of mediating genes. Applications in higher eukaryotes can be valuable for inferring causal molecular pathways underlying complex dynamic processes, such as development, physiology and disease progression. PMID:26039065

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatiblemore » with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.« less

Drug resistance prevalence in human immunodeficiency virus type 1 infected pediatric populations in Honduras and El Salvador during 1989-2009.

PubMed

Holguín, Africa; Erazo, Karen; Escobar, Gustavo; de Mulder, Miguel; Yebra, Gonzalo; Martín, Leticia; Jovel, Luis Enrique; Castaneda, Luis; Pérez, Elsy

2011-05-01

Emergence of viral resistance is a major obstacle for antiretroviral treatment (ART) effectiveness. Human immunodeficiency virus type-1 (HIV-1) variants and drug-resistance mutations were identified in naive and antiretroviral drug-experienced children with virologic failure, in Honduras and El Salvador. Dried blood spots (DBS) from 80 individuals (54 from Honduras, 26 from El Salvador) infected during their childhood between 1989 and 2009 were collected in 2009. The HIV pol region was amplified and sequenced to identify antiretroviral-resistant mutations according to the 2009 International AIDS Society. The genotypic drug resistance interpretation was performed using the Stanford algorithm. HIV-1 variants were characterized by phylogenetic analysis and subtyping tools. HIV-1 protease and reverse transcription sequences were obtained from DBS specimens in 71 and 66 patients, respectively, of the 80 patients. All children were native Central Americans carrying subtype B, with a mean age of 9 years, most were male (65%), perinatally infected (96%), with moderate/severe AIDS symptoms (70%), and receiving first line ART at the time of sequencing (65%). Diagnostic delay was frequently observed. Infected children from Honduras presented longer ART experience and clinical outcomes, and more frequent severe symptoms. Resistant variants infected 1 of 11 naive children from El Salvador but none of the perinatally infected naive children from Honduras. Resistance was higher among ART-exposed individuals in both countries and similar for protease inhibitors (16%), nucleoside reverse transcription inhibitors (44%-52%), and nonnucleoside reverse-transcription inhibitors (66.7%). One in 10 pretreated children in each country was infected with resistant viruses to the 3 drug families. Our data support the need for continued surveillance of resistance patterns using DBS at national levels among naive and pretreated children to optimize the ART regimens.

Distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges.

PubMed

Piasecka, Barbara; Duffy, Darragh; Urrutia, Alejandra; Quach, Hélène; Patin, Etienne; Posseme, Céline; Bergstedt, Jacob; Charbit, Bruno; Rouilly, Vincent; MacPherson, Cameron R; Hasan, Milena; Albaud, Benoit; Gentien, David; Fellay, Jacques; Albert, Matthew L; Quintana-Murci, Lluis

2018-01-16

The contribution of host genetic and nongenetic factors to immunological differences in humans remains largely undefined. Here, we generated bacterial-, fungal-, and viral-induced immune transcriptional profiles in an age- and sex-balanced cohort of 1,000 healthy individuals and searched for the determinants of immune response variation. We found that age and sex affected the transcriptional response of most immune-related genes, with age effects being more stimulus-specific relative to sex effects, which were largely shared across conditions. Although specific cell populations mediated the effects of age and sex on gene expression, including CD8 + T cells for age and CD4 + T cells and monocytes for sex, we detected a direct effect of these intrinsic factors for the majority of immune genes. The mapping of expression quantitative trait loci (eQTLs) revealed that genetic factors had a stronger effect on immune gene regulation than age and sex, yet they affected a smaller number of genes. Importantly, we identified numerous genetic variants that manifested their regulatory effects exclusively on immune stimulation, including a Candida albicans -specific master regulator at the CR1 locus. These response eQTLs were enriched in disease-associated variants, particularly for autoimmune and inflammatory disorders, indicating that differences in disease risk may result from regulatory variants exerting their effects only in the presence of immune stress. Together, this study quantifies the respective effects of age, sex, genetics, and cellular heterogeneity on the interindividual variability of immune responses and constitutes a valuable resource for further exploration in the context of different infection risks or disease outcomes. Copyright © 2018 the Author(s). Published by PNAS.

Identification and characterization of novel peroxisome proliferator-activated receptor-gamma (PPAR-gamma) transcriptional variants in pig and human.

PubMed

Omi, T; Brenig, B; Spilar Kramer, S; Iwamoto, S; Stranzinger, G; Neuenschwander, S

2005-04-01

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the steroid/thyroid/retinoid receptor superfamily, and is primarily expressed in fat tissue. To date, two major PPAR-gamma isoforms have been identified in pig, PPAR-gamma1 and PPAR-gamma2. Porcine PPAR-gamma1a consists of two leader exons, designated A1 and A2, followed by six exons containing the open reading frame. Here, we report the isolation and characterization of three novel PPAR-gamma1 transcripts. PPAR-gamma1b is derived from exon A1, with exon A2 spliced out. PPAR-gamma1c and PPAR-gamma1d are derived from the new exon, A', containing exon A2 (gamma1c) or without exon A2 (gamma1d). Based on PCR analysis of PAC clones that included sequences from the 5'-untranslated region of the PPAR-gamma gene, the new A' exon is located between the known exons A1 and A2. We also isolated the human homologue to exon A', as well as the two new PPAR-gamma1c and -gamma1d splice variants, from human adipose tissue. Studies of the expression of porcine PPAR-gamma by real time reverse transcription-polymerase chain reaction analysis show that transcripts derived from exon A1 were not expressed at significantly different levels in visceral fat (lamina subserosa) or subcutaneous fat (back fat, inner and outer layer). In contrast, exon A'-derived transcripts were expressed at progressively higher levels in the inner and outer layers of subcutaneous fat than in visceral fat. The same expression pattern was also observed for PPAR-gamma2. We hypothesize that there are three promoters, which differentially regulate PPAR-gamma1 and PPAR-gamma2 gene expression, depending on the specific localization of the fat tissue.

Genetic and environmental risk factors for atherosclerosis regulate transcription of phosphatase and actin regulating gene PHACTR1.

PubMed

Reschen, Michael E; Lin, Da; Chalisey, Anil; Soilleux, Elizabeth J; O'Callaghan, Christopher A

2016-07-01

Coronary artery disease (CAD) risk is associated with non-coding genetic variants at the phosphatase and actin regulating protein 1(PHACTR1) gene locus. The PHACTR1 gene encodes an actin-binding protein with phosphatase regulating activity. The mechanism whereby PHACTR1 influences CAD risk is unknown. We hypothesized that PHACTR1 would be expressed in human cell types relevant to CAD and regulated by atherogenic or genetic factors. Using immunohistochemistry, we demonstrate that PHACTR1 protein is expressed strongly in human atherosclerotic plaque macrophages, lipid-laden foam cells, adventitial lymphocytes and endothelial cells. Using a combination of genomic analysis and molecular techniques, we demonstrate that PHACTR1 is expressed as multiple previously uncharacterized transcripts in macrophages, foam cells, lymphocytes and endothelial cells. Immunoblotting confirmed a total absence of PHACTR1 in vascular smooth muscle cells. Real-time quantitative PCR showed that PHACTR1 is regulated by atherogenic and inflammatory stimuli. In aortic endothelial cells, oxLDL and TNF-alpha both upregulated an intermediate length transcript. A short transcript expressed only in immune cells was upregulated in macrophages by oxidized low-density lipoprotein, and oxidized phospholipids but suppressed by lipopolysaccharide or TNF-alpha. In primary human macrophages, we identified a novel expression quantitative trait locus (eQTL) specific for this short transcript, whereby the risk allele at CAD risk SNP rs9349379 is associated with reduced PHACTR1 expression, similar to the effect of an inflammatory stimulus. Our data demonstrate that PHACTR1 is a key atherosclerosis candidate gene since it is regulated by atherogenic stimuli in macrophages and endothelial cells and we identify an effect of the genetic risk variant on PHACTR1 expression in macrophages that is similar to that of an inflammatory stimulus. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Relationship between variant forms of estrogen receptor RNA and an apoptosis-related RNA, TRPM-2, with survival in patients with breast cancer.

PubMed

Rennie, P S; Mawji, N R; Coldman, A J; Godolphin, W; Jones, E C; Vielkind, J R; Bruchovsky, N

1993-12-15

Although smaller variant forms of estrogen receptor (ER) messenger RNA (mRNA) have been detected in breast tumors, neither their prevalence nor their prognostic significance have been evaluated. Similarly, TRPM-2 mRNA, the product of a gene induced principally during the onset of apoptosis, is present in mouse and human breast cancer cell lines, but whether it also occurs in primary breast tumors and is related to disease outcome is unknown. The relative expression and transcript size of ER mRNA and TRPM-2 mRNA in 126 primary breast tumors were measured by Northern analysis and compared with tumor grade, hormone receptor status, extent of tumor necrosis, and survival. In ER-positive tumors, 64% of the tumors had only the normal 6.5 kb ER mRNA, an additional 9% had the normal plus smaller ER mRNA, and 2% had variant forms. Only 8% of ER-negative tumors had ER mRNA transcripts. There were significant relationships between the occurrence of ER mRNA and low tumor grade, ER-positive receptor status, and better survival. In contrast, TRPM-2 mRNA was found in only 17% of breast tumors, none of which could be grouped with respect to grade, hormone receptor status, or survival. The presence of smaller variant forms of ER mRNA either alone or in association with the normal ER transcript is not indicative of an unfavorable prognosis, whereas TRPM-2 mRNA occurs in many primary breast tumors, but has no apparent relationship to survival.

An Ultraconserved Brain-specific Enhancer within ADGRL3 (LPHN3) Underpins ADHD Susceptibility

PubMed Central

Martinez, Ariel F.; Abe, Yu; Hong, Sungkook; Molyneux, Kevin; Yarnell, David; Löhr, Heiko; Driever, Wolfgang; Acosta, Maria T.; Arcos-Burgos, Mauricio; Muenke, Maximilian

2016-01-01

BACKGROUND Genetic factors predispose to attention deficit/hyperactivity disorder (ADHD). Previous studies have reported linkage and association to ADHD of gene variants within ADGRL3. In this study, we functionally analyzed non-coding variants in this gene as likely pathological contributors. METHODS In silico, in vitro and in vivo approaches were used to identify and characterize evolutionary conserved elements within the ADGRL3 linkage region (~207 Kb). Family-based genetic analyses on 838 individuals (372 affected and 466 unaffected) identified ADHD-associated SNPs harbored in some of these conserved elements. Luciferase assays and zebrafish GFP transgenesis tested conserved elements for transcriptional enhancer activity. Electromobility shift assays were used to verify transcription factor binding disruption by ADHD risk alleles. RESULTS An ultraconserved element was discovered (ECR47) that functions as a transcriptional enhancer. A three-variant ADHD risk haplotype in ECR47, formed by rs17226398, rs56038622 and rs2271338, reduced enhancer activity by 40% in neuroblastoma and astrocytoma cells (PBonferroni<0.0001). This enhancer also drove GFP expression in the zebrafish brain in a tissue-specific manner, sharing aspects of endogenous ADGRL3 expression. The rs2271338 risk allele disrupts binding of YY1, an important factor in the development and function of the central nervous system. Expression quantitative trait loci analysis of post-mortem human brain tissues revealed an association between rs2271338 and reduced ADGRL3 expression in the thalamus. CONCLUSIONS These results uncover the first functional evidence of common non-coding variants with potential implications for the pathology of ADHD. PMID:27692237

Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction.

PubMed

Sun, Bing; Tao, Lian; Zheng, Yun-Ling

2014-06-01

Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency.

Heterozygous KIDINS220/ARMS nonsense variants cause spastic paraplegia, intellectual disability, nystagmus, and obesity.

PubMed

Josifova, Dragana J; Monroe, Glen R; Tessadori, Federico; de Graaff, Esther; van der Zwaag, Bert; Mehta, Sarju G; Harakalova, Magdalena; Duran, Karen J; Savelberg, Sanne M C; Nijman, Isaäc J; Jungbluth, Heinz; Hoogenraad, Casper C; Bakkers, Jeroen; Knoers, Nine V; Firth, Helen V; Beales, Philip L; van Haaften, Gijs; van Haelst, Mieke M

2016-06-01

We identified de novo nonsense variants in KIDINS220/ARMS in three unrelated patients with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). KIDINS220 is an essential scaffold protein coordinating neurotrophin signal pathways in neurites and is spatially and temporally regulated in the brain. Molecular analysis of patients' variants confirmed expression and translation of truncated transcripts similar to recently characterized alternative terminal exon splice isoforms of KIDINS220 KIDINS220 undergoes extensive alternative splicing in specific neuronal populations and developmental time points, reflecting its complex role in neuronal maturation. In mice and humans, KIDINS220 is alternative spliced in the middle region as well as in the last exon. These full-length and KIDINS220 splice variants occur at precise moments in cortical, hippocampal, and motor neuron development, with splice variants similar to the variants seen in our patients and lacking the last exon of KIDINS220 occurring in adult rather than in embryonic brain. We conducted tissue-specific expression studies in zebrafish that resulted in spasms, confirming a functional link with disruption of the KIDINS220 levels in developing neurites. This work reveals a crucial physiological role of KIDINS220 in development and provides insight into how perturbation of the complex interplay of KIDINS220 isoforms and their relative expression can affect neuron control and human metabolism. Altogether, we here show that de novo protein-truncating KIDINS220 variants cause a new syndrome, SINO. This is the first report of KIDINS220 variants causing a human disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Proteogenomic characterization of human colon and rectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bing; Wang, Jing; Wang, Xiaojing

2014-09-18

We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Protein sequence variants encoded by somatic genomic variations displayed reduced expression compared to protein variants encoded by germline variations. mRNA transcript abundance did not reliably predict protein expression differences between tumors. Proteomics identified five protein expression subtypes, two of which were associated with the TCGA "MSI/CIMP" transcriptional subtype, but had distinct mutation and methylation patterns and associated with different clinical outcomes. Although CNAs showed strong cis- and trans-effects on mRNA expression, relatively few of these extend to the proteinmore » level. Thus, proteomics data enabled prioritization of candidate driver genes. Our analyses identified HNF4A, a novel candidate driver gene in tumors with chromosome 20q amplifications. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords novel insights into cancer biology.« less

Epigenetic Regulation of Transcription in Trypanosomatid Protozoa.

PubMed

Martínez-Calvillo, Santiago; Romero-Meza, Gabriela; Vizuet-de-Rueda, Juan C; Florencio-Martínez, Luis E; Manning-Cela, Rebeca; Nepomuceno-Mejía, Tomás

2018-02-01

The Trypanosomatid family includes flagellated parasites that cause fatal human diseases. Remarkably, protein-coding genes in these organisms are positioned in long tandem arrays that are transcribed polycistronically. However, the knowledge about regulation of transcription initiation and termination in trypanosomatids is scarce. The importance of epigenetic regulation in these processes has become evident in the last years, as distinctive histone modifications and histone variants have been found in transcription initiation and termination regions. Moreover, multiple chromatin-related proteins have been identified and characterized in trypanosomatids, including histone-modifying enzymes, effector complexes, chromatin-remodelling enzymes and histone chaperones. Notably, base J, a modified thymine residue present in the nuclear DNA of trypanosomatids, has been implicated in transcriptional regulation. Here we review the current knowledge on epigenetic control of transcription by all three RNA polymerases in this group of early-diverged eukaryotes.

Diabetes-induced changes in the alternative splicing of the slo gene in corporal tissue.

PubMed

Davies, Kelvin P; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C; Desai, Pratik; Verselis, Vytas K; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J

2007-10-01

Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

Intergenic disease-associated regions are abundant in novel transcripts.

PubMed

Bartonicek, N; Clark, M B; Quek, X C; Torpy, J R; Pritchard, A L; Maag, J L V; Gloss, B S; Crawford, J; Taft, R J; Hayward, N K; Montgomery, G W; Mattick, J S; Mercer, T R; Dinger, M E

2017-12-28

Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

A trans-acting Variant within the Transcription Factor RIM101 Interacts with Genetic Background to Determine its Regulatory Capacity.

PubMed

Read, Timothy; Richmond, Phillip A; Dowell, Robin D

2016-01-01

Most genetic variants associated with disease occur within regulatory regions of the genome, underscoring the importance of defining the mechanisms underlying differences in regulation of gene expression between individuals. We discovered a pair of co-regulated, divergently oriented transcripts, AQY2 and ncFRE6, that are expressed in one strain of Saccharomyces cerevisiae, ∑1278b, but not in another, S288c. By combining classical genetics techniques with high-throughput sequencing, we identified a trans-acting single nucleotide polymorphism within the transcription factor RIM101 that causes the background-dependent expression of both transcripts. Subsequent RNA-seq experiments revealed that RIM101 regulates many more targets in S288c than in ∑1278b and that deletion of RIM101 in both backgrounds abrogates the majority of differential expression between the strains. Strikingly, only three transcripts undergo a significant change in expression after swapping RIM101 alleles between backgrounds, implying that the differences in the RIM101 allele lead to a remarkably focused transcriptional response. However, hundreds of RIM101-dependent targets undergo a subtle but consistent shift in expression in the S288c RIM101-swapped strain, but not its ∑1278b counterpart. We conclude that ∑1278b may harbor a variant(s) that buffers against widespread transcriptional dysregulation upon introduction of a non-native RIM101 allele, emphasizing the importance of accounting for genetic background when assessing the impact of a regulatory variant.

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Epigenetics, chromatin and genome organization: recent advances from the ENCODE project.

PubMed

Siggens, L; Ekwall, K

2014-09-01

The organization of the genome into functional units, such as enhancers and active or repressed promoters, is associated with distinct patterns of DNA and histone modifications. The Encyclopedia of DNA Elements (ENCODE) project has advanced our understanding of the principles of genome, epigenome and chromatin organization, identifying hundreds of thousands of potential regulatory regions and transcription factor binding sites. Part of the ENCODE consortium, GENCODE, has annotated the human genome with novel transcripts including new noncoding RNAs and pseudogenes, highlighting transcriptional complexity. Many disease variants identified in genome-wide association studies are located within putative enhancer regions defined by the ENCODE project. Understanding the principles of chromatin and epigenome organization will help to identify new disease mechanisms, biomarkers and drug targets, particularly as ongoing epigenome mapping projects generate data for primary human cell types that play important roles in disease. © 2014 The Association for the Publication of the Journal of Internal Medicine.

Mining the topography and dynamics of the 4D Nucleome to identify novel CNS drug pathways.

PubMed

Higgins, Gerald A; Allyn-Feuer, Ari; Georgoff, Patrick; Nikolian, Vahagn; Alam, Hasan B; Athey, Brian D

2017-07-01

The pharmacoepigenome can be defined as the active, noncoding province of the genome including canonical spatial and temporal regulatory mechanisms of gene regulation that respond to xenobiotic stimuli. Many psychotropic drugs that have been in clinical use for decades have ill-defined mechanisms of action that are beginning to be resolved as we understand the transcriptional hierarchy and dynamics of the nucleus. In this review, we describe spatial, temporal and biomechanical mechanisms mediated by psychotropic medications. Focus is placed on a bioinformatics pipeline that can be used both for detection of pharmacoepigenomic variants that discretize drug response and adverse events to improve pharmacogenomic testing, and for the discovery of novel CNS therapeutics. This approach integrates the functional topology and dynamics of the transcriptional hierarchy of the pharmacoepigenome, gene variant-driven identification of pharmacogenomic regulatory domains, and mesoscale mapping for the discovery of novel CNS pharmacodynamic pathways in human brain. Examples of the application of this pipeline are provided, including the discovery of valproic acid (VPA) mediated transcriptional reprogramming of neuronal cell fate following injury, and mapping of a CNS pathway glutamatergic pathway for the mood stabilizer lithium. These examples in regulatory pharmacoepigenomics illustrate how ongoing research using the 4D nucleome provides a foundation to further insight into previously unrecognized psychotropic drug pharmacodynamic pathways in the human CNS. Copyright © 2017. Published by Elsevier Inc.

Molecular Cloning and Characterization of the Human ErbB4 Gene: Identification of Novel Splice Isoforms in the Developing and Adult Brain

PubMed Central

Tan, Wei; Dean, Michael; Law, Amanda J.

2010-01-01

ErbB4 is a growth factor receptor tyrosine kinase essential for neurodevelopment. Genetic variation in ErbB4 is associated with schizophrenia and risk-associated polymorphisms predict overexpression of ErbB4 CYT-1 isoforms in the brain in the disorder. The molecular mechanism of association is unclear because the polymorphisms flank exon 3 of the gene and reside 700 kb distal to the CYT-1 defining exon. We hypothesized that the polymorphisms are indirectly associated with ErbB4 CYT-1 via splicing of exon 3 on the CYT-1 background. We report via cloning and sequencing of adult and fetal human brain cDNA libraries the identification of novel splice isoforms of ErbB4, whereby exon 3 is skipped (del.3). ErbB4 del.3 transcripts exist as CYT-2 isoforms and are predicted to produce truncated proteins. Furthermore, our data refine the structure of the human ErbB4 gene, clarify that juxtamembrane (JM) splice variants of ErbB4, JM-a and JM-b respectively, are characterized by the replacement of a 75 nucleotide (nt) sequence with a 45-nt insertion, and demonstrate that there are four alternative exons in the gene. Our analyses reveal that novel splice variants of ErbB4 exist in the developing and adult human brain and, given the failure to identify ErbB4 del.3 CYT-1 transcripts, suggest that the association of risk polymorphisms in the ErbB4 gene with CYT-1 transcript levels is not mediated via an exon 3 splicing event. PMID:20886074

Sequence Variants and Haplotype Analysis of Cat ERBB2 Gene: A Survey on Spontaneous Cat Mammary Neoplastic and Non-Neoplastic Lesions

PubMed Central

Santos, Sara; Bastos, Estela; Baptista, Cláudia S.; Sá, Daniela; Caloustian, Christophe; Guedes-Pinto, Henrique; Gärtner, Fátima; Gut, Ivo G.; Chaves, Raquel

2012-01-01

The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine. PMID:22489125

Molecular Characterization of the Lipid Genome-Wide Association Study Signal on Chromosome 18q11.2 Implicates HNF4A-Mediated Regulation of the TMEM241 Gene.

PubMed

Rodríguez, Alejandra; Gonzalez, Luis; Ko, Arthur; Alvarez, Marcus; Miao, Zong; Bhagat, Yash; Nikkola, Elina; Cruz-Bautista, Ivette; Arellano-Campos, Olimpia; Muñoz-Hernández, Linda L; Ordóñez-Sánchez, Maria-Luisa; Rodriguez-Guillen, Rosario; Mohlke, Karen L; Laakso, Markku; Tusie-Luna, Teresa; Aguilar-Salinas, Carlos A; Pajukanta, Päivi

2016-07-01

We recently identified a locus on chromosome 18q11.2 for high serum triglycerides in Mexicans. We hypothesize that the lead genome-wide association study single-nucleotide polymorphism rs9949617, or its linkage disequilibrium proxies, regulates 1 of the 5 genes in the triglyceride-associated region. We performed a linkage disequilibrium analysis and found 9 additional variants in linkage disequilibrium (r(2)>0.7) with the lead single-nucleotide polymorphism. To select the variants for functional analyses, we annotated the 10 variants using DNase I hypersensitive sites, transcription factor and chromatin states and identified rs17259126 as the lead candidate variant for functional in vitro validation. Using luciferase transcriptional reporter assay in liver HepG2 cells, we found that the G allele exhibits a significantly lower effect on transcription (P<0.05). The electrophoretic mobility shift and ChIPqPCR (chromatin immunoprecipitation coupled with quantitative polymerase chain reaction) assays confirmed that the minor G allele of rs17259126 disrupts an hepatocyte nuclear factor 4 α-binding site. To find the regional candidate gene, we performed a local expression quantitative trait locus analysis and found that rs17259126 and its linkage disequilibrium proxies alter expression of the regional transmembrane protein 241 (TMEM241) gene in 795 adipose RNAs from the Metabolic Syndrome In Men (METSIM) cohort (P=6.11×10(-07)-5.80×10(-04)). These results were replicated in expression profiles of TMEM241 from the Multiple Tissue Human Expression Resource (MuTHER; n=856). The Mexican genome-wide association study signal for high serum triglycerides on chromosome 18q11.2 harbors a regulatory single-nucleotide polymorphism, rs17259126, which disrupts normal hepatocyte nuclear factor 4 α binding and decreases the expression of the regional TMEM241 gene. Our data suggest that decreased transcript levels of TMEM241 contribute to increased triglyceride levels in Mexicans. © 2016 American Heart Association, Inc.

Epigenetic profiling of growth plate chondrocytes sheds insight into regulatory genetic variation influencing height.

PubMed

Guo, Michael; Liu, Zun; Willen, Jessie; Shaw, Cameron P; Richard, Daniel; Jagoda, Evelyn; Doxey, Andrew C; Hirschhorn, Joel; Capellini, Terence D

2017-12-05

GWAS have identified hundreds of height-associated loci. However, determining causal mechanisms is challenging, especially since height-relevant tissues (e.g. growth plates) are difficult to study. To uncover mechanisms by which height GWAS variants function, we performed epigenetic profiling of murine femoral growth plates. The profiled open chromatin regions recapitulate known chondrocyte and skeletal biology, are enriched at height GWAS loci, particularly near differentially expressed growth plate genes, and enriched for binding motifs of transcription factors with roles in chondrocyte biology. At specific loci, our analyses identified compelling mechanisms for GWAS variants. For example, at CHSY1 , we identified a candidate causal variant (rs9920291) overlapping an open chromatin region. Reporter assays demonstrated that rs9920291 shows allelic regulatory activity, and CRISPR/Cas9 targeting of human chondrocytes demonstrates that the region regulates CHSY1 expression. Thus, integrating biologically relevant epigenetic information (here, from growth plates) with genetic association results can identify biological mechanisms important for human growth.

Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

PubMed

Kjellman, Christian; Honeth, Gabriella; Järnum, Sofia; Lindvall, Magnus; Darabi, Anna; Nilsson, Ingar; Edvardsen, Klaus; Salford, Leif G; Widegren, Bengt

2004-03-03

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

Extent, Causes, and Consequences of Small RNA Expression Variation in Human Adipose Tissue

PubMed Central

Knights, Andrew J.; Abreu-Goodger, Cei; van de Bunt, Martijn; Guerra-Assunção, José Afonso; Bartonicek, Nenad; van Dongen, Stijn; Mägi, Reedik; Nisbet, James; Barrett, Amy; Rantalainen, Mattias; Nica, Alexandra C.; Quail, Michael A.; Small, Kerrin S.; Glass, Daniel; Enright, Anton J.; Winn, John; Deloukas, Panos; Dermitzakis, Emmanouil T.; McCarthy, Mark I.; Spector, Timothy D.; Durbin, Richard; Lindgren, Cecilia M.

2012-01-01

Small RNAs are functional molecules that modulate mRNA transcripts and have been implicated in the aetiology of several common diseases. However, little is known about the extent of their variability within the human population. Here, we characterise the extent, causes, and effects of naturally occurring variation in expression and sequence of small RNAs from adipose tissue in relation to genotype, gene expression, and metabolic traits in the MuTHER reference cohort. We profiled the expression of 15 to 30 base pair RNA molecules in subcutaneous adipose tissue from 131 individuals using high-throughput sequencing, and quantified levels of 591 microRNAs and small nucleolar RNAs. We identified three genetic variants and three RNA editing events. Highly expressed small RNAs are more conserved within mammals than average, as are those with highly variable expression. We identified 14 genetic loci significantly associated with nearby small RNA expression levels, seven of which also regulate an mRNA transcript level in the same region. In addition, these loci are enriched for variants significant in genome-wide association studies for body mass index. Contrary to expectation, we found no evidence for negative correlation between expression level of a microRNA and its target mRNAs. Trunk fat mass, body mass index, and fasting insulin were associated with more than twenty small RNA expression levels each, while fasting glucose had no significant associations. This study highlights the similar genetic complexity and shared genetic control of small RNA and mRNA transcripts, and gives a quantitative picture of small RNA expression variation in the human population. PMID:22589741

ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator.

PubMed

Jahn, Arne; Rane, Grishma; Paszkowski-Rogacz, Maciej; Sayols, Sergi; Bluhm, Alina; Han, Chung-Ting; Draškovič, Irena; Londoño-Vallejo, José Arturo; Kumar, Alan Prem; Buchholz, Frank; Butter, Falk; Kappei, Dennis

2017-06-01

Telomeres constitute the ends of linear chromosomes and together with the shelterin complex form a structure essential for genome maintenance and stability. In addition to the constitutive binding of the shelterin complex, other direct, yet more transient interactions are mediated by the CST complex and HOT1/HMBOX1, while subtelomeric variant repeats are recognized by NR2C/F transcription factors. Recently, the Kruppel-like zinc finger protein ZBTB48/HKR3/TZAP has been described as a novel telomere-associated factor in the vertebrate lineage. Here, we show that ZBTB48 binds directly both to telomeric and to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR.

PubMed

Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan

2014-01-01

New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

PXR (NR1I2): splice variants in human tissues, including brain, and identification of neurosteroids and nicotine as PXR activators.

PubMed

Lamba, Vishal; Yasuda, Kazuto; Lamba, Jatinder K; Assem, Mahfoud; Davila, Julio; Strom, Stephen; Schuetz, Erin G

2004-09-15

To gain insight on the expression of pregnane X receptor (PXR), we analyzed PXR.1 and PXR alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nt, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. Quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Brain expression of PXR prompted analysis of whether some brain acting chemicals were PXR ligands. The neurosteroids allopregnanolone and pregnanolone activated PXR and induced transcription of a CYP3A4-luciferase reporter. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, was an efficacious activator of PXR and inducer of CYP3A4 transcription. Because nicotine activation of PXR will enhance metabolism of nicotine to the non-psychoactive cotinine, these results provide one molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues.

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing.

PubMed

Lee, Mei-Chong Wendy; Lopez-Diaz, Fernando J; Khan, Shahid Yar; Tariq, Muhammad Akram; Dayn, Yelena; Vaske, Charles Joseph; Radenbaugh, Amie J; Kim, Hyunsung John; Emerson, Beverly M; Pourmand, Nader

2014-11-04

The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drug-tolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

PubMed Central

Lee, Mei-Chong Wendy; Lopez-Diaz, Fernando J.; Khan, Shahid Yar; Tariq, Muhammad Akram; Dayn, Yelena; Vaske, Charles Joseph; Radenbaugh, Amie J.; Kim, Hyunsung John; Emerson, Beverly M.; Pourmand, Nader

2014-01-01

The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drug-tolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy. PMID:25339441

TCF7L2 is a master regulator of insulin production and processing.

PubMed

Zhou, Yuedan; Park, Soo-Young; Su, Jing; Bailey, Kathleen; Ottosson-Laakso, Emilia; Shcherbina, Liliya; Oskolkov, Nikolay; Zhang, Enming; Thevenin, Thomas; Fadista, João; Bennet, Hedvig; Vikman, Petter; Wierup, Nils; Fex, Malin; Rung, Johan; Wollheim, Claes; Nobrega, Marcelo; Renström, Erik; Groop, Leif; Hansson, Ola

2014-12-15

Genome-wide association studies have revealed >60 loci associated with type 2 diabetes (T2D), but the underlying causal variants and functional mechanisms remain largely elusive. Although variants in TCF7L2 confer the strongest risk of T2D among common variants by presumed effects on islet function, the molecular mechanisms are not yet well understood. Using RNA-sequencing, we have identified a TCF7L2-regulated transcriptional network responsible for its effect on insulin secretion in rodent and human pancreatic islets. ISL1 is a primary target of TCF7L2 and regulates proinsulin production and processing via MAFA, PDX1, NKX6.1, PCSK1, PCSK2 and SLC30A8, thereby providing evidence for a coordinated regulation of insulin production and processing. The risk T-allele of rs7903146 was associated with increased TCF7L2 expression, and decreased insulin content and secretion. Using gene expression profiles of 66 human pancreatic islets donors', we also show that the identified TCF7L2-ISL1 transcriptional network is regulated in a genotype-dependent manner. Taken together, these results demonstrate that not only synthesis of proinsulin is regulated by TCF7L2 but also processing and possibly clearance of proinsulin and insulin. These multiple targets in key pathways may explain why TCF7L2 has emerged as the gene showing one of the strongest associations with T2D. © The Author 2014. Published by Oxford University Press.

H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements

PubMed Central

Zink, Lisa-Maria; Delbarre, Erwan; Eberl, H. Christian; Keilhauer, Eva C.; Bönisch, Clemens; Pünzeler, Sebastian; Bartkuhn, Marek; Collas, Philippe; Mann, Matthias

2017-01-01

Abstract Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin. PMID:28334823

A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity.

PubMed

Gervois, P; Torra, I P; Chinetti, G; Grötzinger, T; Dubois, G; Fruchart, J C; Fruchart-Najib, J; Leitersdorf, E; Staels, B

1999-09-01

The peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARalpha activators. This incited us to screen for PPARalpha variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARalpha mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARalpha protein (PPARalphatr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARalphatr mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARalpha mRNA. By contrast, PPARalphatr mRNA could not be detected in rodent tissues. Western blot analysis using PPARalpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARalphatr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARalphatr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARalphawt was mainly nuclear localized, the majority of PPARalphatr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARalphatr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARalphawt transactivation function, PPARalphatr interfered with PPARalphatr transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARalphawt by PPARalphatr, suggesting that the dominant negative effect of PPARalphatr might occur through competition for essential coactivators. In addition, PPARalphatr interfered with transcriptional activity of other nuclear receptors such as PPARgamma, hepatic nuclear factor-4, and glucocorticoid receptor-alpha, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARalpha splice variant that may negatively interfere with PPARalphawt function. Factors regulating either the ratio of PPARalphawt vs. PPARalphatr mRNA or the nuclear entry of PPARalphatr protein should therefore lead to altered signaling via the PPARalpha and, possibly also, other nuclear receptor pathways.

Differential Expression Profile of ZFX Variants Discriminates Breast Cancer Subtypes

PubMed

Pourkeramati, Fatemeh; Asadi, Malek Hossein; Shakeri, Shahryar; Farsinejad, Alireza

2018-05-13

ZFX is a transcriptional regulator in embryonic stem cells that plays an important role in pluripotency and self-renewal. ZFX is widely expressed in pluripotent stem cells and is down-regulated during differentiation of embryonic stem cells. ZFX has five different variants that encode three different protein isoforms. While several reports have determined the overexpression of ZFX in a variety of somatic cancers, the expression of ZFX-spliced variants in cancer cells is not well-understood. We investigated the expression of ZFX variants in a series of breast cancer tissues and cell lines using quantitative PCR. The expression of ZFX variant 1/3 was higher in tumor tissue compared to marginal tissue. In contrast, the ZFX variant 5 was down-regulated in tumor tissues. While the ZFX variant 1/3 and ZFX variant 5 expression significantly increased in low-grade tumors, ZFX variant 4 was strongly expressed in high-grade tumors and demonstrating lymphatic invasion. In addition, our result revealed a significant association between the HER2 status and the expression of ZFX-spliced variants. Our data suggest that the expression of ZFX-spliced transcripts varies between different types of breast cancer and may contribute to their tumorigenesis process. Hence, ZFX-spliced transcripts could be considered as novel tumor markers with a probable value in diagnosis, prognosis, and therapy of breast cancer.

D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein profile

PubMed Central

Valleix, Sophie; Verona, Guglielmo; Jourde-Chiche, Noémie; Nédelec, Brigitte; Mangione, P. Patrizia; Bridoux, Frank; Mangé, Alain; Dogan, Ahmet; Goujon, Jean-Michel; Lhomme, Marie; Dauteuille, Carolane; Chabert, Michèle; Porcari, Riccardo; Waudby, Christopher A.; Relini, Annalisa; Talmud, Philippa J.; Kovrov, Oleg; Olivecrona, Gunilla; Stoppini, Monica; Christodoulou, John; Hawkins, Philip N.; Grateau, Gilles; Delpech, Marc; Kontush, Anatol; Gillmore, Julian D.; Kalopissis, Athina D.; Bellotti, Vittorio

2016-01-01

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients. PMID:26790392

Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

PubMed Central

Sharma, Neeraj; Sosnay, Patrick R.; Ramalho, Anabela S.; Douville, Christopher; Franca, Arianna; Gottschalk, Laura B.; Park, Jeenah; Lee, Melissa; Vecchio-Pagan, Briana; Raraigh, Karen S.; Amaral, Margarida D.; Karchin, Rachel; Cutting, Garry R.

2015-01-01

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. PMID:25066652

Glucocorticoid receptor gene expression and promoter CpG modifications throughout the human brain.

PubMed

Cao-Lei, Lei; Suwansirikul, Songkiet; Jutavijittum, Prapan; Mériaux, Sophie B; Turner, Jonathan D; Muller, Claude P

2013-11-01

Glucocorticoids and the glucocorticoid (GR) and mineralocorticoid (MR) receptors have been implicated in many processes, particularly in negative feedback regulation of the hypothalamic-pituitary-adrenal axis. Epigenetically programmed GR alternative promoter usage underlies transcriptional control of GR levels, generation of GR 3' splice variants, and the overall GC response in the brain. No detailed analysis of GR first exons or GR transcript variants throughout the human brain has been reported. Therefore we investigated post mortem tissues from 28 brain regions of 5 individuals. GR first exons were expressed throughout the healthy human brain with no region-specific usage patterns. First exon levels were highly inter-correlated suggesting that they are co-regulated. GR 3' splice variants (GRα and GR-P) were equally distributed in all regions, and GRβ expression was always low. GR/MR ratios showed significant differences between the 28 tissues with the highest ratio in the pituitary gland. Modification levels of individual CpG dinucleotides, including 5-mC and 5-hmC, in promoters 1D, 1E, 1F, and 1H were low, and diffusely clustered; despite significant heterogeneity between the donors. In agreement with this clustering, sum modification levels rather than individual CpG modifications correlated with GR expression. Two-way ANOVA showed that this sum modification was both promoter and brain region specific, but that there was however no promoter*tissue interaction. The heterogeneity between donors may however hide such an interaction. In both promoters 1F and 1H modification levels correlated with GRα expression suggesting that 5-mC and 5-hmC play an important role in fine tuning GR expression levels throughout the brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny

2006-06-09

Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivatormore » activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.« less

Diabetes-Induced Changes in the Alternative Splicing of the Slo Gene in Corporal Tissue

PubMed Central

Davies, Kelvin P.; Zhao, Weixin; Tar, Moses; Figueroa, Johanna C.; Desai, Pratik; Verselis, Vytas K.; Kronengold, Jack; Wang, Hong-Zhan; Melman, Arnold; Christ, George J.

2007-01-01

Objectives Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel α-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. Methods Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. Results A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. Conclusions Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity. PMID:17150299

A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

PubMed Central

Saxena, Pratik; Heng, Boon Chin; Bai, Peng; Folcher, Marc; Zulewski, Henryk; Fussenegger, Martin

2016-01-01

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. PMID:27063289

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

PubMed

Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

2015-12-17

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

An Integrated Encyclopedia of DNA Elements in the Human Genome

PubMed Central

2012-01-01

Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

Designing oligo libraries taking alternative splicing into account

NASA Astrophysics Data System (ADS)

Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

2001-06-01

We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

Principles of regulatory information conservation between mouse and human.

PubMed

Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun; Wu, Weisheng; Cayting, Philip; Boyle, Alan P; Sundaram, Vasavi; Xing, Xiaoyun; Dogan, Nergiz; Li, Jingjing; Euskirchen, Ghia; Lin, Shin; Lin, Yiing; Visel, Axel; Kawli, Trupti; Yang, Xinqiong; Patacsil, Dorrelyn; Keller, Cheryl A; Giardine, Belinda; Kundaje, Anshul; Wang, Ting; Pennacchio, Len A; Weng, Zhiping; Hardison, Ross C; Snyder, Michael P

2014-11-20

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress

PubMed Central

Lehman, Stacey L.; Cerniglia, George J.; Johannes, Gregg J.; Ye, Jiangbin; Ryeom, Sandra; Koumenis, Constantinos

2015-01-01

Multiple transcripts encode for the cell cycle inhibitor p21Cip1. These transcripts produce identical proteins but differ in their 5’ untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through 35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5’ upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress. PMID:26102367

A novel transcript of cyclin-dependent kinase-like 5 (CDKL5) has an alternative C-terminus and is the predominant transcript in brain.

PubMed

Williamson, Sarah L; Giudici, Laura; Kilstrup-Nielsen, Charlotte; Gold, Wendy; Pelka, Gregory J; Tam, Patrick P L; Grimm, Andrew; Prodi, Dionigio; Landsberger, Nicoletta; Christodoulou, John

2012-02-01

The X-linked cyclin-dependent kinase-like 5 (CDKL5) gene is an important molecular determinant of early-onset intractable seizures with infantile spasms and Rett syndrome-like phenotype. The gene encodes a kinase that may influence components of molecular pathways associated with MeCP2. In humans there are two previously reported splice variants that differ in the 5' untranslated exons and produce the same 115 kDa protein. Furthermore, very recently, a novel transcript including a novel exon (16b) has been described. By aligning both the human and mouse CDKL5 proteins to the orthologs of other species, we identified a theoretical 107 kDa isoform with an alternative C-terminus that terminates in intron 18. In human brain and all other tissues investigated except the testis, this novel isoform is the major CDKL5 transcript. The detailed characterisation of this novel isoform of CDKL5 reveals functional and subcellular localisation attributes that overlap greatly, but not completely, with that of the previously studied human CDKL5 protein. Considering its predominant expression in the human and mouse brain, we believe that this novel isoform is likely to be of primary pathogenic importance in human diseases associated with CDKL5 deficiency, and suggest that screening of the related intronic sequence should be included in the molecular genetic analyses of patients with a suggestive clinical phenotype.

Changes in the transcriptional profile in response to overexpression of the osteopontin-c splice isoform in ovarian (OvCar-3) and prostate (PC-3) cancer cell lines

PubMed Central

2014-01-01

Background Especially in human tumor cells, the osteopontin (OPN) primary transcript is subject to alternative splicing, generating three isoforms termed OPNa, OPNb and OPNc. We previously demonstrated that the OPNc splice variant activates several aspects of the progression of ovarian and prostate cancers. The goal of the present study was to develop cell line models to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods Human ovarian and prostate cancer cell lines, OvCar-3 and PC-3 cells, respectively, were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses. Results Among 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34 and 16, respectively, were differentially expressed between OvCar-3 and PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Based on marked up-regulation of Vegfa transcript in response to OPNc overexpression, we partially validated the array data by demonstrating that conditioned medium (CM) secreted from OvCar-3 and PC-3 OPNc-overexpressing cells significantly induced endothelial cell adhesion, proliferation and migration, compared to CM secreted from control cells. Conclusions Overall, the present study elucidated transcriptional changes of OvCar-3 and PC-3 cancer cell lines in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. PMID:24928374

Streptococcal Adhesin P (SadP) contributes to Streptococcus suis adhesion to the human intestinal epithelium.

PubMed

Ferrando, Maria Laura; Willemse, Niels; Zaccaria, Edoardo; Pannekoek, Yvonne; van der Ende, Arie; Schultsz, Constance

2017-01-01

Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of Streptococcal adhesin Protein (SadP) to host-pathogen interaction at GIT level. SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro. Expression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC. SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential.

De Novo Truncating Variants in ASXL2 Are Associated with a Unique and Recognizable Clinical Phenotype.

PubMed

Shashi, Vandana; Pena, Loren D M; Kim, Katherine; Burton, Barbara; Hempel, Maja; Schoch, Kelly; Walkiewicz, Magdalena; McLaughlin, Heather M; Cho, Megan; Stong, Nicholas; Hickey, Scott E; Shuss, Christine M; Freemark, Michael S; Bellet, Jane S; Keels, Martha Ann; Bonner, Melanie J; El-Dairi, Maysantoine; Butler, Megan; Kranz, Peter G; Stumpel, Constance T R M; Klinkenberg, Sylvia; Oberndorff, Karin; Alawi, Malik; Santer, Rene; Petrovski, Slavé; Kuismin, Outi; Korpi-Heikkilä, Satu; Pietilainen, Olli; Aarno, Palotie; Kurki, Mitja I; Hoischen, Alexander; Need, Anna C; Goldstein, David B; Kortüm, Fanny

2016-10-06

The ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers syndromes, which result in overlapping features of severe intellectual disability and dysmorphic features. ASXL2 has not yet been associated with a human Mendelian disorder. In this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. All six had de novo truncating variants in ASXL2. A careful review enabled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia, and developmental disabilities. Although overlapping features with Bohring-Opitz and Bainbridge-Ropers syndromes exist, features that distinguish the ASXL2-associated condition from ASXL1- and ASXL3-related disorders are macrocephaly, absence of growth retardation, and more variability in the degree of intellectual disabilities. We were also able to demonstrate with mRNA studies that these variants are likely to exert a dominant-negative effect, given that both alleles are expressed in blood and the mutated ASXL2 transcripts escape nonsense-mediated decay. In conclusion, de novo truncating variants in ASXL2 underlie a neurodevelopmental syndrome with a clinically recognizable phenotype. This report expands the germline disorders that are linked to the ASXL genes. Copyright © 2016. Published by Elsevier Inc.

The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences

PubMed Central

Xue, Cheng; Raveendran, Muthuswamy; Harris, R. Alan; Fawcett, Gloria L.; Liu, Xiaoming; White, Simon; Dahdouli, Mahmoud; Rio Deiros, David; Below, Jennifer E.; Salerno, William; Cox, Laura; Fan, Guoping; Ferguson, Betsy; Horvath, Julie; Johnson, Zach; Kanthaswamy, Sree; Kubisch, H. Michael; Liu, Dahai; Platt, Michael; Smith, David G.; Sun, Binghua; Vallender, Eric J.; Wang, Feng; Wiseman, Roger W.; Chen, Rui; Muzny, Donna M.; Gibbs, Richard A.; Yu, Fuli; Rogers, Jeffrey

2016-01-01

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease. PMID:27934697

Characterization of murine SIRT3 transcript variants and corresponding protein products

USDA-ARS?s Scientific Manuscript database

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast SIR2 gene. SIRT3 possesses NAD(+)-dependent protein deacetylase activity. Recent studies indicate that the murine SIRT3 gene expresses different transcript variants, resulting in three possible SIRT3 protein isoforms with various leng...

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

PubMed Central

Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

2010-01-01

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

A comprehensive collection of annotations to interpret sequence variation in human mitochondrial transfer RNAs.

PubMed

Diroma, Maria Angela; Lubisco, Paolo; Attimonelli, Marcella

2016-11-08

The abundance of biological data characterizing the genomics era is contributing to a comprehensive understanding of human mitochondrial genetics. Nevertheless, many aspects are still unclear, specifically about the variability of the 22 human mitochondrial transfer RNA (tRNA) genes and their involvement in diseases. The complex enrichment and isolation of tRNAs in vitro leads to an incomplete knowledge of their post-transcriptional modifications and three-dimensional folding, essential for correct tRNA functioning. An accurate annotation of mitochondrial tRNA variants would be definitely useful and appreciated by mitochondrial researchers and clinicians since the most of bioinformatics tools for variant annotation and prioritization available so far cannot shed light on the functional role of tRNA variations. To this aim, we updated our MToolBox pipeline for mitochondrial DNA analysis of high throughput and Sanger sequencing data by integrating tRNA variant annotations in order to identify and characterize relevant variants not only in protein coding regions, but also in tRNA genes. The annotation step in the pipeline now provides detailed information for variants mapping onto the 22 mitochondrial tRNAs. For each mt-tRNA position along the entire genome, the relative tRNA numbering, tRNA type, cloverleaf secondary domains (loops and stems), mature nucleotide and interactions in the three-dimensional folding were reported. Moreover, pathogenicity predictions for tRNA and rRNA variants were retrieved from the literature and integrated within the annotations provided by MToolBox, both in the stand-alone version and web-based tool at the Mitochondrial Disease Sequence Data Resource (MSeqDR) website. All the information available in the annotation step of MToolBox were exploited to generate custom tracks which can be displayed in the GBrowse instance at MSeqDR website. To the best of our knowledge, specific data regarding mitochondrial variants in tRNA genes were introduced for the first time in a tool for mitochondrial genome analysis, supporting the interpretation of genetic variants in specific genomic contexts.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

PubMed

Sato, Akira; Takano, Takeshi; Hiramoto, Akiko; Naito, Tomoharu; Matsuda, Akira; Fukushima, Masakazu; Wataya, Yusuke; Kim, Hye-Sook

2017-08-01

A nucleosidic medicine, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

PubMed

Sethi, Isha; Romano, Rose-Anne; Gluck, Christian; Smalley, Kirsten; Vojtesek, Borivoj; Buck, Michael J; Sinha, Satrajit

2015-08-07

The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

PubMed Central

Leong, Ivone U.S.; Dryland, Philippa A.; Prosser, Debra O.; Lai, Stella W.-S.; Graham, Mandy; Stiles, Martin; Crawford, Jackie; Skinner, Jonathan R.; Love, Donald R.

2017-01-01

Background Approximately 75% of clinically definite long QT syndrome (LQTS) cases are caused by mutations in the KCNQ1, KCNH2 and SCN5A genes. Of these mutations, a small proportion (3.2-9.2%) are predicted to affect splicing. These mutations present a particular challenge in ascribing pathogenicity. Methods Here we report an analysis of the transcriptional consequences of two mutations, one in the KCNQ1 gene (c.781_782delinsTC) and one in the SCN5A gene (c.2437-5C>A), which are predicted to affect splicing. We isolated RNA from lymphocytes and used a directed PCR amplification strategy of cDNA to show mis-spliced transcripts in mutation-positive patients. Results The loss of an exon in each mis-spliced transcript had no deduced effect on the translational reading frame. The clinical phenotype corresponded closely with genotypic status in family members carrying the KCNQ1 splice variant, but not in family members with the SCN5A splice variant. These results are put in the context of a literature review, where only 20% of all splice variants reported in the KCNQ1, KCNH2 and SCN5A gene entries in the HGMDPro 2015.4 database have been evaluated using transcriptional assays. Conclusions Prediction programmes play a strong role in most diagnostic laboratories in classifying variants located at splice sites; however, transcriptional analysis should be considered critical to confirm mis-splicing. Critically, this study shows that genuine mis- splicing may not always imply clinical significance, and genotype/phenotype cosegregation remains important even when mis-splicing is confirmed. PMID:28725320

Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer's disease.

