H3 K79 dimethylation marks developmental activation of the beta-globin gene but is reduced upon LCR-mediated high-level transcription.

PubMed

Sawado, Tomoyuki; Halow, Jessica; Im, Hogune; Ragoczy, Tobias; Bresnick, Emery H; Bender, M A; Groudine, Mark

2008-07-15

Genome-wide analyses of the relationship between H3 K79 dimethylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels of wild-type (WT) and transcriptionally impaired beta-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/Delta locus control region (LCR) heterozygous mice reveals no significant H3 K79 dimethylation of the beta-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and DeltaLCR alleles, and both alleles are located in proximity to H3 K79 dimethylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the DeltaLCR allele compared with the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 dimethylation is inversely correlated with beta-globin gene expression levels. Thus, while our results support a link between H3 K79 dimethylation and gene expression, high levels of this mark are not essential for high level beta-globin gene transcription. We propose that H3 K79 dimethylation is destabilized on a highly transcribed template.

Top-level dynamics and the regulated gene response of feed-forward loop transcriptional motifs.

PubMed

Mayo, Michael; Abdelzaher, Ahmed; Perkins, Edward J; Ghosh, Preetam

2014-09-01

Feed-forward loops are hierarchical three-node transcriptional subnetworks, wherein a top-level protein regulates the activity of a target gene via two paths: a direct-regulatory path, and an indirect route, whereby the top-level proteins act implicitly through an intermediate transcription factor. Using a transcriptional network of the model bacterium Escherichia coli, we confirmed that nearly all types of feed-forward loop were significantly overrepresented in the bacterial network. We then used mathematical modeling to study their dynamics by manipulating the rise times of the top-level protein concentration, termed the induction time, through alteration of the protein destruction rates. Rise times of the regulated proteins exhibited two qualitatively different regimes, depending on whether top-level inductions were "fast" or "slow." In the fast regime, rise times were nearly independent of rapid top-level inductions, indicative of biological robustness, and occurred when RNA production rate-limits the protein yield. Alternatively, the protein rise times were dependent upon slower top-level inductions, greater than approximately one bacterial cell cycle. An equation is given for this crossover, which depends upon three parameters of the direct-regulatory path: transcriptional cooperation at the DNA-binding site, a protein-DNA dissociation constant, and the relative magnitude of the top-level protien concentration.

YY1 binding association with sex-biased transcription revealed through X-linked transcript levels and allelic binding analyses.

PubMed

Chen, Chih-Yu; Shi, Wenqiang; Balaton, Bradley P; Matthews, Allison M; Li, Yifeng; Arenillas, David J; Mathelier, Anthony; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Brown, Carolyn J; Wasserman, Wyeth W

2016-11-18

Sex differences in susceptibility and progression have been reported in numerous diseases. Female cells have two copies of the X chromosome with X-chromosome inactivation imparting mono-allelic gene silencing for dosage compensation. However, a subset of genes, named escapees, escape silencing and are transcribed bi-allelically resulting in sexual dimorphism. Here we conducted in silico analyses of the sexes using human datasets to gain perspectives into such regulation. We identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 CAGE data. Significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of Xi-specific superloops. Our study suggests a role for YY1 in transcriptional activity on Xi in general through sequence-specific binding, and its involvement at superloop anchors.

Polycomb Responds to Low Levels of Transcription.

PubMed

Berrozpe, Georgina; Bryant, Gene O; Warpinski, Katherine; Spagna, Dan; Narayan, Santosh; Shah, Shivangi; Ptashne, Mark

2017-07-25

How is Polycomb (Pc), a eukaryotic negative regulator of transcription, targeted to specific mammalian genes? Our genome-wide analysis of the Pc mark H3K27me3 in murine cells revealed that Pc is preferentially associated with CpG island promoters of genes that are transcribed at a low level and less so with promoters of genes that are either silent or more highly expressed. Studies of the CpG island promoter of the Kit gene demonstrate that Pc is largely absent when the gene is silent in myeloid cells, as well as when the gene is highly expressed in mast cells. Manipulations that increase transcription in the former case, and reduce it in the latter, increase Pc occupancy. The average negative effect of Pc, we infer, is about 2-fold. We suggest possible biological roles for such negative effects and propose a mechanism by which Pc might be recruited to weakly transcribed genes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

YY1 binding association with sex-biased transcription revealed through X-linked transcript levels and allelic binding analyses

PubMed Central

Chen, Chih-yu; Shi, Wenqiang; Balaton, Bradley P.; Matthews, Allison M.; Li, Yifeng; Arenillas, David J.; Mathelier, Anthony; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.; Brown, Carolyn J.; Wasserman, Wyeth W.

2016-01-01

Sex differences in susceptibility and progression have been reported in numerous diseases. Female cells have two copies of the X chromosome with X-chromosome inactivation imparting mono-allelic gene silencing for dosage compensation. However, a subset of genes, named escapees, escape silencing and are transcribed bi-allelically resulting in sexual dimorphism. Here we conducted in silico analyses of the sexes using human datasets to gain perspectives into such regulation. We identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 CAGE data. Significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of Xi-specific superloops. Our study suggests a role for YY1 in transcriptional activity on Xi in general through sequence-specific binding, and its involvement at superloop anchors. PMID:27857184

Transcriptional regulation of the novel monoamine oxidase renalase: Crucial roles of transcription factors Sp1, STAT3, and ZBP89.

PubMed

Sonawane, Parshuram J; Gupta, Vinayak; Sasi, Binu K; Kalyani, Ananthamohan; Natarajan, Bhargavi; Khan, Abrar A; Sahu, Bhavani S; Mahapatra, Nitish R

2014-11-11

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5'-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.

Pervasive, Coordinated Protein-Level Changes Driven by Transcript Isoform Switching during Meiosis.

PubMed

Cheng, Ze; Otto, George Maxwell; Powers, Emily Nicole; Keskin, Abdurrahman; Mertins, Philipp; Carr, Steven Alfred; Jovanovic, Marko; Brar, Gloria Ann

2018-02-22

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 5' extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced. Copyright © 2018 Elsevier Inc. All rights reserved.

Congruence of Additive and Non-Additive Effects on Gene Expression Estimated from Pedigree and SNP Data

PubMed Central

Powell, Joseph E.; Henders, Anjali K.; McRae, Allan F.; Kim, Jinhee; Hemani, Gibran; Martin, Nicholas G.; Dermitzakis, Emmanouil T.; Gibson, Greg

2013-01-01

There is increasing evidence that heritable variation in gene expression underlies genetic variation in susceptibility to disease. Therefore, a comprehensive understanding of the similarity between relatives for transcript variation is warranted—in particular, dissection of phenotypic variation into additive and non-additive genetic factors and shared environmental effects. We conducted a gene expression study in blood samples of 862 individuals from 312 nuclear families containing MZ or DZ twin pairs using both pedigree and genotype information. From a pedigree analysis we show that the vast majority of genetic variation across 17,994 probes is additive, although non-additive genetic variation is identified for 960 transcripts. For 180 of the 960 transcripts with non-additive genetic variation, we identify expression quantitative trait loci (eQTL) with dominance effects in a sample of 339 unrelated individuals and replicate 31% of these associations in an independent sample of 139 unrelated individuals. Over-dominance was detected and replicated for a trans association between rs12313805 and ETV6, located 4MB apart on chromosome 12. Surprisingly, only 17 probes exhibit significant levels of common environmental effects, suggesting that environmental and lifestyle factors common to a family do not affect expression variation for most transcripts, at least those measured in blood. Consistent with the genetic architecture of common diseases, gene expression is predominantly additive, but a minority of transcripts display non-additive effects. PMID:23696747

Congruence of additive and non-additive effects on gene expression estimated from pedigree and SNP data.

PubMed

Powell, Joseph E; Henders, Anjali K; McRae, Allan F; Kim, Jinhee; Hemani, Gibran; Martin, Nicholas G; Dermitzakis, Emmanouil T; Gibson, Greg; Montgomery, Grant W; Visscher, Peter M

2013-05-01

There is increasing evidence that heritable variation in gene expression underlies genetic variation in susceptibility to disease. Therefore, a comprehensive understanding of the similarity between relatives for transcript variation is warranted--in particular, dissection of phenotypic variation into additive and non-additive genetic factors and shared environmental effects. We conducted a gene expression study in blood samples of 862 individuals from 312 nuclear families containing MZ or DZ twin pairs using both pedigree and genotype information. From a pedigree analysis we show that the vast majority of genetic variation across 17,994 probes is additive, although non-additive genetic variation is identified for 960 transcripts. For 180 of the 960 transcripts with non-additive genetic variation, we identify expression quantitative trait loci (eQTL) with dominance effects in a sample of 339 unrelated individuals and replicate 31% of these associations in an independent sample of 139 unrelated individuals. Over-dominance was detected and replicated for a trans association between rs12313805 and ETV6, located 4MB apart on chromosome 12. Surprisingly, only 17 probes exhibit significant levels of common environmental effects, suggesting that environmental and lifestyle factors common to a family do not affect expression variation for most transcripts, at least those measured in blood. Consistent with the genetic architecture of common diseases, gene expression is predominantly additive, but a minority of transcripts display non-additive effects.

V(D)J recombination on minichromosomes is not affected by transcription.

PubMed

Hsieh, C L; McCloskey, R P; Lieber, M R

1992-08-05

It has been shown previously by others that transcription is temporally correlated with the onset of V(D)J recombination at the endogenous antigen receptor loci. We have been interested in determining whether this temporal correlation indicates a causal connection between these two processes. We have compared V(D)J recombination minichromosome substrates that have transcripts running through the recombination zone with substrates that do not in a transient transfection assay. In this system, the substrates acquire a minichromosome conformation within the first several hours after transfection. We find that the substrates recombine equally well over a 100-fold range in transcriptional variation. In additional studies, we have taken substrates that have low levels of transcription and inhibited transcription further by methylating the substrate DNA or by treating the cells with a general transcription inhibitor (alpha-amanitin). Although these treatments decrease the level of expression an additional 10-100-fold, there is still no observable effect on V(D)J recombination. Based on these results, we conclude that transcription is not necessary for the V(D)J reaction mechanism and does not alter substrate structure at the DNA level or at the simplest levels of chromatin structure in a way that affects the reaction.

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references

CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

PubMed Central

El Mouali, Youssef; Gaviria-Cantin, Tania; Gibert, Marta; Westermann, Alexander J.; Vogel, Jörg

2018-01-01

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression. PMID:29879120

Transcript levels of ten-eleven translocation type 1–3 in cervical cancer and non-cancerous cervical tissues

PubMed Central

Bronowicka-Kłys, Dorota Ewa; Roszak, Andrzej; Pawlik, Piotr; Sajdak, Stefan; Sowińska, Anna; Jagodziński, Paweł Piotr

2017-01-01

Decreased expression of ten-eleven translocation (TET1, TET2 and TET3) proteins has been reported in various types of cancer. However, the expression levels of TET proteins in cervical cancer (CC) remain to be elucidated. The present study determined the levels of TET1, TET2 and TET3 transcripts in cancerous (n=80) and non-cancerous cervical tissues (n=41). The results revealed a significant reduction in TET1 transcripts (P=0.0000001) in cervical tissue samples from patients with primary CC compared with samples from control patients. Significantly decreased TET1 transcript levels, as compared to non-cancerous cervical tissues, were also observed in tissue samples with the following characteristics: Stage I (P=0.016), II (P<0.0001), III (P=0.00007) and grade of differentiation G1 (P=0.026), G2 (P=0.00006), G3 (P=0.0007) and Gx (P=0.0004) and squamous histological type (P<0.00001). TET1 transcript levels were significantly lower in patients aged 45–60 years (P=0.0002) and patients age >60 years (P=0.003), as compared with non-cancerous cervical tissues. TET2 transcript levels were lower in cervical cancer tissues classified as stage II (P=0.043) and TET3 transcript levels were lower in stage III samples (P=0.010), tissue samples with a grade of differentiation of G3 (P=0.025) and tissue with squamous type histology (P=0.047), all compared with non-cancerous cervical tissues. The present study demonstrated a significantly reduced level of TET1 transcripts in cancerous cervical tissues, as compared with non-cancerous tissues. Furthermore, decreased TET1-3 transcript levels were identified when patients with CC were stratified by clinicopathological variables, as compared with non-cancerous cervical tissues. PMID:28521490

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

PubMed Central

Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

PubMed Central

Galasinski, Shelly K.; Lively, Tricia N.; Grebe de Barron, Alexandra; Goodrich, James A.

2000-01-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression. PMID:10688640

Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones.

PubMed

Galasinski, S K; Lively, T N; Grebe De Barron, A; Goodrich, J A

2000-03-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression.

NUCLEAR FACTOR Y Transcription Factors Have Both Opposing and Additive Roles in ABA-Mediated Seed Germination

PubMed Central

Kumimoto, Roderick W.; Siriwardana, Chamindika L.; Gayler, Krystal K.; Risinger, Jan R.; Siefers, Nicholas; Holt, Ben F.

2013-01-01

In the model organism Arabidopsis thaliana the heterotrimeric transcription factor NUCLEAR FACTOR Y (NF-Y) has been shown to play multiple roles in facilitating plant growth and development. Although NF-Y itself represents a multi-protein transcriptional complex, recent studies have shown important interactions with other transcription factors, especially those in the bZIP family. Here we add to the growing evidence that NF-Y and bZIP form common complexes to affect many processes. We carried out transcriptional profiling on nf-yc mutants and through subsequent analyses found an enrichment of bZIP binding sites in the promoter elements of misregulated genes. Using NF-Y as bait, yeast two hybrid assays yielded interactions with bZIP proteins that are known to control ABA signaling. Accordingly, we find that plants mutant for several NF-Y subunits show characteristic phenotypes associated with the disruption of ABA signaling. While previous reports have shown additive roles for NF-YC family members in photoperiodic flowering, we found that they can have opposing roles in ABA signaling. Collectively, these results demonstrated the importance and complexity of NF-Y in the integration of environmental and hormone signals. PMID:23527203

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

PubMed Central

Tiziano, Francesco Danilo; Pinto, Anna Maria; Fiori, Stefania; Lomastro, Rosa; Messina, Sonia; Bruno, Claudio; Pini, Antonella; Pane, Marika; D'Amico, Adele; Ghezzo, Alessandro; Bertini, Enrico; Mercuri, Eugenio; Neri, Giovanni; Brahe, Christina

2010-01-01

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I–III) on the basis of clinical severity. All patients have at least one or more (usually 2–4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II–III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials. PMID:19603064

Dual transcriptional-translational cascade permits cellular level tuneable expression control

PubMed Central

Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

2016-01-01

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

Using Poisson mixed-effects model to quantify transcript-level gene expression in RNA-Seq.

PubMed

Hu, Ming; Zhu, Yu; Taylor, Jeremy M G; Liu, Jun S; Qin, Zhaohui S

2012-01-01

RNA sequencing (RNA-Seq) is a powerful new technology for mapping and quantifying transcriptomes using ultra high-throughput next-generation sequencing technologies. Using deep sequencing, gene expression levels of all transcripts including novel ones can be quantified digitally. Although extremely promising, the massive amounts of data generated by RNA-Seq, substantial biases and uncertainty in short read alignment pose challenges for data analysis. In particular, large base-specific variation and between-base dependence make simple approaches, such as those that use averaging to normalize RNA-Seq data and quantify gene expressions, ineffective. In this study, we propose a Poisson mixed-effects (POME) model to characterize base-level read coverage within each transcript. The underlying expression level is included as a key parameter in this model. Since the proposed model is capable of incorporating base-specific variation as well as between-base dependence that affect read coverage profile throughout the transcript, it can lead to improved quantification of the true underlying expression level. POME can be freely downloaded at http://www.stat.purdue.edu/~yuzhu/pome.html. yuzhu@purdue.edu; zhaohui.qin@emory.edu Supplementary data are available at Bioinformatics online.

Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

PubMed

Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

2004-12-01

The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

Complex transcriptional and post-transcriptional regulation of an enzyme for Lipopolysaccharide modification

PubMed Central

Moon, Kyung; Six, David A.; Lee, Hyun-Jung; Raetz, Christian R.H.; Gottesman, Susan

2013-01-01

Summary The PhoQ/PhoP two-component system activates many genes for lipopolysaccharide (LPS) modification when cells are grown at low Mg2+ concentrations. An additional target of PhoQ and PhoP is MgrR, an Hfq-dependent small RNA that negatively regulates expression of eptB, also encoding a protein that carries out LPS modification. Examination of LPS confirmed that MgrR effectively silences EptB; the phosphoethanolamine modification associated with EptB is found in ΔmgrR::kan but not mgrR+ cells. Sigma E has been reported to positively regulate eptB, although the eptB promoter does not have the expected Sigma E recognition motifs. The effects of Sigma E and deletion of mgrR on levels of eptB mRNA were independent, and the same 5′ end was found in both cases. In vitro transcription and the behavior of transcriptional and translational fusions demonstrate that Sigma E acts directly at the level of transcription initiation for eptB, from the same start point as Sigma 70. The results suggest that when Sigma E is active, synthesis of eptB transcript outstrips MgrR-dependent degradation; presumably the modification of LPS is important under these conditions. Adding to the complexity of eptB regulation is a second sRNA, ArcZ, which also directly and negatively regulates eptB. PMID:23659637

Transcriptional bursting explains the noise–versus–mean relationship in mRNA and protein levels

DOE PAGES

Dar, Roy; Shaffer, Sydney M.; Singh, Abhyudai; ...

2016-07-28

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-tocell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: thatmore » increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. In conclusion, the data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.« less

PEPCase Transcript Levels in Mesembryanthemum crystallinum Decline Rapidly upon Relief from Salt Stress 1

PubMed Central

Vernon, Daniel M.; Ostrem, James A.; Schmitt, Juergen M.; Bohnert, Hans J.

1988-01-01

Mesembryanthemum crystallinum plants respond to water stress by changing their pathway of carbon assimilation from C3 to Crassulacean acid metabolism (CAM). Stressed plants are characterized by elevated levels of phosphoenolpyruvate carboxylase (PEPCase) mRNA, protein, and enzyme activity. We wanted to determine whether CAM is a reversible response to environmental conditions or a developmentally programmed adaptation that is irreversibly expressed once induced. Plants were osmotically stressed by irrigation with 500 millimolar NaCl for 12 days to elicit CAM. Salt was then thoroughly flushed from the soil and PEPCase protein and transcript levels were monitored. PEPCase mRNA levels dropped by 77% within 2.5 hours after salt removal. PEPCase activity and polypeptide levels declined more slowly, with a half-life of 2 to 3 days. These results show that PEPCase expression in M. crystallinum is a reversible response to stress that is regulated at the level of transcription or stability of the PEPCase mRNA. Images Fig. 2 Fig. 3 PMID:16666021

47. MAIN WAREHOUSE SECOND LEVEL ADDITION Second level was ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

47. MAIN WAREHOUSE - SECOND LEVEL ADDITION Second level was added in 1941. Note the variety of building materials used in the wall: cement, bricks and finally cement blocks, with wood topping the entire wall. - Hovden Cannery, 886 Cannery Row, Monterey, Monterey County, CA

Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)(2)Cys(6) transcriptional activator and induced by kojic acid at the transcriptional level.

PubMed

Marui, Junichiro; Yamane, Noriko; Ohashi-Kunihiro, Sumiko; Ando, Tomohiro; Terabayashi, Yasunobu; Sano, Motoaki; Ohashi, Shinichi; Ohshima, Eiji; Tachibana, Kuniharu; Higa, Yoshitaka; Nishimura, Marie; Koike, Hideaki; Machida, Masayuki

2011-07-01

A gene encoding the Zn(II)(2)Cys(6) transcriptional factor is clustered with two genes involved in biosynthesis of a secondary metabolite, kojic acid (KA), in Aspergillus oryzae. We determined that the gene was essential for KA production and the transcriptional activation of KA biosynthetic genes, which were triggered by the addition of KA. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Inflammation response at the transcriptional level of HepG2 cells induced by multi-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Piret, Jean-Pascal; Vankoningsloo, Sébastien; Noël, Florence; Mejia Mendoza, Jorge; Lucas, Stéphane; Saout, Christelle; Toussaint, Olivier

2011-07-01

Poor information are currently available about the biological effects of multi-walled carbon nanotubes (MWCNT) on the liver. In this study, we evaluated the effects of MWCNT at the transcriptional level on the classical in vitro model of HepG2 hepatocarcinoma cells. The expression levels of 96 transcript species implicated in the inflammatory and immune responses was studied after a 24h incubation of HepG2 cells in presence of raw MWCNT dispersed in water by stirring. Among the 46 transcript species detected, only a few transcripts including mRNA coding for interleukine-7, chemokines receptor of the C-C families CCR7, as well as Endothelin-1, were statistically more abundant after treatment with MWCNT. Altogether, these data indicate that MWCNT can only induce a weak inflammatory response in HepG2 cells.

Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

PubMed

Dorrell, Richard G; Hinksman, George A; Howe, Christopher J

2016-02-01

Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3' poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNA (Met)-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5' end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes.

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

PubMed

Stapleton, P C; Dobson, A D W

2003-04-25

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

PubMed

Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

2012-07-01

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Mechanical Properties of Transcription

NASA Astrophysics Data System (ADS)

Sevier, Stuart A.; Levine, Herbert

2017-06-01

The mechanical properties of transcription have recently been shown to play a central role in gene expression. However, a full physical characterization of this central biological process is lacking. In this Letter, we introduce a simple description of the basic physical elements of transcription where RNA elongation, RNA polymerase rotation, and DNA supercoiling are coupled. The resulting framework describes the relative amount of RNA polymerase rotation and DNA supercoiling that occurs during RNA elongation. Asymptotic behavior is derived and can be used to experimentally extract unknown mechanical parameters of transcription. Mechanical limits to transcription are incorporated through the addition of a DNA supercoiling-dependent RNA polymerase velocity. This addition can lead to transcriptional stalling and resulting implications for gene expression, chromatin structure and genome organization are discussed.

Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2.

PubMed

Tabor, D E; Kim, J B; Spiegelman, B M; Edwards, P A

1999-07-16

We previously identified stearoyl-CoA desaturase 2 (SCD2) as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear sterol regulatory element binding proteins (SREBPs) or adipocyte determination and differentiation factor 1 (ADD1). A novel sterol regulatory element (SRE) (5'-AGCAGATTGTG-3') identified in the proximal promoter of the mouse SCD2 gene is required for induction of SCD2 promoter-reporter genes in response to cellular sterol depletion (Tabor, D. E., Kim, J. B., Spiegelman, B. M., and Edwards, P. A. (1998) J. Biol. Chem. 273, 22052-22058). In this report, we demonstrate that this novel SRE is both present in the promoter of the SCD1 gene and is critical for the sterol-dependent transcription of SCD1 promoter-reporter genes. Two conserved cis elements (5'-CCAAT-3') lie 5 and 48 base pairs 3' of the novel SREs in the promoters of both the SCD1 and SCD2 murine genes. Mutation of either of these putative NF-Y binding sites attenuates the transcriptional activation of SCD1 or SCD2 promoter-reporter genes in response to cellular sterol deprivation. Induction of both reporter genes is also attenuated when cells are cotransfected with dominant-negative forms of either NF-Y or SREBP. In addition, we demonstrate that the induction of SCD1 and SCD2 mRNAs that occurs during the differentiation of 3T3-L1 preadipocytes to adipocytes is paralleled by an increase in the levels of ADD1/SREBP-1c and that the SCD1 and SCD2 mRNAs are induced to even higher levels in response to ectopic expression of ADD1/SREBP-1c. We conclude that transcription of both SCD1 and SCD2 genes is responsive to cellular sterol levels and to the levels of nuclear SREBP/ADD1 and that transcriptional induction requires three spatially conserved cis elements, that bind SREBP and NF-Y. Additional studies demonstrate that maximal transcriptional repression of SCD2 reporter genes in response to an exogenous polyunsaturated fatty acid is

Transcriptional regulators of Na,K-ATPase subunits

PubMed Central

Li, Zhiqin; Langhans, Sigrid A.

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

Deciphering principles of transcription regulation in eukaryotic genomes

PubMed Central

Nguyen, Dat H; D'haeseleer, Patrik

2006-01-01

Transcription regulation has been responsible for organismal complexity and diversity in the course of biological evolution and adaptation, and it is determined largely by the context-dependent behavior of cis-regulatory elements (CREs). Therefore, understanding principles underlying CRE behavior in regulating transcription constitutes a fundamental objective of quantitative biology, yet these remain poorly understood. Here we present a deterministic mathematical strategy, the motif expression decomposition (MED) method, for deriving principles of transcription regulation at the single-gene resolution level. MED operates on all genes in a genome without requiring any a priori knowledge of gene cluster membership, or manual tuning of parameters. Applying MED to Saccharomyces cerevisiae transcriptional networks, we identified four functions describing four different ways that CREs can quantitatively affect gene expression levels. These functions, three of which have extrema in different positions in the gene promoter (short-, mid-, and long-range) whereas the other depends on the motif orientation, are validated by expression data. We illustrate how nature could use these principles as an additional dimension to amplify the combinatorial power of a small set of CREs in regulating transcription. PMID:16738557

Enzymes Involved in Post-transcriptional RNA Metabolism in Gram-negative bacteria

PubMed Central

Mohanty, Bijoy K.

2018-01-01

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways. PMID:29676246

Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101

PubMed Central

Post, Anton F; Rihtman, Branko; Wang, Qingfeng

2012-01-01

Nitrogen (N) physiology in the marine cyanobacterium Trichodesmium IMS101 was studied along with transcript accumulation of the N-regulatory gene ntcA and of two of its target genes: napA (nitrate assimilation) and nifH (N2 fixation). N2 fixation was impaired in the presence of nitrite, nitrate and urea. Strain IMS101 was capable of growth on these combined N sources at <2 μ but growth rates declined at elevated concentrations. Assimilation of nitrate and urea was impaired in the presence of ammonium. Whereas ecologically relevant N concentrations (2–20 μ) suppressed growth and assimilation, much higher concentrations were required to affect transcript levels. Transcripts of nifH accumulated under nitrogen-fixing conditions; these transcript levels were maintained in the presence of nitrate (100 μ) and ammonium (20 μ). However, nifH transcript levels were below detection at ammonium concentrations >20 μ. napA mRNA was found at low levels in both N2-fixing and ammonium-utilizing filaments, and it accumulated in filaments grown with nitrate. The positive effect of nitrate on napA transcription was abolished by ammonium additions of >200 μ. This effect was restored upon addition of the glutamine synthetase inhibitor -methionin--sulfoximine. Surprisingly, ntcA transcript levels remained high in the presence of ammonium, even at elevated concentrations. These findings indicate that ammonium repression is decoupled from transcriptional activation of ntcA in Trichodesmium IMS101. PMID:21938021

Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101.

PubMed

Post, Anton F; Rihtman, Branko; Wang, Qingfeng

2012-03-01

Nitrogen (N) physiology in the marine cyanobacterium Trichodesmium IMS101 was studied along with transcript accumulation of the N-regulatory gene ntcA and of two of its target genes: napA (nitrate assimilation) and nifH (N(2) fixation). N(2) fixation was impaired in the presence of nitrite, nitrate and urea. Strain IMS101 was capable of growth on these combined N sources at <2 μM but growth rates declined at elevated concentrations. Assimilation of nitrate and urea was impaired in the presence of ammonium. Whereas ecologically relevant N concentrations (2-20 μM) suppressed growth and assimilation, much higher concentrations were required to affect transcript levels. Transcripts of nifH accumulated under nitrogen-fixing conditions; these transcript levels were maintained in the presence of nitrate (100 μM) and ammonium (20 μM). However, nifH transcript levels were below detection at ammonium concentrations >20 μM. napA mRNA was found at low levels in both N(2)-fixing and ammonium-utilizing filaments, and it accumulated in filaments grown with nitrate. The positive effect of nitrate on napA transcription was abolished by ammonium additions of >200 μM. This effect was restored upon addition of the glutamine synthetase inhibitor L-methionin-DL-sulfoximine. Surprisingly, ntcA transcript levels remained high in the presence of ammonium, even at elevated concentrations. These findings indicate that ammonium repression is decoupled from transcriptional activation of ntcA in Trichodesmium IMS101.

Alteration at transcriptional level of cardiac renin-angiotensin system by letrozole treatment.

PubMed

Shekarforoush, Shahnaz; Koohpeyma, Farhad; Safari, Fatemeh

2018-06-17

The use of aromatase inhibitors (AIs) for breast cancer led to a marked change in ventricular function. Since accumulating evidence indicates that overactivation of the cardiac renin-angiotensin system (RAS) plays an important role in the development of cardiovascular diseases such as hypertrophy and remodelling, we aimed to investigate whether letrozole alters the transcription level of RAS related genes in the cardiac tissue. Twenty four rats were randomly divided into four groups (n = 6 per group): two groups were letrozole treated (1 and 2 mg/kg/day orally), one group was vehicle treated (DMSO) and one group was the control group without any treatment. 12 weeks after beginning treatment with letrozole, we examined the rate of transcription of renin, angiotensinogen, AngII type 1a and 1b (AT1a and AT1b) and type 2 receptors (AT2) in the rat heart using real-time polymerase chain reaction. The cardiac mRNA levels of several components of the RAS in the rats treated with letrozole were significantly increased including AT1a receptor (80%), renin (51%), and angiotensinogen (33%). Though not significant, AT2 receptor levels were observed to decrease with increasing doses of letrozole. Letrozole can induce significant changes in some RAS related genes. These alterations are important to understand the pathways and consequences beyond cardiac events induced by breast cancer treatments.

Sodium selenite increases the transcript levels of iodothyronine deiodinases I and II in ovine and bovine fetal thyrocytes in vitro.

PubMed

Foroughi, Mohammad Ali; Dehghani, Hesam; Mahdavi-Shahri, Naser; Bassami, Mohammad Reza

2013-07-01

Selenium is essential for thyroid hormone homeostasis. Selenium is co-translationally incorporated into the protein backbone of 5' deiodinase enzymes, which are responsible for the intra- and extra-thyroidal activation of thyroid hormones. The objective of this study was to evaluate the effects of sodium selenite on the transcript levels of type I (DIO1) and II (DIO2) deiodinases in the primary culture of ovine and bovine fetal thyroid. By culture of fetal thyrocytes in the presence or absence of sodium selenite, and quantification of DIO1 and DIO2 transcripts using real-time reverse transcription polymerase chain reaction (RT-qPCR), we found that sodium selenite is able to increase the abundance of transcripts for DIO1 and DIO2 genes. We also found that cultured thyrocytes in the presence of sodium selenite compared to control cultured thyrocytes release more T3 into the culture medium. This indicates that in the presence of sodium selenite higher levels of DIO1 and DIO2 enzymes are produced, which are able to convert T4 to T3. In conclusion, we have shown that sodium selenite is increasing the abundance of DIO1 and DIO2 transcripts and increasing the production and release of T3 from cultured fetal thyrocytes. This finding emphasizes the role of selenium in transcriptional and expression processes during development and suggests that selenium deficiency during pregnancy in sheep and cattle may lead to the lower levels of DIO1 and DIO2 transcription in fetal thyroid, and thus, lower level of thyroidal T3 release into the fetal serum. Copyright © 2013 Elsevier GmbH. All rights reserved.

Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

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

Long, Cong; Wang, Jingchao; Guo, Wei

Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated thatmore » triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.« less

Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I

PubMed Central

Kadota, Shinichi; Nagata, Kyosuke

2014-01-01

Chromatin structure and its alteration play critical roles in the regulation of transcription. However, the transcriptional silencing mechanism with regard to the chromatin structure at an unstimulated state of the interferon (IFN)-stimulated gene (ISG) remains unclear. Here we investigated the role of template activating factor-I (TAF-I, also known as SET) in ISG transcription. Knockdown (KD) of TAF-I increased ISG transcript and simultaneously reduced the histone H1 level on the ISG promoters during the early stages of transcription after IFN stimulation from the unstimulated state. The transcription factor levels on the ISG promoters were increased in TAF-I KD cells only during the early stages of transcription. Furthermore, histone H1 KD also increased ISG transcript. TAF-I and histone H1 double KD did not show the additive effect in ISG transcription, suggesting that TAF-I and histone H1 may act on the same regulatory pathway to control ISG transcription. In addition, TAF-I KD and histone H1 KD affected the chromatin structure near the ISG promoters. On the basis of these findings, we propose that TAF-I and its target histone H1 are key regulators of the chromatin structure at the ISG promoter to maintain the silent state of ISG transcription. PMID:24878923

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos.

PubMed

Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

2017-04-20

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

WRKY transcription factors

PubMed Central

Bakshi, Madhunita; Oelmüller, Ralf

2014-01-01

WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes. PMID:24492469

Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture.

PubMed

Girard, Beatrice M; Merriam, Laura A; Tompkins, John D; Vizzard, Margaret A; Parsons, Rodney L

2013-11-15

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, β4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.

Chromatin potentiates transcription

PubMed Central

Nagai, Shigeki; Davis, Ralph E.; Mattei, Pierre Jean; Eagen, Kyle Patrick; Kornberg, Roger D.