PubMed

Gjoneska, Elizabeta; Pfenning, Andreas R; Mathys, Hansruedi; Quon, Gerald; Kundaje, Anshul; Tsai, Li-Huei; Kellis, Manolis

2015-02-19

Alzheimer's disease (AD) is a severe age-related neurodegenerative disorder characterized by accumulation of amyloid-β plaques and neurofibrillary tangles, synaptic and neuronal loss, and cognitive decline. Several genes have been implicated in AD, but chromatin state alterations during neurodegeneration remain uncharacterized. Here we profile transcriptional and chromatin state dynamics across early and late pathology in the hippocampus of an inducible mouse model of AD-like neurodegeneration. We find a coordinated downregulation of synaptic plasticity genes and regulatory regions, and upregulation of immune response genes and regulatory regions, which are targeted by factors that belong to the ETS family of transcriptional regulators, including PU.1. Human regions orthologous to increasing-level enhancers show immune-cell-specific enhancer signatures as well as immune cell expression quantitative trait loci, while decreasing-level enhancer orthologues show fetal-brain-specific enhancer activity. Notably, AD-associated genetic variants are specifically enriched in increasing-level enhancer orthologues, implicating immune processes in AD predisposition. Indeed, increasing enhancers overlap known AD loci lacking protein-altering variants, and implicate additional loci that do not reach genome-wide significance. Our results reveal new insights into the mechanisms of neurodegeneration and establish the mouse as a useful model for functional studies of AD regulatory regions.

Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant.

PubMed

Yandell, C A; Francis, G L; Wheldrake, J F; Upton, Z

1999-06-01

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

PubMed

Karyagina, A S; Boksha, I S; Grunina, T M; Demidenko, A V; Poponova, M S; Sergienko, O V; Lyashchuk, A M; Galushkina, Z M; Soboleva, L A; Osidak, E O; Bartov, M S; Gromov, A V; Lunin, V G

2017-05-01

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

ATOH7 mutations cause autosomal recessive persistent hyperplasia of the primary vitreous

PubMed Central

Prasov, Lev; Masud, Tehmina; Khaliq, Shagufta; Mehdi, S. Qasim; Abid, Aiysha; Oliver, Edward R.; Silva, Eduardo D.; Lewanda, Amy; Brodsky, Michael C.; Borchert, Mark; Kelberman, Daniel; Sowden, Jane C.; Dattani, Mehul T.; Glaser, Tom

2012-01-01

The vertebrate basic helix–loop–helix (bHLH) transcription factor ATOH7 (Math5) is specifically expressed in the embryonic neural retina and is required for the genesis of retinal ganglion cells (RGCs) and optic nerves. In Atoh7 mutant mice, the absence of trophic factors secreted by RGCs prevents the development of the intrinsic retinal vasculature and the regression of fetal blood vessels, causing persistent hyperplasia of the primary vitreous (PHPV). We therefore screened patients with hereditary PHPV, as well as bilateral optic nerve aplasia (ONA) or hypoplasia (ONH), for mutations in ATOH7. We identified a homozygous ATOH7 mutation (N46H) in a large family with an autosomal recessive PHPV disease trait linked to 10q21, and a heterozygous variant (R65G, p.Arg65Gly) in one of five sporadic ONA patients. High-density single-nucleotide polymorphism analysis also revealed a CNTN4 duplication and an OTX2 deletion in the ONA cohort. Functional analysis of ATOH7 bHLH domain substitutions, by electrophoretic mobility shift and luciferase cotransfection assays, revealed that the N46H variant cannot bind DNA or activate transcription, consistent with structural modeling. The N46H variant also failed to rescue RGC development in mouse Atoh7−/− retinal explants. The R65G variant retains all of these activities, similar to wild-type human ATOH7. Our results strongly suggest that autosomal recessive persistent hyperplastic primary vitreous is caused by N46H and is etiologically related to nonsyndromic congenital retinal nonattachment. The R65G allele, however, cannot explain the ONA phenotype. Our study firmly establishes ATOH7 as a retinal disease gene and provides a functional basis to analyze new coding variants. PMID:22645276

Structure-Function Relationships in Human Testis-determining Factor SRY

PubMed Central

Racca, Joseph D.; Chen, Yen-Shan; Maloy, James D.; Wickramasinghe, Nalinda; Phillips, Nelson B.; Weiss, Michael A.

2014-01-01

Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46 XY gonadal dysgenesis with female somatic phenotype (Swyer syndrome) and confer a high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high mobility group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast one-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through the following: (i) in vitro assays of specific DNA binding and protein stability, and (ii) cell culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic “buttress” beneath its specific DNA-bending surface. PMID:25258310

Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints

PubMed Central

Suciu, Maria C.; Telenius, Jelena

2017-01-01

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k-mer-based analysis of DNase footprints to determine any k-mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome. PMID:28904015

Identification of interleukin-26 in the dromedary camel (Camelus dromedarius): Evidence of alternative splicing and isolation of novel splice variants.

PubMed

Premraj, Avinash; Nautiyal, Binita; Aleyas, Abi G; Rasool, Thaha Jamal

2015-10-01

Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Insights into the association of Gla-rich protein and osteoarthritis, novel splice variants and γ-carboxylation status.

PubMed

Rafael, Marta S; Cavaco, Sofia; Viegas, Carla S B; Santos, Sofia; Ramos, Acácio; Willems, Brecht A G; Herfs, Marjolein; Theuwissen, Elke; Vermeer, Cees; Simes, Dina C

2014-08-01

Gla-rich protein (GRP) is a vitamin K dependent protein, characterized by a high density of γ-carboxylated Glu residues, shown to accumulate in mouse and sturgeon cartilage and at sites of skin and vascular calcification in humans. Therefore, we investigated the involvement of GRP in pathological calcification in osteoarthritis (OA). Comparative analysis of GRP patterning at transcriptional and translational levels was performed between controls and OA patients. Using a RT-PCR strategy we unveiled two novel splice variants in human-GRP-F5 and F6-potentially characterized by the loss of full γ-carboxylation and secretion functional motifs. GRP-F1 is shown to be the predominant splice variant expressed in mouse and human adult tissues, particularly in OA cartilage, while an overexpressing human cell model points it as the major γ-carboxylated isoform. Using validated conformational antibodies detecting carboxylated or undercarboxylated GRP (c/uc GRP), we have demonstrated cGRP accumulation in controls, whereas ucGRP was the predominant form in OA-affected tissues, colocalizing at sites of ectopic calcification. Overall, our results indicate the predominance of GRP-F1, and a clear association of ucGRP with OA cartilage and synovial membrane. Levels of vitamin K should be further assessed in these patients to determine its potential therapeutic use as a supplement in OA treatment. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

APADB: a database for alternative polyadenylation and microRNA regulation events

PubMed Central

Müller, Sören; Rycak, Lukas; Afonso-Grunz, Fabian; Winter, Peter; Zawada, Adam M.; Damrath, Ewa; Scheider, Jessica; Schmäh, Juliane; Koch, Ina; Kahl, Günter; Rotter, Björn

2014-01-01

Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3′ untranslated region (3′UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3′UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3′ end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3′ end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3′UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/ PMID:25052703

Functional characterization of an oxytocin receptor gene variant (rs2268498) previously associated with social cognition by expression analysis in vitro and in human brain biopsy.

PubMed

Reuter, Martin; Montag, Christian; Altmann, Steffen; Bendlow, Fabian; Elger, Christian; Kirsch, Peter; Becker, Albert; Schoch-McGovern, Susanne; Simon, Matthias; Weber, Bernd; Felten, Andrea

2017-10-01

The oxytocin system plays a prominent role in social behavior across species, and numerous genetic studies in humans have reported associations between polymorphisms on the oxytocin receptor (OXTR) gene and phenotypes related to social cognition, affiliation, perspective taking, and sociability in healthy subjects and in patients with atypical social behavior, such as in autism spectrum disorders (ASD). Recently, the first study demonstrating altered agonist-induced OXTR internalization and recycling for the exonic variant rs35062132 emerged. Beside this, there has been no further demonstration of the functionality of the OXTR variants especially there does not exist any for the regulatory units. To address this gap in the literature, we tested the functionality of the promoter flanking single nucleotide polymorphism (SNP) rs2268498, which has proven an interesting candidate for predicting social behavior in recent association studies. Results of genetic expression analyses in human hippocampal tissue showed a twofold difference in messenger RNA transcription, dependent on the presence or absence of the C-allele. This finding was corroborated by cloning, i.e., in vitro reporter gene expression analysis after transfection of OXTR promoter plasmids into HEK-293 cells. Our results underline the importance of OXTR rs2268498 for genetic research in social behavior and ASD.

Complex nature of SNP genotype effects on gene expression in primary human leucocytes.

PubMed

Heap, Graham A; Trynka, Gosia; Jansen, Ritsert C; Bruinenberg, Marcel; Swertz, Morris A; Dinesen, Lotte C; Hunt, Karen A; Wijmenga, Cisca; Vanheel, David A; Franke, Lude

2009-01-07

Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown. We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease - a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects. In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, cis expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed cis-eQTLs. Celiac associated risk variants from two regions, containing genes IL18RAP and CCR3, showed significant cis genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected. In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.

Toll-Like Receptor-3 and Geographic Atrophy in Age-Related Macular Degeneration

PubMed Central

Yang, Zhenglin; Stratton, Charity; Francis, Peter J.; Kleinman, Mark E.; Tan, Perciliz L.; Gibbs, Daniel; Tong, Zongzhong; Chen, Haoyu; Constantine, Ryan; Yang, Xian; Chen, Yuhong; Zeng, Jiexi; Davey, Lisa; Ma, Xiang; Hau, Vincent S.; Wang, Chi; Harmon, Jennifer; Buehler, Jeanette; Pearson, Erik; Patel, Shrena; Kaminoh, Yuuki; Watkins, Scott; Luo, Ling; Zabriskie, Norman A.; Bernstein, Paul S.; Cho, Wongil; Schwager, Andrea; Hinton, David R; Klein, Michael L; Hamon, Sara C.; Simmons, Emily; Yu, Beifeng; Campochiaro, Betsy; Sunness, Janet S.; Campochiaro, Peter; Jorde, Lynn; Parmigiani, Giovanni; Zack, Donald J.; Katsanis, Nicholas; Ambati, Jayakrishna; Zhang, Kang

2008-01-01

BACKGROUND Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world. Advanced AMD is comprised of geographic atrophy (GA) and choroidal neovascularization (CNV). Specific genetic variants that predispose for GA are largely unknown. METHODS We tested (i) for association between the functional toll-like receptor-3 (TLR3) variant rs3775291 (L412F) and AMD in European Americans and (ii) the effect of TLR3 L and F variants on the viability of human retinal pigment epithelium (RPE) cells in vitro and on RPE cell apoptosis in wildtype and Tlr3−/− mice. RESULTS The F variant (or T allele at single nucleotide polymorphism at rs3775291) was associated with protection against GA (P=0.005); this association was replicated in two independent GA case-control series (P=5.43×10−4 and P=0.002, respectively. We observed no association between TLR3 variants and CNV. The rs377291 variant is probably critical to the function of TLR3, because a prototypic TLR3 ligand induced cell death and apoptosis in human RPE cells with the LL genotype to a greater extent than it did RPE cells with the LF genotype. Moreover, the ligand induced more RPE cell death and apoptosis in wild-type than in Tlr3−/− mice. CONCLUSIONS The TLR3 412F variant confers protection against GA, probably by suppressing RPE cell death. Given that double stranded RNA can activate TLR3-mediated apoptosis, our results suggest a possible role for viral dsRNA transcripts in the development of GA and raise awareness of potential toxicity induced by short interfering RNA (siRNA) therapeutics in the eye. PMID:18753640

A few nucleotide polymorphisms are sufficient to recruit nuclear factors differentially to the intron 1 of HPV-16 intratypic variants.

PubMed

López-Urrutia, Eduardo; Valdés, Jesús; Bonilla-Moreno, Raúl; Martínez-Salazar, Martha; Martínez-Garcia, Martha; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

2012-06-01

The HPV-16 E6/E7 genes, which contain intron 1, are processed by alternative splicing and its transcripts are detected with a heterogeneous profile in tumours cells. Frequently, the HPV-16 positive carcinoma cells bear viral variants that contain single nucleotide polymorphisms into its DNA sequence. We were interested in analysing the contribution of this polymorphism to the heterogeneity in the pattern of the E6/E7 spliced transcripts. Using the E6/E7 sequences from three closely related HPV-16 variants, we have shown that a few nucleotide changes are sufficient to produce heterogeneity in the splicing profile. Furthermore, using mutants that contained a single SNP, we also showed that one nucleotide change was sufficient to reproduce the heterogeneous splicing profile. Additionally, a difference of two or three SNPs among these viral sequences was sufficient to recruit differentially several splicing factors to the polymorphic E6/E7 transcripts. Moreover, only one SNP was sufficient to alter the binding site of at least one splicing factor, changing the ability of splicing factors to bind the transcript. Finally, the factors that were differentially bound to the short form of intron 1 of one of these E6/E7 variants were identified as TIA1 and/or TIAR and U1-70k, while U2AF65, U5-52k and PTB were preferentially bound to the transcript of the other variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Retinoic acid-induced nNOS expression depends on a novel PI3K/Akt/DAX1 pathway in human TGW-nu-I neuroblastoma cells.

PubMed

Nagl, Florian; Schönhofer, Katrin; Seidler, Barbara; Mages, Jörg; Allescher, Hans-Dieter; Schmid, Roland M; Schneider, Günter; Saur, Dieter

2009-11-01

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon 1a-1l, transcribed from distinct promoters, leading to nNOS variants with different 5'-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NR0B1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment.

Equivalent missense variant in the FOXP2 and FOXP1 transcription factors causes distinct neurodevelopmental disorders.

PubMed

Sollis, Elliot; Deriziotis, Pelagia; Saitsu, Hirotomo; Miyake, Noriko; Matsumoto, Naomichi; Hoffer, Mariëtte J V; Ruivenkamp, Claudia A L; Alders, Mariëlle; Okamoto, Nobuhiko; Bijlsma, Emilia K; Plomp, Astrid S; Fisher, Simon E

2017-11-01

The closely related paralogues FOXP2 and FOXP1 encode transcription factors with shared functions in the development of many tissues, including the brain. However, while mutations in FOXP2 lead to a speech/language disorder characterized by childhood apraxia of speech (CAS), the clinical profile of FOXP1 variants includes a broader neurodevelopmental phenotype with global developmental delay, intellectual disability, and speech/language impairment. Using clinical whole-exome sequencing, we report an identical de novo missense FOXP1 variant identified in three unrelated patients. The variant, p.R514H, is located in the forkhead-box DNA-binding domain and is equivalent to the well-studied p.R553H FOXP2 variant that cosegregates with CAS in a large UK family. We present here for the first time a direct comparison of the molecular and clinical consequences of the same mutation affecting the equivalent residue in FOXP1 and FOXP2. Detailed functional characterization of the two variants in cell model systems revealed very similar molecular consequences, including aberrant subcellular localization, disruption of transcription factor activity, and deleterious effects on protein interactions. Nonetheless, clinical manifestations were broader and more severe in the three cases carrying the p.R514H FOXP1 variant than in individuals with the p.R553H variant related to CAS, highlighting divergent roles of FOXP2 and FOXP1 in neurodevelopment. © 2017 Wiley Periodicals, Inc.

Modeling gene-environment interactions in oral cavity and esophageal cancers demonstrates a role for the p53 R72P polymorphism in modulating susceptibility.

PubMed

Sarkar, Jayanta; Dominguez, Emily; Li, Guojun; Kusewitt, Donna F; Johnson, David G

2014-08-01

A large number of epidemiological studies have linked a common single-nucleotide polymorphism (SNP) in the human p53 gene to risk for developing a variety of cancers. This SNP encodes either an arginine or proline at position 72 (R72P) of the p53 protein, which can alter the apoptotic activity of p53 via transcriptional and non-transcriptional mechanisms. This SNP has also been reported to modulate the development of human papilloma virus (HPV)-driven cancers through differential targeting of the p53 variant proteins by the E6 viral oncoprotein. Mouse models for the p53 R72P polymorphism have recently been developed but a role for this SNP in modifying cancer risk in response to viral and chemical carcinogens has yet to be established experimentally. Here, we demonstrate that the p53 R72P polymorphism modulates the hyperprolferative, apoptotic and inflammatory phenotypes caused by expression of the HPV16 E6 and E7 oncoproteins. Moreover, the R72P SNP also modifies the carcinogenic response to the chemical carcinogen 4NQO, in the presence and absence of the HPV16 transgene. Our findings confirm several human epidemiological studies associating the codon 72 proline variant with increased risk for certain cancers but also suggest that there are tissue-specific differences in how the R72P polymorphism influences the response to environmental carcinogens. © 2013 Wiley Periodicals, Inc.

Histone Lysine Methylases and Demethylases in the Landscape of Human Developmental Disorders.

PubMed

Faundes, Víctor; Newman, William G; Bernardini, Laura; Canham, Natalie; Clayton-Smith, Jill; Dallapiccola, Bruno; Davies, Sally J; Demos, Michelle K; Goldman, Amy; Gill, Harinder; Horton, Rachel; Kerr, Bronwyn; Kumar, Dhavendra; Lehman, Anna; McKee, Shane; Morton, Jenny; Parker, Michael J; Rankin, Julia; Robertson, Lisa; Temple, I Karen; Banka, Siddharth

2018-01-04

Histone lysine methyltransferases (KMTs) and demethylases (KDMs) underpin gene regulation. Here we demonstrate that variants causing haploinsufficiency of KMTs and KDMs are frequently encountered in individuals with developmental disorders. Using a combination of human variation databases and existing animal models, we determine 22 KMTs and KDMs as additional candidates for dominantly inherited developmental disorders. We show that KMTs and KDMs that are associated with, or are candidates for, dominant developmental disorders tend to have a higher level of transcription, longer canonical transcripts, more interactors, and a higher number and more types of post-translational modifications than other KMT and KDMs. We provide evidence to firmly associate KMT2C, ASH1L, and KMT5B haploinsufficiency with dominant developmental disorders. Whereas KMT2C or ASH1L haploinsufficiency results in a predominantly neurodevelopmental phenotype with occasional physical anomalies, KMT5B mutations cause an overgrowth syndrome with intellectual disability. We further expand the phenotypic spectrum of KMT2B-related disorders and show that some individuals can have severe developmental delay without dystonia at least until mid-childhood. Additionally, we describe a recessive histone lysine-methylation defect caused by homozygous or compound heterozygous KDM5B variants and resulting in a recognizable syndrome with developmental delay, facial dysmorphism, and camptodactyly. Collectively, these results emphasize the significance of histone lysine methylation in normal human development and the importance of this process in human developmental disorders. Our results demonstrate that systematic clinically oriented pathway-based analysis of genomic data can accelerate the discovery of rare genetic disorders. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

ΔN-TRPV1: A Molecular Co-detector of Body Temperature and Osmotic Stress.

PubMed

Zaelzer, Cristian; Hua, Pierce; Prager-Khoutorsky, Masha; Ciura, Sorana; Voisin, Daniel L; Liedtke, Wolfgang; Bourque, Charles W

2015-10-06

Thirst and antidiuretic hormone secretion occur during hyperthermia or hypertonicity to preserve body hydration. These vital responses are triggered when hypothalamic osmoregulatory neurons become depolarized by ion channels encoded by an unknown product of the transient receptor potential vanilloid-1 gene (Trpv1). Here, we show that rodent osmoregulatory neurons express a transcript of Trpv1 that mediates the selective translation of a TRPV1 variant that lacks a significant portion of the channel's amino terminus (ΔN-TRPV1). The mRNA transcript encoding this variant (Trpv1dn) is widely expressed in the brains of osmoregulating vertebrates, including the human hypothalamus. Transfection of Trpv1dn into heterologous cells induced the expression of ion channels that could be activated by either hypertonicity or by heating in the physiological range. Moreover, expression of Trpv1dn rescued the osmosensory and thermosensory responses of single hypothalamic neurons obtained from Trpv1 knockout mice. ΔN-TRPV1 is therefore a co-detector of core body temperature and fluid tonicity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Integrating evolutionary and regulatory information with a multispecies approach implicates genes and pathways in obsessive-compulsive disorder.

PubMed

Noh, Hyun Ji; Tang, Ruqi; Flannick, Jason; O'Dushlaine, Colm; Swofford, Ross; Howrigan, Daniel; Genereux, Diane P; Johnson, Jeremy; van Grootheest, Gerard; Grünblatt, Edna; Andersson, Erik; Djurfeldt, Diana R; Patel, Paresh D; Koltookian, Michele; M Hultman, Christina; Pato, Michele T; Pato, Carlos N; Rasmussen, Steven A; Jenike, Michael A; Hanna, Gregory L; Stewart, S Evelyn; Knowles, James A; Ruhrmann, Stephan; Grabe, Hans-Jörgen; Wagner, Michael; Rück, Christian; Mathews, Carol A; Walitza, Susanne; Cath, Daniëlle C; Feng, Guoping; Karlsson, Elinor K; Lindblad-Toh, Kerstin

2017-10-17

Obsessive-compulsive disorder is a severe psychiatric disorder linked to abnormalities in glutamate signaling and the cortico-striatal circuit. We sequenced coding and regulatory elements for 608 genes potentially involved in obsessive-compulsive disorder in human, dog, and mouse. Using a new method that prioritizes likely functional variants, we compared 592 cases to 560 controls and found four strongly associated genes, validated in a larger cohort. NRXN1 and HTR2A are enriched for coding variants altering postsynaptic protein-binding domains. CTTNBP2 (synapse maintenance) and REEP3 (vesicle trafficking) are enriched for regulatory variants, of which at least six (35%) alter transcription factor-DNA binding in neuroblastoma cells. NRXN1 achieves genome-wide significance (p = 6.37 × 10 -11 ) when we include 33,370 population-matched controls. Our findings suggest synaptic adhesion as a key component in compulsive behaviors, and show that targeted sequencing plus functional annotation can identify potentially causative variants, even when genomic data are limited.Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder with symptoms including intrusive thoughts and time-consuming repetitive behaviors. Here Noh and colleagues identify genes enriched for functional variants associated with increased risk of OCD.

The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans.

PubMed

Bai, JinLi; Qu, YuJin; Cao, YanYan; Yang, Lan; Ge, Lin; Jin, YuWei; Wang, Hong; Song, Fang

2018-02-20

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%-10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor-Up-frameshift protein 1 (UPF1)-was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (fl-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged from 2.5±0.4 to 8.3±0.1, 1.9±0.2 to 5.0±0.7 and 2.2±0.1 to 4.9±0.2 for two lymphoblastoid cell lines and one fibroblasts cell line, respectively. For these cell lines, the fold increases of the SMN1 transcript levels of puromycin were as follows: 5.5±0.2 to 19.5±4.0, 3.1±0.3 to 9.9±1.8 and 1.5±0.2 to 6.5±0.5. Meanwhile, the SC35 1.7-kb transcript levels were markedly increased in all 3 cell lines. In addition, lentivirus-mediated UPF1 knockdown lead to a reduction of the UPF1 protein level to 22.5% compared to the negative control lentivirus. Additionally, knockdown of the UPF1 gene also promoted mRNA expression of the SC35 1.7kb and fl-SMN1 genes. The increases of the SMN1 and SC35 1.7-kb mRNA levels reached about 4- and 6.5-fold in fibroblasts derived from the patient 2, respectively. Altogether, our study provides the first evidence that the c.22 dupA variant in the SMN1 gene triggers NMD. SMA pathogenesis in the patient is associated with mRNA degradation of SMN1, but not the truncated SMN protein. Copyright © 2017. Published by Elsevier B.V.

Global analysis of A-to-I RNA editing reveals association with common disease variants

PubMed Central

Jain, Rajeev; Jain, Anamika; Betsholtz, Christer; Giannarelli, Chiara; Kovacic, Jason C.; Ruusalepp, Arno; Skogsberg, Josefin; Hao, Ke; Schadt, Eric E.

2018-01-01

RNA editing modifies transcripts and may alter their regulation or function. In humans, the most common modification is adenosine to inosine (A-to-I). We examined the global characteristics of RNA editing in 4,301 human tissue samples. More than 1.6 million A-to-I edits were identified in 62% of all protein-coding transcripts. mRNA recoding was extremely rare; only 11 novel recoding sites were uncovered. Thirty single nucleotide polymorphisms from genome-wide association studies were associated with RNA editing; one that influences type 2 diabetes (rs2028299) was associated with editing in ARPIN. Twenty-five genes, including LRP11 and PLIN5, had editing sites that were associated with plasma lipid levels. Our findings provide new insights into the genetic regulation of RNA editing and establish a rich catalogue for further exploration of this process. PMID:29527417

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

PubMed Central

Ronander, Elena; Bengtsson, Dominique C.; Joergensen, Louise; Jensen, Anja T. R.; Arnot, David E.

2012-01-01

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1. Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types. PMID:23070076

Association of regulatory TPH2 polymorphisms with higher reduction in depressive symptoms in children and adolescents treated with fluoxetine.

PubMed

Gassó, Patricia; Rodríguez, Natalia; Boloc, Daniel; Blázquez, Ana; Torres, Teresa; Gortat, Ana; Plana, Maria Teresa; Lafuente, Amalia; Mas, Sergi; Arnaiz, Joan Albert; Lázaro, Luisa

2017-07-03

Genetic variability related to the brain serotonergic system has a significant impact on both the susceptibility to psychiatric disorders, such as major depressive disorder (MDD), and the response to antidepressant drugs, such as fluoxetine. TPH2 is one of the most important serotonergic candidate genes in selective serotonin reuptake inhibitors (SSRIs) pharmacogenetic studies. The aim of the present study was to evaluate the influence of regulatory polymorphisms that are specifically located in human TPH2 transcription factor binding sites (TFBSs), and therefore could be functional by altering gene expression, on clinical improvement in children and adolescents treated with fluoxetine. The selection of SNPs was also based on their linkage disequilibrium with TPH2 rs4570625, a genetic variant with questionable functionality, which was previously associated with clinical response in our pediatric population. A total of 83 children and adolescents were clinically evaluated 12weeks after initiating antidepressant treatment with fluoxetine for the first time. Clinical improvement was assessed by reductions in depressive symptoms measured using the Children's Depression Inventory (CDI) scale. The polymorphisms rs11179002, rs60032326 and rs34517220 were, for the first time in the literature, significantly associated with higher clinical improvement. The strongest association was found for rs34517220. In particular, minor allele homozygotes showed higher score reductions on the CDI scale compared with the major allele carriers. Interestingly, this polymorphism is located in a human TPH2 TFBS for two relevant transcription factors in the serotoninergic neurons, Foxa1 and Foxa2, which together with the high level of significance found for this SNP, could indicate that rs34517220 is in fact the crucial functional genetic variant related to the fluoxetine response. These results provide new evidence for the role of regulatory genetic variants that could modulate human TPH2 expression in the SSRI antidepressant response. Copyright © 2017 Elsevier Inc. All rights reserved.

Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.

PubMed Central

Scherf, A; Hernandez-Rivas, R; Buffet, P; Bottius, E; Benatar, C; Pouvelle, B; Gysin, J; Lanzer, M

1998-01-01

Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM-1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run-on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation. PMID:9736619

A unique H2A histone variant occupies the transcriptional start site of active genes.

PubMed

Soboleva, Tatiana A; Nekrasov, Maxim; Pahwa, Anuj; Williams, Rohan; Huttley, Gavin A; Tremethick, David J

2011-12-04

Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis.

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1

PubMed Central

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-01-01

AIM: To study the expression of HBV enhancer II by transcription factor COUP-TF1. METHODS: In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS: Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. CONCLUSION: Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes. PMID:17009409

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1.

PubMed

Fischer, Silke F; Schmidt, Katja; Fiedler, Nicola; Glebe, Dieter; Schüttler, Christian; Sun, Jianguang; Gerlich, Wolfram H; Repp, Reinald; Schaefer, Stephan

2006-10-07

To study the expression of HBV enhancer II by transcription factor COUP-TF1. In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.

Two splice variants of the bovine lactoferrin gene identified in Staphylococcus aureus isolated from mastitis in dairy cattle.

PubMed

Huang, J M; Wang, Z Y; Ju, Z H; Wang, C F; Li, Q L; Sun, T; Hou, Q L; Hang, S Q; Hou, M H; Zhong, J F

2011-12-21

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

PubMed

Schoch, Kelly; Meng, Linyan; Szelinger, Szabolcs; Bearden, David R; Stray-Pedersen, Asbjorg; Busk, Oyvind L; Stong, Nicholas; Liston, Eriskay; Cohn, Ronald D; Scaglia, Fernando; Rosenfeld, Jill A; Tarpinian, Jennifer; Skraban, Cara M; Deardorff, Matthew A; Friedman, Jeremy N; Akdemir, Zeynep Coban; Walley, Nicole; Mikati, Mohamad A; Kranz, Peter G; Jasien, Joan; McConkie-Rosell, Allyn; McDonald, Marie; Wechsler, Stephanie Burns; Freemark, Michael; Kansagra, Sujay; Freedman, Sharon; Bali, Deeksha; Millan, Francisca; Bale, Sherri; Nelson, Stanley F; Lee, Hane; Dorrani, Naghmeh; Goldstein, David B; Xiao, Rui; Yang, Yaping; Posey, Jennifer E; Martinez-Agosto, Julian A; Lupski, James R; Wangler, Michael F; Shashi, Vandana

2017-02-02

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10 -14 ). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1. Copyright © 2017 American Society of Human Genetics. All rights reserved.

A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Koshi, E-mail: khashi@med.gunma-u.ac.jp; Ishida, Emi; Matsumoto, Shunichi

2009-12-25

We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced tomore » be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.« less

GeneiASE: Detection of condition-dependent and static allele-specific expression from RNA-seq data without haplotype information

PubMed Central

Edsgärd, Daniel; Iglesias, Maria Jesus; Reilly, Sarah-Jayne; Hamsten, Anders; Tornvall, Per; Odeberg, Jacob; Emanuelsson, Olof

2016-01-01

Allele-specific expression (ASE) is the imbalance in transcription between maternal and paternal alleles at a locus and can be probed in single individuals using massively parallel DNA sequencing technology. Assessing ASE within a single sample provides a static picture of the ASE, but the magnitude of ASE for a given transcript may vary between different biological conditions in an individual. Such condition-dependent ASE could indicate a genetic variation with a functional role in the phenotypic difference. We investigated ASE through RNA-sequencing of primary white blood cells from eight human individuals before and after the controlled induction of an inflammatory response, and detected condition-dependent and static ASE at 211 and 13021 variants, respectively. We developed a method, GeneiASE, to detect genes exhibiting static or condition-dependent ASE in single individuals. GeneiASE performed consistently over a range of read depths and ASE effect sizes, and did not require phasing of variants to estimate haplotypes. We observed condition-dependent ASE related to the inflammatory response in 19 genes, and static ASE in 1389 genes. Allele-specific expression was confirmed by validation of variants through real-time quantitative RT-PCR, with RNA-seq and RT-PCR ASE effect-size correlations r = 0.67 and r = 0.94 for static and condition-dependent ASE, respectively. PMID:26887787

Novel mechanism of conjoined gene formation in the human genome.

PubMed

Kim, Ryong Nam; Kim, Aeri; Choi, Sang-Haeng; Kim, Dae-Soo; Nam, Seong-Hyeuk; Kim, Dae-Won; Kim, Dong-Wook; Kang, Aram; Kim, Min-Young; Park, Kun-Hyang; Yoon, Byoung-Ha; Lee, Kang Seon; Park, Hong-Seog

2012-03-01

Recently, conjoined genes (CGs) have emerged as important genetic factors necessary for understanding the human genome. However, their formation mechanism and precise structures have remained mysterious. Based on a detailed structural analysis of 57 human CG transcript variants (CGTVs, discovered in this study) and all (833) known CGs in the human genome, we discovered that the poly(A) signal site from the upstream parent gene region is completely removed via the skipping or truncation of the final exon; consequently, CG transcription is terminated at the poly(A) signal site of the downstream parent gene. This result led us to propose a novel mechanism of CG formation: the complete removal of the poly(A) signal site from the upstream parent gene is a prerequisite for the CG transcriptional machinery to continue transcribing uninterrupted into the intergenic region and downstream parent gene. The removal of the poly(A) signal sequence from the upstream gene region appears to be caused by a deletion or truncation mutation in the human genome rather than post-transcriptional trans-splicing events. With respect to the characteristics of CG sequence structures, we found that intergenic regions are hot spots for novel exon creation during CGTV formation and that exons farther from the intergenic regions are more highly conserved in the CGTVs. Interestingly, many novel exons newly created within the intergenic and intragenic regions originated from transposable element sequences. Additionally, the CGTVs showed tumor tissue-biased expression. In conclusion, our study provides novel insights into the CG formation mechanism and expands the present concepts of the genetic structural landscape, gene regulation, and gene formation mechanisms in the human genome.

Genomic variants in an inbred mouse model predict mania-like behaviors.

PubMed

Saul, Michael C; Stevenson, Sharon A; Zhao, Changjiu; Driessen, Terri M; Eisinger, Brian E; Gammie, Stephen C

2018-01-01

Contemporary rodent models for bipolar disorders split the bipolar spectrum into complimentary behavioral endophenotypes representing mania and depression. Widely accepted mania models typically utilize single gene transgenics or pharmacological manipulations, but inbred rodent strains show great potential as mania models. Their acceptance is often limited by the lack of genotypic data needed to establish construct validity. In this study, we used a unique strategy to inexpensively explore and confirm population allele differences in naturally occurring candidate variants in a manic rodent strain, the Madison (MSN) mouse strain. Variants were identified using whole exome resequencing on a small population of animals. Interesting candidate variants were confirmed in a larger population with genotyping. We enriched these results with observations of locomotor behavior from a previous study. Resequencing identified 447 structural variants that are mostly fixed in the MSN strain relative to control strains. After filtering and annotation, we found 11 non-synonymous MSN variants that we believe alter protein function. The allele frequencies for 6 of these variants were consistent with explanatory variants for the Madison strain's phenotype. The variants are in the Npas2, Cp, Polr3c, Smarca4, Trpv1, and Slc5a7 genes, and many of these genes' products are in pathways implicated in human bipolar disorders. Variants in Smarca4 and Polr3c together explained over 40% of the variance in locomotor behavior in the Hsd:ICR founder strain. These results enhance the MSN strain's construct validity and implicate altered nucleosome structure and transcriptional regulation as a chief molecular system underpinning behavior.

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

PubMed Central

Lim, Kwang-il; Klimczak, Ryan; Yu, Julie H.; Schaffer, David V.

2010-01-01

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS. PMID:20616052

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2011-09-01

the ETS family of transcription factors showing diverse expression patterns in human tissues (Turner and Watson, 2008). ERG, similar to other...and adult mouse tissues . Most striking of these observations was highly selective and abundant expression of erg protein in endothelial cells of...mouse tissues . We for the first time clarified that endogenous ERG was not expressed in normal mouse prostate epithelium (Mohamed et al., 2010

TFIIH Subunit Alterations Causing Xeroderma Pigmentosum and Trichothiodystrophy Specifically Disturb Several Steps during Transcription

PubMed Central

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-01-01

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. PMID:25620205

Surfactant Protein-C Promoter Variants Associated with Neonatal Respiratory Distress Syndrome Reduce Transcription

PubMed Central

Wambach, Jennifer A.; Yang, Ping; Wegner, Daniel J.; An, Ping; Hackett, Brian P.; Cole, F. S.; Hamvas, Aaron

2010-01-01

Dominant mutations in coding regions of the surfactant protein-C gene (SFTPC) cause respiratory distress syndrome (RDS) in infants. However, the contribution of variants in noncoding regions of SFTPC to pulmonary phenotypes is unknown. Using a case-control group of infants ≥34 weeks gestation (n=538), we used complete resequencing of SFTPC and its promoter, genotyping, and logistic regression to identify 80 single nucleotide polymorphisms (SNPs). Three promoter SNPs were statistically associated with neonatal RDS among European descent infants. To assess the transcriptional effects of these three promoter SNPs, we selectively mutated the SFTPC promoter and performed transient transfection using MLE-15 cells and a firefly luciferase reporter vector. Each promoter SNP decreased SFTPC transcription. The combination of two variants in high linkage dysequilibrium also decreased SFTPC transcription. In silico evaluation of transcription factor binding demonstrated that the rare allele at g.-1167 disrupts a SOX (SRY-related high mobility group box) consensus motif and introduces a GATA-1 site, at g.-2385 removes a MZF-1 (myeloid zinc finger) binding site, and at g.-1647 removes a potential methylation site. This combined statistical, in vitro, and in silico approach suggests that reduced SFTPC transcription contributes to the genetic risk for neonatal RDS in developmentally susceptible infants. PMID:20539253

Decoding the role of regulatory element polymorphisms in complex disease.

PubMed

Vockley, Christopher M; Barrera, Alejandro; Reddy, Timothy E

2017-04-01

Genetic variation in gene regulatory elements contributes to diverse human diseases, ranging from rare and severe developmental defects to common and complex diseases such as obesity and diabetes. Early examples of regulatory mechanisms of human diseases involve large chromosomal rearrangements that change the regulatory connections within the genome. Single nucleotide variants in regulatory elements can also contribute to disease, potentially via demonstrated associations with changes in transcription factor binding, enhancer activity, post-translational histone modifications, long-range enhancer-promoter interactions, or RNA polymerase recruitment. Establishing causality between non-coding genetic variants, gene regulation, and disease has recently become more feasible with advances in genome-editing and epigenome-editing technologies. As establishing causal regulatory mechanisms of diseases becomes routine, functional annotation of target genes is likely to emerge as a major bottleneck for translation into patient benefits. In this review, we discuss the history and recent advances in understanding the regulatory mechanisms of human disease, and new challenges likely to be encountered once establishing those mechanisms becomes rote. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lynch syndrome associated with two MLH1 promoter variants and allelic imbalance of MLH1 expression.

PubMed

Hesson, Luke B; Packham, Deborah; Kwok, Chau-To; Nunez, Andrea C; Ng, Benedict; Schmidt, Christa; Fields, Michael; Wong, Jason W H; Sloane, Mathew A; Ward, Robyn L

2015-06-01

Lynch syndrome is a hereditary cancer syndrome caused by a constitutional mutation in one of the mismatch repair genes. The implementation of predictive testing and targeted preventative surveillance is hindered by the frequent finding of sequence variants of uncertain significance in these genes. We aimed to determine the pathogenicity of previously reported variants (c.-28A>G and c.-7C>T) within the MLH1 5'untranslated region (UTR) in two individuals from unrelated suspected Lynch syndrome families. We investigated whether these variants were associated with other pathogenic alterations using targeted high-throughput sequencing of the MLH1 locus. We also determined their relationship to gene expression and epigenetic alterations at the promoter. Sequencing revealed that the c.-28A>G and c.-7C>T variants were the only potentially pathogenic alterations within the MLH1 gene. In both individuals, the levels of transcription from the variant allele were reduced to 50% compared with the wild-type allele. Partial loss of expression occurred in the absence of constitutional epigenetic alterations within the MLH1 promoter. We propose that these variants may be pathogenic due to constitutional partial loss of MLH1 expression, and that this may be associated with intermediate penetrance of a Lynch syndrome phenotype. Our findings provide further evidence of the potential importance of noncoding variants in the MLH1 5'UTR in the pathogenesis of Lynch syndrome. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

Discovery of stimulation-responsive immune enhancers with CRISPR activation

PubMed Central

Simeonov, Dimitre R.; Gowen, Benjamin G.; Boontanrart, Mandy; Roth, Theodore L.; Gagnon, John D.; Mumbach, Maxwell R.; Satpathy, Ansuman T.; Lee, Youjin; Bray, Nicolas L.; Chan, Alice Y.; Lituiev, Dmytro S.; Nguyen, Michelle L.; Gate, Rachel E.; Subramaniam, Meena; Li, Zhongmei; Woo, Jonathan M.; Mitros, Therese; Ray, Graham J.; Curie, Gemma L.; Naddaf, Nicki; Chu, Julia S.; Ma, Hong; Boyer, Eric; Van Gool, Frederic; Huang, Hailiang; Liu, Ruize; Tobin, Victoria R.; Schumann, Kathrin; Daly, Mark J.; Farh, Kyle K; Ansel, K. Mark; Ye, Chun J.; Greenleaf, William J.; Anderson, Mark S.; Bluestone, Jeffrey A.; Chang, Howard Y.; Corn, Jacob E.; Marson, Alexander

2017-01-01

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues1–3. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption4–6, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa)7 to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs. PMID:28854172

Discovery of stimulation-responsive immune enhancers with CRISPR activation.

PubMed

Simeonov, Dimitre R; Gowen, Benjamin G; Boontanrart, Mandy; Roth, Theodore L; Gagnon, John D; Mumbach, Maxwell R; Satpathy, Ansuman T; Lee, Youjin; Bray, Nicolas L; Chan, Alice Y; Lituiev, Dmytro S; Nguyen, Michelle L; Gate, Rachel E; Subramaniam, Meena; Li, Zhongmei; Woo, Jonathan M; Mitros, Therese; Ray, Graham J; Curie, Gemma L; Naddaf, Nicki; Chu, Julia S; Ma, Hong; Boyer, Eric; Van Gool, Frederic; Huang, Hailiang; Liu, Ruize; Tobin, Victoria R; Schumann, Kathrin; Daly, Mark J; Farh, Kyle K; Ansel, K Mark; Ye, Chun J; Greenleaf, William J; Anderson, Mark S; Bluestone, Jeffrey A; Chang, Howard Y; Corn, Jacob E; Marson, Alexander

2017-09-07

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T H 17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Discovery of stimulation-responsive immune enhancers with CRISPR activation

NASA Astrophysics Data System (ADS)

Simeonov, Dimitre R.; Gowen, Benjamin G.; Boontanrart, Mandy; Roth, Theodore L.; Gagnon, John D.; Mumbach, Maxwell R.; Satpathy, Ansuman T.; Lee, Youjin; Bray, Nicolas L.; Chan, Alice Y.; Lituiev, Dmytro S.; Nguyen, Michelle L.; Gate, Rachel E.; Subramaniam, Meena; Li, Zhongmei; Woo, Jonathan M.; Mitros, Therese; Ray, Graham J.; Curie, Gemma L.; Naddaf, Nicki; Chu, Julia S.; Ma, Hong; Boyer, Eric; van Gool, Frederic; Huang, Hailiang; Liu, Ruize; Tobin, Victoria R.; Schumann, Kathrin; Daly, Mark J.; Farh, Kyle K.; Ansel, K. Mark; Ye, Chun J.; Greenleaf, William J.; Anderson, Mark S.; Bluestone, Jeffrey A.; Chang, Howard Y.; Corn, Jacob E.; Marson, Alexander

2017-09-01

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

PubMed Central

Tsotakos, Nikolaos; Silveyra, Patricia; Lin, Zhenwu; Thomas, Neal; Vaid, Mudit

2014-01-01

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication. PMID:25326576

Evolutionary expansion and divergence in a large family of primate-specific zinc finger transcription factor genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, A T; Huntley, S; Tran-Gyamfi, M

Although most genes are conserved as one-to-one orthologs in different mammalian orders, certain gene families have evolved to comprise different numbers and types of protein-coding genes through independent series of gene duplications, divergence and gene loss in each evolutionary lineage. One such family encodes KRAB-zinc finger (KRAB-ZNF) genes, which are likely to function as transcriptional repressors. One KRAB-ZNF subfamily, the ZNF91 clade, has expanded specifically in primates to comprise more than 110 loci in the human genome, yielding large gene clusters in human chromosomes 19 and 7 and smaller clusters or isolated copies at other chromosomal locations. Although phylogenetic analysismore » indicates that many of these genes arose before the split between old world monkeys and new world monkeys, the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. The paralogous loci are distinguished by sequence divergence within their zinc finger arrays indicating a selection for proteins with different DNA binding specificities. RT-PCR and in situ hybridization data show that some of these ZNF genes can have tissue-specific expression patterns, however many KRAB-ZNFs that are near-ubiquitous could also be playing very specific roles in halting target pathways in all tissues except for a few, where the target is released by the absence of its repressor. The number of variant KRAB-ZNF proteins is increased not only because of the large number of loci, but also because many loci can produce multiple splice variants, which because of the modular structure of these genes may have separate and perhaps even conflicting regulatory roles. The lineage-specific duplication and rapid divergence of this family of transcription factor genes suggests a role in determining species-specific biological differences and the evolution of novel primate traits.« less

A novel EML4-ALK variant: exon 6 of EML4 fused to exon 19 of ALK.

PubMed

Penzel, Roland; Schirmacher, Peter; Warth, Arne

2012-07-01

Cytotoxic chemotherapy remains the mainstay of treatment for most patients with advanced disease. Recently, anaplastic lymphoma kinase (ALK) expression as a major target for successful treatment with ALK inhibitors was detected in a subset of non-small-cell lung carcinomas, usually as a result of echinoderm microtubule-associated protein-like 4 (EML4)-ALK rearrangements. Although the chromosomal breakpoint within the EML4 gene varied, the breakpoint within ALK was most frequently reported within intron 19 or rarely in exon 20. Therefore, the different EML4-ALK variants so far contain the same 3' portion of ALK starting with exon 20. Here, we report a novel EML4-ALK variant detected by reverse transcription polymerase chain reaction analysis. Subsequent sequencing revealed an EML4-ALK fusion variant in which exon 6 of EML4 was fused to exon 19 of ALK. It occurred in a predominant solid pulmonary adenocarcinoma of a 65-year-old woman with a clear split signal of ALK in fluorescence in situ hybridization analysis and a weakly homogeneous ALK expression in immunohistochemical staining. Because of the growing number of fusion variants a primary reverse transcription polymerase chain reaction-based screening for ALK-positive non-small-cell lung carcinoma patients may not be sufficient for predictive diagnostics but transcript-based approaches and sequencing of ALK fusion variants might finally contribute to an optimized selection of patients.

A Common CYFIP1 Variant at the 15q11.2 Disease Locus Is Associated with Structural Variation at the Language-Related Left Supramarginal Gyrus

PubMed Central

Woo, Young Jae; Wang, Tao; Guadalupe, Tulio; Nebel, Rebecca A.; Vino, Arianna; Del Bene, Victor A.; Molholm, Sophie; Ross, Lars A.; Zwiers, Marcel P.; Fisher, Simon E.; Foxe, John J.; Abrahams, Brett S.

2016-01-01

Copy number variants (CNVs) at the Breakpoint 1 to Breakpoint 2 region at 15q11.2 (BP1-2) are associated with language-related difficulties and increased risk for developmental disorders in which language is compromised. Towards underlying mechanisms, we investigated relationships between single nucleotide polymorphisms (SNPs) across the region and quantitative measures of human brain structure obtained by magnetic resonance imaging of healthy subjects. We report an association between rs4778298, a common variant at CYFIP1, and inter-individual variation in surface area across the left supramarginal gyrus (lh.SMG), a cortical structure implicated in speech and language in independent discovery (n = 100) and validation cohorts (n = 2621). In silico analyses determined that this same variant, and others nearby, is also associated with differences in levels of CYFIP1 mRNA in human brain. One of these nearby polymorphisms is predicted to disrupt a consensus binding site for FOXP2, a transcription factor implicated in speech and language. Consistent with a model where FOXP2 regulates CYFIP1 levels and in turn influences lh.SMG surface area, analysis of publically available expression data identified a relationship between expression of FOXP2 and CYFIP1 mRNA in human brain. We propose that altered CYFIP1 dosage, through aberrant patterning of the lh.SMG, may contribute to language-related difficulties associated with BP1-2 CNVs. More generally, this approach may be useful in clarifying the contribution of individual genes at CNV risk loci. PMID:27351196

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smirnova, Anna S.; Morgun, Andrey; Shulzhenko, Natalia

Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice formsmore » in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.« less

Alternative Splicing of NOX4 in the Failing Human Heart

PubMed Central

Varga, Zoltán V.; Pipicz, Márton; Baán, Júlia A.; Baranyai, Tamás; Koncsos, Gábor; Leszek, Przemyslaw; Kuśmierczyk, Mariusz; Sánchez-Cabo, Fátima; García-Pavía, Pablo; Brenner, Gábor J.; Giricz, Zoltán; Csont, Tamás; Mendler, Luca; Lara-Pezzi, Enrique; Pacher, Pál; Ferdinandy, Péter

2017-01-01

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5′-3′ end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure. PMID:29204124

Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints.

PubMed

Schwessinger, Ron; Suciu, Maria C; McGowan, Simon J; Telenius, Jelena; Taylor, Stephen; Higgs, Doug R; Hughes, Jim R

2017-10-01

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k -mer-based analysis of DNase footprints to determine any k -mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome. © 2017 Schwessinger et al.; Published by Cold Spring Harbor Laboratory Press.

Intersection of diverse neuronal genomes and neuropsychiatric disease: The Brain Somatic Mosaicism Network.

PubMed

McConnell, Michael J; Moran, John V; Abyzov, Alexej; Akbarian, Schahram; Bae, Taejeong; Cortes-Ciriano, Isidro; Erwin, Jennifer A; Fasching, Liana; Flasch, Diane A; Freed, Donald; Ganz, Javier; Jaffe, Andrew E; Kwan, Kenneth Y; Kwon, Minseok; Lodato, Michael A; Mills, Ryan E; Paquola, Apua C M; Rodin, Rachel E; Rosenbluh, Chaggai; Sestan, Nenad; Sherman, Maxwell A; Shin, Joo Heon; Song, Saera; Straub, Richard E; Thorpe, Jeremy; Weinberger, Daniel R; Urban, Alexander E; Zhou, Bo; Gage, Fred H; Lehner, Thomas; Senthil, Geetha; Walsh, Christopher A; Chess, Andrew; Courchesne, Eric; Gleeson, Joseph G; Kidd, Jeffrey M; Park, Peter J; Pevsner, Jonathan; Vaccarino, Flora M

2017-04-28

Neuropsychiatric disorders have a complex genetic architecture. Human genetic population-based studies have identified numerous heritable sequence and structural genomic variants associated with susceptibility to neuropsychiatric disease. However, these germline variants do not fully account for disease risk. During brain development, progenitor cells undergo billions of cell divisions to generate the ~80 billion neurons in the brain. The failure to accurately repair DNA damage arising during replication, transcription, and cellular metabolism amid this dramatic cellular expansion can lead to somatic mutations. Somatic mutations that alter subsets of neuronal transcriptomes and proteomes can, in turn, affect cell proliferation and survival and lead to neurodevelopmental disorders. The long life span of individual neurons and the direct relationship between neural circuits and behavior suggest that somatic mutations in small populations of neurons can significantly affect individual neurodevelopment. The Brain Somatic Mosaicism Network has been founded to study somatic mosaicism both in neurotypical human brains and in the context of complex neuropsychiatric disorders. Copyright © 2017, American Association for the Advancement of Science.

The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

PubMed Central

Iezzi, Manuela; Zenobi, Santa; Montani, Maura; Pietrella, Lucia; Kalogris, Cristina; Rossini, Anna; Ciravolo, Valentina; Castagnoli, Lorenzo; Tagliabue, Elda; Pupa, Serenella M.; Musiani, Piero; Monaci, Paolo; Menard, Sylvie; Amici, Augusto

2011-01-01

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform “per se” mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein. PMID:21559085

Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing.

PubMed

Nakabayashi, Kazumi; Bartsch, Melanie; Ding, Jia; Soppe, Wim J J

2015-12-01

The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1) is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

Deciphering molecular circuits from genetic variation underlying transcriptional responsiveness to stimuli

PubMed Central

Gat-Viks, Irit; Chevrier, Nicolas; Wilentzik, Roni; Eisenhaure, Thomas; Raychowdhury, Raktima; Steuerman, Yael; Shalek, Alex; Hacohen, Nir; Amit, Ido; Regev, Aviv

2013-01-01

Individual genetic variation affects gene expression in response to stimuli, often by influencing complex molecular circuits. Here we combine genomic and intermediate-scale transcriptional profiling with computational methods to identify variants that affect the responsiveness of genes to stimuli (responsiveness QTLs; reQTLs) and to position these variants in molecular circuit diagrams. We apply this approach to study variation in transcriptional responsiveness to pathogen components in dendritic cells from recombinant inbred mouse strains. We identify reQTLs that correlate with particular stimuli and position them in known pathways. For example, in response to a virus-like stimulus, a trans-acting variant acts as an activator of the antiviral response; using RNAi, we identify Rgs16 as the likely causal gene. Our approach charts an experimental and analytic path to decipher the mechanisms underlying genetic variation in circuits that control responses to stimuli. PMID:23503680

Deciphering molecular circuits from genetic variation underlying transcriptional responsiveness to stimuli.

PubMed

Gat-Viks, Irit; Chevrier, Nicolas; Wilentzik, Roni; Eisenhaure, Thomas; Raychowdhury, Raktima; Steuerman, Yael; Shalek, Alex K; Hacohen, Nir; Amit, Ido; Regev, Aviv

2013-04-01

Individual genetic variation affects gene responsiveness to stimuli, often by influencing complex molecular circuits. Here we combine genomic and intermediate-scale transcriptional profiling with computational methods to identify variants that affect the responsiveness of genes to stimuli (responsiveness quantitative trait loci or reQTLs) and to position these variants in molecular circuit diagrams. We apply this approach to study variation in transcriptional responsiveness to pathogen components in dendritic cells from recombinant inbred mouse strains. We identify reQTLs that correlate with particular stimuli and position them in known pathways. For example, in response to a virus-like stimulus, a trans-acting variant responds as an activator of the antiviral response; using RNA interference, we identify Rgs16 as the likely causal gene. Our approach charts an experimental and analytic path to decipher the mechanisms underlying genetic variation in circuits that control responses to stimuli.

Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters.

PubMed

Javierre, Biola M; Burren, Oliver S; Wilder, Steven P; Kreuzhuber, Roman; Hill, Steven M; Sewitz, Sven; Cairns, Jonathan; Wingett, Steven W; Várnai, Csilla; Thiecke, Michiel J; Burden, Frances; Farrow, Samantha; Cutler, Antony J; Rehnström, Karola; Downes, Kate; Grassi, Luigi; Kostadima, Myrto; Freire-Pritchett, Paula; Wang, Fan; Stunnenberg, Hendrik G; Todd, John A; Zerbino, Daniel R; Stegle, Oliver; Ouwehand, Willem H; Frontini, Mattia; Wallace, Chris; Spivakov, Mikhail; Fraser, Peter

2016-11-17

Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The genomic basis of circadian and circalunar timing adaptations in a midge.

PubMed

Kaiser, Tobias S; Poehn, Birgit; Szkiba, David; Preussner, Marco; Sedlazeck, Fritz J; Zrim, Alexander; Neumann, Tobias; Nguyen, Lam-Tung; Betancourt, Andrea J; Hummel, Thomas; Vogel, Heiko; Dorner, Silke; Heyd, Florian; von Haeseler, Arndt; Tessmar-Raible, Kristin

2016-12-01

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

Effects of ubiquilin 1 on the unfolded protein response.

PubMed

Lu, Alice; Hiltunen, Mikko; Romano, Donna M; Soininen, Hilkka; Hyman, Bradley T; Bertram, Lars; Tanzi, Rudolph E

2009-05-01

Previous studies have implicated the unfolded protein response (UPR) in the pathogenesis of Alzheimer's disease (AD). We previously reported that DNA variants in the ubiquilin 1 (UBQLN1) gene increase the risk for AD. Since UBQLN1 has been shown to play a role in the UPR, we assessed the effects of overexpression and downregulation of UBQLN1 splice variants during tunicamycin-induced ER stress. In addition to previously described transcript variants, TV1 and TV2, we identified two novel transcript variants of UBQLN1 in brain: TV3 (lacking exons 2-4) and TV4 (lacking exon 4). Overexpression of TV1-3, but not TV4 significantly decreased the mRNA induction of UPR-inducible genes, C/EBP homologous protein (CHOP), BiP/GRP78, and protein disulfide isomerase (PDI) during the UPR. Stable overexpression of TV1-3, but not TV4, also significantly decreased the induction of CHOP protein and increased cell viability during the UPR. In contrast, downregulation of UBQLN1 did not affect CHOP mRNA induction, but instead increased PDI mRNA levels. These findings suggest that overexpression UBQLN1 transcript variants TV1-3, but not TV4, exert a protective effect during the UPR by attenuating CHOP induction and potentially increasing cell viability.

Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

PubMed Central

Boddu, Ravindra; Yang, Chaozhe; O’Connor, Amber K.; Hendrickson, Robert Curtis; Boone, Braden; Cui, Xiangqin; Garcia-Gonzalez, Miguel; Igarashi, Peter; Onuchic, Luiz F.; Germino, Gregory G.

2014-01-01

Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. PMID:24984783

Efficient analysis of mouse genome sequences reveal many nonsense variants

PubMed Central

Steeland, Sophie; Timmermans, Steven; Van Ryckeghem, Sara; Hulpiau, Paco; Saeys, Yvan; Van Montagu, Marc; Vandenbroucke, Roosmarijn E.; Libert, Claude

2016-01-01

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1. PMID:27147605

Promoter variant -204A > C of the cholesterol 7α-hydroxylase gene: association with response to plant sterols in humans and increased transcriptional activity in transfected HepG2 cells.

PubMed

De Castro-Orós, Isabel; Pampín, Sandra; Cofán, Montserrat; Mozas, Pilar; Pintó, Xavier; Salas-Salvadó, Jordi; Rodríguez-Rey, Jose C; Ros, Emilio; Civeira, Fernando; Pocoví, Miguel

2011-04-01

The bile acid pool influences intestinal cholesterol absorption because this process is strictly dependent on micellar solubilization, which is disrupted by plant sterols (PS). Plasma lipid variation relates to promoter variant -204A > C (rs3808607) of the CYP7A1 gene encoding for 7α-hydroxylase, an enzyme for bile acid synthesis. We hypothesized that this polymorphism would be associated with variability in lipid responses to PS. We investigated 67 subjects (31 AA and 36 AC + CC) with lipid responses to PS documented in two studies. To assess the functionality of the -204A > C variant, electrophoretic mobility gel shift assays were performed and luciferase reporter plasmids containing the promoter were transfected into HepG2 cells. Compared to AA-subjects, C-carriers showed significantly higher adjusted mean reductions in total cholesterol (0.14 versus 0.43 mmol/L, P = 0.042) and increases in lathosterol-to-cholesterol ratios (0.10 versus 0.75, P = 0.013). The C-construct caused a 78% promoter activity increase and gel-shift assays showed lower affinity for nuclear transcription factors, while in silico experiments predicted a binding site for inhibitory nuclear factors RXR-CAR. Results suggest that promoter -204A > C variant is associated with enhanced CYP7A1 activity. Increased intestinal bile acids and ensuing more efficient cholesterol absorption might explain why C-allele carriers show enhanced cholesterol lowering and increased feedback cholesterol synthesis to PS intervention. Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Histone H3 Variants in Trichomonas vaginalis

PubMed Central

Zubáčová, Zuzana; Hostomská, Jitka

2012-01-01

The parabasalid protist Trichomonas vaginalis is a widespread parasite that affects humans, frequently causing vaginitis in infected women. Trichomonad mitosis is marked by the persistence of the nuclear membrane and the presence of an asymmetric extranuclear spindle with no obvious direct connection to the chromosomes. No centromeric markers have been described in T. vaginalis, which has prevented a detailed analysis of mitotic events in this organism. In other eukaryotes, nucleosomes of centromeric chromatin contain the histone H3 variant CenH3. The principal aim of this work was to identify a CenH3 homolog in T. vaginalis. We performed a screen of the T. vaginalis genome to retrieve sequences of canonical and variant H3 histones. Three variant histone H3 proteins were identified, and the subcellular localization of their epitope-tagged variants was determined. The localization of the variant TVAG_185390 could not be distinguished from that of the canonical H3 histone. The sequence of the variant TVAG_087830 closely resembled that of histone H3. The tagged protein colocalized with sites of active transcription, indicating that the variant TVAG_087830 represented H3.3 in T. vaginalis. The third H3 variant (TVAG_224460) was localized to 6 or 12 distinct spots at the periphery of the nucleus, corresponding to the number of chromosomes in G1 phase and G2 phase, respectively. We propose that this variant represents the centromeric marker CenH3 and thus can be employed as a tool to study mitosis in T. vaginalis. Furthermore, we suggest that the peripheral distribution of CenH3 within the nucleus results from the association of centromeres with the nuclear envelope throughout the cell cycle. PMID:22408228

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

PubMed Central

Johnson, Amanda N.; Weil, P. Anthony

2017-01-01

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways. PMID:28196871

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions.

PubMed

Tiscornia, A C; Cayota, A; Landoni, A I; Brito, C; Oppezzo, P; Vuillier, F; Robello, C; Dighiero, G; Gabús, R; Pritsch, O

2004-01-01

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

SpliceCenter: A suite of web-based bioinformatic applications for evaluating the impact of alternative splicing on RT-PCR, RNAi, microarray, and peptide-based studies

PubMed Central

Ryan, Michael C; Zeeberg, Barry R; Caplen, Natasha J; Cleland, James A; Kahn, Ari B; Liu, Hongfang; Weinstein, John N

2008-01-01

Background Over 60% of protein-coding genes in vertebrates express mRNAs that undergo alternative splicing. The resulting collection of transcript isoforms poses significant challenges for contemporary biological assays. For example, RT-PCR validation of gene expression microarray results may be unsuccessful if the two technologies target different splice variants. Effective use of sequence-based technologies requires knowledge of the specific splice variant(s) that are targeted. In addition, the critical roles of alternative splice forms in biological function and in disease suggest that assay results may be more informative if analyzed in the context of the targeted splice variant. Results A number of contemporary technologies are used for analyzing transcripts or proteins. To enable investigation of the impact of splice variation on the interpretation of data derived from those technologies, we have developed SpliceCenter. SpliceCenter is a suite of user-friendly, web-based applications that includes programs for analysis of RT-PCR primer/probe sets, effectors of RNAi, microarrays, and protein-targeting technologies. Both interactive and high-throughput implementations of the tools are provided. The interactive versions of SpliceCenter tools provide visualizations of a gene's alternative transcripts and probe target positions, enabling the user to identify which splice variants are or are not targeted. The high-throughput batch versions accept user query files and provide results in tabular form. When, for example, we used SpliceCenter's batch siRNA-Check to process the Cancer Genome Anatomy Project's large-scale shRNA library, we found that only 59% of the 50,766 shRNAs in the library target all known splice variants of the target gene, 32% target some but not all, and 9% do not target any currently annotated transcript. Conclusion SpliceCenter provides unique, user-friendly applications for assessing the impact of transcript variation on the design and interpretation of RT-PCR, RNAi, gene expression microarrays, antibody-based detection, and mass spectrometry proteomics. The tools are intended for use by bench biologists as well as bioinformaticists. PMID:18638396

A de novo FOXP1 variant in a patient with autism, intellectual disability and severe speech and language impairment.

PubMed

Lozano, Reymundo; Vino, Arianna; Lozano, Cristina; Fisher, Simon E; Deriziotis, Pelagia

2015-12-01

FOXP1 (forkhead box protein P1) is a transcription factor involved in the development of several tissues, including the brain. An emerging phenotype of patients with protein-disrupting FOXP1 variants includes global developmental delay, intellectual disability and mild to severe speech/language deficits. We report on a female child with a history of severe hypotonia, autism spectrum disorder and mild intellectual disability with severe speech/language impairment. Clinical exome sequencing identified a heterozygous de novo FOXP1 variant c.1267_1268delGT (p.V423Hfs*37). Functional analyses using cellular models show that the variant disrupts multiple aspects of FOXP1 activity, including subcellular localization and transcriptional repression properties. Our findings highlight the importance of performing functional characterization to help uncover the biological significance of variants identified by genomics approaches, thereby providing insight into pathways underlying complex neurodevelopmental disorders. Moreover, our data support the hypothesis that de novo variants represent significant causal factors in severe sporadic disorders and extend the phenotype seen in individuals with FOXP1 haploinsufficiency.

VAT: a computational framework to functionally annotate variants in personal genomes within a cloud-computing environment.

PubMed

Habegger, Lukas; Balasubramanian, Suganthi; Chen, David Z; Khurana, Ekta; Sboner, Andrea; Harmanci, Arif; Rozowsky, Joel; Clarke, Declan; Snyder, Michael; Gerstein, Mark

2012-09-01

The functional annotation of variants obtained through sequencing projects is generally assumed to be a simple intersection of genomic coordinates with genomic features. However, complexities arise for several reasons, including the differential effects of a variant on alternatively spliced transcripts, as well as the difficulty in assessing the impact of small insertions/deletions and large structural variants. Taking these factors into consideration, we developed the Variant Annotation Tool (VAT) to functionally annotate variants from multiple personal genomes at the transcript level as well as obtain summary statistics across genes and individuals. VAT also allows visualization of the effects of different variants, integrates allele frequencies and genotype data from the underlying individuals and facilitates comparative analysis between different groups of individuals. VAT can either be run through a command-line interface or as a web application. Finally, in order to enable on-demand access and to minimize unnecessary transfers of large data files, VAT can be run as a virtual machine in a cloud-computing environment. VAT is implemented in C and PHP. The VAT web service, Amazon Machine Image, source code and detailed documentation are available at vat.gersteinlab.org.

Characterization of Smoc-1 uncovers two transcript variants showing differential tissue and age specific expression in Bubalus bubalis

PubMed Central

Srivastava, Jyoti; Premi, Sanjay; Kumar, Sudhir; Parwez, Iqbal; Ali, Sher

2007-01-01

Background Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCACCTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. Results We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual increment in expression of variant-02. Immunohistochemically, Smoc-1 was localized in the basement membrane zones and extracellular matrices of various tissues. Conclusion These data added to our understandings about the tissue, age and species specific functions of the Smoc-1. It also enabled us to demonstrate varying expression of the two transcript variants of Smoc-1 amongst different somatic tissues/gonads and ages, in spite of their identical coding frames. Pursuance of these variants for their roles in various disease phenotypes such as hepatocellular carcinoma and angiogenesis is envisaged to establish broader biological significance of this gene. PMID:18042303

A Splice Defect in the EDA Gene in Dogs with an X-Linked Hypohidrotic Ectodermal Dysplasia (XLHED) Phenotype.

PubMed

Waluk, Dominik P; Zur, Gila; Kaufmann, Ronnie; Welle, Monika M; Jagannathan, Vidhya; Drögemüller, Cord; Müller, Eliane J; Leeb, Tosso; Galichet, Arnaud

2016-09-08

X-linked hypohidrotic ectodermal dysplasia (XLHED) caused by variants in the EDA gene represents the most common ectodermal dysplasia in humans. We investigated three male mixed-breed dogs with an ectodermal dysplasia phenotype characterized by marked hypotrichosis and multifocal complete alopecia, almost complete absence of sweat and sebaceous glands, and altered dentition with missing and abnormally shaped teeth. Analysis of SNP chip genotypes and whole genome sequence data from the three affected dogs revealed that the affected dogs shared the same haplotype on a large segment of the X-chromosome, including the EDA gene. Unexpectedly, the whole genome sequence data did not reveal any nonsynonymous EDA variant in the affected dogs. We therefore performed an RNA-seq experiment on skin biopsies to search for changes in the transcriptome. This analysis revealed that the EDA transcript in the affected dogs lacked 103 nucleotides encoded by exon 2. We speculate that this exon skipping is caused by a genetic variant located in one of the large introns flanking this exon, which was missed by whole genome sequencing with the illumina short read technology. The altered EDA transcript splicing most likely causes the observed ectodermal dysplasia in the affected dogs. These dogs thus offer an excellent opportunity to gain insights into the complex splicing processes required for expression of the EDA gene, and other genes with large introns. Copyright © 2016 Waluk et al.

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers

PubMed Central

Udugama, Maheshi; Sanij, Elaine; Voon, Hsiao P. J.; Son, Jinbae; Hii, Linda; Henson, Jeremy D.; Chan, F. Lyn; Chang, Fiona T. M.; Liu, Yumei; Pearson, Richard B.; Kalitsis, Paul; Mann, Jeffrey R.; Collas, Philippe; Hannan, Ross D.; Wong, Lee H.

2018-01-01

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers. PMID:29669917

Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.

PubMed

Sun, Bing; Zheng, Yun-Ling

2018-01-01

Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

PubMed

Colombo, Mara; Lòpez-Perolio, Irene; Meeks, Huong D; Caleca, Laura; Parsons, Michael T; Li, Hongyan; De Vecchi, Giovanna; Tudini, Emma; Foglia, Claudia; Mondini, Patrizia; Manoukian, Siranoush; Behar, Raquel; Garcia, Encarna B Gómez; Meindl, Alfons; Montagna, Marco; Niederacher, Dieter; Schmidt, Ane Y; Varesco, Liliana; Wappenschmidt, Barbara; Bolla, Manjeet K; Dennis, Joe; Michailidou, Kyriaki; Wang, Qin; Aittomäki, Kristiina; Andrulis, Irene L; Anton-Culver, Hoda; Arndt, Volker; Beckmann, Matthias W; Beeghly-Fadel, Alicia; Benitez, Javier; Boeckx, Bram; Bogdanova, Natalia V; Bojesen, Stig E; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Burwinkel, Barbara; Chang-Claude, Jenny; Conroy, Don M; Couch, Fergus J; Cox, Angela; Cross, Simon S; Czene, Kamila; Devilee, Peter; Dörk, Thilo; Eriksson, Mikael; Fasching, Peter A; Figueroa, Jonine; Fletcher, Olivia; Flyger, Henrik; Gabrielson, Marike; García-Closas, Montserrat; Giles, Graham G; González-Neira, Anna; Guénel, Pascal; Haiman, Christopher A; Hall, Per; Hamann, Ute; Hartman, Mikael; Hauke, Jan; Hollestelle, Antoinette; Hopper, John L; Jakubowska, Anna; Jung, Audrey; Kosma, Veli-Matti; Lambrechts, Diether; Le Marchand, Loid; Lindblom, Annika; Lubinski, Jan; Mannermaa, Arto; Margolin, Sara; Miao, Hui; Milne, Roger L; Neuhausen, Susan L; Nevanlinna, Heli; Olson, Janet E; Peterlongo, Paolo; Peto, Julian; Pylkäs, Katri; Sawyer, Elinor J; Schmidt, Marjanka K; Schmutzler, Rita K; Schneeweiss, Andreas; Schoemaker, Minouk J; See, Mee Hoong; Southey, Melissa C; Swerdlow, Anthony; Teo, Soo H; Toland, Amanda E; Tomlinson, Ian; Truong, Thérèse; van Asperen, Christi J; van den Ouweland, Ans M W; van der Kolk, Lizet E; Winqvist, Robert; Yannoukakos, Drakoulis; Zheng, Wei; Dunning, Alison M; Easton, Douglas F; Henderson, Alex; Hogervorst, Frans B L; Izatt, Louise; Offitt, Kenneth; Side, Lucy E; van Rensburg, Elizabeth J; Embrace, Study; Hebon, Study; McGuffog, Lesley; Antoniou, Antonis C; Chenevix-Trench, Georgia; Spurdle, Amanda B; Goldgar, David E; Hoya, Miguel de la; Radice, Paolo

2018-05-01

Although the spliceogenic nature of the BRCA2 c.68-7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real-time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10 -115 . There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24) nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68-7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants. © 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.

KCC2a expression in a human fetal lens epithelial cell line.

PubMed

Lauf, Peter K; Di Fulvio, Mauricio; Srivastava, Vinita; Sharma, Neelima; Adragna, Norma C

2012-01-01

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin. Copyright © 2012 S. Karger AG, Basel.

Defining functional DNA elements in the human genome

PubMed Central

Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

2014-01-01

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

Multilayered Genetic and Omics Dissection of Mitochondrial Activity in a Mouse Reference Population

PubMed Central

Wu, Yibo; Williams, Evan G.; Dubuis, Sébastien; Mottis, Adrienne; Jovaisaite, Virginija; Houten, Sander M.; Argmann, Carmen A.; Faridi, Pouya; Wolski, Witold; Kutalik, Zoltán; Zamboni, Nicola; Auwerx, Johan; Aebersold, Ruedi

2014-01-01

SUMMARY The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome—a subset of the metabolome—and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPRmt). UPRmt shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes. PMID:25215496

Experimental annotation of the human genome using microarray technology.

PubMed

Shoemaker, D D; Schadt, E E; Armour, C D; He, Y D; Garrett-Engele, P; McDonagh, P D; Loerch, P M; Leonardson, A; Lum, P Y; Cavet, G; Wu, L F; Altschuler, S J; Edwards, S; King, J; Tsang, J S; Schimmack, G; Schelter, J M; Koch, J; Ziman, M; Marton, M J; Li, B; Cundiff, P; Ward, T; Castle, J; Krolewski, M; Meyer, M R; Mao, M; Burchard, J; Kidd, M J; Dai, H; Phillips, J W; Linsley, P S; Stoughton, R; Scherer, S; Boguski, M S

2001-02-15

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Principles of regulatory information conservation between mouse and human

DOE PAGES

Cheng, Yong; Ma, Zhihai; Kim, Bong-Hyun; ...

2014-11-19

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and withmore » genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Lastly, single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.« less

Trans-ancestry Fine Mapping and Molecular Assays Identify Regulatory Variants at the ANGPTL8 HDL-C GWAS Locus

PubMed Central

Cannon, Maren E.; Duan, Qing; Wu, Ying; Zeynalzadeh, Monica; Xu, Zheng; Kangas, Antti J.; Soininen, Pasi; Ala-Korpela, Mika; Civelek, Mete; Lusis, Aldons J.; Kuusisto, Johanna; Collins, Francis S.; Boehnke, Michael; Tang, Hua; Laakso, Markku; Li, Yun; Mohlke, Karen L.

2017-01-01

Recent genome-wide association studies (GWAS) have identified variants associated with high-density lipoprotein cholesterol (HDL-C) located in or near the ANGPTL8 gene. Given the extensive sharing of GWAS loci across populations, we hypothesized that at least one shared variant at this locus affects HDL-C. The HDL-C–associated variants are coincident with expression quantitative trait loci for ANGPTL8 and DOCK6 in subcutaneous adipose tissue; however, only ANGPTL8 expression levels are associated with HDL-C levels. We identified a 400-bp promoter region of ANGPTL8 and enhancer regions within 5 kb that contribute to regulating expression in liver and adipose. To identify variants functionally responsible for the HDL-C association, we performed fine-mapping analyses and selected 13 candidate variants that overlap putative regulatory regions to test for allelic differences in regulatory function. Of these variants, rs12463177-G increased transcriptional activity (1.5-fold, P = 0.004) and showed differential protein binding. Six additional variants (rs17699089, rs200788077, rs56322906, rs3760782, rs737337, and rs3745683) showed evidence of allelic differences in transcriptional activity and/or protein binding. Taken together, these data suggest a regulatory mechanism at the ANGPTL8 HDL-C GWAS locus involving tissue-selective expression and at least one functional variant. PMID:28754724

Sir2a regulates rDNA transcription and multiplication rate in the human malaria parasite Plasmodium falciparum

PubMed Central

Mancio-Silva, Liliana; Lopez-Rubio, Jose Juan; Claes, Aurélie; Scherf, Artur

2013-01-01

The Plasmodium falciparum histone deacetylase Sir2a localizes at telomeric regions where it contributes to epigenetic silencing of clonally variant virulence genes. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. Here we investigate the nucleolar function of PfSir2a and demonstrate that PfSir2a fine-tunes ribosomal RNA gene transcription. Using a parasite line in which PfSir2a has been disrupted, we observe that histones near the transcription start sites of all ribosomal RNA genes are hyperacetylated and that transcription of ribosomal RNA genes is upregulated. Complementation of the PfSir2a-disrupted parasites restores the ribosomal RNA levels, whereas PfSir2a overexpression in wild-type parasites decreases ribosomal RNA synthesis. Furthermore, we observe that PfSir2a modulation of ribosomal RNA synthesis is linked to an altered number of daughter merozoites and the parasite multiplication rate. These findings provide new insights into an epigenetic mechanism that controls malaria parasite proliferation and virulence. PMID:23443558

Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression

PubMed Central

Lu, Xiaoming; Zoller, Erin E.; Weirauch, Matthew T.; Wu, Zhiguo; Namjou, Bahram; Williams, Adrienne H.; Ziegler, Julie T.; Comeau, Mary E.; Marion, Miranda C.; Glenn, Stuart B.; Adler, Adam; Shen, Nan; Nath, Swapan K.; Stevens, Anne M.; Freedman, Barry I.; Tsao, Betty P.; Jacob, Chaim O.; Kamen, Diane L.; Brown, Elizabeth E.; Gilkeson, Gary S.; Alarcón, Graciela S.; Reveille, John D.; Anaya, Juan-Manuel; James, Judith A.; Sivils, Kathy L.; Criswell, Lindsey A.; Vilá, Luis M.; Alarcón-Riquelme, Marta E.; Petri, Michelle; Scofield, R. Hal; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Joo, Young Bin; Choi, Jeongim; Bae, Sang-Cheol; Boackle, Susan A.; Graham, Deborah Cunninghame; Vyse, Timothy J.; Guthridge, Joel M.; Gaffney, Patrick M.; Langefeld, Carl D.; Kelly, Jennifer A.; Greis, Kenneth D.; Kaufman, Kenneth M.; Harley, John B.; Kottyan, Leah C.

2015-01-01

Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1. PMID:25865496

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features

PubMed Central

Low, Karen J; Ansari, Morad; Abou Jamra, Rami; Clarke, Angus; El Chehadeh, Salima; FitzPatrick, David R; Greenslade, Mark; Henderson, Alex; Hurst, Jane; Keller, Kory; Kuentz, Paul; Prescott, Trine; Roessler, Franziska; Selmer, Kaja K; Schneider, Michael C; Stewart, Fiona; Tatton-Brown, Katrina; Thevenon, Julien; Vigeland, Magnus D; Vogt, Julie; Willems, Marjolaine; Zonana, Jonathan; Study, D D D; Smithson, Sarah F

2017-01-01

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function. PMID:28327570

Mutations in the BAF-Complex Subunit DPF2 Are Associated with Coffin-Siris Syndrome.

PubMed

Vasileiou, Georgia; Vergarajauregui, Silvia; Endele, Sabine; Popp, Bernt; Büttner, Christian; Ekici, Arif B; Gerard, Marion; Bramswig, Nuria C; Albrecht, Beate; Clayton-Smith, Jill; Morton, Jenny; Tomkins, Susan; Low, Karen; Weber, Astrid; Wenzel, Maren; Altmüller, Janine; Li, Yun; Wollnik, Bernd; Hoganson, George; Plona, Maria-Renée; Cho, Megan T; Thiel, Christian T; Lüdecke, Hermann-Josef; Strom, Tim M; Calpena, Eduardo; Wilkie, Andrew O M; Wieczorek, Dagmar; Engel, Felix B; Reis, André

2018-03-01

Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Upregulation of the splice variant MUC4/Y in the pancreatic cancer cell line MIA PaCa-2 potentiates proliferation and suppresses apoptosis: new insight into the presence of the transcript variant of MUC4.

PubMed

Xie, Kunling; Zhi, Xiaofei; Tang, Jie; Zhu, Yi; Zhang, Jingjing; Li, Zheng; Tao, Jinqiu; Xu, Zekuan

2014-05-01

MUC4/Y, the transcript variant 4 of MUC4, lacks exon 2 as compared with the transcript variant 1 of MUC4. To date, direct evidence for the function of MU4/Y remains to be reported. Previous studies based their hypotheses regarding the function of MUC4/Y on the characteristic structure domains of this variant. The aim of the present study was to investigate the specific function of MUC4/Y. The pancreatic cancer cell line MIA PaCa-2 with low MUC4/Y expression was used to establish a stable cell model of MUC4/Y upregulation using a lentivirus vector system. Results showed that MUC4/Y anchored on the cytomembrane and affected cell morphology and cell cycle. Functional analyses indicated that MUC4/Y upregulation slightly potentiated cell proliferation and significantly suppressed apoptosis both in vivo and in vitro. Further studies revealed that the JNK and AKT signalling pathways were activated. Meanwhile, MUC4/Y upregulation elicited minimal effect on the phosphorylation level of HER2, a membrane partner of MUC4. These results suggest that MUC4/Y promotes tumour progression through its anti-apoptotic and weak mitogenic effect on MIA PaCa-2 cells.

Insights into Mutation Effect in Three Poikiloderma with Neutropenia Patients by Transcript Analysis and Disease Evolution of Reported Patients with the Same Pathogenic Variants.

PubMed

Colombo, Elisa A; Elcioglu, Nursel H; Graziano, Claudio; Farinelli, Pamela; Di Fede, Elisabetta; Neri, Iria; Facchini, Elena; Greco, Mariangela; Gervasini, Cristina; Larizza, Lidia

2018-05-16

Poikiloderma with neutropenia (PN) is a genodermatosis currently described in 77 patients, all presenting with early-onset poikiloderma, neutropenia, and several additional signs. Biallelic loss-of-function mutations in USB1 gene are detected in all molecularly tested patients but genotype-phenotype correlation remains elusive. Cancer predisposition is recognized among PN features and pathogenic variants found in patients who developed early in life myelodysplasia (n = 12), acute myeloid leukemia (n = 2), and squamous cell carcinoma (n = 2) should be kept into account in management and follow-up of novel patients. This will hopefully allow achieving data clustered on specific mutations relevant to oncological surveillance of the carrier patients. We describe the clinical features of three unreported PN patients and characterize their USB1 pathogenic variants by transcript analysis to get insights into the effect on the overall phenotype and disease evolution. A Turkish boy is homozygous for the c.531delA deletion, a recurrent mutation in Turkey; an adult Italian male is compound heterozygous for two nonsense mutations, c.243G>A and c.541C>T, while an Italian boy is homozygous for the splicing c.683_693+1del variant. The identified mutations have already been reported in PN patients who developed hematologic or skin cancer. Aberrant mRNAs of all four mutated alleles could be identified confirming that transcripts of USB1 main isoform either carrying stop codons or mis-spliced may at least partially escape nonsense-mediated decay. Our study addresses the need of gathering insights on genotype-phenotype correlations in newly described PN patients, by transcript analysis and information on disease evolution of reported patients with the same pathogenic variants.

ATM splicing variants as biomarkers for low dose dexamethasone treatment of A-T.

PubMed

Menotta, Michele; Biagiotti, Sara; Spapperi, Chiara; Orazi, Sara; Rossi, Luigia; Chessa, Luciana; Leuzzi, Vincenzo; D'Agnano, Daniela; Soresina, Annarosa; Micheli, Roberto; Magnani, Mauro

2017-07-05

Ataxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1. In the present study, the new ATMdexa1 transcript was also identified in vivo, specifically in the PMBCs of AT patients treated with intra-erythrocyte Dexamethasone (EryDex). In these patients it was also possible to isolate new "ATMdexa1 variants" originating from canonical and non-canonical splicing, each containing the coding sequence for the ATM kinase domain. The expression of the ATMdexa1 transcript family was directly related to treatment and higher expression levels of the transcript in patients' blood correlated with a positive response to Dexamethasone therapy. Neither untreated AT patients nor untreated healthy volunteers possessed detectable levels of the transcripts. ATMdexa1 transcript expression was found to be elevated 8 days after the drug infusion, while it decreased 21 days after treatment. For the first time, the expression of ATM splicing variants, similar to those previously observed in vitro, has been found in the PBMCs of patients treated with EryDex. These findings show a correlation between the expression of ATMdexa1 transcripts and the clinical response to low dose dexamethasone administration.

Functional Variants at the 11q13 Risk Locus for Breast Cancer Regulate Cyclin D1 Expression through Long-Range Enhancers

PubMed Central

French, Juliet D.; Ghoussaini, Maya; Edwards, Stacey L.; Meyer, Kerstin B.; Michailidou, Kyriaki; Ahmed, Shahana; Khan, Sofia; Maranian, Mel J.; O’Reilly, Martin; Hillman, Kristine M.; Betts, Joshua A.; Carroll, Thomas; Bailey, Peter J.; Dicks, Ed; Beesley, Jonathan; Tyrer, Jonathan; Maia, Ana-Teresa; Beck, Andrew; Knoblauch, Nicholas W.; Chen, Constance; Kraft, Peter; Barnes, Daniel; González-Neira, Anna; Alonso, M. Rosario; Herrero, Daniel; Tessier, Daniel C.; Vincent, Daniel; Bacot, Francois; Luccarini, Craig; Baynes, Caroline; Conroy, Don; Dennis, Joe; Bolla, Manjeet K.; Wang, Qin; Hopper, John L.; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Verhoef, Senno; Cornelissen, Sten; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Loehberg, Christian R.; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Aitken, Zoe; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marme, Frederik; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Milne, Roger L.; Zamora, M. Pilar; Arias Perez, Jose Ignacio; Benitez, Javier; Anton-Culver, Hoda; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Lichtner, Peter; Schmutzler, Rita K.; Engel, Christoph; Brauch, Hiltrud; Hamann, Ute; Justenhoven, Christina; Aaltonen, Kirsimari; Heikkilä, Päivi; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Sueta, Aiko; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Wu, Anna H.; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O.; Lambrechts, Diether; Peeters, Stephanie; Smeets, Ann; Floris, Giuseppe; Chang-Claude, Jenny; Rudolph, Anja; Nickels, Stefan; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Sardella, Domenico; Couch, Fergus J.; Wang, Xianshu; Pankratz, Vernon S.; Lee, Adam; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo Hwang; Yip, Cheng Har; Ng, Char-Hong; Vithana, Eranga Nishanthie; Kristensen, Vessela; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Mulligan, Anna Marie; Devilee, Peter; Seynaeve, Caroline; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J.; Lissowska, Jolanta; Czene, Kamila; Klevebring, Daniel; Schoof, Nils; Hooning, Maartje J.; Martens, John W.M.; Collée, J. Margriet; Tilanus-Linthorst, Madeleine; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Balasubramanian, Sabapathy P.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Healey, Catherine S.; Shah, Mitul; Pooley, Karen A.; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Hartman, Mikael; Miao, Hui; Sng, Jen-Hwei; Sim, Xueling; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; McKay, James; Toland, Amanda E.; Ambrosone, Christine B.; Yannoukakos, Drakoulis; Godwin, Andrew K.; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Chen, Shou-Tung; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J.; Ponder, Bruce A.J.; Nevanlinna, Heli; Brown, Melissa A.; Chenevix-Trench, Georgia; Easton, Douglas F.; Dunning, Alison M.

2013-01-01

Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1. PMID:23540573

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1.

PubMed

Pelekanou, Vasiliki; Kampa, Marilena; Kiagiadaki, Foteini; Deli, Alexandra; Theodoropoulos, Panayiotis; Agrogiannis, George; Patsouris, Efstratios; Tsapis, Andreas; Castanas, Elias; Notas, George

2016-02-01

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-β-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes. © Society for Leukocyte Biology.

Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.

PubMed

Lee, Melissa; Vecchio-Pagán, Briana; Sharma, Neeraj; Waheed, Abdul; Li, Xiaopeng; Raraigh, Karen S; Robbins, Sarah; Han, Sangwoo T; Franca, Arianna L; Pellicore, Matthew J; Evans, Taylor A; Arcara, Kristin M; Nguyen, Hien; Luan, Shan; Belchis, Deborah; Hertecant, Jozef; Zabner, Joseph; Sly, William S; Cutting, Garry R

2016-05-15

Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fusion of the Human Gene for the Polyubiquitination Coeffector UEV1 with Kua, a Newly Identified Gene

PubMed Central

Thomson, Timothy M.; Lozano, Juan José; Loukili, Noureddine; Carrió, Roberto; Serras, Florenci; Cormand, Bru; Valeri, Marta; Díaz, Víctor M.; Abril, Josep; Burset, Moisés; Merino, Jesús; Macaya, Alfons; Corominas, Montserrat; Guigó, Roderic

2000-01-01

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination. [Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).] PMID:11076860

Natural rpoS mutations contribute to population heterogeneity in Escherichia coli O157:H7 strains linked to the 2006 US spinach-associated outbreak.

PubMed

Carter, Michelle Qiu; Louie, Jacqueline W; Huynh, Steven; Parker, Craig T

2014-12-01

We previously reported significantly different acid resistance between curli variants derived from the same Escherichia coli O157:H7 strain, although the curli fimbriae were not associated with this phenotypic divergence. Here we investigated the underlying molecular mechanism by examining the genes encoding the common transcriptional regulators of curli biogenesis and acid resistance. rpoS null mutations were detected in all curli-expressing variants of the 2006 spinach-associated outbreak strains, whereas a wild-type rpoS was present in all curli-deficient variants. Consequently curli-expressing variants were much more sensitive to various stress challenges than curli-deficient variants. This loss of general stress fitness appeared solely to be the result of rpoS mutation since the stress resistances could be restored in curli-expressing variants by a functional rpoS. Comparative transcriptomic analyses between the curli variants revealed a large number of differentially expressed genes, characterized by the enhanced expression of metabolic genes in curli-expressing variants, but a marked decrease in transcription of genes related to stress resistances. Unlike the curli-expressing variants of the 1993 US hamburger-associated outbreak strains (Applied Environmental Microbiology 78: 7706-7719), all curli-expressing variants of the 2006 spinach-associated outbreak strains carry a functional rcsB gene, suggesting an alternative mechanism governing intra-strain phenotypic divergence in E. coli O157:H7. Published by Elsevier Ltd.

Differential expression and localisation of gasdermin-like (GSDML), a novel member of the cancer-associated GSDMDC protein family, in neoplastic and non-neoplastic gastric, hepatic, and colon tissues.

PubMed

Carl-McGrath, Stacy; Schneider-Stock, Regine; Ebert, Matthias; Röcken, Christoph

2008-01-01

Gasdermin-like (GSDML) is a novel member of the cancer associated gasdermin-domain containing (GSDMDC) protein family. The GSDMDC family has been linked to cancer development and progression, and this is the first study analysing the expression and intracellular localisation of GSDML. GSDML gene transcription was analysed using quantitative real-time RT-PCR. Anti-peptide antibodies against GSDML were developed in rabbits, and an in vitro transcription-translation reaction was used to verify specificity. The Protein-G affinity purified antibodies were used in immunohistochemistry and immunoblotting on hepatocellular, gastric, and colorectal carcinomas and non-lesional tissues. The GSDML gene was transcribed in human gastric, liver and colon cell lines, carcinomas and non-lesional tissues. The GSDML protein was localised to the cytoplasm of cells in both tumour and non-lesional tissues. The GSDML protein splicing variants range in molecular weight from 35 to 50 kDa, and the expression profile varies between tumour and non-tumour. A distinctive vesicular staining pattern was exhibited by GSDML in the apical region of gastric chief cells and colonic surface mucous cells, and the basal region of neuroendocrine cells. GSDML may be a secretory or metabolic product involved in a secretory pathway, and changes in the regulation of GSDML splicing variant transcription and translation may be seen in the development and/or progression of gastrointestinal and hepatic cancers.

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q

PubMed Central

Marinovic-Terzic, Ivana; Yoshioka-Yamashita, Atsuko; Shimodaira, Hideki; Avdievich, Elena; Hunton, Irina C.; Kolodner, Richard D.; Edelmann, Winfried; Wang, Jean Y. J.

2008-01-01

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients. PMID:18768816

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q.

PubMed

Marinovic-Terzic, Ivana; Yoshioka-Yamashita, Atsuko; Shimodaira, Hideki; Avdievich, Elena; Hunton, Irina C; Kolodner, Richard D; Edelmann, Winfried; Wang, Jean Y J

2008-09-16

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma

PubMed Central

Bharti, Brij; Shukla, Sachin; Tripathi, Ratnakar; Mishra, Suman; Kumar, Mohan; Pandey, Manoj; Mishra, Rajnikant

2016-01-01

Background & objectives: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. Methods: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, Western blot analysis, and lactate dehydrogenase (LDH) specific staining. Results: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, ZAP70, HIF1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. Interpretation & conclusions: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy. PMID:27748274

Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

PubMed

He, Xian-hui; Xu, Li-hui; Liu, Yi

2005-04-01

To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

Transcriptional Response of Human Cells to Microbeam Irradiation with 2.1 MeV Alpha Particles

NASA Astrophysics Data System (ADS)

Hellweg, C. E.; Bogner, S.; Spitta, L.; Arenz, A.; Baumstark-Khan, C.; Greif, K. D.; Giesen, U.

Within the next decades an increasing number of human beings in space will be simultaneously exposed to different stimuli especially microgravity and radiation To assess the risks for humans during long-duration space missions the complex interplay of these parameters at the cellular level must be understood Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors Activation of the Nuclear Factor kappa B NF- kappa B pathway as a possible anti-apoptotic route represents such an important cellular stress response A screening assay for detection of NF- kappa B-dependent gene activation using the destabilized variant of Enhanced Green Fluorescent Protein d2EGFP as reporter protein had been developed It consists of Human Embryonic Kidney HEK 293 Cells stably transfected with a receptor-reporter-construct carrying d2EGFP under the control of a NF- kappa B response element Clones positive for Tumor Necrosis Factor alpha TNF- alpha inducible d2EGFP expression were selected as cellular reporters Irradiation was performed either with X-rays 150 kV 19 mA at DLR Cologne or with 2 1 MeV alpha particles LET sim 160 keV mu m at PTB Braunschweig After irradiation the following biological endpoints were determined i cell survival via the colony forming ability test ii time-dependent activation of NF- kappa B dependent d2EGFP gene expression using flow cytometry iii quantitative RT-PCR

1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

PubMed

Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

2017-10-01

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D 3 , the biologically active form of vitamin D 3 , there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Metabolic Modifiers That Underlie Phenotypic Variations in Energy-Balance Regulation

PubMed Central

Chang, Chia Lin; Cai, James J.; Cheng, Po Jen; Chueh, Ho Yen; Hsu, Sheau Yu Teddy

2011-01-01

OBJECTIVE Although recent studies have shown that human genomes contain hundreds of loci that exhibit signatures of positive selection, variants that are associated with adaptation in energy-balance regulation remain elusive. We reasoned that the difficulty in identifying such variants could be due to heterogeneity in selection pressure and that an integrative approach that incorporated experiment-based evidence and population genetics-based statistical judgments would be needed to reveal important metabolic modifiers in humans. RESEARCH DESIGN AND METHODS To identify common metabolic modifiers that underlie phenotypic variation in diabetes-associated or obesity-associated traits in humans, or both, we screened 207 candidate loci for regulatory single nucleotide polymorphisms (SNPs) that exhibited evidence of gene–environmental interactions. RESULTS Three SNPs (rs3895874, rs3848460, and rs937301) at the 5′ gene region of human GIP were identified as prime metabolic-modifier candidates at the enteroinsular axis. Functional studies have shown that GIP promoter reporters carrying derived alleles of these three SNPs (haplotype GIP−1920A) have significantly lower transcriptional activities than those with ancestral alleles at corresponding positions (haplotype GIP−1920G). Consistently, studies of pregnant women who have undergone a screening test for gestational diabetes have shown that patients with a homozygous GIP−1920A/A genotype have significantly lower serum concentrations of glucose-dependent insulinotropic polypeptide (GIP) than those carrying an ancestral GIP−1920G haplotype. After controlling for a GIPR variation, we showed that serum glucose concentrations of patients carrying GIP−1920A/A homozygotes are significantly higher than that of those carrying an ancestral GIP−1920G haplotype (odds ratio 3.53). CONCLUSIONS Our proof-of-concept study indicates that common regulatory GIP variants impart a difference in GIP and glucose metabolism. The study also provides a rare example that identified the common variant-common phenotypic variation pattern based on evidence of moderate gene–environmental interactions. PMID:21300845

Regulatory variation: an emerging vantage point for cancer biology.

PubMed

Li, Luolan; Lorzadeh, Alireza; Hirst, Martin

2014-01-01

Transcriptional regulation involves complex and interdependent interactions of noncoding and coding regions of the genome with proteins that interact and modify them. Genetic variation/mutation in coding and noncoding regions of the genome can drive aberrant transcription and disease. In spite of accounting for nearly 98% of the genome comparatively little is known about the contribution of noncoding DNA elements to disease. Genome-wide association studies of complex human diseases including cancer have revealed enrichment for variants in the noncoding genome. A striking finding of recent cancer genome re-sequencing efforts has been the previously underappreciated frequency of mutations in epigenetic modifiers across a wide range of cancer types. Taken together these results point to the importance of dysregulation in transcriptional regulatory control in genesis of cancer. Powered by recent technological advancements in functional genomic profiling, exploration of normal and transformed regulatory networks will provide novel insight into the initiation and progression of cancer and open new windows to future prognostic and diagnostic tools. © 2013 Wiley Periodicals, Inc.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

PubMed

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-04-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis

PubMed Central

Bielli, Pamela; Busà, Roberta; Di Stasi, Savino M; Munoz, Manuel J; Botti, Flavia; Kornblihtt, Alberto R; Sette, Claudio

2014-01-01

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. PMID:24514149

Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

PubMed

Lahm, H; Hoeflich, A; Andre, S; Sordat, B; Kaltner, H; Wolf, E; Gabius, H J

2000-09-01

The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

PubMed

Gamonet, Clémentine; Bole-Richard, Elodie; Delherme, Aurélia; Aubin, François; Toussirot, Eric; Garnache-Ottou, Francine; Godet, Yann; Ysebaert, Loïc; Tournilhac, Olivier; Caroline, Dartigeas; Larosa, Fabrice; Deconinck, Eric; Saas, Philippe; Borg, Christophe; Deschamps, Marina; Ferrand, Christophe

2015-01-01

CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.

Extremely High Peak Power Pulsed RF and UWB EMR Effects on Genomic Transcription - Microarray Assessment

DTIC Science & Technology

2008-06-26

Homo sapiens decorin variant C mRNA, complete cds. 2.117 PKNOX2 HUM408A08B Human fetal brain (TFujiwara) Homo sapiens cDNA clone GEN -408A08 5’, mRNA...mRNA, complete cds. 2.117 PKNOX2 HUM408A08B Human fetal brain (TFujiwara) Homo sapiens cDNA clone GEN -408A08 5’, mRNA sequence. 2.076 SEC23B...RAS oncogene family ; RAB33B, member RAS oncogene family 205300_s_at 0.37 U1SNRNPBP U11/U12 snRNP 35K 220728_at 0.349 218689_at 0.342 FANCF Fanconi

VAT: a computational framework to functionally annotate variants in personal genomes within a cloud-computing environment

PubMed Central

Habegger, Lukas; Balasubramanian, Suganthi; Chen, David Z.; Khurana, Ekta; Sboner, Andrea; Harmanci, Arif; Rozowsky, Joel; Clarke, Declan; Snyder, Michael; Gerstein, Mark

2012-01-01

Summary: The functional annotation of variants obtained through sequencing projects is generally assumed to be a simple intersection of genomic coordinates with genomic features. However, complexities arise for several reasons, including the differential effects of a variant on alternatively spliced transcripts, as well as the difficulty in assessing the impact of small insertions/deletions and large structural variants. Taking these factors into consideration, we developed the Variant Annotation Tool (VAT) to functionally annotate variants from multiple personal genomes at the transcript level as well as obtain summary statistics across genes and individuals. VAT also allows visualization of the effects of different variants, integrates allele frequencies and genotype data from the underlying individuals and facilitates comparative analysis between different groups of individuals. VAT can either be run through a command-line interface or as a web application. Finally, in order to enable on-demand access and to minimize unnecessary transfers of large data files, VAT can be run as a virtual machine in a cloud-computing environment. Availability and Implementation: VAT is implemented in C and PHP. The VAT web service, Amazon Machine Image, source code and detailed documentation are available at vat.gersteinlab.org. Contact: lukas.habegger@yale.edu or mark.gerstein@yale.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22743228

A Pan-Cancer Proteogenomic Atlas of PI3K/AKT/mTOR Pathway Alterations | Office of Cancer Genomics

Cancer.gov

Molecular alterations involving the PI3K/Akt/mTOR pathway (including mutation, copy number, protein, or RNA) were examined across 11,219 human cancers representing 32 major types. Within specific mutated genes, frequency, mutation hotspot residues, in silico predictions, and functional assays were all informative in distinguishing the subset of genetic variants more likely to have functional relevance. Multiple oncogenic pathways including PI3K/Akt/mTOR converged on similar sets of downstream transcriptional targets.

BDNF Variants May Modulate Long-Term Visual Memory Performance in a Healthy Cohort

PubMed Central

Avgan, Nesli; Sutherland, Heidi G.; Spriggens, Lauren K.; Yu, Chieh; Ibrahim, Omar; Bellis, Claire; Haupt, Larisa M.; Shum, David H. K.; Griffiths, Lyn R.

2017-01-01

Brain-derived neurotrophic factor (BDNF) is involved in numerous cognitive functions including learning and memory. BDNF plays an important role in synaptic plasticity in humans and rats with BDNF shown to be essential for the formation of long-term memories. We previously identified a significant association between the BDNF Val66Met polymorphism (rs6265) and long-term visual memory (p-value = 0.003) in a small cohort (n = 181) comprised of healthy individuals who had been phenotyped for various aspects of memory function. In this study, we have extended the cohort to 597 individuals and examined multiple genetic variants across both the BDNF and BDNF-AS genes for association with visual memory performance as assessed by the Wechsler Memory Scale—Fourth Edition subtests Visual Reproduction I and II (VR I and II). VR I assesses immediate visual memory, whereas VR II assesses long-term visual memory. Genetic association analyses were performed for 34 single nucleotide polymorphisms genotyped on Illumina OmniExpress BeadChip arrays with the immediate and long-term visual memory phenotypes. While none of the BDNF and BDNF-AS variants were shown to be significant for immediate visual memory, we found 10 variants (including the Val66Met polymorphism (p-value = 0.006)) that were nominally associated, and three variants (two variants in BDNF and one variant in the BDNF-AS locus) that were significantly associated with long-term visual memory. Our data therefore suggests a potential role for BDNF, and its anti-sense transcript BDNF-AS, in long-term visual memory performance. PMID:28304362

BDNF Variants May Modulate Long-Term Visual Memory Performance in a Healthy Cohort.