2017-01-01

Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription. PMID:28137832

A detailed transcript-level probe annotation reveals alternative splicing based microarray platform differences

PubMed Central

Lee, Joseph C; Stiles, David; Lu, Jun; Cam, Margaret C

2007-01-01

Background Microarrays are a popular tool used in experiments to measure gene expression levels. Improving the reproducibility of microarray results produced by different chips from various manufacturers is important to create comparable and combinable experimental results. Alternative splicing has been cited as a possible cause of differences in expression measurements across platforms, though no study to this point has been conducted to show its influence in cross-platform differences. Results Using probe sequence data, a new microarray probe/transcript annotation was created based on the AceView Aug05 release that allowed for the categorization of genes based on their expression measurements' susceptibility to alternative splicing differences across microarray platforms. Examining gene expression data from multiple platforms in light of the new categorization, genes unsusceptible to alternative splicing differences showed higher signal agreement than those genes most susceptible to alternative splicing differences. The analysis gave rise to a different probe-level visualization method that can highlight probe differences according to transcript specificity. Conclusion The results highlight the need for detailed probe annotation at the transcriptome level. The presence of alternative splicing within a given sample can affect gene expression measurements and is a contributing factor to overall technical differences across platforms. PMID:17708771

trans-Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

PubMed Central

2012-01-01

An in vitro model of ischemic cerebral stroke [oxygen-glucose deprivation (OGD) for 6 h followed by 24 h reoxygenation (R)] with PC12 cells increases Ca2+ influx by upregulating native L-type Ca2+ channels and reactive oxygen species (ROS) generation. This reactive oxygen species generation and increase in intracellular Ca2+ triggers the expression of hypoxic homeostasis transcription factors such as hypoxia induced factor-1 alpha (HIF-1α), Cav-beta 3 (Cav β3), signal transducer and activator of transcription 3 (STAT3), heat shock protein 27 (hsp-27), and cationic channel transient receptor potential melastatin 7 (TRPM7). OGD insulted PC12 cells were subjected to biologically safe doses (5, 10, and 25 μM) of trans-resveratrol in three different treatment groups: 24 h prior to OGD (pre-treatment); 24 h post OGD (post-treatment); and from 24 h before OGD to end of reoxygenation period (whole-treatment). Here, we demonstrated that OGD-R-induced neuronal injury/death is by reactive oxygen species generation, increase in intracellular calcium levels, and decrease in antioxidant defense enzymes. trans-Resveratrol increases the viability of OGD-R insulted PC12 cells, which was assessed by using MTT, NRU, and LDH release assay. In addition, trans-resveratrol significantly decreases reactive oxygen species generation, intracellular Ca2+ levels, and hypoxia associated transcription factors and also increases the level of antioxidant defense enzymes. Our data shows that the whole-treatment group of trans-resveratrol is most efficient in decreasing hypoxia induced cell death through its antioxidant properties. PMID:23421680

Predicting phonetic transcription agreement: Insights from research in infant vocalizations

PubMed Central

RAMSDELL, HEATHER L.; OLLER, D. KIMBROUGH; ETHINGTON, CORINNA A.

2010-01-01

The purpose of this study is to provide new perspectives on correlates of phonetic transcription agreement. Our research focuses on phonetic transcription and coding of infant vocalizations. The findings are presumed to be broadly applicable to other difficult cases of transcription, such as found in severe disorders of speech, which similarly result in low reliability for a variety of reasons. We evaluated the predictiveness of two factors not previously documented in the literature as influencing transcription agreement: canonicity and coder confidence. Transcribers coded samples of infant vocalizations, judging both canonicity and confidence. Correlation results showed that canonicity and confidence were strongly related to agreement levels, and regression results showed that canonicity and confidence both contributed significantly to explanation of variance. Specifically, the results suggest that canonicity plays a major role in transcription agreement when utterances involve supraglottal articulation, with coder confidence offering additional power in predicting transcription agreement. PMID:17882695

Widespread antisense transcription of Populus genome under drought.

PubMed

Yuan, Yinan; Chen, Su

2018-06-06

Antisense transcription is widespread in many genomes and plays important regulatory roles in gene expression. The objective of our study was to investigate the extent and functional relevance of antisense transcription in forest trees. We employed Populus, a model tree species, to probe the antisense transcriptional response of tree genome under drought, through stranded RNA-seq analysis. We detected nearly 48% of annotated Populus gene loci with antisense transcripts and 44% of them with co-transcription from both DNA strands. Global distribution of reads pattern across annotated gene regions uncovered that antisense transcription was enriched in untranslated regions while sense reads were predominantly mapped in coding exons. We further detected 1185 drought-responsive sense and antisense gene loci and identified a strong positive correlation between the expression of antisense and sense transcripts. Additionally, we assessed the antisense expression in introns and found a strong correlation between intronic expression and exonic expression, confirming antisense transcription of introns contributes to transcriptional activity of Populus genome under drought. Finally, we functionally characterized drought-responsive sense-antisense transcript pairs through gene ontology analysis and discovered that functional groups including transcription factors and histones were concordantly regulated at both sense and antisense transcriptional level. Overall, our study demonstrated the extensive occurrence of antisense transcripts of Populus genes under drought and provided insights into genome structure, regulation pattern and functional significance of drought-responsive antisense genes in forest trees. Datasets generated in this study serve as a foundation for future genetic analysis to improve our understanding of gene regulation by antisense transcription.

Alteration of respiration capacity and transcript accumulation level of alternative oxidase genes in necrosis lines of common wheat.

PubMed

Sugie, Atsushi; Murai, Koji; Takumi, Shigeo

2007-06-01

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in plants. AOX is a key enzyme of the alternative respiration pathway. To study the effects of necrotic cell death on the mitochondrial function, production of reactive oxygen species (ROS), respiration capacities and accumulation patterns of mitochondria-targeted protein-encoding gene transcripts were compared between wild-type, lesion-mimic mutant and hybrid necrosis wheat plants. Around cells with the necrosis symptom, ROS accumulated abundantly in the intercellular spaces. The ratio of the alternative pathway to the cytochrome pathway was markedly enhanced in the necrotic leaves. Transcripts of a wheat AOX gene, Waox1a, were more abundant in a novel lesion-mimic mutant of common wheat than in the wild-type plants. An increased level of the Waox1a transcripts was also observed in hybrid plants containing Ne1 and Ne2 genes. These results indicated that an increase of the wheat AOX transcript level resulted in enhancement of respiration capacity of the alternative pathway in the necrotic cells.

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

PubMed Central

Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

2017-01-01

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001 PMID:28425915

HALO--a Java framework for precise transcript half-life determination.

PubMed

Friedel, Caroline C; Kaufmann, Stefanie; Dölken, Lars; Zimmer, Ralf

2010-05-01

Recent improvements in experimental technologies now allow measurements of de novo transcription and/or RNA decay at whole transcriptome level and determination of precise transcript half-lives. Such transcript half-lives provide important insights into the regulation of biological processes and the relative contributions of RNA decay and de novo transcription to differential gene expression. In this article, we present HALO (Half-life Organizer), the first software for the precise determination of transcript half-lives from measurements of RNA de novo transcription or decay determined with microarrays or RNA-seq. In addition, methods for quality control, filtering and normalization are supplied. HALO provides a graphical user interface, command-line tools and a well-documented Java application programming interface (API). Thus, it can be used both by biologists to determine transcript half-lives fast and reliably with the provided user interfaces as well as software developers integrating transcript half-life analysis into other gene expression profiling pipelines. Source code, executables and documentation are available at http://www.bio.ifi.lmu.de/software/halo.

Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

PubMed

Li, Guojing; Meng, Xiangzong; Wang, Ruigang; Mao, Guohong; Han, Ling; Liu, Yidong; Zhang, Shuqun

2012-06-01

Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

Silencing of IFN-stimulated gene transcription is regulated by histone H1 and its chaperone TAF-I.

PubMed

Kadota, Shinichi; Nagata, Kyosuke

2014-07-01

Chromatin structure and its alteration play critical roles in the regulation of transcription. However, the transcriptional silencing mechanism with regard to the chromatin structure at an unstimulated state of the interferon (IFN)-stimulated gene (ISG) remains unclear. Here we investigated the role of template activating factor-I (TAF-I, also known as SET) in ISG transcription. Knockdown (KD) of TAF-I increased ISG transcript and simultaneously reduced the histone H1 level on the ISG promoters during the early stages of transcription after IFN stimulation from the unstimulated state. The transcription factor levels on the ISG promoters were increased in TAF-I KD cells only during the early stages of transcription. Furthermore, histone H1 KD also increased ISG transcript. TAF-I and histone H1 double KD did not show the additive effect in ISG transcription, suggesting that TAF-I and histone H1 may act on the same regulatory pathway to control ISG transcription. In addition, TAF-I KD and histone H1 KD affected the chromatin structure near the ISG promoters. On the basis of these findings, we propose that TAF-I and its target histone H1 are key regulators of the chromatin structure at the ISG promoter to maintain the silent state of ISG transcription. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Natural antisense transcript-targeted regulation of inducible nitric oxide synthase mRNA levels.

PubMed

Yoshigai, Emi; Hara, Takafumi; Araki, Yoshiro; Tanaka, Yoshito; Oishi, Masaharu; Tokuhara, Katsuji; Kaibori, Masaki; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2013-04-01

Natural antisense transcripts (asRNAs) are frequently transcribed from mammalian genes. Recently, we found that non-coding asRNAs are transcribed from the 3' untranslated region (3'UTR) of the rat and mouse genes encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide. The iNOS asRNA stabilizes iNOS mRNA by interacting with the mRNA 3'UTR. Furthermore, single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence were found to reduce iNOS mRNA levels by interfering with mRNA-asRNA interactions in rat hepatocytes. This method was named natural antisense transcript-targeted regulation (NATRE) technology. In this study, we detected human iNOS asRNA expressed in hepatocarcinoma and colon carcinoma tissues. The human iNOS asRNA harbored a sequence complementary to an evolutionarily conserved region of the iNOS mRNA 3'UTR. When introduced into hepatocytes, iNOS sense oligonucleotides that were modified by substitution with partial phosphorothioate bonds and locked nucleic acids or 2'-O-methyl nucleic acids greatly reduced levels of iNOS mRNA and iNOS protein. Moreover, sense oligonucleotides and short interfering RNAs decreased iNOS mRNA to comparable levels. These results suggest that NATRE technology using iNOS sense oligonucleotides could potentially be used to treat human inflammatory diseases and cancers by reducing iNOS mRNA levels. Copyright © 2013 Elsevier Inc. All rights reserved.

TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I.

PubMed

Schnapp, G; Schnapp, A; Rosenbauer, H; Grummt, I

1994-09-01

We have characterized a transcription factor from Ehrlich ascites cells that is required for ribosomal gene transcription by RNA polymerase I (Pol I). This factor, termed TIF-IC, has a native molecular mass of 65 kDa, associates with Pol I, and is required both for the assembly of Sarkosyl-resistant initiation complexes and for the formation of the first internucleotide bonds. In addition to its function in transcription initiation, TIF-IC also plays a role in elongation of nascent RNA chains. At suboptimal levels of TIF-IC, transcripts with heterogeneous 3' ends are formed which are chased into full-length transcripts by the addition of more TIF-IC. Moreover, on a tailed template, which allows initiation in the absence of auxiliary factors, TIF-IC was found to stimulate the overall rate of transcription elongation and suppress pausing of Pol I. Thus TIF-IC appears to serve a function similar to the Pol II-specific factor TFIIF which is required for Pol II transcription initiation and elongation.

The legume miR1514a modulates a NAC transcription factor transcript to trigger phasiRNA formation in response to drought

PubMed Central

Sosa-Valencia, Guadalupe; Palomar, Miguel; Covarrubias, Alejandra A.

2017-01-01

Abstract Recent studies have identified microRNAs as post-transcriptional regulators involved in stress responses. miR1514a is a legume microRNA that is induced in response to drought stress in Phaseolus vulgaris (common bean) and shows differential accumulation levels in roots during water deficit in two cultivars with different drought tolerance phenotypes. A recent degradome analysis revealed that miR1514a targets the transcripts of two NAC transcription factors (TFs), Phvul.010g121000 and Phvul.010g120700. Furthermore, expression studies and small RNA-seq data indicate that only Phvul.010g120700 generates phasiRNAs, which also accumulate under water deficit conditions. To confirm these results, we over-expressed miR1514a in transgenic hairy roots, and observed a reduced accumulation of Phvul.010g120700 and an increase in NAC-derived phasiRNAs; inhibition of miR1514a activity resulted in the opposite effect. Moreover, we determined that a NAC-derived phasiRNA associates with ARGONAUTE 1 (AGO1), suggesting that it is functional. In addition, a transcriptome analysis of transgenic hairy roots with reduced miR1514a levels revealed several differentially expressed transcripts, mainly involved in metabolism and stress responses, suggesting they are regulated by the NAC TF and/or by phasiRNAs. This work therefore demonstrates the participation of miR1514 in the regulation of a NAC transcription factor transcript through phasiRNA production during the plant response to water deficit. PMID:28338719

Transcript of the Joint FAA/Industry Symposium on Level B Airplane simulator aeromodel validation requirements

DOT National Transportation Integrated Search

1996-03-13

"This is the transcript of the Joint FAA/Industry Symposium on Level B Airplane Simulator Aeromodel Validation Requirements held on March 13-14, 1996, at the Washington Dulles Airport Hilton. The purpose of the meeting was to discuss the aeromodeling...

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

PubMed

Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

2014-07-01

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

PubMed

Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C; Zhang, Zhen; Young, Randee E; Verheyden, Jamie M; Sun, Xin

2016-07-05

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.

Evolution of Chloroplast Transcript Processing in Plasmodium and Its Chromerid Algal Relatives

PubMed Central

Dorrell, Richard G.; Drew, James; Nisbet, R. Ellen R.; Howe, Christopher J.

2014-01-01

It is well understood that apicomplexan parasites, such as the malaria pathogen Plasmodium, are descended from free-living algae, and maintain a vestigial chloroplast that has secondarily lost all genes of photosynthetic function. Recently, two fully photosynthetic relatives of parasitic apicomplexans have been identified, the ‘chromerid’ algae Chromera velia and Vitrella brassicaformis, which retain photosynthesis genes within their chloroplasts. Elucidating the processes governing gene expression in chromerid chloroplasts might provide valuable insights into the origins of parasitism in the apicomplexans. We have characterised chloroplast transcript processing pathways in C. velia, V. brassicaformis and P. falciparum with a focus on the addition of an unusual, 3′ poly(U) tail. We demonstrate that poly(U) tails in chromerids are preferentially added to transcripts that encode proteins that are directly involved in photosynthetic electron transfer, over transcripts for proteins that are not involved in photosynthesis. To our knowledge, this represents the first chloroplast transcript processing pathway to be associated with a particular functional category of genes. In contrast, Plasmodium chloroplast transcripts are not polyuridylylated. We additionally present evidence that poly(U) tail addition in chromerids is involved in the alternative processing of polycistronic precursors covering multiple photosynthesis genes, and appears to be associated with high levels of transcript abundance. We propose that changes to the chloroplast transcript processing machinery were an important step in the loss of photosynthesis in ancestors of parasitic apicomplexans. PMID:24453981

Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs

NASA Astrophysics Data System (ADS)

Che, You; Liang, Peixin; Gong, Ting; Cao, Xiangyu; Zhao, Ying; Yang, Chao; Song, Cunjiang

2017-03-01

We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal.

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

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

Gao, Yuen; Wang, Yuan; Feng, Jinyan

2015-04-03

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within itsmore » 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.« less

A structure-based kinetic model of transcription.

PubMed

Zuo, Yuhong; Steitz, Thomas A

2017-01-01

During transcription, RNA polymerase moves downstream along the DNA template and maintains a transcription bubble. Several recent structural studies of transcription complexes with a complete transcription bubble provide new insights into how RNAP couples the nucleotide addition reaction to its directional movement.

[Relationship between the expression levels of PAPP-A metalloproteinase and growth and transcriptional factors in endometrial cancer].

PubMed

Iunusova, N V; Spirina, L V; Kondakova, L A; Kolomiets, A L; Chernyshova, A L; Koval', V D; Nedosekov, V V; Savenkova, O V

2013-01-01

We have examined for the first time the relationship between the expression of PAPP-A metalloproteinase and insulin-like growth factors (IGF-I, IGF-II, VEGF) and transcription factors (NF-kappaB, HIF-1) playing an important role in pathogenesis of cancer. We also demonstrated a positive association between the level of PAPP-A metalloproteinase and the level of growth (VEGF and IGF-I) and transcription factors (NF-kappaB p50, NF-kappaB p65, HIF-1alpha). The current findings suggest an important role of PAPP-A in regulation of bioavailability of IGF-I, VEGF, activated forms of NF-kappaB, and alpha-subunits of HIF-1 in endometrial tumors.

A structure-based kinetic model of transcription

PubMed Central

Steitz, Thomas A.

2017-01-01

ABSTRACT During transcription, RNA polymerase moves downstream along the DNA template and maintains a transcription bubble. Several recent structural studies of transcription complexes with a complete transcription bubble provide new insights into how RNAP couples the nucleotide addition reaction to its directional movement. PMID:27656764

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

PubMed Central

2010-01-01

also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels. Conclusions This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies. PMID:20385001

TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I.

PubMed Central

Schnapp, G; Schnapp, A; Rosenbauer, H; Grummt, I

1994-01-01

We have characterized a transcription factor from Ehrlich ascites cells that is required for ribosomal gene transcription by RNA polymerase I (Pol I). This factor, termed TIF-IC, has a native molecular mass of 65 kDa, associates with Pol I, and is required both for the assembly of Sarkosyl-resistant initiation complexes and for the formation of the first internucleotide bonds. In addition to its function in transcription initiation, TIF-IC also plays a role in elongation of nascent RNA chains. At suboptimal levels of TIF-IC, transcripts with heterogeneous 3' ends are formed which are chased into full-length transcripts by the addition of more TIF-IC. Moreover, on a tailed template, which allows initiation in the absence of auxiliary factors, TIF-IC was found to stimulate the overall rate of transcription elongation and suppress pausing of Pol I. Thus TIF-IC appears to serve a function similar to the Pol II-specific factor TFIIF which is required for Pol II transcription initiation and elongation. Images PMID:8076598

Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

PubMed

Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

2013-06-01

To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner

Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)

USGS Publications Warehouse

Ferrante, Jason; Hunter, Margaret; Wellehan, James F.X.

2018-01-01

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher (P<0.05) in manatees from the east coast of Florida than they were from those from the west coast. We found IL-10 and β-actin to be consistent between sites and identified β-actin as a good candidate for use as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

DEVELOPMENT AND VALIDATION OF QUANTITATIVE PCR ASSAYS TO MEASURE CYTOKINE TRANSCRIPT LEVELS IN THE FLORIDA MANATEE ( TRICHECHUS MANATUS LATIROSTRIS).

PubMed

Ferrante, Jason A; Hunter, Margaret E; Wellehan, James F X

2018-04-01

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees ( Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α; and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida, US. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher ( P<0.05) in manatees from the east coast of Florida than they were from those from the west coast. We found IL-10 and β-actin to be consistent between sites and identified β-actin as a good candidate for use as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

RNA-Skim: a rapid method for RNA-Seq quantification at transcript level

PubMed Central

Zhang, Zhaojun; Wang, Wei

2014-01-01

Motivation: RNA-Seq technique has been demonstrated as a revolutionary means for exploring transcriptome because it provides deep coverage and base pair-level resolution. RNA-Seq quantification is proven to be an efficient alternative to Microarray technique in gene expression study, and it is a critical component in RNA-Seq differential expression analysis. Most existing RNA-Seq quantification tools require the alignments of fragments to either a genome or a transcriptome, entailing a time-consuming and intricate alignment step. To improve the performance of RNA-Seq quantification, an alignment-free method, Sailfish, has been recently proposed to quantify transcript abundances using all k-mers in the transcriptome, demonstrating the feasibility of designing an efficient alignment-free method for transcriptome quantification. Even though Sailfish is substantially faster than alternative alignment-dependent methods such as Cufflinks, using all k-mers in the transcriptome quantification impedes the scalability of the method. Results: We propose a novel RNA-Seq quantification method, RNA-Skim, which partitions the transcriptome into disjoint transcript clusters based on sequence similarity, and introduces the notion of sig-mers, which are a special type of k-mers uniquely associated with each cluster. We demonstrate that the sig-mer counts within a cluster are sufficient for estimating transcript abundances with accuracy comparable with any state-of-the-art method. This enables RNA-Skim to perform transcript quantification on each cluster independently, reducing a complex optimization problem into smaller optimization tasks that can be run in parallel. As a result, RNA-Skim uses <4% of the k-mers and <10% of the CPU time required by Sailfish. It is able to finish transcriptome quantification in <10 min per sample by using just a single thread on a commodity computer, which represents >100 speedup over the state-of-the-art alignment-based methods, while delivering

Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

PubMed

Funaba, Masayuki; Ikeda, Teruo; Murakami, Masaru; Ogawa, Kenji; Tsuchida, Kunihiro; Sugino, Hiromu; Abe, Matanobu

2003-12-26

Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

Changes in neuropeptide Y immunoreactivity and transcript levels in circadian system structures of the diurnal rodent, the thirteen-lined ground squirrel

PubMed Central

Vidal, Luis; Lugo, Nidza

2007-01-01

The intergeniculate leaflet (IGL) and its neuropeptide Y (NPY) projection to the main circadian clock, the suprachiasmatic nucleus (SCN), have been the focus of extensive research conducted, for the most part, on nocturnal rodent species. However, a variety of anatomical and physiological differences between the circadian system of diurnal and nocturnal species have been reported. These differences led us to question whether the role of NPY in the circadian system of the diurnal ground squirrel differs from that in nocturnal rodents. We used semi-quantitative immunohistochemistry to analyze NPY content in SCN terminals of squirrels sacrificed at specific times of the day and compared the data to previous published results from the rat. Additionally, NPY mRNA was quantified using real-time PCR to determine if varying NPY-immunoreactivity (-ir) levels could be the result of changes in peptide transcription. Our results demonstrate that NPY-ir levels in the ground squirrel SCN peak during the middle of the night unlike what is observed in the rat. Cell counts of NPY-ir neurons in the IGL revealed a pattern of variation 6 hr out of phase compared to what was observed in the SCN. NPY mRNA levels showed only one sharp increase in the middle of the night, coinciding with increases in NPY-ir levels observed in the SCN. Differences in the pattern of fluctuation of NPY in the SCN between the rat and squirrel suggest that this peptide may serve distinct roles in the circadian system of diurnal and nocturnal species. Our data provide the first evidence of the relationship between transcript and peptide levels in the circadian system of a diurnal species. PMID:17109825

Transcript Levels of Major Interleukins in Relation to the Clinicopathological Profile of Patients with Tuberculous Intervertebral Discs and Healthy Controls

PubMed Central

Liu, Chong; Zhan, Xinli; Xiao, Zengming; Fan, Qie; Deng, Li; Cui, Mingxing; Xiong, Chunxiang; Xue, Jingbo; Xie, Xiangtao

2014-01-01

Objectives The purpose of the present study was to simultaneously examine the transcript levels of a large number of interleukins (ILs; IL-9, IL-10, IL-12, IL-13, IL-16, IL-17, IL-18, IL-26, and IL-27) and investigate their correlation with the clinicopathological profiles of patients with tuberculous intervertebral discs. Methods Clinical data were collected from 150 patients participating in the study from January 2013 to December 2013. mRNA expression levels in 70 tuberculous, 70 herniated, and 10 control intervertebral disc specimens were determined by real-time polymerase chain reaction. Results IL-10, IL-16, IL-17, IL-18, and IL-27 displayed stronger expression in tuberculous spinal disc tissue than in normal intervertebral disc tissue (P<0.05). Our results illustrated multiple correlations among IL-10, IL-16, IL-17, IL-18, and IL-27 mRNA expression in tuberculous samples. Smoking habits were found to have a positive correlation with IL-17 transcript levels and a negative correlation with IL-10 transcript levels (P<0.05). Pain intensity, symptom duration, C-reactive protein levels, and the erythrocyte sedimentation rate exhibited multiple correlations with the transcript levels of several ILs (P<0.05). Conclusions The experimental data imply a double-sided effect on the activity of ILs in tuberculous spinal intervertebral discs, suggesting that they may be involved in intervertebral discs destruction. Our findings also suggest that smoking may affect the intervertebral discs destruction process of spinal tuberculosis. However, further studies are necessary to elucidate the exact role of ILs in the intervertebral discs destruction process of spinal tuberculosis. PMID:24971599

Nitrite reductase expression is regulated at the post-transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana.

PubMed

Crété, P; Caboche, M; Meyer, C

1997-04-01

Higher plant nitrite reductase (NiR) is a monomeric chloroplastic protein catalysing the reduction of nitrite, the product of nitrate reduction, to ammonium. The expression of this enzyme is controlled at the transcriptional level by light and by the nitrogen source. In order to study the post-transcriptional regulation of NiR, Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed with a chimaeric NiR construct containing the tobacco leaf NiR1 coding sequence driven by the CaMV 35S RNA promoter. Transformed plants did not show any phenotypic difference when compared with the wild-type, although they overexpressed NiR activity in the leaves. When these plants were grown in vitro on media containing either nitrate or ammonium as sole nitrogen source, NiR mRNA derived from transgene expression was constitutively expressed, whereas NiR activity and protein level were strongly reduced on ammonium-containing medium. These results suggest that, together with transcriptional control, post-transcriptional regulation by the nitrogen source is operating on NiR expression. This post-transcriptional regulation of tobacco leaf NiR1 expression was observed not only in the closely related species N. plumbaginifolia but also in the more distant species A. thaliana.

Intergenic Transcriptional Interference Is Blocked by RNA Polymerase III Transcription Factor TFIIIB in Saccharomyces cerevisiae

PubMed Central

Korde, Asawari; Rosselot, Jessica M.; Donze, David

2014-01-01

The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such “extratranscriptional” activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467–ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins. PMID:24336746

Global isoform-specific transcript alterations and deregulated networks in clear cell renal cell carcinoma

PubMed Central

Hamilton, Michael J.; Girke, Thomas; Martinez, Ernest

2018-01-01

Extensive genome-wide analyses of deregulated gene expression have now been performed for many types of cancer. However, most studies have focused on deregulation at the gene-level, which may overlook the alterations of specific transcripts for a given gene. Clear cell renal cell carcinoma (ccRCC) is one of the best-characterized and most pervasive renal cancers, and ccRCCs are well-documented to have aberrant RNA processing. In the present study, we examine the extent of aberrant isoform-specific RNA expression by reporting a comprehensive transcript-level analysis, using the new kallisto-sleuth-RATs pipeline, investigating coding and non-coding differential transcript expression in ccRCC. We analyzed 50 ccRCC tumors and their matched normal samples from The Cancer Genome Altas datasets. We identified 7,339 differentially expressed transcripts and 94 genes exhibiting differential transcript isoform usage in ccRCC. Additionally, transcript-level coexpression network analyses identified vasculature development and the tricarboxylic acid cycle as the most significantly deregulated networks correlating with ccRCC progression. These analyses uncovered several uncharacterized transcripts, including lncRNAs FGD5-AS1 and AL035661.1, as potential regulators of the tricarboxylic acid cycle associated with ccRCC progression. As ccRCC still presents treatment challenges, our results provide a new resource of potential therapeutics targets and highlight the importance of exploring alternative methodologies in transcriptome-wide studies.

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition.

PubMed

Morey, Jeanine S; Monroe, Emily A; Kinney, Amanda L; Beal, Marion; Johnson, Jillian G; Hitchcock, Gary L; Van Dolah, Frances M

2011-07-05

The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' trans-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of K. brevis is responsive to nitrogen and phosphorus and is informative of nutrient status. Microarray analysis of N-depleted K. brevis cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes. Microarray analysis provided transcriptomic

The Impact of Promoting Transcription on Early Text Production: Effects on Bursts and Pauses, Levels of Written Language, and Writing Performance

ERIC Educational Resources Information Center

Alves, Rui A.; Limpo, Teresa; Fidalgo, Raquel; Carvalhais, Lénia; Pereira, Luísa Álvares; Castro, São Luís

2016-01-01

Writing development seems heavily dependent upon the automatization of transcription. This study aimed to further investigate the link between transcription and writing by examining the effects of promoting handwriting and spelling skills on a comprehensive set of writing measures (viz., bursts and pauses, levels of written language, and writing…

DNA damage mediated transcription arrest: Step back to go forward.

PubMed

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

PubMed

Yan, Xiaodong; Liu, Juanfang; Zhang, Zhengping; Li, Wenhao; Sun, Siguo; Zhao, Jian; Dong, Xin; Qian, Jixian; Sun, Honghui

2017-01-01

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca 2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca 2+ ] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca 2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca 2+ ) stores. Blockade of Ca 2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca 2+ -ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca 2+ -dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans.

PubMed

Grishok, Alla; Sinskey, Jina L; Sharp, Phillip A

2005-03-15

The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma.

PubMed

Parker, Nicole R; Hudson, Amanda L; Khong, Peter; Parkinson, Jonathon F; Dwight, Trisha; Ikin, Rowan J; Zhu, Ying; Cheng, Zhangkai Jason; Vafaee, Fatemeh; Chen, Jason; Wheeler, Helen R; Howell, Viive M

2016-03-04

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

PubMed

Todor, Horia; Gooding, Jessica; Ilkayeva, Olga R; Schmid, Amy K

2015-01-01

Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Action of boron at the molecular level: effects on transcription and translation in an acellular system.

PubMed

Dzondo-Gadet, M; Mayap-Nzietchueng, R; Hess, K; Nabet, P; Belleville, F; Dousset, B

2002-01-01

It has been shown that boric acid has well-defined biological effects such as stimulation of wound healing in vivo, release of growth factors and cytokines, and increase of the extracellular matrice turnover. We examined its action at the molecular level, using cell-free systems of transcription (isolated placenta nuclei) and translation (wheat germ extract). We found that 10 mM boric acid greatly increased RNA synthesis, measured by absorbance at 260 nm (x 6.4) or by [3H]-UTP uptake (x 11). Full-length functional mRNA was produced because proteins of 14-80 kDa were translated. Among these proteins, factors involved in angiogenesis and, subsequently, in wound healing (VEGF and TGFbeta) were identified by slot blot, whereas growth factors such as FGF1 and TNFalpha were not detected. These results demonstrate that boron may contribute to biological cell activities at both the transcription and translation levels. However, the mechanism of action is still not known.

The transcription factor titration effect dictates level of gene expression.

PubMed

Brewster, Robert C; Weinert, Franz M; Garcia, Hernan G; Song, Dan; Rydenfelt, Mattias; Phillips, Rob

2014-03-13

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number-in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

[Construction of Corynebacterium crenatum AS 1.542 δ argR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway].

PubMed

Chen, Xuelan; Tang, Li; Jiao, Haitao; Xu, Feng; Xiong, Yonghua

2013-01-04

ArgR, coded by the argR gene from Corynebacterium crenatum AS 1.542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene: C. crenatum AS 1.542 Delta argR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. We used marker-less knockout technology to construct C. crenatum AS 1.542 Delta argR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. C. crenatum AS 1.542 Delta argR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162.1 folds. The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.

Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana

DOE PAGES

Smith, Sarah R.; Gle, Corine; Abbriano, Raffaela M.; ...

2016-02-04

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterizedmore » alongside lipid accumulation. Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels.« less

Assembly of transcriptionally inactive chromatin in vitro.

PubMed

Shanahan, M M; Kmiec, E B

1989-07-01

We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

FLO11 gene length and transcriptional level affect biofilm-forming ability of wild flor strains of Saccharomyces cerevisiae.

PubMed

Zara, Giacomo; Zara, Severino; Pinna, Claudia; Marceddu, Salvatore; Budroni, Marilena

2009-12-01

In Saccharomyces cerevisiae, FLO11 encodes an adhesin that is associated with different phenotypes, such as adherence to solid surfaces, hydrophobicity, mat and air-liquid biofilm formation. In the present study, we analysed FLO11 allelic polymorphisms and FLO11-associated phenotypes of 20 flor strains. We identified 13 alleles of different lengths, varying from 3.0 to 6.1 kb, thus demonstrating that FLO11 is highly polymorphic. Two alleles of 3.1 and 5.0 kb were cloned into strain BY4742 to compare the FLO11-associated phenotypes in the same genetic background. We show that there is a significant correlation between biofilm-forming ability and FLO11 length both in different and in the same genetic backgrounds. Moreover, we propose a multiple regression model that allows prediction of air-liquid biofilm-forming ability on the basis of transcription levels and lengths of FLO11 alleles in a population of S. cerevisiae flor strains. Considering that transcriptional differences are only partially explained by the differences in the promoter sequences, our results are consistent with the hypothesis that FLO11 transcription levels are strongly influenced by genetic background and affect biofilm-forming ability.

Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase.

PubMed

Zheng, D; MacLean, P S; Pohnert, S C; Knight, J B; Olson, A L; Winder, W W; Dohm, G L

2001-09-01

Skeletal muscle GLUT-4 transcription in response to treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMP-activated protein kinase (AMPK), was studied in rats and mice. The increase in GLUT-4 mRNA levels in response to a single subcutaneous injection of AICAR, peaked at 13 h in white and red quadriceps muscles but not in the soleus muscle. The mRNA level of chloramphenicol acyltransferase reporter gene which is driven by 1,154 or 895 bp of the human GLUT-4 proximal promoter was increased in AICAR-treated transgenic mice, demonstrating the transcriptional upregulation of the GLUT-4 gene by AICAR. However, this induction of transcription was not apparent with 730 bp of the promoter. In addition, nuclear extracts from AICAR-treated mice bound to the consensus sequence of myocyte enhancer factor-2 (from -473 to -464) to a greater extent than from saline-injected mice. Thus AMP-activated protein kinase activation by AICAR increases GLUT-4 transcription by a mechanism that requires response elements within 895 bp of human GLUT-4 proximal promoter and that may be cooperatively mediated by myocyte enhancer factor-2.