PubMed

Avgan, Nesli; Sutherland, Heidi G; Spriggens, Lauren K; Yu, Chieh; Ibrahim, Omar; Bellis, Claire; Haupt, Larisa M; Shum, David H K; Griffiths, Lyn R

2017-03-17

Brain-derived neurotrophic factor (BDNF) is involved in numerous cognitive functions including learning and memory. BDNF plays an important role in synaptic plasticity in humans and rats with BDNF shown to be essential for the formation of long-term memories. We previously identified a significant association between the BDNF Val66Met polymorphism (rs6265) and long-term visual memory ( p -value = 0.003) in a small cohort ( n = 181) comprised of healthy individuals who had been phenotyped for various aspects of memory function. In this study, we have extended the cohort to 597 individuals and examined multiple genetic variants across both the BDNF and BDNF-AS genes for association with visual memory performance as assessed by the Wechsler Memory Scale-Fourth Edition subtests Visual Reproduction I and II (VR I and II). VR I assesses immediate visual memory, whereas VR II assesses long-term visual memory. Genetic association analyses were performed for 34 single nucleotide polymorphisms genotyped on Illumina OmniExpress BeadChip arrays with the immediate and long-term visual memory phenotypes. While none of the BDNF and BDNF-AS variants were shown to be significant for immediate visual memory, we found 10 variants (including the Val66Met polymorphism ( p -value = 0.006)) that were nominally associated, and three variants (two variants in BDNF and one variant in the BDNF-AS locus) that were significantly associated with long-term visual memory. Our data therefore suggests a potential role for BDNF , and its anti-sense transcript BDNF-AS , in long-term visual memory performance.

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

PubMed

Li-Korotky, Ha-Sheng; Lo, Chia-Yee; Zeng, Fan-Rui; Lo, David; Banks, Juliane M

2009-10-01

Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa. Human ME epithelial cells were preconditioned for 24h under one of the three gas/pressure conditions designed to simulate those for (1) normal ME (NME), (2) ME with Eustachian tube obstruction (ETO) and (3) ME with tympanostomy tube placement (TT), and then were incubated with ∼ 10(7)CFU/ml of either T or O variants of S. pneumoniae (6A) for 3h. Relative expression levels of genes encoding virulence factors, PsaA (surface adhesion), SpxB (pyruvate oxidase), Ply (pneumolysin), and LytA (autolysin) were assessed separately in epithelium-attached and supernatant bacteria 3h post infection using real-time PCR. Basal levels of the virulence molecules in inocula were comparable between two variants. However, relative expression levels of the gene transcripts were significantly induced in epithelium-attached T variants 3h after infection. Comparing with NME and TT conditions, ETO environment produced the largest effect on the differential expression of the virulence genes in the infected ME epithelial cells between T (induced) and O (suppressed) phenotypic pneumococci. T variant is a predominant phenotype responsible for the pathogenesis of pneumococcal OM.

De novo nonsense and frameshift variants of TCF20 in individuals with intellectual disability and postnatal overgrowth.

PubMed

Schäfgen, Johanna; Cremer, Kirsten; Becker, Jessica; Wieland, Thomas; Zink, Alexander M; Kim, Sarah; Windheuser, Isabelle C; Kreiß, Martina; Aretz, Stefan; Strom, Tim M; Wieczorek, Dagmar; Engels, Hartmut

2016-12-01

Recently, germline variants of the transcriptional co-regulator gene TCF20 have been implicated in the aetiology of autism spectrum disorders (ASD). However, the knowledge about the associated clinical picture remains fragmentary. In this study, two individuals with de novo TCF20 sequence variants were identified in a cohort of 313 individuals with intellectual disability of unknown aetiology, which was analysed by whole exome sequencing using a child-parent trio design. Both detected variants - one nonsense and one frameshift variant - were truncating. A comprehensive clinical characterisation of the patients yielded mild intellectual disability, postnatal tall stature and macrocephaly, obesity and muscular hypotonia as common clinical signs while ASD was only present in one proband. The present report begins to establish the clinical picture of individuals with de novo nonsense and frameshift variants of TCF20 which includes features such as proportionate overgrowth and muscular hypotonia. Furthermore, intellectual disability/developmental delay seems to be fully penetrant amongst known individuals with de novo nonsense and frameshift variants of TCF20, whereas ASD is shown to be incompletely penetrant. The transcriptional co-regulator gene TCF20 is hereby added to the growing number of genes implicated in the aetiology of both ASD and intellectual disability. Furthermore, such de novo variants of TCF20 may represent a novel differential diagnosis in the overgrowth syndrome spectrum.

A PIT-1 Homeodomain Mutant Blocks the Intranuclear Recruitment Of the CCAAT/Enhancer Binding Protein α Required for Prolactin Gene Transcription

PubMed Central

ENWRIGHT, JOHN F.; KAWECKI-CROOK, MARGARET A.; VOSS, TY C.; SCHAUFELE, FRED; DAY, RICHARD N.

2010-01-01

The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, in cluding CCAAT/enhancer binding protein α (C/ EBPα), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPα with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPα induced PRL gene transcription in pituitary GHFT1–5 cells, whereas the coexpression of Pit-1 and C/EBPα in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPα, fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPα from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPα. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPα. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPα, was itself recruited by C/EBPα to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity. PMID:12554749

Multiple splicing events involved in regulation of human aromatase expression by a novel promoter, I.6.

PubMed

Shozu, M; Zhao, Y; Bulun, S E; Simpson, E R

1998-04-01

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and 1f. Recently, we have found a new putative exon I in a RACE-generated library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a 1-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, I.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon I.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon I.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon I.6 in THP-1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon I.6 is regulated mainly via an AP-1 binding site that is found upstream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testis and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon I.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a significant level of exon I.6 expression, but not so much as testicular tumor and hepatic tumor. Several splicing variants of exon I.6 were detected especially in THP-1 and JEG-3 cells, and to a lesser extent in primary cultures and tissue samples. These variants were identified as an unspliced form, a form spliced at the end of exon I.4, a form spliced at the end of exon I.3 (truncated) and a form spliced 220 bp downstream of the 3' end of exon I.6. The last variant revealed a new splicing site. Because most of the splicing variants contain the sequence specific for exon I.3, RT-PCR specific for exon I.3 can coamplify these splicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are known to express exon I.3 such as breast adipose tissue, in which promoter usage of exon I of the aromatase gene switches from exon I.4 to I.3 in the course of malignant transformation.

The histone variant H2A.X is a regulator of the epithelial-mesenchymal transition.

PubMed

Weyemi, Urbain; Redon, Christophe E; Choudhuri, Rohini; Aziz, Towqir; Maeda, Daisuke; Boufraqech, Myriem; Parekh, Palak R; Sethi, Taresh K; Kasoji, Manjula; Abrams, Natalie; Merchant, Anand; Rajapakse, Vinodh N; Bonner, William M

2016-02-15

The epithelial-mesenchymal transition (EMT), considered essential for metastatic cancer, has been a focus of much research, but important questions remain. Here, we show that silencing or removing H2A.X, a histone H2A variant involved in cellular DNA repair and robust growth, induces mesenchymal-like characteristics including activation of EMT transcription factors, Slug and ZEB1, in HCT116 human colon cancer cells. Ectopic H2A.X re-expression partially reverses these changes, as does silencing Slug and ZEB1. In an experimental metastasis model, the HCT116 parental and H2A.X-null cells exhibit a similar metastatic behaviour, but the cells with re-expressed H2A.X are substantially more metastatic. We surmise that H2A.X re-expression leads to partial EMT reversal and increases robustness in the HCT116 cells, permitting them to both form tumours and to metastasize. In a human adenocarcinoma panel, H2A.X levels correlate inversely with Slug and ZEB1 levels. Together, these results point to H2A.X as a regulator of EMT.

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology.

PubMed

Van Vaerenbergh, Matthias; De Smet, Lina; Rafei-Shamsabadi, David; Blank, Simon; Spillner, Edzard; Ebo, Didier G; Devreese, Bart; Jakob, Thilo; de Graaf, Dirk C

2015-02-01

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4. Copyright © 2014 Elsevier Ltd. All rights reserved.

Alzheimer's disease susceptibility variants in the MS4A6A gene are associated with altered levels of MS4A6A expression in blood.

PubMed

Proitsi, Petroula; Lee, Sang Hyuck; Lunnon, Katie; Keohane, Aoife; Powell, John; Troakes, Claire; Al-Sarraj, Safa; Furney, Simon; Soininen, Hilkka; Kłoszewska, Iwona; Mecocci, Patrizia; Tsolaki, Magda; Vellas, Bruno; Lovestone, Simon; Hodges, Angela

2014-02-01

An increased risk of developing Alzheimer's disease (AD) has previously been found to be associated with variants at the MS4A6A locus. We sought to identify which genes and transcripts in this region have altered expression in AD and mild cognitive impairment (MCI) and are influenced by the AD risk variant(s), as a first step to understanding the molecular basis of AD susceptibility at this locus. Common variants located within highly expressed MS4A6A transcripts were significantly associated with AD and MS4A6A expression levels in blood from MCI and AD subjects (p < 0.05, rs610932, rs7232, rs583791). More copies of the protective (minor) allele were associated with lower MS4A6A expression of each transcript (e.g., p = 0.019; rs610932-total MS4A6A). Furthermore, in heterozygous AD subjects, relative expression of the protective allele of V4-MS4A6A transcripts was lower (p < 0.008). Irrespective of genotype, MS4A6A transcripts were increased in blood from people with AD (p < 0.003), whereas lower expression of full length V1-MS4A6A (p = 0.002) and higher expression of V4-MS4A6A (p = 1.8 × 10(-4)) were observed in MCI, relative to elderly controls. The association between genotype and expression was less consistent in brain, although BA9 did have a similar genotype association with V4-MS4A6A transcripts as in blood. MS4A6A transcripts were widely expressed in tissues and cells, with the exception of V4-MS4A6A, which was not expressed in neuronal cells. Together these results suggest that high levels of MS4A6A in emerging AD pathology are detrimental. Persons with MCI may lower MS4A6A expression to minimize detrimental disease associated MS4A6A activity. However, those with the susceptibility allele appear unable to decrease expression sufficiently, which may explain their increased risk for developing AD. Inhibiting MS4A6A may therefore promote a more neuroprotective phenotype, although further work is needed to establish whether this is the case. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

PubMed Central

Neve, Bernadette; Fernandez-Zapico, Martin E.; Ashkenazi-Katalan, Vered; Dina, Christian; Hamid, Yasmin H.; Joly, Erik; Vaillant, Emmanuel; Benmezroua, Yamina; Durand, Emmanuelle; Bakaher, Nicolas; Delannoy, Valerie; Vaxillaire, Martine; Cook, Tiffany; Dallinga-Thie, Geesje M.; Jansen, Hans; Charles, Marie-Aline; Clément, Karine; Galan, Pilar; Hercberg, Serge; Helbecque, Nicole; Charpentier, Guillaume; Prentki, Marc; Hansen, Torben; Pedersen, Oluf; Urrutia, Raul; Melloul, Danielle; Froguel, Philippe

2005-01-01

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes. PMID:15774581

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences.

PubMed

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-06-09

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences

PubMed Central

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-01-01

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics. PMID:27279482

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

PubMed

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases. © 2017 American Heart Association, Inc.

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

PubMed Central

Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B.; Gersbach, Charles A.

2014-01-01

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

Central nervous system involvement in severe congenital neutropenia: neurological and neuropsychological abnormalities associated with specific HAX1 mutations.

PubMed

Carlsson, G; van't Hooft, I; Melin, M; Entesarian, M; Laurencikas, E; Nennesmo, I; Trebińska, A; Grzybowska, E; Palmblad, J; Dahl, N; Nordenskjöld, M; Fadeel, B; Henter, J-I

2008-10-01

Homozygous mutations in the HAX1 gene were recently identified in severe congenital neutropenia patients belonging to the original Kostmann family in northern Sweden. Our observations suggested that these patients also develop neurological and neuropsychological symptoms. Detailed clinical studies and mutation analyses were performed in the surviving patients belonging to the Kostmann kindred and in two patients not related to this family, along with studies of HAX1 splice variant expression in normal human tissues. Five of six Kostmann family patients and one other patient from northern Sweden harboured homozygous HAX1 mutations (568C-->T, Q190X) and one carried a heterozygous ELA2 gene mutation. One Swedish patient of Kurdish extraction carried alternative homozygous HAX1 mutations (131G-->A, W44X). All the three patients with Q190X mutations who were alive and available for evaluation developed neurological disease with decreased cognitive function, and three of four patients who reached 10 years developed epilepsy. In contrast, the patients with the ELA2 and W44X HAX1 mutations, respectively, showed no obvious neurological abnormalities. Moreover, two alternative HAX1 splice variants were identified in normal human tissues, including the brain. Both transcripts contained exon 5, harbouring the Q190X mutation, whereas the 5' end of exon 2 containing the W44X mutation was spliced out from the second transcript. We describe neurological and neuropsychological abnormalities for the first time in Kostmann disease patients. These central nervous system symptoms appear to be associated with specific HAX1 mutations.

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies.

PubMed

Powell, Christopher A; Kopajtich, Robert; D'Souza, Aaron R; Rorbach, Joanna; Kremer, Laura S; Husain, Ralf A; Dallabona, Cristina; Donnini, Claudia; Alston, Charlotte L; Griffin, Helen; Pyle, Angela; Chinnery, Patrick F; Strom, Tim M; Meitinger, Thomas; Rodenburg, Richard J; Schottmann, Gudrun; Schuelke, Markus; Romain, Nadine; Haller, Ronald G; Ferrero, Ileana; Haack, Tobias B; Taylor, Robert W; Prokisch, Holger; Minczuk, Michal

2015-08-06

Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Mapping cis- and trans-regulatory effects across multiple tissues in twins

PubMed Central

Grundberg, Elin; Small, Kerrin S.; Hedman, Åsa K.; Nica, Alexandra C.; Buil, Alfonso; Keildson, Sarah; Bell, Jordana T.; Yang, Tsun-Po; Meduri, Eshwar; Barrett, Amy; Nisbett, James; Sekowska, Magdalena; Wilk, Alicja; Shin, So-Youn; Glass, Daniel; Travers, Mary; Min, Josine L.; Ring, Sue; Ho, Karen; Thorleifsson, Gudmar; Kong, Augustine; Thorsteindottir, Unnur; Ainali, Chrysanthi; Dimas, Antigone S.; Hassanali, Neelam; Ingle, Catherine; Knowles, David; Krestyaninova, Maria; Lowe, Christopher E.; Di Meglio, Paola; Montgomery, Stephen B.; Parts, Leopold; Potter, Simon; Surdulescu, Gabriela; Tsaprouni, Loukia; Tsoka, Sophia; Bataille, Veronique; Durbin, Richard; Nestle, Frank O.; O’Rahilly, Stephen; Soranzo, Nicole; Lindgren, Cecilia M.; Zondervan, Krina T.; Ahmadi, Kourosh R.; Schadt, Eric E.; Stefansson, Kari; Smith, George Davey; McCarthy, Mark I.; Deloukas, Panos; Dermitzakis, Emmanouil T.; Spector, Tim D.

2013-01-01

Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many eQTL studies typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis-effect on expression cannot be accounted for by common cis-variants, a finding which exposes the contribution of low frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene and identify several replicating trans-variants which act predominantly in a tissue-restricted manner and may regulate the transcription of many genes. PMID:22941192

The efficacy of 9-cis retinoic acid in experimental models of cancer.

PubMed

Gottardis, M M; Lamph, W W; Shalinsky, D R; Wellstein, A; Heyman, R A

1996-01-01

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

A novel de novo activating mutation in STAT3 identified in a patient with common variable immunodeficiency (CVID).

PubMed

Russell, Mark A; Pigors, Manuela; Houssen, Maha E; Manson, Ania; Kelsell, David; Longhurst, Hilary; Morgan, Noel G

2018-02-01

Common variable immunodeficiency (CVID) is characterised by repeated infection associated with primary acquired hypogammaglobulinemia. CVID frequently has a complex aetiology but, in certain cases, it has a monogenic cause. Recently, variants within the gene encoding the transcription factor STAT3 were implicated in monogenic CVID. Here, we describe a patient presenting with symptoms synonymous with CVID, who displayed reduced levels of IgG and IgA, repeated viral infections and multiple additional co-morbidities. Whole-exome sequencing revealed a de novo novel missense mutation in the coiled-coil domain of STAT3 (c.870A>T; p.K290N). Accordingly, the K290N variant of STAT3 was generated, and a STAT3 responsive dual-luciferase reporter assay revealed that the variant strongly enhances STAT3 transcriptional activity both under basal and stimulated (with IL-6) conditions. Overall, these data complement earlier studies in which CVID-associated STAT3 mutations are predicted to enhance transcriptional activity, suggesting that such patients may respond favourably to IL-6 receptor antagonists (e.g. tocilizumab). Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of genetic risk factors in the Chinese population implicates a role of immune system in Alzheimer's disease pathogenesis.

PubMed

Zhou, Xiaopu; Chen, Yu; Mok, Kin Y; Zhao, Qianhua; Chen, Keliang; Chen, Yuewen; Hardy, John; Li, Yun; Fu, Amy K Y; Guo, Qihao; Ip, Nancy Y

2018-02-20

Alzheimer's disease (AD) is a leading cause of mortality among the elderly. We performed a whole-genome sequencing study of AD in the Chinese population. In addition to the variants identified in or around the APOE locus (sentinel variant rs73052335, P = 1.44 × 10 -14 ), two common variants, GCH1 (rs72713460, P = 4.36 × 10 -5 ) and KCNJ15 (rs928771, P = 3.60 × 10 -6 ), were identified and further verified for their possible risk effects for AD in three small non-Asian AD cohorts. Genotype-phenotype analysis showed that KCNJ15 variant rs928771 affects the onset age of AD, with earlier disease onset in minor allele carriers. In addition, altered expression level of the KCNJ15 transcript can be observed in the blood of AD subjects. Moreover, the risk variants of GCH1 and KCNJ15 are associated with changes in their transcript levels in specific tissues, as well as changes of plasma biomarkers levels in AD subjects. Importantly, network analysis of hippocampus and blood transcriptome datasets suggests that the risk variants in the APOE , GCH1 , and KCNJ15 loci might exert their functions through their regulatory effects on immune-related pathways. Taking these data together, we identified common variants of GCH1 and KCNJ15 in the Chinese population that contribute to AD risk. These variants may exert their functional effects through the immune system. Copyright © 2018 the Author(s). Published by PNAS.

Large-scale screening and molecular characterization of EML4-ALK fusion variants in archival non-small-cell lung cancer tumor specimens using quantitative reverse transcription polymerase chain reaction assays.

PubMed

Li, Tianhong; Maus, Martin K H; Desai, Sonal J; Beckett, Laurel A; Stephens, Craig; Huang, Eric; Hsiang, Jack; Zeger, Gary; Danenberg, Kathleen D; Astrow, Stephanie H; Gandara, David R

2014-01-01

The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non-small-cell lung cancer (NSCLC) patients. We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23-89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes.

PubMed

Sadowska, Agnieszka; Paukszto, Lukasz; Nynca, Anna; Szczerbal, Izabela; Orlowska, Karina; Swigonska, Sylwia; Ruszkowska, Monika; Molcan, Tomasz; Jastrzebski, Jan P; Panasiewicz, Grzegorz; Ciereszko, Renata E

2017-03-01

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

PubMed Central

Festari, María Florencia; Trajtenberg, Felipe; Berois, Nora; Pantano, Sergio; Revoredo, Leslie; Kong, Yun; Solari-Saquieres, Patricia; Narimatsu, Yoshiki; Freire, Teresa; Bay, Sylvie; Robello, Carlos; Bénard, Jean; Gerken, Thomas A; Clausen, Henrik; Osinaga, Eduardo

2017-01-01

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain. PMID:27913570

Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations using human MCF10A isogenic cell lines

PubMed Central

Stolze, Britta; Reinhart, Stefanie; Bulllinger, Lars; Fröhling, Stefan; Scholl, Claudia

2015-01-01

KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. We used MCF10A human mammary epithelial cells to establish isogenic cell lines that express different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological or elevated levels, and investigated the biochemical and functional consequences of the different variants. The overall effects of low-expressing mutants were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes, migratory abilities, or increased phosphorylation of ERK, PDK1, and AKT. KRAS-G12D, G12V, G13D, and K117N mediated EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by K117N and Q61H. Both codon 13 mutations were associated with increased EGFR expression. Finally, global gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells. PMID:25705018

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

PubMed Central

Saravanaperumal, Siva Arumugam; Pediconi, Dario; Renieri, Carlo; La Terza, Antonietta

2012-01-01

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals. PMID:22719917

Human IRGM gene "to be or not to be".

PubMed

Bekpen, Cemaletin; Xavier, Ramnik J; Eichler, Evan E

2010-12-01

The immunity-related GTPases (IRG proteins) are one of the strongest early resistance systems against intracellular pathogens. The IRG gene family contains 21 copies arranged as tandem gene clusters on two chromosomes in the C57BL/6 mouse genome but has been reduced to only two copies in humans: IRGC and IRGM. IRGC is not involved in immunity, but the human IRGM gene plays a role in autophagy-targeted destruction of Mycobacterium tuberculosis (BCG) and Salmonella typhimurium. Variant IRGM haplotypes have been associated with increased risk for Crohn's disease and correlated with differential expression of IRGM transcripts. This article reviews in detail the studies performed on human samples, in vitro, and in sequence analyses that provide evidence for the unusual evolutionary history of the IRGM locus and the important role of the IRGM gene in autophagy and Crohn's disease in response to pathogenesis.

Disruption of an Evolutionarily Novel Synaptic Expression Pattern in Autism

PubMed Central

Jiang, Xi; Hu, Haiyang; Guijarro, Patricia; Mitchell, Amanda; Ely, John J.; Sherwood, Chet C.; Hof, Patrick R.; Qiu, Zilong; Pääbo, Svante; Akbarian, Schahram; Khaitovich, Philipp

2016-01-01

Cognitive defects in autism spectrum disorder (ASD) include socialization and communication: key behavioral capacities that separate humans from other species. Here, we analyze gene expression in the prefrontal cortex of 63 autism patients and control individuals, as well as 62 chimpanzees and macaques, from natal to adult age. We show that among all aberrant expression changes seen in ASD brains, a single aberrant expression pattern overrepresented in genes involved synaptic-related pathways is enriched in nucleotide variants linked to autism. Furthermore, only this pattern contains an excess of developmental expression features unique to humans, thus resulting in the disruption of human-specific developmental programs in autism. Several members of the early growth response (EGR) transcription factor family can be implicated in regulation of this aberrant developmental change. Our study draws a connection between the genetic risk architecture of autism and molecular features of cortical development unique to humans. PMID:27685936

Rare Coding Variants in ANGPTL6 Are Associated with Familial Forms of Intracranial Aneurysm.

PubMed

Bourcier, Romain; Le Scouarnec, Solena; Bonnaud, Stéphanie; Karakachoff, Matilde; Bourcereau, Emmanuelle; Heurtebise-Chrétien, Sandrine; Menguy, Céline; Dina, Christian; Simonet, Floriane; Moles, Alexis; Lenoble, Cédric; Lindenbaum, Pierre; Chatel, Stéphanie; Isidor, Bertrand; Génin, Emmanuelle; Deleuze, Jean-François; Schott, Jean-Jacques; Le Marec, Hervé; Loirand, Gervaise; Desal, Hubert; Redon, Richard

2018-01-04

Intracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)-which encodes a circulating pro-angiogenic factor mainly secreted from the liver-shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity

PubMed Central

Song, Lingyun; Zhang, Zhancheng; Grasfeder, Linda L.; Boyle, Alan P.; Giresi, Paul G.; Lee, Bum-Kyu; Sheffield, Nathan C.; Gräf, Stefan; Huss, Mikael; Keefe, Damian; Liu, Zheng; London, Darin; McDaniell, Ryan M.; Shibata, Yoichiro; Showers, Kimberly A.; Simon, Jeremy M.; Vales, Teresa; Wang, Tianyuan; Winter, Deborah; Zhang, Zhuzhu; Clarke, Neil D.; Birney, Ewan; Iyer, Vishwanath R.; Crawford, Gregory E.; Lieb, Jason D.; Furey, Terrence S.

2011-01-01

The human body contains thousands of unique cell types, each with specialized functions. Cell identity is governed in large part by gene transcription programs, which are determined by regulatory elements encoded in DNA. To identify regulatory elements active in seven cell lines representative of diverse human cell types, we used DNase-seq and FAIRE-seq (Formaldehyde Assisted Isolation of Regulatory Elements) to map “open chromatin.” Over 870,000 DNaseI or FAIRE sites, which correspond tightly to nucleosome-depleted regions, were identified across the seven cell lines, covering nearly 9% of the genome. The combination of DNaseI and FAIRE is more effective than either assay alone in identifying likely regulatory elements, as judged by coincidence with transcription factor binding locations determined in the same cells. Open chromatin common to all seven cell types tended to be at or near transcription start sites and to be coincident with CTCF binding sites, while open chromatin sites found in only one cell type were typically located away from transcription start sites and contained DNA motifs recognized by regulators of cell-type identity. We show that open chromatin regions bound by CTCF are potent insulators. We identified clusters of open regulatory elements (COREs) that were physically near each other and whose appearance was coordinated among one or more cell types. Gene expression and RNA Pol II binding data support the hypothesis that COREs control gene activity required for the maintenance of cell-type identity. This publicly available atlas of regulatory elements may prove valuable in identifying noncoding DNA sequence variants that are causally linked to human disease. PMID:21750106

N-terminal nesprin-2 variants regulate β-catenin signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

Tissue-specific promoter utilisation of the kallikrein-related peptidase genes, KLK5 and KLK7, and cellular localisation of the encoded proteins suggest roles in exocrine pancreatic function.

PubMed

Dong, Ying; Matigian, Nick; Harvey, Tracey J; Samaratunga, Hemamali; Hooper, John D; Clements, Judith A

2008-02-01

Abstract Tissue kallikrein (kallikrein 1) was first identified in pancreas and is the namesake of the kallikrein-related peptidase (KLK) family. KLK1 and the other 14 members of the human KLK family are encoded by 15 serine protease genes clustered at chromosome 19q13.4. Our Northern blot analysis of 19 normal human tissues for expression of KLK4 to KLK15 identified pancreas as a common expression site for the gene cluster spanning KLK5 to KLK13, as well as for KLK15 which is located adjacent to KLK1. Consistent with previous reports detailing the ability of KLK genes to generate organ- and disease-specific transcripts, detailed molecular and in silico analyses indicated that KLK5 and KLK7 generate transcripts in pancreas variant from those in skin or ovary. Consistently, we identified in the promoters of these KLK genes motifs which conform with consensus binding sites for transcription factors conferring pancreatic expression. In addition, immunohistochemical analysis revealed predominant localisation of KLK5 and KLK7 in acinar cells of the exocrine pancreas, suggesting roles for these enzymes in digestion. Our data also support expression patterns derived from gene duplication events in the human KLK cluster. These findings suggest that, in addition to KLK1, other related KLK enzymes will function in the exocrine pancreas.

Redefining the MED13L syndrome

PubMed Central

Adegbola, Abidemi; Musante, Luciana; Callewaert, Bert; Maciel, Patricia; Hu, Hao; Isidor, Bertrand; Picker-Minh, Sylvie; Le Caignec, Cedric; Delle Chiaie, Barbara; Vanakker, Olivier; Menten, Björn; Dheedene, Annelies; Bockaert, Nele; Roelens, Filip; Decaestecker, Karin; Silva, João; Soares, Gabriela; Lopes, Fátima; Najmabadi, Hossein; Kahrizi, Kimia; Cox, Gerald F; Angus, Steven P; Staropoli, John F; Fischer, Ute; Suckow, Vanessa; Bartsch, Oliver; Chess, Andrew; Ropers, Hans-Hilger; Wienker, Thomas F; Hübner, Christoph; Kaindl, Angela M; Kalscheuer, Vera M

2015-01-01

Congenital cardiac and neurodevelopmental deficits have been recently linked to the mediator complex subunit 13-like protein MED13L, a subunit of the CDK8-associated mediator complex that functions in transcriptional regulation through DNA-binding transcription factors and RNA polymerase II. Heterozygous MED13L variants cause transposition of the great arteries and intellectual disability (ID). Here, we report eight patients with predominantly novel MED13L variants who lack such complex congenital heart malformations. Rather, they depict a syndromic form of ID characterized by facial dysmorphism, ID, speech impairment, motor developmental delay with muscular hypotonia and behavioral difficulties. We thereby define a novel syndrome and significantly broaden the clinical spectrum associated with MED13L variants. A prominent feature of the MED13L neurocognitive presentation is profound language impairment, often in combination with articulatory deficits. PMID:25758992

Redefining the MED13L syndrome.

PubMed

Adegbola, Abidemi; Musante, Luciana; Callewaert, Bert; Maciel, Patricia; Hu, Hao; Isidor, Bertrand; Picker-Minh, Sylvie; Le Caignec, Cedric; Delle Chiaie, Barbara; Vanakker, Olivier; Menten, Björn; Dheedene, Annelies; Bockaert, Nele; Roelens, Filip; Decaestecker, Karin; Silva, João; Soares, Gabriela; Lopes, Fátima; Najmabadi, Hossein; Kahrizi, Kimia; Cox, Gerald F; Angus, Steven P; Staropoli, John F; Fischer, Ute; Suckow, Vanessa; Bartsch, Oliver; Chess, Andrew; Ropers, Hans-Hilger; Wienker, Thomas F; Hübner, Christoph; Kaindl, Angela M; Kalscheuer, Vera M

2015-10-01

Congenital cardiac and neurodevelopmental deficits have been recently linked to the mediator complex subunit 13-like protein MED13L, a subunit of the CDK8-associated mediator complex that functions in transcriptional regulation through DNA-binding transcription factors and RNA polymerase II. Heterozygous MED13L variants cause transposition of the great arteries and intellectual disability (ID). Here, we report eight patients with predominantly novel MED13L variants who lack such complex congenital heart malformations. Rather, they depict a syndromic form of ID characterized by facial dysmorphism, ID, speech impairment, motor developmental delay with muscular hypotonia and behavioral difficulties. We thereby define a novel syndrome and significantly broaden the clinical spectrum associated with MED13L variants. A prominent feature of the MED13L neurocognitive presentation is profound language impairment, often in combination with articulatory deficits.

Mutations in SNRPB, encoding components of the core splicing machinery, cause cerebro-costo-mandibular syndrome.

PubMed

Bacrot, Séverine; Doyard, Mathilde; Huber, Céline; Alibeu, Olivier; Feldhahn, Niklas; Lehalle, Daphné; Lacombe, Didier; Marlin, Sandrine; Nitschke, Patrick; Petit, Florence; Vazquez, Marie-Paule; Munnich, Arnold; Cormier-Daire, Valérie

2015-02-01

Cerebro-costo-mandibular syndrome (CCMS) is a developmental disorder characterized by the association of Pierre Robin sequence and posterior rib defects. Exome sequencing and Sanger sequencing in five unrelated CCMS patients revealed five heterozygous variants in the small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene. This gene includes three transcripts, namely transcripts 1 and 2, encoding components of the core spliceosomal machinery (SmB' and SmB) and transcript 3 undergoing nonsense-mediated mRNA decay. All variants were located in the premature termination codon (PTC)-introducing alternative exon of transcript 3. Quantitative RT-PCR analysis revealed a significant increase in transcript 3 levels in leukocytes of CCMS individuals compared to controls. We conclude that CCMS is due to heterozygous mutations in SNRPB, enhancing inclusion of a SNRPB PTC-introducing alternative exon, and show that this developmental disease is caused by defects in the splicing machinery. Our finding confirms the report of SNRPB mutations in CCMS patients by Lynch et al. (2014) and further extends the clinical and molecular observations. © 2014 WILEY PERIODICALS, INC.

Microgranular variant of acute promyelocytic leukemia with der(17) ins(17;15): A case report and review of the literature

PubMed Central

GUAN, HONGZAI; LIU, JING; GUO, XIAOFANG; WU, CHUNMEI; YU, HUAWEI

2015-01-01

Acute promyelocytic leukemia (APL) with variant translocations is rare. The patient of the present case report, a 2-year-old male with a microgranular variant of APL carrying der(17) ins(17;15) translocation, exhibited fever and epistaxis. The complete blood count showed marked leukocytosis with 72% atypical promyelocytes, anemia and thrombocytopenia. Conventional cytogenetic analysis of the bone marrow cells revealed a karyotype of 47, XY, add(3)(q29), −7, ins(17;15)(q12;q14q22),+21,+mar. The promyelocytic leukemia/retinoic acid receptor α (PML/RARα) rearrangement and insertion were confirmed by fluorescence in situ hybridization. The PML/RARα transcripts were not detected by the reverse transcription polymerase chain reaction, and the patient was diagnosed with microgranular variant M3 APL. The patient achieved remission after a 30-day treatment and was still in remission during a recent follow-up. The present findings suggest that the ins(17;15) variant in APL may not be associated with an unfavorable prognosis. In summary, we reported an extremely rare case of APL with der(17) ins(17;15) abnormality in a pediatric patient and reviewed the literature. PMID:26622430

Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

PubMed

Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

2017-06-01

Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

eMelanoBase: an online locus-specific variant database for familial melanoma.

PubMed

Fung, David C Y; Holland, Elizabeth A; Becker, Therese M; Hayward, Nicholas K; Bressac-de Paillerets, Brigitte; Mann, Graham J

2003-01-01

A proportion of melanoma-prone individuals in both familial and non-familial contexts has been shown to carry inactivating mutations in either CDKN2A or, rarely, CDK4. CDKN2A is a complex locus that encodes two unrelated proteins from alternately spliced transcripts that are read in different frames. The alpha transcript (exons 1alpha, 2, and 3) produces the p16INK4A cyclin-dependent kinase inhibitor, while the beta transcript (exons 1beta and 2) is translated as p14ARF, a stabilizing factor of p53 levels through binding to MDM2. Mutations in exon 2 can impair both polypeptides and insertions and deletions in exons 1alpha, 1beta, and 2, which can theoretically generate p16INK4A-p14ARF fusion proteins. No online database currently takes into account all the consequences of these genotypes, a situation compounded by some problematic previous annotations of CDKN2A-related sequences and descriptions of their mutations. As an initiative of the international Melanoma Genetics Consortium, we have therefore established a database of germline variants observed in all loci implicated in familial melanoma susceptibility. Such a comprehensive, publicly accessible database is an essential foundation for research on melanoma susceptibility and its clinical application. Our database serves two types of data as defined by HUGO. The core dataset includes the nucleotide variants on the genomic and transcript levels, amino acid variants, and citation. The ancillary dataset includes keyword description of events at the transcription and translation levels and epidemiological data. The application that handles users' queries was designed in the model-view-controller architecture and was implemented in Java. The object-relational database schema was deduced using functional dependency analysis. We hereby present our first functional prototype of eMelanoBase. The service is accessible via the URL www.wmi.usyd.edu.au:8080/melanoma.html. Copyright 2002 Wiley-Liss, Inc.

Identification of OCTN2 variants and their association with phenotypes of Crohn’s disease in a Korean population

PubMed Central

Park, Hyo Jin; Jung, Eun Suk; Kong, Kyoung Ae; Park, Eun-Mi; Cheon, Jae Hee; Choi, Ji Ha

2016-01-01

Crohn’s disease (CD) is a chronic inflammatory bowel disease and a genetic variant in the OCTN2, g.-207G > C is significantly associated with CD susceptibility. This study was aimed to identify novel OCTN2 functional promoter variants and their roles in transcriptional regulation using various in vitro assays. In addition, we investigated the association between OCTN2 genotypes and CD through genetic analysis using DNA samples from 193 patients with CD and 281 healthy controls. Among the three major promoter haplotypes of OCTN2 identified, one haplotype, H3, showed a significant decrease in promoter activity: two polymorphisms in H3 were associated with a significant reduction in promoter activity. In particular, we found that the reduced transcriptional activity of those two polymorphisms results from a reduction in the binding affinity of the activators, NF-E2 and YY1, to the OCTN2 promoter. The functional haplotype of the OCTN2 promoter was associated with clinical course of CD such as the disease behavior and need for surgery. However, genetic variants or haplotypes of OCTN2 did not affect the susceptibility to CD. Our results suggest that a common promoter haplotype of OCTN2 regulates the transcriptional rate of OCTN2 and influences the clinical course of CD. PMID:26965072

Isolation and characterization of novel RECK tumor suppressor gene splice variants

PubMed Central

Trombetta-Lima, Marina; Winnischofer, Sheila Maria Brochado; Demasi, Marcos Angelo Almeida; Filho, Renato Astorino; Carreira, Ana Claudia Oliveira; Wei, Beiyang; de Assis Ribas, Thais; Konig, Michelle Silberspitz; Bowman-Colin, Christian; Oba-Shinjo, Sueli Mieko; Marie, Suely Kazue Nagahashi; Stetler-Stevenson, William; Sogayar, Mari Cleide

2015-01-01

Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients. PMID:26431549

Structural insights into alternative splicing-mediated desensitization of jasmonate signaling.

PubMed

Zhang, Feng; Ke, Jiyuan; Zhang, Li; Chen, Rongzhi; Sugimoto, Koichi; Howe, Gregg A; Xu, H Eric; Zhou, Mingguo; He, Sheng Yang; Melcher, Karsten

2017-02-14

Jasmonate ZIM-domain (JAZ) transcriptional repressors play a key role in regulating jasmonate (JA) signaling in plants. Below a threshold concentration of jasmonoyl isoleucine (JA-Ile), the active form of JA, the C-terminal Jas motif of JAZ proteins binds MYC transcription factors to repress JA signaling. With increasing JA-Ile concentration, the Jas motif binds to JA-Ile and the COI1 subunit of the SCF COI1 E3 ligase, which mediates ubiquitination and proteasomal degradation of JAZ repressors, resulting in derepression of MYC transcription factors. JA signaling subsequently becomes desensitized, in part by feedback induction of JAZ splice variants that lack the C-terminal Jas motif but include an N-terminal cryptic MYC-interaction domain (CMID). The CMID sequence is dissimilar to the Jas motif and is incapable of recruiting SCF COI1 , allowing CMID-containing JAZ splice variants to accumulate in the presence of JA and to re-repress MYC transcription factors as an integral part of reestablishing signal homeostasis. The mechanism by which the CMID represses MYC transcription factors remains elusive. Here we describe the crystal structure of the MYC3-CMID JAZ10 complex. In contrast to the Jas motif, which forms a single continuous helix when bound to MYC3, the CMID adopts a loop-helix-loop-helix architecture with modular interactions with both the Jas-binding groove and the backside of the Jas-interaction domain of MYC3. This clamp-like interaction allows the CMID to bind MYC3 tightly and block access of MED25 (a subunit of the Mediator coactivator complex) to the MYC3 transcriptional activation domain, shedding light on the enigmatic mechanism by which JAZ splice variants desensitize JA signaling.

A variant in the CHEK2 promoter at a methylation site relieves transcriptional repression and confers reduced risk of lung cancer.

PubMed

Zhang, Shuyu; Lu, Juan; Zhao, Xueying; Wu, Wenting; Wang, Huibo; Lu, Jun; Wu, Qihan; Chen, Xin; Fan, Weiwei; Chen, Hongyan; Wang, Feng; Hu, Zhibin; Jin, Li; Wei, Qingyi; Shen, Hongbing; Huang, Wei; Lu, Daru

2010-07-01

Checkpoint kinase (CHEK) 2, a tumor suppressor gene, plays an essential role in the DNA damage checkpoint response cascade. We first investigated two polymorphisms in the proximal promoter of the CHEK2 gene and evaluated their associations with the risk of lung cancer in a case-control study using 500 incident lung cancer cases and 517 cancer-free controls. We found that CHEK2 rs2236141 -48 G > A was significantly associated with lung cancer risk (P = 0.0018). Similar results were obtained in a follow-up replication study in 575 lung cancer patients and 589 controls (P = 0.042). Quantitative polymerase chain reaction showed that individuals with the G allele had lower levels of CHEK2 transcripts in peripheral blood mononuclear cells and normal lung tissues. The -48 G-->A variant eliminated a methylation site and thereby relieve the transcriptional repression of CHEK2. Therefore, this polymorphism affected downstream transcription through genetic and epigenetic modifications. Luciferase reporter assays demonstrated that the major G allele significantly attenuated reporter gene expression when methylated. Electrophoretic Mobility shift assays and surface plasmon resonance revealed that the methylated G allele increased transcription factor accessibility. We used in vivo chromatin immunoprecipitation to confirm that the relevant transcription factor was Sp1. Using lung tissue heterozygous for the G/A single-nucleotide polymorphism, we found that Sp1 acted as a repressor and had a stronger binding affinity for the G allele. These results support our hypothesis that the CHEK2 rs2236141 variant modifies lung cancer susceptibility in the Chinese population by affecting CHEK2 expression.

α6-Integrin alternative splicing: distinct cytoplasmic variants in stem cell fate specification and niche interaction.

PubMed

Zhou, Zijing; Qu, Jing; He, Li; Peng, Hong; Chen, Ping; Zhou, Yong

2018-05-02

α6-Integrin subunit (also known as CD49f) is a stemness signature that has been found on the plasma membrane of more than 30 stem cell populations. A growing body of studies have focused on the critical role of α6-containing integrins (α6β1 and α6β4) in the regulation of stem cell properties, lineage-specific differentiation, and niche interaction. α6-Integrin subunit can be alternatively spliced at the post-transcriptional level, giving rise to divergent isoforms which differ in the cytoplasmic and/or extracellular domains. The cytoplasmic domain of integrins is an important functional part of integrin-mediated signals. Structural changes in the cytoplasmic domain of α6 provide an efficient means for the regulation of stem cell responses to biochemical stimuli and/or biophysical cues in the stem cell niche, thus impacting stem cell fate determination. In this review, we summarize the current knowledge on the structural variants of the α6-integrin subunit and spatiotemporal expression of α6 cytoplasmic variants in embryonic and adult stem/progenitor cells. We highlight the roles of α6 cytoplasmic variants in stem cell fate decision and niche interaction, and discuss the potential mechanisms involved. Understanding of the distinct functions of α6 splicing variants in stem cell biology may inform the rational design of novel stem cell-based therapies for a range of human diseases.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array

PubMed Central

Gardina, Paul J; Clark, Tyson A; Shimada, Brian; Staples, Michelle K; Yang, Qing; Veitch, James; Schweitzer, Anthony; Awad, Tarif; Sugnet, Charles; Dee, Suzanne; Davies, Christopher; Williams, Alan; Turpaz, Yaron

2006-01-01

Background Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. Results We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. Conclusion Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility. PMID:17192196

Comprehensive analysis of the MLH1 promoter region in 480 patients with colorectal cancer and 1150 controls reveals new variants including one with a heritable constitutional MLH1 epimutation.

PubMed

Morak, Monika; Ibisler, Ayseguel; Keller, Gisela; Jessen, Ellen; Laner, Andreas; Gonzales-Fassrainer, Daniela; Locher, Melanie; Massdorf, Trisari; Nissen, Anke M; Benet-Pagès, Anna; Holinski-Feder, Elke

2018-04-01

Germline defects in MLH1 , MSH2 , MSH6 and PMS2 predisposing for Lynch syndrome (LS) are mainly based on sequence changes, whereas a constitutional epimutation of MLH1 (CEM) is exceptionally rare. This abnormal MLH1 promoter methylation is not hereditary when arising de novo, whereas a stably heritable and variant-induced CEM was described for one single allele. We searched for MLH1 promoter variants causing a germline or somatic methylation induction or transcriptional repression. We analysed the MLH1 promoter sequence in five different patient groups with colorectal cancer (CRC) (n=480) composed of patients with i) CEM (n=16), ii) unsolved loss of MLH1 expression in CRC (n=37), iii) CpG-island methylator-phenotype CRC (n=102), iv) patients with LS (n=83) and v) MLH1-proficient CRC (n=242) as controls. 1150 patients with non-LS tumours also served as controls to correctly judge the results. We detected 10 rare MLH1 promoter variants. One novel, complex MLH1 variant c.-63_-58delins18 is present in a patient with CRC with CEM and his sister, both showing a complete allele-specific promoter methylation and transcriptional silencing. The other nine promoter variants detected in 17 individuals were not associated with methylation. For four of these, a normal, biallelic MLH1 expression was found in the patients' cDNA. We report the second promoter variant stably inducing a hereditary CEM. Concerning the classification of promoter variants, we discuss contradictory results from the literature for two variants, describe classification discrepancies between existing rules for five variants, suggest the (re-)classification of five promoter variants to (likely) benign and regard four variants as functionally unclear. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Expression of ZNF804A in human brain and alterations in schizophrenia, bipolar disorder, and major depressive disorder: a novel transcript fetally regulated by the psychosis risk variant rs1344706.

PubMed

Tao, Ran; Cousijn, Helena; Jaffe, Andrew E; Burnet, Philip W J; Edwards, Freya; Eastwood, Sharon L; Shin, Joo Heon; Lane, Tracy A; Walker, Mary A; Maher, Brady J; Weinberger, Daniel R; Harrison, Paul J; Hyde, Thomas M; Kleinman, Joel E

2014-10-01

The single-nucleotide polymorphism rs1344706 in the zinc finger protein 804A gene (ZNF804A) shows genome-wide association with schizophrenia and bipolar disorder. Little is known regarding the expression of ZNF804A and the functionality of rs1344706. To characterize ZNF804A expression in human brain and to investigate how it changes across the life span and how it is affected by rs1344706, schizophrenia, bipolar disorder, and major depressive disorder. Molecular and immunochemical methods were used to study ZNF804A messenger RNA (mRNA) and ZNF804A protein, respectively. ZNF804A transcripts were investigated using next-generation sequencing and polymerase chain reaction-based methods, and ZNF804A protein was investigated using Western blots and immunohistochemistry. Samples of dorsolateral prefrontal cortex and inferior parietal lobe tissue were interrogated from 697 participants between 14 weeks' gestational age and age 85 years, including patients with schizophrenia, bipolar disorder, or major depressive disorder. Quantitative measurements of ZNF804A mRNA and immunoreactivity, and the effect of diagnosis and rs1344706 genotype. ZNF804A was expressed across the life span, with highest expression prenatally. An abundant and developmentally regulated truncated ZNF804A transcript was identified, missing exons 1 and 2 (ZNF804AE3E4) and predicted to encode a protein lacking the zinc finger domain. rs1344706 influenced expression of ZNF804AE3E4 mRNA in fetal brain (P = .02). In contrast, full-length ZNF804A showed no association with genotype (P > .05). ZNF804AE3E4 mRNA expression was decreased in patients with schizophrenia (P = .006) and increased in those with major depressive disorder (P < .001), and there was a genotype-by-diagnosis interaction in bipolar disorder (P = .002). ZNF804A immunoreactivity was detected in fetal and adult human cerebral cortex. It was localized primarily to pyramidal neurons, with cytoplasmic as well as dendritic and nuclear staining. No differences in ZNF804A-immunoreactive neurons were seen in schizophrenia or related to rs1344706 (P > .05). rs1344706 influences the expression of ZNF804AE3E4, a novel splice variant. The effect is limited to fetal brain and to this isoform. It may be part of the mechanism by which allelic variation in ZNF804A affects risk of psychosis. ZNF804A is translated in human brain, where its functions may extend beyond its predicted role as a transcription factor.

C282Y-HFE Gene Variant Affects Cholesterol Metabolism in Human Neuroblastoma Cells

PubMed Central

Ali-Rahmani, Fatima; Huang, Michael A.; Schengrund, C.-L.; Connor, James R.; Lee, Sang Y.

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells. PMID:24533143

C282Y-HFE gene variant affects cholesterol metabolism in human neuroblastoma cells.

PubMed

Ali-Rahmani, Fatima; Huang, Michael A; Schengrund, C-L; Connor, James R; Lee, Sang Y

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions

PubMed Central

Boudreault, Simon; Martenon-Brodeur, Camille; Caron, Marie; Garant, Jean-Michel; Tremblay, Marie-Pier; Armero, Victoria E. S.; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S.; Lemay, Guy; Bisaillon, Martin

2016-01-01

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection. PMID:27598998

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions.

PubMed

Boudreault, Simon; Martenon-Brodeur, Camille; Caron, Marie; Garant, Jean-Michel; Tremblay, Marie-Pier; Armero, Victoria E S; Durand, Mathieu; Lapointe, Elvy; Thibault, Philippe; Tremblay-Létourneau, Maude; Perreault, Jean-Pierre; Scott, Michelle S; Lemay, Guy; Bisaillon, Martin

2016-01-01

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.

Natural genetic variation of the cardiac transcriptome in non-diseased donors and patients with dilated cardiomyopathy.

PubMed

Heinig, Matthias; Adriaens, Michiel E; Schafer, Sebastian; van Deutekom, Hanneke W M; Lodder, Elisabeth M; Ware, James S; Schneider, Valentin; Felkin, Leanne E; Creemers, Esther E; Meder, Benjamin; Katus, Hugo A; Rühle, Frank; Stoll, Monika; Cambien, François; Villard, Eric; Charron, Philippe; Varro, Andras; Bishopric, Nanette H; George, Alfred L; Dos Remedios, Cristobal; Moreno-Moral, Aida; Pesce, Francesco; Bauerfeind, Anja; Rüschendorf, Franz; Rintisch, Carola; Petretto, Enrico; Barton, Paul J; Cook, Stuart A; Pinto, Yigal M; Bezzina, Connie R; Hubner, Norbert

2017-09-14

Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases. Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts. RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.

The 2p21 deletion syndrome: characterization of the transcription content.