In vitro and in vivo effects of 2,4 diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS: Context-specific modulation of SMN transcript levels

PubMed Central

Androphy, Elliot J.; Calo, Alessandro; Potter, Kyle; Custer, Sara K.; Du, Sarah; Foley, Timothy L.; Gopalsamy, Ariamala; Reedich, Emily J.; Gordo, Susana M.; Gordon, William; Hosea, Natalie; Jones, Lyn H.; Krizay, Daniel K.; LaRosa, Gregory; Li, Hongxia; Mathur, Sachin; Menard, Carol A.; Patel, Paraj; Ramos-Zayas, Rebeca; Rietz, Anne; Rong, Haojing; Zhang, Baohong; Tones, Michael A.

2017-01-01

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation. PMID:28945765

Investigating transcription reinitiation through in vitro approaches

PubMed Central

Dieci, Giorgio; Fermi, Beatrice; Bosio, Maria Cristina

2014-01-01

By influencing the number of RNA molecules repeatedly synthesized from the same gene, the control of transcription reinitiation has the potential to shape the transcriptome. Transcription reinitiation mechanisms have been mainly addressed in vitro, through approaches based on both crude and reconstituted systems. These studies support the notion that transcription reinitiation and its regulation rely on dedicated networks of molecular interactions within transcription machineries. At the same time, comparison with in vivo transcription rates suggests that additional mechanisms, factors and conditions must exist in the nucleus, whose biochemical elucidation is a fascinating challenge for future in vitro transcription studies. PMID:25764113

Clk post-transcriptional control denoises circadian transcription both temporally and spatially.

PubMed

Lerner, Immanuel; Bartok, Osnat; Wolfson, Victoria; Menet, Jerome S; Weissbein, Uri; Afik, Shaked; Haimovich, Daniel; Gafni, Chen; Friedman, Nir; Rosbash, Michael; Kadener, Sebastian

2015-05-08

The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.

Cocoa procyanidins and human cytokine transcription and secretion.

PubMed

Mao, T; Van De Water, J; Keen, C L; Schmitz, H H; Gershwin, M E

2000-08-01

We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions

PubMed Central

Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W.; Oegema, Karen; Desai, Arshad

2016-01-01

SUMMARY Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

Sequences required for transcription termination at the intrinsic lambdatI terminator.

PubMed

Martínez-Trujillo, Miguel; Sánchez-Trujillo, Alejandra; Ceja, Víctor; Avila-Moreno, Federico; Bermúdez-Cruz, Rosa María; Court, Donald; Montañez, Cecilia

2010-02-01

The lambdatI terminator is located approximately 280 bp beyond the lambdaint gene, and it has a typical structure of an intrinsic terminator. To identify sequences required for lambdatI transcription termination a set of deletion mutants were generated, either from the 5' or the 3' end onto the lambdatI region. The termination efficiency was determined by measuring galactokinase (galK) levels by Northern blot assays and by in vitro transcription termination. The importance of the uridines and the stability of the stem structure in the termination were demonstrated. The nontranscribed DNA beyond the 3' end also affects termination. Additionally, sequences upstream have a small effect on transcription termination. The in vivo RNA termination sites at lambdatI were determined by S1 mapping and were located at 8 different positions. Processing of transcripts from the 3' end confirmed the importance of the hairpin stem in protection against exonuclease.

Butyrate-Induced Transcriptional Changes in Human Colonic Mucosa

PubMed Central

Vanhoutvin, Steven A. L. W.; Troost, Freddy J.; Hamer, Henrike M.; Lindsey, Patrick J.; Koek, Ger H.; Jonkers, Daisy M. A. E.; Kodde, Andrea; Venema, Koen; Brummer, Robert J. M.

2009-01-01

Background Fermentation of dietary fiber in the colon results in the production of short chain fatty acids (mainly propionate, butyrate and acetate). Butyrate modulates a wide range of processes, but its mechanism of action is mostly unknown. This study aimed to determine the effects of butyrate on the transcriptional regulation of human colonic mucosa in vivo. Methodology/Principal Findings Five hundred genes were found to be differentially expressed after a two week daily butyrate administration with enemas. Pathway analysis showed that the butyrate intervention mainly resulted in an increased transcriptional regulation of the pathways representing fatty acid oxidation, electron transport chain and oxidative stress. In addition, several genes associated with epithelial integrity and apoptosis, were found to be differentially expressed after the butyrate intervention. Conclusions/Significance Colonic administration of butyrate in concentrations that can be achieved by consumption of a high-fiber diet enhances the maintenance of colonic homeostasis in healthy subjects, by regulating fatty acid metabolism, electron transport and oxidative stress pathways on the transcriptional level and provide for the first time, detailed molecular insight in the transcriptional response of gut mucosa to butyrate. PMID:19707587

The alpaca melanocortin 1 receptor: gene mutations, transcripts, and relative levels of expression in ventral skin biopsies.

PubMed

Chandramohan, Bathrachalam; Renieri, Carlo; La Manna, Vincenzo; La Terza, Antonietta

2015-01-01

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5'-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3'UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation.

Additional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation on histone H3K4 and H3K27 during transcription of hsp70.

PubMed

Li, Taosui; Hodgson, Jacob W; Petruk, Svetlana; Mazo, Alexander; Brock, Hugh W

2017-09-19

Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear. We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repression of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation. We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70.

FBXW10 is negatively regulated in transcription and expression level by protein O-GlcNAcylation.

PubMed

Feng, Zhou; Hui, Yan; Ling, Li; Xiaoyan, Liu; Yuqiu, Wang; Peng, Wang; Lianwen, Zhang

2013-08-23

Intricate cross-talks exist among multiple post-translational modifications that play critical roles in various cellular events, such as the control of gene expression and regulation of protein function. Here, the cross-talk between O-GlcNAcylation and ubiquitination was investigated in HEK293T cells. By PCR array, 84 ubiquitination-related genes were explored in transcription level in response to the elevation of total protein O-GlcNAcylation due to over-expression of OGT, inhibition of OGA or GlcN treatment. Varied genes were transcriptionally regulated by using different method. But FBXW10, an F-box protein targeting specific proteins for ubiquitination, could be negatively regulated in all ways, suggesting its regulation by protein O-GlcNAcylation. By RT-PCR and Western blot analysis, it was found that FBXW10 could be sharply down-regulated in mRNA and protein level in GlcN-treated cells in a time-dependent way, in line with the enhancement of protein O-GlcNAcylation. It was also found that endogenous FBXW10 was modified by O-GlcNAc in HEK293T cells, implying O-GlcNAcylation might regulate FBXW10 in multiple levels. These findings indicate that O-GlcNAcylation is involved in the regulation of ubiquitination-related genes, and help us understand the cross-talk between O-GlcNAcylation and ubiquitination. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcript level of the porcine ME1 gene is affected by SNP in its 3'UTR, which is also associated with subcutaneous fat thickness.

PubMed

Bartz, M; Kociucka, B; Mankowska, M; Switonski, M; Szydlowski, M

2014-08-01

Pork quality depends on multiple factors, including fatty acid composition in muscle and fat tissues. The ME1 gene is a strong candidate for fat accumulation, as it encodes the malic enzyme, which is required for fatty acid synthesis. We identified seven new polymorphisms in 3'UTR of the ME1 gene and moreover confirmed the presence of 4 polymorphisms detected previously. Interestingly, the studied Duroc pigs were monomorphic at all these polymorphic sites, while in 3 other breeds (Pietrain, Polish Landrace and Polish Large White), the polymorphisms were unevenly distributed. One of the novel SNPs (c.*488A>G) was found in the Polish Large White and the Polish Landrace only, and the association studies revealed that it was significantly associated with backfat thickness and average daily weight gain in the Polish Landrace (N = 207) and the Polish Large White (N = 157). This SNP was differently associated with ME1 transcript level in muscle and backfat. The in silico analysis of another novel SNP (c.*548C>T) indicated that it is located within a binding sequence conserved among vertebrates for the miR-30 family in 3'UTR of the ME1. It was shown that in the longissimus muscle, but not in adipose tissue, CT gilts compared with CC ones had significantly lower levels of the ME1 transcript. This polymorphism, however, was not associated with production traits. Additionally, we observed that transcript level of the ME1 was significantly higher in subcutaneous fat than in the longissimus muscle, as well as both investigated tissues of the Polish Landrace when compared to the other breeds. However, no association was found between this polymorphism and fatty acid profiles. We conclude that the ME1 gene polymorphism (c.*488A>G) is a potential marker for porcine backfat thickness. © 2013 Blackwell Verlag GmbH.

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

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

Wang, Dang; Fang, Liurong; Luo, Rui

2010-08-13

Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reductionmore » of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.« less

Separation of replication and transcription domains in nucleoli.

PubMed

Smirnov, E; Borkovec, J; Kováčik, L; Svidenská, S; Schröfel, A; Skalníková, M; Švindrych, Z; Křížek, P; Ovesný, M; Hagen, G M; Juda, P; Michalová, K; Cardoso, M C; Cmarko, D; Raška, I

2014-12-01

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation. Copyright © 2014 Elsevier Inc. All rights reserved.

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida 1

PubMed Central

Weiss, David; van Blokland, Rik; Kooter, Jan M.; Mol, Joseph N. M.; van Tunen, Arjen J.

1992-01-01

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA3). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA3 in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA3 in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA3 was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA3 controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA3 resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA3. The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA3 suggest that GA3 controls chs transcription in an indirect manner. Our data support a model in which GA3 induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668613

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

PubMed

Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

2013-05-21

Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR

PubMed Central

2013-01-01

Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071

A Successful Automated Online Transcript System.

ERIC Educational Resources Information Center

Barnfather, Tony; Rosmanitz, Fred

1981-01-01

The academic transcript system at the University of Calgary, a successful application that combines online and batch processing to generate student transcripts, is described. In addition to improved service to students and alumni, the registrar's operating budget has been reduced and productivity has increased. (Author/MLW)

Association of Neuropeptide Y (NPY), Interleukin-1B (IL1B) Genetic Variants and Correlation of IL1B Transcript Levels with Vitiligo Susceptibility

PubMed Central

Laddha, Naresh C.; Dwivedi, Mitesh; Mansuri, Mohmmad Shoab; Singh, Mala; Patel, Hetanshi H.; Agarwal, Nishtha; Shah, Anish M.; Begum, Rasheedunnisa

2014-01-01

Background Vitiligo is a depigmenting disorder resulting from loss of functional melanocytes in the skin. NPY plays an important role in induction of immune response by acting on a variety of immune cells. NPY synthesis and release is governed by IL1B. Moreover, genetic variability in IL1B is reported to be associated with elevated NPY levels. Objectives Aim of the present study was to explore NPY promoter −399T/C (rs16147) and exon2 +1128T/C (rs16139) polymorphisms as well as IL1B promoter −511C/T (rs16944) polymorphism and to correlate IL1B transcript levels with vitiligo. Methods PCR-RFLP method was used to genotype NPY -399T/C SNP in 454 patients and 1226 controls; +1128T/C SNP in 575 patients and 1279 controls and IL1B −511C/T SNP in 448 patients and 785 controls from Gujarat. IL1B transcript levels in blood were also assessed in 105 controls and 95 patients using real-time PCR. Results Genotype and allele frequencies for NPY −399T/C, +1128T/C and IL1B −511C/T SNPs differed significantly (p<0.0001, p<0.0001; p = 0.0161, p = 0.0035 and p<0.0001, p<0.0001) between patients and controls. ‘TC’ haplotype containing minor alleles of NPY polymorphisms was significantly higher in patients and increased the risk of vitiligo by 2.3 fold (p<0.0001). Transcript levels of IL1B were significantly higher, in patients compared to controls (p = 0.0029), in patients with active than stable vitiligo (p = 0.015), also in female patients than male patients (p = 0.026). Genotype-phenotype correlation showed moderate association of IL1B -511C/T polymorphism with higher IL1B transcript levels. Trend analysis revealed significant difference between patients and controls for IL1B transcript levels with respect to different genotypes. Conclusion Our results suggest that NPY −399T/C, +1128T/C and IL1B −511C/T polymorphisms are associated with vitiligo and IL1B −511C/T SNP influences its transcript levels leading to increased risk for vitiligo in

The relationship between gene transcription and combinations of histone modifications

NASA Astrophysics Data System (ADS)

Cui, Xiangjun; Li, Hong; Luo, Liaofu

2012-09-01

Histone modification is an important subject of epigenetics which plays an intrinsic role in transcriptional regulation. It is known that multiple histone modifications act in a combinatorial fashion. In this study, we demonstrated that the pathways within constructed Bayesian networks can give an indication for the combinations among 12 histone modifications which have been studied in the TSS+1kb region in S. cerevisiae. After Bayesian networks for the genes with high transcript levels (H-network) and low transcript levels (L-network) were constructed, the combinations of modifications within the two networks were analyzed from the view of transcript level. The results showed that different combinations played dissimilar roles in the regulation of gene transcription when there exist differences for gene expression at transcription level.

Shift from posttranscriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human Jurkat cells.

PubMed

Razanajaona, D; Maroc, C; Lopez, M; Mannoni, P; Gabert, J

1992-05-01

The expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is differentially regulated in various cell types. We investigated the mechanisms controlling its expression in 12-O-tetradecanoylphorbol-13-acetate plus phytohemagglutinin-stimulated Jurkat cells, a human T-cell line. In unstimulated cells, GM-CSF mRNA was undetectable by Northern blot. Upon activation, it was detected from 3 h onward, with a progressive increase in the levels of the transcript up to 24 h of stimulation. Whereas cycloheximide treatment at the time of stimulation blocked mRNA induction, its addition at later times resulted in a marked increase in transcript levels. Run-on analysis showed that transcription of the GM-CSF gene was low to undetectable in unstimulated cells; stimulation led to transcriptional activation, which was weak at 6 h but had increased 16-fold at 24 h. In addition, the mRNA half-life decreased during activation, from 2.5 h at 6 h down to 45 min at 24 h. Cycloheximide treatment increased GM-CSF mRNA half-life (3- and 4-fold, respectively). Our results show: (a) both transcriptional and posttranscriptional signals regulate GM-CSF mRNA levels in activated Jurkat cells, (b) de novo protein synthesis is required for mRNA induction, whereas destabilizing labile proteins control the transcript stability, and (c) a shift from a posttranscriptional to a predominant transcriptional control of GM-CSF gene expression occurs during activation.

Alterations in the neuropeptide galanin system in major depressive disorder involve levels of transcripts, methylation, and peptide

PubMed Central

Barde, Swapnali; Rüegg, Joelle; Prud’homme, Josée; Ekström, Tomas J.; Palkovits, Miklos; Turecki, Gustavo; Bagdy, Gyorgy; Ihnatko, Robert; Theodorsson, Elvar; Juhasz, Gabriella; Diaz-Heijtz, Rochellys; Mechawar, Naguib; Hökfelt, Tomas G. M.

2016-01-01

Major depressive disorder (MDD) is a substantial burden to patients, families, and society, but many patients cannot be treated adequately. Rodent experiments suggest that the neuropeptide galanin (GAL) and its three G protein-coupled receptors, GAL1–3, are involved in mood regulation. To explore the translational potential of these results, we assessed the transcript levels (by quantitative PCR), DNA methylation status (by bisulfite pyrosequencing), and GAL peptide by RIA of the GAL system in postmortem brains from depressed persons who had committed suicide and controls. Transcripts for all four members were detected and showed marked regional variations, GAL and galanin receptor 1 (GALR1) being most abundant. Striking increases in GAL and GALR3 mRNA levels, especially in the noradrenergic locus coeruleus and the dorsal raphe nucleus, in parallel with decreased DNA methylation, were found in both male and female suicide subjects as compared with controls. In contrast, GAL and GALR3 transcript levels were decreased, GALR1 was increased, and DNA methylation was increased in the dorsolateral prefrontal cortex of male suicide subjects, however, there were no changes in the anterior cingulate cortex. Thus, GAL and its receptor GALR3 are differentially methylated and expressed in brains of MDD subjects in a region- and sex-specific manner. Such an epigenetic modification in GALR3, a hyperpolarizing receptor, might contribute to the dysregulation of noradrenergic and serotonergic neurons implicated in the pathogenesis of MDD. Thus, one may speculate that a GAL3 antagonist could have antidepressant properties by disinhibiting the firing of these neurons, resulting in increased release of noradrenaline and serotonin in forebrain areas involved in mood regulation. PMID:27940914

The Alpaca Melanocortin 1 Receptor: Gene Mutations, Transcripts, and Relative Levels of Expression in Ventral Skin Biopsies

PubMed Central

Renieri, Carlo; La Terza, Antonietta

2015-01-01

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5′-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3′UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation. PMID:25685836

tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level.

PubMed

Dill, Kariena K; Amacher, Sharon L

2005-11-15

We have identified the zebrafish tortuga (tor) gene by an ENU-induced mutation that disrupts the presomitic mesoderm (PSM) expression of Notch pathway genes. In tor mutants, Notch pathway gene expression persists in regions of the PSM where expression is normally off in wild type embryos. The expression of hairy/Enhancer of split-related 1 (her1) is affected first, followed by the delta genes deltaC and deltaD, and finally, by another hairy/Enhancer of split-related gene, her7. In situ hybridization with intron-specific probes for her1 and deltaC indicates that transcriptional bursts of expression are normal in tor mutants, suggesting that tor normally functions to refine her1 and deltaC message levels downstream of transcription. Despite the striking defects in Notch pathway gene expression, somite boundaries form normally in tor mutant embryos, although somitic mesoderm defects are apparent later, when cells mature to form muscle fibers. Thus, while the function of Notch pathway genes is required for proper somite formation, the tor mutant phenotype suggests that precise oscillations of Notch pathway transcripts are not essential for establishing segmental pattern in the presomitic mesoderm.

The C2H2-type transcription factor, FlbC, is involved in the transcriptional regulation of Aspergillus oryzae glucoamylase and protease genes specifically expressed in solid-state culture.

PubMed

Tanaka, Mizuki; Yoshimura, Midori; Ogawa, Masahiro; Koyama, Yasuji; Shintani, Takahiro; Gomi, Katsuya

2016-07-01

Aspergillus oryzae produces a large amount of secreted proteins in solid-state culture, and some proteins such as glucoamylase (GlaB) and acid protease (PepA) are specifically produced in solid-state culture, but rarely in submerged culture. From the disruption mutant library of A. oryzae transcriptional regulators, we successfully identified a disruption mutant showing an extremely low production level of GlaB but a normal level of α-amylase production. This strain was a disruption mutant of the C2H2-type transcription factor, FlbC, which is reported to be involved in the regulation of conidiospore development. Disruption mutants of other upstream regulators comprising a conidiation regulatory network had no apparent effect on GlaB production in solid-state culture. In addition to GlaB, the production of acid protease in solid-state culture was also markedly decreased by flbC disruption. Northern blot analyses revealed that transcripts of glaB and pepA were significantly decreased in the flbC disruption strain. These results suggested that FlbC is involved in the transcriptional regulation of genes specifically expressed under solid-state cultivation conditions, possibly independent of the conidiation regulatory network.

PlantTFDB: a comprehensive plant transcription factor database

PubMed Central

Guo, An-Yuan; Chen, Xin; Gao, Ge; Zhang, He; Zhu, Qi-Hui; Liu, Xiao-Chuan; Zhong, Ying-Fu; Gu, Xiaocheng; He, Kun; Luo, Jingchu

2008-01-01

Transcription factors (TFs) play key roles in controlling gene expression. Systematic identification and annotation of TFs, followed by construction of TF databases may serve as useful resources for studying the function and evolution of transcription factors. We developed a comprehensive plant transcription factor database PlantTFDB (http://planttfdb.cbi.pku.edu.cn), which contains 26 402 TFs predicted from 22 species, including five model organisms with available whole genome sequence and 17 plants with available EST sequences. To provide comprehensive information for those putative TFs, we made extensive annotation at both family and gene levels. A brief introduction and key references were presented for each family. Functional domain information and cross-references to various well-known public databases were available for each identified TF. In addition, we predicted putative orthologs of those TFs among the 22 species. PlantTFDB has a simple interface to allow users to search the database by IDs or free texts, to make sequence similarity search against TFs of all or individual species, and to download TF sequences for local analysis. PMID:17933783

A novel GhBEE1-Like gene of cotton causes anther indehiscence in transgenic Arabidopsis under uncontrolled transcription level.

PubMed

Chen, Eryong; Wang, Xiaoqian; Gong, Qian; Butt, Hamama Islam; Chen, Yanli; Zhang, Chaojun; Yang, Zuoren; Wu, Zhixia; Ge, Xiaoyang; Zhang, Xianlong; Li, Fuguang; Zhang, Xueyan

2017-09-05

Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton (Gossypium hirsutum), named GhBEE1-Like. Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding. Copyright © 2017. Published by Elsevier B.V.

Regulation of IL-17 in autoimmune diseases by transcriptional factors and microRNAs

PubMed Central

Khan, Deena; Ansar Ahmed, S.

2015-01-01

In recent years, IL-17A (IL-17), a pro-inflammatory cytokine, has received intense attention of researchers and clinicians alike with documented effects in inflammation and autoimmune diseases. IL-17 mobilizes, recruits and activates different cells to increase inflammation. Although protective in infections, overproduction of IL-17 promotes inflammation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, among others. Regulating IL-17 levels or action by using IL-17-blocking antibodies or IL-17R antagonist has shown to attenuate experimental autoimmune diseases. It is now known that in addition to IL-17-specific transcription factor, RORγt, several other transcription factors and select microRNAs (miRNA) regulate IL-17. Given that miRNAs are dysregulated in autoimmune diseases, a better understanding of transcriptional factors and miRNA regulation of IL-17 expression and function will be essential for devising potential new therapies. In this review, we will overview IL-17 induction and function in relation to autoimmune diseases. In addition, current findings on transcriptional regulation of IL-17 induction and plausible interplay between IL-17 and miRNA in autoimmune diseases are highlighted. PMID:26236331

Archaeal RNA polymerase and transcription regulation

PubMed Central

Jun, Sung-Hoon; Reichlen, Matthew J.; Tajiri, Momoko; Murakami, Katsuhiko S.

2010-01-01

To elucidate the mechanism of transcription by cellular RNA polymerases (RNAPs), high resolution X-ray crystal structures together with structure-guided biochemical, biophysical and genetics studies are essential. The recently-solved X-ray crystal structures of archaeal RNA polymerase (RNAP) allow a structural comparison of the transcription machinery among all three domains of life. The archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukaryote at the molecular level than bacteria. According to these structures, the archaeal transcription apparatus, which includes RNAP and general transcription factors, is similar to the eukaryotic transcription machinery. Yet, the transcription regulators, activators and repressors, encoded by archaeal genomes are closely related to bacterial factors. Therefore, archaeal transcription appears to possess an intriguing hybrid of eukaryotic-type transcription apparatus and bacterial-like regulatory mechanisms. Elucidating the transcription mechanism in archaea, which possesses a combination of bacterial and eukaryotic transcription mechanisms that are commonly regarded as separate and mutually exclusive, can provide data that will bring basic transcription mechanisms across all three domains of life. PMID:21250781

The significance of alternative transcripts for Caenorhabditis elegans transcription factor genes, based on expression pattern analysis

PubMed Central

2013-01-01

Background Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal’s genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. Results Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. Conclusions Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms. PMID:23586691

The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

PubMed

Gasanov, N B; Toshchakov, S V; Georgiev, P G; Maksimenko, O G

2015-01-01

Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

ICE1 of Pyrus ussuriensis functions in cold tolerance by enhancing PuDREBa transcriptional levels through interacting with PuHHP1

NASA Astrophysics Data System (ADS)

Huang, Xiaosan; Li, Kongqing; Jin, Cong; Zhang, Shaoling

2015-12-01

ICE1 transcription factor plays an important role in plant cold stress via regulating the expression of stress-responsive genes. In this study, a PuICE1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. The expression levels of the PuICE1 were induced by cold, dehydration and salt, with the greatest induction under cold conditions. PuICE1 was localized in the nucleus and could bind specifically to the MYC element in the PuDREBa promoter. The PuICE1 fused to the GAL4 DNA-binding domain to have transcriptional activation activity. Ectopic expression of the PuICE1 in tomato conferred enhanced tolerance to cold stress at cold temperatures, less electrolyte leakage, less MDA content, higher chlorophyll content, higher survival rate, higher proline content, higher activities of enzymes. In additon, steady-state mRNA levels of six stress-responsive genes coding for either functional or regulatory genes were induced to higher levels in the transgenic lines by cold stress. Yeast two-hybrid, transient assay, split luciferase complementation and BiFC assays all revealed that PuHHP1 protein can physically interact with PuICE1. Taken together, these results demonstrated that PuICE1 plays a positive role in cold tolerance, which may be due to enhancement of PuDREBa transcriptional levels through interacting with the PuHHP1.

Reduced BRCA1 transcript levels in freshly isolated blood leukocytes from BRCA1 mutation carriers is mutation specific.

PubMed

Chehade, Rania; Pettapiece-Phillips, Rachael; Salmena, Leonardo; Kotlyar, Max; Jurisica, Igor; Narod, Steven A; Akbari, Mohammad R; Kotsopoulos, Joanne

2016-08-17

BRCA1 mutation carriers face a high lifetime risk of developing both breast and ovarian cancer. Haploinsufficiency is thought to predispose these women to cancer by reducing the pool of available BRCA1 transcript and protein, thereby compromising BRCA1 function. Whether or not cancer-free BRCA1 mutation carriers have lower messenger (m)RNA transcript levels in peripheral blood leukocytes has not been evaluated. The primary aim of this study was to characterize an association between BRCA1 mutation status and BRCA1 mRNA leukocyte expression levels among healthy women with a BRCA1 mutation. RNA was extracted from freshly isolated peripheral blood leukocytes of 58 cancer-free, female participants (22 BRCA1 mutation carriers and 36 non-carriers). The expression levels of 236 cancer-associated genes, including BRCA1, were quantified using the Human Cancer Reference gene panel from the Nanostring Technologies nCounter Analysis System. Multivariate modeling demonstrated that carrying a BRCA1 mutation was the most significant predictor of BRCA1 mRNA levels. BRCA1 mRNA levels were significantly lower in BRCA1 mutation carriers compared to non-carriers (146.7 counts vs. 175.1 counts; P = 0.002). Samples with BRCA1 mutations within exon 11 had lower BRCA1 mRNA levels than samples with mutations within the 5' and 3' regions of the BRCA1 gene (122.1 counts vs. 138.9 and 168.6 counts, respectively; P = 0.003). Unsupervised hierarchical clustering of gene expression profiles from freshly isolated blood leukocytes revealed that BRCA1 mutation carriers cluster more closely with other BRCA1 mutation carriers than with BRCA1 wild-type samples. Moreover, a set of 17 genes (including BRCA1) previously shown to be involved in carcinogenesis, were differentially expressed between BRCA1 mutation carriers and non-carriers. Overall, these findings support the concept of BRCA1 haploinsufficiency wherein a specific mutation results in dosage-dependent alteration of BRCA1 at the

Post-transcriptional bursting in genes regulated by small RNA molecules

NASA Astrophysics Data System (ADS)

Rodrigo, Guillermo

2018-03-01

Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Intrinsic disorder in transcription factors†

PubMed Central

Liu, Jiangang; Perumal, Narayanan B.; Oldfield, Christopher J.; Su, Eric W.; Uversky, Vladimir N.; Dunker, A. Keith

2008-01-01

Intrinsic disorder (ID) is highly abundant in eukaryotes, which reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported, no systematic analysis has been reported so far. To test for a general prevalence of intrinsic disorder in transcriptional regulation, we used the Predictor Of Natural Disorder Regions (PONDR) to analyze the abundance of intrinsic disorder in three transcription factor datasets and two control sets. This analysis revealed that from 94.13% to 82.63% of transcription factors posses extended regions of intrinsic disorder, relative to 54.51% and 18.64% of the proteins in two control datasets, which indicates the significant prevalence of intrinsic disorder in transcription factors. This propensity of transcription factors for intrinsic disorder was confirmed by cumulative distribution function analysis and charge-hydropathy plots. The amino acid composition analysis showed that all three transcription factor datasets were substantially depleted in order-promoting residues, and significantly enriched in disorder-promoting residues. Our analysis of the distribution of disorder within the transcription factor datasets revealed that: (a) The AT-hooks and basic regions of transcription factor DNA-binding domains are highly disordered; (b) The degree of disorder in transcription factor activation regions is much higher than that in DNA-binding domains; (c) The degree of disorder is significantly higher in eukaryotic transcription factors than in prokaryotic transcription factors; (d) The level of α-MoRFs (molecular recognition feature) prediction is much higher in transcription factors. Overall, our data reflected the fact that the eukaryotes with well-developed gene transcription machinery require transcription factor flexibility to be more efficient. PMID:16734424

Transcriptional regulation of hepatic lipogenesis.

PubMed

Wang, Yuhui; Viscarra, Jose; Kim, Sun-Joong; Sul, Hei Sook

2015-11-01

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

Transcriptional Mechanisms Underlying Hemoglobin Synthesis

PubMed Central

Katsumura, Koichi R.; DeVilbiss, Andrew W.; Pope, Nathaniel J.; Johnson, Kirby D.; Bresnick, Emery H.

2013-01-01

The physiological switch in expression of the embryonic, fetal, and adult β-like globin genes has garnered enormous attention from investigators interested in transcriptional mechanisms and the molecular basis of hemoglobinopathies. These efforts have led to the discovery of cell type-specific transcription factors, unprecedented mechanisms of transcriptional coregulator function, genome biology principles, unique contributions of nuclear organization to transcription and cell function, and promising therapeutic targets. Given the vast literature accrued on this topic, this article will focus on the master regulator of erythroid cell development and function GATA-1, its associated proteins, and its frontline role in controlling hemoglobin synthesis. GATA-1 is a crucial regulator of genes encoding hemoglobin subunits and heme biosynthetic enzymes. GATA-1-dependent mechanisms constitute an essential regulatory core that nucleates additional mechanisms to achieve the physiological control of hemoglobin synthesis. PMID:23838521

Neurotoxocarosis alters myelin protein gene transcription and expression.

PubMed

Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

2015-06-01

Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

Sucrose-induced anthocyanin accumulation in vegetative tissue of Petunia plants requires anthocyanin regulatory transcription factors.

PubMed

Ai, Trinh Ngoc; Naing, Aung Htay; Arun, Muthukrishnan; Lim, Sun-Hyung; Kim, Chang Kil

2016-11-01

The effects of three different sucrose concentrations on plant growth and anthocyanin accumulation were examined in non-transgenic (NT) and transgenic (T 2 ) specimens of the Petunia hybrida cultivar 'Mirage rose' that carried the anthocyanin regulatory transcription factors B-Peru+mPAP1 or RsMYB1. Anthocyanin accumulation was not observed in NT plants in any treatments, whereas a range of anthocyanin accumulation was observed in transgenic plants. The anthocyanin content detected in transgenic plants expressing the anthocyanin regulatory transcription factors (B-Peru+mPAP1 or RsMYB1) was higher than that in NT plants. In addition, increasing sucrose concentration strongly enhanced anthocyanin content as shown by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, wherein increased concentrations of sucrose enhanced transcript levels of the transcription factors that are responsible for the induction of biosynthetic genes involved in anthocyanin synthesis; this pattern was not observed in NT plants. In addition, sucrose affected plant growth, although the effects were different between NT and transgenic plants. Taken together, the application of sucrose could enhance anthocyanin production in vegetative tissue of transgenic Petunia carrying anthocyanin regulatory transcription factors, and this study provides insights about interactive effects of sucrose and transcription factors in anthocyanin biosynthesis in the transgenic plant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Genome-wide Analysis Reveals Extensive Functional Interaction between DNA Replication Initiation and Transcription in the Genome of Trypanosoma brucei

PubMed Central

Tiengwe, Calvin; Marcello, Lucio; Farr, Helen; Dickens, Nicholas; Kelly, Steven; Swiderski, Michal; Vaughan, Diane; Gull, Keith; Barry, J. David; Bell, Stephen D.; McCulloch, Richard

2012-01-01

Summary Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion. PMID:22840408

Up-Regulation of Bcl-xl by Hepatocyte Growth Factor in Human Mesothelioma Cells Involves ETS Transcription Factors

PubMed Central

Cao, Xiaobo; Littlejohn, James; Rodarte, Charles; Zhang, Lidong; Martino, Benjamin; Rascoe, Philip; Hamid, Kamran; Jupiter, Daniel; Smythe, W. Roy

2009-01-01

Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl. PMID:19834061

Mediator-regulated transcription through the +1 nucleosome.

PubMed

Nock, Adam; Ascano, Janice M; Barrero, Maria J; Malik, Sohail

2012-12-28

Many genes are regulated at the level of a Pol II that is recruited to a nucleosome-free region upstream of the +1 nucleosome. How the Mediator coactivator complex, which functions at multiple steps, affects transcription through the promoter proximal region, including this nucleosome, remains largely unaddressed. We have established a fully defined in vitro assay system to delineate mechanisms for Pol II transit across the +1 nucleosome. Our results reveal cooperative functions of multiple cofactors, particularly of Mediator and elongation factor SII, in transcribing into this nucleosome. This is achieved, in part, through an unusual activity of SII that alters the intrinsic catalytic properties of promoter-proximal Pol II and, in concert with the Mediator, leads to enhancement in transcription of nucleosomal DNA. Our data provide additional mechanistic bases for Mediator function after recruitment of Pol II and, potentially, for regulation of genes controlled via nucleosome-mediated promoter-proximal pausing. Copyright © 2012 Elsevier Inc. All rights reserved.

SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

PubMed

Almeida, Luciana O; Neto, Marinaldo P C; Sousa, Lucas O; Tannous, Maryna A; Curti, Carlos; Leopoldino, Andreia M

2017-04-18

Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

Low-level expression of human ACAT2 gene in monocytic cells is regulated by the C/EBP transcription factors

PubMed Central

Guo, Dongqing; Lu, Ming; Hu, Xihan; Xu, Jiajia; Hu, Guangjing; Zhu, Ming; Zhang, Xiaowei; Li, Qin; Chang, Catherine C. Y.; Chang, Tayuan; Song, Baoliang; Xiong, Ying; Li, Boliang

2016-01-01

Acyl-coenzyme A:cholesterol acyltransferases (ACATs) are the exclusive intracellular enzymes that catalyze the formation of cholesteryl/steryl esters (CE/SE). In our previous work, we found that the high-level expression of human ACAT2 gene with the CpG hypomethylation of its whole promoter was synergistically regulated by two transcription factors Cdx2 and HNF1α in the intestine and fetal liver. Here, we first observed that the specific CpG-hypomethylated promoter was correlated with the low expression of human ACAT2 gene in monocytic cell line THP-1. Then, two CCAAT/enhancer binding protein (C/EBP) elements within the activation domain in the specific CpG-hypomethylation promoter region were identified, and the expression of ACAT2 in THP-1 cells was evidently decreased when the C/EBP transcription factors were knock-downed using RNAi technology. Furthermore, ChIP assay confirmed that C/EBPs directly bind to their elements for low-level expression of human ACAT2 gene in THP-1 cells. Significantly, the increased expressions of ACAT2 and C/EBPs were also found in macrophages differentiated from both ATRA-treated THP-1 cells and cultured human blood monocytes. These results demonstrate that the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the C/EBP transcription factors in monocytic cells, and imply that the lowly expressed ACAT2 catalyzes the synthesis of certain CE/SE that are assembled into lipoproteins for the secretion. PMID:27688151

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

PubMed Central

Pacifico, D.; Galetto, L.; Rashidi, M.; Abbà, S.; Palmano, S.; Firrao, G.; Bosco, D.

2015-01-01

To highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of “Candidatus Phytoplasma asteris,” chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatus and Euscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection of A. thaliana and M. quadripunctulatus but were more constant in E. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant. PMID:25636844

The cre-inducer doxycycline lowers cytokine and chemokine transcript levels in the gut of mice.

PubMed

Hansen, Axel Kornerup; Malm, Sara Astrup; Metzdorff, Stine B

2017-11-01

The antibiotic doxycycline is used as an inducer of recombinase (cre)-based conditional gene knockout in mice, which is a common tool to show the effect of disrupted gene functions only in one period of a research animal's life. However, other types of such antibiotics have been shown to have a strong impact on the immune system. Here we show that in C57BL/6 mice, the most commonly applied strain for genetic modification, doxycycline treatment lowered transcription of the genes Il1b, Il10, Il18, Tnf, Cxcl1, and Cxcl2 in the ileum, and of the gene Il18 in colon. Cytokines and chemokines encoded by these genes are important in the disease expression in a range of mouse models. Although protein abundances only rarely correlate 100% to transcript levels, and the net result, therefore, may be less dramatic, it seems reasonable to be aware that a broad spectrum antibiotic, such as doxycycline, may impact the transgenic animal in ways unrelated to the activation of the gene deletion.

Changes in the level of perforin and its transcript during effector and target cell interactions.

PubMed

Kim, K K; Blakely, A; Zhou, Z; Davis, J; Clark, W; Kwon, B S

1993-05-01

Perforin is a cytoplasmic granule protein expressed in cytotoxic lymphocytes, and is capable of lysing target cells. This protein is induced as cytotoxic T cells are activated, and the mRNA expression is modulated by various stimulators. These observations suggest possible changes in the level of perforin transcripts and protein when killer lymphocytes meet specific target cells leading to target cell death. To address this question, we examined three murine T-cell clones and primary human NK cells in perforin expression. When the cytotoxic lymphocytes were exposed to sensitive targets, perforin mRNA disappeared within 5 to 30 min and appeared within an hour thereafter. Among the murine T cell clones, L3 and OE4 showed two phases of mRNA decrease while human NK cells and the third murine T cell clone, AB.1, showed only one phase of mRNA loss during a 240 min period. The data indicate that when cytotoxic lymphocytes receive signals from a sensitive target, the cells rapidly degrade previously accumulated perforin mRNA and synthesize new transcripts. Interestingly, heat shock protein 70 mRNA was induced as the perforin mRNA levels recovered, while P55 Il-2 receptor mRNA was downregulated within 5 min after exposure to targets. The perforin protein level also rapidly decreased immediately after the interaction with the target, followed by a recovery, and then another decrease as seen in primary human NK cells, OE4 and L3 cells. However, in the AB.1 clone, no change in perforin content was detectable, despite the loss of perforin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

PubMed

Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

2011-07-01

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Impact of nitrogen concentration on validamycin A production and related gene transcription in fermentation of Streptomyces hygroscopicus 5008.

PubMed

Wei, Zhen-Hua; Bai, Linquan; Deng, Zixin; Zhong, Jian-Jiang

2012-09-01

Validamycin A (VAL-A) is an important and widely used agricultural antibiotic. In this study, statistical screening designs were applied to identify significant medium variables for VAL-A production and to find their optimal levels. The optimized medium caused 70% enhancement of VAL-A production. The difference between optimized medium and original medium suggested that low nitrogen source level might attribute to the enhancement of VAL-A production. The addition of different nitrogen sources to the optimized medium inhibited VAL-A production, which confirmed the importance of nitrogen concentration for VAL-A production. Furthermore, differences in structural gene transcription and enzyme activity between the two media were assayed. The results showed that lower nitrogen level in the optimized medium could regulate VAL-A production in gene transcriptional level. Our previous study indicated that the transcription of VAL-A structural genes could be enhanced at elevated temperature. In this work, the increased fermentation temperature from 37 to 42 °C with the optimized medium enhanced VAL-A production by 39%, which testified to the importance of structural gene transcription in VAL-A production. The information is useful for further VAL-A production enhancement.

Endocrine regulation of gonadotropin and growth hormone gene transcription in fish.

PubMed

Melamed, P; Rosenfeld, H; Elizur, A; Yaron, Z

1998-06-01

recrudescing male coho salmon injected with T or E2. T or E2 administered in vivo has dramatic stimulatory effects on the II beta transcript levels in immature fish of a number of species, while less powerful effects are seen in vitro. A response is also seen in cells from early maturing rainbow trout or tilapia, or regressed tilapia, but not in cells from late maturing or spawning fish. These results are substantiated by the finding that the promoter of the salmon II beta gene contains several estrogen responsive elements (EREs) which react and interact differently when exposed to varying levels of E2. In addition, activator protein-1 (AP-1) and steroidogenic factor-1 (SF-1) response elements are also found in the salmon II beta promoter; the AP-1 site is located close to a half ERE, while the SF-1 acts synergistically with the E2 receptor. The mRNA levels of both AP-1 and SP-1 are elevated, at least in mammals, by GnRH, suggesting possible sites for cross-talk between GnRH and steroid activated pathways. Reports of the effects of T or E2 on GH transcription differ. No effect is seen in vitro in pituitaries of tilapia, juvenile rainbow trout or common carp, but T does increase the transcript levels in pituitaries of both immature and mature goldfish. Reasons for these discrepancies are unclear, but other systemic hormones may be more instrumental than the gonadal steroids in regulating GH transcription. These include T3 which increases both GH mRNA levels and de novo synthesis (in tilapia and common carp) and insulin-like growth factor-I (IGF-I) which reduces GH transcript levels as well as inhibiting GH release.

Flp and Cre expressed from Flp–2A–Cre and Flp–IRES–Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange

PubMed Central

Anderson, Rachelle P.; Voziyanova, Eugenia; Voziyanov, Yuri

2012-01-01

Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35–45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp–2A–Cre and Flp–IRES–Cre transcription units. In contrast, the lowest level of dual RMCE (∼1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp–to–Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes. PMID:22270085

Annotation of Alternatively Spliced Proteins and Transcripts with Protein-Folding Algorithms and Isoform-Level Functional Networks.

PubMed

Li, Hongdong; Zhang, Yang; Guan, Yuanfang; Menon, Rajasree; Omenn, Gilbert S

2017-01-01

Tens of thousands of splice isoforms of proteins have been catalogued as predicted sequences from transcripts in humans and other species. Relatively few have been characterized biochemically or structurally. With the extensive development of protein bioinformatics, the characterization and modeling of isoform features, isoform functions, and isoform-level networks have advanced notably. Here we present applications of the I-TASSER family of algorithms for folding and functional predictions and the IsoFunc, MIsoMine, and Hisonet data resources for isoform-level analyses of network and pathway-based functional predictions and protein-protein interactions. Hopefully, predictions and insights from protein bioinformatics will stimulate many experimental validation studies.

Relationships between Translation and Transcription Processes during fMRI Connectivity Scanning and Coded Translation and Transcription in Writing Products after Scanning in Children with and without Transcription Disabilities

PubMed Central

Wallis, Peter; Richards, Todd; Boord, Peter; Abbott, Robert; Berninger, Virginia

2018-01-01

Students with transcription disabilities (dysgraphia/impaired handwriting, n = 13 or dyslexia/impaired word spelling, n = 16) or without transcription disabilities (controls) completed transcription and translation (idea generating, planning, and creating) writing tasks during fMRI connectivity scanning and compositions after scanning, which were coded for transcription and translation variables. Compositions in both groups showed diversity in genre beyond usual narrative-expository distinction; groups differed in coded transcription but not translation variables. For the control group specific transcription or translation tasks during scanning correlated with corresponding coded transcription or translation skills in composition, but connectivity during scanning was not correlated with coded handwriting during composing in dysgraphia group and connectivity during translating was not correlated with any coded variable during composing in dyslexia group. Results are discussed in reference to the trend in neuroscience to use connectivity from relevant seed points while performing tasks and trends in education to recognize the generativity (creativity) of composing at both the genre and syntax levels. PMID:29600113

Growth-dependent regulation of rRNA synthesis is mediated by a transcription initiation factor (TIF-IA).

PubMed

Buttgereit, D; Pflugfelder, G; Grummt, I

1985-11-25

Mouse RNA polymerase I requires at least two chromatographically distinct transcription factors (designated TIF-IA and TIF-IB) to initiate transcription accurately and efficiently in vitro. In this paper we describe the partial purification of TIF-IA by a four-step fractionation procedure. The amount or activity of TIF-IA fluctuates in response to the physiological state of the cells. Extracts from quiescent cells are incapable of specific transcription and do not contain detectable levels of TIF-IA. Transcriptionally inactive extracts can be restored by the addition of TIF-IA preparations that have been highly purified from exponentially growing cells. During the fractionating procedure TIF-IA co-purifies with RNA polymerase I, suggesting that it is functionally associated with the transcribing enzyme. We suggest that only those enzyme molecules that are associated with TIF-IA are capable to interact with TIF-IB and to initiate transcription.

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells.

PubMed

Mina, Marco; Magi, Shigeyuki; Jurman, Giuseppe; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Arner, Erik; Forrest, Alistair R R; Carninci, Piero; Hayashizaki, Yoshihide; Daub, Carsten O; Okada-Hatakeyama, Mariko; Furlanello, Cesare

2015-07-16

The analysis of CAGE (Cap Analysis of Gene Expression) time-course has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. To identify the most prominent transcriptional regulations induced by growth factors in human breast cancer, we apply here the Complexity Invariant Dynamic Time Warping motif EnRichment (CIDER) analysis approach to the CAGE time-course datasets of MCF-7 cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We identify a multi-level cascade of regulations rooted by the Serum Response Factor (SRF) transcription factor, connecting the MAPK-mediated transduction of the HRG stimulus to the negative regulation of the MAPK pathway by the members of the DUSP family phosphatases. The finding confirms the known primary role of FOS and FOSL1, members of AP-1 family, in shaping gene expression in response to HRG induction. Moreover, we identify a new potential regulation of DUSP5 and RARA (known to antagonize the transcriptional regulation induced by the estrogen receptors) by the activity of the AP-1 complex, specific to HRG response. The results indicate that a divergence in AP-1 regulation determines cellular changes of breast cancer cells stimulated by ErbB receptors.

Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants.

PubMed

Dollhofer, Veronika; Callaghan, Tony M; Griffith, Gareth W; Lebuhn, Michael; Bauer, Johann

2017-07-01

Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester. Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcript levels of genes implicated in steroidogenesis in the testes and fat tissue in relation to androstenone accumulation in fat of pubertal pigs.

PubMed

Robic, A; Feve, K; Riquet, J; Prunier, A

2016-10-01

The present study was performed to measure messenger RNA levels of steroidogenic enzymes in testes and fat tissue and determine whether they are related to fat androstenone level. Real-time polymerase chain reaction experiments were performed on 26 testes and 12 adipose tissue samples from pubertal boars using 21 genes. The absence of significant correlations between fat androstenone and the transcriptional activity of the SRD5A2 and SRD5A3 genes but the high correlation coefficient with that of the SRD5A1 gene (r = 0.62, P < 0.05) suggests that the enzyme coded by SRD5A1 is mainly responsible for the last step of androstenone synthesis. The testicular transcriptional activities of CYP17, CYP11A1, CYP19A, AKR1C-pig6, SRD5A1, LHCGR, and AR were significantly correlated. Only transcriptional levels of CYP17, CYP11A1, CYP19A, SRD5A1, and AKR1C-pig6 were correlated with the fat concentration of androstenone (0.57 < r < 0.70, P < 0.05) confirming that the amount of androstenone stored in fat is related to the production in testes of androstenone and more generally to all sex steroids. Altogether, our data are in favor of a preponderant role of AKR1C-pig6 instead of HSD17B3 for testicular synthesis of steroids. Concerning fat tissue, our data do not support a significant de novo biosynthesis of steroids in porcine adipose tissues. The presence of transcripts coding for steroid enzymes, especially those of AKR1C-pig6, suggests that steroids can be transformed. None of transcript abundance was related to androstenone accumulation (P > 0.1). Therefore, steroids synthesized elsewhere can be transformed in fat tissue but synthesis of androstenone is unlikely. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantifying Temporal Autocorrelations for the Expression of Geobacter species mRNA Gene Transcripts at Variable Ammonium Levels during in situ U(VI) Bioremediation

NASA Astrophysics Data System (ADS)

Mouser, P. J.

2010-12-01

In order to develop decision-making tools for the prediction and optimization of subsurface bioremediation strategies, we must be able to link the molecular-scale activity of microorganisms involved in remediation processes with biogeochemical processes observed at the field-scale. This requires the ability to quantify changes in the in situ metabolic condition of dominant microbes and associate these changes to fluctuations in nutrient levels throughout the bioremediation process. It also necessitates a need to understand the spatiotemporal variability of the molecular-scale information to develop meaningful parameters and constraint ranges in complex bio-physio-chemical models. The expression of three Geobacter species genes (ammonium transporter (amtB), nitrogen fixation (nifD), and a housekeeping gene (recA)) were tracked at two monitoring locations that differed significantly in ammonium (NH4+) concentrations during a field-scale experiment where acetate was injected into the subsurface to simulate Geobacteraceae in a uranium-contaminated aquifer. Analysis of amtB and nifD mRNA transcript levels indicated that NH4+ was the primary form of fixed nitrogen during bioremediation. Overall expression levels of amtB were on average 8-fold higher at NH4+ concentrations of 300 μM or more than at lower NH4+ levels (average 60 μM). The degree of temporal correlation in Geobacter species mRNA expression levels was calculated at both locations using autocorrelation methods that describe the relationship between sample semi-variance and time lag. At the monitoring location with lower NH4+, a temporal correlation lag of 8 days was observed for both amtB and nifD transcript patterns. At the location where higher NH4+ levels were observed, no discernable temporal correlation lag above the sampling frequency (approximately every 2 days) was observed for amtB or nifD transcript fluctuations. Autocorrelation trends in recA expression levels at both locations indicated that

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

PubMed Central

Plunkett, Mary H.; Natarajan, Velmurugan; Mus, Florence; Knutson, Carolann M.; Peters, John W.

2017-01-01

ABSTRACT Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results

Association of CD30 transcripts with Th1 responses and proinflammatory cytokines in patients with end-stage renal disease.

PubMed

Velásquez, Sonia Y; Opelz, Gerhard; Rojas, Mauricio; Süsal, Caner; Alvarez, Cristiam M

2016-05-01

High serum sCD30 levels are associated with inflammatory disorders and poor outcome in renal transplantation. The contribution to these phenomena of transcripts and proteins related to CD30-activation and -cleavage is unknown. We assessed in peripheral blood of end-stage renal disease patients (ESRDP) transcripts of CD30-activation proteins CD30 and CD30L, CD30-cleavage proteins ADAM10 and ADAM17, and Th1- and Th2-type immunity-related factors t-bet and GATA3. Additionally, we evaluated the same transcripts and release of sCD30 and 32 cytokines after allogeneic and polyclonal T-cell activation. In peripheral blood, ESRDP showed increased levels of t-bet and GATA3 transcripts compared to healthy controls (HC) (both P<0.01) whereas levels of CD30, CD30L, ADAM10 and ADAM17 transcripts were similar. Polyclonal and allogeneic stimulation induced higher levels of CD30 transcripts in ESRDP than in HC (both P<0.001). Principal component analysis (PCA) in allogeneic cultures of ESRDP identified two correlation clusters, one consisting of sCD30, the Th-1 cytokine IFN-γ, MIP-1α, RANTES, sIL-2Rα, MIP-1β, TNF-β, MDC, GM-CSF and IL-5, and another one consisting of CD30 and t-bet transcripts, IL-13 and proinflammatory proteins IP-10, IL-8, IL-1Rα and MCP-1. Reflecting an activated immune state, ESRDP exhibited after allostimulation upregulation of CD30 transcripts in T cells, which was associated with Th1 and proinflammatory responses. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Transcriptional and catalytic responses of antioxidant and biotransformation pathways in mussels, Mytilus galloprovincialis, exposed to chemical mixtures.

PubMed

Giuliani, Maria Elisa; Benedetti, Maura; Arukwe, Augustine; Regoli, Francesco

2013-06-15

Antioxidant and biotransformation pathways are widely studied in marine organisms exposed to environmental stressors. However, mechanisms of responses and links between different intracellular levels are not always easy to elucidate and conflicting results are frequently observed between molecular and enzymatic data. In this study, transcriptional and catalytic responses of antioxidant and biotransformation parameters were analyzed after a 4-week exposure of a marine invertebrate, Mytilus galloprovincialis, to chemical mixtures from low polluted and highly polluted sediments. A significant, dose-dependent bioaccumulation was observed for polycyclic aromatic hydrocarbons, especially low molecular weight compounds. Among antioxidant defences, catalase and glutathione peroxidases did not exhibit variations in enzymatic activity, while the corresponding gene transcriptions were up- and down-regulated, respectively; unchanged mRNA levels of superoxide dismutase confirmed the non-synchronous pathways of variations for such antioxidants. Biotransformation responses also revealed inconsistent trends between transcriptional and catalytic variations of glutathione S-transferases, and a significant increase in mRNA levels for cytochrome P450 3A1. The overall results indicated that transcriptional responses might be sensitive but do not necessarily correspond to functional changes, being more useful as "exposure" rather than "effect" biomarkers. Data on gene transcription and catalytic activities should be carefully interpreted when assessing the impact of chemical pollutants and additional studies are needed on modulation of post-transcriptional mechanisms by environmental stressors. Copyright © 2013 Elsevier B.V. All rights reserved.

Epigenetics regulates transcription and pathogenesis in the parasite Trichomonas vaginalis.

PubMed

Pachano, Tomas; Nievas, Yesica R; Lizarraga, Ayelen; Johnson, Patricia J; Strobl-Mazzulla, Pablo H; de Miguel, Natalia

2017-06-01

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T. vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T. vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T. vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T. vaginalis. © 2017 John Wiley & Sons Ltd.

Transcriptional regulation induced by cAMP elevation in mouse Schwann cells

PubMed Central

Schmid, Daniela; Zeis, Thomas; Schaeren-Wiemers, Nicole

2014-01-01

In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be modulated by forskolin. One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1), whose mRNA expression levels were reduced in treated Schwann cells. Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage. Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix), underlining its importance during Schwann cell differentiation in vitro. Comparison of samples derived from postnatal sciatic nerves and from both treated and untreated Schwann cell cultures showed considerable differences in gene expression between in vivo and in vitro, allowing us to separate Schwann cell autonomous from tissue-related changes. The whole data set of the cell culture microarray study is provided to offer an interactive search tool for genes of interest. PMID:24641305

Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.

PubMed

Thiel, Gerald; Rössler, Oliver G

2017-03-01

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulus-responsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulus-responsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Absence of 60-Hz, 0.1-mT magnetic field-induced changes in oncogene transcription rates or levels in CEM-CM3 cells.

PubMed

Jahreis, G P; Johnson, P G; Zhao, Y L; Hui, S W

1998-12-22

Our objective was to assess the reproducibility of the 60-Hz magnetic field-induced, time-dependent transcription changes of c-fos, c-jun and c-myc oncogenes in CEM-CM3 cells reported by Phillips et al. (Biochim. Biophys. Acta, 1132 (1992) 140-144). Cells were exposed to a 60-Hz magnetic field (MF) at 0.1 mT (rms), generated by a pair of Helmholtz coils energized in a reinforcing (MF) mode, or to a null magnetic field when the coils were energized in a bucking (sham) mode. After MF or sham exposure for 15, 30, 60 or 120 min, nuclei and cytoplasmic RNA were extracted. Transcription rates were measured by a nuclear run-on assay, and values were normalized against either their zero-time exposure values, or against those of the c-G3PDH (housekeeping) gene at the same time points. There was no significant difference, at P=0.05, detected between MF and either sham-exposed or control cells at any time point. Transcript levels of the oncogenes were measured by Northern analysis and normalized as above. No significant difference (P=0.05) in transcript levels between MF and either sham-exposed or control cells was detected.

The Wheat NAC Transcription Factor TaNAC2L Is Regulated at the Transcriptional and Post-Translational Levels and Promotes Heat Stress Tolerance in Transgenic Arabidopsis.

PubMed

Guo, Weiwei; Zhang, Jinxia; Zhang, Ning; Xin, Mingming; Peng, Huiru; Hu, Zhaorong; Ni, Zhongfu; Du, Jinkun

2015-01-01

Heat stress poses a serious threat to global crop production. In efforts that aim to mitigate the adverse effects of heat stress on crops, a variety of genetic tools are being used to develop plants with improved thermotolerance. The characterization of important regulators of heat stress tolerance provides essential information for this aim. In this study, we examine the wheat (Triticum aestivum) NAC transcription factor gene TaNAC2L. High temperature induced TaNAC2L expression in wheat and overexpression of TaNAC2L in Arabidopsis thaliana enhanced acquired heat tolerance without causing obvious alterations in phenotype compared with wild type under normal conditions. TaNAC2L overexpression also activated the expression of heat-related genes in the transgenic Arabidopsis plants, suggesting that TaNAC2L may improve heat tolerance by regulating the expression of stress-responsive genes. Notably, TaNAC2L is also regulated at the post-translational level and might be degraded via a proteasome-mediated pathway. Thus, this wheat transcription factor may have potential uses in enhancing thermotolerance in crops.

Transcriptional Control in Marine Copiotrophic and Oligotrophic Bacteria with Streamlined Genomes.

PubMed

Cottrell, Matthew T; Kirchman, David L

2016-10-01

Bacteria often respond to environmental stimuli using transcriptional control, but this may not be the case for marine bacteria such as "Candidatus Pelagibacter ubique," a cultivated representative of the SAR11 clade, the most abundant organism in the ocean. This bacterium has a small, streamlined genome and an unusually low number of transcriptional regulators, suggesting that transcriptional control is low in Pelagibacter and limits its response to environmental conditions. Transcriptome sequencing during batch culture growth revealed that only 0.1% of protein-encoding genes appear to be under transcriptional control in Pelagibacter and in another oligotroph (SAR92) whereas >10% of genes were under transcriptional control in the copiotrophs Polaribacter sp. strain MED152 and Ruegeria pomeroyi When growth levels changed, transcript levels remained steady in Pelagibacter and SAR92 but shifted in MED152 and R. pomeroyi Transcript abundances per cell, determined using an internal RNA sequencing standard, were low (<1 transcript per cell) for all but a few of the most highly transcribed genes in all four taxa, and there was no correlation between transcript abundances per cell and shifts in the levels of transcription. These results suggest that low transcriptional control contributes to the success of Pelagibacter and possibly other oligotrophic microbes that dominate microbial communities in the oceans. Diverse heterotrophic bacteria drive biogeochemical cycling in the ocean. The most abundant types of marine bacteria are oligotrophs with small, streamlined genomes. The metabolic controls that regulate the response of oligotrophic bacteria to environmental conditions remain unclear. Our results reveal that transcriptional control is lower in marine oligotrophic bacteria than in marine copiotrophic bacteria. Although responses of bacteria to environmental conditions are commonly regulated at the level of transcription, metabolism in the most abundant bacteria in the

POEM: Identifying Joint Additive Effects on Regulatory Circuits.

PubMed

Botzman, Maya; Nachshon, Aharon; Brodt, Avital; Gat-Viks, Irit

2016-01-01

Expression Quantitative Trait Locus (eQTL) mapping tackles the problem of identifying variation in DNA sequence that have an effect on the transcriptional regulatory network. Major computational efforts are aimed at characterizing the joint effects of several eQTLs acting in concert to govern the expression of the same genes. Yet, progress toward a comprehensive prediction of such joint effects is limited. For example, existing eQTL methods commonly discover interacting loci affecting the expression levels of a module of co-regulated genes. Such "modularization" approaches, however, are focused on epistatic relations and thus have limited utility for the case of additive (non-epistatic) effects. Here we present POEM (Pairwise effect On Expression Modules), a methodology for identifying pairwise eQTL effects on gene modules. POEM is specifically designed to achieve high performance in the case of additive joint effects. We applied POEM to transcription profiles measured in bone marrow-derived dendritic cells across a population of genotyped mice. Our study reveals widespread additive, trans-acting pairwise effects on gene modules, characterizes their organizational principles, and highlights high-order interconnections between modules within the immune signaling network. These analyses elucidate the central role of additive pairwise effect in regulatory circuits, and provide computational tools for future investigations into the interplay between eQTLs. The software described in this article is available at csgi.tau.ac.il/POEM/.

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

PubMed Central

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

An overview of transcriptional regulation in response to toxicological insult.

PubMed

Jennings, Paul; Limonciel, Alice; Felice, Luca; Leonard, Martin O

2013-01-01

The completion of the human genome project and the subsequent advent of DNA microarray and high-throughput sequencing technologies have led to a renaissance in molecular toxicology. Toxicogenomic data sets, from both in vivo and in vitro studies, are growing exponentially, providing a wealth of information on regulation of stress pathways at the transcriptome level. Through such studies, we are now beginning to appreciate the diversity and complexity of biological responses to xenobiotics. In this review, we aim to consolidate and summarise the major toxicologically relevant transcription factor-governed molecular pathways. It is becoming clear that different chemical entities can cause oxidative, genotoxic and proteotoxic stress, which induce cellular responses in an effort to restore homoeostasis. Primary among the response pathways involved are NFE2L2 (Nrf2), NFE2L1 (Nrf1), p53, heat shock factor and the unfolded protein response. Additionally, more specific mechanisms exist where xenobiotics act as ligands, including the aryl hydrocarbon receptor, metal-responsive transcription factor-1 and the nuclear receptor family of transcription factors. Other pathways including the immunomodulatory transcription factors NF-κB and STAT together with the hypoxia-inducible transcription factor HIF are also implicated in cellular responses to xenobiotic exposure. A less specific but equally important aspect to cellular injury controlled by transcriptional activity is loss of tissue-specific gene expression, resulting in dedifferentiation of target cells and compromise of tissue function. Here, we review these pathways and the genes they regulate in order to provide an overview of this growing field of molecular toxicology.

Identification of transcripts related to high egg production in the chicken hypothalamus and pituitary gland.

PubMed

Shiue, Yow-Ling; Chen, Lih-Ren; Chen, Chih-Feng; Chen, Yi-Ling; Ju, Jhy-Phen; Chao, Ching-Hsien; Lin, Yuan-Ping; Kuo, Yu-Ming; Tang, Pin-Chi; Lee, Yen-Pai

2006-09-15

To identify transcripts related to high egg production expressed specifically in the hypothalamus and pituitary gland of the chicken, two subtracted cDNA libraries were constructed. Two divergently selected strains of Taiwan Country Chickens (TCCs), B (sire line) and L2 (dam line) were used; they had originated from a single population and were further subjected (since 1982) to selection for egg production to 40 wk of age and body weight/comb size, respectively. A total of 324 and 370 clones were identified from the L2-B (L2-subtract-B) and the B-L2 subtracted cDNA libraries, respectively. After sequencing and annotation, 175 and 136 transcripts that represented 53 known and 65 unknown non-redundant sequences were characterized in the L2-B subtracted cDNA library. Quantitative reverse-transcription (RT)-PCR was used to screen the mRNA expression levels of 32 randomly selected transcripts in another 78 laying hens from five different strains. These strains included the two original strains (B and L2) used to construct the subtracted cDNA libraries and an additional three commercial strains, i.e., Black- and Red-feather TCCs and Single-Comb White Leghorn (WL) layer. The mRNA expression levels of 16 transcripts were significantly higher in the L2 than in the B strain, whereas the mRNA expression levels of nine transcripts, BDH, NCAM1, PCDHA@, PGDS, PLAG1, PRL, SAR1A, SCG2 and STMN2, were significantly higher in two high egg production strains, L2 and Single-Comb WL; this indicated their usefulness as molecular markers of high egg production.

Comparative transcriptional profiling of two wheat genotypes, with contrasting levels of minerals in grains, shows expression differences during grain filling.

PubMed

Singh, Sudhir P; Jeet, Raja; Kumar, Jitendra; Shukla, Vishnu; Srivastava, Rakesh; Mantri, Shrikant S; Tuli, Rakesh

2014-01-01

Wheat is one of the most important cereal crops in the world. To identify the candidate genes for mineral accumulation, it is important to examine differential transcriptome between wheat genotypes, with contrasting levels of minerals in grains. A transcriptional comparison of developing grains was carried out between two wheat genotypes- Triticum aestivum Cv. WL711 (low grain mineral), and T. aestivum L. IITR26 (high grain mineral), using Affymetrix GeneChip Wheat Genome Array. The study identified a total of 580 probe sets as differentially expressed (with log2 fold change of ≥2 at p≤0.01) between the two genotypes, during grain filling. Transcripts with significant differences in induction or repression between the two genotypes included genes related to metal homeostasis, metal tolerance, lignin and flavonoid biosynthesis, amino acid and protein transport, vacuolar-sorting receptor, aquaporins, and stress responses. Meta-analysis revealed spatial and temporal signatures of a majority of the differentially regulated transcripts.

Comparative Transcriptional Profiling of Two Wheat Genotypes, with Contrasting Levels of Minerals in Grains, Shows Expression Differences during Grain Filling

PubMed Central

Singh, Sudhir P.; Jeet, Raja; Kumar, Jitendra; Shukla, Vishnu; Srivastava, Rakesh; Mantri, Shrikant S.; Tuli, Rakesh

2014-01-01

Wheat is one of the most important cereal crops in the world. To identify the candidate genes for mineral accumulation, it is important to examine differential transcriptome between wheat genotypes, with contrasting levels of minerals in grains. A transcriptional comparison of developing grains was carried out between two wheat genotypes- Triticum aestivum Cv. WL711 (low grain mineral), and T. aestivum L. IITR26 (high grain mineral), using Affymetrix GeneChip Wheat Genome Array. The study identified a total of 580 probe sets as differentially expressed (with log2 fold change of ≥2 at p≤0.01) between the two genotypes, during grain filling. Transcripts with significant differences in induction or repression between the two genotypes included genes related to metal homeostasis, metal tolerance, lignin and flavonoid biosynthesis, amino acid and protein transport, vacuolar-sorting receptor, aquaporins, and stress responses. Meta-analysis revealed spatial and temporal signatures of a majority of the differentially regulated transcripts. PMID:25364903

Multiple circadian transcriptional elements cooperatively regulate cell-autonomous transcriptional oscillation of Period3, a mammalian clock gene.