PubMed

Parvari, Ruti; Gonen, Yael; Alshafee, Ismael; Buriakovsky, Sophia; Regev, Kfir; Hershkovitz, Eli

2005-08-01

The vast majority of small-deletion syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We have previously identified a homozygous deletion of 179,311 bp on chromosome 2p21 as the cause of a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria. We now present the transcription content of this region: Multiple splicing variants of the genes protein phosphatase 1B (formerly 2C) magnesium-dependent, beta isoform (PPM1B), SLC3A1, and KIAA0436 (approved gene symbol PREPL) were identified and their patterns of expression analyzed. The spliced variants are predicted to have additional functions compared to the known variants and their patterns of expression fit the tissues affected by the syndrome. The first exon of an additional gene (C2orf34) is encoded in the deleted region and the gene is not expressed in the patients. In addition several transcripts with very short open reading frames are also encoded in the deletion. The identification of all transcripts encoded in the region deleted in the patients is the first step in the study of the genotype-phenotype correlation of the 2p21 patients.

Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant.

PubMed

Almeida, Rodrigo; Ricaño-Ponce, Isis; Kumar, Vinod; Deelen, Patrick; Szperl, Agata; Trynka, Gosia; Gutierrez-Achury, Javier; Kanterakis, Alexandros; Westra, Harm-Jan; Franke, Lude; Swertz, Morris A; Platteel, Mathieu; Bilbao, Jose Ramon; Barisani, Donatella; Greco, Luigi; Mearin, Luisa; Wolters, Victorien M; Mulder, Chris; Mazzilli, Maria Cristina; Sood, Ajit; Cukrowska, Bozena; Núñez, Concepción; Pratesi, Riccardo; Withoff, Sebo; Wijmenga, Cisca

2014-05-01

Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of ∼1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 × 10(-49)), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 × 10(-44)). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 × 10(-49)), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD.

Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome.

PubMed

Matilainen, Sanna; Carroll, Christopher J; Richter, Uwe; Euro, Liliya; Pohjanpelto, Max; Paetau, Anders; Isohanni, Pirjo; Suomalainen, Anu

2017-09-01

Leigh syndrome is a severe infantile encephalopathy with an exceptionally variable genetic background. We studied the exome of a child manifesting with Leigh syndrome at one month of age and progressing to death by the age of 2.4 years, and identified novel compound heterozygous variants in PNPT1, encoding the polynucleotide phosphorylase (PNPase). Expression of the wild type PNPT1 in the subject's myoblasts functionally complemented the defects, and the pathogenicity was further supported by structural predictions and protein and RNA analyses. PNPase is a key enzyme in mitochondrial RNA metabolism, with suggested roles in mitochondrial RNA import and degradation. The variants were predicted to locate in the PNPase active site and disturb the RNA processing activity of the enzyme. The PNPase trimer formation was not affected, but specific RNA processing intermediates derived from mitochondrial transcripts of the ND6 subunit of Complex I, as well as small mRNA fragments, accumulated in the subject's myoblasts. Mitochondrial RNA processing mediated by the degradosome consisting of hSUV3 and PNPase is poorly characterized, and controversy on the role and location of PNPase within human mitochondria exists. Our evidence indicates that PNPase activity is essential for the correct maturation of the ND6 transcripts, and likely for the efficient removal of degradation intermediates. Loss of its activity will result in combined respiratory chain deficiency, and a classic respiratory chain-deficiency-associated disease, Leigh syndrome, indicating an essential role for the enzyme for normal function of the mitochondrial respiratory chain. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Nucleosome displacement in transcription.

PubMed

Workman, Jerry L

2006-08-01

Recent reports reinforce the notion that nucleosomes are highly dynamic in response to the process of transcription. Nucleosomes are displaced at promoters during gene activation in a process that involves histone modification, ATP-dependent nucleosome remodeling complexes, histone chaperones and perhaps histone variants. During transcription elongation nucleosomes are acetylated and transferred behind RNA polymerase II where they are required to suppress spurious transcription initiation within the body of the gene. It is becoming increasingly clear that the eukaryotic transcriptional machinery is adapted to exploit the presence of nucleosomes in very sophisticated ways.

Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

PubMed

Asenjo, Ana; Villanueva, Nieves

2016-01-04

The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel splice mutation in microthalmia-associated transcription factor in Waardenburg Syndrome.

PubMed

Brenner, Laura; Burke, Kelly; Leduc, Charles A; Guha, Saurav; Guo, Jiancheng; Chung, Wendy K

2011-01-01

Waardenburg Syndrome (WS) is a syndromic form of hearing loss associated with mutations in six different genes. We identified a large family with WS that had previously undergone clinical testing, with no reported pathogenic mutation. Using linkage analysis, a region on 3p14.1 with an LOD score of 6.6 was identified. Microthalmia-Associated Transcription Factor, a gene known to cause WS, is located within this region of linkage. Sequencing of Microthalmia-Associated Transcription Factor demonstrated a c.1212 G>A synonymous variant that segregated with the WS in the family and was predicted to cause a novel splicing site that was confirmed with expression analysis of the mRNA. This case illustrates the need to computationally analyze novel synonymous sequence variants for possible effects on splicing to maximize the clinical sensitivity of sequence-based genetic testing.

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

PubMed

Lakshmikumaran, M; Negi, M S

1994-03-01

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha1A1-adrenoceptors.

PubMed

Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork; Pickering, Darryl S; Kristensen, Torsten

2008-10-31

Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant which was relevant, we used quantitative real-time polymerase chain reaction (qPCR) to determine the prevalence in human subcutaneous small arteries of three of the five splice variants ADRA1A_v1-5, which encode functional protein: alpha(1A1)-, alpha(1A3)-, alpha(1A4)-adrenoceptors. Our statistical analysis showed higher transcription levels of alpha(1A1)- than of alpha(1A3)- and alpha(1A4)-adrenoceptors (1.6 and 5.8 times, respectively). We therefore chose to study the alpha(1A1)-adrenoceptor, and the cDNA encoding it was transfected into the Flp-In-293 (modified from HEK-293) cell line to produce a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors to yield pK(B) values of 8.40, 8.05, 8.26 and 7.38, respectively. In [7-methoxy-(3)H]-prazosin binding experiments, high expression was seen (B(max)=48.5+/-16.7 pmol/mg protein, +/-S.E.M.) along with high affinity binding to a single site (K(d)=0.210+/-0.034 nM). The pharmacological profiles of recombinant human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol.

Involvement of dominant-negative spliced variants of the intermediate conductance Ca2+-activated K+ channel, K(Ca)3.1, in immune function of lymphoid cells.

PubMed

Ohya, Susumu; Niwa, Satomi; Yanagi, Ayano; Fukuyo, Yuka; Yamamura, Hisao; Imaizumi, Yuji

2011-05-13

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Cyclin D1b splice variant promotes αvβ3-mediated adhesion and invasive migration of breast cancer cells.

PubMed

Wu, Feng-Hua; Luo, Li-Qiong; Liu, Yi; Zhan, Qiu-Xiao; Luo, Chao; Luo, Jing; Zhang, Gui-Mei; Feng, Zuo-Hua

2014-12-01

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes

PubMed Central

Atilano, Shari R.; Malik, Deepika; Chwa, Marilyn; Cáceres-Del-Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin; Kenney, M. Cristina

2015-01-01

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2′-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. PMID:25964427

BISQUE: locus- and variant-specific conversion of genomic, transcriptomic and proteomic database identifiers.

PubMed

Meyer, Michael J; Geske, Philip; Yu, Haiyuan

2016-05-15

Biological sequence databases are integral to efforts to characterize and understand biological molecules and share biological data. However, when analyzing these data, scientists are often left holding disparate biological currency-molecular identifiers from different databases. For downstream applications that require converting the identifiers themselves, there are many resources available, but analyzing associated loci and variants can be cumbersome if data is not given in a form amenable to particular analyses. Here we present BISQUE, a web server and customizable command-line tool for converting molecular identifiers and their contained loci and variants between different database conventions. BISQUE uses a graph traversal algorithm to generalize the conversion process for residues in the human genome, genes, transcripts and proteins, allowing for conversion across classes of molecules and in all directions through an intuitive web interface and a URL-based web service. BISQUE is freely available via the web using any major web browser (http://bisque.yulab.org/). Source code is available in a public GitHub repository (https://github.com/hyulab/BISQUE). haiyuan.yu@cornell.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Variant Ionotropic Receptors in the Malaria Vector Mosquito Anopheles gambiae Tuned to Amines and Carboxylic Acids

PubMed Central

Pitts, R. Jason; Derryberry, Stephen L.; Zhang, Zhiwei; Zwiebel, Laurence J.

2017-01-01

The principal Afrotropical human malaria vector mosquito, Anopheles gambiae, remains a significant threat to global health. A critical component in the transmission of malaria is the ability of An. gambiae females to detect and respond to human-derived chemical kairomones in their search for blood meal hosts. The basis for host odor responses resides in olfactory receptor neurons (ORNs) that express chemoreceptors encoded by large gene families, including the odorant receptors (ORs) and the variant ionotropic receptors (IRs). While ORs have been the focus of extensive investigation, functional IR complexes and the chemical compounds that activate them have not been identified in An. gambiae. Here we report the transcriptional profiles and functional characterization of three An. gambiae IR (AgIr) complexes that specifically respond to amines or carboxylic acids - two classes of semiochemicals that have been implicated in mediating host-seeking by adult females but are not known to activate An. gambiae ORs (AgOrs). Our results suggest that AgIrs play critical roles in the detection and behavioral responses to important classes of host odors that are underrepresented in the AgOr chemical space. PMID:28067294

Whole Transcriptome Sequencing Enables Discovery and Analysis of Viruses in Archived Primary Central Nervous System Lymphomas

PubMed Central

DeBoever, Christopher; Reid, Erin G.; Smith, Erin N.; Wang, Xiaoyun; Dumaop, Wilmar; Harismendy, Olivier; Carson, Dennis; Richman, Douglas; Masliah, Eliezer; Frazer, Kelly A.

2013-01-01

Primary central nervous system lymphomas (PCNSL) have a dramatically increased prevalence among persons living with AIDS and are known to be associated with human Epstein Barr virus (EBV) infection. Previous work suggests that in some cases, co-infection with other viruses may be important for PCNSL pathogenesis. Viral transcription in tumor samples can be measured using next generation transcriptome sequencing. We demonstrate the ability of transcriptome sequencing to identify viruses, characterize viral expression, and identify viral variants by sequencing four archived AIDS-related PCNSL tissue samples and analyzing raw sequencing reads. EBV was detected in all four PCNSL samples and cytomegalovirus (CMV), JC polyomavirus (JCV), and HIV were also discovered, consistent with clinical diagnoses. CMV was found to express three long non-coding RNAs recently reported as expressed during active infection. Single nucleotide variants were observed in each of the viruses observed and three indels were found in CMV. No viruses were found in several control tumor types including 32 diffuse large B-cell lymphoma samples. This study demonstrates the ability of next generation transcriptome sequencing to accurately identify viruses, including DNA viruses, in solid human cancer tissue samples. PMID:24023918

The human RHOX gene cluster: target genes and functional analysis of gene variants in infertile men.

PubMed

Borgmann, Jennifer; Tüttelmann, Frank; Dworniczak, Bernd; Röpke, Albrecht; Song, Hye-Won; Kliesch, Sabine; Wilkinson, Miles F; Laurentino, Sandra; Gromoll, Jörg

2016-11-15

The X-linked reproductive homeobox (RHOX) gene cluster encodes transcription factors preferentially expressed in reproductive tissues. This gene cluster has important roles in male fertility based on phenotypic defects of Rhox-mutant mice and the finding that aberrant RHOX promoter methylation is strongly associated with abnormal human sperm parameters. However, little is known about the molecular mechanism of RHOX function in humans. Using gene expression profiling, we identified genes regulated by members of the human RHOX gene cluster. Some genes were uniquely regulated by RHOXF1 or RHOXF2/2B, while others were regulated by both of these transcription factors. Several of these regulated genes encode proteins involved in processes relevant to spermatogenesis; e.g. stress protection and cell survival. One of the target genes of RHOXF2/2B is RHOXF1, suggesting cross-regulation to enhance transcriptional responses. The potential role of RHOX in human infertility was addressed by sequencing all RHOX exons in a group of 250 patients with severe oligozoospermia. This revealed two mutations in RHOXF1 (c.515G > A and c.522C > T) and four in RHOXF2/2B (-73C > G, c.202G > A, c.411C > T and c.679G > A), of which only one (c.202G > A) was found in a control group of men with normal sperm concentration. Functional analysis demonstrated that c.202G > A and c.679G > A significantly impaired the ability of RHOXF2/2B to regulate downstream genes. Molecular modelling suggested that these mutations alter RHOXF2/F2B protein conformation. By combining clinical data with in vitro functional analysis, we demonstrate how the X-linked RHOX gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility.

A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease.

PubMed

Bečanović, Kristina; Nørremølle, Anne; Neal, Scott J; Kay, Chris; Collins, Jennifer A; Arenillas, David; Lilja, Tobias; Gaudenzi, Giulia; Manoharan, Shiana; Doty, Crystal N; Beck, Jessalyn; Lahiri, Nayana; Portales-Casamar, Elodie; Warby, Simon C; Connolly, Colúm; De Souza, Rebecca A G; Tabrizi, Sarah J; Hermanson, Ola; Langbehn, Douglas R; Hayden, Michael R; Wasserman, Wyeth W; Leavitt, Blair R

2015-06-01

Cis-regulatory variants that alter gene expression can modify disease expressivity, but none have previously been identified in Huntington disease (HD). Here we provide in vivo evidence in HD patients that cis-regulatory variants in the HTT promoter are bidirectional modifiers of HD age of onset. HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A) variant on the HD disease allele was associated with delayed age of onset in familial cases, whereas the presence of the rs13102260 (A) variant on the wild-type HTT allele was associated with earlier age of onset in HD patients in an extreme case-based cohort. Our findings suggest a previously unknown mechanism linking allele-specific effects of rs13102260 on HTT expression to HD age of onset and have implications for HTT silencing treatments that are currently in development.

Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype.

PubMed

Camats, Núria; Fernández-Cancio, Mónica; Audí, Laura; Mullis, Primus E; Moreno, Francisca; González Casado, Isabel; López-Siguero, Juan Pedro; Corripio, Raquel; Bermúdez de la Vega, José Antonio; Blanco, José Antonio; Flück, Christa E

2015-01-01

MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life.

Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype

PubMed Central

Audí, Laura; Mullis, Primus E.; Moreno, Francisca; González Casado, Isabel; López-Siguero, Juan Pedro; Corripio, Raquel; Bermúdez de la Vega, José Antonio; Blanco, José Antonio; Flück, Christa E.

2015-01-01

MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life. PMID:26580071

Mutation detection of E6 and LCR genes from HPV 16 associated with carcinogenesis.

PubMed

Mosmann, Jessica P; Monetti, Marina S; Frutos, Maria C; Kiguen, Ana X; Venezuela, Raul F; Cuffini, Cecilia G

2015-01-01

Human papillomavirus (HPV) is responsible for one of the most frequent sexually transmitted infections. The first phylogenetic analysis was based on a LCR region fragment. Nowadays, 4 variants are known: African (Af-1, Af-2), Asian-American (AA) and European (E). However the existence of sub-lineages of the European variant havs been proposed, specific mutations in the E6 and LCR sequences being possibly related to persistent viral infections. The aim of this study was a phylogenetic study of HPV16 sequences of endocervical samples from Cordoba, in order to detect the circulating lineages and analyze the presence of mutations that could be correlated with malignant disease. The phylogenetic analysis determined that 86% of the samples belonged to the E variant, 7% to AF-1 and the remaining 7% to AF-2. The most frequent mutation in LCR sequences was G7521A, in 80% of the analyzed samples; it affects the binding site of a transcription factor that could contribute to carcinogenesis. In the E6 sequences, the most common mutation was T350G (L83V), detected in 67% of the samples, associated with increased risk of persistent infection. The high detection rate of the European lineage correlated with patterns of human migration. This study emphasizes the importance of recognizing circulating lineages, as well as the detection of mutations associated with high-grade neoplastic lesions that could be correlated to the development of carcinogenic lesions.

Cavβ2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes.

PubMed

Moreno, Cristian; Hermosilla, Tamara; Morales, Danna; Encina, Matías; Torres-Díaz, Leandro; Díaz, Pablo; Sarmiento, Daniela; Simon, Felipe; Varela, Diego

2015-12-01

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

Histone H1 depletion triggers an interferon response in cancer cells via activation of heterochromatic repeats

PubMed Central

Izquierdo-Bouldstridge, Andrea; Bustillos, Alberto; Bonet-Costa, Carles; Aribau-Miralbés, Patricia; García-Gomis, Daniel; Dabad, Marc; Esteve-Codina, Anna; Pascual-Reguant, Laura; Peiró, Sandra; Esteller, Manel; Murtha, Matthew; Millán-Ariño, Lluís

2017-01-01

Abstract Histone H1 has seven variants in human somatic cells and contributes to chromatin compaction and transcriptional regulation. Knock-down (KD) of each H1 variant in breast cancer cells results in altered gene expression and proliferation differently in a variant specific manner with H1.2 and H1.4 KDs being most deleterious. Here we show combined depletion of H1.2 and H1.4 has a strong deleterious effect resulting in a strong interferon (IFN) response, as evidenced by an up-regulation of many IFN-stimulated genes (ISGs) not seen in individual nor in other combinations of H1 variant KDs. Although H1 participates to repress ISG promoters, IFN activation upon H1.2 and H1.4 KD is mainly generated through the activation of the IFN response by cytosolic nucleic acid receptors and IFN synthesis, and without changes in histone modifications at induced ISG promoters. H1.2 and H1.4 co-KD also promotes the appearance of accessibility sites genome wide and, particularly, at satellites and other repeats. The IFN response may be triggered by the expression of noncoding RNA generated from heterochromatic repeats or endogenous retroviruses upon H1 KD. In conclusion, redundant H1-mediated silencing of heterochromatin is important to maintain cell homeostasis and to avoid an unspecific IFN response. PMID:28977426

Genome-wide common and rare variant analysis provides novel insights into clozapine-associated neutropenia.

PubMed

Legge, S E; Hamshere, M L; Ripke, S; Pardinas, A F; Goldstein, J I; Rees, E; Richards, A L; Leonenko, G; Jorskog, L F; Chambert, K D; Collier, D A; Genovese, G; Giegling, I; Holmans, P; Jonasdottir, A; Kirov, G; McCarroll, S A; MacCabe, J H; Mantripragada, K; Moran, J L; Neale, B M; Stefansson, H; Rujescu, D; Daly, M J; Sullivan, P F; Owen, M J; O'Donovan, M C; Walters, J T R

2017-10-01

The antipsychotic clozapine is uniquely effective in the management of schizophrenia; however, its use is limited by its potential to induce agranulocytosis. The causes of this, and of its precursor neutropenia, are largely unknown, although genetic factors have an important role. We sought risk alleles for clozapine-associated neutropenia in a sample of 66 cases and 5583 clozapine-treated controls, through a genome-wide association study (GWAS), imputed human leukocyte antigen (HLA) alleles, exome array and copy-number variation (CNV) analyses. We then combined associated variants in a meta-analysis with data from the Clozapine-Induced Agranulocytosis Consortium (up to 163 cases and 7970 controls). In the largest combined sample to date, we identified a novel association with rs149104283 (odds ratio (OR)=4.32, P=1.79 × 10 -8 ), intronic to transcripts of SLCO1B3 and SLCO1B7, members of a family of hepatic transporter genes previously implicated in adverse drug reactions including simvastatin-induced myopathy and docetaxel-induced neutropenia. Exome array analysis identified gene-wide associations of uncommon non-synonymous variants within UBAP2 and STARD9. We additionally provide independent replication of a previously identified variant in HLA-DQB1 (OR=15.6, P=0.015, positive predictive value=35.1%). These results implicate biological pathways through which clozapine may act to cause this serious adverse effect.

Genome-wide common and rare variant analysis provides novel insights into clozapine-associated neutropenia

PubMed Central

Legge, S E; Hamshere, M L; Ripke, S; Pardinas, A F; Goldstein, J I; Rees, E; Richards, A L; Leonenko, G; Jorskog, L F; Goldstein, Jacqueline I; Jarskog, L Fredrik; Hilliard, Chris; Alfirevic, Ana; Duncan, Laramie; Fourches, Denis; Huang, Hailiang; Lek, Monkol; Neale, Benjamin M; Ripke, Stephan; Shianna, Kevin; Szatkiewicz, Jin P; Tropsha, Alexander; van den Oord, Edwin JCG; Cascorbi, Ingolf; Dettling, Michael; Gazit, Ephraim; Goff, Donald C; Holden, Arthur L; Kelly, Deanna L; Malhotra, Anil K; Nielsen, Jimmi; Pirmohamed, Munir; Rujescu, Dan; Werge, Thomas; Levy, Deborah L; Josiassen, Richard C; Kennedy, James L; Lieberman, Jeffrey A; Daly, Mark J; Sullivan, Patrick F; Chambert, K D; Collier, D A; Genovese, G; Giegling, I; Holmans, P; Jonasdottir, A; Kirov, G; McCarroll, S A; MacCabe, J H; Mantripragada, K; Moran, J L; Neale, B M; Stefansson, H; Rujescu, D; Daly, M J; Sullivan, P F; Owen, M J; O'Donovan, M C; Walters, J T R

2017-01-01

The antipsychotic clozapine is uniquely effective in the management of schizophrenia; however, its use is limited by its potential to induce agranulocytosis. The causes of this, and of its precursor neutropenia, are largely unknown, although genetic factors have an important role. We sought risk alleles for clozapine-associated neutropenia in a sample of 66 cases and 5583 clozapine-treated controls, through a genome-wide association study (GWAS), imputed human leukocyte antigen (HLA) alleles, exome array and copy-number variation (CNV) analyses. We then combined associated variants in a meta-analysis with data from the Clozapine-Induced Agranulocytosis Consortium (up to 163 cases and 7970 controls). In the largest combined sample to date, we identified a novel association with rs149104283 (odds ratio (OR)=4.32, P=1.79 × 10−8), intronic to transcripts of SLCO1B3 and SLCO1B7, members of a family of hepatic transporter genes previously implicated in adverse drug reactions including simvastatin-induced myopathy and docetaxel-induced neutropenia. Exome array analysis identified gene-wide associations of uncommon non-synonymous variants within UBAP2 and STARD9. We additionally provide independent replication of a previously identified variant in HLA-DQB1 (OR=15.6, P=0.015, positive predictive value=35.1%). These results implicate biological pathways through which clozapine may act to cause this serious adverse effect. PMID:27400856

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus

DOE PAGES

Muchero, Wellington; Guo, Jianjun; Difazio, Stephen P.; ...

2015-01-23

We report the identification of six genetic loci and the allelic-variants associated with Populus cell wall phenotypes determined independently using pyrolysis Molecular Beam Mass Spectrometry (pyMBMS), saccharification assay and wet chemistry in two partially overlapping populations of P. trichocarpa genotypes sampled from multiple environments in the Pacific Northwest of North America. All 6 variants co-located with a quantitative trait locus (QTL) hotspot on chromosome XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6- carbon sugars identified in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree. Genomic intervals containing an amino acid transporter, a MYB transcriptionmore » factor, an angustifolia CtBP transcription factor, a copper transport protein ATOX1-related, a Ca 2+ transporting ATPase and a protein kinase were identified within 5 QTL regions. Each interval contained single nucleotide polymorphisms (SNPs) that were significantly associated to cell-wall phenotypes, with associations exceeding the chromosome-wise Bonferroni-adjusted p-values in at least one environment. cDNA sequencing for allelic variants of 3 of the 6 genes identified polymorphisms leading to premature stop codons in the MYB transcription factor and protein kinase. On the other hand, variants of the Angustifolia CtBP transcription factor exhibited a polyglutamine (PolyQ) length polymorphism. Results from transient protoplast assays suggested that each of the polymorphisms conferred allelic differences in activation of cellulose, hemicelluloses and lignin pathway marker genes, with truncated and short PolyQ alleles exhibiting significantly reduced marker gene activation. Genes identified in this study represent novel targets for reducing cell wall recalcitrance for lignocellulosic biofuels production using plant biomass.« less

Identification and functional analysis of glycemic trait loci in the China Health and Nutrition Survey

PubMed Central

Wu, Ying; Zou, Meng; Raulerson, Chelsea K.; Jackson, Kayla; Yuan, Wentao; Wang, Haifeng; Shou, Weihua; Wang, Ying; Luo, Jingchun; Lange, Leslie A.; Lange, Ethan M.; Gordon-Larsen, Penny; Du, Shufa; Huang, Wei; Mohlke, Karen L.

2018-01-01

To identify genetic contributions to type 2 diabetes (T2D) and related glycemic traits (fasting glucose, fasting insulin, and HbA1c), we conducted genome-wide association analyses (GWAS) in up to 7,178 Chinese subjects from nine provinces in the China Health and Nutrition Survey (CHNS). We examined patterns of population structure within CHNS and found that allele frequencies differed across provinces, consistent with genetic drift and population substructure. We further validated 32 previously described T2D- and glycemic trait-loci, including G6PC2 and SIX3-SIX2 associated with fasting glucose. At G6PC2, we replicated a known fasting glucose-associated variant (rs34177044) and identified a second signal (rs2232326), a low-frequency (4%), probably damaging missense variant (S324P). A variant within the lead fasting glucose-associated signal at SIX3-SIX2 co-localized with pancreatic islet expression quantitative trait loci (eQTL) for SIX3, SIX2, and three noncoding transcripts. To identify variants functionally responsible for the fasting glucose association at SIX3-SIX2, we tested five candidate variants for allelic differences in regulatory function. The rs12712928-C allele, associated with higher fasting glucose and lower transcript expression level, showed lower transcriptional activity in reporter assays and increased binding to GABP compared to the rs12712928-G, suggesting that rs12712928-C contributes to elevated fasting glucose levels by disrupting an islet enhancer, resulting in reduced gene expression. Taken together, these analyses identified multiple loci associated with glycemic traits across China, and suggest a regulatory mechanism at the SIX3-SIX2 fasting glucose GWAS locus. PMID:29621232

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano

2015-07-15

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colonmore » cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.« less

A splicing mutation in Aryl Hydrocarbon Receptor associated with retinitis pigmentosa.

PubMed

Zhou, Yu; Li, Shijin; Huang, Lulin; Yang, Yeming; Zhang, Lin; Yang, Mu; Liu, Wenjing; Ramasamy, Kim; Jiang, Zhilin; Sundaresan, Periasamy; Zhu, Xianjun; Yang, Zhenglin

2018-05-02

Retinitis pigmentosa (RP) refers to a group of retinal degenerative diseases, which often lead to vision loss. Although 70 genes have been identified in RP patients, the genetic cause of approximately 30% of RP cases remains unknown. We aimed to identify the cause of the disease in a cohort of RP families by whole exome sequencing. A rare homozygous splicing variant, c.1160 + 1G>A, which introduced skipping of exon 9 of the aryl hydrocarbon receptor (AHR), was identified in family RD-134. This variant is very rare in several exome databases and leads to skipping of exon 9 in the transcript. AHR is expressed in the human retina and is a ligand-activated transcription factor with multiple functions. Mutant AHR failed to promote 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced Xenobiotic Responsive Element (XRE) luciferase activity. In parallel, mutation in AHR abolished activation of its downstream target gene, such as CYP1A1 and CYP1A2. To investigate the in vivo roles of Ahr in the retina, we generated a retina-specific conditional knockout mouse model of Ahr. Comparing with wildtype mouse, Ahr knockout mice exhibited reduced electroretinogram responses at 9 months of age. Retinal histology revealed Retinal histology showed the degeneration of photoreceptors with a thinner outer nuclear layer. Thus, our data demonstrate that AHR is associated with RP.

Putative bovine topological association domains and CTCF binding motifs can reduce the search space for causative regulatory variants of complex traits.

PubMed

Wang, Min; Hancock, Timothy P; Chamberlain, Amanda J; Vander Jagt, Christy J; Pryce, Jennie E; Cocks, Benjamin G; Goddard, Mike E; Hayes, Benjamin J

2018-05-24

Topological association domains (TADs) are chromosomal domains characterised by frequent internal DNA-DNA interactions. The transcription factor CTCF binds to conserved DNA sequence patterns called CTCF binding motifs to either prohibit or facilitate chromosomal interactions. TADs and CTCF binding motifs control gene expression, but they are not yet well defined in the bovine genome. In this paper, we sought to improve the annotation of bovine TADs and CTCF binding motifs, and assess whether the new annotation can reduce the search space for cis-regulatory variants. We used genomic synteny to map TADs and CTCF binding motifs from humans, mice, dogs and macaques to the bovine genome. We found that our mapped TADs exhibited the same hallmark properties of those sourced from experimental data, such as housekeeping genes, transfer RNA genes, CTCF binding motifs, short interspersed elements, H3K4me3 and H3K27ac. We showed that runs of genes with the same pattern of allele-specific expression (ASE) (either favouring paternal or maternal allele) were often located in the same TAD or between the same conserved CTCF binding motifs. Analyses of variance showed that when averaged across all bovine tissues tested, TADs explained 14% of ASE variation (standard deviation, SD: 0.056), while CTCF explained 27% (SD: 0.078). Furthermore, we showed that the quantitative trait loci (QTLs) associated with gene expression variation (eQTLs) or ASE variation (aseQTLs), which were identified from mRNA transcripts from 141 lactating cows' white blood and milk cells, were highly enriched at putative bovine CTCF binding motifs. The linearly-furthermost, and most-significant aseQTL and eQTL for each genic target were located within the same TAD as the gene more often than expected (Chi-Squared test P-value < 0.001). Our results suggest that genomic synteny can be used to functionally annotate conserved transcriptional components, and provides a tool to reduce the search space for causative regulatory variants in the bovine genome.

Functional Impact and Evolution of a Novel Human Polymorphic Inversion That Disrupts a Gene and Creates a Fusion Transcript

PubMed Central

Puig, Marta; Castellano, David; Pantano, Lorena; Giner-Delgado, Carla; Izquierdo, David; Gayà-Vidal, Magdalena; Lucas-Lledó, José Ignacio; Esko, Tõnu; Terao, Chikashi; Matsuda, Fumihiko; Cáceres, Mario

2015-01-01

Despite many years of study into inversions, very little is known about their functional consequences, especially in humans. A common hypothesis is that the selective value of inversions stems in part from their effects on nearby genes, although evidence of this in natural populations is almost nonexistent. Here we present a global analysis of a new 415-kb polymorphic inversion that is among the longest ones found in humans and is the first with clear position effects. This inversion is located in chromosome 19 and has been generated by non-homologous end joining between blocks of transposable elements with low identity. PCR genotyping in 541 individuals from eight different human populations allowed the detection of tag SNPs and inversion genotyping in multiple populations worldwide, showing that the inverted allele is mainly found in East Asia with an average frequency of 4.7%. Interestingly, one of the breakpoints disrupts the transcription factor gene ZNF257, causing a significant reduction in the total expression level of this gene in lymphoblastoid cell lines. RNA-Seq analysis of the effects of this expression change in standard homozygotes and inversion heterozygotes revealed distinct expression patterns that were validated by quantitative RT-PCR. Moreover, we have found a new fusion transcript that is generated exclusively from inverted chromosomes around one of the breakpoints. Finally, by the analysis of the associated nucleotide variation, we have estimated that the inversion was generated ~40,000–50,000 years ago and, while a neutral evolution cannot be ruled out, its current frequencies are more consistent with those expected for a deleterious variant, although no significant association with phenotypic traits has been found so far. PMID:26427027

Functional Impact and Evolution of a Novel Human Polymorphic Inversion That Disrupts a Gene and Creates a Fusion Transcript.

PubMed

Puig, Marta; Castellano, David; Pantano, Lorena; Giner-Delgado, Carla; Izquierdo, David; Gayà-Vidal, Magdalena; Lucas-Lledó, José Ignacio; Esko, Tõnu; Terao, Chikashi; Matsuda, Fumihiko; Cáceres, Mario

2015-10-01

Despite many years of study into inversions, very little is known about their functional consequences, especially in humans. A common hypothesis is that the selective value of inversions stems in part from their effects on nearby genes, although evidence of this in natural populations is almost nonexistent. Here we present a global analysis of a new 415-kb polymorphic inversion that is among the longest ones found in humans and is the first with clear position effects. This inversion is located in chromosome 19 and has been generated by non-homologous end joining between blocks of transposable elements with low identity. PCR genotyping in 541 individuals from eight different human populations allowed the detection of tag SNPs and inversion genotyping in multiple populations worldwide, showing that the inverted allele is mainly found in East Asia with an average frequency of 4.7%. Interestingly, one of the breakpoints disrupts the transcription factor gene ZNF257, causing a significant reduction in the total expression level of this gene in lymphoblastoid cell lines. RNA-Seq analysis of the effects of this expression change in standard homozygotes and inversion heterozygotes revealed distinct expression patterns that were validated by quantitative RT-PCR. Moreover, we have found a new fusion transcript that is generated exclusively from inverted chromosomes around one of the breakpoints. Finally, by the analysis of the associated nucleotide variation, we have estimated that the inversion was generated ~40,000-50,000 years ago and, while a neutral evolution cannot be ruled out, its current frequencies are more consistent with those expected for a deleterious variant, although no significant association with phenotypic traits has been found so far.

Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos).

PubMed

Wang, Cui; Li, Shi-Jun; Yu, Wen-Hua; Xin, Qing-Wu; Li, Chuang; Feng, Yan-Ping; Peng, Xiu-Li; Gong, Yan-Zhang

2011-08-05

The very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce. Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package. In duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05). Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks.

Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos)

PubMed Central

2011-01-01

Background The very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce. Methods Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package. Results In duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05). Conclusions Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks. PMID:21819592

Distinct Transcriptional Profiles and Phenotypes Exhibited by Escherichia coli O157:H7 Isolates Related to the 2006 Spinach-Associated Outbreak

PubMed Central

Kyle, Jennifer L.; Huynh, Steven; Carter, Michelle Q.; Brandl, Maria T.; Mandrell, Robert E.

2012-01-01

In 2006, a large outbreak of Escherichia coli O157:H7 was linked to the consumption of ready-to-eat bagged baby spinach in the United States. The likely sources of preharvest spinach contamination were soil and water that became contaminated via cattle or feral pigs in the proximity of the spinach fields. In this study, we compared the transcriptional profiles of 12 E. coli O157:H7 isolates that possess the same two-enzyme pulsed-field gel electrophoresis (PFGE) profile and are related temporally or geographically to the above outbreak. These E. coli O157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig. The three clinical isolates and two spinach bag isolates grown in cultures to stationary phase showed decreased expression of many σS-regulated genes, including gadA, osmE, osmY, and katE, compared with the soil, water, cow, feral pig, and the other three spinach bag isolates. The decreased expression of these σS-regulated genes was correlated with the decreased resistance of the isolates to acid stress, osmotic stress, and oxidative stress but increases in scavenging ability. We also observed that intraisolate variability was much more pronounced among the clinical and spinach isolates than among the environmental isolates. Together, the transcriptional and phenotypic differences of the spinach outbreak isolates of E. coli O157:H7 support the hypothesis that some variants within the spinach bag retained characteristics of the preharvest isolates, whereas other variants with altered gene expression and phenotypes infected the human host. PMID:22081562

The nuclear proteome of Trypanosoma brucei

PubMed Central

Goos, Carina; Dejung, Mario; Janzen, Christian J.; Butter, Falk

2017-01-01

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. PMID:28727848

GATA4 Variants in Individuals With a 46,XY Disorder of Sex Development (DSD) May or May Not Be Associated With Cardiac Defects Depending on Second Hits in Other DSD Genes.

PubMed

Martinez de LaPiscina, Idoia; de Mingo, Carmen; Riedl, Stefan; Rodriguez, Amaia; Pandey, Amit V; Fernández-Cancio, Mónica; Camats, Nuria; Sinclair, Andrew; Castaño, Luis; Audi, Laura; Flück, Christa E

2018-01-01

Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms.

GATA4 Variants in Individuals With a 46,XY Disorder of Sex Development (DSD) May or May Not Be Associated With Cardiac Defects Depending on Second Hits in Other DSD Genes

PubMed Central

Martinez de LaPiscina, Idoia; de Mingo, Carmen; Riedl, Stefan; Rodriguez, Amaia; Pandey, Amit V.; Fernández-Cancio, Mónica; Camats, Nuria; Sinclair, Andrew; Castaño, Luis; Audi, Laura; Flück, Christa E.

2018-01-01

Disorders of sex development (DSD) consist of a wide range of conditions involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. GATA4 gene variants, mainly related to congenital heart defects (CHD), have also been recently associated with 46,XY DSD. In this study, we characterized three individuals presenting with 46,XY DSD with or without CHD and GATA4 variants in order to understand the phenotypical variability. We studied one patient presenting CHD and 46,XY gonadal dysgenesis, and two patients with a history of genetically unsolved 46,XY DSD, also known as male primary hypogonadism. Mutation analysis was carried out by candidate gene approach or targeted gene panel sequencing. Functional activity of GATA4 variants was tested in vitro on the CYP17 promoter involved in sex development using JEG3 cells. We found two novel and one previously described GATA4 variants located in the N-terminal zinc finger domain of the protein. Cys238Arg variant lost transcriptional activity on the CYP17 promoter reporter, while Trp228Cys and Pro226Leu behaved similar to wild type. These results were in line with bioinformatics simulation studies. Additional DSD variations, in the LRP4 and LHCGR genes, respectively, were identified in the two 46,XY individuals without CHD. Overall, our study shows that human GATA4 mutations identified in patients with 46,XY DSD may or may not be associated with CHD. Possible explanations for phenotypical variability may comprise incomplete penetrance, variable sensitivity of partner genes, and oligogenic mechanisms. PMID:29670578

Genome-wide association study of red blood cell traits in Hispanics/Latinos: The Hispanic Community Health Study/Study of Latinos

PubMed Central

Morrison, Jean V.; Brown, Lisa; Schurmann, Claudia; Chen, Diane D.; Liu, Yong Mei; Auer, Paul L.; Taylor, Kent D.; Papanicolaou, George; Kurita, Ryo; Nakamura, Yukio; Loos, Ruth J. F.; North, Kari E.; Thornton, Timothy A.; Pankratz, Nathan; Bauer, Daniel E.

2017-01-01

Prior GWAS have identified loci associated with red blood cell (RBC) traits in populations of European, African, and Asian ancestry. These studies have not included individuals with an Amerindian ancestral background, such as Hispanics/Latinos, nor evaluated the full spectrum of genomic variation beyond single nucleotide variants. Using a custom genotyping array enriched for Amerindian ancestral content and 1000 Genomes imputation, we performed GWAS in 12,502 participants of Hispanic Community Health Study and Study of Latinos (HCHS/SOL) for hematocrit, hemoglobin, RBC count, RBC distribution width (RDW), and RBC indices. Approximately 60% of previously reported RBC trait loci generalized to HCHS/SOL Hispanics/Latinos, including African ancestral alpha- and beta-globin gene variants. In addition to the known 3.8kb alpha-globin copy number variant, we identified an Amerindian ancestral association in an alpha-globin regulatory region on chromosome 16p13.3 for mean corpuscular volume and mean corpuscular hemoglobin. We also discovered and replicated three genome-wide significant variants in previously unreported loci for RDW (SLC12A2 rs17764730, PSMB5 rs941718), and hematocrit (PROX1 rs3754140). Among the proxy variants at the SLC12A2 locus we identified rs3812049, located in a bi-directional promoter between SLC12A2 (which encodes a red cell membrane ion-transport protein) and an upstream anti-sense long-noncoding RNA, LINC01184, as the likely causal variant. We further demonstrate that disruption of the regulatory element harboring rs3812049 affects transcription of SLC12A2 and LINC01184 in human erythroid progenitor cells. Together, these results reinforce the importance of genetic study of diverse ancestral populations, in particular Hispanics/Latinos. PMID:28453575

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells.

PubMed

Li, Yantao; Fu, Tuo; Liu, Tao; Guo, Huaizu; Guo, Qingcheng; Xu, Jin; Zhang, Dapeng; Qian, Weizhu; Dai, Jianxin; Li, Bohua; Guo, Yajun; Hou, Sheng; Wang, Hao

2016-07-01

Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.

Clarifying the impact of polycomb complex component disruption in human cancers.

PubMed

Yamamoto, Yukiya; Abe, Akihiro; Emi, Nobuhiko

2014-04-01

The dysregulation of proper transcriptional control is a major cause of developmental diseases and cancers. Polycomb proteins form chromatin-modifying complexes that transcriptionally silence genome regions in higher eukaryotes. The BCL6 corepressor (BCOR) complex comprises ring finger protein 1B (RNF2/RING1B), polycomb group ring finger 1 (PCGF1), and lysine-specific demethylase 2B (KDM2B) and is uniquely recruited to nonmethylated CpG islands, where it removes histone H3K36me2 and induces repressive histone H2A monoubiquitylation. Germline BCOR mutations have been detected in patients with oculofaciocardiodental and Lenz microphthalmia syndromes, which are inherited conditions. Recently, several variants of BCOR and BCOR-like 1 (BCORL1) chimeric fusion transcripts were reported in human cancers, including acute promyelocytic leukemia, bone sarcoma, and hepatocellular carcinoma. In addition, massively parallel sequencing has identified inactivating somatic BCOR and BCORL1 mutations in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia, medulloblastoma, and retinoblastoma. More importantly, patients with AML and MDS with BCOR mutations exhibit poor prognosis. This perspective highlights the detection of BCOR mutations and fusion transcripts of BCOR and BCORL1 and discusses their importance for diagnosing cancer subtypes and estimating the treatment responses of patients. Furthermore, this perspective proposes the need for additional functional studies to clarify the oncogenic mechanism by which BCOR and BCORL1 are disrupted in cancers, and how this may lead to the development of novel therapeutics. Mol Cancer Res; 12(4); 479-84. ©2014 AACR.

Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Taylor J.; Wozniak, Ryan J.; Arizona Cancer Center, University of Arizona, Tucson, AZ 85724

2009-02-15

Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modificationsmore » and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.« less

Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein.

PubMed Central

Kolpakova, E; Frengen, E; Stokke, T; Olsnes, S

2000-01-01

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region. PMID:11104667

Of mice and men: molecular genetics of congenital heart disease.

PubMed

Andersen, Troels Askhøj; Troelsen, Karin de Linde Lind; Larsen, Lars Allan

2014-04-01

Congenital heart disease (CHD) affects nearly 1 % of the population. It is a complex disease, which may be caused by multiple genetic and environmental factors. Studies in human genetics have led to the identification of more than 50 human genes, involved in isolated CHD or genetic syndromes, where CHD is part of the phenotype. Furthermore, mapping of genomic copy number variants and exome sequencing of CHD patients have led to the identification of a large number of candidate disease genes. Experiments in animal models, particularly in mice, have been used to verify human disease genes and to gain further insight into the molecular pathology behind CHD. The picture emerging from these studies suggest that genetic lesions associated with CHD affect a broad range of cellular signaling components, from ligands and receptors, across down-stream effector molecules to transcription factors and co-factors, including chromatin modifiers.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

PubMed Central

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

2015-01-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei.

PubMed

Zhao, Shan; Lu, Xin; Zhang, Yueling; Zhao, Xianliang; Zhong, Mingqi; Li, Shengkang; Lun, Jingsheng

2013-01-01

Recent evidences suggest that invertebrates express families of immune molecules with high levels of sequence diversity. Hemocyanin is an important non-specific immune molecule present in the hemolymph of both mollusks and arthropods. In the present study, we characterized a novel alternative splicing variant of hemocyanin (cHE1) from Litopenaeus vannamei that produced mRNA transcript of 2579 bp in length. The isoform contained two additional sequences of 296 and 267 bp in the 5'- and 3'-terminus respectively, in comparison to that of wild type hemocyanin (cHE). Sequence of cHE1 shows 100% identity to that of hemocyanin genomic DNA (HE, which does not form an open reading frame), suggesting that cHE1 might be an alternative splicing variant due to intron retention. Moreover, cHE1 could be detected by RT-PCR from five tissues (heart, gill, stomach, intestine and brain), and from shrimps at stages from nauplius to mysis larva. Further, cHE1 mRNA transcripts were significantly increased in hearts after 12h of infection with Vibrio parahemolyticus or poly I: C, while no significant difference in the transcript levels of hepatopancreas cHE was detected in the pathogen-treated shrimps during the period. In summary, these studies suggested a novel splicing variant of hemocyanin in shrimp, which might be involved in shrimp resistance to pathogenic infection. Copyright © 2013 Elsevier B.V. All rights reserved.

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

PubMed

Pfurr, Sabrina; Chu, Yu-Hsuan; Bohrer, Christian; Greulich, Franziska; Beattie, Robert; Mammadzada, Könül; Hils, Miriam; Arnold, Sebastian J; Taylor, Verdon; Schachtrup, Kristina; Uhlenhaut, N Henriette; Schachtrup, Christian

2017-11-01

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2). © 2017. Published by The Company of Biologists Ltd.

Capturing the biological impact of CDKN2A and MC1R genes as an early predisposing event in melanoma and non melanoma skin cancer

PubMed Central

Puig-Butille, Joan Anton; Escámez, María José; Garcia-Garcia, Francisco; Tell-Marti, Gemma; Fabra, Àngels; Martínez-Santamaría, Lucía; Badenas, Celia; Aguilera, Paula; Pevida, Marta; Dopazo, Joaquín; del Río, Marcela; Puig, Susana

2014-01-01

Germline mutations in CDKN2A and/or red hair color variants in MC1R genes are associated with an increased susceptibility to develop cutaneous melanoma or non melanoma skin cancer. We studied the impact of the CDKN2A germinal mutation p.G101W and MC1R variants on gene expression and transcription profiles associated with skin cancer. To this end we set-up primary skin cell co-cultures from siblings of melanoma prone-families that were later analyzed using the expression array approach. As a result, we found that 1535 transcripts were deregulated in CDKN2A mutated cells, with over-expression of immunity-related genes (HLA-DPB1, CLEC2B, IFI44, IFI44L, IFI27, IFIT1, IFIT2, SP110 and IFNK) and down-regulation of genes playing a role in the Notch signaling pathway. 3570 transcripts were deregulated in MC1R variant carriers. In particular, genes related to oxidative stress and DNA damage pathways were up-regulated as well as genes associated with neurodegenerative diseases such as Parkinson’s, Alzheimer and Huntington. Finally, we observed that the expression signatures indentified in phenotypically normal cells carrying CDKN2A mutations or MC1R variants are maintained in skin cancer tumors (melanoma and squamous cell carcinoma). These results indicate that transcriptome deregulation represents an early event critical for skin cancer development. PMID:24742402

Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

PubMed Central

Braun, Terry A.; Mullins, Robert F.; Wagner, Alex H.; Andorf, Jeaneen L.; Johnston, Rebecca M.; Bakall, Benjamin B.; Deluca, Adam P.; Fishman, Gerald A.; Lam, Byron L.; Weleber, Richard G.; Cideciyan, Artur V.; Jacobson, Samuel G.; Sheffield, Val C.; Tucker, Budd A.; Stone, Edwin M.

2013-01-01

Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing. PMID:23918662

Proteogenomic characterization of human colon and rectal cancer

PubMed Central

Zhang, Bing; Wang, Jing; Wang, Xiaojing; Zhu, Jing; Liu, Qi; Shi, Zhiao; Chambers, Matthew C.; Zimmerman, Lisa J.; Shaddox, Kent F.; Kim, Sangtae; Davies, Sherri R.; Wang, Sean; Wang, Pei; Kinsinger, Christopher R.; Rivers, Robert C.; Rodriguez, Henry; Townsend, R. Reid; Ellis, Matthew J.C.; Carr, Steven A.; Tabb, David L.; Coffey, Robert J.; Slebos, Robbert J.C.; Liebler, Daniel C.