PubMed

Matsumura, Ritsuko; Akashi, Makoto

2017-09-29

Cell-autonomous oscillation in clock gene expression drives circadian rhythms. The development of comprehensive analytical techniques, such as bioinformatics and ChIP-sequencing, has enabled the genome-wide identification of potential circadian transcriptional elements that regulate the transcriptional oscillation of clock genes. However, detailed analyses using traditional biochemical and molecular-biological approaches, such as binding and reporter assays, are still necessary to determine whether these potential circadian transcriptional elements are actually functional and how significantly they contribute to driving transcriptional oscillation. Here, we focused on the molecular mechanism of transcriptional oscillations in the mammalian clock gene Period3 ( Per3 ). The PER3 protein is essential for robust peripheral clocks and is a key component in circadian output processes. We found three E box-like elements located upstream of human Per3 transcription start sites that additively contributed to cell-autonomous transcriptional oscillation. However, we also found that Per3 is still expressed in a circadian manner when all three E box-like elements are functionally impaired. We noted that Per3 transcription was activated by the synergistic actions of two D box-like elements and the three E box-like elements, leading to a drastic increase in circadian amplitude. Interestingly, circadian expression of Per3 was completely disrupted only when all five transcriptional elements were functionally impaired. These results indicate that three E box-like and two D box-like elements cooperatively and redundantly regulate cell-autonomous transcriptional oscillation of Per3 . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Brief Report: Effect of CMV and HIV Transcription on CD57 and PD-1 T-Cell Expression During Suppressive ART

PubMed Central

Massanella, Marta; Smith, Davey M.; Spina, Celsa A.; Schrier, Rachel; Daar, Eric S.; Dube, Michael P.; Morris, Sheldon R.; Gianella, Sara

2016-01-01

Abstract: HIV-infected men who have sex with men are nearly universally coinfected with cytomegalovirus (CMV). In this study of 45 HIV-infected men who have sex with men virologically suppressed on ART, we found that presence of seminal CMV DNA shedding and higher levels of systemic cellular HIV RNA transcription were both independently associated with increased PD-1 expression on circulating CD4+ T cells, but not with higher levels of senescent (CD57+) T cells. In addition, greater HIV RNA transcription was associated with lower CD57 expression on CD8 T cells. Although causality cannot be inferred from this retrospective study, these results suggest that asymptomatic CMV replication and residual cellular HIV transcription may contribute to persistent immune dysregulation during suppressive ART. PMID:26818740

Nascent Transcription Affected by RNA Polymerase IV in Zea mays

PubMed Central

Erhard, Karl F.; Talbot, Joy-El R. B.; Deans, Natalie C.; McClish, Allison E.; Hollick, Jay B.

2015-01-01

All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3ʹ-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

Parasite Specific Antibody Levels, Interferon-γ and TLR2 and TLR4 Transcripts in Blood from Dogs with Different Clinical Stages of Leishmaniosis

PubMed Central

Montserrat-Sangrà, Sara; Ordeix, Laura; Martínez-Orellana, Pamela; Solano-Gallego, Laia

2018-01-01

Canine leishmaniosis has a wide range of disease severity from mild (stage I), to severe (stages II–III), or very severe disease (stage IV). The objective of the study was to evaluate and compare serum antibody levels, Leishmania infantum specific IFN-γ production and TLR2 and TLR4 transcripts in non-stimulated blood from dogs with different clinical stages at the time of diagnosis as well as blood parasitemia. Enzyme-Linked ImmunoSorbent Assay (ELISAs) were performed to determine serum antibody levels and IFN-γ production and quantitative polymerase chain reaction (qPCRs) in order to determine blood parasite load and TLR2 and TLR4 transcripts. Older dogs were significantly affected by more severe disease with higher antibody levels and blood parasitemia than dogs with mild disease. IFN-γ production was significantly higher in dogs with stage I disease when compared to dogs with more severe disease. Relative quantification of TLR2 in dogs with mild disease was similar to that of control dogs. On the other hand, TLR2 transcripts were significantly higher in dogs with severe disease as compared with that from control healthy dogs. No differences were found in TLR4 relative quantification between groups. This study demonstrates that dogs with different clinical stages of leishmaniosis present different levels of biological markers indicative of different immune responses. PMID:29547503

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

PubMed

Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription

Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages.

PubMed

Basler, Tina; Jeckstadt, Sabine; Valentin-Weigand, Peter; Goethe, Ralph

2006-03-01

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

Oral Quercetin Supplementation Enhances Adiponectin Receptor Transcript Expression in Polycystic Ovary Syndrome Patients: A Randomized Placebo-Controlled Double-Blind Clinical Trial

PubMed Central

Rezvan, Neda; Moini, Ashraf; Gorgani-Firuzjaee, Sattar; Hosseinzadeh-Attar, Mohammad Javad

2018-01-01

Objective Polycystic ovary syndrome (PCOS), an ovarian-pituitary axis androgen disorder, is a common endocrine disease in women. Obesity-induced androgenesis and imbalance of adipokine secretion may lead to some metabolic features of PCOS. The beneficial effects of polyphenolic compounds such as quercetin have been reported, however, the underlying molecular mechanism is not entirely understood. In the present study, we investigated the effect of quercetin supplementation on the expression of adiponectin receptors at the transcript level in peripheral blood mononuclear cells (PBMC) samples of PCOS patients. Materials and Methods In this randomized clinical trial, 84 PCOS subjects were randomly assigned to two groups; the treatment group received 1 g quercetin (two 500 mg capsules) daily for 12 weeks and the control group received placebo. To examine the effect of quercetin supplementation on PCOS patients in addition to biochemical and anthropometric assessments, the expression of ADIPOR1 and ADIPOR2 at the transcript level and AMPK level were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and ELISA assays respectively. Results Oral quercetin supplementation significantly increased ADIPOR1 and ADIPOR2 transcript expression by 1.32- and 1.46-fold respecetively (P<0.01). In addition, quercetin supplementation enhanced AMPK level by 12.3% compared with the control group (P<0.05). Conclusion Oral quercetin supplementation improves the metabolic features of PCOS patients by upregulating the expression of adiponectin receptors and AMPK (Registration Number: IRCT2013112515536N1). PMID:29105398

Transcriptional response of dermal fibroblasts in direct current electric fields.

PubMed

Jennings, Jessica; Chen, Dongquan; Feldman, Dale

2008-07-01

During the course of normal wound healing, fibroblasts at the wound edge are exposed to electric fields (EFs) ranging from 40 to 200 mV/mm. Various forms of EFs influence fibroblast migration, proliferation, and protein synthesis. Thus, EFs may contribute to fibroblast activation during wound repair. To elucidate the role of EFs during the normal progression of healing, this study compares gene expression in normal adult dermal fibroblasts exposed to a 100 mV/mm EF for 1 h to non-stimulated controls. Significantly increased expression of 162 transcripts and decreased expression of 302 transcripts was detected using microarrays, with 126 transcripts above the level of 1.4-fold increases or decreases compared to the controls. Above the level of twofold, only 11 genes were significantly increased or decreased compared to controls. Many of these significantly regulated genes are associated with wound repair through the processes of matrix production, cellular signaling, and growth. Activity within specific cellular signaling pathways is noted, including TGF-beta, G-proteins, and inhibition of apoptosis. In addition, RT-PCR analysis of the expression of KLF6, FN1, RGS2, and JMJD1C over continued stimulation and at different field strengths suggests that there are specific windows of field characteristics for maximum induction of these genes. EFs thus appear to have an important role in controlling fibroblast activity in the process of wound healing.

Equipment boxes (later addition) one level down from antenna cab, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Equipment boxes (later addition) one level down from antenna cab, looking southwest. - Western Union Telegraph Company, Jennerstown Relay, Laurel Summit Road off U.S. 30, Laughlintown, Westmoreland County, PA

Mitochondrial run-on transcription assay using biotin labeling.

PubMed

Kühn, Kristina

2015-01-01

RNA synthesis and different posttranscriptional processes shape the transcriptome of plant mitochondria. It is believed that mitochondrial transcription in plants is not stringently controlled, and that RNA degradation has a major impact on mitochondrial steady-state transcript levels. Nevertheless, the presence of two RNA polymerases with different gene specificities in mitochondria of dicotyledonous species indicates that transcriptional mechanisms may provide a means to control mitochondrial steady-state RNA pools and gene expression. To experimentally assess transcriptional activities in mitochondria, run-on transcription assays have been developed. These assays measure elongation rates for endogenous transcripts in freshly prepared mitochondrial extracts. The mitochondrial run-on transcription protocol described here has been optimized for the model plant Arabidopsis (Arabidopsis thaliana). It uses mitochondria prepared from soil-grown Arabidopsis plants and employs nonradioactive labeling for the subsequent detection of run-on transcripts.

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

PubMed Central

Tell, G; Perrone, L; Fabbro, D; Pellizzari, L; Pucillo, C; De Felice, M; Acquaviva, R; Formisano, S; Damante, G

1998-01-01

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development. PMID:9425125

Cloning and characterization of aquaglyceroporin genes from rainbow smelt (Osmerus mordax) and transcript expression in response to cold temperature.

PubMed

Hall, Jennifer R; Clow, Kathy A; Rise, Matthew L; Driedzic, William R

2015-09-01

Aquaglyceroporins (GLPs) are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. In this study, GLP-encoding genes were characterized in rainbow smelt (Osmerus mordax mordax), an anadromous teleost that accumulates high glycerol and modest urea levels in plasma and tissues as an adaptive cryoprotectant mechanism in sub-zero temperatures. We report the gene and promoter sequences for two aqp10b paralogs (aqp10ba, aqp10bb) that are 82% identical at the predicted amino acid level, and aqp9b. Aqp10bb and aqp9b have the 6 exon structure common to vertebrate GLPs. Aqp10ba has 8 exons; there are two additional exons at the 5' end, and the promoter sequence is different from aqp10bb. Molecular phylogenetic analysis suggests that the aqp10b paralogs arose from a gene duplication event specific to the smelt lineage. Smelt GLP transcripts are ubiquitously expressed; however, aqp10ba transcripts were highest in kidney, aqp10bb transcripts were highest in kidney, intestine, pyloric caeca and brain, and aqp9b transcripts were highest in spleen, liver, red blood cells and kidney. In cold-temperature challenge experiments, plasma glycerol and urea levels were significantly higher in cold- compared to warm-acclimated smelt; however, GLP transcript levels were generally either significantly lower or remained constant. The exception was significantly higher aqp10ba transcript levels in kidney. High aqp10ba transcripts in smelt kidney that increase significantly in response to cold temperature in congruence with plasma urea suggest that this gene duplicate may have evolved to allow the re-absorption of urea to concomitantly conserve nitrogen and prevent freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

PubMed

Sun, Shuhong; Zhang, Xiaotian; Lyu, Lin; Li, Xixi; Yao, Siliang; Zhang, Junjie

2016-12-09

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

PubMed Central

Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

2015-01-01

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

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

Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8more » is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.« less

Advanced Glycated End-Products Affect HIF-Transcriptional Activity in Renal Cells

PubMed Central

Bondeva, Tzvetanka; Heinzig, Juliane; Ruhe, Carola

2013-01-01

Advanced glycated end-products (AGEs) are ligands of the receptor for AGEs and increase in diabetic disease. MAPK organizer 1 (Morg1) via its binding partner prolyl-hydroxylase domain (PHD)-3 presumably plays a role in the regulation of hypoxia-inducible factor (HIF)-1α and HIF-2α transcriptional activation. The purpose of this study was to analyze the influence of AGEs on Morg1 expression and its correlation to PHD3 activity and HIF-transcriptional activity in various renal cell types. The addition of glycated BSA (AGE-BSA) significantly up-regulated Morg1 mRNA levels in murine mesangial cells and down-regulated it in murine proximal tubular cells and differentiated podocytes. These effects were reversible when the cells were preincubated with a receptor for α-AGE antibody. AGE-BSA treatment induced a relocalization of the Morg1 cellular distribution compared with nonglycated control-BSA. Analysis of PHD3 activity demonstrated an elevated PHD3 enzymatic activity in murine mesangial cells but an inhibition in murine proximal tubular cells and podocytes after the addition of AGE-BSA. HIF-transcriptional activity was also affected by AGE-BSA treatment. Reporter gene assays and EMSAs showed that AGEs regulate HIF- transcriptional activity under nonhypoxic conditions in a cell type-specific manner. In proximal tubular cells, AGE-BSA stimulation elevated mainly HIF-1α transcriptional activity and to a lesser extent HIF-2α. We also detected an increased expression of the HIF-1α and the HIF-2α proteins in kidneys from Morg1 heterozygous (HZ) placebo mice compared with the Morg1 wild-type (WT) placebo-treated mice, and the HIF-1α protein expression in the Morg1 HZ streptozotocin-treated mice was significantly higher than the WT streptozotocin-treated mice. Analysis of isolated mesangial cells from Morg1 HZ (±) and WT mice showed an inhibited PHD3 activity and an increased HIF-transcriptional activity in cells with only one Morg1 allele. These findings are

Identification and Classification of New Transcripts in Dorper and Small-Tailed Han Sheep Skeletal Muscle Transcriptomes.

PubMed

Chao, Tianle; Wang, Guizhi; Wang, Jianmin; Liu, Zhaohua; Ji, Zhibin; Hou, Lei; Zhang, Chunlan

2016-01-01

High-throughput mRNA sequencing enables the discovery of new transcripts and additional parts of incompletely annotated transcripts. Compared with the human and cow genomes, the reference annotation level of the sheep genome is still low. An investigation of new transcripts in sheep skeletal muscle will improve our understanding of muscle development. Therefore, applying high-throughput sequencing, two cDNA libraries from the biceps brachii of small-tailed Han sheep and Dorper sheep were constructed, and whole-transcriptome analysis was performed to determine the unknown transcript catalogue of this tissue. In this study, 40,129 transcripts were finally mapped to the sheep genome. Among them, 3,467 transcripts were determined to be unannotated in the current reference sheep genome and were defined as new transcripts. Based on protein-coding capacity prediction and comparative analysis of sequence similarity, 246 transcripts were classified as portions of unannotated genes or incompletely annotated genes. Another 1,520 transcripts were predicted with high confidence to be long non-coding RNAs. Our analysis also revealed 334 new transcripts that displayed specific expression in ruminants and uncovered a number of new transcripts without intergenus homology but with specific expression in sheep skeletal muscle. The results confirmed a complex transcript pattern of coding and non-coding RNA in sheep skeletal muscle. This study provided important information concerning the sheep genome and transcriptome annotation, which could provide a basis for further study.

Transcription Factor Information System (TFIS): A Tool for Detection of Transcription Factor Binding Sites.

PubMed

Narad, Priyanka; Kumar, Abhishek; Chakraborty, Amlan; Patni, Pranav; Sengupta, Abhishek; Wadhwa, Gulshan; Upadhyaya, K C

2017-09-01

Transcription factors are trans-acting proteins that interact with specific nucleotide sequences known as transcription factor binding site (TFBS), and these interactions are implicated in regulation of the gene expression. Regulation of transcriptional activation of a gene often involves multiple interactions of transcription factors with various sequence elements. Identification of these sequence elements is the first step in understanding the underlying molecular mechanism(s) that regulate the gene expression. For in silico identification of these sequence elements, we have developed an online computational tool named transcription factor information system (TFIS) for detecting TFBS for the first time using a collection of JAVA programs and is mainly based on TFBS detection using position weight matrix (PWM). The database used for obtaining position frequency matrices (PFM) is JASPAR and HOCOMOCO, which is an open-access database of transcription factor binding profiles. Pseudo-counts are used while converting PFM to PWM, and TFBS detection is carried out on the basis of percent score taken as threshold value. TFIS is equipped with advanced features such as direct sequence retrieving from NCBI database using gene identification number and accession number, detecting binding site for common TF in a batch of gene sequences, and TFBS detection after generating PWM from known raw binding sequences in addition to general detection methods. TFIS can detect the presence of potential TFBSs in both the directions at the same time. This feature increases its efficiency. And the results for this dual detection are presented in different colors specific to the orientation of the binding site. Results obtained by the TFIS are more detailed and specific to the detected TFs as integration of more informative links from various related web servers are added in the result pages like Gene Ontology, PAZAR database and Transcription Factor Encyclopedia in addition to NCBI and Uni

Transcriptional regulation of drought response: a tortuous network of transcriptional factors

PubMed Central

Singh, Dhriti; Laxmi, Ashverya

2015-01-01

Drought is one of the leading factors responsible for the reduction in crop yield worldwide. Due to climate change, in future, more areas are going to be affected by drought and for prolonged periods. Therefore, understanding the mechanisms underlying the drought response is one of the major scientific concerns for improving crop yield. Plants deploy diverse strategies and mechanisms to respond and tolerate drought stress. Expression of numerous genes is modulated in different plants under drought stress that help them to optimize their growth and development. Plant hormone abscisic acid (ABA) plays a major role in plant response and tolerance by regulating the expression of many genes under drought stress. Transcription factors being the major regulator of gene expression play a crucial role in stress response. ABA regulates the expression of most of the target genes through ABA-responsive element (ABRE) binding protein/ABRE binding factor (AREB/ABF) transcription factors. Genes regulated by AREB/ABFs constitute a regulon termed as AREB/ABF regulon. In addition to this, drought responsive genes are also regulated by ABA-independent mechanisms. In ABA-independent regulation, dehydration-responsive element binding protein (DREB), NAM, ATAF, and CUC regulons play an important role by regulating many drought-responsive genes. Apart from these major regulons, MYB/MYC, WRKY, and nuclear factor-Y (NF-Y) transcription factors are also involved in drought response and tolerance. Our understanding about transcriptional regulation of drought is still evolving. Recent reports have suggested the existence of crosstalk between different transcription factors operating under drought stress. In this article, we have reviewed various regulons working under drought stress and their crosstalk with each other. PMID:26579147

Chromatin Redistribution of the DEK Oncoprotein Represses hTERT Transcription in Leukemias12

PubMed Central

Karam, Maroun; Thenoz, Morgan; Capraro, Valérie; Robin, Jean-Philippe; Pinatel, Christiane; Lancon, Agnès; Galia, Perrine; Sibon, David; Thomas, Xavier; Ducastelle-Lepretre, Sophie; Nicolini, Franck; El-Hamri, Mohamed; Chelghoun, Youcef; Wattel, Eric; Mortreux, Franck

2014-01-01

Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis. PMID:24563617

Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

PubMed Central

Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

2015-01-01

Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

Integration of transcriptomic and metabolic data reveals hub transcription factors involved in drought stress response in sunflower (Helianthus annuus L.).

PubMed

Moschen, Sebastián; Di Rienzo, Julio A; Higgins, Janet; Tohge, Takayuki; Watanabe, Mutsumi; González, Sergio; Rivarola, Máximo; García-García, Francisco; Dopazo, Joaquin; Hopp, H Esteban; Hoefgen, Rainer; Fernie, Alisdair R; Paniego, Norma; Fernández, Paula; Heinz, Ruth A

2017-07-01

By integration of transcriptional and metabolic profiles we identified pathways and hubs transcription factors regulated during drought conditions in sunflower, useful for applications in molecular and/or biotechnological breeding. Drought is one of the most important environmental stresses that effects crop productivity in many agricultural regions. Sunflower is tolerant to drought conditions but the mechanisms involved in this tolerance remain unclear at the molecular level. The aim of this study was to characterize and integrate transcriptional and metabolic pathways related to drought stress in sunflower plants, by using a system biology approach. Our results showed a delay in plant senescence with an increase in the expression level of photosynthesis related genes as well as higher levels of sugars, osmoprotectant amino acids and ionic nutrients under drought conditions. In addition, we identified transcription factors that were upregulated during drought conditions and that may act as hubs in the transcriptional network. Many of these transcription factors belong to families implicated in the drought response in model species. The integration of transcriptomic and metabolomic data in this study, together with physiological measurements, has improved our understanding of the biological responses during droughts and contributes to elucidate the molecular mechanisms involved under this environmental condition. These findings will provide useful biotechnological tools to improve stress tolerance while maintaining crop yield under restricted water availability.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level.

PubMed

Prielhofer, Roland; Cartwright, Stephanie P; Graf, Alexandra B; Valli, Minoska; Bill, Roslyn M; Mattanovich, Diethard; Gasser, Brigitte

2015-03-11

The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation.

PubMed

Castro-Prego, Raquel; Lamas-Maceiras, Mónica; Soengas, Pilar; Carneiro, Isabel; González-Siso, Isabel; Cerdán, M Esperanza

2009-12-14

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides -557 to -376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

Transcriptional Regulation of Pattern-Triggered Immunity in Plants.

PubMed

Li, Bo; Meng, Xiangzong; Shan, Libo; He, Ping

2016-05-11

Perception of microbe-associated molecular patterns (MAMPs) by cell-surface-resident pattern recognition receptors (PRRs) induces rapid, robust, and selective transcriptional reprogramming, which is central for launching effective pattern-triggered immunity (PTI) in plants. Signal relay from PRR complexes to the nuclear transcriptional machinery via intracellular kinase cascades rapidly activates primary immune response genes. The coordinated action of gene-specific transcription factors and the general transcriptional machinery contribute to the selectivity of immune gene activation. In addition, PRR complexes and signaling components are often transcriptionally upregulated upon MAMP perception to ensure the robustness and sustainability of PTI outputs. In this review, we discuss recent advances in deciphering the signaling pathways and regulatory mechanisms that coordinately lead to timely and accurate MAMP-induced gene expression in plants. Copyright © 2016 Elsevier Inc. All rights reserved.

The transcriptional diversity of 25 Drosophila cell lines

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

Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signalingmore » pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines

Divergent transcription is associated with promoters of transcriptional regulators

PubMed Central

2013-01-01

Background Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. Results We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. Conclusions We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. PMID:24365181

A systems biology perspective on the role of WRKY transcription factors in drought responses in plants.

PubMed

Tripathi, Prateek; Rabara, Roel C; Rushton, Paul J

2014-02-01

Drought is one of the major challenges affecting crop productivity and yield. However, water stress responses are notoriously multigenic and quantitative with strong environmental effects on phenotypes. It is also clear that water stress often does not occur alone under field conditions but rather in conjunction with other abiotic stresses such as high temperature and high light intensities. A multidisciplinary approach with successful integration of a whole range of -omics technologies will not only define the system, but also provide new gene targets for both transgenic approaches and marker-assisted selection. Transcription factors are major players in water stress signaling and some constitute major hubs in the signaling webs. The main transcription factors in this network include MYB, bHLH, bZIP, ERF, NAC, and WRKY transcription factors. The role of WRKY transcription factors in abiotic stress signaling networks is just becoming apparent and systems biology approaches are starting to define their places in the signaling network. Using systems biology approaches, there are now many transcriptomic analyses and promoter analyses that concern WRKY transcription factors. In addition, reports on nuclear proteomics have identified WRKY proteins that are up-regulated at the protein level by water stress. Interactomics has started to identify different classes of WRKY-interacting proteins. What are often lacking are connections between metabolomics, WRKY transcription factors, promoters, biosynthetic pathways, fluxes and downstream responses. As more levels of the system are characterized, a more detailed understanding of the roles of WRKY transcription factors in drought responses in crops will be obtained.

The Severity of Plasmodium falciparum Infection Is Associated with Transcript Levels of var Genes Encoding Endothelial Protein C Receptor-Binding P. falciparum Erythrocyte Membrane Protein 1.

PubMed

Mkumbaye, Sixbert I; Wang, Christian W; Lyimo, Eric; Jespersen, Jakob S; Manjurano, Alphaxard; Mosha, Jacklin; Kavishe, Reginald A; Mwakalinga, Steven B; Minja, Daniel T R; Lusingu, John P; Theander, Thor G; Lavstsen, Thomas

2017-04-01

By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDRα1 domains, we compared Pf emp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria ( n = 42), children with mild malaria not requiring hospitalization ( n = 10), and children with parasitemia and no ongoing fever ( n = 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDRα1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBLβ or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDRα1 subtypes. Copyright © 2017 American Society for Microbiology.

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

Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

PubMed Central

Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

2011-01-01

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome.

PubMed

Wu, Jia Qian; Du, Jiang; Rozowsky, Joel; Zhang, Zhengdong; Urban, Alexander E; Euskirchen, Ghia; Weissman, Sherman; Gerstein, Mark; Snyder, Michael

2008-01-03

Recent studies of the mammalian transcriptome have revealed a large number of additional transcribed regions and extraordinary complexity in transcript diversity. However, there is still much uncertainty regarding precisely what portion of the genome is transcribed, the exact structures of these novel transcripts, and the levels of the transcripts produced. We have interrogated the transcribed loci in 420 selected ENCyclopedia Of DNA Elements (ENCODE) regions using rapid amplification of cDNA ends (RACE) sequencing. We analyzed annotated known gene regions, but primarily we focused on novel transcriptionally active regions (TARs), which were previously identified by high-density oligonucleotide tiling arrays and on random regions that were not believed to be transcribed. We found RACE sequencing to be very sensitive and were able to detect low levels of transcripts in specific cell types that were not detectable by microarrays. We also observed many instances of sense-antisense transcripts; further analysis suggests that many of the antisense transcripts (but not all) may be artifacts generated from the reverse transcription reaction. Our results show that the majority of the novel TARs analyzed (60%) are connected to other novel TARs or known exons. Of previously unannotated random regions, 17% were shown to produce overlapping transcripts. Furthermore, it is estimated that 9% of the novel transcripts encode proteins. We conclude that RACE sequencing is an efficient, sensitive, and highly accurate method for characterization of the transcriptome of specific cell/tissue types. Using this method, it appears that much of the genome is represented in polyA+ RNA. Moreover, a fraction of the novel RNAs can encode protein and are likely to be functional.

Ribosome profiling reveals changes in translational status of soybean transcripts during immature cotyledon development

PubMed Central

Shamimuzzaman, Md.

2018-01-01

To understand translational capacity on a genome-wide scale across three developmental stages of immature soybean seed cotyledons, ribosome profiling was performed in combination with RNA sequencing and cluster analysis. Transcripts representing 216 unique genes demonstrated a higher level of translational activity in at least one stage by exhibiting higher translational efficiencies (TEs) in which there were relatively more ribosome footprint sequence reads mapping to the transcript than were present in the control total RNA sample. The majority of these transcripts were more translationally active at the early stage of seed development and included 12 unique serine or cysteine proteases and 16 2S albumin and low molecular weight cysteine-rich proteins that may serve as substrates for turnover and mobilization early in seed development. It would appear that the serine proteases and 2S albumins play a vital role in the early stages. In contrast, our investigation of profiles of 19 genes encoding high abundance seed storage proteins, such as glycinins, beta-conglycinins, lectin, and Kunitz trypsin inhibitors, showed that they all had similar patterns in which the TE values started at low levels and increased approximately 2 to 6-fold during development. The highest levels of these seed protein transcripts were found at the mid-developmental stage, whereas the highest ribosome footprint levels of only up to 1.6 TE were found at the late developmental stage. These experimental findings suggest that the major seed storage protein coding genes are primarily regulated at the transcriptional level during normal soybean cotyledon development. Finally, our analyses also identified a total of 370 unique gene models that showed very low TE values including over 48 genes encoding ribosomal family proteins and 95 gene models that are related to energy and photosynthetic functions, many of which have homology to the chloroplast genome. Additionally, we showed that genes of the

FOXM1 promotes the progression of prostate cancer by regulating PSA gene transcription.

PubMed

Liu, Youhong; Liu, Yijun; Yuan, Bowen; Yin, Linglong; Peng, Yuchong; Yu, Xiaohui; Zhou, Weibing; Gong, Zhicheng; Liu, Jianye; He, Leye; Li, Xiong

2017-03-07

Androgen/AR is the primary contributor to prostate cancer (PCa) progression by regulating Prostate Specific Antigen (PSA) gene transcription. The disease inevitably evolves to androgen-independent (AI) status. Other mechanisms by which PSA is regulated and develops to AI have not yet been fully determined. FOXM1 is a cell proliferation-specific transcription factor highly expressed in PCa cells compared to non-malignant prostate epithelial cells, suggesting that the aberrant overexpression of FOXM1 contributes to PCa development. In addition to regulating AR gene transcription and cell cycle-regulatory genes, FOXM1 selectively regulates the gene transcription of KLK2 and PSA, typical androgen responsive genes. Screening the potential FOXM1-binding sites by ChIP-PCR, we found that FOXM1 directly binds to the FHK binding motifs in the PSA promoter/enhancer regions. AI C4-2 cells have more FOXM1 binding sites than androgen dependent LNCaP cells. The depletion of FOXM1 by small molecular inhibitors significantly improves the suppression of PSA gene transcription by the anti-AR agent Cadosax. This is the first report showing that FOXM1 promotes PCa progression by regulating PSA gene transcription, particularly in AI PCa cells. The combination of anti-AR agents and FOXM1 inhibitors has the potential to greatly improve therapy for late-stage PCa patients by suppressing PSA levels.

Cold acclimation induces distinctive changes in the chromatin state and transcript levels of COR genes in Cannabis sativa varieties with contrasting cold acclimation capacities.

PubMed

Mayer, Boris F; Ali-Benali, Mohamed Ali; Demone, Jordan; Bertrand, Annick; Charron, Jean-Benoit

2015-11-01

Little is known about the capacity of Cannabis sativa to cold-acclimate and develop freezing tolerance. This study investigates the cold acclimation (CA) capacity of nine C. sativa varieties and the underlying genetic and epigenetic responses. The varieties were divided into three groups based on their contrasting CA capacities by comparing the survival of non-acclimated and cold-acclimated plants in whole-plant freeze tests. In response to the CA treatment, all varieties accumulated soluble sugars but only the varieties with superior capacity for CA could maintain higher levels throughout the treatment. In addition, the varieties that acclimated most efficiently accumulated higher transcript levels of cold-regulated (COR) genes and genes involved in de novo DNA methylation while displaying locus- and variety-specific changes in the levels of H3K9ac, H3K27me3 and methylcytosine (MeC) during CA. Furthermore, these hardy C. sativa varieties displayed significant increases in MeC levels at COR gene loci when deacclimated, suggesting a role for locus-specific DNA methylation in deacclimation. This study uncovers the molecular mechanisms underlying CA in C. sativa and reveals higher levels of complexity regarding how genetic, epigenetic and environmental factors intertwine. © 2014 Scandinavian Plant Physiology Society.

Bacterial antisense RNAs are mainly the product of transcriptional noise.

PubMed

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

2016-03-01

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

Bacterial Transcription as a Target for Antibacterial Drug Development

PubMed Central

Ma, Cong; Yang, Xiao

2016-01-01

SUMMARY Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design. PMID:26764017

Microarray analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice

PubMed Central

Garg, Rohini; Tyagi, Akhilesh K.; Jain, Mukesh

2012-01-01

Hormones exert pleiotropic effects on plant growth and development throughout the life cycle. Many of these effects are mediated at molecular level via altering gene expression. In this study, we investigated the exogenous effect of plant hormones, including auxin, cytokinin, abscisic acid, ethylene, salicylic acid and jasmonic acid, on the transcription of rice genes at whole genome level using microarray. Our analysis identified a total of 4171 genes involved in several biological processes, whose expression was altered significantly in the presence of different hormones. Further, 28% of these genes exhibited overlapping transcriptional responses in the presence of any two hormones, indicating crosstalk among plant hormones. In addition, we identified genes showing only a particular hormone-specific response, which can be used as hormone-specific markers. The results of this study will facilitate further studies in hormone biology in rice. PMID:22827941

Transcriptional regulation of podoplanin expression by Prox1 in lymphatic endothelial cells.

PubMed

Pan, Yanfang; Wang, Wen-di; Yago, Tadayuki

2014-07-01

Transcription factor prospero homeobox 1 (Prox-1) and podoplanin (PDPN), mucin-type transmembane protein, are both constantly expressed in lymphatic endothelial cells (LECs) and appear to function in an LEC-autonomous manner. Mice globally lacking PDPN (Pdpn(-/-)) develop abnormal and blood-filled lymphatic vessels that highly resemble those in inducible mice lacking Prox-1 (Prox1(-/-)). Prox1 has also been reported to induce PDPN expression in cultured ECs. Thus, we hypothesize that PDPN functions downstream of Prox1 and that its expression is regulated by Prox1 in LECs at the transcriptional level. We first identified four putative binding elements for Prox1 in the 5' upstream regulatory region of Pdpn gene and found that Prox1 directly binds to the 5' regulatory sequence of Pdpn gene in LECs by chromatin immunoprecipitation assay. DNA pull down assay confirmed that Prox1 binds to the putative binding element. In addition, luciferase reporter assay indicated that Prox1 binding to the 5' regulatory sequence of Pdpn regulates Pdpn gene expression. We are therefore the first to experimentally demonstrate that Prox1 regulates PDPN expression at the transcriptional level in the lymphatic vascular system. Copyright © 2014 Elsevier Inc. All rights reserved.

POEM: Identifying Joint Additive Effects on Regulatory Circuits

PubMed Central

Botzman, Maya; Nachshon, Aharon; Brodt, Avital; Gat-Viks, Irit

2016-01-01

Motivation: Expression Quantitative Trait Locus (eQTL) mapping tackles the problem of identifying variation in DNA sequence that have an effect on the transcriptional regulatory network. Major computational efforts are aimed at characterizing the joint effects of several eQTLs acting in concert to govern the expression of the same genes. Yet, progress toward a comprehensive prediction of such joint effects is limited. For example, existing eQTL methods commonly discover interacting loci affecting the expression levels of a module of co-regulated genes. Such “modularization” approaches, however, are focused on epistatic relations and thus have limited utility for the case of additive (non-epistatic) effects. Results: Here we present POEM (Pairwise effect On Expression Modules), a methodology for identifying pairwise eQTL effects on gene modules. POEM is specifically designed to achieve high performance in the case of additive joint effects. We applied POEM to transcription profiles measured in bone marrow-derived dendritic cells across a population of genotyped mice. Our study reveals widespread additive, trans-acting pairwise effects on gene modules, characterizes their organizational principles, and highlights high-order interconnections between modules within the immune signaling network. These analyses elucidate the central role of additive pairwise effect in regulatory circuits, and provide computational tools for future investigations into the interplay between eQTLs. Availability: The software described in this article is available at csgi.tau.ac.il/POEM/. PMID:27148351

Global transcriptional responses of Bacillus subtilis to xenocoumacin 1.

PubMed

Zhou, T; Zeng, H; Qiu, D; Yang, X; Wang, B; Chen, M; Guo, L; Wang, S

2011-09-01

To determine the global transcriptional response of Bacillus subtilis to an antimicrobial agent, xenocoumacin 1 (Xcn1). Subinhibitory concentration of Xcn1 applied to B. subtilis was measured according to Hutter's method for determining optimal concentrations. cDNA microarray technology was used to study the global transcriptional response of B. subtilis to Xcn1. Real-time RT-PCR was employed to verify alterations in the transcript levels of six genes. The subinhibitory concentration was determined to be 1 μg ml(-1). The microarray data demonstrated that Xcn1 treatment of B. subtilis led to more than a 2.0-fold up-regulation of 480 genes and more than a 2.0-fold down-regulation of 479 genes (q ≤ 0.05). The transcriptional responses of B. subtilis to Xcn1 were determined, and several processes were affected by Xcn1. Additionally, cluster analysis of gene expression profiles after treatment with Xcn1 or 37 previously studied antibiotics indicated that Xcn1 has similar mechanisms of action to protein synthesis inhibitors. These microarray data showed alterations of gene expression in B. subtilis after exposure to Xcn1. From the results, we identified various processes affected by Xcn1. This study provides a whole-genome perspective to elucidate the action of Xcn1 as a potential antimicrobial agent. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Writing an Independently Composed Sentence by Spanish-Speaking Children with and without Poor Transcription Skills: A Writing-Level Match Design

ERIC Educational Resources Information Center

Garcia, Eduardo; Crespo, Patricia; Bermúdez, Ivana

2017-01-01

The main objective of this research was to analyze the impact of transcription skills of Spanish writers when writing an independently composed sentence within a writing-level design. The free-writing sentence task from the "Early Grade Writing Assessment" (Jiménez, in press) was used to examine the production, accuracy, speed, syntactic…

Polycomb repressive complex 1 modifies transcription of active genes

PubMed Central

Pherson, Michelle; Misulovin, Ziva; Gause, Maria; Mihindukulasuriya, Kathie; Swain, Amanda; Dorsett, Dale

2017-01-01

This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences. PMID:28782042

Expression levels of long non-coding RNA HOXA distal transcript antisense RNA and metabotropic glutamate receptor 1 in pancreatic carcinoma, and their prognostic values.

PubMed

Wang, Xiaoqing; Xiao, Lili; Yu, Haitao

2018-06-01

As a type of malignant tumor developed at the pancreas, the prognosis of pancreatic carcinoma is usually poor, and >90% patients will sucumb to this disease <5 years after diagnosis. Therefore, early detection and treatment of this disease are important for improving the prognosis of patients. Long non-coding RNAs (lncRNAs) have been proven to serve pivotal functions in the development and progression of various tumors. The lncRNA HOXA distal transcript antisense RNA (HOTTIP), which serves an oncogenic role in different types of malignant tumors, has also been reported to be closely correlated with the migration and invasion of pancreatic carcinoma. In addition, the metabotropic glutamate receptor 1 (mGluR1) is also associated with the progression of various types of human cancer; however, its functionality in pancreatic carcinoma is largely unknown. In the present study, the expression levels of HOTTIP and mGluR1 were compared between pancreatic carcinoma and adjacent normal healthy tissues, and the correlation between these expression levels was analyzed. The prognostic value of HOTTIP and mGluR1 in pancreatic carcinoma was also examined. It was observed that the expression levels of HOTTIP and mGluR1 were upregulated in pancreatic carcinoma tissues and pancreatic carcinoma cells, while the expression of HOTTIP was able to positively affect the expression of mGluR1. In addition, high expression levels of HOTTIP were significantly correlated with the tumor size and distant metastasis. These data suggested that HOTTIP and mGluR1 may potentially serve as biomarkers for the prognosis of pancreatic carcinoma.

saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

PubMed

Portnoy, Victoria; Lin, Szu Hua Sharon; Li, Kathy H; Burlingame, Alma; Hu, Zheng-Hui; Li, Hao; Li, Long-Cheng

2016-03-01

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction.

PubMed

Singh, N K; Eliash, N; Stein, I; Kamer, Y; Ilia, Z; Rafaeli, A; Soroker, V

2016-04-01

The ectoparasitic mite Varroa destructor is one of the major threats to apiculture. Using a behavioural choice bioassay, we determined that phoretic mites were more successful in reaching a bee than reproductive mites, suggesting an energy trade-off between reproduction and host selection. We used both chemo-ecological and molecular strategies to identify the regulation of the olfactory machinery of Varroa and its association with reproduction. We focused on transcription regulation. Using primers designed to the conserved DNA binding region of transcription factors, we identified a gene transcript in V. destructor homologous to the pheromone receptor transcription factor (PRTF) gene of Pediculus humanus corporis. Quantitative PCR (qPCR) revealed that this PRTF-like gene transcript is expressed in the forelegs at higher levels than in the body devoid of forelegs. Subsequent comparative qPCR analysis showed that transcript expression was significantly higher in the phoretic as compared to the reproductive stage. Electrophysiological and behavioural studies revealed a reduction in the sensitivity of PRTF RNA interference-silenced mites to bee headspace, consistent with a reduction in the mites' ability to reach a host. In addition, vitellogenin expression was stimulated in PRTF-silenced mites to similar levels as found in reproductive mites. These data shed light upon the regulatory mechanism of host chemosensing in V. destructor. © 2016 The Royal Entomological Society.

Maize Iranian mosaic virus shows a descending transcript accumulation order in plant and insect hosts.

PubMed

Hortamani, Mozhgan; Massah, Amir; Izadpanah, Keramat

2018-04-01

Maize Iranian mosaic virus (MIMV) is a distinct member of the genus Nucleorhabdovirus. In this study, expression of all MIMV genes in maize for four weeks after inoculation and in inoculative planthoppers was examined using a quantitative RT-PCR (RT-qPCR) assay. Accumulation of MIMV P, gene 3, M, G and L transcripts relative to N transcripts was measured and normalized to 18S rRNA in maize plants and to the ribosomal protein S13 gene (RPS13) in planthoppers using the comparative C T method. In plants, higher levels of MIMV N transcripts were found relative to other transcripts, while MIMV L transcripts were at the lowest levels. The highest accumulation of MIMV transcripts was found at 14 days postinoculation (dpi). At 21 dpi, we found the lowest transcript levels for all genes, which increased again at 28 dpi, although in lower amounts than at 14 dpi. In Laodelphax striatellus, MIMV M, G and L transcripts accumulated at lower levels than other transcripts. The gene 3 transcript level was high in both plants and planthoppers. Our results showed that transcript accumulation for the MIMV genes was similar in both hosts and followed the pattern of sequential transcriptional attenuation from the 3' to the 5' end of the genome, similar to vertebrate rhabdoviruses. These results indicate that the regulation of virus gene transcription for this plant-infecting rhabdovirus is similar to that of some vertebrate-infecting rhabdoviruses.

Transcriptional Response of Nitrifying Communities to Wetting of Dry Soil

PubMed Central

Firestone, Mary K.

2013-01-01

The first rainfall following a severe dry period provides an abrupt water potential change that is both an acute physiological stress and a defined stimulus for the reawakening of soil microbial communities. We followed the responses of indigenous communities of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and nitrite-oxidizing bacteria to the addition of water to laboratory incubations of soils taken from two California annual grasslands following a typically dry Mediterranean summer. By quantifying transcripts for a subunit of bacterial and archaeal ammonia monooxygenases (amoA) and a bacterial nitrite oxidoreductase (nxrA) in soil from 15 min to 72 h after water addition, we identified transcriptional response patterns for each of these three groups of nitrifiers. An increase in quantity of bacterial amoA transcripts was detectable within 1 h of wet-up and continued until the size of the ammonium pool began to decrease, reflecting a possible role of transcription in upregulation of nitrification after drought-induced stasis. In one soil, the pulse of amoA transcription lasted for less than 24 h, demonstrating the transience of transcriptional pools and the tight coupling of transcription to the local soil environment. Analysis of 16S rRNA using a high-density microarray suggested that nitrite-oxidizing Nitrobacter spp. respond in tandem with ammonia-oxidizing bacteria while nitrite-oxidizing Nitrospina spp. and Nitrospira bacteria may not. Archaeal ammonia oxidizers may respond slightly later than bacterial ammonia oxidizers but may maintain elevated transcription longer. Despite months of desiccation-induced inactivation, we found rapid transcriptional response by all three groups of soil nitrifiers. PMID:23524666

O-GlcNAc transferase regulates transcriptional activity of human Oct4.

PubMed

Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

2017-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bacterial antisense RNAs are mainly the product of transcriptional noise

PubMed Central

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

2016-01-01

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

Imatinib discontinuation in chronic myeloid leukaemia patients with undetectable BCR-ABL transcript level: A systematic review and a meta-analysis.

PubMed

Campiotti, Leonardo; Suter, Matteo Basilio; Guasti, Luigina; Piazza, Rocco; Gambacorti-Passerini, Carlo; Grandi, Anna Maria; Squizzato, Alessandro

2017-05-01

Tyrosine kinase inhibitors (TKIs) are the cornerstones of treatment for patients with chronic myeloid leukaemia (CML). In recent years, several studies were conducted to evaluate the safety of TKIs discontinuation. We performed a systematic review of the literature to determine the incidence of CML relapse, to identify possible factors relapse rates and to evaluate the long-term safety in CML patients with stable undetectable BCR-ABL transcript level who discontinued TKIs. Studies evaluating TKIs discontinuation in CML patients with undetectable BCR-ABL transcript level were identified by electronic search of MEDLINE and EMBASE database until May 2015. Weighted mean proportion and 95% confidence intervals (CIs) of CML relapse was calculated using a fixed-effects and a random-effects model. Statistical heterogeneity was evaluated using the I 2 statistic. Fifteen cohort studies, for a total of 509 patients, were included. Nine studies were at low-risk of bias. All 15 studies included only patients on imatinib. Overall weighted mean molecular relapse rate of CML was 51% (95% CI 44-58%; I 2  = 55). Weighted mean molecular relapse rate at 6-month follow-up was 41% (95% CI 32-51%; I 2  = 78). Eighty percent of molecular relapses occurred in the first 6 months. All 509 patients were alive at 2-year follow-up and only one patient (0.8%, 95% CI 0.2-1.8%; I 2  = 0) has progressed to a blastic crisis. Our findings suggest that imatinib discontinuation is feasible for the majority of CML patients with stable undetectable BCR-ABL transcript level. Approximately 50% of patients remain therapy-free after imatinib discontinuation. Restarting TKIs therapy was followed by a very high rate of molecular response, with no deaths 2 years after discontinuation. Copyright © 2017 Elsevier Ltd. All rights reserved.

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

PubMed

Maekawa, T; Sudo, T; Kurimoto, M; Ishii, S

1991-09-11

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

PPAR{gamma} activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

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

Mogilenko, Denis A., E-mail: denis@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Shavva, Vladimir S.

Research highlights: {yields} PPAR{gamma} activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. {yields} Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1-LXR{beta} complex. {yields} Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex. {yields} Activation of PPAR{gamma} leads to increasing of the level of LXR{beta} associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPAR{gamma} is known as activator of ABCA1 expression, but details of PPAR{gamma}-mediatedmore » regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPAR{gamma} activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXR{beta} binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1/LXR{beta} complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex, but does not block PPAR{gamma}-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPAR{gamma} may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPAR{gamma}, LXR{beta} and MEK1/2 in regulation of ABCA1 mRNA and protein expression.« less

The effect of resveratrol on angiotensin II levels and the rate of transcription of its receptors in the rat cardiac hypertrophy model.

PubMed

Dorri Mashhadi, Fahimeh; Zavvar Reza, Javad; Jamhiri, Mohabbat; Hafizi, Zeinab; Zare Mehrjardi, Fatemeh; Safari, Fatemeh

2017-03-01

This study investigated the effect of resveratrol on serum and cardiac levels of angiotensin II and transcription of its main receptors following pressure overload induced-hypertrophy. Rats were divided into untreated (Hyp) and resveratrol treated hypertrophied groups (H + R). Intact animals served as the control (Ctl). Cardiac hypertrophy was induced by abdominal aortic banding. Blood pressure (BP) was recorded via left carotid artery cannula. Fibrosis was confirmed by Masson trichrome staining. Angiotensin II level was measured using an ELIZA test. Gene expression was assessed by a real time PCR (RT-PCR) technique. We observed that in the H + R group BP and heart weight/body weight were decreased significantly (p < 0.001, p < 0.05, respectively vs Hyp). The cardiac levels of angiotensin II and AT1a mRNA were increased in the Hyp group (p < 0.01 vs Ctl). In the H + R group the AT1a mRNA level was decreased significantly (p < 0.05 vs Hyp). It could be concluded that resveratrol protects the heart against hypertrophy progression in part by affecting cardiac AT1a transcription.

Genes on a Wire: The Nucleoid-Associated Protein HU Insulates Transcription Units in Escherichia coli

PubMed Central

Berger, Michael; Gerganova, Veneta; Berger, Petya; Rapiteanu, Radu; Lisicovas, Viktoras; Dobrindt, Ulrich

2016-01-01

The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling. PMID:27545593

38. VIEW SOUTHWEST OF STREET LEVEL IN SHOWROOM ADDITION (NOW ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

38. VIEW SOUTHWEST OF STREET LEVEL IN SHOWROOM ADDITION (NOW USED AS FINISHING ROOM) WITH FRESHLY PAINTED DORY BOATS ON DISPLAY. 2X4 STUD WALL AND ROOF FRAMING CAN BE SEEN. - Lowell's Boat Shop, 459 Main Street, Amesbury, Essex County, MA

Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit.

PubMed

Ho, Emily V; Klenotich, Stephanie J; McMurray, Matthew S; Dulawa, Stephanie C

2016-01-01

Anorexia nervosa (AN) is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA) paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF), an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1) to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC), increased NCAM1 mRNA expression in the ventral tegmental area (VTA), and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC). In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc). Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

PubMed

Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

2018-01-01

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.

PubMed

Lara, Alvaro R; Leal, Lidia; Flores, Noemí; Gosset, Guillermo; Bolívar, Francisco; Ramírez, Octavio T

2006-02-05

Escherichia coli, expressing recombinant green fluorescent protein (GFP), was subjected to dissolved oxygen tension (DOT) oscillations in a two-compartment system for simulating gradients that can occur in large-scale bioreactors. Cells were continuously circulated between the anaerobic (0% DOT) and aerobic (10% DOT) vessels of the scale-down system to mimic an overall circulation time of 50 s, and a mean residence time in the anaerobic and aerobic compartments of 33 and 17 s, respectively. Transcription levels of mixed acid fermentation genes (ldhA, poxB, frdD, ackA, adhE, pflD, and fdhF), measured by quantitative RT-PCR, increased between 1.5- to over 6-fold under oscillatory DOT compared to aerobic cultures (constant 10% DOT). In addition, the transcription level of fumB increased whereas it decreased for sucA and sucB, suggesting that the tricarboxylic acid cycle was functioning as two open branches. Gene transcription levels revealed that cytrochrome bd, which has higher affinity to oxygen but lower energy efficiency, was preferred over cytochrome bO3 in oscillatory DOT cultures. Post-transcriptional processing limited heterologous protein production in the scale-down system, as inferred from similar gfp transcription but 19% lower GFP concentration compared to aerobic cultures. Simulated DOT gradients also affected the transcription of genes of the glyoxylate shunt (aceA), of global regulators of aerobic and anaerobic metabolism (fnr, arcA, and arcB), and other relevant genes (luxS, sodA, fumA, and sdhB). Transcriptional changes explained the observed alterations in overall stoichiometric and kinetic parameters, and production of ethanol and organic acids. Differences in transcription levels between aerobic and anaerobic compartments were also observed, indicating that E. coli can respond very fast to intermittent DOT conditions. The transcriptional responses of E. coli to DOT gradients reported here are useful for establishing rational scale-up criteria and

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

PubMed

Park, Jeong-Woong; Song, Ki-Duk; Kim, Nam Young; Choi, Jae-Young; Hong, Seul A; Oh, Jin Hyeog; Kim, Si Won; Lee, Jeong Hyo; Park, Tae Sub; Kim, Jin-Kyoo; Kim, Jong Geun; Cho, Byung-Wook

2017-10-01

Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase ( AXL ) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

Transcriptional regulation of mammalian selenoprotein expression

PubMed Central

Stoytcheva, Zoia R.; Berry, Marla J.

2009-01-01

Background Selenoproteins contain the twenty-first amino acid, selenocysteine, and are involved in cellular defenses against oxidative damage, important metabolic and developmental pathways, and responses to environmental challenges. Elucidating the mechanisms regulating selenoprotein expression at the transcriptional level is key to understanding how these mechanisms are called into play to respond to the changing environment. Methods This review summarizes published studies on transcriptional regulation of selenoprotein genes, focused primarily on genes whose encoded protein functions are at least partially understood. This is followed by in silico analysis of predicted regulatory elements in selenoprotein genes, including those in the aforementioned category as well as the genes whose functions are not known. Results Our findings reveal regulatory pathways common to many selenoprotein genes, including several involved in stress-responses. In addition, tissue-specific regulatory factors are implicated in regulating many selenoprotein genes. Conclusions These studies provide new insights into how selenoprotein genes respond to environmental and other challenges, and the roles these proteins play in allowing cells to adapt to these changes. General Significance Elucidating the regulatory mechanisms affecting selenoprotein expression is essential for understanding their roles in human diseases, and for developing diagnostic and potential therapeutic approaches to address dysregulation of members of this gene family. PMID:19465084

DcpS is a transcript-specific modulator of RNA in mammalian cells

PubMed Central

Zhou, Mi; Bail, Sophie; Plasterer, Heather L.; Rusche, James

2015-01-01

The scavenger decapping enzyme DcpS is a multifunctional protein initially identified by its property to hydrolyze the resulting cap structure following 3′ end mRNA decay. In Saccharomyces cerevisiae, the DcpS homolog Dcs1 is an obligate cofactor for the 5′-3′ exoribonuclease Xrn1 while the Caenorhabditis elegans homolog Dcs-1, facilitates Xrn1 mediated microRNA turnover. In both cases, this function is independent of the decapping activity. Whether DcpS and its decapping activity can affect mRNA steady state or stability in mammalian cells remains unknown. We sought to determine DcpS target genes in mammalian cells using a cell-permeable DcpS inhibitor compound, RG3039 initially developed for therapeutic treatment of spinal muscular atrophy. Global mRNA levels were examined following DcpS decapping inhibition with RG3039. The steady-state levels of 222 RNAs were altered upon RG3039 treatment. Of a subset selected for validation, two transcripts that appear to be long noncoding RNAs HS370762 and BC011766, were dependent on DcpS and its scavenger decapping catalytic activity and referred to as DcpS-responsive noncoding transcripts (DRNT) 1 and 2, respectively. Interestingly, only the increase in DRNT1 transcript was accompanied with an increase of its RNA stability and this increase was dependent on both DcpS and Xrn1. Importantly, unlike in yeast where the DcpS homolog is an obligate cofactor for Xrn1, stability of additional Xrn1 dependent RNAs were not altered by a reduction in DcpS levels. Collectively, our data demonstrate that DcpS in conjunction with Xrn1 has the potential to regulate RNA stability in a transcript-selective manner in mammalian cells. PMID:26001796

Redox-dependent transcriptional regulation.

PubMed

Liu, Hongjun; Colavitti, Renata; Rovira, Ilsa I; Finkel, Toren

2005-11-11

Reactive oxygen species contribute to the pathogenesis of a number of disparate disorders including tissue inflammation, heart failure, hypertension, and atherosclerosis. In response to oxidative stress, cells activate expression of a number of genes, including those required for the detoxification of reactive molecules as well as for the repair and maintenance of cellular homeostasis. In many cases, these induced genes are regulated by transcription factors whose structure, subcellular localization, or affinity for DNA is directly or indirectly regulated by the level of oxidative stress. This review summarizes the recent progress on how cellular redox status can regulate transcription-factor activity and the implications of this regulation for cardiovascular disease.

Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops.

PubMed

Kar, Anirban; Willcox, Smaranda; Griffith, Jack D

2016-11-02

The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Different types of pausing modes during transcription initiation.

PubMed

Lerner, Eitan; Ingargiola, Antonino; Lee, Jookyung J; Borukhov, Sergei; Michalet, Xavier; Weiss, Shimon

2017-08-08

In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript.

Structural Basis of Mitochondrial Transcription Initiation.

PubMed

Hillen, Hauke S; Morozov, Yaroslav I; Sarfallah, Azadeh; Temiakov, Dmitry; Cramer, Patrick

2017-11-16

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcription factor levels enable metabolic diversification of single cells of environmental bacteria.

PubMed

Guantes, Raúl; Benedetti, Ilaria; Silva-Rocha, Rafael; de Lorenzo, Víctor

2016-05-01

Transcriptional noise is a necessary consequence of the molecular events that drive gene expression in prokaryotes. However, some environmental microorganisms that inhabit polluted sites, for example, the m-xylene degrading soil bacterium Pseudomonas putida mt-2 seem to have co-opted evolutionarily such a noise for deploying a metabolic diversification strategy that allows a cautious exploration of new chemical landscapes. We have examined this phenomenon under the light of deterministic and stochastic models for activation of the main promoter of the master m-xylene responsive promoter of the system (Pu) by its cognate transcriptional factor (XylR). These analyses consider the role of co-factors for Pu activation and determinants of xylR mRNA translation. The model traces the onset and eventual disappearance of the bimodal distribution of Pu activity along time to the growth-phase dependent abundance of XylR itself, that is, very low in exponentially growing cells and high in stationary. This tenet was validated by examining the behaviour of a Pu-GFP fusion in a P. putida strain in which xylR expression was engineered under the control of an IPTG-inducible system. This work shows how a relatively simple regulatory scenario (for example, growth-phase dependent expression of a limiting transcription factor) originates a regime of phenotypic diversity likely to be advantageous in competitive environmental settings.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

PubMed

Müller-Tidow, Carsten; Klein, Hans-Ulrich; Hascher, Antje; Isken, Fabienne; Tickenbrock, Lara; Thoennissen, Nils; Agrawal-Singh, Shuchi; Tschanter, Petra; Disselhoff, Christine; Wang, Yipeng; Becker, Anke; Thiede, Christian; Ehninger, Gerhard; zur Stadt, Udo; Koschmieder, Steffen; Seidl, Matthias; Müller, Frank U; Schmitz, Wilhelm; Schlenke, Peter; McClelland, Michael; Berdel, Wolfgang E; Dugas, Martin; Serve, Hubert

2010-11-04

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia

PubMed Central

Klein, Hans-Ulrich; Hascher, Antje; Isken, Fabienne; Tickenbrock, Lara; Thoennissen, Nils; Agrawal-Singh, Shuchi; Tschanter, Petra; Disselhoff, Christine; Wang, Yipeng; Becker, Anke; Thiede, Christian; Ehninger, Gerhard; zur Stadt, Udo; Koschmieder, Steffen; Seidl, Matthias; Müller, Frank U.; Schmitz, Wilhelm; Schlenke, Peter; McClelland, Michael; Berdel, Wolfgang E.; Dugas, Martin; Serve, Hubert

2010-01-01

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34+ cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML. PMID:20498303

Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

PubMed

Clifford, Jacob; Adami, Christoph

2015-09-02

Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors

PubMed Central

Duong, Vanessa; Licznar, Anne; Margueron, Raphaël; Boulle, Nathalie; Busson, Muriel; Lacroix, Matthieu; Katzenellenbogen, Benita S.; Cavaillès, Vincent; Lazennec, Gwendal

2006-01-01

The proliferative action of ERα largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERβ displays tumor-suppressor properties, thus supporting the interest to identify compounds which could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) up-regulated ERβ protein levels, whereas it decreased ERα expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERβ more strongly than that of ERα. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERβ expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERβ and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy. PMID:16158045

Effects of the lipid regulating drug clofibric acid on PPARα-regulated gene transcript levels in common carp (Cyprinus carpio) at pharmacological and environmental exposure levels.

PubMed

Corcoran, Jenna; Winter, Matthew J; Lange, Anke; Cumming, Rob; Owen, Stewart F; Tyler, Charles R

2015-04-01

In mammals, the peroxisome proliferator-activated receptor α (PPARα) plays a key role in regulating various genes involved in lipid metabolism, bile acid synthesis and cholesterol homeostasis, and is activated by a diverse group of compounds collectively termed peroxisome proliferators (PPs). Specific PPs have been detected in the aquatic environment; however little is known on their pharmacological activity in fish. We investigated the bioavailability and persistence of the human PPARα ligand clofibric acid (CFA) in carp, together with various relevant endpoints, at a concentration similar to therapeutic levels in humans (20mg/L) and for an environmentally relevant concentration (4μg/L). Exposure to pharmacologically-relevant concentrations of CFA resulted in increased transcript levels of a number of known PPARα target genes together with increased acyl-coA oxidase (Acox1) activity, supporting stimulation of lipid metabolism pathways in carp which are known to be similarly activated in mammals. Although Cu,Zn-superoxide dismutase (Sod1) activity was not affected, mRNA levels of several biotransformation genes were also increased, paralleling previous reports in mammals and indicating a potential role in hepatic detoxification for PPARα in carp. Importantly, transcription of some of these genes (and Acox1 activity) were affected at exposure concentrations comparable with those reported in effluent discharges. Collectively, these data suggest that CFA is pharmacologically active in carp and has the potential to invoke PPARα-related responses in fish exposed in the environment, particularly considering that CFA may represent just one of a number of PPAR-active compounds present to which wild fish may be exposed. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of the lipid regulating drug clofibric acid on PPARα-regulated gene transcript levels in common carp (Cyprinus carpio) at pharmacological and environmental exposure levels

PubMed Central

Corcoran, Jenna; Winter, Matthew J.; Lange, Anke; Cumming, Rob; Owen, Stewart F.; Tyler, Charles R.

2015-01-01

In mammals, the peroxisome proliferator-activated receptor α (PPARα) plays a key role in regulating various genes involved in lipid metabolism, bile acid synthesis and cholesterol homeostasis, and is activated by a diverse group of compounds collectively termed peroxisome proliferators (PPs). Specific PPs have been detected in the aquatic environment; however little is known on their pharmacological activity in fish. We investigated the bioavailability and persistence of the human PPARα ligand clofibric acid (CFA) in carp, together with various relevant endpoints, at a concentration similar to therapeutic levels in humans (20 mg/L) and for an environmentally relevant concentration (4 μg/L). Exposure to pharmacologically-relevant concentrations of CFA resulted in increased transcript levels of a number of known PPARα target genes together with increased acyl-coA oxidase (Acox1) activity, supporting stimulation of lipid metabolism pathways in carp which are known to be similarly activated in mammals. Although Cu,Zn-superoxide dismutase (Sod1) activity was not affected, mRNA levels of several biotransformation genes were also increased, paralleling previous reports in mammals and indicating a potential role in hepatic detoxification for PPARα in carp. Importantly, transcription of some of these genes (and Acox1 activity) were affected at exposure concentrations comparable with those reported in effluent discharges. Collectively, these data suggest that CFA is pharmacologically active in carp and has the potential to invoke PPARα-related responses in fish exposed in the environment, particularly considering that CFA may represent just one of a number of PPAR-active compounds present to which wild fish may be exposed. PMID:25749508

Transcription through enhancers suppresses their activity in Drosophila

PubMed Central

2013-01-01

Background Enhancer elements determine the level of target gene transcription in a tissue-specific manner, providing for individual patterns of gene expression in different cells. Knowledge of the mechanisms controlling enhancer action is crucial for understanding global regulation of transcription. In particular, enhancers are often localized within transcribed regions of the genome. A number of experiments suggest that transcription can have both positive and negative effects on regulatory elements. In this study, we performed direct tests for the effect of transcription on enhancer activity. Results Using a transgenic reporter system, we investigated the relationship between the presence of pass-through transcription and the activity of Drosophila enhancers controlling the expression of the white and yellow genes. The results show that transcription from different promoters affects the activity of enhancers, counteracting their ability to activate the target genes. As expected, the presence of a transcriptional terminator between the inhibiting promoter and the affected enhancer strongly reduces the suppression. Moreover, transcription leads to dislodging of the Zeste protein that is responsible for the enhancer-dependent regulation of the white gene, suggesting a 'transcription interference’ mechanism for this regulation. Conclusions Our findings suggest a role for pass-through transcription in negative regulation of enhancer activity. PMID:24279291

Berry Flesh and Skin Ripening Features in Vitis vinifera as Assessed by Transcriptional Profiling

PubMed Central

Grimplet, Jérôme; Bravo, Gema; Flores, Pilar; Fenoll, José; Hellín, Pilar; Oliveros, Juan Carlos; Martínez-Zapater, José M.

2012-01-01

Background Ripening of fleshy fruit is a complex developmental process involving the differentiation of tissues with separate functions. During grapevine berry ripening important processes contributing to table and wine grape quality take place, some of them flesh- or skin-specific. In this study, transcriptional profiles throughout flesh and skin ripening were followed during two different seasons in a table grape cultivar ‘Muscat Hamburg’ to determine tissue-specific as well as common developmental programs. Methodology/Principal Findings Using an updated GrapeGen Affymetrix GeneChip® annotation based on grapevine 12×v1 gene predictions, 2188 differentially accumulated transcripts between flesh and skin and 2839 transcripts differentially accumulated throughout ripening in the same manner in both tissues were identified. Transcriptional profiles were dominated by changes at the beginning of veraison which affect both pericarp tissues, although frequently delayed or with lower intensity in the skin than in the flesh. Functional enrichment analysis identified the decay on biosynthetic processes, photosynthesis and transport as a major part of the program delayed in the skin. In addition, a higher number of functional categories, including several related to macromolecule transport and phenylpropanoid and lipid biosynthesis, were over-represented in transcripts accumulated to higher levels in the skin. Functional enrichment also indicated auxin, gibberellins and bHLH transcription factors to take part in the regulation of pre-veraison processes in the pericarp, whereas WRKY and C2H2 family transcription factors seems to more specifically participate in the regulation of skin and flesh ripening, respectively. Conclusions/Significance A transcriptomic analysis indicates that a large part of the ripening program is shared by both pericarp tissues despite some components are delayed in the skin. In addition, important tissue differences are present from early

The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway

PubMed Central

2013-01-01

Background The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized. Results Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors. Conclusions Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude. PMID:24099172

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

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

Lau, Wen Min; Doucet, Michele; Huang, David

2013-07-26

Highlights: •The effects of elevated CITED2 on ER function in breast cancer cells are examined. •CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. •CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found thatmore » CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a

Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.

PubMed

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J; Considine, Robert V; Sethi, Jaswinder K; Vidal-Puig, Antonio; O'Rahilly, Stephen

2004-03-19

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

Transcription as a Threat to Genome Integrity.

PubMed

Gaillard, Hélène; Aguilera, Andrés

2016-06-02

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs

PubMed Central

Nepal, Chirag; Coolen, Marion; Hadzhiev, Yavor; Cussigh, Delphine; Mydel, Piotr; Steen, Vidar M.; Carninci, Piero; Andersen, Jesper B.; Bally-Cuif, Laure; Müller, Ferenc; Lenhard, Boris

2016-01-01

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9–5 was validated by 5′ RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3′-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay. PMID:26673698

A support vector machine model provides an accurate transcript-level-based diagnostic for major depressive disorder

PubMed Central

Yu, J S; Xue, A Y; Redei, E E; Bagheri, N

2016-01-01

Major depressive disorder (MDD) is a critical cause of morbidity and disability with an economic cost of hundreds of billions of dollars each year, necessitating more effective treatment strategies and novel approaches to translational research. A notable barrier in addressing this public health threat involves reliable identification of the disorder, as many affected individuals remain undiagnosed or misdiagnosed. An objective blood-based diagnostic test using transcript levels of a panel of markers would provide an invaluable tool for MDD as the infrastructure—including equipment, trained personnel, billing, and governmental approval—for similar tests is well established in clinics worldwide. Here we present a supervised classification model utilizing support vector machines (SVMs) for the analysis of transcriptomic data readily obtained from a peripheral blood specimen. The model was trained on data from subjects with MDD (n=32) and age- and gender-matched controls (n=32). This SVM model provides a cross-validated sensitivity and specificity of 90.6% for the diagnosis of MDD using a panel of 10 transcripts. We applied a logistic equation on the SVM model and quantified a likelihood of depression score. This score gives the probability of a MDD diagnosis and allows the tuning of specificity and sensitivity for individual patients to bring personalized medicine closer in psychiatry. PMID:27779627

Structural analysis of nucleosomal barrier to transcription.

PubMed

Gaykalova, Daria A; Kulaeva, Olga I; Volokh, Olesya; Shaytan, Alexey K; Hsieh, Fu-Kai; Kirpichnikov, Mikhail P; Sokolova, Olga S; Studitsky, Vasily M

2015-10-27

Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA-histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone-histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA-protein and protein-protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed.

Structural analysis of nucleosomal barrier to transcription

PubMed Central

Gaykalova, Daria A.; Kulaeva, Olga I.; Volokh, Olesya; Shaytan, Alexey K.; Hsieh, Fu-Kai; Kirpichnikov, Mikhail P.; Sokolova, Olga S.; Studitsky, Vasily M.

2015-01-01

Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA–histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone–histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA–protein and protein–protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed. PMID:26460019

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

PubMed

Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

PubMed Central

Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi

2013-01-01

Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

Transcriptional networks in plant immunity.