2014-01-01

Summary We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. mRNA transcript abundance did not reliably predict protein abundance differences between tumors. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA “MSI/CIMP” transcriptomic subtype, but had distinct mutation, methylation, and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates including HNF4A, TOMM34 and SRC. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology. PMID:25043054

IRF4 haploinsufficiency in a family with Whipple’s disease

PubMed Central

Guérin, Antoine; Kerner, Gaspard; Marr, Nico; Markle, Janet G; Fenollar, Florence; Wong, Natalie; Boughorbel, Sabri; Avery, Danielle T; Ma, Cindy S; Bougarn, Salim; Bouaziz, Matthieu; Béziat, Vivien; Della Mina, Erika; Oleaga-Quintas, Carmen; Lazarov, Tomi; Worley, Lisa; Nguyen, Tina; Patin, Etienne; Deswarte, Caroline; Martinez-Barricarte, Rubén; Boucherit, Soraya; Ayral, Xavier; Edouard, Sophie; Boisson-Dupuis, Stéphanie; Rattina, Vimel; Bigio, Benedetta; Vogt, Guillaume; Geissmann, Frédéric; Quintana-Murci, Lluis; Chaussabel, Damien; Tangye, Stuart G; Raoult, Didier; Abel, Laurent; Bustamante, Jacinta

2018-01-01

Most humans are exposed to Tropheryma whipplei (Tw). Whipple’s disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance. PMID:29537367

Intestinal DMBT1 Expression Is Modulated by Crohn’s Disease-Associated IL23R Variants and by a DMBT1 Variant Which Influences Binding of the Transcription Factors CREB1 and ATF-2

PubMed Central

Le Bras, Emmanuelle; Zimmermann, Eva; Olszak, Torsten; Bedynek, Andrea; Göke, Burkhard; Franke, Andre

2013-01-01

Objectives DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression. Methods Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn’s disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays. Results IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0×10−7, OR 1.42; 95% CI 1.24–1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1×10−18). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele. Conclusion We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis. PMID:24223725

Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

PubMed

Diegelmann, Julia; Czamara, Darina; Le Bras, Emmanuelle; Zimmermann, Eva; Olszak, Torsten; Bedynek, Andrea; Göke, Burkhard; Franke, Andre; Glas, Jürgen; Brand, Stephan

2013-01-01

DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression. Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays. IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele. We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

A genetic variant in the LDLR promoter is responsible for part of the LDL-cholesterol variability in primary hypercholesterolemia

PubMed Central

2014-01-01

Background GWAS have consistently revealed that LDLR locus variability influences LDL-cholesterol in general population. Severe LDLR mutations are responsible for familial hypercholesterolemia (FH). However, most primary hypercholesterolemias are polygenic diseases. Although Cis-regulatory regions might be the cause of LDL-cholesterol variability; an extensive analysis of the LDLR distal promoter has not yet been performed. We hypothesized that genetic variants in this region are responsible for the LDLR association with LDL-cholesterol found in GWAS. Methods Four-hundred seventy-seven unrelated subjects with polygenic hypercholesterolemia (PH) and without causative FH-mutations and 525 normolipemic subjects were selected. A 3103 pb from LDLR (-625 to +2468) was sequenced in 125 subjects with PH. All subjects were genotyped for 4 SNPs (rs17242346, rs17242739, rs17248720 and rs17249120) predicted to be potentially involved in transcription regulation by in silico analysis. EMSA and luciferase assays were carried out for the rs17248720 variant. Multivariable linear regression analysis using LDL-cholesterol levels as the dependent variable were done in order to find out the variables that were independently associated with LDL-cholesterol. Results The sequencing of the 125 PH subjects did not show variants with minor allele frequency ≥ 10%. The T-allele from g.3131C > T (rs17248720) had frequencies of 9% (PH) and 16.4% (normolipemic), p < 0.00001. Studies of this variant with EMSA and luciferase assays showed a higher affinity for transcription factors and an increase of 2.5 times in LDLR transcriptional activity (T-allele vs C-allele). At multivariate analysis, this polymorphism with the lipoprotein(a) and age explained ≈ 10% of LDL-cholesterol variability. Conclusion Our results suggest that the T-allele at the g.3131 T > C SNP is associated with LDL-cholesterol levels, and explains part of the LDL-cholesterol variability. As a plausible cause, the T-allele produces an increase in LDLR transcriptional activity and lower LDL-cholesterol levels. PMID:24708769

Identification and Functional Characterization of G6PC2 Coding Variants Influencing Glycemic Traits Define an Effector Transcript at the G6PC2-ABCB11 Locus

PubMed Central

Mahajan, Anubha; Sim, Xueling; Ng, Hui Jin; Manning, Alisa; Rivas, Manuel A.; Highland, Heather M.; Locke, Adam E.; Grarup, Niels; Im, Hae Kyung; Cingolani, Pablo; Flannick, Jason; Fontanillas, Pierre; Fuchsberger, Christian; Gaulton, Kyle J.; Teslovich, Tanya M.; Rayner, N. William; Robertson, Neil R.; Beer, Nicola L.; Rundle, Jana K.; Bork-Jensen, Jette; Ladenvall, Claes; Blancher, Christine; Buck, David; Buck, Gemma; Burtt, Noël P.; Gabriel, Stacey; Gjesing, Anette P.; Groves, Christopher J.; Hollensted, Mette; Huyghe, Jeroen R.; Jackson, Anne U.; Jun, Goo; Justesen, Johanne Marie; Mangino, Massimo; Murphy, Jacquelyn; Neville, Matt; Onofrio, Robert; Small, Kerrin S.; Stringham, Heather M.; Syvänen, Ann-Christine; Trakalo, Joseph; Abecasis, Goncalo; Bell, Graeme I.; Blangero, John; Cox, Nancy J.; Duggirala, Ravindranath; Hanis, Craig L.; Seielstad, Mark; Wilson, James G.; Christensen, Cramer; Brandslund, Ivan; Rauramaa, Rainer; Surdulescu, Gabriela L.; Doney, Alex S. F.; Lannfelt, Lars; Linneberg, Allan; Isomaa, Bo; Tuomi, Tiinamaija; Jørgensen, Marit E.; Jørgensen, Torben; Kuusisto, Johanna; Uusitupa, Matti; Salomaa, Veikko; Spector, Timothy D.; Morris, Andrew D.; Palmer, Colin N. A.; Collins, Francis S.; Mohlke, Karen L.; Bergman, Richard N.; Ingelsson, Erik; Lind, Lars; Tuomilehto, Jaakko; Hansen, Torben; Watanabe, Richard M.; Prokopenko, Inga; Dupuis, Josee; Karpe, Fredrik; Groop, Leif; Laakso, Markku; Pedersen, Oluf; Florez, Jose C.; Morris, Andrew P.; Altshuler, David; Meigs, James B.; Boehnke, Michael; McCarthy, Mark I.; Lindgren, Cecilia M.; Gloyn, Anna L.

2015-01-01

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights. PMID:25625282

TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC.

PubMed

Robesova, Blanka; Bajerova, Monika; Liskova, Kvetoslava; Skrickova, Jana; Tomiskova, Marcela; Pospisilova, Sarka; Mayer, Jiri; Dvorakova, Dana

2014-07-01

Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus.

PubMed

Mahajan, Anubha; Sim, Xueling; Ng, Hui Jin; Manning, Alisa; Rivas, Manuel A; Highland, Heather M; Locke, Adam E; Grarup, Niels; Im, Hae Kyung; Cingolani, Pablo; Flannick, Jason; Fontanillas, Pierre; Fuchsberger, Christian; Gaulton, Kyle J; Teslovich, Tanya M; Rayner, N William; Robertson, Neil R; Beer, Nicola L; Rundle, Jana K; Bork-Jensen, Jette; Ladenvall, Claes; Blancher, Christine; Buck, David; Buck, Gemma; Burtt, Noël P; Gabriel, Stacey; Gjesing, Anette P; Groves, Christopher J; Hollensted, Mette; Huyghe, Jeroen R; Jackson, Anne U; Jun, Goo; Justesen, Johanne Marie; Mangino, Massimo; Murphy, Jacquelyn; Neville, Matt; Onofrio, Robert; Small, Kerrin S; Stringham, Heather M; Syvänen, Ann-Christine; Trakalo, Joseph; Abecasis, Goncalo; Bell, Graeme I; Blangero, John; Cox, Nancy J; Duggirala, Ravindranath; Hanis, Craig L; Seielstad, Mark; Wilson, James G; Christensen, Cramer; Brandslund, Ivan; Rauramaa, Rainer; Surdulescu, Gabriela L; Doney, Alex S F; Lannfelt, Lars; Linneberg, Allan; Isomaa, Bo; Tuomi, Tiinamaija; Jørgensen, Marit E; Jørgensen, Torben; Kuusisto, Johanna; Uusitupa, Matti; Salomaa, Veikko; Spector, Timothy D; Morris, Andrew D; Palmer, Colin N A; Collins, Francis S; Mohlke, Karen L; Bergman, Richard N; Ingelsson, Erik; Lind, Lars; Tuomilehto, Jaakko; Hansen, Torben; Watanabe, Richard M; Prokopenko, Inga; Dupuis, Josee; Karpe, Fredrik; Groop, Leif; Laakso, Markku; Pedersen, Oluf; Florez, Jose C; Morris, Andrew P; Altshuler, David; Meigs, James B; Boehnke, Michael; McCarthy, Mark I; Lindgren, Cecilia M; Gloyn, Anna L

2015-01-01

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights.

Classification of BRCA1 missense variants of unknown clinical significance

PubMed Central

Phelan, C; Dapic, V; Tice, B; Favis, R; Kwan, E; Barany, F; Manoukian, S; Radice, P; van der Luijt, R B; van Nesselrooij, B P M; Chenevix-Trench, G; kConFab; Caldes, T; de La Hoya, M; Lindquist, S; Tavtigian, S; Goldgar, D; Borg, A; Narod, S; Monteiro, A

2005-01-01

Background: BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast–ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk. Objective: To investigate a panel of missense variants. Methods and results: The panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396–1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated. Conclusions: While individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability. PMID:15689452

Three novel variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro.

PubMed

Zong, Yanan; Liu, Ning; Ma, Shanshan; Bai, Ying; Guan, Fangxia; Kong, Xiangdong

2018-08-20

Phenylketonuria (PKU) is the most common inherited metabolic disease, an autosomal recessive disorder affecting >10,000 newborns each year globally. It can be caused by over 1000 different naturally occurring mutations in the phenylalanine hydroxylase (PAH) gene. We analyzed three novel naturally occurring PAH gene variants: p.Glu178Lys (c.532G>A), p.Val245Met (c.733G>A) and p.Ser250Phe (c.749C>T). The mutant effect on the PAH enzyme structure and function was predicted by bioinformatics software. Vectors expressing the corresponding PAH variants were generated for expression in E. coli and in HEK293T cells. The RNA expression of the three PAH variants was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The mutant PAH protein levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA). All three variants were predicted to be pathogenic by bioinformatics analysis. The transcription of the three PAH variants was similar to the wild type PAH gene in HEK293T cells. In contrast, the levels of mutant PAH proteins decreased significantly compared to the wild type control, in both E. coli and HEK293T cells. Our results indicate that the three novel PAH gene variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) impair PAH protein expression and function in prokaryotic and eukaryotic cells. Copyright © 2018. Published by Elsevier B.V.

The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

PubMed

Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

2017-10-30

Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

Exploring the sequence-function relationship in transcriptional regulation by the lac O1 operator.

PubMed

Maity, Tuhin S; Jha, Ramesh K; Strauss, Charlie E M; Dunbar, John

2012-07-01

Understanding how binding of a transcription factor to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternative binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O(1) operator variants. The 114 variants examined contained a mean of 2.9 (range 0-4) mutations at positions -4, -2, +2 and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying expression of a GFP reporter gene and Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed variations in the GFP fluorescent signal among the operator variants of more than an order of magnitude under both uninduced and induced conditions. We found that a single nucleotide change may result in changes of up to six- and 12-fold in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that nucleotide G at position -4 is strongly correlated with strong repression. By Rosetta modeling, we found a significant correlation between the calculated binding energy and the experimentally observed transcriptional repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein-DNA interactions. © 2012 The Authors Journal compilation © 2012 FEBS.

Genetic Predictors of Interindividual Variability in Hepatic CYP3A4 ExpressionS⃞

PubMed Central

Lamba, Vishal; Panetta, John C.; Strom, Stephen

2010-01-01

Variability in hepatic CYP3A4 cannot be explained by common CYP3A4 coding variants. We previously identified polymorphisms in pregnane X receptor (PXR) and ATP-binding cassette subfamily B member 1 (ABCB1) associated with CYP3A4 mRNA levels in small cohorts of human livers. However, the relative contributions of these genetic variations or of polymorphisms in other CYP3A4 regulators to variable CYP3A4 expression were not known. We phenotyped livers from white donors (n = 128) by quantitative real-time polymerase chain reaction for expression of CYP3A4, CYP3A5, and CYP3A7 and nine transcriptional regulators, coactivators, and corepressors. We resequenced hepatic nuclear factor-3-β (HNF3β, FoxA2), HNF4α, HNF3γ (FoxA3), nuclear receptor corepressor 2 (NCoR2), and regions of the CYP3A4 promoter and genotyped informative single-nucleotide polymorphisms in PXR and ABCB1 in the same livers. CYP3A4 mRNA was positively correlated with PXR and FoxA2 and negatively correlated with NCoR2 mRNA. A common silent polymorphism and a polymorphic trinucleotide (CCT) repeat in FoxA2 were associated with CYP3A4 expression. The transcriptional activity of the FoxA2 polymorphic CCT repeat alleles (wild-type, n = 14 and variant, n = 13, 15, and 19) when assayed by luciferase reporter transactivation assays was greatest for the wild-type repeat, with deviations from this number having decreased transcriptional activity. This corresponded with higher expression of FoxA2 mRNA and its targets PXR and CYP3A4 in human livers with (CCT) n = 14 genotypes. Multiple linear regression analysis was used to quantify the contributions of selected genetic polymorphisms to variable CYP3A4 expression. This approach identified sex and polymorphisms in FoxA2, HNF4α, FoxA3, PXR, ABCB1, and the CYP3A4 promoter that together explained as much as 24.6% of the variation in hepatic CYP3A4 expression. PMID:19934400

Association between an alternative promoter polymorphism and sperm deformity rate is due to modulation of the expression of KATNAL1 transcripts in Chinese Holstein bulls.

PubMed

Zhang, X; Wang, C; Zhang, Y; Ju, Z; Qi, C; Wang, X; Huang, J; Zhang, S; Li, J; Zhong, J; Shi, F

2014-10-01

Katanin p60 subunit A-like 1 (KATNAL1) is an ATPase that regulates Sertoli cell microtubule dynamics and sperm retention. We evaluated one novel splice variant and characterized the promoter and a functional single nucleotide polymorphism (SNP) of the bovine KATNAL1 gene to explore its expression pattern, possible regulatory mechanism and relationship with semen traits in Chinese Holstein bulls. A novel splice variant, KATNAL1 transcript variant 2 (KATNAL1-TV2) of the retained 68 bp in intron 2, was identified by RT-PCR and compared with KATNAL1 transcript variant 1 (KATNAL1-TV1, NM 001192918.1) in various tissues. Bioinformatics analyses predicted that KATNAL1 transcription was regulated by two promoters: P1 in KATNAL1-TV1 and P2 in KATNAL1-TV2. Results of qRT-PCR revealed that KATNAL1-TV1 had higher expression than did KATNAL1-TV2 in testes of adult bulls (P < 0.05). Promoter luciferase activity analysis suggested that the core sequences of P1 and P2 were mapped to the region of c.-575˜c.-180 and c.163-40˜c.333+59 respectively. One novel SNP (c.163-210T>C, ss836312085) located in intron 1 was found using sequence alignment. The SNP in P2 resulted in the presence of the DeltaE binding site, improving its basal promoter activity (P < 0.05); and we observed a greater sperm deformity rate in bulls with the genotype CC than in those with the genotype TT (P < 0.05), which indicated that different genotypes were associated with the bovine semen traits. Bioinformatics analysis of the KATNAL1 protein sequence predicted that the loss of the MIT domain in the KATNAL1-TV2 transcript resulted in protein dysfunction. These findings help us to understand that a functional SNP in P2 and subsequent triggering of expression diversity of KATNAL1 transcripts are likely to play an important role with regard to semen traits in bull breeding programs. © 2014 Stichting International Foundation for Animal Genetics.

Macaca specific exon creation event generates a novel ZKSCAN5 transcript.

PubMed

Kim, Young-Hyun; Choe, Se-Hee; Song, Bong-Seok; Park, Sang-Je; Kim, Myung-Jin; Park, Young-Ho; Yoon, Seung-Bin; Lee, Youngjeon; Jin, Yeung Bae; Sim, Bo-Woong; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Sun-Uk; Lee, Sang-Rae; Park, Young-Il; Huh, Jae-Won; Chang, Kyu-Tae

2016-02-15

ZKSCAN5 (also known as ZFP95) is a zinc-finger protein belonging to the Krűppel family. ZKSCAN5 contains a SCAN box and a KRAB A domain and is proposed to play a distinct role during spermatogenesis. In humans, alternatively spliced ZKSCAN5 transcripts with different 5'-untranslated regions (UTRs) have been identified. However, investigation of our Macaca UniGene Database revealed novel alternative ZKSCAN5 transcripts that arose due to an exon creation event. Therefore, in this study, we identified the full-length sequences of ZKSCAN5 and its alternative transcripts in Macaca spp. Additionally, we investigated different nonhuman primate sequences to elucidate the molecular mechanism underlying the exon creation event. We analyzed the evolutionary features of the ZKSCAN5 transcripts by reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR, and by sequencing various nonhuman primate DNA and RNA samples. The exon-created transcript was only detected in the Macaca lineage (crab-eating monkey and rhesus monkey). Full-length sequence analysis by rapid amplification of cDNA ends (RACE) identified ten full-length transcripts and four functional isoforms of ZKSCAN5. Protein sequence analyses revealed the presence of two groups of isoforms that arose because of differences in start-codon usage. Together, our results demonstrate that there has been specific selection for a discrete set of ZKSCAN5 variants in the Macaca lineage. Furthermore, study of this locus (and perhaps others) in Macaca spp. might facilitate our understanding of the evolutionary pressures that have shaped the mechanism of exon creation in primates. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of different functional isoforms in haematopoiesis.

PubMed

Grech, Godfrey; Pollacco, Joel; Portelli, Mark; Sacco, Keith; Baldacchino, Shawn; Grixti, Justine; Saliba, Christian

2014-01-01

Haematopoiesis is a complex process regulated at various levels facilitating rapid responses to external factors including stress, modulation of lineage commitment and terminal differentiation of progenitors. Although the transcription program determines the RNA pool of a cell, various mRNA strands can be obtained from the same template, giving rise to multiple protein isoforms. The majority of variants and isoforms co-occur in normal haematopoietic cells or are differentially expressed at various maturity stages of progenitor maturation and cellular differentiation within the same lineage or across lineages. Genetic aberrations or specific cellular states result in the predominant expression of abnormal isoforms leading to deregulation and disease. The presence of upstream open reading frames (uORF) in 5' untranslated regions (UTRs) of a transcript, couples the utilization of start codons with the cellular status and availability of translation initiation factors (eIFs). In addition, tissue-specific and cell lineage-specific alternative promoter use, regulates several transcription factors producing transcript variants with variable 5' exons. In this review, we propose to give a detailed account of the differential isoform formation, causing haematological malignancies.

Analysis of predicted loss-of-function variants in UK Biobank identifies variants protective for disease.

PubMed

Emdin, Connor A; Khera, Amit V; Chaffin, Mark; Klarin, Derek; Natarajan, Pradeep; Aragam, Krishna; Haas, Mary; Bick, Alexander; Zekavat, Seyedeh M; Nomura, Akihiro; Ardissino, Diego; Wilson, James G; Schunkert, Heribert; McPherson, Ruth; Watkins, Hugh; Elosua, Roberto; Bown, Matthew J; Samani, Nilesh J; Baber, Usman; Erdmann, Jeanette; Gupta, Namrata; Danesh, John; Chasman, Daniel; Ridker, Paul; Denny, Joshua; Bastarache, Lisa; Lichtman, Judith H; D'Onofrio, Gail; Mattera, Jennifer; Spertus, John A; Sheu, Wayne H-H; Taylor, Kent D; Psaty, Bruce M; Rich, Stephen S; Post, Wendy; Rotter, Jerome I; Chen, Yii-Der Ida; Krumholz, Harlan; Saleheen, Danish; Gabriel, Stacey; Kathiresan, Sekar

2018-04-24

Less than 3% of protein-coding genetic variants are predicted to result in loss of protein function through the introduction of a stop codon, frameshift, or the disruption of an essential splice site; however, such predicted loss-of-function (pLOF) variants provide insight into effector transcript and direction of biological effect. In >400,000 UK Biobank participants, we conduct association analyses of 3759 pLOF variants with six metabolic traits, six cardiometabolic diseases, and twelve additional diseases. We identified 18 new low-frequency or rare (allele frequency < 5%) pLOF variant-phenotype associations. pLOF variants in the gene GPR151 protect against obesity and type 2 diabetes, in the gene IL33 against asthma and allergic disease, and in the gene IFIH1 against hypothyroidism. In the gene PDE3B, pLOF variants associate with elevated height, improved body fat distribution and protection from coronary artery disease. Our findings prioritize genes for which pharmacologic mimics of pLOF variants may lower risk for disease.

Interactive web-based identification and visualization of transcript shared sequences.

PubMed

Azhir, Alaleh; Merino, Louis-Henri; Nauen, David W

2018-05-12

We have developed TraC (Transcript Consensus), a web-based tool for detecting and visualizing shared sequences among two or more mRNA transcripts such as splice variants. Results including exon-exon boundaries are returned in a highly intuitive, data-rich, interactive plot that permits users to explore the similarities and differences of multiple transcript sequences. The online tool (http://labs.pathology.jhu.edu/nauen/trac/) is free to use. The source code is freely available for download (https://github.com/nauenlab/TraC). Copyright © 2018 Elsevier Inc. All rights reserved.

Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal

Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable themore » differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i.e. cerebellum versus heart for differential variation at the gene, isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the “electron transport chain” and neuronal differentiation, emphasizing that “tissue important” genes are regulated at several levels. Furthermore, our analysis shows that the “across tissue approach” has a promising potential when screening for possible explanations for variations, such as those observed at the gene expression levels.« less

RB1CC1 protein suppresses type II collagen synthesis in chondrocytes and causes dwarfism.

PubMed

Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

2011-12-23

RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations.

RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

PubMed Central

Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

2011-01-01

RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

PGC1α -1 Nucleosome Position and Splice Variant Expression and Cardiovascular Disease Risk in Overweight and Obese Individuals.

PubMed

Henagan, Tara M; Stewart, Laura K; Forney, Laura A; Sparks, Lauren M; Johannsen, Neil; Church, Timothy S

2014-01-01

PGC1α, a transcriptional coactivator, interacts with PPARs and others to regulate skeletal muscle metabolism. PGC1α undergoes splicing to produce several mRNA variants, with the NTPGC1α variant having a similar biological function to the full length PGC1α (FLPGC1α). CVD is associated with obesity and T2D and a lower percentage of type 1 oxidative fibers and impaired mitochondrial function in skeletal muscle, characteristics determined by PGC1α expression. PGC1α expression is epigenetically regulated in skeletal muscle to determine mitochondrial adaptations, and epigenetic modifications may regulate mRNA splicing. We report in this paper that skeletal muscle PGC1α  -1 nucleosome (-1N) position is associated with splice variant NTPGC1α but not FLPGC1α expression. Division of participants based on the -1N position revealed that those individuals with a -1N phased further upstream from the transcriptional start site (UP) expressed lower levels of NTPGC1α than those with the -1N more proximal to TSS (DN). UP showed an increase in body fat percentage and serum total and LDL cholesterol. These findings suggest that the -1N may be a potential epigenetic regulator of NTPGC1α splice variant expression, and -1N position and NTPGC1α variant expression in skeletal muscle are linked to CVD risk. This trial is registered with clinicaltrials.gov, identifier NCT00458133.

Modulation of transcription factor binding and epigenetic regulation of the MLH1 CpG island and shore by polymorphism rs1800734 in colorectal cancer

PubMed Central

Savio, Andrea J.; Bapat, Bharati

2017-01-01

ABSTRACT The MLH1 promoter polymorphism rs1800734 is associated with MLH1 CpG island hypermethylation and expression loss in colorectal cancer (CRC). Conversely, variant rs1800734 is associated with MLH1 shore, but not island, hypomethylation in peripheral blood mononuclear cell DNA. To explore these distinct patterns, MLH1 CpG island and shore methylation was assessed in CRC cell lines stratified by rs1800734 genotype. Cell lines containing the variant A allele demonstrated MLH1 shore hypomethylation compared to wild type (GG). There was significant enrichment of transcription factor AP4 at the MLH1 promoter in GG and GA cell lines, but not the AA cell line, by chromatin immunoprecipitation studies. Preferential binding to the G allele was confirmed by sequencing in the GA cell line. The enhancer-associated histone modification H3K4me1 was enriched at the MLH1 shore; however, H3K27ac was not, indicating the shore is an inactive enhancer. These results demonstrate the role of variant rs1800734 in altering transcription factor binding as well as epigenetics at regions beyond the MLH1 CpG island in which it is located. PMID:28304185

Modulation of transcription factor binding and epigenetic regulation of the MLH1 CpG island and shore by polymorphism rs1800734 in colorectal cancer.

PubMed

Savio, Andrea J; Bapat, Bharati

2017-06-03

The MLH1 promoter polymorphism rs1800734 is associated with MLH1 CpG island hypermethylation and expression loss in colorectal cancer (CRC). Conversely, variant rs1800734 is associated with MLH1 shore, but not island, hypomethylation in peripheral blood mononuclear cell DNA. To explore these distinct patterns, MLH1 CpG island and shore methylation was assessed in CRC cell lines stratified by rs1800734 genotype. Cell lines containing the variant A allele demonstrated MLH1 shore hypomethylation compared to wild type (GG). There was significant enrichment of transcription factor AP4 at the MLH1 promoter in GG and GA cell lines, but not the AA cell line, by chromatin immunoprecipitation studies. Preferential binding to the G allele was confirmed by sequencing in the GA cell line. The enhancer-associated histone modification H3K4me1 was enriched at the MLH1 shore; however, H3K27ac was not, indicating the shore is an inactive enhancer. These results demonstrate the role of variant rs1800734 in altering transcription factor binding as well as epigenetics at regions beyond the MLH1 CpG island in which it is located.

Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity

PubMed Central

Cork, David M.W.; Darby, Steven; Ryan-Munden, Claudia A.; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke

2017-01-01

Abstract The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway. PMID:27903893

Mechanistic insights into the link between visfatin gene C-1535T polymorphism and coronary artery disease: an in vitro study.

PubMed

Wang, Yong-Sheng; Gao, Wei; Li, Hong-Fen; Wang, Ze-Mu; Zhu, Jun; Zhao, Huan; Yan, Jian-Jun; Jia, En-Zhi; Yang, Zhi-Jian; Wang, Lian-Sheng

2012-04-01

Visfatin, a pro-inflammatory cytokine predominantly released from leucocytes, is correlated with coronary artery disease (CAD). We have previously reported that the -1535C>T polymorphism (rs1330082), which located on the promoter region of visfatin, was associated with decreased risk of CAD. Here, we investigated the underlying mechanism by which this polymorphism affects the genetic susceptibility to CAD. The difference of the promoter activities between -1535T variant and -1535C allele was tested by luciferase reporter gene assay. The difference of transcription factor binding activities between T and C allele was evaluated by electrophoretic mobility shift assay. In reporter gene assay, we showed that the T variant had a significantly reduced transcriptional activity compared with the C allele. The T-variant significantly attenuated the promoter binding affinity to nuclear transcription factors and this effect became much obvious after treatment with TNF-α. Moreover, competition experiment revealed that the retarded complex formed by T-1535- or C-1535-probe binding to nuclear extracts was nearly completely inhibited by unlabeled activator protein-1 (AP-1) specific probe, indicating that AP-1 might be the target nuclear effector. Taken together, our data provided potential mechanistic link between the visfatin -1535C>T polymorphism and reduced CAD risk.

Path from schizophrenia genomics to biology: gene regulation and perturbation in neurons derived from induced pluripotent stem cells and genome editing.

PubMed

Duan, Jubao

2015-02-01

Schizophrenia (SZ) is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies (GWASs) of SZ have identified >100 risk loci. However, the causal variants/genes and the causal mechanisms remain largely unknown, which hinders the translation of GWAS findings into disease biology and drug targets. Most risk variants are noncoding, thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling, and open chromatin is a versatile predictor of regulatory sequences. MicroRNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specific to cell type and/or developmental stage. The emerging genome-editing technology enables the creation of isogenic iPSCs and neurons to efficiently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic, glutamatergic, and GABAergic neurotransmissions. SZ GWAS findings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.

KCNQ1 p.L353L affects splicing and modifies the phenotype in a founder population with long QT syndrome type 1

PubMed Central

Kapplinger, Jamie D; Erickson, Anders; Asuri, Sirisha; Tester, David J; McIntosh, Sarah; Kerr, Charles R; Morrison, Julie; Tang, Anthony; Sanatani, Shubhayan; Arbour, Laura; Ackerman, Michael J

2017-01-01

Background Variable expressivity and incomplete penetrance between individuals with identical long QT syndrome (LQTS) causative mutations largely remain unexplained. Founder populations provide a unique opportunity to explore modifying genetic effects. We examined the role of a novel synonymous KCNQ1 p.L353L variant on the splicing of exon 8 and on heart rate corrected QT interval (QTc) in a population known to have a pathogenic LQTS type 1 (LQTS1) causative mutation, p.V205M, in KCNQ1-encoded Kv7.1. Methods 419 adults were genotyped for p.V205M, p.L353L and a previously described QTc modifier (KCNH2-p.K897T). Adjusted linear regression determined the effect of each variant on QTc, alone and in combination. In addition, peripheral blood RNA was extracted from three controls and three p.L353L-positive individuals. The mutant transcript levels were assessed via qPCR and normalised to overall KCNQ1 transcript levels to assess the effect on splicing. Results For women and men, respectively, p.L353L alone conferred a 10.0 (p=0.064) ms and 14.0 (p=0.014) ms increase in QTc and in men only a significant interaction effect in combination with the p.V205M (34.6 ms, p=0.003) resulting in a QTc of ∼500 ms. The mechanism of p.L353L's effect was attributed to approximately threefold increase in exon 8 exclusion resulting in ∼25% mutant transcripts of the total KCNQ1 transcript levels. Conclusions Our results provide the first evidence that synonymous variants outside the canonical splice sites in KCNQ1 can alter splicing and clinically impact phenotype. Through this mechanism, we identified that p.L353L can precipitate QT prolongation by itself and produce a clinically relevant interactive effect in conjunction with other LQTS variants. PMID:28264985

Guidelines for investigating causality of sequence variants in human disease

PubMed Central

MacArthur, D. G.; Manolio, T. A.; Dimmock, D. P.; Rehm, H. L.; Shendure, J.; Abecasis, G. R.; Adams, D. R.; Altman, R. B.; Antonarakis, S. E.; Ashley, E. A.; Barrett, J. C.; Biesecker, L. G.; Conrad, D. F.; Cooper, G. M.; Cox, N. J.; Daly, M. J.; Gerstein, M. B.; Goldstein, D. B.; Hirschhorn, J. N.; Leal, S. M.; Pennacchio, L. A.; Stamatoyannopoulos, J. A.; Sunyaev, S. R.; Valle, D.; Voight, B. F.; Winckler, W.; Gunter, C.

2014-01-01

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development. PMID:24759409

Guidelines for investigating causality of sequence variants in human disease.

PubMed

MacArthur, D G; Manolio, T A; Dimmock, D P; Rehm, H L; Shendure, J; Abecasis, G R; Adams, D R; Altman, R B; Antonarakis, S E; Ashley, E A; Barrett, J C; Biesecker, L G; Conrad, D F; Cooper, G M; Cox, N J; Daly, M J; Gerstein, M B; Goldstein, D B; Hirschhorn, J N; Leal, S M; Pennacchio, L A; Stamatoyannopoulos, J A; Sunyaev, S R; Valle, D; Voight, B F; Winckler, W; Gunter, C

2014-04-24

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.

Identification of Candidate Transcription Factor Binding Sites in the Cattle Genome

PubMed Central

Bickhart, Derek M.; Liu, George E.

2013-01-01

A resource that provides candidate transcription factor binding sites (TFBSs) does not currently exist for cattle. Such data is necessary, as predicted sites may serve as excellent starting locations for future omics studies to develop transcriptional regulation hypotheses. In order to generate this resource, we employed a phylogenetic footprinting approach—using sequence conservation across cattle, human and dog—and position-specific scoring matrices to identify 379,333 putative TFBSs upstream of nearly 8000 Mammalian Gene Collection (MGC) annotated genes within the cattle genome. Comparisons of our predictions to known binding site loci within the PCK1, ACTA1 and G6PC promoter regions revealed 75% sensitivity for our method of discovery. Additionally, we intersected our predictions with known cattle SNP variants in dbSNP and on the Illumina BovineHD 770k and Bos 1 SNP chips, finding 7534, 444 and 346 overlaps, respectively. Due to our stringent filtering criteria, these results represent high quality predictions of putative TFBSs within the cattle genome. All binding site predictions are freely available at http://bfgl.anri.barc.usda.gov/BovineTFBS/ or http://199.133.54.77/BovineTFBS. PMID:23433959

Involvement of Clathrin-Mediated Endocytosis in Human Immunodeficiency Virus Type 1 Entry

PubMed Central

Daecke, Jessica; Fackler, Oliver T.; Dittmar, Matthias T.; Kräusslich, Hans-Georg

2005-01-01

Productive entry of human immunodeficiency virus (HIV) is believed to occur by direct fusion at the plasma membrane. Endocytic uptake of HIV particles has been observed in several studies but is considered to be nonproductive, leading to virus degradation in the lysosome. We show here that endocytosis contributes significantly to productive HIV entry in HeLa cells by using trans dominant-negative mutants of dynamin and Eps15. Inducible expression of a dominant-negative mutant of dynamin in a CD4-positive HeLa cell line reduced HIV infection by 40 to 80%. This effect was independent of the infectious dose and was observed for three different isolates. Analysis of reverse transcription products by real-time PCR and of virus entry by delivery of a virion-associated Vpr-β-lactamase fusion protein revealed a similar reduction, indicating that the block occurred at the entry stage. A strong reduction of HIV entry was also observed upon transient transfection of a different trans dominant-negative variant of dynamin, and this reduction correlated with the relative inhibition of transferrin endocytosis. Expression of a dominant-negative variant of Eps15, which is specific for clathrin-dependent endocytosis, reduced HIV entry in HeLa cells by ca 95%, confirming the role of endocytosis for productive infection. In contrast, no effect was observed for a dominant-negative variant of caveolin. We conclude that dynamin-dependent, clathrin-mediated endocytosis can lead to productive entry of HIV in HeLa cells, suggesting this pathway as an alternative route of virus entry. PMID:15650184

Evaluation of 10 genes encoding cardiac proteins in Doberman Pinschers with dilated cardiomyopathy.

PubMed

O'Sullivan, M Lynne; O'Grady, Michael R; Pyle, W Glen; Dawson, John F

2011-07-01

To identify a causative mutation for dilated cardiomyopathy (DCM) in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes known to be associated with familial DCM in humans. 5 Doberman Pinschers with DCM and congestive heart failure and 5 control mixed-breed dogs that were euthanized or died. RNA was extracted from frozen ventricular myocardial samples from each dog, and first-strand cDNA was synthesized via reverse transcription, followed by PCR amplification with gene-specific primers. Ten cardiac genes were analyzed: cardiac actin, α-actinin, α-tropomyosin, β-myosin heavy chain, metavinculin, muscle LIM protein, myosinbinding protein C, tafazzin, titin-cap (telethonin), and troponin T. Sequences for DCM-affected and control dogs and the published canine genome were compared. None of the coding sequences yielded a common causative mutation among all Doberman Pinscher samples. However, 3 variants were identified in the α-actinin gene in the DCM-affected Doberman Pinschers. One of these variants, identified in 2 of the 5 Doberman Pinschers, resulted in an amino acid change in the rod-forming triple coiled-coil domain. Mutations in the coding regions of several genes associated with DCM in humans did not appear to consistently account for DCM in Doberman Pinschers. However, an α-actinin variant was detected in some Doberman Pinschers that may contribute to the development of DCM given its potential effect on the structure of this protein. Investigation of additional candidate gene coding and noncoding regions and further evaluation of the role of α-actinin in development of DCM in Doberman Pinschers are warranted.

Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction.

PubMed

Ronsard, Larance; Ganguli, Nilanjana; Singh, Vivek K; Mohankumar, Kumaravel; Rai, Tripti; Sridharan, Subhashree; Pajaniradje, Sankar; Kumar, Binod; Rai, Devesh; Chaudhuri, Suhnrita; Coumar, Mohane S; Ramachandran, Vishnampettai G; Banerjea, Akhil C

2017-01-01

HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans -activator of transcription (Tat), a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans -activation response (TAR) RNA. In this study, HIV-1 infected patients ( n = 120) from North India revealed Ser46Phe (20%) and Ser61Arg (2%) mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

Resequencing three candidate genes discovers seven potentially deleterious variants susceptibility to major depressive disorder and suicide attempts in Chinese.

PubMed

Rao, Shitao; Leung, Cherry She Ting; Lam, Macro Hb; Wing, Yun Kwok; Waye, Mary Miu Yee; Tsui, Stephen Kwok Wing

2017-03-01

To date almost 200 genes were found to be associated with major depressive disorder (MDD) or suicide attempts (SA), but very few genes were reported for their molecular mechanisms. This study aimed to find out whether there were common or rare variants in three candidate genes altering the risk for MDD and SA in Chinese. Three candidate genes (HOMER1, SLC6A4 and TEF) were chosen for resequencing analysis and association studies as they were reported to be involved in the etiology of MDD and SA. Following that, bioinformatics analyses were applied on those variants of interest. After resequencing analysis and alignment for the amplicons, a total of 34 common or rare variants were found in the randomly selected 36 Hong Kong Chinese patients with both MDD and SA. Among those, seven variants show potentially deleterious features. Rs60029191 and a rare variant located in regulatory region of the HOMER1 gene may affect the promoter activities through interacting with predicted transcription factors. Two missense mutations existed in the SLC6A4 coding regions were firstly reported in Hong Kong Chinese MDD and SA patients, and both of them could affect the transport efficiency of SLC6A4 to serotonin. Moreover, a common variant rs6354 located in the untranslated region of this gene may affect the expression level or exonic splicing of serotonin transporter. In addition, both of a most studied polymorphism rs738499 and a low-frequency variant in the promoter region of the TEF gene were found to be located in potential transcription factor binding sites, which may let the two variants be able to influence the promoter activities of the gene. This study elucidated the potentially molecular mechanisms of the three candidate genes altering the risk for MDD and SA. These findings implied that not only common variants but rare variants could make contributions to the genetic susceptibility to MDD and SA in Chinese. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

PubMed

Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

2015-01-01

Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

Sperm RNA elements as markers of health.

PubMed

Burl, Rayanne B; Clough, Stephanie; Sendler, Edward; Estill, Molly; Krawetz, Stephen A

2018-02-01

Idiopathic infertility, an etiology not identified as part of standard clinical assessment, represents approximately 20% of all infertility cases. Current male infertility diagnosis focuses on the concentration, motility, and morphology of spermatozoa. This is of limited value when predicting birth success and of limited utility when selecting the optimum treatment. At fertilization, spermatozoa provide their genomic contribution, as well as a set of RNAs and proteins that have distinct roles in development. The potential of spermatozoal RNAs to be used as a prognostic of live birth has been shown [Jodar et al. (2015) Science Translational Medicine 7(295):295re6]. This relied on a set of 648 sperm RNA elements derived from 285 genes that are perhaps indicative of future health status. To address this tenet, the present study correlated the levels of each transcript among all samples to assess linkage between transcript absence, birth success, and possible disease association. Correlations between transcript levels of the 285 genes were analyzed amongst themselves, and within the context of the entire transcript population for these samples. The transcripts ACE, GIGYF2, and ODF2 had many negative correlations and form the majority of correlations, suggesting an important function for these transcripts. Eleven of the 285 queried genes had disease-associated variants within a sperm RNA element. Three genes, GPX4, NDRG1, and RPS24 had SREs were absent in at least one individual from the test cohort. GPX4 and RPS24 are associated with developmental defects and/or neonatal lethality. This leaves the intriguing possibility that, while sperm RNAs delivered to the oocyte inform the success of live birth, they may also be predictors of human health. GO: Gene Ontology; ART: assisted reproductive technology; IVF: in vitro fertilization; ICSI: intra-cytoplasmic sperm injection; RNA-seq: RNA-sequencing; TIC: timed intercourse; IUI: intrauterine insemination; SRE: sperm RNA elements; HPA: Human Protein Atlas; SMDS: sedaghatian-type spondylometaphyseal dysplasia; DBA: Diamond-Blackfan anemia; RPKM: reads per kilobase per million; TPM: transcripts per million; IPA: Ingenuity Pathway Analysis; OMIM: Online Mendelian Inheritance in Man.

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

PubMed Central

Takahashi, Melissa K.; Watters, Kyle E.; Gasper, Paul M.; Abbott, Timothy R.; Carlson, Paul D.; Chen, Alan A.

2016-01-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure–function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure–function design principles for a diverse array of natural and synthetic RNA regulators. PMID:27103533

The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana.

PubMed

Wollmann, Heike; Stroud, Hume; Yelagandula, Ramesh; Tarutani, Yoshiaki; Jiang, Danhua; Jing, Li; Jamge, Bhagyshree; Takeuchi, Hidenori; Holec, Sarah; Nie, Xin; Kakutani, Tetsuji; Jacobsen, Steven E; Berger, Frédéric

2017-05-18

Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. We propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

Transcriptional expression analysis of survivin splice variants reveals differential expression of survivin-3α in breast cancer.

PubMed

Moniri Javadhesari, Solmaz; Gharechahi, Javad; Hosseinpour Feizi, Mohammad Ali; Montazeri, Vahid; Halimi, Monireh

2013-04-01

Survivin, which is a novel member of the inhibitor of apoptosis family proteins, is known to play an important role in the regulation of cell cycle and apoptosis. Differential expression of survivin in tumor tissues introduces it as a new candidate molecular marker for cancer. Here we investigated the expression of survivin and its splice variants in breast tumors, as well as normal adjacent tissues obtained from the same patients. Thirty five tumors and 17 normal adjacent tissues from women diagnosed with breast cancer were explored in this study. Differential expression of different survivin splice variants was detected and semiquantitatively analyzed using reverse transcription-polymerase chain reaction. Results showed that survivin and its splice variants were differentially expressed in tumor specimens compared with normal adjacent tissues. The expression of survivin-3B and survivin-3α was specifically detected in tumor tissues compared with normal adjacent ones (53% in tumor tissues compared to 5% in normal adjacent for survivin-3B and 65% in tumor tissues and 0.0% in normal adjacent tissues for survivin-3α). Statistical analysis showed that survivin and survivin-ΔEx3 were upregulated in benign (90%, p<0.034) and malignant (76%, p<0.042) tumors, respectively. On the other hand, our results showed that survivin-2α (100% of the cases) was the dominant expressed variant of survivin in breast cancer. The data presented here showed that survivin splice variants were differentially expressed in benign and malignant breast cancer tissues, suggesting their potential role in breast cancer development. Differential expression of survivin-2α and survivin-3α splice variants highlights their usefulness as new candidate markers for breast cancer diagnosis and prognosis.

Enhanced expression of Rubisco activase splicing variants differentially affects Rubisco activity during low temperature treatment in Lolium perenne.

PubMed

Jurczyk, Barbara; Pociecha, Ewa; Grzesiak, Maciej; Kalita, Katarzyna; Rapacz, Marcin

2016-07-01

Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold. Copyright © 2016 Elsevier GmbH. All rights reserved.

Comparative transcriptome analysis of three color variants of the sea cucumber Apostichopus japonicus.

PubMed

Jo, Jihoon; Park, Jongsun; Lee, Hyun-Gwan; Kern, Elizabeth M A; Cheon, Seongmin; Jin, Soyeong; Park, Joong-Ki; Cho, Sung-Jin; Park, Chungoo

2016-08-01

The sea cucumber Apostichopus japonicus Selenka 1867 represents an important resource in biomedical research, traditional medicine, and the seafood industry. Much of the commercial value of A. japonicus is determined by dorsal/ventral color variation (red, green, and black), yet the taxonomic relationships between these color variants are not clearly understood. We performed the first comparative analysis of de novo assembled transcriptome data from three color variants of A. japonicus. Using the Illumina platform, we sequenced nearly 177,596,774 clean reads representing a total of 18.2Gbp of sea cucumber transcriptome. A comparison of over 0.3 million transcript scaffolds against the Uniprot/Swiss-Prot database yielded 8513, 8602, and 8588 positive matches for green, red, and black body color transcriptomes, respectively. Using the Panther gene classification system, we assessed an extensive and diverse set of expressed genes in three color variants and found that (1) among the three color variants of A. japonicus, genes associated with RNA binding protein, oxidoreductase, nucleic acid binding, transferase, and KRAB box transcription factor were most commonly expressed; and (2) the main protein functional classes are differently regulated in all three color variants (extracellular matrix protein and phosphatase for green color, transporter and potassium channel for red color, and G-protein modulator and enzyme modulator for black color). This work will assist in the discovery and annotation of novel genes that play significant morphological and physiological roles in color variants of A. japonicus, and these sequence data will provide a useful set of resources for the rapidly growing sea cucumber aquaculture industry. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel polymorphism in the PAI-1 gene promoter enhances gene expression. A novel pro-thrombotic risk factor?

PubMed

Liguori, Renato; Quaranta, Sandro; Di Fiore, Rosanna; Elce, Ausilia; Castaldo, Giuseppe; Amato, Felice

2014-12-01

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue-type plasminogen activator in plasma and the most important regulator of the fibrinolytic pathway. The 4G/5G polymorphism (rs1799889) in the PAI-1 promoter is associated with altered PAI-1 transcription. We have identified a new 4G/5G allele, in which a T is inserted near the 4G tract or replaces a G in the 5G tract, forming a T plus 4G (T4G) region. This new variant was first identified in two women, one had experienced juvenile myocardial infarction, the other repeated miscarriage; both had increased PAI-1 plasma activity. In view of the important influence of this promoter region on PAI-1 protein plasma level, we performed in vitro evaluation of the effects of the T4G variant on the transcription activity of the PAI-1 gene promoter. In silico prediction analysis showed that presence of the T4G allele disrupts the E-Box region upstream of the T4G variant, altering the affinity of the target sequence for E-Box binding factors like upstream stimulatory factor-1 (USF-1). Basal T4G promoter activity was 50% higher compared to 4G and 5G variants, but it was less stimulated by USF-1 overexpression. We also analyzed the effects of IL-1β and IL-6 on the PAI-1 promoter activity of our three constructs and showed that the T4G variant was less affected by IL-1β than the other variants. These findings indicate that the T4G variant may be a novel risk factor for thrombotic events. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sox11 gene disruption causes congenital anomalies of the kidney and urinary tract (CAKUT).

PubMed

Neirijnck, Yasmine; Reginensi, Antoine; Renkema, Kirsten Y; Massa, Filippo; Kozlov, Vladimir M; Dhib, Haroun; Bongers, Ernie M H F; Feitz, Wout F; van Eerde, Albertien M; Lefebvre, Veronique; Knoers, Nine V A M; Tabatabaei, Mansoureh; Schulz, Herbert; McNeill, Helen; Schaefer, Franz; Wegner, Michael; Sock, Elisabeth; Schedl, Andreas

2018-05-01

Congenital abnormalities of the kidney and the urinary tract (CAKUT) belong to the most common birth defects in human, but the molecular basis for the majority of CAKUT patients remains unknown. Here we show that the transcription factor SOX11 is a crucial regulator of kidney development. SOX11 is expressed in both mesenchymal and epithelial components of the early kidney anlagen. Deletion of Sox11 in mice causes an extension of the domain expressing Gdnf within rostral regions of the nephrogenic cord and results in duplex kidney formation. On the molecular level SOX11 directly binds and regulates a locus control region of the protocadherin B cluster. At later stages of kidney development, SOX11 becomes restricted to the intermediate segment of the developing nephron where it is required for the elongation of Henle's loop. Finally, mutation analysis in a cohort of patients suffering from CAKUT identified a series of rare SOX11 variants, one of which interferes with the transactivation capacity of the SOX11 protein. Taken together these data demonstrate a key role for SOX11 in normal kidney development and may suggest that variants in this gene predispose to CAKUT in humans. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

De Novo Mutations in SON Disrupt RNA Splicing of Genes Essential for Brain Development and Metabolism, Causing an Intellectual-Disability Syndrome.

PubMed

Kim, Jung-Hyun; Shinde, Deepali N; Reijnders, Margot R F; Hauser, Natalie S; Belmonte, Rebecca L; Wilson, Gregory R; Bosch, Daniëlle G M; Bubulya, Paula A; Shashi, Vandana; Petrovski, Slavé; Stone, Joshua K; Park, Eun Young; Veltman, Joris A; Sinnema, Margje; Stumpel, Connie T R M; Draaisma, Jos M; Nicolai, Joost; Yntema, Helger G; Lindstrom, Kristin; de Vries, Bert B A; Jewett, Tamison; Santoro, Stephanie L; Vogt, Julie; Bachman, Kristine K; Seeley, Andrea H; Krokosky, Alyson; Turner, Clesson; Rohena, Luis; Hempel, Maja; Kortüm, Fanny; Lessel, Davor; Neu, Axel; Strom, Tim M; Wieczorek, Dagmar; Bramswig, Nuria; Laccone, Franco A; Behunova, Jana; Rehder, Helga; Gordon, Christopher T; Rio, Marlène; Romana, Serge; Tang, Sha; El-Khechen, Dima; Cho, Megan T; McWalter, Kirsty; Douglas, Ganka; Baskin, Berivan; Begtrup, Amber; Funari, Tara; Schoch, Kelly; Stegmann, Alexander P A; Stevens, Servi J C; Zhang, Dong-Er; Traver, David; Yao, Xu; MacArthur, Daniel G; Brunner, Han G; Mancini, Grazia M; Myers, Richard M; Owen, Laurie B; Lim, Ssang-Taek; Stachura, David L; Vissers, Lisenka E L M; Ahn, Eun-Young Erin

2016-09-01

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Mammalian genomic regulatory regions predicted by utilizing human genomics, transcriptomics, and epigenetics data

PubMed Central

Nguyen, Quan H; Tellam, Ross L; Naval-Sanchez, Marina; Porto-Neto, Laercio R; Barendse, William; Reverter, Antonio; Hayes, Benjamin; Kijas, James; Dalrymple, Brian P

2018-01-01

Abstract Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers, and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits and identifying potential genome editing targets. PMID:29618048

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Finniss, Susan; Bögler, Oliver; Duchrow, Michael

2004-04-15

The Ki-67 antigen, pKi-67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi-67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi-67 N-terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi-67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi-67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi-67 N-terminus is differentially spliced resulting in at least five different isoforms with different functions. Copyright 2004 Wiley-Liss, Inc.

Mammalian genomic regulatory regions predicted by utilizing human genomics, transcriptomics, and epigenetics data.

PubMed

Nguyen, Quan H; Tellam, Ross L; Naval-Sanchez, Marina; Porto-Neto, Laercio R; Barendse, William; Reverter, Antonio; Hayes, Benjamin; Kijas, James; Dalrymple, Brian P

2018-03-01

Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers, and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits and identifying potential genome editing targets.

The Accumulation of Versican in the Nodules of Benign Prostatic Hyperplasia

PubMed Central

True, Lawrence D.; Hawley, Sarah; Norwood, Thomas H.; Braun, Kathleen R.; Evanko, Stephen P.; Chan, Christina K.; LeBaron, Richard C.; Wight, Thomas N.

2014-01-01

Background Proteoglycans, a complex group of extracellular matrix (ECM) molecules, are elevated in benign prostatic hyperplasia (BPH). Versican is a stromal proteoglycan present in prostate tissue. Versican expression is elevated in tissues with increased proliferation. Based on these observations, we determined the extent and distribution of versican expression in prostates with BPH. Methods The involvement of versican in BPH nodules was compared with levels in non-nodular transition (TZ) and peripheral zone (PZ) tissues from 18 human prostate glands using immunohistochemistry, Northern blots and/or QRTPCR to localize versican and quantify versican mRNA transcript levels, and Western blots to assess gene product levels. Results Increased versican immunoreactivity was observed in the stroma of BPH nodules. Higher steady state levels of versican variants V0, V1, and V3 mRNA transcript and gene product were detected in the nodular tissues than in the non-nodular TZ or PZ parenchyma. Conclusions These results suggest that versican may play a role in nodule formation in BPH. PMID:18819099

Regulation of cerebral cortical neurogenesis by the Pax6 transcription factor

PubMed Central

Manuel, Martine N.; Mi, Da; Mason, John O.; Price, David J.