PubMed

Tsuda, Kenichi; Somssich, Imre E

2015-05-01

Next to numerous abiotic stresses, plants are constantly exposed to a variety of pathogens within their environment. Thus, their ability to survive and prosper during the course of evolution was strongly dependent on adapting efficient strategies to perceive and to respond to such potential threats. It is therefore not surprising that modern plants have a highly sophisticated immune repertoire consisting of diverse signal perception and intracellular signaling pathways. This signaling network is intricate and deeply interconnected, probably reflecting the diverse lifestyles and infection strategies used by the multitude of invading phytopathogens. Moreover it allows signal communication between developmental and defense programs thereby ensuring that plant growth and fitness are not significantly retarded. How plants integrate and prioritize the incoming signals and how this information is transduced to enable appropriate immune responses is currently a major research area. An important finding has been that pathogen-triggered cellular responses involve massive transcriptional reprogramming within the host. Additional key observations emerging from such studies are that transcription factors (TFs) are often sites of signal convergence and that signal-regulated TFs act in concert with other context-specific TFs and transcriptional co-regulators to establish sensory transcription regulatory networks required for plant immunity. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

PubMed

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-03-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

PubMed Central

Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173

The global regulatory architecture of transcription during the Caulobacter cell cycle.

PubMed

Zhou, Bo; Schrader, Jared M; Kalogeraki, Virginia S; Abeliuk, Eduardo; Dinh, Cong B; Pham, James Q; Cui, Zhongying Z; Dill, David L; McAdams, Harley H; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

PubMed

Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

2014-01-01

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

PubMed Central

Joo, Yoo Jin; Ficarro, Scott B.; Soares, Luis M.; Chun, Yujin; Marto, Jarrod A.; Buratowski, Stephen

2017-01-01

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations. PMID:29203645

Mechanisms of specificity in neuronal activity-regulated gene transcription

PubMed Central

Lyons, Michelle R.; West, Anne E.

2011-01-01

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. PMID:21620929

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

PubMed Central

Naughton, Catherine; Avlonitis, Nicolaos; Corless, Samuel; Prendergast, James G.; Mati, Ioulia K.; Eijk, Paul P.; Cockroft, Scott L.; Bradley, Mark; Ylstra, Bauke; Gilbert, Nick

2013-01-01

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes. PMID:23416946

Transcription termination factor Rho and microbial phenotypic heterogeneity.

PubMed

Bidnenko, Elena; Bidnenko, Vladimir

2018-06-01

Populations of genetically identical microorganisms exhibit high degree of cell-to-cell phenotypic diversity even when grown in uniform environmental conditions. Heterogeneity is a genetically determined trait, which ensures bacterial adaptation and survival in the ever changing environmental conditions. Fluctuations in gene expression (noise) at the level of transcription initiation largely contribute to cell-to-cell variability within population. Not surprisingly, the analyses of the mechanisms driving phenotypic heterogeneity are mainly focused on the activity of promoters and transcriptional factors. Less attention is currently given to a role of intrinsic and factor-dependent transcription terminators. Here, we discuss recent advances in understanding the regulatory role of the multi-functional transcription termination factor Rho, the major inhibitor of pervasive transcription in bacteria and the emerging global regulator of gene expression. We propose that termination activity of Rho might be among the mechanisms by which cells manage the intensity of transcriptional noise, thus affecting population heterogeneity.

The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

PubMed Central

Balajee, Adayabalam S.; Machwe, Amrita; May, Alfred; Gray, Matthew D.; Oshima, Junko; Martin, George M.; Nehlin, Jan O.; Brosh, Robert; Orren, David K.; Bohr, Vilhelm A.

1999-01-01

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype

Post-transcriptional regulation mediated by specific neurofilament introns in vivo.

PubMed

Wang, Chen; Szaro, Ben G

2016-04-01

Neurons regulate genes post-transcriptionally to coordinate the supply of cytoskeletal proteins, such as the medium neurofilament (NEFM), with demand for structural materials in response to extracellular cues encountered by developing axons. By using a method for evaluating functionality of cis-regulatory gene elements in vivo through plasmid injection into Xenopus embryos, we discovered that splicing of a specific nefm intron was required for robust transgene expression, regardless of promoter or cell type. Transgenes utilizing the nefm 3'-UTR but substituting other nefm introns expressed little or no protein owing to defects in handling of the messenger (m)RNA as opposed to transcription or splicing. Post-transcriptional events at multiple steps, but mainly during nucleocytoplasmic export, contributed to these varied levels of protein expression. An intron of the β-globin gene was also able to promote expression in a manner identical to that of the nefm intron, implying a more general preference for certain introns in controlling nefm expression. These results expand our knowledge of intron-mediated gene expression to encompass neurofilaments, indicating an additional layer of complexity in the control of a cytoskeletal gene needed for developing and maintaining healthy axons. © 2016. Published by The Company of Biologists Ltd.

Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

PubMed

Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

2016-02-18

Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator

PubMed Central

2012-01-01

Background Glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNAAsp. Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. Results The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. Conclusions The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri. PMID:23035718

Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator.

PubMed

Caballero, Valeria C; Toledo, Viviana P; Maturana, Cristian; Fisher, Carolyn R; Payne, Shelley M; Salazar, Juan Carlos

2012-10-05

Glutamyl queuosine-tRNA(Asp) synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNA(Asp). Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.

Transcriptional regulation of ceruloplasmin gene expression during inflammation

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

Gitlin, J.D.

1988-05-05

Mixed sequence oligonucleotides were used to isolate a series of acute-phase human liver cDNA clones corresponding to the serum ..cap alpha../sub 2/-globulin ceruloplasmin. These clones were characterized, sequenced, and used to analyze changes in hepatic ceruloplasmin mRNA content during inflammation. In all species examined, hepatic ceruloplasmin mRNA content increased approximately 6-10-fold over control values within 24 h following the induction of inflammation. The mechanisms leading to this increase in hepatic ceruloplasmin mRNA content were studied following turpentine-induced inflammation in Syrian hamsters. Nuclear run-on assays demonstrated an increase in the relative rate of transcription of the ceruloplasmin gene within 3 hmore » following induction, reaching maximum values by 18 h. Hepatic ceruloplasmin mRNA content increased 2-fold within 12 h following induction, reached maximum values by 24 h, and returned to control within 72 h. In contrast, serum ceruloplasmin concentration did not increase until 36 h, reached maximal levels by 120 h, and remained elevated for the course of the study. These data indicate that inflammation leads to a rapid increase in hepatic ceruloplasmin mRNA content. This increase is largely the result of increased ceruloplasmin gene transcription, but comparison of the relative rate of transcription and mRNA accumulation suggests that changes in ceruloplasmin mRNA turnover are also involved. In addition, translational and/or post-translational mechanisms must account for the observed changes in serum ceruloplasmin concentration seen during inflammation.« less

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

PubMed

Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

2017-08-30

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the

Light-mediated control of DNA transcription in yeast

PubMed Central

Hughes, Robert M.; Bolger, Steven; Tapadia, Hersh; Tucker, Chandra L.

2012-01-01

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light / cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems. PMID:22922268

A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.

PubMed

Zhao, Cui; Zhang, Chen; Chen, Bin; Shi, Yanghui; Quan, Yanping; Nie, Zuoming; Zhang, Yaozhou; Yu, Wei

2016-01-01

A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.

Transcriptional and post-transcriptional regulation of NK cell development and function

PubMed Central

Leong, Jeffrey W.; Wagner, Julia A.; Ireland, Aaron R.; Fehniger, Todd A.

2016-01-01

Natural killer (NK) cells are specialized innate lymphoid cells that survey against viral infections and malignancy. Numerous advances have improved our understanding of the molecular mechanisms that control NK cell development and function over the past decade. These include both studies on the regulatory effects of transcription factors and translational repression via microRNAs. In this review, we summarize our current knowledge of DNA-binding transcription factors that regulate gene expression and thereby orchestrate NK cell development and activation, with an emphasis on recent discoveries. Additionally, we highlight our understanding of how RNA-bindings microRNAs fine tune the NK cell molecular program. We also underscore the large number of open questions in field that are now being addressed using new technological approaches and genetically engineered model organisms. Ultimately, a deeper understanding of the basic molecular biology of NK cells will facilitate new strategies to manipulate NK cells for the treatment of human disease. PMID:26948928

Gene-specific changes in alpha-tubulin transcript accumulation in developing cotton fibers.

PubMed

Whittaker, D J; Triplett, B A

1999-09-01

The fibers of cotton (Gossypium hirsutum) are single-cell trichomes that undergo rapid and synchronous elongation. Cortical microtubules provide spatial information necessary for the alignment of cellulose microfibrils that confine and regulate cell elongation. We used gene-specific probes to investigate alpha-tubulin transcript levels in elongating cotton fibers. Two discrete patterns of transcript accumulation were observed. Whereas transcripts of alpha-tubulin genes GhTua2/3 and GhTua4 increased in abundance from 10 to 20 d post anthesis (DPA), GhTua1 and GhTua5 transcripts were abundant only through to 14 DPA, and dropped significantly at 16 DPA with the onset of secondary wall synthesis. This is the first report, to our knowledge, of gene-specific changes in tubulin transcript levels during the development of a terminally differentiated plant cell. The decrease in abundance of GhTua1 and GhTua5 transcripts was correlated with pronounced changes in cell wall structure, suggesting that alpha-tubulin isoforms may be functionally distinct in elongating fiber cells. Although total alpha-tubulin transcript levels were much higher in fiber than several other tissues, including the hypocotyl and pollen, none of the alpha-tubulins was specific to fiber cells.

Post-transcriptional Mechanisms Contribute Little to Phenotypic Variation in Snake Venoms.

PubMed

Rokyta, Darin R; Margres, Mark J; Calvin, Kate

2015-09-09

Protein expression is a major link in the genotype-phenotype relationship, and processes affecting protein abundances, such as rates of transcription and translation, could contribute to phenotypic evolution if they generate heritable variation. Recent work has suggested that mRNA abundances do not accurately predict final protein abundances, which would imply that post-transcriptional regulatory processes contribute significantly to phenotypes. Post-transcriptional processes also appear to buffer changes in transcriptional patterns as species diverge, suggesting that the transcriptional changes have little or no effect on the phenotypes undergoing study. We tested for concordance between mRNA and protein expression levels in snake venoms by means of mRNA-seq and quantitative mass spectrometry for 11 snakes representing 10 species, six genera, and three families. In contrast to most previous work, we found high correlations between venom gland transcriptomes and venom proteomes for 10 of our 11 comparisons. We tested for protein-level buffering of transcriptional changes during species divergence by comparing the difference between transcript abundance and protein abundance for three pairs of species and one intraspecific pair. We found no evidence for buffering during divergence of our three species pairs but did find evidence for protein-level buffering for our single intraspecific comparison, suggesting that buffering, if present, was a transient phenomenon in venom divergence. Our results demonstrated that post-transcriptional mechanisms did not contribute significantly to phenotypic evolution in venoms and suggest a more prominent and direct role for cis-regulatory evolution in phenotypic variation, particularly for snake venoms. Copyright © 2015 Rokyta et al.

Technical Advance: Transcription factor, promoter, and enhancer utilization in human myeloid cells.

PubMed

Joshi, Anagha; Pooley, Christopher; Freeman, Tom C; Lennartsson, Andreas; Babina, Magda; Schmidl, Christian; Geijtenbeek, Teunis; Michoel, Tom; Severin, Jessica; Itoh, Masayoshi; Lassmann, Timo; Kawaji, Hideya; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Rehli, Michael; Hume, David A

2015-05-01

The generation of myeloid cells from their progenitors is regulated at the level of transcription by combinatorial control of key transcription factors influencing cell-fate choice. To unravel the global dynamics of this process at the transcript level, we generated transcription profiles for 91 human cell types of myeloid origin by use of CAGE profiling. The CAGE sequencing of these samples has allowed us to investigate diverse aspects of transcription control during myelopoiesis, such as identification of novel transcription factors, miRNAs, and noncoding RNAs specific to the myeloid lineage. We further reconstructed a transcription regulatory network by clustering coexpressed transcripts and associating them with enriched cis-regulatory motifs. With the use of the bidirectional expression as a proxy for enhancers, we predicted over 2000 novel enhancers, including an enhancer 38 kb downstream of IRF8 and an intronic enhancer in the KIT gene locus. Finally, we highlighted relevance of these data to dissect transcription dynamics during progressive maturation of granulocyte precursors. A multifaceted analysis of the myeloid transcriptome is made available (www.myeloidome.roslin.ed.ac.uk). This high-quality dataset provides a powerful resource to study transcriptional regulation during myelopoiesis and to infer the likely functions of unannotated genes in human innate immunity. © The Author(s).

Amphetamine manipulates monoamine oxidase-A level and behavior using theranostic aptamers of transcription factors AP-1/NF-kB.

PubMed

Liu, Christina H; Ren, Jiaqian; Liu, Philip K

2016-02-03

Monoamine oxidase (MAO) enzymes play a critical role in controlling the catabolism of monoamine neurotransmitters and biogenic trace amines and behavior in humans. However, the mechanisms that regulate MAO are unclear. Several transcription factor proteins are proposed to modulate the transcription of MAO gene, but evidence supporting these hypotheses is controversial. We aimed to investigate the mechanism of gene transcription regulator proteins on amphetamine-induced behavior. We applied aptamers containing a DNA binding sequence, as well as a random sequence (without target) to study the modulation of amphetamine-induced MAO levels and hyperactivity in living mice. We pretreated in adult male C57black6 mice (Taconic Farm, Germantown, NY) (n ≥ 3 litters at a time), 2 to 3 months of age (23 ± 2 gm body weight) with double-stranded (ds) DNA aptamers with sequence specific to activator protein-1 (5ECdsAP1), nuclear factor-kappa beta (5ECdsNF-kB), special protein-1 (5ECdsSP-1) or cyclicAMP responsive element binding (5ECdsCreB) protein binding regions, 5ECdsRan [a random sequence without target], single-stranded AP-1 (5ECssAP-1) (8 nmol DNA per kg) or saline (5 μl, intracerebroventricular [icv] injection) control before amphetamine administration (4 mg/kg, i.p.). We then measured and analyzed locomotor activities and the level of MAO-A and MAO-B activity. In the pathological condition of amphetamine exposure, we showed here that pretreatment with 5ECdsAP1 and 5ECdsNF-kB reversed the decrease of MAO-A activity (p < 0.05, t test), but not activity of the B isomer (MAO-B), in the ventral tegmental area (VTA) and substantia nigra (SN) of C57black6 mice. The change in MAO-A level coincided with a reversed amphetamine-induced restless behavior of mice. Pretreatments with saline, 5ECdsCreB, 5ECdsSP-1, 5ECdsRan or 5ECssAP-1 had no effect. Our data lead us to conclude that elevation of AP-1 or NF-kB indirectly decreases MAO-A protein levels which, in turn

Transcriptional profiling of the pea shoot apical meristem reveals processes underlying its function and maintenance

PubMed Central

Wong, Chui E; Bhalla, Prem L; Ottenhof, Harald; Singh, Mohan B

2008-01-01

Background Despite the importance of the shoot apical meristem (SAM) in plant development and organ formation, our understanding of the molecular mechanisms controlling its function is limited. Genomic tools have the potential to unravel the molecular mysteries of the SAM, and legume systems are increasingly being used in plant-development studies owing to their unique characteristics such as nitrogen fixation, secondary metabolism, and pod development. Garden pea (Pisum sativum) is a well-established classic model species for genetics studies that has been used since the Mendel era. In addition, the availability of a plethora of developmental mutants makes pea an ideal crop legume for genomics studies. This study aims to utilise genomics tools in isolating genes that play potential roles in the regulation of SAM activity. Results In order to identify genes that are differentially expressed in the SAM, we generated 2735 ESTs from three cDNA libraries derived from freshly micro-dissected SAMs from 10-day-old garden peas (Pisum sativum cv Torsdag). Custom-designed oligonucleotide arrays were used to compare the transcriptional profiles of pea SAMs and non-meristematic tissues. A total of 184 and 175 transcripts were significantly up- or down-regulated in the pea SAM, respectively. As expected, close to 61% of the transcripts down-regulated in the SAM were found in the public database, whereas sequences from the same source only comprised 12% of the genes that were expressed at higher levels in the SAM. This highlights the under-representation of transcripts from the meristematic tissues in the current public pea protein database, and demonstrates the utility of our SAM EST collection as an essential genetic resource for revealing further information on the regulation of this developmental process. In addition to unknowns, many of the up-regulated transcripts are known to encode products associated with cell division and proliferation, epigenetic regulation, auxin

ANKRD1 Acts as a Transcriptional Repressor of MMP13 via the AP-1 Site

PubMed Central

Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E.

2014-01-01

The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1−/− (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1fl/fl (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

77 FR 16817 - Request for Comment on Payday Lending Hearing Transcript

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-22

... Lending Hearing Transcript AGENCY: Bureau of Consumer Financial Protection. ACTION: Notice; request for... hearing on payday lending. The Bureau invites the public to review the transcript and provide additional..., 2012 in Birmingham, Alabama. Payday lending products are typically marketed to bridge a cash flow...

The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila

PubMed Central

Kwok, Rosanna S.; Li, Ying H.; Lei, Anna J.; Edery, Isaac; Chiu, Joanna C.

2015-01-01

Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription. PMID:26132408

Distinct contributions of replication and transcription to mutation rate variation of human genomes.

PubMed

Cui, Peng; Ding, Feng; Lin, Qiang; Zhang, Lingfang; Li, Ang; Zhang, Zhang; Hu, Songnian; Yu, Jun

2012-02-01

Here, we evaluate the contribution of two major biological processes--DNA replication and transcription--to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes. Copyright © 2012 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

Additional Remarks on Designing Category-Level Attributes for Discriminative Visual Recognition

DTIC Science & Technology

2013-01-01

Discriminative Visual Recognition ∗ Felix X. Yu†, Liangliang Cao§, Rogerio S. Feris§, John R. Smith§, Shih-Fu Chang† † Columbia University § IBM T. J...for Designing Category-Level Attributes for Dis- criminative Visual Recognition [3]. We first provide an overview of the proposed ap- proach in...2013 to 00-00-2013 4. TITLE AND SUBTITLE Additional Remarks on Designing Category-Level Attributes for Discriminative Visual Recognition 5a

(S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level.

PubMed Central

Wang, Y L; Beach, M J; Rodwell, V W

1989-01-01

We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon. Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivo was induced by mevalonate. Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms. The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level. Images PMID:2477360

RNA-seq analysis of the transcriptional response to blue and red light in the extremophilic red alga, Cyanidioschyzon merolae.

PubMed

Tardu, Mehmet; Dikbas, Ugur Meric; Baris, Ibrahim; Kavakli, Ibrahim Halil

2016-11-01

Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red lights were found to regulate 35 % of the total genes in C. merolae. Blue light affected the transcription of genes involved in protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae.

Post-transcriptional inducible gene regulation by natural antisense RNA.

PubMed

Nishizawa, Mikio; Ikeya, Yukinobu; Okumura, Tadayoshi; Kimura, Tominori

2015-01-01

Accumulating data indicate the existence of natural antisense transcripts (asRNAs), frequently transcribed from eukaryotic genes and do not encode proteins in many cases. However, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. A low-copy-number asRNA may post-transcriptionally regulate gene expression with cis-controlling elements on the mRNA. The asRNA itself may act as regulatory RNA in concert with trans-acting factors, including various RNA-binding proteins that bind to cis-controlling elements, microRNAs, and drugs. A novel mechanism that regulates mRNA stability includes the interaction of asRNA with mRNA by hybridization to loops in secondary structures. Furthermore, recent studies have shown that the functional network of mRNAs, asRNAs, and microRNAs finely tunes the levels of mRNA expression. The post-transcriptional mechanisms via these RNA-RNA interactions may play pivotal roles to regulate inducible gene expression and present the possibility of the involvement of asRNAs in various diseases.

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

HIV-1 transcription and latency: an update

PubMed Central

2013-01-01

Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs. PMID:23803414

Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta.

PubMed

Konopiński, R; Nowak, R; Siedlecki, J A

1996-10-17

Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

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

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

DOE PAGES

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna; ...

2018-05-25

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Enhancing Listening with Captions and Transcripts: Exploring Learner Differences

ERIC Educational Resources Information Center

Danan, Martine

2016-01-01

Through an analysis of students' reactions to transcription exercises, this exploratory study examined some of the differences between two forms of written help enhancing listening passages--second language (L2) captions and transcripts. This primarily qualitative analysis highlighted the role that student proficiency levels may have played in…

Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

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

Kumar, S.; Cole, R.; Chiappelli, F.

During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase inmore » mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.« less

Metabolic and transcriptional regulatory mechanisms underlying the anoxic adaptation of rice coleoptile

PubMed Central

Lakshmanan, Meiyappan; Mohanty, Bijayalaxmi; Lim, Sun-Hyung; Ha, Sun-Hwa; Lee, Dong-Yup

2014-01-01

The ability of rice to germinate under anoxia by extending the coleoptile is a highly unusual characteristic and a key feature underpinning the ability of rice seeds to establish in such a stressful environment. The process has been a focal point for research for many years. However, the molecular mechanisms underlying the anoxic growth of the coleoptile still remain largely unknown. To unravel the key regulatory mechanisms of rice germination under anoxic stress, we combined in silico modelling with gene expression data analysis. Our initial modelling analysis via random flux sampling revealed numerous changes in rice primary metabolism in the absence of oxygen. In particular, several reactions associated with sucrose metabolism and fermentation showed a significant increase in flux levels, whereas reaction fluxes across oxidative phosphorylation, the tricarboxylic acid cycle and the pentose phosphate pathway were down-regulated. The subsequent comparative analysis of the differences in calculated fluxes with previously published gene expression data under air and anoxia identified at least 37 reactions from rice central metabolism that are transcriptionally regulated. Additionally, cis-regulatory content analyses of these transcriptionally controlled enzymes indicate a regulatory role for transcription factors such as MYB, bZIP, ERF and ZnF in transcriptional control of genes that are up-regulated during rice germination and coleoptile elongation under anoxia. PMID:24894389

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

PubMed Central

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-01-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation. PMID:9062372

HnRNP-like proteins as post-transcriptional regulators.

PubMed

Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

2014-10-01

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

PubMed

Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

2014-02-01

In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rapid Myeloid Cell Transcriptional and Proteomic Responses to Periodontopathogenic Porphyromonas gingivalis

PubMed Central

Nares, Salvador; Moutsopoulos, Niki M.; Angelov, Nikola; Rangel, Zoila G.; Munson, Peter J.; Sinha, Neha; Wahl, Sharon M.

2009-01-01

Long-lived monocytes, macrophages, and dendritic cells (DCs) are Toll-like receptor-expressing, antigen-presenting cells derived from a common myeloid lineage that play key roles in innate and adaptive immune responses. Based on immunohistochemical and molecular analyses of inflamed tissues from patients with chronic destructive periodontal disease, these cells, found in the inflammatory infiltrate, may drive the progressive periodontal pathogenesis. To investigate early transcriptional signatures and subsequent proteomic responses to the periodontal pathogen, Porphyromonas gingivalis, donor-matched human blood monocytes, differentiated DCs, and macrophages were exposed to P. gingivalis lipopolysaccharide (LPS) and gene expression levels were measured by oligonucleotide microarrays. In addition to striking differences in constitutive transcriptional profiles between these myeloid populations, we identify a P. gingivalis LPS-inducible convergent, transcriptional core response of more than 400 annotated genes/ESTs among these populations, reflected by a shared, but quantitatively distinct, proteomic response. Nonetheless, clear differences emerged between the monocytes, DCs, and macrophages. The finding that long-lived myeloid inflammatory cells, particularly DCs, rapidly and aggressively respond to P. gingivalis LPS by generating chemokines, proteases, and cytokines capable of driving T-helper cell lineage polarization without evidence of corresponding immunosuppressive pathways highlights their prominent role in host defense and progressive tissue pathogenesis. The shared, unique, and/or complementary transcriptional and proteomic profiles may frame the context of the host response to P. gingivalis, contributing to the destructive nature of periodontal inflammation. PMID:19264901

Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations

PubMed Central

Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

2017-01-01

Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators. PMID:28301511

Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations.

PubMed

Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

2017-01-01

Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

An evolutionarily conserved RNase-based mechanism for repression of transcriptional positive autoregulation

PubMed Central

Wurtmann, Elisabeth J.; Ratushny, Alexander V.; Pan, Min; Beer, Karlyn D.; Aitchison, John D.; Baliga, Nitin S.

2014-01-01

Summary It is known that environmental context influences the degree of regulation at the transcriptional and post-transcriptional levels. However, the principles governing the differential usage and interplay of regulation at these two levels are not clear. Here, we show that the integration of transcriptional and post-transcriptional regulatory mechanisms in a characteristic network motif drives efficient environment-dependent state transitions. Through phenotypic screening, systems analysis, and rigorous experimental validation, we discovered an RNase (VNG2099C) in Halobacterium salinarum that is transcriptionally co-regulated with genes of the aerobic physiologic state but acts on transcripts of the anaerobic state. Through modeling and experimentation we show that this arrangement generates an efficient state-transition switch, within which RNase-repression of a transcriptional positive autoregulation (RPAR) loop is critical for shutting down ATP-consuming active potassium uptake to reserve energy required for salinity adaptation under aerobic, high potassium, or dark conditions. Subsequently, we discovered that many Escherichia coli operons with energy-associated functions are also putatively controlled by RPAR indicating that this network motif may have evolved independently in phylogenetically distant organisms. Thus, our data suggest that interplay of transcriptional and post-transcriptional regulation in the RPAR motifis a generalized principle for efficient environment-dependent state transitions across prokaryotes. PMID:24612392

MRD assessed by WT1 and NPM1 transcript levels identifies distinct outcomes in AML patients and is influenced by gemtuzumab ozogamicin

PubMed Central

Lambert, Juliette; Lambert, Jérôme; Nibourel, Olivier; Pautas, Cécile; Hayette, Sandrine; Cayuela, Jean-Michel; Terré, Christine; Rousselot, Philippe; Dombret, Hervé; Chevret, Sylvie; Preudhomme, Claude; Castaigne, Sylvie; Renneville, Aline

2014-01-01

We analysed the prognostic significance of minimal residual disease (MRD) level in adult patients with acute myeloid leukemia (AML) treated in the randomized gemtuzumab ozogamicin (GO) ALFA-0701 trial. Levels of WT1 and NPM1 gene transcripts were assessed using cDNA-based real-time quantitative PCR in 183 patients with WT1 overexpression and in 77 patients with NMP1 mutation (NPM1mut) at diagnosis. Positive WT1 MRD (defined as > 0.5% in the peripheral blood) after induction and at the end of treatment were both significantly associated with a higher risk of relapse and a shorter overall survival (OS). Positive NPM1mut MRD (defined as > 0.1% in the bone marrow) after induction and at the end of treatment also predicted a higher risk of relapse, but did not influence OS. Interestingly, the achievement of a negative NPM1mut MRD was significantly more frequent in patients treated in the GO arm compared to those treated in control arm (39% versus 7% (p=0.006) after induction and 91% versus 61% (p=0.028) at the end of treatment). However, GO did not influence WT1 MRD levels. Our study supports the prognostic significance of MRD assessed by WT1 and NPM1mut transcript levels and show that NPM1 MRD is decreased by GO treatment. PMID:25026287

MRD assessed by WT1 and NPM1 transcript levels identifies distinct outcomes in AML patients and is influenced by gemtuzumab ozogamicin.

PubMed

Lambert, Juliette; Lambert, Jérôme; Nibourel, Olivier; Pautas, Cécile; Hayette, Sandrine; Cayuela, Jean-Michel; Terré, Christine; Rousselot, Philippe; Dombret, Hervé; Chevret, Sylvie; Preudhomme, Claude; Castaigne, Sylvie; Renneville, Aline

2014-08-15

We analysed the prognostic significance of minimal residual disease (MRD) level in adult patients with acute myeloid leukemia (AML) treated in the randomized gemtuzumab ozogamicin (GO) ALFA-0701 trial. Levels of WT1 and NPM1 gene transcripts were assessed using cDNA-based real-time quantitative PCR in 183 patients with WT1 overexpression and in 77 patients with NMP1 mutation (NPM1mut) at diagnosis. Positive WT1 MRD (defined as > 0.5% in the peripheral blood) after induction and at the end of treatment were both significantly associated with a higher risk of relapse and a shorter overall survival (OS). Positive NPM1mut MRD (defined as > 0.1% in the bone marrow) after induction and at the end of treatment also predicted a higher risk of relapse, but did not influence OS. Interestingly, the achievement of a negative NPM1mut MRD was significantly more frequent in patients treated in the GO arm compared to those treated in control arm (39 % versus 7% (p=0.006) after induction and 91% versus 61% (p=0.028) at the end of treatment). However, GO did not influence WT1 MRD levels. Our study supports the prognostic significance of MRD assessed by WT1 and NPM1mut transcript levels and show that NPM1 MRD is decreased by GO treatment.

Plant Mediator complex and its critical functions in transcription regulation.

PubMed

Yang, Yan; Li, Ling; Qu, Li-Jia

2016-02-01

The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted. © 2015 Institute of Botany, Chinese Academy of Sciences.

Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis

PubMed Central

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J.; Considine, Robert V.; Sethi, Jaswinder K.; Vidal-Puig, Antonio; O’Rahilly, Stephen

2015-01-01

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation. PMID:14701838

Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis.

PubMed

Li, S; Zhang, P; Zhang, M; Fu, C; Yu, L

2013-01-01

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

Reanalysis of RNA-Sequencing Data Reveals Several Additional Fusion Genes with Multiple Isoforms

PubMed Central

Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

2012-01-01

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts. PMID:23119097

Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

PubMed

Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

2012-01-01

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.

PubMed Central

Frieman, M; Chen, Z J; Saez-Vasquez, J; Shen, L A; Pikaard, C S

1999-01-01

In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level. PMID:10224274

Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c.

PubMed

Kim, Kang Ho; Song, Min Jeong; Yoo, Eung Jae; Choe, Sung Sik; Park, Sang Dai; Kim, Jae Bum

2004-12-10

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3beta down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism.

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

PubMed

Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

2001-07-06

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Chromatin insulation by a transcriptional activator

PubMed Central

Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

2003-01-01

In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

Regulation of steroid hormone receptors and coregulators during the cell cycle highlights potential novel function in addition to roles as transcription factors

PubMed Central

Zheng, Yingfeng; Murphy, Leigh C.

2016-01-01

Cell cycle progression is tightly controlled by several kinase families including Cyclin-Dependent Kinases, Polo-Like Kinases, and Aurora Kinases. A large amount of data show that steroid hormone receptors and various components of the cell cycle, including cell cycle regulated kinases, interact, and this often results in altered transcriptional activity of the receptor. Furthermore, steroid hormones, through their receptors, can also regulate the transcriptional expression of genes that are required for cell cycle regulation. However, emerging data suggest that steroid hormone receptors may have roles in cell cycle progression independent of their transcriptional activity. The following is a review of how steroid receptors and their coregulators can regulate or be regulated by the cell cycle machinery, with a particular focus on roles independent of transcription in G2/M. PMID:26778927

The glucose sensor Snf1 and the transcription factors Msn2 and Msn4 regulate transcription of the vacuolar iron importer gene CCC1 and iron resistance in yeast.

PubMed

Li, Liangtao; Kaplan, Jerry; Ward, Diane M

2017-09-15

The budding yeast Saccharomyces cerevisiae stores iron in the vacuole, which is a major resistance mechanism against iron toxicity. One key protein involved in vacuolar iron storage is the iron importer Ccc1, which facilitates iron entry into the vacuole. Transcription of the CCC1 gene is largely regulated by the binding of iron-sulfur clusters to the activator domain of the transcriptional activator Yap5. Additional evidence, however, suggests that Yap5-independent transcriptional activation of CCC1 also contributes to iron resistance. Here, we demonstrate that components of the signaling pathway involving the low-glucose sensor Snf1 regulate CCC1 transcription and iron resistance. We found that SNF1 deletion acts synergistically with YAP5 deletion to regulate CCC1 transcription and iron resistance. A kinase-dead mutation of Snf1 lowered iron resistance as did deletion of SNF4 , which encodes a partner protein of Snf1. Deletion of all three alternative partners of Snf1 encoded by SIT1 , SIT2 , and GAL83 decreased both CCC1 transcription and iron resistance. The Snf1 complex is known to activate the general stress transcription factors Msn2 and Msn4. We show that Msn2 and Msn4 contribute to Snf1-mediated CCC1 transcription. Of note, SNF1 deletion in combination with MSN2 and MSN4 deletion resulted in additive effects on CCC1 transcription, suggesting that other activators contribute to the regulation of CCC1 transcription. In conclusion, we show that yeast have developed multiple transcriptional mechanisms to regulate Ccc1 expression and to protect against high cytosolic iron toxicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA residence time is a regulatory factor of transcription repression

PubMed Central

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

PubMed

Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

2012-01-01

Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

Dynamical behavior of psb gene transcripts in greening wheat seedlings. I. Time course of accumulation of the pshA through psbN gene transcripts during light-induced greening.

PubMed

Kawaguchi, H; Fukuda, I; Shiina, T; Toyoshima, Y

1992-11-01

The time course of the accumulation of the transcripts from 13 psb genes encoding a major part of the proteins composing photosystem II during light-induced greening of dark-grown wheat seedlings was examined focusing on early stages of plastid development (0.5 h through 72 h). The 13 genes can be divided into three groups. (1) The psbA gene is transcribed as a single transcript of 1.3 kb in the dark-grown seedlings, but its level increases 5- to 7-fold in response to light due to selective increase in RNA stability as well as in transcription activity. (2) The psbE-F-L-J operon, psbM and psbN genes are transcribed as a single transcript of 1.1 kb, two transcripts of 0.5 and 0.7 kb and a single transcript of 0.3 kb, respectively, in the dark-grown seedlings. The levels of accumulation of every transcript remain unchanged or rather decrease during plastid development under illumination. (3) The psbK-I-D-C gene cluster and psbB-H operon exhibit fairly complicated northern hybridization patterns during the greening process. When a psbC or psbD gene probe was used for northern hybridization, five transcripts differing in length were detected in the etioplasts from 5-day old dark-grown seedlings. After 2 h illumination, two new transcripts of different length appeared. Light induction of new transcripts was also observed in the psbB-H operon.

Reduction in cab and psb A RNA transcripts in response to supplementary ultraviolet-B radiation.

PubMed

Jordan, B R; Chow, W S; Strid, A; Anderson, J M

1991-06-17

The cab and psb A RNA transcript levels have been determined in Pisum sativum leaves exposed to supplementary ultraviolet-B radiation. The nuclear-encoded cab transcripts are reduced to low levels after only 4 h of UV-B treatment and are undetectable after 3 days exposure. In contrast, the chloroplast-encoded psb A transcript levels, although reduced, are present for at least 3 days. After short periods of UV-B exposure (4 h or 8 h), followed by recovery under control conditions, cab RNA transcript levels had not recovered after 1 day, but were re-established to ca. 60% of control levels after 2 more days. Increased irradiance during exposure to UV-B reduced the effect upon cab transcripts, although the decrease was still substantial. These results indicate rapid changes in the cellular regulation of gene expression in response to supplementary UV-B and suggest increased UV-B radiation may have profound consequences for future productivity of sensitive crop species.

Post-translational regulation of WRKY transcription factors in plant immunity.

PubMed

Ishihama, Nobuaki; Yoshioka, Hirofumi

2012-08-01

Plants have evolved immune system to protect themselves against invading pathogens. Recent research has illustrated that signaling networks, after perception of diverse pathogen-derived signals, facilitate transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. WRKY proteins, which comprise a large family of plant transcription factors, are key players in plant immune responses. WRKY transcription factors participate in the control of defense-related genes either as positive or as negative regulators, and essentially are regulated at the transcriptional level. Emerging evidence emphasizes that group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are also activated by MAPK-dependent phosphorylation, underlining their importance in plant immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks.

PubMed

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-10

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

NASA Astrophysics Data System (ADS)

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-01

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

Concordance of transcriptional and apical benchmark dose levels for conazole-induced liver effects in mice.

PubMed

Bhat, Virunya S; Hester, Susan D; Nesnow, Stephen; Eastmond, David A

2013-11-01

The ability to anchor chemical class-based gene expression changes to phenotypic lesions and to describe these changes as a function of dose and time informs mode-of-action determinations and improves quantitative risk assessments. Previous global expression profiling identified a 330-probe cluster differentially expressed and commonly responsive to 3 hepatotumorigenic conazoles (cyproconazole, epoxiconazole, and propiconazole) at 30 days. Extended to 2 more conazoles (triadimefon and myclobutanil), the present assessment encompasses 4 tumorigenic and 1 nontumorigenic conazole. Transcriptional benchmark dose levels (BMDL(T)) were estimated for a subset of the cluster with dose-responsive behavior and a ≥ 5-fold increase or decrease in signal intensity at the highest dose. These genes primarily encompassed CAR/RXR activation, P450 metabolism, liver hypertrophy- glutathione depletion, LPS/IL-1-mediated inhibition of RXR, and NRF2-mediated oxidative stress pathways. Median BMDL(T) estimates from the subset were concordant (within a factor of 2.4) with apical benchmark doses (BMDL(A)) for increased liver weight at 30 days for the 5 conazoles. The 30-day median BMDL(T) estimates were within one-half order of magnitude of the chronic BMDLA for hepatocellular tumors. Potency differences seen in the dose-responsive transcription of certain phase II metabolism, bile acid detoxification, and lipid oxidation genes mirrored each conazole's tumorigenic potency. The 30-day BMDL(T) corresponded to tumorigenic potency on a milligram per kilogram day basis with cyproconazole > epoxiconazole > propiconazole > triadimefon > myclobutanil (nontumorigenic). These results support the utility of measuring short-term gene expression changes to inform quantitative risk assessments from long-term exposures.

Roles of CAMTA transcription factors and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis.

PubMed

Kim, YongSig; Park, Sunchung; Gilmour, Sarah J; Thomashow, Michael F

2013-08-01

Previous studies in Arabidopsis thaliana established roles for CALMODULIN BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) in the rapid cold induction of CRT/DRE BINDING FACTOR (CBF) genes CBF1 and CBF2, and the repression of salicylic acid (SA) biosynthesis at warm temperature. Here we show that CAMTA1 and CAMTA2 work in concert with CAMTA3 at low temperature (4°C) to induce peak transcript levels of CBF1, CBF2 and CBF3 at 2 h, contribute to up-regulation of approximately 15% of the genes induced at 24 h, most of which fall outside the CBF pathway, and increase plant freezing tolerance. In addition, CAMTA1, CAMTA2 and CAMTA3 function together to inhibit SA biosynthesis at warm temperature (22°C). However, SA levels increase in Arabidopsis plants that are exposed to low temperature for more than 1 week. We show that this chilling-induced SA biosynthesis proceeds through the isochorismate synthase (ICS) pathway, with cold induction of ICS1 (which encodes ICS), and two genes encoding transcription factors that positively regulate ICS1 - CBP60g and SARD1 -, paralleling SA accumulation. The three CAMTA proteins effectively repress the accumulation of ICS1, CBP60g and SARD1 transcripts at warm temperature but not at low temperature. This impairment of CAMTA function may involve post-transcriptional regulation, as CAMTA transcript levels did not decrease at low temperature. Salicylic acid biosynthesis at low temperature did not contribute to freezing tolerance, but had a major role in configuring the transcriptome, including the induction of 'defense response' genes, suggesting the possible existence of a pre-emptive defense strategy programmed by prolonged chilling temperatures. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A hyperactive transcriptional state marks genome reactivation at the mitosis–G1 transition

PubMed Central

Hsiung, Chris C.-S.; Bartman, Caroline R.; Huang, Peng; Ginart, Paul; Stonestrom, Aaron J.; Keller, Cheryl A.; Face, Carolyne; Jahn, Kristen S.; Evans, Perry; Sankaranarayanan, Laavanya; Giardine, Belinda; Hardison, Ross C.; Raj, Arjun; Blobel, Gerd A.

2016-01-01

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis–G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis–G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer–promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis–G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis–G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis–G1 transition might predispose cells to diverge in gene expression states. PMID:27340175

Multisite Light-Induced Phosphorylation of the Transcription Factor PIF3 Is Necessary for Both Its Rapid Degradation and Concomitant Negative Feedback Modulation of Photoreceptor phyB Levels in Arabidopsis[C][W

PubMed Central

Ni, Weimin; Xu, Shou-Ling; Chalkley, Robert J.; Pham, Thao Nguyen D.; Guan, Shenheng; Maltby, Dave A.; Burlingame, Alma L.; Wang, Zhi-Yong; Quail, Peter H.

2013-01-01

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure. PMID:23903316

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

PubMed Central

Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

2015-01-01

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants.

PubMed

Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

2015-01-01

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5' transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5'UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5'end can modulate protein levels up to 160%-300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for

Silencers, silencing, and heritable transcriptional states.

PubMed Central

Laurenson, P; Rine, J

1992-01-01

Three copies of the mating-type genes, which determine cell type, are found in the budding yeast Saccharomyces cerevisiae. The copy at the MAT locus is transcriptionally active, whereas identical copies of the mating-type genes at the HML and HMR loci are transcriptionally silent. Hence, HML and HMR, also known as the silent mating-type loci, are subject to a position effect. Regulatory sequences flank the silent mating-type loci and mediate repression of HML and HMR. These regulatory sequences are called silencers for their ability to repress the transcription of nearby genes in a distance- and orientation-independent fashion. In addition, a number of proteins, including the four SIR proteins, histone H4, and an alpha-acetyltransferase, are required for the complete repression of HML and HMR. Because alterations in the amino-terminal domain of histone H4 result in the derepression of the silent mating-type loci, the mechanism of repression may involve the assembly of a specific chromatin structure. A number of additional clues permit insight into the nature of repression at HML and HMR. First, an S phase event is required for the establishment of repression. Second, at least one gene appears to play a role in the establishment mechanism yet is not essential for the stable propagation of repression through many rounds of cell division. Third, certain aspects of repression are linked to aspects of replication. The silent mating-type loci share many similarities with heterochromatin. Furthermore, regions of S. cerevisiae chromosomes, such as telomeres, which are known to be heterochromatic in other organisms, require a subset of SIR proteins for repression. Further analysis of the transcriptional repression at the silent mating-type loci may lend insight into heritable repression in other eukaryotes. PMID:1480108

A critical role for topoisomerase IIb and DNA double strand breaks in transcription

PubMed Central

Calderwood, Stuart K.

2016-01-01

ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

PubMed

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

MENA is a transcriptional target of the Wnt/beta-catenin pathway.

PubMed

Najafov, Ayaz; Seker, Tuncay; Even, Ipek; Hoxhaj, Gerta; Selvi, Osman; Ozel, Duygu Esen; Koman, Ahmet; Birgül-İyison, Necla

2012-01-01

Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify MENA (ENAH, Mammalian enabled homologue) as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor--the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.

Statistical mechanical model of coupled transcription from multiple promoters due to transcription factor titration

PubMed Central

Rydenfelt, Mattias; Cox, Robert Sidney; Garcia, Hernan; Phillips, Rob

2014-01-01

Transcription factors (TFs) with regulatory action at multiple promoter targets is the rule rather than the exception, with examples ranging from the cAMP receptor protein (CRP) in E. coli that regulates hundreds of different genes simultaneously to situations involving multiple copies of the same gene, such as plasmids, retrotransposons, or highly replicated viral DNA. When the number of TFs heavily exceeds the number of binding sites, TF binding to each promoter can be regarded as independent. However, when the number of TF molecules is comparable to the number of binding sites, TF titration will result in correlation (“promoter entanglement”) between transcription of different genes. We develop a statistical mechanical model which takes the TF titration effect into account and use it to predict both the level of gene expression for a general set of promoters and the resulting correlation in transcription rates of different genes. Our results show that the TF titration effect could be important for understanding gene expression in many regulatory settings. PMID:24580252

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

PubMed Central

Zhong, Xuehua; Hale, Christopher J.; Nguyen, Minh; Ausin, Israel; Groth, Martin; Hetzel, Jonathan; Vashisht, Ajay A.; Henderson, Ian R.; Wohlschlegel, James A.; Jacobsen, Steven E.

2015-01-01

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation. PMID:25561521

Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei.

PubMed

He, Ronglin; Ma, Lijuan; Li, Chen; Jia, Wendi; Li, Demao; Zhang, Dongyuan; Chen, Shulin

2014-12-01

Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei. Copyright © 2014 Elsevier Inc. All rights reserved.

45. VIEW OF UPPER LEVEL CRUSHER ADDITION FROM CRUSHED OXIDIZED ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

45. VIEW OF UPPER LEVEL CRUSHER ADDITION FROM CRUSHED OXIDIZED ORE BIN. 18 INCH BELT CONVEYOR BIN FEED, LOWER CENTER, WITH STEPHENS-ADAMSON 25 TON/HR ELEVATOR SPLIT DISCHARGE (OXIDIZED/UNOXIDIZED) IN CENTER. CRUDE ORE BINS AND MACHINE SHOP BEYOND. NOTE TOP OF CRUSHED OXIDIZED ORE BIN IS BELOW TOP OF CRUDE ORE BINS. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

Gene transcription patterns in response to low level petroleum contaminants in Mytilus trossulus from field sites and harbors in southcentral Alaska

NASA Astrophysics Data System (ADS)

Bowen, Lizabeth; Miles, A. Keith; Ballachey, Brenda; Waters, Shannon; Bodkin, James; Lindeberg, Mandy; Esler, Daniel

2018-01-01

The 1989 Exxon Valdez oil spill damaged a wide range of natural resources, including intertidal communities, and post-spill studies demonstrated acute and chronic exposure and injury to an array of species. Standard toxicological methods to evaluate petroleum contaminants have assessed tissue burdens, with fewer assays providing indicators of health or physiology, particularly when contaminant levels are low and chronic. Marine mussels are a ubiquitous and crucial component of the nearshore environment, and new genomic technologies exist to quantify molecular responses of individual mussels to stimuli, including exposure to polycyclic aromatic hydrocarbons (PAHs). We used gene-based assays of exposure and physiological function to assess chronic oil contamination using the Pacific blue mussel, Mytilus trossulus. We developed a diagnostic gene transcription panel to investigate exposure to PAHs and other contaminants and its effects on mussel physiology and health. During 2012-2015, we analyzed mussels from five field sites in western Prince William Sound, Alaska, with varying oil histories from the 1989 Exxon Valdez oil spill, and from three boat harbors in the area. Gene transcription patterns of mussels from harbors were consistent with elevated exposure to PAHs or other contaminants, whereas transcription patterns of mussels sampled from shorelines in areas affected by the oil spill indicated no PAH exposure.

Gene transcription patterns in response to low level petroleum contaminants in Mytilus trossulus from field sites and harbors in southcentral Alaska

USGS Publications Warehouse

Bowen, Lizabeth; Miles, A. Keith; Ballachey, Brenda E.; Waters, Shannon C.; Bodkin, James L.; Lindeberg, Mandy; Esler, Daniel N.

2017-01-01

The 1989 Exxon Valdez oil spill damaged a wide range of natural resources, including intertidal communities, and post-spill studies demonstrated acute and chronic exposure and injury to an array of species. Standard toxicological methods to evaluate petroleum contaminants have assessed tissue burdens, with fewer assays providing indicators of health or physiology, particularly when contaminant levels are low and chronic. Marine mussels are a ubiquitous and crucial component of the nearshore environment, and new genomic technologies exist to quantify molecular responses of individual mussels to stimuli, including exposure to polycyclic aromatic hydrocarbons (PAHs). We used gene-based assays of exposure and physiological function to assess chronic oil contamination using the Pacific blue mussel, Mytilus trossulus. We developed a diagnostic gene transcription panel to investigate exposure to PAHs and other contaminants and its effects on mussel physiology and health. During 2012–2015, we analyzed mussels from five field sites in western Prince William Sound, Alaska, with varying oil histories from the 1989 Exxon Valdez oil spill, and from three boat harbors in the area. Gene transcription patterns of mussels from harbors were consistent with elevated exposure to PAHs or other contaminants, whereas transcription patterns of mussels sampled from shorelines in areas affected by the oil spill indicated no PAH exposure.

Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers.

PubMed

Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Josse, Claire; Jerusalem, Guy

2018-01-02

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.

Highly efficient Cas9-mediated transcriptional programming

DOE PAGES

Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani; ...

2015-03-02

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. Here we describe an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. Here, we demonstrate its utility in activating endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

Nascent RNA kinetics: Transient and steady state behavior of models of transcription

NASA Astrophysics Data System (ADS)

Choubey, Sandeep

2018-02-01

Regulation of transcription is a vital process in cells, but mechanistic details of this regulation still remain elusive. The dominant approach to unravel the dynamics of transcriptional regulation is to first develop mathematical models of transcription and then experimentally test the predictions these models make for the distribution of mRNA and protein molecules at the individual cell level. However, these measurements are affected by a multitude of downstream processes which make it difficult to interpret the measurements. Recent experimental advancements allow for counting the nascent mRNA number of a gene as a function of time at the single-inglr cell level. These measurements closely reflect the dynamics of transcription. In this paper, we consider a general mechanism of transcription with stochastic initiation and deterministic elongation and probe its impact on the temporal behavior of nascent RNA levels. Using techniques from queueing theory, we derive exact analytical expressions for the mean and variance of the nascent RNA distribution as functions of time. We apply these analytical results to obtain the mean and variance of nascent RNA distribution for specific models of transcription. These models of initiation exhibit qualitatively distinct transient behaviors for both the mean and variance which further allows us to discriminate between them. Stochastic simulations confirm these results. Overall the analytical results presented here provide the necessary tools to connect mechanisms of transcription initiation to single-cell measurements of nascent RNA.

Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots

PubMed Central

Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

2013-01-01

Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g−1 of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l−1 EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. PMID:23170978

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

PubMed

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J; Jopling, Catherine L

2015-04-01

MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

The forkhead-like transcription factor (Fhl1p) maintains yeast replicative lifespan by regulating ribonucleotide reductase 1 (RNR1) gene transcription

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

Tai, Akiko; Kamei, Yuka; Mukai, Yukio

In eukaryotes, numerous genetic factors contribute to the lifespan including metabolic enzymes, signal transducers, and transcription factors. As previously reported, the forkhead-like transcription factor (FHL1) gene was required for yeast replicative lifespan and cell proliferation. To determine how Fhl1p regulates the lifespan, we performed a DNA microarray analysis of a heterozygous diploid strain deleted for FHL1. We discovered numerous Fhl1p-target genes, which were then screened for lifespan-regulating activity. We identified the ribonucleotide reductase (RNR) 1 gene (RNR1) as a regulator of replicative lifespan. RNR1 encodes a large subunit of the RNR complex, which consists of two large (Rnr1p/Rnr3p) and twomore » small (Rnr2p/Rnr4p) subunits. Heterozygous deletion of FHL1 reduced transcription of RNR1 and RNR3, but not RNR2 and RNR4. Chromatin immunoprecipitation showed that Fhl1p binds to the promoter regions of RNR1 and RNR3. Cells harboring an RNR1 deletion or an rnr1-C428A mutation, which abolishes RNR catalytic activity, exhibited a short lifespan. In contrast, cells with a deletion of the other RNR genes had a normal lifespan. Overexpression of RNR1, but not RNR3, restored the lifespan of the heterozygous FHL1 mutant to the wild-type (WT) level. The Δfhl1/FHL1 mutant conferred a decrease in dNTP levels and an increase in hydroxyurea (HU) sensitivity. These findings reveal that Fhl1p regulates RNR1 gene transcription to maintain dNTP levels, thus modulating longevity by protection against replication stress. - Highlights: • Fhl1p regulates replicative lifespan and transcription of RNR large subunit genes. • Rnr1p uniquely acts as a lifespan regulator independent of the RNR complex. • dNTP levels modulate longevity by protection against replication stress.« less

WRKY transcription factors.

PubMed

Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

2010-05-01

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.

Trps1 activates a network of secreted Wnt inhibitors and transcription factors crucial to vibrissa follicle morphogenesis

PubMed Central

Fantauzzo, Katherine A.; Christiano, Angela M.

2012-01-01

Mutations in TRPS1 cause trichorhinophalangeal syndrome types I and III, which are characterized by sparse scalp hair in addition to craniofacial and skeletal abnormalities. Trps1 is a vertebrate transcription factor that contains nine zinc-finger domains, including a GATA-type zinc finger through which it binds DNA. Mice in which the GATA domain of Trps1 has been deleted (Trps1Δgt/Δgt) have a reduced number of pelage follicles and lack vibrissae follicles postnatally. To identify the transcriptional targets of Trps1 in the developing vibrissa follicle, we performed microarray hybridization analysis, comparing expression patterns in the whisker pads of wild-type versus Trps1Δgt/Δgt embryos. We identified a number of transcription factors and Wnt inhibitors among transcripts downregulated in the mutant embryos and several extracellular matrix proteins that were upregulated in the mutant samples, and demonstrated that target gene expression levels were altered in vivo in Trps1Δgt/Δgt vibrissae. Unexpectedly, we discovered that Trps1 can directly bind the promoters of its target genes to activate transcription, expanding upon its established role as a transcriptional repressor. Our findings identify Trps1 as a novel regulator of the Wnt signaling pathway and of early hair follicle progenitors in the developing vibrissa follicle. PMID:22115758

Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons.

PubMed

Guzowski, John F; Miyashita, Teiko; Chawla, Monica K; Sanderson, Jennifer; Maes, Levi I; Houston, Frank P; Lipa, Peter; McNaughton, Bruce L; Worley, Paul F; Barnes, Carol A

2006-01-24

The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate-early gene Arc. Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at approximately 40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

PubMed

Wen, Zewen; Liu, Zhiyong; Hou, Yuyong; Liu, Chenfeng; Gao, Feng; Zheng, Yubin; Chen, Fangjian

2015-10-01

Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis. Copyright © 2015 Elsevier Inc. All rights reserved.

Non-Equilibrium Thermodynamics of Transcriptional Bursts

NASA Astrophysics Data System (ADS)

Hernández-Lemus, Enrique

Gene transcription or Gene Expression (GE) is the process which transforms the information encoded in DNA into a functional RNA message. It is known that GE can occur in bursts or pulses. Transcription is irregular, with strong periods of activity, interspersed by long periods of inactivity. If we consider the average behavior over millions of cells, this process appears to be continuous. But at the individual cell level, there is considerable variability, and for most genes, very little activity at any one time. Some have claimed that GE bursting can account for the high variability in gene expression occurring between cells in isogenic populations. This variability has a big impact on cell behavior and thus on phenotypic conditions and disease. In view of these facts, the development of a thermodynamic framework to study gene expression and transcriptional regulation to integrate the vast amount of molecular biophysical GE data is appealing. Application of such thermodynamic formalism is useful to observe various dissipative phenomena in GE regulatory dynamics. In this chapter we will examine at some detail the complex phenomena of transcriptional bursts (specially of a certain class of anomalous bursts) in the context of a non-equilibrium thermodynamics formalism and will make some initial comments on the relevance of some irreversible processes that may be connected to anomalous transcriptional bursts.

A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition.

PubMed

Hsiung, Chris C-S; Bartman, Caroline R; Huang, Peng; Ginart, Paul; Stonestrom, Aaron J; Keller, Cheryl A; Face, Carolyne; Jahn, Kristen S; Evans, Perry; Sankaranarayanan, Laavanya; Giardine, Belinda; Hardison, Ross C; Raj, Arjun; Blobel, Gerd A

2016-06-15

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states. © 2016 Hsiung et al.; Published by Cold Spring Harbor Laboratory Press.

Antisense transcription is pervasive but rarely conserved in enteric bacteria.

PubMed

Raghavan, Rahul; Sloan, Daniel B; Ochman, Howard

2012-01-01

Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing

Analysis of gene expression and Ig transcription in PU.1/Spi-B-deficient progenitor B cell lines.

PubMed

Schweitzer, Brock L; DeKoter, Rodney P

2004-01-01

A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1(-/-)Spi-B(-/-) IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1(-/-)Spi-B(-/-) pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Igkappa transcription are induced after IL-7 withdrawal of wild-type or PU.1(-/-)Spi-B(-/-) pro-B cells. In contrast, we found that Iglambda transcription is reduced in PU.1(-/-)Spi-B(-/-) pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Iglambda, but not IgH or Igkappa, transcription, is dependent on PU.1 and/or Spi-B. The PU.1(-/-)Spi-B(-/-) pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.

Piano Transcription with Convolutional Sparse Lateral Inhibition

DOE PAGES

Cogliati, Andrea; Duan, Zhiyao; Wohlberg, Brendt Egon

2017-02-08

This paper extends our prior work on contextdependent piano transcription to estimate the length of the notes in addition to their pitch and onset. This approach employs convolutional sparse coding along with lateral inhibition constraints to approximate a musical signal as the sum of piano note waveforms (dictionary elements) convolved with their temporal activations. The waveforms are pre-recorded for the specific piano to be transcribed in the specific environment. A dictionary containing multiple waveforms per pitch is generated by truncating a long waveform for each pitch to different lengths. During transcription, the dictionary elements are fixed and their temporal activationsmore » are estimated and post-processed to obtain the pitch, onset and note length estimation. A sparsity penalty promotes globally sparse activations of the dictionary elements, and a lateral inhibition term penalizes concurrent activations of different waveforms corresponding to the same pitch within a temporal neighborhood, to achieve note length estimation. Experiments on the MAPS dataset show that the proposed approach significantly outperforms a state-of-the-art music transcription method trained in the same context-dependent setting in transcription accuracy.« less

Piano Transcription with Convolutional Sparse Lateral Inhibition

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

Cogliati, Andrea; Duan, Zhiyao; Wohlberg, Brendt Egon

This paper extends our prior work on contextdependent piano transcription to estimate the length of the notes in addition to their pitch and onset. This approach employs convolutional sparse coding along with lateral inhibition constraints to approximate a musical signal as the sum of piano note waveforms (dictionary elements) convolved with their temporal activations. The waveforms are pre-recorded for the specific piano to be transcribed in the specific environment. A dictionary containing multiple waveforms per pitch is generated by truncating a long waveform for each pitch to different lengths. During transcription, the dictionary elements are fixed and their temporal activationsmore » are estimated and post-processed to obtain the pitch, onset and note length estimation. A sparsity penalty promotes globally sparse activations of the dictionary elements, and a lateral inhibition term penalizes concurrent activations of different waveforms corresponding to the same pitch within a temporal neighborhood, to achieve note length estimation. Experiments on the MAPS dataset show that the proposed approach significantly outperforms a state-of-the-art music transcription method trained in the same context-dependent setting in transcription accuracy.« less

Genetic regulation of adipose tissue transcript expression is involved in modulating serum triglyceride and HDL-cholesterol.

PubMed

Sajuthi, Satria P; Sharma, Neeraj K; Comeau, Mary E; Chou, Jeff W; Bowden, Donald W; Freedman, Barry I; Langefeld, Carl D; Parks, John S; Das, Swapan K

2017-10-20

Dyslipidemia is a major contributor to the increased cardiovascular disease and mortality associated with obesity and type 2 diabetes. We hypothesized that variation in expression of adipose tissue transcripts is associated with serum lipid concentrations in African Americans (AAs), and common genetic variants regulate expression levels of these transcripts. Fasting serum lipid levels, genome-wide transcript expression profiles of subcutaneous adipose tissue, and genome-wide SNP genotypes were analyzed in a cohort of non-diabetic AAs (N=250). Serum triglyceride (TRIG) and high density lipoprotein-cholesterol (HDL-C) levels were associated (FDR<0.01) with expression level of 1021 and 1875 adipose tissue transcripts, respectively, but none associated with total cholesterol or LDL-C levels. Serum HDL-C-associated transcripts were enriched for salient biological pathways, including branched-chain amino acid degradation, and oxidative phosphorylation. Genes in immuno-inflammatory pathways were activated among individuals with higher serum TRIG levels. We identified significant cis-regulatory SNPs (cis-eSNPs) for 449 serum lipid-associated transcripts in adipose tissue. The cis-eSNPs of 12 genes were nominally associated (p<0.001) with serum lipid level in genome wide association studies in Global Lipids Genetics Consortium (GLGC) cohorts. Allelic effect direction of cis-eSNPs on expression of MARCH2, BEST1 and TMEM258 matched with effect direction of these SNP alleles on serum TRIG or HDL-C levels in GLGC cohorts. These data suggest that expressions of serum lipid-associated transcripts in adipose tissue are dependent on common cis-eSNPs in African Americans. Thus, genetically-mediated transcriptional regulation in adipose tissue may play a role in reducing HDL-C and increasing TRIG in serum. Copyright © 2017 Elsevier B.V. All rights reserved.

Combinatorial Effects of the Glucocorticoid Receptor and Krüppel-Like Transcription Factor 15 on Bovine Herpesvirus 1 Transcription and Productive Infection.

PubMed

El-Mayet, Fouad S; Sawant, Laximan; Thunuguntla, Prasanth; Jones, Clinton

2017-11-01

Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes

Combinatorial Effects of the Glucocorticoid Receptor and Krüppel-Like Transcription Factor 15 on Bovine Herpesvirus 1 Transcription and Productive Infection

PubMed Central

El-mayet, Fouad S.; Sawant, Laximan; Thunuguntla, Prasanth

2017-01-01

ABSTRACT Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bovine ICP0 (bICP0) and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency, and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same trigeminal ganglion (TG) neuron during reactivation and cooperatively stimulated productive infection and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are transactivated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL52; a component of the DNA primase/helicase complex), bICP4, IEtu2, and the unique short region were stimulated by KLF15 and the GR. Chromatin immunoprecipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and the UL52 fragment. Coimmunoprecipitation studies demonstrated that KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest that these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that

Snail1 transcription factor controls telomere transcription and integrity

PubMed Central

Mazzolini, Rocco; Gonzàlez, Núria; Garcia-Garijo, Andrea; Millanes-Romero, Alba; Peiró, Sandra; Smith, Susan

2018-01-01

Abstract Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes. PMID:29059385

Def1 interacts with TFIIH and modulates RNA polymerase II transcription.

PubMed

Damodaren, Nivedita; Van Eeuwen, Trevor; Zamel, Joanna; Lin-Shiao, Enrique; Kalisman, Nir; Murakami, Kenji

2017-12-12

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

Factor requirements for transcription in the Archaeon Sulfolobus shibatae.

PubMed

Qureshi, S A; Bell, S D; Jackson, S P

1997-05-15

Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes). Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems. Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is homologous to eukaryotic TFIIB. Here, we investigate the factor requirements for transcription of several promoters of the archaeon Sulfolobus shibatae and its associated virus SSV. Through in vitro transcription and immunodepletion, we demonstrate that S. shibatae TBP, TFB and RNA polymerase are not complexed tightly with one another and that each is required for efficient transcription of all promoters tested. Furthermore, full transcription is restored by supplementing respective depleted extracts with recombinant TBP or TFB, indicating that TBP-associated factors or TFB-associated factors are not required. Indeed, gel-filtration suggests that Sulfolobus TBP and TFB are not associated stably with other proteins. Finally, all promoters analysed are transcribed accurately and efficiently in an in vitro system comprising recombinant TBP and TFB, together with essentially homogeneous preparation of RNA polymerase. Transcription in Archaea is therefore fundamentally homologous to that in eukaryotes, although factor requirements appear to be much less complex.

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

PubMed

Luque-Almagro, Victor M; Manso, Isabel; Sullivan, Matthew J; Rowley, Gary; Ferguson, Stuart J; Moreno-Vivián, Conrado; Richardson, David J; Gates, Andrew J; Roldán, M Dolores

2017-05-10

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans , by which nitrate induction operates at both transcriptional and translational levels, is proposed. © 2017 The Author(s).

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans

PubMed Central

Luque-Almagro, Victor M.; Manso, Isabel; Sullivan, Matthew J.; Rowley, Gary; Ferguson, Stuart J.; Moreno-Vivián, Conrado; Richardson, David J.; Gates, Andrew J.

2017-01-01

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH–nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed. PMID:28385879

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca).

PubMed

Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra T; McBain, James; Dold, Christopher; Stott, Jeffrey L

2016-07-01

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify "insults" and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca)

USGS Publications Warehouse

Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra; McBain, James; Dold, Christopher; Stott, Jeffrey L.

2016-01-01

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify “insults” and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The

Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation.

PubMed

Matheis, S; Yemelin, A; Scheps, D; Andresen, K; Jacob, S; Thines, E; Foster, A J

2017-03-01

The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Sex differences in depressive, anxious behaviors and hippocampal transcript levels in a genetic rat model.

PubMed

Mehta, N S; Wang, L; Redei, E E

2013-10-01

Major depressive disorder (MDD) is a common, debilitating illness with high prevalence of comorbid anxiety. The incidence of depression and of comorbid anxiety is much higher in women than in men. These gender biases appear after puberty and their etiology is mostly unknown. Selective breeding of the Wistar Kyoto (WKY) rat strain, an accepted model of adult and adolescent depression, resulted in two fully inbred substrains. Adult WKY more immobile (WMI) rats of both sexes consistently show increased depression-like behavior in the forced swim test when compared with the control WKY less immobile (WLI) strain. In contrast, here we show that while adult female WMIs and WLIs both display high anxiety-like behaviors, only WLI males, but not WMI males, show this behavior. Moreover, the behavioral profile of WMI males is consistent from early adolescence to adulthood, but the high depression- and anxiety-like behaviors of the female WMIs appear only in adulthood. These sex-specific behavioral patterns are paralleled by marked sex differences in hippocampal gene expression differences established by genome-wide transcriptional analyses of 13th generation WMIs and WLIs. Moreover, sex- and age-specific differences in transcript levels of selected genes are present in the hippocampus of the current, fully inbred WMIs and WLIs. Thus, the contribution of specific genes and/or the influence of the gonadal hormonal environment to depression- and anxiety-like behaviors may differ between male and female WMIs, resulting in their distinct behavioral and transcriptomic profiles despite shared sequences of the somatic chromosomes. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis

PubMed Central

Zhang, Zhiyong; Zheng, Xixi; Yang, Jun; Messing, Joachim; Wu, Yongrui

2016-01-01

The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors. PMID:27621432

Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine

PubMed Central

Pellegrini, Kathryn L.; Patil, Dattatraya; Douglas, Kristen J.S.; Lee, Grace; Wehrmeyer, Kathryn; Torlak, Mersiha; Clark, Jeremy; Cooper, Colin S.; Moreno, Carlos S.; Sanda, Martin G.

2018-01-01

Background The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient’s risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs. Methods Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing. Results RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG. Conclusions Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection. PMID:28419548

Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine.

PubMed

Pellegrini, Kathryn L; Patil, Dattatraya; Douglas, Kristen J S; Lee, Grace; Wehrmeyer, Kathryn; Torlak, Mersiha; Clark, Jeremy; Cooper, Colin S; Moreno, Carlos S; Sanda, Martin G

2017-06-01

The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs. Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing. RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG. Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection. © 2017 Wiley Periodicals, Inc.

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

PubMed

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptional dynamics with time-dependent reaction rates