2015-01-01

Understanding brain development remains a major challenge at the heart of understanding what makes us human. The neocortex, in evolutionary terms the newest part of the cerebral cortex, is the seat of higher cognitive functions. Its normal development requires the production, positioning, and appropriate interconnection of very large numbers of both excitatory and inhibitory neurons. Pax6 is one of a relatively small group of transcription factors that exert high-level control of cortical development, and whose mutation or deletion from developing embryos causes major brain defects and a wide range of neurodevelopmental disorders. Pax6 is very highly conserved between primate and non-primate species, is expressed in a gradient throughout the developing cortex and is essential for normal corticogenesis. Our understanding of Pax6’s functions and the cellular processes that it regulates during mammalian cortical development has significantly advanced in the last decade, owing to the combined application of genetic and biochemical analyses. Here, we review the functional importance of Pax6 in regulating cortical progenitor proliferation, neurogenesis, and formation of cortical layers and highlight important differences between rodents and primates. We also review the pathological effects of PAX6 mutations in human neurodevelopmental disorders. We discuss some aspects of Pax6’s molecular actions including its own complex transcriptional regulation, the distinct molecular functions of its splice variants and some of Pax6’s known direct targets which mediate its actions during cortical development. PMID:25805971

CRIMEtoYHU: a new web tool to develop yeast-based functional assays for characterizing cancer-associated missense variants.

PubMed

Mercatanti, Alberto; Lodovichi, Samuele; Cervelli, Tiziana; Galli, Alvaro

2017-12-01

Evaluation of the functional impact of cancer-associated missense variants is more difficult than for protein-truncating mutations and consequently standard guidelines for the interpretation of sequence variants have been recently proposed. A number of algorithms and software products were developed to predict the impact of cancer-associated missense mutations on protein structure and function. Importantly, direct assessment of the variants using high-throughput functional assays using simple genetic systems can help in speeding up the functional evaluation of newly identified cancer-associated variants. We developed the web tool CRIMEtoYHU (CTY) to help geneticists in the evaluation of the functional impact of cancer-associated missense variants. Humans and the yeast Saccharomyces cerevisiae share thousands of protein-coding genes although they have diverged for a billion years. Therefore, yeast humanization can be helpful in deciphering the functional consequences of human genetic variants found in cancer and give information on the pathogenicity of missense variants. To humanize specific positions within yeast genes, human and yeast genes have to share functional homology. If a mutation in a specific residue is associated with a particular phenotype in humans, a similar substitution in the yeast counterpart may reveal its effect at the organism level. CTY simultaneously finds yeast homologous genes, identifies the corresponding variants and determines the transferability of human variants to yeast counterparts by assigning a reliability score (RS) that may be predictive for the validity of a functional assay. CTY analyzes newly identified mutations or retrieves mutations reported in the COSMIC database, provides information about the functional conservation between yeast and human and shows the mutation distribution in human genes. CTY analyzes also newly found mutations and aborts when no yeast homologue is found. Then, on the basis of the protein domain localization and functional conservation between yeast and human, the selected variants are ranked by the RS. The RS is assigned by an algorithm that computes functional data, type of mutation, chemistry of amino acid substitution and the degree of mutation transferability between human and yeast protein. Mutations giving a positive RS are highly transferable to yeast and, therefore, yeast functional assays will be more predictable. To validate the web application, we have analyzed 8078 cancer-associated variants located in 31 genes that have a yeast homologue. More than 50% of variants are transferable to yeast. Incidentally, 88% of all transferable mutations have a reliability score >0. Moreover, we analyzed by CTY 72 functionally validated missense variants located in yeast genes at positions corresponding to the human cancer-associated variants. All these variants gave a positive RS. To further validate CTY, we analyzed 3949 protein variants (with positive RS) by the predictive algorithm PROVEAN. This analysis shows that yeast-based functional assays will be more predictable for the variants with positive RS. We believe that CTY could be an important resource for the cancer research community by providing information concerning the functional impact of specific mutations, as well as for the design of functional assays useful for decision support in precision medicine. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcriptional regulation of the MET receptor tyrosine kinase gene by MeCP2 and sex-specific expression in autism and Rett syndrome

PubMed Central

Plummer, J T; Evgrafov, O V; Bergman, M Y; Friez, M; Haiman, C A; Levitt, P; Aldinger, K A

2013-01-01

Single nucleotide variants (SNV) in the gene encoding the MET receptor tyrosine kinase have been associated with an increased risk for autism spectrum disorders (ASD). The MET promoter SNV rs1858830 C ‘low activity' allele is enriched in ASD, associated with reduced protein expression, and impacts functional and structural circuit connectivity in humans. To gain insight into the transcriptional regulation of MET on ASD-risk etiology, we examined an interaction between the methyl CpG-binding protein 2 (MeCP2) and the MET 5′ promoter region. Mutations in MeCP2 cause Rett syndrome (RTT), a predominantly female neurodevelopmental disorder sharing some ASD clinical symptoms. MeCP2 binds to a region of the MET promoter containing the ASD-risk SNV, and displays rs1858830 genotype-specific binding in human neural progenitor cells derived from the olfactory neuroepithelium. MeCP2 binding enhances MET expression in the presence of the rs1858830 C allele, but MET transcription is attenuated by RTT-specific mutations in MeCP2. In the postmortem temporal cortex, a region normally enriched in MET, gene expression is reduced dramatically in females with RTT, although not due to enrichment of the rs1858830 C ‘low activity' allele. We newly identified a sex-based reduction in MET expression, with male ASD cases, but not female ASD cases compared with sex-matched controls. The experimental data reveal a prominent allele-specific regulation of MET transcription by MeCP2. The mechanisms underlying the pronounced reduction of MET in ASD and RTT temporal cortex are distinct and likely related to factors unique to each disorder, including a noted sex bias. PMID:24150225

Transcriptional regulation of the MET receptor tyrosine kinase gene by MeCP2 and sex-specific expression in autism and Rett syndrome.

PubMed

Plummer, J T; Evgrafov, O V; Bergman, M Y; Friez, M; Haiman, C A; Levitt, P; Aldinger, K A

2013-10-22

Single nucleotide variants (SNV) in the gene encoding the MET receptor tyrosine kinase have been associated with an increased risk for autism spectrum disorders (ASD). The MET promoter SNV rs1858830 C 'low activity' allele is enriched in ASD, associated with reduced protein expression, and impacts functional and structural circuit connectivity in humans. To gain insight into the transcriptional regulation of MET on ASD-risk etiology, we examined an interaction between the methyl CpG-binding protein 2 (MeCP2) and the MET 5' promoter region. Mutations in MeCP2 cause Rett syndrome (RTT), a predominantly female neurodevelopmental disorder sharing some ASD clinical symptoms. MeCP2 binds to a region of the MET promoter containing the ASD-risk SNV, and displays rs1858830 genotype-specific binding in human neural progenitor cells derived from the olfactory neuroepithelium. MeCP2 binding enhances MET expression in the presence of the rs1858830 C allele, but MET transcription is attenuated by RTT-specific mutations in MeCP2. In the postmortem temporal cortex, a region normally enriched in MET, gene expression is reduced dramatically in females with RTT, although not due to enrichment of the rs1858830 C 'low activity' allele. We newly identified a sex-based reduction in MET expression, with male ASD cases, but not female ASD cases compared with sex-matched controls. The experimental data reveal a prominent allele-specific regulation of MET transcription by MeCP2. The mechanisms underlying the pronounced reduction of MET in ASD and RTT temporal cortex are distinct and likely related to factors unique to each disorder, including a noted sex bias.

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

PubMed

Semler, Matthew R; Wiseman, Roger W; Karl, Julie A; Graham, Michael E; Gieger, Samantha M; O'Connor, David H

2018-06-01

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

Transcription factor ERG and joint and articular cartilage formation during mouse limb and spine skeletogenesis.

PubMed

Iwamoto, Masahiro; Tamamura, Yoshihiro; Koyama, Eiki; Komori, Toshihisa; Takeshita, Nobuo; Williams, Julie A; Nakamura, Takashi; Enomoto-Iwamoto, Motomi; Pacifici, Maurizio

2007-05-01

Articular cartilage and synovial joints are critical for skeletal function, but the mechanisms regulating their development are largely unknown. In previous studies we found that the ets transcription factor ERG and its alternatively-spliced variant C-1-1 have roles in joint formation in chick. Here, we extended our studies to mouse. We found that ERG is also expressed in developing mouse limb joints. To test regulation of ERG expression, beads coated with the joint master regulator protein GDF-5 were implanted close to incipient joints in mouse limb explants; this led to rapid and strong ectopic ERG expression. We cloned and characterized several mammalian ERG variants and expressed a human C-1-1 counterpart (hERG3Delta81) throughout the cartilaginous skeleton of transgenic mice, using Col2a1 gene promoter/enhancer sequences. The skeletal phenotype was severe and neonatal lethal, and the transgenic mice were smaller than wild type littermates and their skeletons were largely cartilaginous. Limb long bone anlagen were entirely composed of chondrocytes actively expressing collagen IX and aggrecan as well as articular markers such as tenascin-C. Typical growth plates were absent and there was very low expression of maturation and hypertrophy markers, including Indian hedgehog, collagen X and MMP-13. The results suggest that ERG is part of molecular mechanisms leading chondrocytes into a permanent developmental path and become joint forming cells, and may do so by acting downstream of GDF-5.

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

PubMed

Johnstone, Daniel; Graham, Ross M; Trinder, Debbie; Delima, Roheeth D; Riveros, Carlos; Olynyk, John K; Scott, Rodney J; Moscato, Pablo; Milward, Elizabeth A

2012-04-11

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Variants in the 3' UTR of General Transcription Factor IIF, polypeptide 2 affect female calving efficiency in Japanese Black cattle

PubMed Central

2013-01-01

Background Calving efficiency can be described as the measure of a cow’s ability to produce viable offspring within a specific period of time. This trait is crucial in beef cattle because calves are necessary both for the production of beef and for heifer replacements. Recently, the number of calves produced at 4 years of age (NCP4) has been used to evaluate the calving efficiency of Japanese Black cattle. To identify variants associated with calving efficiency in Japanese Black cattle, we conducted a genome-wide association study (GWAS) using 688 animals with extreme NCP4 values selected from 15,225 animals. Results We identified genetic variants on bovine chromosome 12 (BTA12) that were associated with NCP4. The General Transcription Factor IIF, polypeptide 2 (GTF2F2), located in the 132 kbp-associated region, proved to be in strong linkage disequilibrium. We found 15 associated variants in the promoter and the 3' UTR regions. Consistent with this finding, transcripts of GTF2F2 derived from the haplotype (Q) with the increased number of calves were 1.33-fold more abundant than q-derived transcripts. Furthermore, luciferase assays revealed that the activity of the 3' UTR, a region that includes nine SNPs, was higher in constructs with the Q haplotype than in those with the q haplotype by approximately 1.35-fold. In contrast, the activity of the promoter region did not differ between haplotypes. The association was replicated in an independent sample of 827 animals that were randomly selected from the remainder of the cohort from the same farms used in the GWAS. In the replicated population, the frequency of the Q haplotype is 0.313, and this haplotype accounts for 2.69% of the total phenotypic variance. The effect of the Q to q haplotype substitution on NCP4 was 0.054 calves. These findings suggest that variants in the 3' UTR of GTF2F2 affect the level of GTF2F2 mRNA, which is associated with calving efficiency. Conclusions This GWAS has identified variants in the 3’ UTR of GTF2F2 that were associated with the NCP4 of Japanese Black cattle, and this association was validated in an independent sample. The Q haplotype will be immediately useful in improving the calving efficiency of Japanese Black cattle. PMID:23663537

Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants.

PubMed

Kao, Liyo; Azimov, Rustam; Shao, Xuesi M; Frausto, Ricardo F; Abuladze, Natalia; Newman, Debra; Aldave, Anthony J; Kurtz, Ira

2016-11-01

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH 2 -terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H + equivalents in both a Na + -independent and Na + -coupled mode. In both transport modes, SLC4A11-C H + flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH 2 -terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH 2 -terminus functions as an autoactivating domain, enhancing Na + -independent and Na + -coupled H + flux without significantly affecting the electrogenic NH 3 -H (n) + cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11. Copyright © 2016 the American Physiological Society.

The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants.

PubMed

Zhang, J; Kuehl, P; Green, E D; Touchman, J W; Watkins, P B; Daly, A; Hall, S D; Maurel, P; Relling, M; Brimer, C; Yasuda, K; Wrighton, S A; Hancock, M; Kim, R B; Strom, S; Thummel, K; Russell, C G; Hudson, J R; Schuetz, E G; Boguski, M S

2001-10-01

The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.

Corepressors: custom tailoring and alterations while you wait

PubMed Central

Goodson, Michael; Jonas, Brian A.; Privalsky, Martin A.

2005-01-01

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages. PMID:16604171

Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

PubMed Central

Verma, Anjali; Rajagopalan, Pavithra; Lotke, Rishikesh; Varghese, Rebu; Selvam, Deepak; Kundu, Tapas K.

2016-01-01

ABSTRACT Of the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-κB binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-κB and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-κB motif binds NF-κB with an affinity 2-fold higher than that of the generic NF-κB site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-κB motif. An additional substitution of the Sp1III motif fully restores viral replication, suggesting that the subtype C-specific Sp1III has evolved to function with the variant, but not generic, NF-κB motif. A change of only two base pairs in the central NF-κB motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to tumor necrosis factor alpha (TNF-α) induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-κB motif and a unique Sp1 site in the context of an HIV-1 promoter. Our work provides important leads as to the evolution of the HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency. IMPORTANCE Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes, if any. Only in subtype C of HIV-1 are two genetically distinct transcription factor binding sites positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-κB binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that the subtype C viral promoter not only is stronger but also is endowed with a qualitative gain-of-function advantage. The genetically variant NF-κB and the Sp1III motifs may be respond differently to specific cell signal pathways, and these mechanisms must be examined. PMID:27194770

Silencing of the Drosophila ortholog of SOX5 leads to abnormal neuronal development and behavioral impairment.

PubMed

Li, Airong; Hooli, Basavaraj; Mullin, Kristina; Tate, Rebecca E; Bubnys, Adele; Kirchner, Rory; Chapman, Brad; Hofmann, Oliver; Hide, Winston; Tanzi, Rudolph E

2017-04-15

SOX5 encodes a transcription factor that is expressed in multiple tissues including heart, lung and brain. Mutations in SOX5 have been previously found in patients with amyotrophic lateral sclerosis (ALS) and developmental delay, intellectual disability and dysmorphic features. To characterize the neuronal role of SOX5, we silenced the Drosophila ortholog of SOX5, Sox102F, by RNAi in various neuronal subtypes in Drosophila. Silencing of Sox102F led to misorientated and disorganized michrochaetes, neurons with shorter dendritic arborization (DA) and reduced complexity, diminished larval peristaltic contractions, loss of neuromuscular junction bouton structures, impaired olfactory perception, and severe neurodegeneration in brain. Silencing of SOX5 in human SH-SY5Y neuroblastoma cells resulted in a significant repression of WNT signaling activity and altered expression of WNT-related genes. Genetic association and meta-analyses of the results in several large family-based and case-control late-onset familial Alzheimer's disease (LOAD) samples of SOX5 variants revealed several variants that show significant association with AD disease status. In addition, analysis for rare and highly penetrate functional variants revealed four novel variants/mutations in SOX5, which taken together with functional prediction analysis, suggests a strong role of SOX5 causing AD in the carrier families. Collectively, these findings indicate that SOX5 is a novel candidate gene for LOAD with an important role in neuronal function. The genetic findings warrant further studies to identify and characterize SOX5 variants that confer risk for AD, ALS and intellectual disability. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Widespread Site-Dependent Buffering of Human Regulatory Polymorphism

PubMed Central

Kutyavin, Tanya; Stamatoyannopoulos, John A.

2012-01-01

The average individual is expected to harbor thousands of variants within non-coding genomic regions involved in gene regulation. However, it is currently not possible to interpret reliably the functional consequences of genetic variation within any given transcription factor recognition sequence. To address this, we comprehensively analyzed heritable genome-wide binding patterns of a major sequence-specific regulator (CTCF) in relation to genetic variability in binding site sequences across a multi-generational pedigree. We localized and quantified CTCF occupancy by ChIP-seq in 12 related and unrelated individuals spanning three generations, followed by comprehensive targeted resequencing of the entire CTCF–binding landscape across all individuals. We identified hundreds of variants with reproducible quantitative effects on CTCF occupancy (both positive and negative). While these effects paralleled protein–DNA recognition energetics when averaged, they were extensively buffered by striking local context dependencies. In the significant majority of cases buffering was complete, resulting in silent variants spanning every position within the DNA recognition interface irrespective of level of binding energy or evolutionary constraint. The prevalence of complex partial or complete buffering effects severely constrained the ability to predict reliably the impact of variation within any given binding site instance. Surprisingly, 40% of variants that increased CTCF occupancy occurred at positions of human–chimp divergence, challenging the expectation that the vast majority of functional regulatory variants should be deleterious. Our results suggest that, even in the presence of “perfect” genetic information afforded by resequencing and parallel studies in multiple related individuals, genomic site-specific prediction of the consequences of individual variation in regulatory DNA will require systematic coupling with empirical functional genomic measurements. PMID:22457641

Identifying the Deleterious Effect of Rare LHX4 Allelic Variants, a Challenging Issue

PubMed Central

Rochette, Claire; Jullien, Nicolas; Saveanu, Alexandru; Caldagues, Emmanuelle; Bergada, Ignacio; Braslavsky, Debora; Pfeifer, Marija; Reynaud, Rachel; Herman, Jean-Paul; Barlier, Anne; Brue, Thierry; Enjalbert, Alain; Castinetti, Frederic

2015-01-01

LHX4 is a LIM homeodomain transcription factor involved in the early steps of pituitary ontogenesis. To date, 8 heterozygous LHX4 mutations have been reported as responsible of combined pituitary hormone deficiency (CPHD) in Humans. We identified 4 new LHX4 heterozygous allelic variants in patients with congenital hypopituitarism: W204X, delK242, N271S and Q346R. Our objective was to determine the role of LHX4 variants in patients’ phenotypes. Heterologous HEK293T cells were transfected with plasmids encoding for wild-type or mutant LHX4. Protein expression was analysed by Western Blot, and DNA binding by electro-mobility shift assay experiments. Target promoters of LHX4 were cotransfected with wild type or mutant LHX4 to test the transactivating abilities of each variant. Our results show that the W204X mutation was associated with early GH and TSH deficiencies and later onset ACTH deficiency. It led to a truncated protein unable to bind to alpha-Gsu promoter binding consensus sequence. W204X was not able to activate target promoters in vitro. Cotransfection experiments did not favour a dominant negative effect. In contrast, all other mutants were able to bind the promoters and led to an activation similar as that observed with wild type LHX4, suggesting that they were likely polymorphisms. To conclude, our study underlines the need for functional in vitro studies to ascertain the role of rare allelic variants of LHX4 in disease phenotypes. It supports the causative role of the W204X mutation in CPHD and adds up childhood onset ACTH deficiency to the clinical spectrum of the various phenotypes related to LHX4 mutations. PMID:25955177

Long genes and genes with multiple splice variants are enriched in pathways linked to cancer and other multigenic diseases.

PubMed

Sahakyan, Aleksandr B; Balasubramanian, Shankar

2016-03-12

The role of random mutations and genetic errors in defining the etiology of cancer and other multigenic diseases has recently received much attention. With the view that complex genes should be particularly vulnerable to such events, here we explore the link between the simple properties of the human genes, such as transcript length, number of splice variants, exon/intron composition, and their involvement in the pathways linked to cancer and other multigenic diseases. We reveal a substantial enrichment of cancer pathways with long genes and genes that have multiple splice variants. Although the latter two factors are interdependent, we show that the overall gene length and splicing complexity increase in cancer pathways in a partially decoupled manner. Our systematic survey for the pathways enriched with top lengthy genes and with genes that have multiple splice variants reveal, along with cancer pathways, the pathways involved in various neuronal processes, cardiomyopathies and type II diabetes. We outline a correlation between the gene length and the number of somatic mutations. Our work is a step forward in the assessment of the role of simple gene characteristics in cancer and a wider range of multigenic diseases. We demonstrate a significant accumulation of long genes and genes with multiple splice variants in pathways of multigenic diseases that have already been associated with de novo mutations. Unlike the cancer pathways, we note that the pathways of neuronal processes, cardiomyopathies and type II diabetes contain genes long enough for topoisomerase-dependent gene expression to also be a potential contributing factor in the emergence of pathologies, should topoisomerases become impaired.

Mutation of ATF6 causes autosomal recessive achromatopsia.

PubMed

Ansar, Muhammad; Santos-Cortez, Regie Lyn P; Saqib, Muhammad Arif Nadeem; Zulfiqar, Fareeha; Lee, Kwanghyuk; Ashraf, Naeem Mahmood; Ullah, Ehsan; Wang, Xin; Sajid, Sundus; Khan, Falak Sher; Amin-ud-Din, Muhammad; Smith, Joshua D; Shendure, Jay; Bamshad, Michael J; Nickerson, Deborah A; Hameed, Abdul; Riazuddin, Saima; Ahmed, Zubair M; Ahmad, Wasim; Leal, Suzanne M

2015-09-01

Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.12-Mb region on chromosome 1q23.1-q24.3 with a maximum LOD score of 3.6. A DNA sample from an affected family member underwent exome sequencing. Within the ATF6 gene, a single-base insertion variant c.355_356dupG (p.Glu119Glyfs*8) was identified, which completely segregates with the ACHM phenotype within the family. The frameshift variant was absent in public variant databases, in 130 exomes from unrelated Pakistani individuals, and in 235 ethnically matched controls. The variant is predicted to result in a truncated protein that lacks the DNA binding and transmembrane domains and therefore affects the function of ATF6 as a transcription factor that initiates the unfolded protein response during endoplasmic reticulum (ER) stress. Immunolabeling with anti-ATF6 antibodies showed localization throughout the mouse neuronal retina, including retinal pigment epithelium, photoreceptor cells, inner nuclear layer, inner and outer plexiform layers, with a more prominent signal in retinal ganglion cells. In contrast to cytoplasmic expression of wild-type protein, in heterologous cells ATF6 protein with the p.Glu119Glyfs*8 variant is mainly confined to the nucleus. Our results imply that response to ER stress as mediated by the ATF6 pathway is essential for color vision in humans.

A study of the role of the FOXP2 and CNTNAP2 genes in persistent developmental stuttering.

PubMed

Han, Tae-Un; Park, John; Domingues, Carlos F; Moretti-Ferreira, Danilo; Paris, Emily; Sainz, Eduardo; Gutierrez, Joanne; Drayna, Dennis

2014-09-01

A number of speech disorders including stuttering have been shown to have important genetic contributions, as indicated by high heritability estimates from twin and other studies. We studied the potential contribution to stuttering from variants in the FOXP2 gene, which have previously been associated with developmental verbal dyspraxia, and from variants in the CNTNAP2 gene, which have been associated with specific language impairment (SLI). DNA sequence analysis of these two genes in a group of 602 unrelated cases, all with familial persistent developmental stuttering, revealed no excess of potentially deleterious coding sequence variants in the cases compared to a matched group of 487 well characterized neurologically normal controls. This was compared to the distribution of variants in the GNPTAB, GNPTG, and NAGPA genes which have previously been associated with persistent stuttering. Using an expanded subject data set, we again found that NAGPA showed significantly different mutation frequencies in North Americans of European descent (p=0.0091) and a significant difference existed in the mutation frequency of GNPTAB in Brazilians (p=0.00050). No significant differences in mutation frequency in the FOXP2 and CNTNAP2 genes were observed between cases and controls. To examine the pattern of expression of these five genes in the human brain, real time quantitative reverse transcription PCR was performed on RNA purified from 27 different human brain regions. The expression patterns of FOXP2 and CNTNAP2 were generally different from those of GNPTAB, GNPTG and NAPGA in terms of relatively lower expression in the cerebellum. This study provides an improved estimate of the contribution of mutations in GNPTAB, GNPTG and NAGPA to persistent stuttering, and suggests that variants in FOXP2 and CNTNAP2 are not involved in the genesis of familial persistent stuttering. This, together with the different brain expression patterns of GNPTAB, GNPTG, and NAGPA compared to that of FOXP2 and CNTNAP2, suggests that the genetic neuropathological origins of stuttering differ from those of verbal dyspraxia and SLI. Published by Elsevier Inc.

An integrated map of structural variation in 2,504 human genomes.

PubMed

Sudmant, Peter H; Rausch, Tobias; Gardner, Eugene J; Handsaker, Robert E; Abyzov, Alexej; Huddleston, John; Zhang, Yan; Ye, Kai; Jun, Goo; Fritz, Markus Hsi-Yang; Konkel, Miriam K; Malhotra, Ankit; Stütz, Adrian M; Shi, Xinghua; Casale, Francesco Paolo; Chen, Jieming; Hormozdiari, Fereydoun; Dayama, Gargi; Chen, Ken; Malig, Maika; Chaisson, Mark J P; Walter, Klaudia; Meiers, Sascha; Kashin, Seva; Garrison, Erik; Auton, Adam; Lam, Hugo Y K; Mu, Xinmeng Jasmine; Alkan, Can; Antaki, Danny; Bae, Taejeong; Cerveira, Eliza; Chines, Peter; Chong, Zechen; Clarke, Laura; Dal, Elif; Ding, Li; Emery, Sarah; Fan, Xian; Gujral, Madhusudan; Kahveci, Fatma; Kidd, Jeffrey M; Kong, Yu; Lameijer, Eric-Wubbo; McCarthy, Shane; Flicek, Paul; Gibbs, Richard A; Marth, Gabor; Mason, Christopher E; Menelaou, Androniki; Muzny, Donna M; Nelson, Bradley J; Noor, Amina; Parrish, Nicholas F; Pendleton, Matthew; Quitadamo, Andrew; Raeder, Benjamin; Schadt, Eric E; Romanovitch, Mallory; Schlattl, Andreas; Sebra, Robert; Shabalin, Andrey A; Untergasser, Andreas; Walker, Jerilyn A; Wang, Min; Yu, Fuli; Zhang, Chengsheng; Zhang, Jing; Zheng-Bradley, Xiangqun; Zhou, Wanding; Zichner, Thomas; Sebat, Jonathan; Batzer, Mark A; McCarroll, Steven A; Mills, Ryan E; Gerstein, Mark B; Bashir, Ali; Stegle, Oliver; Devine, Scott E; Lee, Charles; Eichler, Evan E; Korbel, Jan O

2015-10-01

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

Mapping of oxidative stress responses of human tumor cells following photodynamic therapy using hexaminolevulinate

PubMed Central

Cekaite, Lina; Peng, Qian; Reiner, Andrew; Shahzidi, Susan; Tveito, Siri; Furre, Ingegerd E; Hovig, Eivind

2007-01-01

Background Photodynamic therapy (PDT) involves systemic or topical administration of a lesion-localizing photosensitizer or its precursor, followed by irradiation of visible light to cause singlet oxygen-induced damage to the affected tissue. A number of mechanisms seem to be involved in the protective responses to PDT, including activation of transcription factors, heat shock proteins, antioxidant enzymes and apoptotic pathways. Results In this study, we address the effects of a destructive/lethal hexaminolevulinate (HAL) mediated PDT dose on the transcriptome by using transcriptional exon evidence oligo microarrays. Here, we confirm deviations in the steady state expression levels of previously identified early defence response genes and extend this to include unreported PDT inducible gene groups, most notably the metallothioneins and histones. HAL-PDT mediated stress also altered expression of genes encoded by mitochondrial DNA (mtDNA). Further, we report PDT stress induced alternative splicing. Specifically, the ATF3 alternative isoform (deltaZip2) was up-regulated, while the full-length variant was not changed by the treatment. Results were independently verified by two different technological microarray platforms. Good microarray, RT-PCR and Western immunoblotting correlation for selected genes support these findings. Conclusion Here, we report new insights into how destructive/lethal PDT alters the transcriptome not only at the transcriptional level but also at post-transcriptional level via alternative splicing. PMID:17692132

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

PubMed

Gregory, L C; Gaston-Massuet, C; Andoniadou, C L; Carreno, G; Webb, E A; Kelberman, D; McCabe, M J; Panagiotakopoulos, L; Saldanha, J W; Spoudeas, H A; Torpiano, J; Rossi, M; Raine, J; Canham, N; Martinez-Barbera, J P; Dattani, M T

2015-05-01

The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH. © 2014 John Wiley & Sons Ltd.

A global evolutionary and metabolic analysis of human obesity gene risk variants.

PubMed

Castillo, Joseph J; Hazlett, Zachary S; Orlando, Robert A; Garver, William S

2017-09-05

It is generally accepted that the selection of gene variants during human evolution optimized energy metabolism that now interacts with our obesogenic environment to increase the prevalence of obesity. The purpose of this study was to perform a global evolutionary and metabolic analysis of human obesity gene risk variants (110 human obesity genes with 127 nearest gene risk variants) identified using genome-wide association studies (GWAS) to enhance our knowledge of early and late genotypes. As a result of determining the mean frequency of these obesity gene risk variants in 13 available populations from around the world our results provide evidence for the early selection of ancestral risk variants (defined as selection before migration from Africa) and late selection of derived risk variants (defined as selection after migration from Africa). Our results also provide novel information for association of these obesity genes or encoded proteins with diverse metabolic pathways and other human diseases. The overall results indicate a significant differential evolutionary pattern for the selection of obesity gene ancestral and derived risk variants proposed to optimize energy metabolism in varying global environments and complex association with metabolic pathways and other human diseases. These results are consistent with obesity genes that encode proteins possessing a fundamental role in maintaining energy metabolism and survival during the course of human evolution. Copyright © 2017. Published by Elsevier B.V.

Variant Histone H2A.Z Is Globally Localized to the Promoters of Inactive Yeast Genes and Regulates Nucleosome Positioning

PubMed Central

Gévry, Nicolas; Adam, Maryse; Blanchette, Mathieu

2005-01-01

H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions—typically one or two nucleosomes wide—decorated with H2A.Z. Those “Z loci” are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed. PMID:16248679

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing.

PubMed

Hong, Yoonki; Kim, Woo Jin; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

2016-04-01

Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

DNA Fragmentation Factor 45 (DFF45) Gene at 1p36.2 Is Homozygously Deleted and Encodes Variant Transcripts in Neuroblastoma Cell Line1

PubMed Central

Yang, Hong Wei; Chen, Ying Zhang; Piao, Hui Ying; Takita, Junko; Soeda, Eiichi; Hayashi, Yasuhide

2001-01-01

Abstract Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region. PMID:11420752

Large-scale mass spectrometric detection of variant peptides resulting from non-synonymous nucleotide differences

PubMed Central

Sheynkman, Gloria M.; Shortreed, Michael R.; Frey, Brian L.; Scalf, Mark; Smith, Lloyd M.

2013-01-01

Each individual carries thousands of non-synonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level in order to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses, due to their absence from the generic databases that are employed for mass spectrometry searching. Here, we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep coverage shotgun MS data obtained from the same sample. This approach enabled detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data, and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs, and found to be comparable in all cases. PMID:24175627

Androgen Receptor and its Splice Variant, AR-V7, Differentially Regulate FOXA1 Sensitive Genes in LNCaP Prostate Cancer Cells

PubMed Central

Krause, William C.; Shafi, Ayesha A.; Nakka, Manjula; Weigel, Nancy L.

2014-01-01

Prostate cancer (PCa) is an androgen-dependent disease, and tumors that are resistant to androgen ablation therapy often remain androgen receptor (AR) dependent. Among the contributors to castration-resistant PCa are AR splice variants that lack the ligand-binding domain (LBD). Instead, they have small amounts of unique sequence derived from cryptic exons or from out of frame translation. The AR-V7 (or AR3) variant is constitutively active and is expressed under conditions consistent with CRPC. AR-V7 is reported to regulate a transcriptional program that is similar but not identical to that of AR. However, it is unknown whether these differences are due to the unique sequence in AR-V7, or simply to loss of the LBD. To examine transcriptional regulation by AR-V7, we have used lentiviruses encoding AR-V7 (amino acids 1-627 of AR with the 16 amino acids unique to the variant) to prepare a derivative of the androgen-dependent LNCaP cells with inducible expression of AR-V7. An additional cell line was generated with regulated expression of AR-NTD (amino acids 1-660 of AR); this mutant lacks the LBD but does not have the AR-V7 specific sequence. We find that AR and AR-V7 have distinct activities on target genes that are co-regulated by FOXA1. Transcripts regulated by AR-V7 were similarly regulated by AR-NTD, indicating that loss of the LBD is sufficient for the observed differences. Differential regulation of target genes correlates with preferential recruitment of AR or AR-V7 to specific cis-regulatory DNA sequences providing an explanation for some of the observed differences in target gene regulation. PMID:25008967

Functional characterization of the novel intronic nucleotide change c.288+9C>T within the BCKDHA gene: understanding a variant presentation of maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Navarrete, Rosa; Weisiger, Kara; Desviat, Lourdes R; Packman, Seymour; Ugarte, Magdalena; Rodríguez-Pombo, Pilar

2010-12-01

Mutations in any of the three different genes--BCKDHA, BCKDHB, and DBT--encoding for the E1α, E1β, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed--in addition to a low but measurable level of normal mRNA product--an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient's cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype-phenotype correlations and as basis for the development of therapeutic interventions.

Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

PubMed

Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

2015-11-01

Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

NASA Astrophysics Data System (ADS)

Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

2014-06-01

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

Old World Hantaviruses in Rodents in New Orleans, Louisiana

PubMed Central

Cross, Robert W.; Waffa, Bradley; Freeman, Ashley; Riegel, Claudia; Moses, Lina M.; Bennett, Andrew; Safronetz, David; Fischer, Elizabeth R.; Feldmann, Heinz; Voss, Thomas G.; Bausch, Daniel G.

2014-01-01

Seoul virus, an Old World hantavirus, is maintained in brown rats and causes a mild form of hemorrhagic fever with renal syndrome (HFRS) in humans. We captured rodents in New Orleans, Louisiana and tested them for the presence of Old World hantaviruses by reverse transcription polymerase chain reaction (RT-PCR) with sequencing, cell culture, and electron microscopy; 6 (3.4%) of 178 rodents captured—all brown rats—were positive for a Seoul virus variant previously coined Tchoupitoulas virus, which was noted in rodents in New Orleans in the 1980s. The finding of Tchoupitoulas virus in New Orleans over 25 years since its first discovery suggests stable endemicity in the city. Although the degree to which this virus causes human infection and disease remains unknown, repeated demonstration of Seoul virus in rodent populations, recent cases of laboratory-confirmed HFRS in some US cities, and a possible link with hypertensive renal disease warrant additional investigation in both rodents and humans. PMID:24639295

Old World hantaviruses in rodents in New Orleans, Louisiana.

PubMed

Cross, Robert W; Waffa, Bradley; Freeman, Ashley; Riegel, Claudia; Moses, Lina M; Bennett, Andrew; Safronetz, David; Fischer, Elizabeth R; Feldmann, Heinz; Voss, Thomas G; Bausch, Daniel G

2014-05-01

Seoul virus, an Old World hantavirus, is maintained in brown rats and causes a mild form of hemorrhagic fever with renal syndrome (HFRS) in humans. We captured rodents in New Orleans, Louisiana and tested them for the presence of Old World hantaviruses by reverse transcription polymerase chain reaction (RT-PCR) with sequencing, cell culture, and electron microscopy; 6 (3.4%) of 178 rodents captured--all brown rats--were positive for a Seoul virus variant previously coined Tchoupitoulas virus, which was noted in rodents in New Orleans in the 1980s. The finding of Tchoupitoulas virus in New Orleans over 25 years since its first discovery suggests stable endemicity in the city. Although the degree to which this virus causes human infection and disease remains unknown, repeated demonstration of Seoul virus in rodent populations, recent cases of laboratory-confirmed HFRS in some US cities, and a possible link with hypertensive renal disease warrant additional investigation in both rodents and humans.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

PubMed

Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

2014-11-07

Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and hence presumably of the GDF8 gene, in both CHO and C2C12 cultured cells. In vitro the MYOD1-A allelic variant could up-regulate the expression of MYOD1 gene. Additionally, we could assess a different response of in vitro gene expression according to cell type used to transfect constructs, suggesting that MyoD activation is regulated by mechanisms that are specific of myoblasts.

Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel

PubMed Central

Beheshti, Afshin; Pilichowska, Monika; Burgess, Kristine; Ricks-Santi, Luisel; McNiel, Elizabeth; London, Cheryl B.; Ravi, Dashnamoorthy; Evens, Andrew M.

2018-01-01

T-cell lymphoma (TCL) is an uncommon and aggressive form of human cancer. Lymphoma is the most common hematopoietic tumor in canines (companion animals), with TCL representing approximately 30% of diagnoses. Collectively, the canine is an appealing model for cancer research given the spontaneous occurrence of cancer, intact immune system, and phytogenetic proximity to humans. We sought to establish mutational congruence of the canine with known human TCL mutations in order to identify potential actionable oncogenic pathways. Following pathologic confirmation, DNA was sequenced in 16 canine TCL (cTCL) cases using a custom Human Cancer Hotspot Panel of 68 genes commonly mutated in human TCL. Sequencing identified 4,527,638 total reads with average length of 229 bases containing 346 unique variants and 1,474 total variants; each sample had an average of 92 variants. Among these, there were 258 germline and 32 somatic variants. Among the 32 somatic variants there were 8 missense variants, 1 splice junction variant and the remaining were intron or synonymous variants. A frequency of 4 somatic mutations per sample were noted with >7 mutations detected in MET, KDR, STK11 and BRAF. Expression of these associated proteins were also detected via Western blot analyses. In addition, Sanger sequencing confirmed three variants of high quality (MYC, MET, and TP53 missense mutation). Taken together, the mutational spectrum and protein analyses showed mutations in signaling pathways similar to human TCL and also identified novel mutations that may serve as drug targets as well as potential biomarkers. PMID:29854308

Competition through dimerization between antiapoptotic and proapoptotic HS-1-associated protein X-1 (Hax-1).

PubMed

Koontz, Jason; Kontrogianni-Konstantopoulos, Aikaterini

2014-02-07

Studies on Hax-1 have mainly focused on variant (v) 1, demonstrating its antiapoptotic properties. However, HAX1 is heavily spliced, generating structurally distinct isoforms. We sought to characterize the Hax-1 isoforms expressed in rat heart before and after insult. We confirmed the presence of at least four Hax-1 transcripts in healthy rat cardiac muscle. These exhibited differential expression before and after induction of myocardial infarction, with v2 being up-regulated 12-fold at the transcript level and 1.5-fold at the protein level post-insult. Contrary to antiapoptotic rat and human v1, overexpression of rat v2 or human v4 (the human homologue of rat v2) in epithelial cells exacerbated cell death by 30% following H2O2 treatment compared with control vector. Coexpression of rat v1 and v2 or human v1 and v4 neutralized the protective effects of rat and human v1 and the proapoptotic effects of rat v2 and human v4 by modulating cytochrome c release. This is, at least partly, mediated by the ability of Hax-1 proteins to form homotypic and heterotypic dimers with binding affinities ranging from ~3.8 nm for v1 dimers to ~97 nm for v1/v2 dimers. The minimal binding region supporting these interactions lies between amino acids 97-278, which are shared by nearly all Hax-1 proteins, indicating that additional factors regulate the preferential formation of Hax-1 homo- or heterodimers. Our studies are the first to show that Hax-1 is a family of anti- and proapoptotic regulators that may modulate cell survival and death through homo- or heterodimerization.

Human primitive brain displays negative mitochondrial-nuclear expression correlation of respiratory genes.

PubMed

Barshad, Gilad; Blumberg, Amit; Cohen, Tal; Mishmar, Dan

2018-06-14

Oxidative phosphorylation (OXPHOS), a fundamental energy source in all human tissues, requires interactions between mitochondrial (mtDNA)- and nuclear (nDNA)-encoded protein subunits. Although such interactions are fundamental to OXPHOS, bi-genomic coregulation is poorly understood. To address this question, we analyzed ∼8500 RNA-seq experiments from 48 human body sites. Despite well-known variation in mitochondrial activity, quantity, and morphology, we found overall positive mtDNA-nDNA OXPHOS genes' co-expression across human tissues. Nevertheless, negative mtDNA-nDNA gene expression correlation was identified in the hypothalamus, basal ganglia, and amygdala (subcortical brain regions, collectively termed the "primitive" brain). Single-cell RNA-seq analysis of mouse and human brains revealed that this phenomenon is evolutionarily conserved, and both are influenced by brain cell types (involving excitatory/inhibitory neurons and nonneuronal cells) and by their spatial brain location. As the "primitive" brain is highly oxidative, we hypothesized that such negative mtDNA-nDNA co-expression likely controls for the high mtDNA transcript levels, which enforce tight OXPHOS regulation, rather than rewiring toward glycolysis. Accordingly, we found "primitive" brain-specific up-regulation of lactate dehydrogenase B ( LDHB ), which associates with high OXPHOS activity, at the expense of LDHA , which promotes glycolysis. Analyses of co-expression, DNase-seq, and ChIP-seq experiments revealed candidate RNA-binding proteins and CEBPB as the best regulatory candidates to explain these phenomena. Finally, cross-tissue expression analysis unearthed tissue-dependent splice variants and OXPHOS subunit paralogs and allowed revising the list of canonical OXPHOS transcripts. Taken together, our analysis provides a comprehensive view of mito-nuclear gene co-expression across human tissues and provides overall insights into the bi-genomic regulation of mitochondrial activities. © 2018 Barshad et al.; Published by Cold Spring Harbor Laboratory Press.

PI16 is a shear stress and inflammation-regulated inhibitor of MMP2

PubMed Central

Hazell, Georgina G. J.; Peachey, Alasdair M. G.; Teasdale, Jack E.; Sala-Newby, Graciela B.; Angelini, Gianni D.; Newby, Andrew C.; White, Stephen J.

2016-01-01

Raised endothelial shear stress is protective against atherosclerosis but such protection may be lost at sites of inflammation. We found that four splice variants of the peptidase inhibitor 16 (PI16) mRNA are among the most highly shear stress regulated transcripts in human coronary artery endothelial cells (HCAECs), in vitro but that expression is reduced by inflammatory mediators TNFα and IL-1β. Immunohistochemistry demonstrated that PI16 is expressed in human coronary endothelium and in a subset of neointimal cells and medial smooth muscle cells. Adenovirus-mediated PI16 overexpression inhibits HCAEC migration and secreted matrix metalloproteinase (MMP) activity. Moreover, PI16 inhibits MMP2 in part by binding an exposed peptide loop above the active site. Our results imply that, at high endothelial shear stress, PI16 contributes to inhibition of protease activity; protection that can be reversed during inflammation. PMID:27996045

Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.

PubMed

Takahashi, Melissa K; Watters, Kyle E; Gasper, Paul M; Abbott, Timothy R; Carlson, Paul D; Chen, Alan A; Lucks, Julius B

2016-06-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators. © 2016 Takahashi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A genetic signature of the evolution of loss of flight in the Galapagos cormorant

USGS Publications Warehouse

Burga, Alejandro; Wang, Weiguang; Ben-David, Eyal; Wolf, Paul C.; Ramey, Andy M.; Verdugo, Claudio; Lyons, Karen; Parker, Patricia G.; Kruglyak, Leonid

2017-01-01

INTRODUCTIONChanges in the size and proportion of limbs and other structures have played a key role in the evolution of species. One common class of limb modification is recurrent wing reduction and loss of flight in birds. Indeed, Darwin used the occurrence of flightless birds as an argument in favor of his theory of natural selection. Loss of flight has evolved repeatedly and is found among 26 families of birds in 17 different orders. Despite the frequency of these modifications, we have a limited understanding of their underpinnings at the genetic and molecular levels.RATIONALETo better understand the evolution of changes in limb size, we studied a classic case of recent loss of flight in the Galapagos cormorant (Phalacrocorax harrisi). Cormorants are large water birds that live in coastal areas or near lakes, and P. harrisi is the only flightless cormorant among approximately 40 extant species. The entire population is distributed along the coastlines of Isabela and Fernandina islands in the Galapagos archipelago. P. harrisi has a pair of short wings, which are smaller than those of any other cormorant. The extreme reduction of the wings and pectoral skeleton observed in P. harrisi is an attractive model for studying the evolution of loss of flight because it occurred very recently; phylogenetic evidence suggests that P. harrisi diverged from its flighted relatives within the past 2 million years. We developed a comparative and predictive genomics approach that uses the genome sequences of P. harrisi and its flighted relatives to find candidate genetic variants that likely contributed to the evolution of loss of flight.RESULTSWe sequenced and de novo assembled the whole genomes of P. harrisi and three closely related flighted cormorant species. We identified thousands of coding variants exclusive to P. harrisi and classified them according to their probability of altering protein function based on conservation. Variants most likely to alter protein function were significantly enriched in genes mutated in human skeletal ciliopathies, including Ofd1, Evc, Wdr34, and Ift122. We carried out experiments in Caenorhabditis elegans to confirm that a missense variant present in the Galapagos cormorant IFT122 protein is sufficient to affect ciliary function. The primary cilium is essential for Hedgehog (Hh) signaling in vertebrates, and individuals affected by ciliopathies have small limbs and ribcages, mirroring the phenotype of P. harrisi. We also identified a 4–amino acid deletion in the regulatory domain of Cux1, a highly conserved transcription factor that has been experimentally shown to regulate limb growth in chicken. The four missing amino acids are perfectly conserved in all birds and mammals sequenced to date. We tested the consequences of this deletion in a chondrogenic cell line and showed that it impairs the ability of CUX1 to transcriptionally up-regulate cilia-related genes (some of which contain function-altering variants in P. harrisi) and to promote chondrogenic differentiation. Finally, we show that positive selection may have played a role in the fixation of the variants associated with loss of flight in P. harrisi.CONCLUSIONOur results indicate that the combined effect of variants in genes necessary for the correct transcriptional regulation and function of the primary cilium likely contributed to the evolution of highly reduced wings and other skeletal adaptations associated with loss of flight in P. harrisi. Our approach may be generally useful for identification of variants underlying evolutionary novelty from genomes of closely related species.

GETPrime 2.0: gene- and transcript-specific qPCR primers for 13 species including polymorphisms

PubMed Central

David, Fabrice P.A.; Rougemont, Jacques; Deplancke, Bart

2017-01-01

GETPrime (http://bbcftools.epfl.ch/getprime) is a database with a web frontend providing gene- and transcript-specific, pre-computed qPCR primer pairs. The primers have been optimized for genome-wide specificity and for allowing the selective amplification of one or several splice variants of most known genes. To ease selection, primers have also been ranked according to defined criteria such as genome-wide specificity (with BLAST), amplicon size, and isoform coverage. Here, we report a major upgrade (2.0) of the database: eight new species (yeast, chicken, macaque, chimpanzee, rat, platypus, pufferfish, and Anolis carolinensis) now complement the five already included in the previous version (human, mouse, zebrafish, fly, and worm). Furthermore, the genomic reference has been updated to Ensembl v81 (while keeping earlier versions for backward compatibility) as a result of re-designing the back-end database and automating the import of relevant sections of the Ensembl database in species-independent fashion. This also allowed us to map known polymorphisms to the primers (on average three per primer for human), with the aim of reducing experimental error when targeting specific strains or individuals. Another consequence is that the inclusion of future Ensembl releases and other species has now become a relatively straightforward task. PMID:28053161

LBH Gene Transcription Regulation by the Interplay of an Enhancer Risk Allele and DNA Methylation in Rheumatoid Arthritis.

PubMed

Hammaker, Deepa; Whitaker, John W; Maeshima, Keisuke; Boyle, David L; Ekwall, Anna-Karin H; Wang, Wei; Firestein, Gary S

2016-11-01

To identify nonobvious therapeutic targets for rheumatoid arthritis (RA), we performed an integrative analysis incorporating multiple "omics" data and the Encyclopedia of DNA Elements (ENCODE) database for potential regulatory regions. This analysis identified the limb bud and heart development (LBH) gene, which has risk alleles associated with RA/celiac disease and lupus, and can regulate cell proliferation in RA. We identified a novel LBH transcription enhancer with an RA risk allele (rs906868 G [Ref]/T) 6 kb upstream of the LBH gene with a differentially methylated locus. The confluence of 3 regulatory elements, rs906868, an RA differentially methylated locus, and a putative enhancer, led us to investigate their effects on LBH regulation in fibroblast-like synoviocytes (FLS). We cloned the 1.4-kb putative enhancer with either the rs906868 Ref allele or single-nucleotide polymorphism (SNP) variant into reporter constructs. The constructs were methylated in vitro and transfected into cultured FLS by nucleofection. We found that both variants increased transcription, thereby confirming the region's enhancer function. Unexpectedly, the transcriptional activity of the Ref risk allele was significantly lower than that of the SNP variant and is consistent with low LBH levels as a risk factor for aggressive FLS behavior. Using RA FLS lines with a homozygous Ref or SNP allele, we confirmed that homozygous Ref lines expressed lower LBH messenger RNA levels than did the SNP lines. Methylation significantly reduced enhancer activity for both alleles, indicating that enhancer function is dependent on its methylation status. This study shows how the interplay between genetics and epigenetics can affect expression of LBH in RA. © 2016, American College of Rheumatology.

Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms.

PubMed

Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward; Grallert, Harald; Glunk, Viktoria; Berulava, Tea; Lee, Heekyoung; Oskolkov, Nikolay; Fadista, Joao; Ehlers, Kerstin; Wahl, Simone; Hoffmann, Christoph; Qian, Kun; Rönn, Tina; Riess, Helene; Müller-Nurasyid, Martina; Bretschneider, Nancy; Schroeder, Timm; Skurk, Thomas; Horsthemke, Bernhard; Spieler, Derek; Klingenspor, Martin; Seifert, Martin; Kern, Michael J; Mejhert, Niklas; Dahlman, Ingrid; Hansson, Ola; Hauck, Stefanie M; Blüher, Matthias; Arner, Peter; Groop, Leif; Illig, Thomas; Suhre, Karsten; Hsu, Yi-Hsiang; Mellgren, Gunnar; Hauner, Hans; Laumen, Helmut

2014-01-16

Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

A common variant upstream of the PAX6 gene influences islet function in man.

PubMed

Ahlqvist, E; Turrini, F; Lang, S T; Taneera, J; Zhou, Y; Almgren, P; Hansson, O; Isomaa, B; Tuomi, T; Eriksson, K; Eriksson, J G; Lyssenko, V; Groop, L

2012-01-01

Impaired glucose tolerance and impaired insulin secretion have been reported in families with PAX6 mutations and it is suggested that they result from defective proinsulin processing due to lack of prohormone convertase 1/3, encoded by PCSK1. We investigated whether a common PAX6 variant would mimic these findings and explored in detail its effect on islet function in man. A PAX6 candidate single nucleotide polymorphism (rs685428) was associated with fasting insulin levels in the Diabetes Genetics Initiative genome-wide association study. We explored its potential association with glucose tolerance and insulin processing and secretion in three Scandinavian cohorts (N = 8,897 individuals). In addition, insulin secretion and the expression of PAX6 and transcriptional target genes were studied in human pancreatic islets. rs685428 G allele carriers had lower islet mRNA expression of PAX6 (p = 0.01) and PCSK1 (p = 0.001) than AA homozygotes. The G allele was associated with increased fasting insulin (p (replication) = 0.02, p (all) = 0.0008) and HOMA-insulin resistance (p (replication) = 0.02, p (all) = 0.001) as well as a lower fasting proinsulin/insulin ratio (p (all) = 0.008) and lower fasting glucagon (p = 0.04) and gastric inhibitory peptide (GIP) (p = 0.05) concentrations. Arginine-stimulated (p = 0.02) insulin secretion was reduced in vivo, which was further reflected by a reduction of glucose- and potassium-stimulated insulin secretion (p = 0.002 and p = 0.04, respectively) in human islets in vitro. A common variant in PAX6 is associated with reduced PAX6 and PCSK1 expression in human islets and reduced insulin response, as well as decreased glucagon and GIP concentrations and decreased insulin sensitivity. These findings emphasise the central role of PAX6 in the regulation of islet function and glucose metabolism in man.

RSPO fusion transcripts in colorectal cancer in Japanese population.

PubMed

Shinmura, Kazuya; Kahyo, Tomoaki; Kato, Hisami; Igarashi, Hisaki; Matsuura, Shun; Nakamura, Satoki; Kurachi, Kiyotaka; Nakamura, Toshio; Ogawa, Hiroshi; Funai, Kazuhito; Tanahashi, Masayuki; Niwa, Hiroshi; Sugimura, Haruhiko

2014-08-01

R-spondin (RSPO) gene fusions have recently been discovered in a subset of human colorectal cancer (CRC) in the U.S. population; however, whether the fusion is recurrent in CRC arising in patients from the other demographic areas and whether it is specific for CRC remain uncertain. In this study, we examined 75 primary CRCs and 121 primary lung cancers in the Japanese population for EIF3E-RSPO2 and PTPRK-RSPO3 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of EIF3E-RSPO2 and PTPRK-RSPO3 was not detected in any of the lung carcinomas, RSPO fusions were detected in three (4%) of the 75 CRCs. Two CRCs contained EIF3E-RSPO2 fusion transcripts, and another CRC contained PTPRK-RSPO3 fusion transcripts. Interestingly, in one of the two EIF3E-RSPO2 fusion-positive CRCs, a novel fusion variant form of EIF3E-RSPO2 was identified: exon 1 of EIF3E was connected to exon 2 of RSPO2 by a 351-bp insertion. A quantitative RT-PCR analysis revealed that RSPO mRNA expression was upregulated in the three CRCs containing RSPO fusion transcripts, while it was downregulated in nearly all of the other CRCs. An immunohistochemical analysis and a mutational analysis revealed that the RSPO fusion-containing CRC had a CDX2 cell lineage, was positive for mismatch repair protein expression, and had the wild-type APC allele. Finally, the forced expression of RSPO fusion proteins were shown to endow colorectal cells with an increased growth ability. These results suggest that the expression of RSPO fusion transcripts is related to a subset of CRCs arising in the Japanese population.

RNAseq versus genome-predicted transcriptomes: a large population of novel transcripts identified in an Illumina-454 Hydra transcriptome.

PubMed

Wenger, Yvan; Galliot, Brigitte

2013-03-25

Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48'909 unique sequences including splice variants, representing approximately 24'450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10'597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11'270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events.

RNAseq versus genome-predicted transcriptomes: a large population of novel transcripts identified in an Illumina-454 Hydra transcriptome

PubMed Central

2013-01-01

Background Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. Results To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48’909 unique sequences including splice variants, representing approximately 24’450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10’597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11’270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. Conclusions We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events. PMID:23530871

Harmonizing the interpretation of genetic variants across the world: the Malaysian experience.

PubMed

Hassan, Nik Norliza Nik; Plazzer, John-Paul; Smith, Timothy D; Halim-Fikri, Hashim; Macrae, Finlay; Zubaidi, A A L; Zilfalil, Bin Alwi

2016-02-26

Databases for gene variants are very useful for sharing genetic data and to facilitate the understanding of the genetic basis of diseases. This report summarises the issues surrounding the development of the Malaysian Human Variome Project Country Node. The focus is on human germline variants. Somatic variants, mitochondrial variants and other types of genetic variation have corresponding databases which are not covered here, as they have specific issues that do not necessarily apply to germline variations. The ethical, legal, social issues, intellectual property, ownership of the data, information technology implementation, and efforts to improve the standards and systems used in data sharing are discussed. An overarching framework such as provided by the Human Variome Project to co-ordinate activities is invaluable. Country Nodes, such as MyHVP, enable human gene variation associated with human diseases to be collected, stored and shared by all disciplines (clinicians, molecular biologists, pathologists, bioinformaticians) for a consistent interpretation of genetic variants locally and across the world.

Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

PubMed

Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth T S; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

2007-01-01

Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer.

Local Adaptation of Sun-Exposure-Dependent Gene Expression Regulation in Human Skin.

PubMed

Kita, Ryosuke; Fraser, Hunter B

2016-10-01

Sun-exposure is a key environmental variable in the study of human evolution. Several skin-pigmentation genes serve as classical examples of positive selection, suggesting that sun-exposure has significantly shaped worldwide genomic variation. Here we investigate the interaction between genetic variation and sun-exposure, and how this impacts gene expression regulation. Using RNA-Seq data from 607 human skin samples, we identified thousands of transcripts that are differentially expressed between sun-exposed skin and non-sun-exposed skin. We then tested whether genetic variants may influence each individual's gene expression response to sun-exposure. Our analysis revealed 10 sun-exposure-dependent gene expression quantitative trait loci (se-eQTLs), including genes involved in skin pigmentation (SLC45A2) and epidermal differentiation (RASSF9). The allele frequencies of the RASSF9 se-eQTL across diverse populations correlate with the magnitude of solar radiation experienced by these populations, suggesting local adaptation to varying levels of sunlight. These results provide the first examples of sun-exposure-dependent regulatory variation and suggest that this variation has contributed to recent human adaptation.

Using whole-exome sequencing to identify variants inherited from mosaic parents

PubMed Central

Rios, Jonathan J; Delgado, Mauricio R

2015-01-01

Whole-exome sequencing (WES) has allowed the discovery of genes and variants causing rare human disease. This is often achieved by comparing nonsynonymous variants between unrelated patients, and particularly for sporadic or recessive disease, often identifies a single or few candidate genes for further consideration. However, despite the potential for this approach to elucidate the genetic cause of rare human disease, a majority of patients fail to realize a genetic diagnosis using standard exome analysis methods. Although genetic heterogeneity contributes to the difficulty of exome sequence analysis between patients, it remains plausible that rare human disease is not caused by de novo or recessive variants. Multiple human disorders have been described for which the variant was inherited from a phenotypically normal mosaic parent. Here we highlight the potential for exome sequencing to identify a reasonable number of candidate genes when dominant disease variants are inherited from a mosaic parent. We show the power of WES to identify a limited number of candidate genes using this disease model and how sequence coverage affects identification of mosaic variants by WES. We propose this analysis as an alternative to discover genetic causes of rare human disorders for which typical WES approaches fail to identify likely pathogenic variants. PMID:24986828

Increased Transcript Complexity in Genes Associated with Chronic Obstructive Pulmonary Disease

PubMed Central

Lackey, Lela; McArthur, Evonne; Laederach, Alain

2015-01-01

Genome-wide association studies aim to correlate genotype with phenotype. Many common diseases including Type II diabetes, Alzheimer’s, Parkinson’s and Chronic Obstructive Pulmonary Disease (COPD) are complex genetic traits with hundreds of different loci that are associated with varied disease risk. Identifying common features in the genes associated with each disease remains a challenge. Furthermore, the role of post-transcriptional regulation, and in particular alternative splicing, is still poorly understood in most multigenic diseases. We therefore compiled comprehensive lists of genes associated with Type II diabetes, Alzheimer’s, Parkinson’s and COPD in an attempt to identify common features of their corresponding mRNA transcripts within each gene set. The SERPINA1 gene is a well-recognized genetic risk factor of COPD and it produces 11 transcript variants, which is exceptional for a human gene. This led us to hypothesize that other genes associated with COPD, and complex disorders in general, are highly transcriptionally diverse. We found that COPD-associated genes have a statistically significant enrichment in transcript complexity stemming from a disproportionately high level of alternative splicing, however, Type II Diabetes, Alzheimer’s and Parkinson’s disease genes were not significantly enriched. We also identified a subset of transcriptionally complex COPD-associated genes (~40%) that are differentially expressed between mild, moderate and severe COPD. Although the genes associated with other lung diseases are not extensively documented, we found preliminary data that idiopathic pulmonary disease genes, but not cystic fibrosis modulators, are also more transcriptionally complex. Interestingly, complex COPD transcripts are more often the product of alternative acceptor site usage. To verify the biological importance of these alternative transcripts, we used RNA-sequencing analyses to determine that COPD-associated genes are frequently expressed in lung and liver tissues and are regulated in a tissue-specific manner. Additionally, many complex COPD-associated genes are spliced differently between COPD and non-COPD patients. Our analysis therefore suggests that post-transcriptional regulation, particularly alternative splicing, is an important feature specific to COPD disease etiology that warrants further investigation. PMID:26480348

JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

PubMed Central

Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

2010-01-01

Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing. PMID:20730051

Different regulation of limb development by p63 transcript variants.

PubMed

Kawata, Manabu; Taniguchi, Yuki; Mori, Daisuke; Yano, Fumiko; Ohba, Shinsuke; Chung, Ung-Il; Shimogori, Tomomi; Mills, Alea A; Tanaka, Sakae; Saito, Taku

2017-01-01

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.

PubMed

Bloyet, Louis-Marie; Brunel, Joanna; Dosnon, Marion; Hamon, Véronique; Erales, Jenny; Gruet, Antoine; Lazert, Carine; Bignon, Christophe; Roche, Philippe; Longhi, Sonia; Gerlier, Denis

2016-12-01

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength

PubMed Central

Hamon, Véronique; Erales, Jenny; Bignon, Christophe; Roche, Philippe

2016-01-01

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3’end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region. PMID:27936158

Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiang, R.; Lidral, A.C.; Ardinger, H.H.

1993-10-01

Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. The authors carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3[prime] untranslated region ofmore » the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using X[sup 2] analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3[prime] untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P. 46 refs., 3 figs., 3 tabs.« less

SCRIB and PUF60 Are Primary Drivers of the Multisystemic Phenotypes of the 8q24.3 Copy-Number Variant

PubMed Central

Dauber, Andrew; Golzio, Christelle; Guenot, Cécile; Jodelka, Francine M.; Kibaek, Maria; Kjaergaard, Susanne; Leheup, Bruno; Martinet, Danielle; Nowaczyk, Malgorzata J.M.; Rosenfeld, Jill A.; Zeesman, Susan; Zunich, Janice; Beckmann, Jacques S.; Hirschhorn, Joel N.; Hastings, Michelle L.; Jacquemont, Sebastien; Katsanis, Nicholas

2013-01-01

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions. PMID:24140112

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription

PubMed Central

Carlson, Jonathan; Yan, Jiyu; Akinsiku, Olusimidele T.; Schaefer, Malinda; Sabbaj, Steffanie; Bet, Anne; Levy, David N.; Heath, Sonya; Tang, Jianming; Kaslow, Richard A.; Walker, Bruce D.; Ndung’u, Thumbi; Goulder, Philip J.; Heckerman, David; Hunter, Eric; Goepfert, Paul A.

2010-01-01

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity. PMID:20065064

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.).

PubMed

McKenzie, Marian J; Chen, Ronan K Y; Leung, Susanna; Joshi, Srishti; Rippon, Paula E; Joyce, Nigel I; McManus, Michael T

2017-12-01

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Insertion and deletion polymorphisms of the ancient AluS family in the human genome.

PubMed

Kryatova, Maria S; Steranka, Jared P; Burns, Kathleen H; Payer, Lindsay M

2017-01-01

Polymorphic Alu elements account for 17% of structural variants in the human genome. The majority of these belong to the youngest AluY subfamilies, and most structural variant discovery efforts have focused on identifying Alu polymorphisms from these currently retrotranspositionally active subfamilies. In this report we analyze polymorphisms from the evolutionarily older AluS subfamily, whose peak activity was tens of millions of years ago. We annotate the AluS polymorphisms, assess their likely mechanism of origin, and evaluate their contribution to structural variation in the human genome. Of 52 previously reported polymorphic AluS elements ascertained for this study, 48 were confirmed to belong to the AluS subfamily using high stringency subfamily classification criteria. Of these, the majority (77%, 37/48) appear to be deletion polymorphisms. Two polymorphic AluS elements (4%) have features of non-classical Alu insertions and one polymorphic AluS element (2%) likely inserted by a mechanism involving internal priming. Seven AluS polymorphisms (15%) appear to have arisen by the classical target-primed reverse transcription (TPRT) retrotransposition mechanism. These seven TPRT products are 3' intact with 3' poly-A tails, and are flanked by target site duplications; L1 ORF2p endonuclease cleavage sites were also observed, providing additional evidence that these are L1 ORF2p endonuclease-mediated TPRT insertions. Further sequence analysis showed strong conservation of both the RNA polymerase III promoter and SRP9/14 binding sites, important for mediating transcription and interaction with retrotransposition machinery, respectively. This conservation of functional features implies that some of these are fairly recent insertions since they have not diverged significantly from their respective retrotranspositionally competent source elements. Of the polymorphic AluS elements evaluated in this report, 15% (7/48) have features consistent with TPRT-mediated insertion, thus suggesting that some AluS elements have been more active recently than previously thought, or that fixation of AluS insertion alleles remains incomplete. These data expand the potential significance of polymorphic AluS elements in contributing to structural variation in the human genome. Future discovery efforts focusing on polymorphic AluS elements are likely to identify more such polymorphisms, and approaches tailored to identify deletion alleles may be warranted.

Fine-mapping inflammatory bowel disease loci to single variant resolution

PubMed Central

Huang, Hailiang; Fang, Ming; Jostins, Luke; Mirkov, Maša Umićević; Boucher, Gabrielle; Anderson, Carl A; Andersen, Vibeke; Cleynen, Isabelle; Cortes, Adrian; Crins, François; D'Amato, Mauro; Deffontaine, Valérie; Dimitrieva, Julia; Docampo, Elisa; Elansary, Mahmoud; Farh, Kyle Kai-How; Franke, Andre; Gori, Ann-Stephan; Goyette, Philippe; Halfvarson, Jonas; Haritunians, Talin; Knight, Jo; Lawrance, Ian C; Lees, Charlie W; Louis, Edouard; Mariman, Rob; Meuwissen, Theo; Mni, Myriam; Momozawa, Yukihide; Parkes, Miles; Spain, Sarah L; Théâtre, Emilie; Trynka, Gosia; Satsangi, Jack; van Sommeren, Suzanne; Vermeire, Severine; Xavier, Ramnik J; Weersma, Rinse K; Duerr, Richard H; Mathew, Christopher G; Rioux, John D; McGovern, Dermot PB; Cho, Judy H; Georges, Michel; Daly, Mark J; Barrett, Jeffrey C

2017-01-01

Summary The inflammatory bowel diseases (IBD) are chronic gastrointestinal inflammatory disorders that affect millions worldwide. Genome-wide association studies have identified 200 IBD-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 IBD loci using high-density genotyping in 67,852 individuals. We pinpointed 18 associations to a single causal variant with >95% certainty, and an additional 27 associations to a single variant with >50% certainty. These 45 variants are significantly enriched for protein-coding changes (n=13), direct disruption of transcription factor binding sites (n=3) and tissue specific epigenetic marks (n=10), with the latter category showing enrichment in specific immune cells among associations stronger in CD and in gut mucosa among associations stronger in UC. The results of this study suggest that high-resolution fine-mapping in large samples can convert many GWAS discoveries into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms. PMID:28658209

Analysis of the entire genomes of torque teno midi virus variants in chimpanzees: infrequent cross-species infection between humans and chimpanzees.

PubMed

Ninomiya, Masashi; Takahashi, Masaharu; Hoshino, Yu; Ichiyama, Koji; Simmonds, Peter; Okamoto, Hiroaki

2009-02-01

Humans are frequently infected with three anelloviruses which have circular DNA genomes of 3.6-3.9 kb [Torque teno virus (TTV)], 2.8-2.9 kb [Torque teno mini virus (TTMV)] and 3.2 kb [a recently discovered anellovirus named Torque teno midi virus (TTMDV)]. Unexpectedly, human TTMDV DNA was not detectable in any of 74 chimpanzees tested, although all but one tested positive for both human TTV and TTMV DNA. Using universal primers for anelloviruses, novel variants of TTMDV that are phylogenetically clearly separate from human TTMDV were identified from chimpanzees, and over the entire genome, three chimpanzee TTMDV variants differed by 17.9-20.3 % from each other and by 40.4-43.6 % from all 18 reported human TTMDVs. A newly developed PCR assay that uses chimpanzee TTMDV-specific primers revealed the high prevalence of chimpanzee TTMDV in chimpanzees (63/74, 85 %) but low prevalence in humans (1/100). While variants of TTV and TTMV from chimpanzees and humans were phylogenetically interspersed, those of TTMDV were monophyletic for each species, with sequence diversity of <33 and <20 % within the 18 human and three chimpanzee TTMDV variants, respectively. Maximum within-group divergence values for TTV and TTMV were 51 and 57 %, respectively; both of these values were substantially greater than the maximum divergence among TTMDV variants (44 %), consistent with a later evolutionary emergence of TTMDV. However, substantiation of this hypothesis will require further analysis of genetic diversity using an expanded dataset of TTMDV variants in humans and chimpanzees. Similarly, the underlying mechanism of observed infrequent cross-species infection of TTMDV between humans and chimpanzees deserves further analysis.

Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

PubMed

Wang, Li-Feng; Tian, Da-Wei; Li, Hai-Juan; Gao, Ya-Bing; Wang, Chang-Zhen; Zhao, Li; Zuo, Hong-Yan; Dong, Ji; Qiao, Si-Mo; Zou, Yong; Xiong, Lu; Zhou, Hong-Mei; Yang, Yue-Feng; Peng, Rui-Yun; Hu, Xiang-Jun

2016-05-01

Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation.

Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation*

PubMed Central

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J.

2014-01-01

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. PMID:24958729

Outbreak of Influenza A(H3N2) Variant Virus Infections Among Persons Attending Agricultural Fairs Housing Infected Swine - Michigan and Ohio, July-August 2016.

PubMed

Schicker, Rebekah S; Rossow, John; Eckel, Seth; Fisher, Nicolas; Bidol, Sally; Tatham, Lilith; Matthews-Greer, Janice; Sohner, Kevin; Bowman, Andrew S; Avrill, James; Forshey, Tony; Blanton, Lenee; Davis, C Todd; Schiltz, John; Skorupski, Susan; Berman, LaShondra; Jang, Yunho; Bresee, Joseph S; Lindstrom, Stephen; Trock, Susan C; Wentworth, David; Fry, Alicia M; de Fijter, Sietske; Signs, Kimberly; DiOrio, Mary; Olsen, Sonja J; Biggerstaff, Matthew

2016-10-28

On August 3, 2016, the Ohio Department of Health Laboratory reported to CDC that a respiratory specimen collected on July 28 from a male aged 13 years who attended an agricultural fair in Ohio during July 22-29, 2016, and subsequently developed a respiratory illness, tested positive by real-time reverse transcription-polymerase chain reaction (rRT-PCR) for influenza A(H3N2) variant* (H3N2v). The respiratory specimen was collected as part of routine influenza surveillance activities. The next day, CDC was notified of a child aged 9 years who was a swine exhibitor at an agricultural fair in Michigan who became ill on July 29, 2016, and tested positive for H3N2v virus at the Michigan Department of Health and Human Services Laboratory. Investigations by Michigan and Ohio health authorities identified 18 human infections linked to swine exhibits at agricultural fairs. To minimize transmission of influenza viruses from infected swine to visitors, agricultural fair organizers should consider prevention measures such as shortening the time swine are on the fairgrounds, isolating ill swine, maintaining a veterinarian on call, providing handwashing stations, and prohibiting food and beverages in animal barns. Persons at high risk for influenza-associated complications should be discouraged from entering swine barns.

A Rare SNP Identified a TCP Transcription Factor Essential for Tendril Development in Cucumber.

PubMed

Wang, Shenhao; Yang, Xueyong; Xu, Mengnan; Lin, Xingzhong; Lin, Tao; Qi, Jianjian; Shao, Guangjin; Tian, Nana; Yang, Qing; Zhang, Zhonghua; Huang, Sanwen

2015-12-07

Rare genetic variants are abundant in genomes but less tractable in genome-wide association study. Here we exploit a strategy of rare variation mapping to discover a gene essential for tendril development in cucumber (Cucumis sativus L.). In a collection of >3000 lines, we discovered a unique tendril-less line that forms branches instead of tendrils and, therefore, loses its climbing ability. We hypothesized that this unusual phenotype was caused by a rare variation and subsequently identified the causative single nucleotide polymorphism. The affected gene TEN encodes a TCP transcription factor conserved within the cucurbits and is expressed specifically in tendrils, representing a new organ identity gene. The variation occurs within a protein motif unique to the cucurbits and impairs its function as a transcriptional activator. Analyses of transcriptomes from near-isogenic lines identified downstream genes required for the tendril's capability to sense and climb a support. This study provides an example to explore rare functional variants in plant genomes. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Fine-scale mapping of the 5q11.2 breast cancer locus reveals at least three independent risk variants regulating MAP3K1.

PubMed

Glubb, Dylan M; Maranian, Mel J; Michailidou, Kyriaki; Pooley, Karen A; Meyer, Kerstin B; Kar, Siddhartha; Carlebur, Saskia; O'Reilly, Martin; Betts, Joshua A; Hillman, Kristine M; Kaufmann, Susanne; Beesley, Jonathan; Canisius, Sander; Hopper, John L; Southey, Melissa C; Tsimiklis, Helen; Apicella, Carmel; Schmidt, Marjanka K; Broeks, Annegien; Hogervorst, Frans B; van der Schoot, C Ellen; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A; Ruebner, Matthias; Ekici, Arif B; Beckmann, Matthias W; Peto, Julian; dos-Santos-Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Pharoah, Paul D P; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Sawyer, Elinor J; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Burwinkel, Barbara; Marme, Frederik; Yang, Rongxi; Surowy, Harald; Guénel, Pascal; Truong, Thérèse; Menegaux, Florence; Sanchez, Marie; Bojesen, Stig E; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; González-Neira, Anna; Benitez, Javier; Zamora, M Pilar; Arias Perez, Jose Ignacio; Anton-Culver, Hoda; Neuhausen, Susan L; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Ko, Yon-Dschun; Brüning, Thomas; Nevanlinna, Heli; Muranen, Taru A; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Tanaka, Hideo; Dörk, Thilo; Bogdanova, Natalia V; Helbig, Sonja; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Wu, Anna H; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O; Lambrechts, Diether; Zhao, Hui; Weltens, Caroline; van Limbergen, Erik; Chang-Claude, Jenny; Flesch-Janys, Dieter; Rudolph, Anja; Seibold, Petra; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Capra, Fabio; Couch, Fergus J; Olson, Janet E; Hallberg, Emily; Vachon, Celine; Giles, Graham G; Milne, Roger L; McLean, Catriona; Haiman, Christopher A; Henderson, Brian E; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S; Labrèche, France; Dumont, Martine; Teo, Soo Hwang; Yip, Cheng Har; See, Mee-Hoong; Cornes, Belinda; Cheng, Ching-Yu; Ikram, M Kamran; Kristensen, Vessela; Zheng, Wei; Halverson, Sandra L; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L; Knight, Julia A; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A E M; Seynaeve, Caroline; Van Asperen, Christi J; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J; Lissowska, Jolanta; Czene, Kamila; Klevebring, Daniel; Darabi, Hatef; Eriksson, Mikael; Hooning, Maartje J; Hollestelle, Antoinette; Martens, John W M; Collée, J Margriet; Hall, Per; Li, Jingmei; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Cross, Simon S; Reed, Malcolm W R; Blot, William; Signorello, Lisa B; Cai, Qiuyin; Shah, Mitul; Ghoussaini, Maya; Kang, Daehee; Choi, Ji-Yeob; Park, Sue K; Noh, Dong-Young; Hartman, Mikael; Miao, Hui; Lim, Wei Yen; Tang, Anthony; Hamann, Ute; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Olswold, Curtis; Slager, Susan; Toland, Amanda E; Yannoukakos, Drakoulis; Shen, Chen-Yang; Wu, Pei-Ei; Yu, Jyh-Cherng; Hou, Ming-Feng; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Pita, Guillermo; Alonso, M Rosario; Álvarez, Nuria; Herrero, Daniel; Tessier, Daniel C; Vincent, Daniel; Bacot, Francois; Luccarini, Craig; Baynes, Caroline; Ahmed, Shahana; Healey, Catherine S; Brown, Melissa A; Ponder, Bruce A J; Chenevix-Trench, Georgia; Thompson, Deborah J; Edwards, Stacey L; Easton, Douglas F; Dunning, Alison M; French, Juliet D

2015-01-08

Genome-wide association studies (GWASs) have revealed SNP rs889312 on 5q11.2 to be associated with breast cancer risk in women of European ancestry. In an attempt to identify the biologically relevant variants, we analyzed 909 genetic variants across 5q11.2 in 103,991 breast cancer individuals and control individuals from 52 studies in the Breast Cancer Association Consortium. Multiple logistic regression analyses identified three independent risk signals: the strongest associations were with 15 correlated variants (iCHAV1), where the minor allele of the best candidate, rs62355902, associated with significantly increased risks of both estrogen-receptor-positive (ER(+): odds ratio [OR] = 1.24, 95% confidence interval [CI] = 1.21-1.27, ptrend = 5.7 × 10(-44)) and estrogen-receptor-negative (ER(-): OR = 1.10, 95% CI = 1.05-1.15, ptrend = 3.0 × 10(-4)) tumors. After adjustment for rs62355902, we found evidence of association of a further 173 variants (iCHAV2) containing three subsets with a range of effects (the strongest was rs113317823 [pcond = 1.61 × 10(-5)]) and five variants composing iCHAV3 (lead rs11949391; ER(+): OR = 0.90, 95% CI = 0.87-0.93, pcond = 1.4 × 10(-4)). Twenty-six percent of the prioritized candidate variants coincided with four putative regulatory elements that interact with the MAP3K1 promoter through chromatin looping and affect MAP3K1 promoter activity. Functional analysis indicated that the cancer risk alleles of four candidates (rs74345699 and rs62355900 [iCHAV1], rs16886397 [iCHAV2a], and rs17432750 [iCHAV3]) increased MAP3K1 transcriptional activity. Chromatin immunoprecipitation analysis revealed diminished GATA3 binding to the minor (cancer-protective) allele of rs17432750, indicating a mechanism for its action. We propose that the cancer risk alleles act to increase MAP3K1 expression in vivo and might promote breast cancer cell survival. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

RNA Futile Cycling in Model Persisters Derived from MazF Accumulation (Open Access)

DTIC Science & Technology

2015-11-17

they remained highly metabolically active . We further uncovered a futile cycle driven by continued transcription and MazF-mediated transcript degradation...presence of antibiotics (7), although exceptions have been noted for prod- rugs that require activation (8). Rather, they are rare phenotypic variants...arise from slowly grow- ing or nongrowing bacteria with reduced metabolic activity , where antibiotic targets are inactive and resilient to antibiotic

TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

PubMed Central

Nyquist, Michael D.; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S.; Vessella, Robert L.; Silverstein, Kevin A. T.; Voytas, Daniel F.; Dehm, Scott M.

2013-01-01

Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC. PMID:24101480

Integrating alternative splicing detection into gene prediction.

PubMed

Foissac, Sylvain; Schiex, Thomas

2005-02-10

Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGENE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline.

Significance of functional disease-causal/susceptible variants identified by whole-genome analyses for the understanding of human diseases.

PubMed

Hitomi, Yuki; Tokunaga, Katsushi

2017-01-01

Human genome variation may cause differences in traits and disease risks. Disease-causal/susceptible genes and variants for both common and rare diseases can be detected by comprehensive whole-genome analyses, such as whole-genome sequencing (WGS), using next-generation sequencing (NGS) technology and genome-wide association studies (GWAS). Here, in addition to the application of an NGS as a whole-genome analysis method, we summarize approaches for the identification of functional disease-causal/susceptible variants from abundant genetic variants in the human genome and methods for evaluating their functional effects in human diseases, using an NGS and in silico and in vitro functional analyses. We also discuss the clinical applications of the functional disease causal/susceptible variants to personalized medicine.

Characterization of Basigin Isoforms and the Inhibitory Function of Basigin-3 in Human Hepatocellular Carcinoma Proliferation and Invasion ▿

PubMed Central

Liao, Cheng-Gong; Kong, Ling-Min; Song, Fei; Xing, Jin-Liang; Wang, Long-Xin; Sun, Zhi-Jian; Tang, Hao; Yao, Hui; Zhang, Yang; Wang, Li; Wang, Yu; Yang, Xiang-Min; Li, Yu; Chen, Zhi-Nan

2011-01-01

Basigin, which has four isoforms, plays an important role in invasion of hepatocellular carcinoma (HCC). Detailed transcriptional regulation and functions of the basigin isoforms have not been reported except in the case of the predominant isoform basigin-2, which act as inducer of matrix metalloproteinases (MMPs). Here we determined that basigin-2, basigin-3, and basigin-4 were the most abundant transcript variants in human cell lines. GeneRacer PCR and luciferase reporter assays showed that basigin-3 and basigin-4 were initiated from an alternative promoter. Basigin-3 and basigin-4 were widely expressed in various normal human tissues at the mRNA level and were upregulated in HCC tissues compared to in normal tissues. Western blotting and confocal imaging showed that glycosylated basigin-3 and basigin-4 were expressed and localized to the plasma membrane. However, in cultured cell lines, only native basigin-3, and not basigin-4, was detected at protein level. Overexpression of basigin-3 inhibited HCC cell proliferation, MMP induction, and cell invasion in vitro and in vivo. Bimolecular fluorescence complementation assays and nuclear magnetic resonance (NMR) analysis indicated that basigin-3 interacted with basigin-2 to form hetero-oligomers. In conclusion, we systematically investigated the alternative splicing of basigin and found that basigin-3 could inhibit HCC proliferation and invasion, probably through interaction with basigin-2 as an endogenous inhibitor via hetero-oligomerization. PMID:21536654

CCAAT-enhancer-binding protein β (C/EBPβ) and downstream human placental growth hormone genes are targets for dysregulation in pregnancies complicated by maternal obesity.

PubMed

Vakili, Hana; Jin, Yan; Menticoglou, Savas; Cattini, Peter A

2013-08-02

Human chorionic somatomammotropin (CS) and placental growth hormone variant (GH-V) act as metabolic adaptors in response to maternal insulin resistance, which occurs in "normal" pregnancy. Maternal obesity can exacerbate this "resistance," suggesting that CS, GH-V, or transcription factors that regulate their production might be targets. The human CS genes, hCS-A and hCS-B, flank the GH-V gene. A significant decrease in pre-term placental CS/GH-V RNA levels was observed in transgenic mice containing the CS/GH-V genes in a model of high fat diet (HFD)-induced maternal obesity. Similarly, a decrease in CS/GH-V RNA levels was detected in term placentas from obese (body mass index (BMI) ≥ 35 kg/m(2)) versus lean (BMI 20-25 kg/m(2)) women. A specific decrease in transcription factor CCAAT-enhancer-binding protein β (C/EBPβ) RNA levels was also seen with obesity; C/EBPβ is required for mouse placenta development and is expressed, like CS and GH-V, in syncytiotrophoblasts. Binding of C/EBPβ to the CS gene downstream enhancer regions, which by virtue of their position distally flank the GH-V gene, was reduced in placenta chromatin from mice on a HFD and in obese women; a corresponding decrease in RNA polymerase II associated with CS/GH-V promoters was also observed. Detection of decreased endogenous CS/GH-V RNA levels in human placental tumor cells treated with C/EBPβ siRNA is consistent with a direct effect. These data provide evidence for CS/GH-V dysregulation in acute HFD-induced obesity in mouse pregnancy and chronic obesity in human pregnancy and implicate C/EBPβ, a factor associated with CS regulation and placental development.

Characterizing genomic alterations in cancer by complementary functional associations.

PubMed

Kim, Jong Wook; Botvinnik, Olga B; Abudayyeh, Omar; Birger, Chet; Rosenbluh, Joseph; Shrestha, Yashaswi; Abazeed, Mohamed E; Hammerman, Peter S; DiCara, Daniel; Konieczkowski, David J; Johannessen, Cory M; Liberzon, Arthur; Alizad-Rahvar, Amir Reza; Alexe, Gabriela; Aguirre, Andrew; Ghandi, Mahmoud; Greulich, Heidi; Vazquez, Francisca; Weir, Barbara A; Van Allen, Eliezer M; Tsherniak, Aviad; Shao, Diane D; Zack, Travis I; Noble, Michael; Getz, Gad; Beroukhim, Rameen; Garraway, Levi A; Ardakani, Masoud; Romualdi, Chiara; Sales, Gabriele; Barbie, David A; Boehm, Jesse S; Hahn, William C; Mesirov, Jill P; Tamayo, Pablo

2016-05-01

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.

Circular RNAs: analysis, expression and potential functions

PubMed Central

Salzman, Julia

2016-01-01

Just a few years ago, it had been assumed that the dominant RNA isoforms produced from eukaryotic genes were variants of messenger RNA, functioning as intermediates in gene expression. In early 2012, however, a surprising discovery was made: circular RNA (circRNA) was shown to be a transcriptional product in thousands of human and mouse genes and in hundreds of cases constituted the dominant RNA isoform. Subsequent studies revealed that the expression of circRNAs is developmentally regulated, tissue and cell-type specific, and shared across the eukaryotic tree of life. These features suggest important functions for these molecules. Here, we describe major advances in the field of circRNA biology, focusing on the regulation of and functional roles played by these molecules. PMID:27246710

Adaptive evolution of the lager brewing yeast Saccharomyces pastorianus for improved growth under hyperosmotic conditions and its influence on fermentation performance.

PubMed

Ekberg, Jukka; Rautio, Jari; Mattinen, Laura; Vidgren, Virve; Londesborough, John; Gibson, Brian R

2013-05-01

An adaptive evolution method to obtain stable Saccharomyces pastorianus brewing yeast variants with improved fermentation capacity is described. The procedure involved selection for rapid growth resumption at high osmotic strength. It was applied to a lager strain and to a previously isolated ethanol-tolerant strain. Fermentation performance of strains was compared at 15 °P wort strength. A selected osmotolerant variant of the ethanol-tolerant strain showed significantly shorter fermentation time than the parent strain, producing 6.45% alcohol by volume beer in 4-5 days with mostly similar organoleptic properties to the original strain. Diacetyl and pentanedione contents were 50-75% and 3-methylbutyl acetate and 2-phenylethyl acetate 50% higher than with the original strain, leading to a small flavour change. The variant contained significantly less intracellular trehalose and glycogen than the parent. Transcriptional analysis of selected genes at 24 h revealed reduced transcription of hexose transport genes and increased transcription of the MALx1 and MALx2 genes, responsible for α-glucoside uptake and metabolism. It is suggested that an attenuated stress response contributes to the improved fermentation performance. Results show that sequential selection for both ethanol tolerance and rapid growth at high osmotic strength can provide strains with enhanced fermentation speed with acceptable product quality. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Selection of antigenically advanced variants of seasonal influenza viruses.

PubMed

Li, Chengjun; Hatta, Masato; Burke, David F; Ping, Jihui; Zhang, Ying; Ozawa, Makoto; Taft, Andrew S; Das, Subash C; Hanson, Anthony P; Song, Jiasheng; Imai, Masaki; Wilker, Peter R; Watanabe, Tokiko; Watanabe, Shinji; Ito, Mutsumi; Iwatsuki-Horimoto, Kiyoko; Russell, Colin A; James, Sarah L; Skepner, Eugene; Maher, Eileen A; Neumann, Gabriele; Klimov, Alexander I; Kelso, Anne; McCauley, John; Wang, Dayan; Shu, Yuelong; Odagiri, Takato; Tashiro, Masato; Xu, Xiyan; Wentworth, David E; Katz, Jacqueline M; Cox, Nancy J; Smith, Derek J; Kawaoka, Yoshihiro

2016-05-23

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

Region-specific expression and hormonal regulation of the first exon variants of rat prolactin receptor mRNA in rat brain and anterior pituitary gland.

PubMed

Nogami, H; Hoshino, R; Ogasawara, K; Miyamoto, S; Hisano, S

2007-08-01

Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

Increased Risk of Interstitial Lung Disease in Children with a Single R288K Variant of ABCA3

PubMed Central

Wittmann, Thomas; Frixel, Sabrina; Höppner, Stefanie; Schindlbeck, Ulrike; Schams, Andrea; Kappler, Matthias; Hegermann, Jan; Wrede, Christoph; Liebisch, Gerhard; Vierzig, Anne; Zacharasiewicz, Angela; Kopp, Matthias Volkmar; Poets, Christian F; Baden, Winfried; Hartl, Dominik; van Kaam, Anton H; Lohse, Peter; Aslanidis, Charalampos; Zarbock, Ralf; Griese, Matthias

2016-01-01

The ABCA3 gene encodes a lipid transporter in type II pneumocytes critical for survival and normal respiratory function. The frequent ABCA3 variant R288K increases the risk for neonatal respiratory distress syndrome among term and late preterm neonates, but its role in children’s interstitial lung disease has not been studied in detail. In a retrospective cohort study of 228 children with interstitial lung disease related to the alveolar surfactant system, the frequency of R288K was assessed and the phenotype of patients carrying a single R288K variant further characterized by clinical course, lung histology, computed tomography and bronchoalveolar lavage phosphatidylcholine PC 32:0. Cell lines stably transfected with ABCA3-R288K were analyzed for intracellular transcription, processing and targeting of the protein. ABCA3 function was assessed by detoxification assay of doxorubicin, and the induction and volume of lamellar bodies. We found nine children with interstitial lung disease carrying a heterozygous R288K variant, a frequency significantly higher than in the general Caucasian population. All identified patients had neonatal respiratory insufficiency, recovered and developed chronic interstitial lung disease with intermittent exacerbations during early childhood. In vitro analysis showed normal transcription, processing, and targeting of ABCA3-R288K, but impaired detoxification function and smaller lamellar bodies. We propose that the R288K variant can underlie interstitial lung disease in childhood due to reduced function of ABCA3, demonstrated by decelerated detoxification of doxorubicin, reduced PC 32:0 content and decreased lamellar body volume. PMID:26928390

Venomics of the Australian eastern brown snake (Pseudonaja textilis): Detection of new venom proteins and splicing variants.

PubMed

Viala, Vincent Louis; Hildebrand, Diana; Trusch, Maria; Fucase, Tamara Mieco; Sciani, Juliana Mozer; Pimenta, Daniel Carvalho; Arni, Raghuvir K; Schlüter, Hartmut; Betzel, Christian; Mirtschin, Peter; Dunstan, Nathan; Spencer, Patrick Jack

2015-12-01

The eastern brown snake is the predominant cause of snakebites in mainland Australia. Its venom induces defibrination coagulopathy, renal failure and microangiopathic hemolytic anemia. Cardiovascular collapse has been described as an early cause of death in patients, but, so far, the mechanisms involved have not been fully identified. In the present work, we analysed the venome of Pseudonaja textilis by combining high throughput proteomics and transcriptomics, aiming to further characterize the components of this venom. The combination of these techniques in the analysis and identification of toxins, venom proteins and putative toxins allowed the sequence description and the identification of the following: prothrombinase coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and "acidic PLA2", three-finger toxins (3FTx) and the Kunitz-type protease inhibitor textilinin, venom metalloproteinase, C-type lectins, cysteine rich secretory proteins, calreticulin, dipeptidase 2, as well as evidences of Heloderma lizard peptides. Deep data-mining analysis revealed the secretion of a new transcript variant of venom coagulation factor 5a and the existence of a splicing variant of PLA2 modifying the UTR and signal peptide from a same mature protein. The transcriptome revealed the diversity of transcripts and mutations, and also indicates that splicing variants can be an important source of toxin variation. Copyright © 2015 Elsevier Ltd. All rights reserved.

The transcriptome of Candida albicans mitochondria and the evolution of organellar transcription units in yeasts.

PubMed

Kolondra, Adam; Labedzka-Dmoch, Karolina; Wenda, Joanna M; Drzewicka, Katarzyna; Golik, Pawel

2015-10-21

Yeasts show remarkable variation in the organization of their mitochondrial genomes, yet there is little experimental data on organellar gene expression outside few model species. Candida albicans is interesting as a human pathogen, and as a representative of a clade that is distant from the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Unlike them, it encodes seven Complex I subunits in its mtDNA. No experimental data regarding organellar expression were available prior to this study. We used high-throughput RNA sequencing and traditional RNA biology techniques to study the mitochondrial transcriptome of C. albicans strains BWP17 and SN148. The 14 protein-coding genes, two ribosomal RNA genes, and 24 tRNA genes are expressed as eight primary polycistronic transcription units. We also found transcriptional activity in the noncoding regions, and antisense transcripts that could be a part of a regulatory mechanism. The promoter sequence is a variant of the nonanucleotide identified in other yeast mtDNAs, but some of the active promoters show significant departures from the consensus. The primary transcripts are processed by a tRNA punctuation mechanism into the monocistronic and bicistronic mature RNAs. The steady state levels of various mature transcripts exhibit large differences that are a result of posttranscriptional regulation. Transcriptome analysis allowed to precisely annotate the positions of introns in the RNL (2), COB (2) and COX1 (4) genes, as well as to refine the annotation of tRNAs and rRNAs. Comparative study of the mitochondrial genome organization in various Candida species indicates that they undergo shuffling in blocks usually containing 2-3 genes, and that their arrangement in primary transcripts is not conserved. tRNA genes with their associated promoters, as well as GC-rich sequence elements play an important role in these evolutionary events. The main evolutionary force shaping the mitochondrial genomes of yeasts is the frequent recombination, constantly breaking apart and joining genes into novel primary transcription units. The mitochondrial transcription units are constantly rearranged in evolution shaping the features of gene expression, such as the presence of secondary promoter sites that are inactive, or act as "booster" promoters, simplified transcriptional regulation and reliance on posttranscriptional mechanisms.

Tissue-Specific and Genetic Regulation of Insulin Sensitivity-Associated Transcripts in African Americans

PubMed Central

Sharma, Neeraj K.; Sajuthi, Satria P.; Chou, Jeff W.; Calles-Escandon, Jorge; Demons, Jamehl; Rogers, Samantha; Ma, Lijun; Palmer, Nicholette D.; McWilliams, David R.; Beal, John; Comeau, Mary E.; Cherry, Kristina; Hawkins, Gregory A.; Menon, Lata; Kouba, Ethel; Davis, Donna; Burris, Marcie; Byerly, Sara J.; Easter, Linda; Bowden, Donald W.; Freedman, Barry I.; Langefeld, Carl D.

2016-01-01

Context: Compared with European Americans, African Americans (AAs) are more insulin resistant, have a higher insulin secretion response to glucose, and develop type 2 diabetes more often. Molecular processes and/or genetic variations contributing to altered glucose homeostasis in high-risk AAs remain uncharacterized. Objective: Adipose and muscle transcript expression profiling and genotyping were performed in 260 AAs to identify genetic regulatory mechanisms associated with insulin sensitivity (SI). We hypothesized that: 1) transcription profiles would reveal tissue-specific modulation of physiologic pathways with SI, and 2) a subset of SI-associated transcripts would be controlled by DNA sequence variants as expression quantitative traits, and these variants in turn would be associated with SI. Design and Settings: The cross-sectional research study was performed in a clinical research unit. Participants: Unrelated nondiabetic AAs were recruited for the study. Main Outcome Measures: SI was measured by frequently sampled iv glucose tolerance test. Results: The expression levels of 2212 transcripts in adipose and 145 transcripts in muscle were associated with SI. Genes involved in eIF2, eIF4-p70S6K, and mTOR signaling were modulated with SI in both tissues. Genes involved in leukocyte extravasation signaling showed adipose-specific regulation, and genes involved in oxidative phosphorylation had discordant regulation between tissues. Intersecting cis-expression quantitative trait loci results with data from transcript-SI association analysis identified cis-regulatory single nucleotide polymorphisms for 363 and 42 SI-associated transcripts in adipose and muscle, respectively. Cis-eSNPs for three SI-associated adipose transcripts, NINJ1, AGA, and CLEC10A were associated with SI. Abrogation of NINJ1 induction in THP1 macrophages modulated expression of genes in chemokine signaling, cell adhesion, and angiogenesis pathways. Conclusion: This study identified multiple pathways associated with SI; particularly discordant tissue-specific regulation of the oxidative phosphorylation pathway, and adipose-specific regulation of transcripts in the leukocyte extravasation signaling pathway that seem to be important in insulin resistance. Identification of single nucleotide polymorphisms associated with SI and with modulation of expression of SI-associated transcripts, including NINJ1, reveals novel genetic regulatory mechanisms of insulin resistance in AAs. PMID:26789776

The predominant WT1 isoform (+KTS) encodes a DNA-binding protein targeting the planar cell polarity gene Scribble in renal podocytes.

PubMed

Wells, Julie; Rivera, Miguel N; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A

2010-07-01

WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three-amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)-regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway.

Kruppel-Like Factor 15 Is Critical for the Development of Left Ventricular Hypertrophy.

PubMed

Patel, Sheila K; Ramchand, Jay; Crocitti, Vincenzo; Burrell, Louise M

2018-04-27

Left ventricular hypertrophy (LVH) is an independent risk factor for adverse cardiovascular events and is often present in patients with hypertension. Treatment to reduce blood pressure and regress LVH is key to improving health outcomes, but currently available drugs have only modest cardioprotective effects. Improved understanding of the molecular mechanisms involved in the development of LVH may lead to new therapeutic targets in the future. There is now compelling evidence that the transcription factor Kruppel-like factor 15 (KLF15) is an important negative regulator of cardiac hypertrophy in both experimental models and in man. Studies have reported that loss or suppression of KLF15 contributes to LVH, through lack of inhibition of pro-hypertrophic transcription factors and stimulation of trophic and fibrotic signaling pathways. This review provides a summary of the experimental and human studies that have investigated the role of KLF15 in the development of cardiac hypertrophy. It also discusses our recent paper that described the contribution of genetic variants in KLF15 to the development of LVH and heart failure in high-risk patients.

Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

PubMed

Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

2018-05-11

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

The Asian-American E6 Variant Protein of Human Papillomavirus 16 Alone Is Sufficient To Promote Immortalization, Transformation, and Migration of Primary Human Foreskin Keratinocytes

PubMed Central

Niccoli, Sarah; Abraham, Suraj; Richard, Christina

2012-01-01

We examined how well the human papillomavirus (HPV) E6 oncogene can function in the absence of the E7 oncogene during the carcinogenic process in human keratinocytes using a common HPV variant strongly associated with cervical cancer: the Asian-American E6 variant (AAE6). This E6 variant is 20 times more frequently detected in cervical cancer than the prototype European E6 variant, as evidenced by independent epidemiological data. Using cell culture and cell-based functional assays, we assessed how this variant can perform crucial carcinogenesis steps compared to the prototype E6 variant. The ability to immortalize and transform primary human foreskin keratinocytes (PHFKs) to acquire resilient phenotypes and the ability to promote cell migration were evaluated. The immortalization capability was assayed based on population doublings, number of passages, surpassing mortality stages 1 and 2, human telomerase reverse transcriptase (hTERT) expression, and the ability to overcome G1 arrest via p53 degradation. Transformation and migration efficiency were analyzed using a combination of functional cell-based assays. We observed that either AAE6 or prototype E6 proteins alone were sufficient to immortalize PHFKs, although AAE6 was more potent in doing so. The AAE6 variant protein alone pushed PHFKs through transformation and significantly increased their migration ability over that of the E6 prototype. Our findings are in line with epidemiological data that the AA variant of HPV16 confers an increased risk over the European prototype for cervical cancer, as evidenced by a superior immortalization, transformation, and metastatic potential. PMID:22951839

AF4 and AF4N protein complexes: recruitment of P-TEFb kinase, their interactome and potential functions

PubMed Central

Scholz, Bastian; Kowarz, Eric; Rössler, Tanja; Ahmad, Khalil; Steinhilber, Dieter; Marschalek, Rolf

2015-01-01

AF4/AFF1 and AF5/AFF4 are the molecular backbone to assemble “super-elongation complexes” (SECs) that have two main functions: (1) control of transcriptional elongation by recruiting the positive transcription elongation factor b (P-TEFb = CyclinT1/CDK9) that is usually stored in inhibitory 7SK RNPs; (2) binding of different histone methyltransferases, like DOT1L, NSD1 and CARM1. This way, transcribed genes obtain specific histone signatures (e.g. H3K79me2/3, H3K36me2) to generate a transcriptional memory system. Here we addressed several questions: how is P-TEFb recruited into SEC, how is the AF4 interactome composed, and what is the function of the naturally occuring AF4N protein variant which exhibits only the first 360 amino acids of the AF4 full-length protein. Noteworthy, shorter protein variants are a specific feature of all AFF protein family members. Here, we demonstrate that full-length AF4 and AF4N are both catalyzing the transition of P-TEFb from 7SK RNP to their N-terminal domain. We have also mapped the protein-protein interaction network within both complexes. In addition, we have first evidence that the AF4N protein also recruits TFIIH and the tumor suppressor MEN1. This indicate that AF4N may have additional functions in transcriptional initiation and in MEN1-dependend transcriptional processes. PMID:26171280

Human NR5A1/SF-1 Mutations Show Decreased Activity on BDNF (Brain-Derived Neurotrophic Factor), an Important Regulator of Energy Balance: Testing Impact of Novel SF-1 Mutations Beyond Steroidogenesis

PubMed Central

Malikova, Jana; Camats, Núria; Fernández-Cancio, Mónica; Heath, Karen; González, Isabel; Caimarí, María; del Campo, Miguel; Albisu, Marian; Kolouskova, Stanislava; Audí, Laura; Flück, Christa E.

2014-01-01

Context Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. Objective To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. Patients 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. Methods SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). Results Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. Conclusions Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations. PMID:25122490

Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis.

PubMed

Malikova, Jana; Camats, Núria; Fernández-Cancio, Mónica; Heath, Karen; González, Isabel; Caimarí, María; del Campo, Miguel; Albisu, Marian; Kolouskova, Stanislava; Audí, Laura; Flück, Christa E

2014-01-01

Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations.