Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

PubMed

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-07-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster

PubMed Central

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-01-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390

The gap gene giant of Rhodnius prolixus is maternally expressed and required for proper head and abdomen formation.

PubMed

Lavore, Andrés; Pagola, Lucía; Esponda-Behrens, Natalia; Rivera-Pomar, Rolando

2012-01-01

The segmentation process in insects depends on a hierarchical cascade of gene activity. The first effectors downstream of the maternal activation are the gap genes, which divide the embryo in broad fields. We discovered a sequence corresponding to the leucine-zipper domain of the orthologue of the gene giant (Rp-gt) in traces from the genome of Rhodnius prolixus, a hemipteran with intermediate germ-band development. We cloned the Rp-gt gene from a normalized cDNA library and characterized its expression and function. Bioinformatic analysis of 12.5 kbp of genomic sequence containing the Rp-gt transcriptional unit shows a cluster of bona fide regulatory binding sites, which is similar in location and structure to the predicted posterior expression domain of the Drosophila orthologue. Rp-gt is expressed in ovaries and maternally supplied in the early embryo. The maternal contribution forms a gradient of scattered patches of mRNA in the preblastoderm embryo. Zygotic Rp-gt is expressed in two domains that after germ band extension are restricted to the head and the posterior growth zone. Parental RNAi shows that Rp-gt is required for proper head and abdomen formation. The head lacks mandibulary and maxillary appendages and shows reduced clypeus-labrum, while the abdomen lacks anterior segments. We conclude that Rp-gt is a gap gene on the head and abdomen and, in addition, has a function in patterning the anterior head capsule suggesting that the function of gt in hemipterans is more similar to dipterans than expected. Copyright © 2011. Published by Elsevier Inc.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

PubMed

Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

2014-02-10

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. Copyright © 2013 Elsevier B.V. All rights reserved.

Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

PubMed

Jiang, Wenzhi; Brueggeman, Andrew J; Horken, Kempton M; Plucinak, Thomas M; Weeks, Donald P

2014-11-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

USDA-ARS?s Scientific Manuscript database

Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

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.

A framework for analyzing the relationship between gene expression and morphological, topological, and dynamical patterns in neuronal networks.

PubMed

de Arruda, Henrique Ferraz; Comin, Cesar Henrique; Miazaki, Mauro; Viana, Matheus Palhares; Costa, Luciano da Fontoura

2015-04-30

A key point in developmental biology is to understand how gene expression influences the morphological and dynamical patterns that are observed in living beings. In this work we propose a methodology capable of addressing this problem that is based on estimating the mutual information and Pearson correlation between the intensity of gene expression and measurements of several morphological properties of the cells. A similar approach is applied in order to identify effects of gene expression over the system dynamics. Neuronal networks were artificially grown over a lattice by considering a reference model used to generate artificial neurons. The input parameters of the artificial neurons were determined according to two distinct patterns of gene expression and the dynamical response was assessed by considering the integrate-and-fire model. As far as single gene dependence is concerned, we found that the interaction between the gene expression and the network topology, as well as between the former and the dynamics response, is strongly affected by the gene expression pattern. In addition, we observed a high correlation between the gene expression and some topological measurements of the neuronal network for particular patterns of gene expression. To our best understanding, there are no similar analyses to compare with. A proper understanding of gene expression influence requires jointly studying the morphology, topology, and dynamics of neurons. The proposed framework represents a first step towards predicting gene expression patterns from morphology and connectivity. Copyright © 2015. Published by Elsevier B.V.

HoxB2, HoxB4 and Alx4 genes are downregulated in the cadmium-induced omphalocele in the chick model.

PubMed

Doi, Takashi; Puri, Prem; Bannigan, John; Thompson, Jennifer

2010-10-01

In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. HoxB2 and HoxB4, expressed in cell types that contribute to ventral body wall (VBW) formation, act together to mediate proper closure of the VBW, involving a key downstream transcription factor, Alx4. HoxB2 and HoxB4 knockout mice display VBW defects with specific downregulation of Alx4 gene expression, while homozygous Alx4 knockouts show omphalocele phenotype. Although the earliest histological changes in the Cd chick model occur commencing at 4H post treatment, the exact timing and molecular mechanism by which Cd acts is still unclear. We hypothesized that HoxB2, HoxB4 and Alx4 genes are downregulated during the critical timing of very early embryogenesis in the Cd-induced omphalocele chick model. After 60H incubation, chick embryos were harvested at 1H, 4H and 8H after treatment with saline or Cd and divided into controls and Cd group (n = 24 for each group). RT-PCR was performed to investigate the gene expression of HoxB2, HoxB4 and Alx4 and statistically analyzed (significance was accepted at p < 0.05). Immunohistochemical staining was also performed to evaluate the protein expression/distribution of HoxB2, HoxB4 and Alx4 in the chick embryo. The expression levels of HoxB2, HoxB4 and Alx4 gene at 4H were significantly downregulated in the Cd group as compared to controls, whereas there were no significant differences at the other time points. Immunoreactivity of HoxB2, HoxB4 and Alx4 at 4H is also markedly decreased in the ectoderm and the dermomyotome in the Cd chick model as compared to controls. Downregulation of HoxB2, HoxB4 and Alx4 expression during the narrow window of early embryogenesis may cause omphalocele in the Cd chick model by interfering with molecular signaling required for proper VBW formation. Furthermore, these results support the concept that HoxB2, HoxB4 and Alx4 genes work together to mediate proper VBW formation.

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation

PubMed Central

Kurat, Christoph F.; Lambert, Jean-Philippe; Petschnigg, Julia; Friesen, Helena; Pawson, Tony; Rosebrock, Adam; Gingras, Anne-Claude; Fillingham, Jeffrey; Andrews, Brenda

2014-01-01

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase–specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APCCdh1) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation. PMID:25228766

Transcription Factor FoxO1 Is Essential for Enamel Biomineralization

PubMed Central

Poché, Ross A.; Sharma, Ramaswamy; Garcia, Monica D.; Wada, Aya M.; Nolte, Mark J.; Udan, Ryan S.; Paik, Ji-Hye; DePinho, Ronald A.; Bartlett, John D.; Dickinson, Mary E.

2012-01-01

The Transforming growth factor β (Tgf-β) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-β signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta. PMID:22291941

The X chromosome in space.

PubMed

Jégu, Teddy; Aeby, Eric; Lee, Jeannie T

2017-06-01

Extensive 3D folding is required to package a genome into the tiny nuclear space, and this packaging must be compatible with proper gene expression. Thus, in the well-hierarchized nucleus, chromosomes occupy discrete territories and adopt specific 3D organizational structures that facilitate interactions between regulatory elements for gene expression. The mammalian X chromosome exemplifies this structure-function relationship. Recent studies have shown that, upon X-chromosome inactivation, active and inactive X chromosomes localize to different subnuclear positions and adopt distinct chromosomal architectures that reflect their activity states. Here, we review the roles of long non-coding RNAs, chromosomal organizational structures and the subnuclear localization of chromosomes as they relate to X-linked gene expression.

Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

PubMed Central

Gordon, Kacy L.; Arthur, Robert K.; Ruvinsky, Ilya

2015-01-01

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements. PMID:26020930

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation.

PubMed

Novakovic, Boris; Fournier, Thierry; Harris, Lynda K; James, Joanna; Roberts, Claire T; Yong, Hannah E J; Kalionis, Bill; Evain-Brion, Danièle; Ebeling, Peter R; Wallace, Euan M; Saffery, Richard; Murthi, Padma

2017-07-03

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris

PubMed Central

2013-01-01

Background Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. Findings Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. Conclusion Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation. PMID:24083672

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

Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B 6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

Identification and characterization of MicroRNAs expressed in chicken skeletal muscle

USDA-ARS?s Scientific Manuscript database

MicroRNAs (miRNAs, miRs) encompass a class of small noncoding RNAs that negatively regulate gene expression. MicroRNAs play an essential role in skeletal muscle, determining the proper development and maintenance of this tissue. In comparison to other organs and tissues, the full set of muscle miRNA...

Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle.

PubMed

Zhu, Wuzheng; Lin, Yaqiu; Liao, Honghai; Wang, Yong

2015-01-01

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

Differential expression of genes during mastitis in Holstein-Zebu crossbreed dairy cows.

PubMed

Fonseca, I; Antunes, G R; Paiva, D S; Lange, C C; Guimarães, S E F; Martins, M F

2011-07-05

Among the potential public health problems of animal production, infectious-contagious diseases stand out. Mastitis is among the main diseases affecting dairy cattle. One of the most promising options to reduce the problems caused by this disease, besides proper sanitary and management practices, is selective breeding of resistant animals. To shed light on the immune response mechanisms involved in the resistance/susceptibility phenotype to this disease, we quantified the relative expression of the genes IL-2, IL-6, IL-8, IL-12, IFN-γ, TNF-α, TLR-2, SEMA5A, and FEZL in cells of crossbreed dairy cows, divided into two groups, one healthy and the other suffering from clinical mastitis. Total RNA was extracted from the cells in the milk from the animals in each group (with and without clinical mastitis). Gene expression was determined using the real-time PCR method. The levels of gene expression were compared, and the cows with mastitis were found to express 2.5 times more TLR-2 than those free of mastitis (P < 0.05). There were no significant differences in the expression of the other genes.

Discovery, characterization and expression of a novel zebrafish gene, znfr, important for notochord formation.

PubMed

Xu, Yan; Zou, Peng; Liu, Yao; Deng, Fengjiao

2010-06-01

Genes specifically expressed in the notochord may be crucial for proper notochord development. Using the digital differential display program offered by the National Center for Biotechnology Information, we identified a novel EST sequence from a zebrafish ovary library (No. XM_701450). The full-length cDNA of this transcript was cloned by performing 3' and 5'-RACE and was further confirmed by PCR and sequencing. The resulting 614 bp gene was found to encode a novel 94 amino acid protein that did not share significant homology with any other known protein. Characterization of the genomic sequence revealed that the gene spanned 4.9 kb and was composed of four exons and three introns. RT-PCR gene expression analysis revealed that our gene of interest was expressed in ovary, kidney, brain, mature oocytes and during the early stages of embryogenesis. During embryonic development, znfr mRNA was found to be expressed in the embryonic shield, chordamesoderm and the vacuolated notochord cells by in situ hybridization. Based on this information, we hypothesize that this novel gene is an important maternal factor required for zebrafish notochord formation during early embryonic development. We have thus named this gene znfr (zebrafish notochord formation related).

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta).

PubMed

Zhang, YiChen; Jiang, Peng; Gao, JiangTao; Liao, JianMin; Sun, ShiJing; Shen, ZiLong; Qin, Song

2008-12-01

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes.

PubMed

Fuentes, Eduardo N; Safian, Diego; Valdés, Juan Antonio; Molina, Alfredo

2013-08-01

In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

PubMed

Flores-Herrera, Patricio; Arredondo-Zelada, Oscar; Marshall, Sergio H; Gómez, Fernando A

2018-06-01

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis. Copyright © 2018. Published by Elsevier B.V.

Complex genomic rearrangement in CCS-LacZ transgenic mice.

PubMed

Stroud, Dina Myers; Darrow, Bruce J; Kim, Sang Do; Zhang, Jie; Jongbloed, Monique R M; Rentschler, Stacey; Moskowitz, Ivan P G; Seidman, Jonathan; Fishman, Glenn I

2007-02-01

The cardiac conduction system (CCS)-lacZ insertional mouse mutant strain genetically labels the developing and mature CCS. This pattern of expression is presumed to reflect the site of transgene integration rather than regulatory elements within the transgene proper. We sought to characterize the genomic structure of the integration locus and identify nearby gene(s) that might potentially confer the observed CCS-specific transcription. We found rearrangement of chromosome 7 between regions D1 and E1 with altered transcription of multiple genes in the D1 region. Several lines of evidence suggested that regulatory elements from at least one gene, Slco3A1, influenced CCS-restricted reporter gene expression. In embryonic hearts, Slco3A1 was expressed in a spatial pattern similar to the CCS-lacZ transgene and was similarly neuregulin-responsive. At later stages, however, expression patterns of the transgene and Slco3A1 diverged, suggesting that the Slco3A1 locus may be necessary, but not sufficient to confer CCS-specific transgene expression in the CCS-lacZ line. (c) 2007 Wiley-Liss, Inc.

Proper development of relay somatic sensory neurons and D2/D4 interneurons requires homeobox genes Rnx/Tlx-3 and Tlx-1

PubMed Central

Qian, Ying; Shirasawa, Senji; Chen, Chih-Li; Cheng, Leping; Ma, Qiufu

2002-01-01

Trigeminal nuclei and the dorsal spinal cord are first-order relay stations for processing somatic sensory information such as touch, pain, and temperature. The origins and development of these neurons are poorly understood. Here we show that relay somatic sensory neurons and D2/D4 dorsal interneurons likely derive from Mash1-positive neural precursors, and depend on two related homeobox genes, Rnx and Tlx-1, for proper formation. Rnx and Tlx-1 maintain expression of Drg11, a homeobox gene critical for the development of pain circuitry, and are essential for the ingrowth of trkA+ nociceptive/thermoceptive sensory afferents to their central targets. We showed previously that Rnx is necessary for proper formation of the nucleus of solitary tract, the target for visceral sensory afferents. Together, our studies demonstrate a central role for Rnx and Tlx-1 in the development of two major classes of relay sensory neurons, somatic and visceral. PMID:12023301

Proper development of relay somatic sensory neurons and D2/D4 interneurons requires homeobox genes Rnx/Tlx-3 and Tlx-1.

PubMed

Qian, Ying; Shirasawa, Senji; Chen, Chih-Li; Cheng, Leping; Ma, Qiufu

2002-05-15

Trigeminal nuclei and the dorsal spinal cord are first-order relay stations for processing somatic sensory information such as touch, pain, and temperature. The origins and development of these neurons are poorly understood. Here we show that relay somatic sensory neurons and D2/D4 dorsal interneurons likely derive from Mash1-positive neural precursors, and depend on two related homeobox genes, Rnx and Tlx-1, for proper formation. Rnx and Tlx-1 maintain expression of Drg11, a homeobox gene critical for the development of pain circuitry, and are essential for the ingrowth of trkA+ nociceptive/thermoceptive sensory afferents to their central targets. We showed previously that Rnx is necessary for proper formation of the nucleus of solitary tract, the target for visceral sensory afferents. Together, our studies demonstrate a central role for Rnx and Tlx-1 in the development of two major classes of relay sensory neurons, somatic and visceral.

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

PubMed

Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

2016-01-01

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

DOE PAGES

Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah; ...

2016-01-28

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B 6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire

PubMed Central

Najafova, Zeynab; Tirado-Magallanes, Roberto; Subramaniam, Malayannan; Hossan, Tareq; Schmidt, Geske; Nagarajan, Sankari; Baumgart, Simon J.; Mishra, Vivek Kumar; Bedi, Upasana; Hesse, Eric; Knapp, Stefan; Hawse, John R.; Johnsen, Steven A.

2017-01-01

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4. PMID:27651452

BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire.

PubMed

Najafova, Zeynab; Tirado-Magallanes, Roberto; Subramaniam, Malayannan; Hossan, Tareq; Schmidt, Geske; Nagarajan, Sankari; Baumgart, Simon J; Mishra, Vivek Kumar; Bedi, Upasana; Hesse, Eric; Knapp, Stefan; Hawse, John R; Johnsen, Steven A

2017-01-09

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.

PubMed

Liu, Mingying; Jiang, Jing; Han, Xiaojiao; Qiao, Guirong; Zhuo, Renying

2014-01-01

Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro. In this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants. geNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.

Tensor decomposition-based and principal-component-analysis-based unsupervised feature extraction applied to the gene expression and methylation profiles in the brains of social insects with multiple castes.

PubMed

Taguchi, Y-H

2018-05-08

Even though coexistence of multiple phenotypes sharing the same genomic background is interesting, it remains incompletely understood. Epigenomic profiles may represent key factors, with unknown contributions to the development of multiple phenotypes, and social-insect castes are a good model for elucidation of the underlying mechanisms. Nonetheless, previous studies have failed to identify genes associated with aberrant gene expression and methylation profiles because of the lack of suitable methodology that can address this problem properly. A recently proposed principal component analysis (PCA)-based and tensor decomposition (TD)-based unsupervised feature extraction (FE) can solve this problem because these two approaches can deal with gene expression and methylation profiles even when a small number of samples is available. PCA-based and TD-based unsupervised FE methods were applied to the analysis of gene expression and methylation profiles in the brains of two social insects, Polistes canadensis and Dinoponera quadriceps. Genes associated with differential expression and methylation between castes were identified, and analysis of enrichment of Gene Ontology terms confirmed reliability of the obtained sets of genes from the biological standpoint. Biologically relevant genes, shown to be associated with significant differential gene expression and methylation between castes, were identified here for the first time. The identification of these genes may help understand the mechanisms underlying epigenetic control of development of multiple phenotypes under the same genomic conditions.

Knockdown of epigenetic transcriptional co-regulator Brd2a disrupts apoptosis and proper formation of hindbrain and midbrain-hindbrain boundary (MHB) region in zebrafish.

PubMed

Murphy, Tami; Melville, Heather; Fradkin, Eliza; Bistany, Giana; Branigan, Gregory; Olsen, Kelly; Comstock, Catharine R; Hanby, Hayley; Garbade, Ellie; DiBenedetto, Angela J

2017-08-01

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

PubMed

Laporta, J; Driver, A; Khatib, H

2011-08-01

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Trefoil factor 2 (TFF2) deficiency in murine digestive tract influences the immune system.

PubMed

Baus-Loncar, Mirela; Schmid, Janinne; Lalani, El-Nasir; Rosewell, Ian; Goodlad, Robert A; Stamp, Gordon W H; Blin, Nikolaus; Kayademir, Tuncay

2005-01-01

The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach. Copyright (c) 2005 S. Karger AG, Basel.

Parabolic flight induces changes in gene expression patterns in Arabidopsis thaliana.

PubMed

Paul, Anna-Lisa; Manak, Michael S; Mayfield, John D; Reyes, Matthew F; Gurley, William B; Ferl, Robert J

2011-10-01

Our primary objective was to evaluate gene expression changes in Arabidopsis thaliana in response to parabolic flight as part of a comprehensive approach to the molecular biology of spaceflight-related adaptations. In addition, we wished to establish parabolic flight as a tractable operations platform for molecular biology studies. In a succession of experiments on NASA's KC-135 and C-9 parabolic aircraft, Arabidopsis plants were presented with replicated exposure to parabolic flight. Transcriptome profiling revealed that parabolic flight caused changes in gene expression patterns that stood the statistical tests of replication on three different flight days. The earliest response, after 20 parabolas, was characterized by a prominence of genes associated with signal transduction. After 40 parabolas, this prominence was largely replaced by genes associated with biotic and abiotic stimuli and stress. Among these responses, three metabolic processes stand out in particular: the induction of auxin metabolism and signaling, the differential expression of genes associated with calcium-mediated signaling, and the repression of genes associated with disease resistance and cell wall biochemistry. Many, but not all, of these responses are known to be involved in gravity sensing in plants. Changes in auxin-related gene expression were also recorded by reporter genes tuned to auxin signal pathways. These data demonstrate that the parabolic flight environment is appropriate for molecular biology research involving the transition to microgravity, in that with replication, proper controls, and analyses, gene expression changes can be observed in the time frames of typical parabolic flight experiments.

The Schizophrenia Risk Gene MIR137 Acts as a Hippocampal Gene Network Node Orchestrating the Expression of Genes Relevant to Nervous System Development and Function

PubMed Central

Loohuis, Nikkie FM Olde; Kasri, Nael Nadif; Glennon, Jeffrey C; van Bokhoven, Hans; Hébert, Sébastien S; Kaplan, Barry B.; Martens, Gerard JM; Aschrafi, Armaz

2016-01-01

MicroRNAs (miRs) are small regulatory molecules, which orchestrate neuronal development and plasticity through modulation of complex gene networks. microRNA-137 (miR-137) is a brain-enriched RNA with a critical role in regulating brain development and in mediating synaptic plasticity. Importantly, mutations in this miR are associated with the pathoetiology of schizophrenia (SZ), and there is a widespread assumption that disruptions in miR-137 expression lead to aberrant expression of gene regulatory networks associated with SZ. To systematically identify the mRNA targets for this miR, we performed miR-137 gain- and loss-of-function experiments in primary rat hippocampal neurons and profiled differentially expressed mRNAs through next-generation sequencing. We identified 500 genes that were bidirectionally activated or repressed in their expression by the modulation of miR-137 levels. Gene ontology analysis using two independent software resources suggested functions for these miR-137-regulated genes in neurodevelopmental processes, neuronal maturation processes and cell maintenance, all of which known to be critical for proper brain circuitry formation. Since many of the putative miR-137 targets identified here also have been previously shown to be associated with SZ, we propose that this miR acts as a critical gene network hub contributing to the pathophysiology of this neurodevelopmental disorder. PMID:26925706

Exercise training attenuated chronic cigarette smoking-induced up-regulation of FIZZ1/RELMα in lung of rats.

PubMed

Ma, Wan-li; Cai, Peng-cheng; Xiong, Xian-zhi; Ye, Hong

2013-02-01

FIZZ/RELM is a new gene family named "found in inflammatory zone" (FIZZ) or "resistin-like molecule" (RELM). FIZZ1/RELMα is specifically expressed in lung tissue and associated with pulmonary inflammation. Chronic cigarette smoking up-regulates FIZZ1/RELMα expression in rat lung tissues, the mechanism of which is related to cigarette smoking-induced airway hyperresponsiveness. To investigate the effect of exercise training on chronic cigarette smoking-induced airway hyperresponsiveness and up-regulation of FIZZ1/RELMα, rat chronic cigarette smoking model was established. The rats were treated with regular exercise training and their airway responsiveness was measured. Hematoxylin and eosin (HE) staining, immunohistochemistry and in situ hybridization of lung tissues were performed to detect the expression of FIZZ1/RELMα. Results revealed that proper exercise training decreased airway hyperresponsiveness and pulmonary inflammation in rat chronic cigarette smoking model. Cigarette smoking increased the mRNA and protein levels of FIZZ1/RELMα, which were reversed by the proper exercise. It is concluded that proper exercise training prevents up-regulation of FIZZ1/RELMα induced by cigarette smoking, which may be involved in the mechanism of proper exercise training modulating airway hyperresponsiveness.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

PubMed

Li, Lishu; Ikezono, Tetsuo; Sekine, Kuwon; Shindo, Susumu; Matsumura, Tomohiro; Pawankar, Ruby; Ichimiya, Issei; Yagi, Toshiaki

2010-08-01

We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Cardiogenic Genes Expressed in Cardiac Fibroblasts Contribute to Heart Development and Repair

PubMed Central

Furtado, Milena B.; Costa, Mauro W.; Pranoto, Edward Adi; Salimova, Ekaterina; Pinto, Alex; Lam, Nicholas T.; Park, Anthony; Snider, Paige; Chandran, Anjana; Harvey, Richard P.; Boyd, Richard; Conway, Simon J.; Pearson, James; Kaye, David M.; Rosenthal, Nadia A.

2014-01-01

Rationale Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers. Objective To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration. Methods and Results High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical MSC and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. Whilst genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, Tbx20, caused marked myocardial dysmorphology and perturbations in scar formation upon myocardial infarction. Conclusions The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs and direct contribution to cardiac development and repair provokes alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies. PMID:24650916

Gene expression patterns in four brain areas associate with quantitative measure of estrous behavior in dairy cows.

PubMed

Kommadath, Arun; Woelders, Henri; Beerda, Bonne; Mulder, Herman A; de Wit, Agnes A C; Veerkamp, Roel F; te Pas, Marinus F W; Smits, Mari A

2011-04-19

The decline noticed in several fertility traits of dairy cattle over the past few decades is of major concern. Understanding of the genomic factors underlying fertility, which could have potential applications to improve fertility, is very limited. Here, we aimed to identify and study those genes that associated with a key fertility trait namely estrous behavior, among genes expressed in four bovine brain areas (hippocampus, amygdala, dorsal hypothalamus and ventral hypothalamus), either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle. An average heat score was calculated for each of 28 primiparous cows in which estrous behavior was recorded for at least two consecutive estrous cycles starting from 30 days post-partum. Gene expression was then measured in brain tissue samples collected from these cows, 14 of which were sacrificed at the start of estrus and 14 around mid cycle. For each brain area, gene expression was modeled as a function of the orthogonally transformed average heat score values using a Bayesian hierarchical mixed model. Genes whose expression patterns showed significant linear or quadratic relationships with heat scores were identified. These included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation (OXT, AVP, POMC, MCHR1), but also genes whose association with estrous behavior is novel and warrants further investigation. Several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. The genes OXT and AVP play major roles in regulating estrous behavior in dairy cows. Genes related to neurotransmission and neuronal plasticity are also involved in estrous regulation, with several genes and processes expressed in mid-cycle probably contributing to proper expression of estrous behavior in the next estrus. Studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression.

Metabolic Adaptation to Nutrients Involves Coregulation of Gene Expression by the RNA Helicase Dbp2 and the Cyc8 Corepressor in Saccharomyces cerevisiae.

PubMed

Wang, Siwen; Xing, Zheng; Pascuzzi, Pete E; Tran, Elizabeth J

2017-07-05

Cells fine-tune their metabolic programs according to nutrient availability in order to maintain homeostasis. This is achieved largely through integrating signaling pathways and the gene expression program, allowing cells to adapt to nutritional change. Dbp2, a member of the DEAD-box RNA helicase family in Saccharomyces cerevisiae , has been proposed to integrate gene expression with cellular metabolism. Prior work from our laboratory has reported the necessity of DBP2 in proper gene expression, particularly for genes involved in glucose-dependent regulation. Here, by comparing differentially expressed genes in dbp2 ∆ to those of 700 other deletion strains from other studies, we find that CYC8 and TUP1 , which form a complex and inhibit transcription of numerous genes, corepress a common set of genes with DBP2 Gene ontology (GO) annotations reveal that these corepressed genes are related to cellular metabolism, including respiration, gluconeogenesis, and alternative carbon-source utilization genes. Consistent with a direct role in metabolic gene regulation, loss of either DBP2 or CYC8 results in increased cellular respiration rates. Furthermore, we find that corepressed genes have a propensity to be associated with overlapping long noncoding RNAs and that upregulation of these genes in the absence of DBP2 correlates with decreased binding of Cyc8 to these gene promoters. Taken together, this suggests that Dbp2 integrates nutrient availability with energy homeostasis by maintaining repression of glucose-repressed, Cyc8-targeted genes across the genome. Copyright © 2017 Wang et al.

Efficiency of introns from various origins in fish cells.

PubMed

Bétancourt, O H; Attal, J; Théron, M C; Puissant, C; Houdebine, L M

1993-06-01

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

PubMed

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors. PMID:29091919

The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

PubMed Central

Deffit, Sarah N; Yee, Brian A; Manning, Aidan C; Rajendren, Suba; Vadlamani, Pranathi; Wheeler, Emily C; Domissy, Alain; Washburn, Michael C

2017-01-01

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression. PMID:28925356

Conditional clustering of temporal expression profiles

PubMed Central

Wang, Ling; Montano, Monty; Rarick, Matt; Sebastiani, Paola

2008-01-01

Background Many microarray experiments produce temporal profiles in different biological conditions but common cluster techniques are not able to analyze the data conditional on the biological conditions. Results This article presents a novel technique to cluster data from time course microarray experiments performed across several experimental conditions. Our algorithm uses polynomial models to describe the gene expression patterns over time, a full Bayesian approach with proper conjugate priors to make the algorithm invariant to linear transformations, and an iterative procedure to identify genes that have a common temporal expression profile across two or more experimental conditions, and genes that have a unique temporal profile in a specific condition. Conclusion We use simulated data to evaluate the effectiveness of this new algorithm in finding the correct number of clusters and in identifying genes with common and unique profiles. We also use the algorithm to characterize the response of human T cells to stimulations of antigen-receptor signaling gene expression temporal profiles measured in six different biological conditions and we identify common and unique genes. These studies suggest that the methodology proposed here is useful in identifying and distinguishing uniquely stimulated genes from commonly stimulated genes in response to variable stimuli. Software for using this clustering method is available from the project home page. PMID:18334028

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

PubMed

Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration

NASA Astrophysics Data System (ADS)

Gibbs, Holly C.; Dodson, Colin R.; Bai, Yuqiang; Lekven, Arne C.; Yeh, Alvin T.

2014-12-01

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration.

PubMed

Gibbs, Holly C; Dodson, Colin R; Bai, Yuqiang; Lekven, Arne C; Yeh, Alvin T

2014-12-01

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

A novel mutation in PGAP2 gene causes developmental delay, intellectual disability, epilepsy and microcephaly in consanguineous Saudi family.

PubMed

Naseer, Muhammad Imran; Rasool, Mahmood; Jan, Mohammed M; Chaudhary, Adeel G; Pushparaj, Peter Natesan; Abuzenadah, Adel M; Al-Qahtani, Mohammad H

2016-12-15

PGAP2 (Post-GPI Attachment to Proteins 2) gene is involved in lipid remodeling steps of Glycosylphosphatidylinositol (GPI)-anchor maturation. At the surface of the cell this gene is required for proper expression of GPI-anchored proteins. Hyperphosphatasia with mental retardation syndrome-3 is an autosomal recessive disorder usually characterized by severe mental retardation. Mutations in the PGAP2 gene cause hyperphosphatasia mental retardation syndrome-3. We have identified a large consanguineous family from Saudi origin segregating developmental delay, intellectual disability, epilepsy and microcephaly. Whole exome sequencing with 100× coverage was performed on two affected siblings of the family. Data analysis in the patient revealed a novel missense mutation c.191C>T in PGAP2 gene resulting in Alanine to Valine substitution (Ala64Val). The mutation was reconfirmed and validated by subsequent Sanger sequencing method. The mutation was ruled out in 100 unrelated healthy controls. We suggest that this pathogenic mutation disrupts the proper function of the gene proteins resulting in the disease state. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetics of Mitochondrial Disease.

PubMed

Saneto, Russell P

2017-01-01

Mitochondria are intracellular organelles responsible for adenosine triphosphate production. The strict control of intracellular energy needs require proper mitochondrial functioning. The mitochondria are under dual controls of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mitochondrial dysfunction can arise from changes in either mtDNA or nDNA genes regulating function. There are an estimated ∼1500 proteins in the mitoproteome, whereas the mtDNA genome has 37 proteins. There are, to date, ∼275 genes shown to give rise to disease. The unique physiology of mitochondrial functioning contributes to diverse gene expression. The onset and range of phenotypic expression of disease is diverse, with onset from neonatal to seventh decade of life. The range of dysfunction is heterogeneous, ranging from single organ to multisystem involvement. The complexity of disease expression has severely limited gene discovery. Combining phenotypes with improvements in gene sequencing strategies are improving the diagnosis process. This chapter focuses on the interplay of the unique physiology and gene discovery in the current knowledge of genetically derived mitochondrial disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL

PubMed Central

Aghajanirefah, Ali; McLaughlin, Jami; Cheng, Donghui; Geng, Huimin; Eggesbø, Linn M.; Smale, Stephen T.; Müschen, Markus

2017-01-01

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre–B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre–B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre–B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre–B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre–B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage–specific expression of multiple genes through chromatin compaction at its target genes. PMID:28190001

Expression of mouse Tla region class I genes in tissues enriched for gamma delta cells.

PubMed

Eghtesady, P; Brorson, K A; Cheroutre, H; Tigelaar, R E; Hood, L; Kronenberg, M

1992-01-01

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to gamma delta T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for gamma delta T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent and transcripts from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain gamma delta T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing gamma delta T cells further extends the list of potential candidates for antigen presentation to gamma delta T cells.

mir-125a-5p-mediated Regulation of Lfng is Essential for the Avian Segmentation Clock

PubMed Central

Riley, Maurisa F.; Bochter, Matthew S.; Wahi, Kanu; Nuovo, Gerard J.; Cole, Susan E.

2013-01-01

Summary Somites are embryonic precursors of the axial skeleton and skeletal muscles, and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated both at the transcriptional and post-transcriptional levels for proper clock function. Here we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3′UTR, and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that miRNAs function in the post-transcriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of miRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight post-transcriptional regulation. PMID:23484856

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

PubMed

Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics.

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

Bock KW; D Honys; JM. Ward

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. About 1269 genes encoding classified transporters were collected from themore » Arabidopsis thaliana genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3 and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9); while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, were developmentally-regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::GUS analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.« less

Integrating membrane transport with male gametophyte development and function through transcriptomics.

PubMed

Bock, Kevin W; Honys, David; Ward, John M; Padmanaban, Senthilkumar; Nawrocki, Eric P; Hirschi, Kendal D; Twell, David; Sze, Heven

2006-04-01

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::beta-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

High expression of ID family and IGJ genes signature as predictor of low induction treatment response and worst survival in adult Hispanic patients with B-acute lymphoblastic leukemia.

PubMed

Cruz-Rodriguez, Nataly; Combita, Alba L; Enciso, Leonardo J; Quijano, Sandra M; Pinzon, Paula L; Lozano, Olga C; Castillo, Juan S; Li, Li; Bareño, Jose; Cardozo, Claudia; Solano, Julio; Herrera, Maria V; Cudris, Jennifer; Zabaleta, Jovanny

2016-04-05

B-Acute lymphoblastic leukemia (B-ALL) represents a hematologic malignancy with poor clinical outcome and low survival rates in adult patients. Remission rates in Hispanic population are almost 30% lower and Overall Survival (OS) nearly two years inferior than those reported in other ethnic groups. Only 61% of Colombian adult patients with ALL achieve complete remission (CR), median overall survival is 11.3 months and event-free survival (EFS) is 7.34 months. Identification of prognostic factors is crucial for the application of proper treatment strategies and subsequently for successful outcome. Our goal was to identify a gene expression signature that might correlate with response to therapy and evaluate the utility of these as prognostic tool in hispanic patients. We included 43 adult patients newly diagnosed with B-ALL. We used microarray analysis in order to identify genes that distinguish poor from good response to treatment using differential gene expression analysis. The expression profile was validated by real-time PCR (RT-PCT). We identified 442 differentially expressed genes between responders and non-responders to induction treatment. Hierarchical analysis according to the expression of a 7-gene signature revealed 2 subsets of patients that differed in their clinical characteristics and outcome. Our study suggests that response to induction treatment and clinical outcome of Hispanic patients can be predicted from the onset of the disease and that gene expression profiles can be used to stratify patient risk adequately and accurately. The present study represents the first that shows the gene expression profiling of B-ALL Colombian adults and its relevance for stratification in the early course of disease.

Transcriptomic response of Drosophila melanogaster pupae developed in hypergravity.

PubMed

Hateley, Shannon; Hosamani, Ravikumar; Bhardwaj, Shilpa R; Pachter, Lior; Bhattacharya, Sharmila

2016-10-01

Altered gravity can perturb normal development and induce corresponding changes in gene expression. Understanding this relationship between the physical environment and a biological response is important for NASA's space travel goals. We use RNA-Seq and qRT-PCR techniques to profile changes in early Drosophila melanogaster pupae exposed to chronic hypergravity (3g, or three times Earth's gravity). During the pupal stage, D. melanogaster rely upon gravitational cues for proper development. Assessing gene expression changes in the pupae under altered gravity conditions helps highlight gravity-dependent genetic pathways. A robust transcriptional response was observed in hypergravity-treated pupae compared to controls, with 1513 genes showing a significant (q<0.05) difference in gene expression. Five major biological processes were affected: ion transport, redox homeostasis, immune response, proteolysis, and cuticle development. This outlines the underlying molecular and biological changes occurring in Drosophila pupae in response to hypergravity; gravity is important for many biological processes on Earth. Published by Elsevier Inc.

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

Leong, JoAnn Ching

A prototype subunit vaccine to IHN virus is being developed by recombinant DNA techniques. The techniques involve the isolation and characterization of the glycoprotein gene, which encodes the viral protein responsible for inducing a protective immune response in fish. The viral glycoprotein gene has been cloned and a restriction map of the cloned gene has been prepared. Preliminary DNA sequence analysis of the cloned gene has been initiated so that manipulation of the gene for maximum expression in appropriate plasmid vectors is possible. A recombinant plasmid containing the viral gene inserted in the proper orientation adjacent to a very strongmore » lambda promoter and ribosome binding site has been constructed. Evaluation of this recombinant plasmid for gene expression is being conducted. Immunization trials with purified viral glycoprotein indicate that fish are protected against lethal doses of IHNV after immersion and intraperitoneal methods of immunization. In addition, cross protection immunization trials indicate that Type 2 and Type 1 IHN virus produce glycoproteins that are cross-protective.« less

Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure.

PubMed

Brown, Jacob D; Dutta, Sunit; Bharti, Kapil; Bonner, Robert F; Munson, Peter J; Dawid, Igor B; Akhtar, Amana L; Onojafe, Ighovie F; Alur, Ramakrishna P; Gross, Jeffrey M; Hejtmancik, J Fielding; Jiao, Xiaodong; Chan, Wai-Yee; Brooks, Brian P

2009-02-03

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. Here, we profile global gene expression during optic fissure closure using laser capture microdissected (LCM) tissue from the margins of the fissure. From these data, we identify a unique role for the C(2)H(2) zinc finger proteins Nlz1 and Nlz2 in normal fissure closure. Gene knockdown of nlz1 and/or nlz2 in zebrafish leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. We also identify misregulation of pax2 in the developing eye of morphant fish, suggesting that Nlz1 and Nlz2 act upstream of the Pax2 pathway in directing proper closure of the optic fissure.

Manganese-Induced Neurotoxicity and Alterations in Gene Expression in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gandhi, Deepa; Sivanesan, Saravanadevi; Kannan, Krishnamurthi

2018-06-01

Manganese (Mn) is an essential trace element required for many physiological functions including proper biochemical and cellular functioning of the central nervous system (CNS). However, exposure to excess level of Mn through occupational settings or from environmental sources has been associated with neurotoxicity. The cellular and molecular mechanism of Mn-induced neurotoxicity remains unclear. In the current study, we investigated the effects of 30-day exposure to a sub-lethal concentration of Mn (100 μM) in human neuroblastoma cells (SH-SY5Y) using transcriptomic approach. Microarray analysis revealed differential expression of 1057 transcripts in Mn-exposed SH-SY5Y cells as compared to control cells. Gene functional annotation cluster analysis exhibited that the differentially expressed genes were associated with several biological pathways. Specifically, genes involved in neuronal pathways including neuron differentiation and development, regulation of neurogenesis, synaptic transmission, and neuronal cell death (apoptosis) were found to be significantly altered. KEGG pathway analysis showed upregulation of p53 signaling pathways and neuroactive ligand-receptor interaction pathways, and downregulation of neurotrophin signaling pathway. On the basis of the gene expression profile, possible molecular mechanisms underlying Mn-induced neuronal toxicity were predicted.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

PubMed Central

Demmerle, Justin; Koch, Adam J.; Holaska, James M.

2016-01-01

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes. PMID:24062260

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

Conservation of regulatory sequences and gene expression patterns in the disintegrating Drosophila Hox gene complex

PubMed Central

Negre, Bárbara; Casillas, Sònia; Suzanne, Magali; Sánchez-Herrero, Ernesto; Akam, Michael; Nefedov, Michael; Barbadilla, Antonio; de Jong, Pieter; Ruiz, Alfredo

2005-01-01

Homeotic (Hox) genes are usually clustered and arranged in the same order as they are expressed along the anteroposterior body axis of metazoans. The mechanistic explanation for this colinearity has been elusive, and it may well be that a single and universal cause does not exist. The Hox-gene complex (HOM-C) has been rearranged differently in several Drosophila species, producing a striking diversity of Hox gene organizations. We investigated the genomic and functional consequences of the two HOM-C splits present in Drosophila buzzatii. Firstly, we sequenced two regions of the D. buzzatii genome, one containing the genes labial and abdominal A, and another one including proboscipedia, and compared their organization with that of D. melanogaster and D. pseudoobscura in order to map precisely the two splits. Then, a plethora of conserved noncoding sequences, which are putative enhancers, were identified around the three Hox genes closer to the splits. The position and order of these enhancers are conserved, with minor exceptions, between the three Drosophila species. Finally, we analyzed the expression patterns of the same three genes in embryos and imaginal discs of four Drosophila species with different Hox-gene organizations. The results show that their expression patterns are conserved despite the HOM-C splits. We conclude that, in Drosophila, Hox-gene clustering is not an absolute requirement for proper function. Rather, the organization of Hox genes is modular, and their clustering seems the result of phylogenetic inertia more than functional necessity. PMID:15867430

Overlapping but distinct topology for zebrafish V2R-like olfactory receptors reminiscent of odorant receptor spatial expression zones.

PubMed

Ahuja, Gaurav; Reichel, Vera; Kowatschew, Daniel; Syed, Adnan S; Kotagiri, Aswani Kumar; Oka, Yuichiro; Weth, Franco; Korsching, Sigrun I

2018-05-23

The sense of smell is unrivaled in terms of molecular complexity of its input channels. Even zebrafish, a model vertebrate system in many research fields including olfaction, possesses several hundred different olfactory receptor genes, organized in four different gene families. For one of these families, the initially discovered odorant receptors proper, segregation of expression into distinct spatial subdomains within a common sensory surface has been observed both in teleost fish and in mammals. However, for the remaining three families, little to nothing was known about their spatial coding logic. Here we wished to investigate, whether the principle of spatial segregation observed for odorant receptors extends to another olfactory receptor family, the V2R-related OlfC genes. Furthermore we thought to examine, how expression of OlfC genes is integrated into expression zones of odorant receptor genes, which in fish share a single sensory surface with OlfC genes. To select representative genes, we performed a comprehensive phylogenetic study of the zebrafish OlfC family, which identified a novel OlfC gene, reduced the number of pseudogenes to 1, and brought the total family size to 60 intact OlfC receptors. We analyzed the spatial pattern of OlfC-expressing cells for seven representative receptors in three dimensions (height within the epithelial layer, horizontal distance from the center of the olfactory organ, and height within the olfactory organ). We report non-random distributions of labeled neurons for all OlfC genes analysed. Distributions for sparsely expressed OlfC genes are significantly different from each other in nearly all cases, broad overlap notwithstanding. For two of the three coordinates analyzed, OlfC expression zones are intercalated with those of odorant receptor zones, whereas in the third dimension some segregation is observed. Our results show that V2R-related OlfC genes follow the same spatial logic of expression as odorant receptors and their expression zones intermingle with those of odorant receptor genes. Thus, distinctly different expression zones for individual receptor genes constitute a general feature shared by teleost and tetrapod V2R/OlfC and odorant receptor families alike.

The super elongation complex (SEC) and MLL in development and disease

PubMed Central

Smith, Edwin; Lin, Chengqi; Shilatifard, Ali

2011-01-01

Transcriptional regulation at the level of elongation is vital for the control of gene expression and metazoan development. The mixed lineage leukemia (MLL) protein and its Drosophila homolog, Trithorax, which exist within COMPASS (complex of proteins associated with Set1)-like complexes, are master regulators of development. They are required for proper homeotic gene expression, in part through methylation of histone H3 on Lys 4. In humans, the MLL gene is involved in a large number of chromosomal translocations that create chimeric proteins, fusing the N terminus of MLL to several proteins that share little sequence similarity. Several frequent translocation partners of MLL were found recently to coexist in a super elongation complex (SEC) that includes known transcription elongation factors such as eleven-nineteen lysine-rich leukemia (ELL) and P-TEFb. Importantly, the SEC is required for HOX gene expression in leukemic cells, suggesting that chromosomal translocations involving MLL could lead to the overexpression of HOX and other genes through the involvement of the SEC. Here, we review the normal developmental roles of MLL and the SEC, and how MLL fusion proteins can mediate leukemogenesis. PMID:21460034

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development.

PubMed

Bedell, Victoria M; Person, Anthony D; Larson, Jon D; McLoon, Anna; Balciunas, Darius; Clark, Karl J; Neff, Kevin I; Nelson, Katie E; Bill, Brent R; Schimmenti, Lisa A; Beiraghi, Soraya; Ekker, Stephen C

2012-02-01

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

PubMed

Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

2015-09-15

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results in studies on the effect of CO in gene expression. Copyright © 2015 Elsevier Inc. All rights reserved.

The synergistic effects of shear stress and cyclic hydrostatic pressure modulate chondrogenic induction of human mesenchymal stem cells.

PubMed

Hosseini, Motahare-Sadat; Tafazzoli-Shadpour, Mohammad; Haghighipour, Nooshin; Aghdami, Naser; Goodarzi, Alireza

2015-10-01

In this study, we examined chondrogenic regulation of 2 types of mesenchymal stem cells seeded on the bioengineered substrate in monolayer cultures under mechanically defined conditions to mimic the in vivo microenvironment of chondrocytes within articular cartilage tissues. Human adipose-derived mesenchymal stem cells (ASCs) and bone marrow mesenchymal stem cells (BSCs) were exposed to 0.2 Pa shear stress, 3 MPa cyclic hydrostatic pressure, and combined loading with different sequences on chemically designed medical-grade silicone rubber, while no soluble growth factors were added to the culture medium. The expression levels of chondrogenic-specific genes of SOX9, aggrecan, and type II collagen (Col II) were measured. Results were compared to those of cells treated by biological growth factor. Gene expression patterns were dependent on the loading regime. Moreover, the source of mesenchymal stem cells (adipose or bone marrow) was influential in gene expression. Overall, enhanced expression of chondrogenic markers was found through application of mechanical stimuli. The response was generally found to be significantly promoted when the 2 loading regimes were superimposed. Differentiation of ASCs was shown by a modest increase in gene expression profiles. In general, BSCs expressed higher levels of chondrogenic gene expression than ASCs after 3 weeks. A greater effect on Col II and SOX9 mRNA expression was observed when combined loadings were applied. Results may be applied in determining the proper loading sequence for obtaining functional target cells in cartilage engineering applications.

Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold.

PubMed

Hasegawa, Tomoya; Hall, Christopher J; Crosier, Philip S; Abe, Gembu; Kawakami, Koichi; Kudo, Akira; Kawakami, Atsushi

2017-02-23

Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta ( il1b ). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration.

Translating natural genetic variation to gene expression in a computational model of the Drosophila gap gene regulatory network

PubMed Central

Kozlov, Konstantin N.; Kulakovskiy, Ivan V.; Zubair, Asif; Marjoram, Paul; Lawrie, David S.; Nuzhdin, Sergey V.; Samsonova, Maria G.

2017-01-01

Annotating the genotype-phenotype relationship, and developing a proper quantitative description of the relationship, requires understanding the impact of natural genomic variation on gene expression. We apply a sequence-level model of gap gene expression in the early development of Drosophila to analyze single nucleotide polymorphisms (SNPs) in a panel of natural sequenced D. melanogaster lines. Using a thermodynamic modeling framework, we provide both analytical and computational descriptions of how single-nucleotide variants affect gene expression. The analysis reveals that the sequence variants increase (decrease) gene expression if located within binding sites of repressors (activators). We show that the sign of SNP influence (activation or repression) may change in time and space and elucidate the origin of this change in specific examples. The thermodynamic modeling approach predicts non-local and non-linear effects arising from SNPs, and combinations of SNPs, in individual fly genotypes. Simulation of individual fly genotypes using our model reveals that this non-linearity reduces to almost additive inputs from multiple SNPs. Further, we see signatures of the action of purifying selection in the gap gene regulatory regions. To infer the specific targets of purifying selection, we analyze the patterns of polymorphism in the data at two phenotypic levels: the strengths of binding and expression. We find that combinations of SNPs show evidence of being under selective pressure, while individual SNPs do not. The model predicts that SNPs appear to accumulate in the genotypes of the natural population in a way biased towards small increases in activating action on the expression pattern. Taken together, these results provide a systems-level view of how genetic variation translates to the level of gene regulatory networks via combinatorial SNP effects. PMID:28898266

Threshold-dependent cooperativity of Pdx1 and Oc1 in pancreatic progenitors establishes competency for endocrine differentiation and β-cell function

PubMed Central

Wright, Christopher V.E.; Won, Kyoung-Jae

2016-01-01

Summary Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature β cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (e)13.5, but defects in specification and differentiation were apparent at e15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for β-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and β-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of β cells. PMID:27292642

Expression analysis of NOS family and HSP genes during thermal stress in goat ( Capra hircus)

NASA Astrophysics Data System (ADS)

Yadav, Vijay Pratap; Dangi, Satyaveer Singh; Chouhan, Vikrant Singh; Gupta, Mahesh; Dangi, Saroj K.; Singh, Gyanendra; Maurya, Vijay Prakash; Kumar, Puneet; Sarkar, Mihir

2016-03-01

Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher ( P < 0.05) during peak summer, and iNOS and eNOS expressions were also observed to be significantly higher ( P < 0.05) during peak winter season as compared with moderate season. The iNOS, eNOS, cNOS, HSP70, and HSP90 were mainly localized in plasma membrane and cytoplasm of PBMCs. To conclude, data generated in the present study indicate the possible involvement of the NOS family genes in amelioration of thermal stress so as to maintain cellular integrity and homeostasis in goats.

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

PubMed Central

Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses. PMID:24927412

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation.

PubMed

Masè, Michela; Grasso, Margherita; Avogaro, Laura; D'Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

2017-01-24

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-C q , GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions.

A C. elegans Hox gene switches on, off, on and off again to regulate proliferation, differentiation and morphogenesis.

PubMed

Salser, S J; Kenyon, C

1996-05-01

Hox genes establish body pattern throughout the animal kingdom, but the role these genes play at the cellular level to modify and shape parts of the body remains a mystery. We find that the C. elegans Antennapedia homolog, mab-5, sequentially programs many independent events within individual cell lineages. In one body region, mab-5 first switches ON in a lineage to stimulate proliferation, then OFF to specify epidermal structures, then ON in just one branch of the lineage to promote neuroblast formation, and finally OFF to permit proper sense organ morphology. In a neighboring lineage, continuous mab-5 expression leads to a different pattern of development. Thus, this Hox gene achieves much of its power to diversify the anteroposterior axis through fine spatiotemporal differences in expression coupled with a changing pattern of cellular response.

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

PubMed Central

Schusser, Benjamin; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley Mettler; Harriman, William D.; Etches, Robert J.; Leighton, Philip A.

2013-01-01

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens. PMID:24278246

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

PubMed

Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard

2009-08-01

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Epigenetics-related genes in prostate cancer: expression profile in prostate cancer tissues, androgen-sensitive and -insensitive cell lines.

PubMed

Shaikhibrahim, Zaki; Lindstrot, Andreas; Ochsenfahrt, Jacqueline; Fuchs, Kerstin; Wernert, Nicolas

2013-01-01

Epigenetic changes have been suggested to drive prostate cancer (PCa) development and progression. Therefore, in this study, we aimed to identify novel epigenetics-related genes in PCa tissues, and to examine their expression in metastatic PCa cell lines. We analyzed the expression of epigenetics-related genes via a clustering analysis based on gene function in moderately and poorly differentiated PCa glands compared to normal glands of the peripheral zone (prostate proper) from PCa patients using Whole Human Genome Oligo Microarrays. Our analysis identified 12 epigenetics-related genes with a more than 2-fold increase or decrease in expression and a p-value <0.01. In modera-tely differentiated tumors compared to normal glands of the peripheral zone, we found the genes, TDRD1, IGF2, DICER1, ADARB1, HILS1, GLMN and TRIM27, to be upregulated, whereas TNRC6A and DGCR8 were found to be downregulated. In poorly differentiated tumors, we found TDRD1, ADARB and RBM3 to be upregulated, whereas DGCR8, PIWIL2 and BC069781 were downregulated. Our analysis of the expression level for each gene in the metastatic androgen-sensitive VCaP and LNCaP, and -insensitive PC3 and DU-145 PCa cell lines revealed differences in expression among the cell lines which may reflect the different biological properties of each cell line, and the potential role of each gene at different metastatic sites. The novel epigenetics-related genes that we identified in primary PCa tissues may provide further insight into the role that epigenetic changes play in PCa. Moreover, some of the genes that we identified may play important roles in primary PCa and metastasis, in primary PCa only, or in metastasis only. Follow-up studies are required to investigate the functional role and the role that the expression of these genes play in the outcome and progression of PCa using tissue microarrays.

Synapsin Is Required to "Boost" Memory Strength for Highly Salient Events

ERIC Educational Resources Information Center

Kleber, Jörg; Chen, Yi-Chun; Michels, Birgit; Saumweber, Timo; Schleyer, Michael; Kähne, Thilo; Buchner, Erich; Gerber, Bertram

2016-01-01

Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is encoded by only one gene in the "Drosophila" genome and is expressed throughout the nervous system. It regulates the balance between reserve and releasable vesicles, is required to maintain transmission upon heavy demand, and is essential for proper memory function…

Tight junction gene expression in gastrointestinal tract of dairy calves with coccidiosis and treated with glucagon-like peptide-2

USDA-ARS?s Scientific Manuscript database

Selective permeability of the intestinal epithelium and efficient nutrient absorption are important functions for proper growth and development of calves. Damage to the intestinal mucosa can give rise to harmful long-term health effects and reduce productivity of the mature animal. Tight junction pr...

The TRANSPARENT TESTA16 Locus Encodes the ARABIDOPSIS BSISTER MADS Domain Protein and Is Required for Proper Development and Pigmentation of the Seed Coat

PubMed Central

Nesi, Nathalie; Debeaujon, Isabelle; Jond, Clarisse; Stewart, Amanda J.; Jenkins, Gareth I.; Caboche, Michel; Lepiniec, Loïc

2002-01-01

Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified “B-sister” (BS) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the BS clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the BS MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat. PMID:12368498

GRHL3/GET1 and Trithorax Group Members Collaborate to Activate the Epidermal Progenitor Differentiation Program

PubMed Central

Hopkin, Amelia Soto; Gordon, William; Klein, Rachel Herndon; Espitia, Francisco; Daily, Kenneth; Zeller, Michael; Baldi, Pierre; Andersen, Bogi

2012-01-01

The antagonistic actions of Polycomb and Trithorax are responsible for proper cell fate determination in mammalian tissues. In the epidermis, a self-renewing epithelium, previous work has shown that release from Polycomb repression only partially explains differentiation gene activation. We now show that Trithorax is also a key regulator of epidermal differentiation, not only through activation of genes repressed by Polycomb in progenitor cells, but also through activation of genes independent of regulation by Polycomb. The differentiation associated transcription factor GRHL3/GET1 recruits the ubiquitously expressed Trithorax complex to a subset of differentiation genes. PMID:22829784

Histone Deacetylase 3 Suppresses Erk Phosphorylation and Matrix Metalloproteinase (Mmp)-13 Activity in Chondrocytes

PubMed Central

Carpio, Lomeli R.; Bradley, Elizabeth W.; Westendorf, Jennifer J.

2017-01-01

Histone deacetylase inhibitors are emerging therapies for many diseases including cancers and neurological disorders; however, these drugs are teratogens to the developing skeleton. Hdac3 is essential for proper endochondral ossification as its deletion in chondrocytes increases cytokine signaling and the expression of matrix remodeling enzymes. Here we explored the mechanism by which Hdac3 controls Mmp13 expression in chondrocytes. In Hdac3-depleted chondrocytes, Erk1/2 as well as its downstream substrate, Runx2, were hyperphosphorylated as a result of decreased expression and activity of the Erk1/2 specific phosphatase, Dusp6. Erk1/2 kinase inhibitors and Dusp6 adenoviruses reduced Mmp13 expression and partially rescued matrix production in Hdac3-deficient chondrocytes. Postnatal chondrocyte-specific deletion of Hdac3 with an inducible Col2a1-Cre caused premature production of pErk1/2 and Mmp13 in the growth plate. Thus, Hdac3 controls the temporal and spatial expression of tissue-remodeling genes in chondrocytes to ensure proper endochondral ossification during development. PMID:27662443

Selective decline of neurotrophin and neurotrophin receptor genes within CA1 pyramidal neurons and hippocampus proper: Correlation with cognitive performance and neuropathology in mild cognitive impairment and Alzheimer's disease.

PubMed

Ginsberg, Stephen D; Malek-Ahmadi, Michael H; Alldred, Melissa J; Che, Shaoli; Elarova, Irina; Chen, Yinghua; Jeanneteau, Freddy; Kranz, Thorsten M; Chao, Moses V; Counts, Scott E; Mufson, Elliott J

2017-09-09

Hippocampal CA1 pyramidal neurons, a major component of the medial temporal lobe memory circuit, are selectively vulnerable during the progression of Alzheimer's disease (AD). The cellular mechanism(s) underlying degeneration of these neurons and the relationship to cognitive performance remains largely undefined. Here, we profiled neurotrophin and neurotrophin receptor gene expression within microdissected CA1 neurons along with regional hippocampal dissections from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD using laser capture microdissection (LCM), custom-designed microarray analysis, and qPCR of CA1 subregional dissections. Gene expression levels were correlated with cognitive test scores and AD neuropathology criteria. We found a significant downregulation of several neurotrophin genes (e.g., Gdnf, Ngfb, and Ntf4) in CA1 pyramidal neurons in MCI compared to NCI and AD subjects. In addition, the neurotrophin receptor transcripts TrkB and TrkC were decreased in MCI and AD compared to NCI. Regional hippocampal dissections also revealed select neurotrophic gene dysfunction providing evidence for vulnerability within the hippocampus proper during the progression of dementia. Downregulation of several neurotrophins of the NGF family and cognate neurotrophin receptor (TrkA, TrkB, and TrkC) genes correlated with antemortem cognitive measures including the Mini-Mental State Exam (MMSE), a composite global cognitive score (GCS), and Episodic, Semantic, and Working Memory, Perceptual Speed, and Visuospatial domains. Significant correlations were found between select neurotrophic expression downregulation and neuritic plaques (NPs) and neurofibrillary tangles (NFTs), but not diffuse plaques (DPs). These data suggest that dysfunction of neurotrophin signaling complexes have profound negative sequelae within vulnerable hippocampal cell types, which play a role in mnemonic and executive dysfunction during the progression of AD. © 2017 Wiley Periodicals, Inc.

Removal of unwanted variation reveals novel patterns of gene expression linked to sleep homeostasis in murine cortex.

PubMed

Gerstner, Jason R; Koberstein, John N; Watson, Adam J; Zapero, Nikolai; Risso, Davide; Speed, Terence P; Frank, Marcos G; Peixoto, Lucia

2016-10-25

Why we sleep is still one of the most perplexing mysteries in biology. Strong evidence indicates that sleep is necessary for normal brain function and that sleep need is a tightly regulated process. Surprisingly, molecular mechanisms that determine sleep need are incompletely described. Moreover, very little is known about transcriptional changes that specifically accompany the accumulation and discharge of sleep need. Several studies have characterized differential gene expression changes following sleep deprivation. Much less is known, however, about changes in gene expression during the compensatory response to sleep deprivation (i.e. recovery sleep). In this study we present a comprehensive analysis of the effects of sleep deprivation and subsequent recovery sleep on gene expression in the mouse cortex. We used a non-traditional analytical method for normalization of genome-wide gene expression data, Removal of Unwanted Variation (RUV). RUV improves detection of differential gene expression following sleep deprivation. We also show that RUV normalization is crucial to the discovery of differentially expressed genes associated with recovery sleep. Our analysis indicates that the majority of transcripts upregulated by sleep deprivation require 6 h of recovery sleep to return to baseline levels, while the majority of downregulated transcripts return to baseline levels within 1-3 h. We also find that transcripts that change rapidly during recovery (i.e. within 3 h) do so on average with a time constant that is similar to the time constant for the discharge of sleep need. We demonstrate that proper data normalization is essential to identify changes in gene expression that are specifically linked to sleep deprivation and recovery sleep. Our results provide the first evidence that recovery sleep is comprised of two waves of transcriptional regulation that occur at different times and affect functionally distinct classes of genes.

Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

Cancer.gov

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic stem (ES) cells from accumulating DNA damage and has been linked to hematopoietic differentiation of ES cells. Satyendra Singh, Ph.D., a postdoctoral fellow working with Philipp Oberdoerffer, Ph.D., in CCR’s Laboratory of Receptor Biology and Gene Expression, and their colleagues set out to determine whether Sirt1 could play a similar protective role in adult HSPCs.

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).

PubMed

Ashish, Shende; Bhure, S K; Harikrishna, Pillai; Ramteke, S S; Muhammed Kutty, V H; Shruthi, N; Ravi Kumar, G V P P S; Manish, Mahawar; Ghosh, S K; Mihir, Sarkar

2017-04-01

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines. Copyright © 2017 Elsevier Inc. All rights reserved.

Unravelling the molecular basis for light modulated cellulase gene expression - the role of photoreceptors in Neurospora crassa

PubMed Central

2012-01-01

Background Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa. Results We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose. Conclusions Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa. PMID:22462823

Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data.

PubMed

Singh, Nitesh Kumar; Ernst, Mathias; Liebscher, Volkmar; Fuellen, Georg; Taher, Leila

2016-10-20

The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.

Expression of the homeotic gene mab-5 during Caenorhabditis elegans embryogenesis.

PubMed

Cowing, D W; Kenyon, C

1992-10-01

mab-5 is a member of a complex of homeobox-containing genes evolutionarily related to the Antennapedia and bithorax complexes of Drosophila melanogaster. Like the homeotic genes in Drosophila, mab-5 is required in a particular region along the anterior-posterior body axis, and acts during postembryonic development to give cells in this region their characteristic identities. We have used a mab-5-lacZ fusion integrated into the C. elegans genome to study the posterior-specific expression of mab-5 during embryogenesis. The mab-5-lacZ fusion was expressed in the posterior of the embryo by 180 minutes after the first cleavage, indicating that the mechanisms responsible for the position-specific expression of mab-5-lacZ act at a relatively early stage of embryogenesis. In embryos homozygous for mutations in the par genes, which disrupt segregation of factors during early cleavages, expression of mab-5-lacZ was no longer localized to the posterior. This suggests that posterior-specific expression of mab-5 depends on the appropriate segregation of developmental factors during early embryogenesis. After extrusion of any blastomere of the four-cell embryo, descendants of the remaining three cells could still express the mab-5-lacZ fusion. In these partial embryos, however, the fusion was often expressed in cells scattered throughout the embryo, suggesting that cell-cell interactions and/or proper positioning of early blastomeres are required for mab-5 expression to be localized to the posterior.

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

PubMed Central

Sellamuthu, Rajendran; Umbright, Christina; Li, Shengqiao; Kashon, Michael; Joseph, Pius

2015-01-01

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure. PMID:22087542

Identification of two genes essential for sperm development in the male tick Amblyomma hebraeum Koch (Acari: Ixodidae).

PubMed

Guo, Xiuyang; Reuben Kaufman, W

2008-07-01

In most ticks of the family Ixodidae, gonad maturation and spermatogenesis are stimulated by the taking of a blood meal. Previous work from this laboratory identified 35 genes that are up-regulated by feeding [Weiss, B.L., Stepczynski, J.M., Wong, P., Kaufman, W.R., 2002. Identification and characterization of genes differentially expressed in the testis/vas deferens of the fed male tick, Amblyomma hebraeum. Insect Biochemistry and Molecular Biology 32, 785-793]. The functions of most of these genes remain unknown. We used RNA interference technology to investigate the consequences of blocking the function of 13 of these genes. Attenuation of the expression of two of these in particular, AhT/VD 8 and AhT/VD 10, correlated with deformities in the testis and abnormalities in spermiogenesis. Furthermore, most females fed in the company of these males did not engorge properly and laid many fewer eggs, most of which were infertile.

GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.

PubMed

Nazari, Fatemeh; Parham, Abbas; Maleki, Adham Fani

2015-01-01

Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. This study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

Epigenetic Regulation of Placenta-Specific 8 Contributes to Altered Function of Endothelial Colony-Forming Cells Exposed to Intrauterine Gestational Diabetes Mellitus.

PubMed

Blue, Emily K; Sheehan, BreAnn M; Nuss, Zia V; Boyle, Frances A; Hocutt, Caleb M; Gohn, Cassandra R; Varberg, Kaela M; McClintick, Jeanette N; Haneline, Laura S

2015-07-01

Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development

PubMed Central

Bedell, Victoria M.; Person, Anthony D.; Larson, Jon D.; McLoon, Anna; Balciunas, Darius; Clark, Karl J.; Neff, Kevin I.; Nelson, Katie E.; Bill, Brent R.; Schimmenti, Lisa A.; Beiraghi, Soraya; Ekker, Stephen C.

2012-01-01

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity. PMID:22274699

A cDNA microarray gene expression data classifier for clinical diagnostics based on graph theory.

PubMed

Benso, Alfredo; Di Carlo, Stefano; Politano, Gianfranco

2011-01-01

Despite great advances in discovering cancer molecular profiles, the proper application of microarray technology to routine clinical diagnostics is still a challenge. Current practices in the classification of microarrays' data show two main limitations: the reliability of the training data sets used to build the classifiers, and the classifiers' performances, especially when the sample to be classified does not belong to any of the available classes. In this case, state-of-the-art algorithms usually produce a high rate of false positives that, in real diagnostic applications, are unacceptable. To address this problem, this paper presents a new cDNA microarray data classification algorithm based on graph theory and is able to overcome most of the limitations of known classification methodologies. The classifier works by analyzing gene expression data organized in an innovative data structure based on graphs, where vertices correspond to genes and edges to gene expression relationships. To demonstrate the novelty of the proposed approach, the authors present an experimental performance comparison between the proposed classifier and several state-of-the-art classification algorithms.

The Chromatin Remodeler BPTF Activates a Stemness Gene-Expression Program Essential for the Maintenance of Adult Hematopoietic Stem Cells.

PubMed

Xu, Bowen; Cai, Ling; Butler, Jason M; Chen, Dongliang; Lu, Xiongdong; Allison, David F; Lu, Rui; Rafii, Shahin; Parker, Joel S; Zheng, Deyou; Wang, Gang Greg

2018-03-13

Self-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs). Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors (Meis1, Pbx1, Mn1, and Lmo2) required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC "stemness" genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of "stemness" gene-expression programs and proper function of adult HSCs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

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

Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji

2009-05-08

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependentmore » on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.« less

Searching for non-transposable targets of planarian nuclear PIWI in pluripotent stem cells and differentiated cells.

PubMed

Kashima, Makoto; Agata, Kiyokazu; Shibata, Norito

2018-06-01

Nuclear PIWIs together with their guide RNAs (piRNAs) epigenetically silence various genes including transposons in many organisms. In planarians, the nuclear piwi family gene, DjpiwiB is specifically transcribed in adult pluripotent stem cells (adult PSC, neoblast), but not in differentiated cells. However, the protein accumulates in the nuclei of both neoblasts and their descendant differentiated cells. Interestingly, PIWI(DjPiwiB)-piRNA complexes are indispensable for the repression of transposable genes at the onset of differentiation from neoblasts. Here, we conducted a comparative transcriptome analysis between control and DjpiwiB(RNAi) animals to identify non-transposable target genes of the DjPiwiB-piRNA complexes. Using bioinformatic analyses and RNAi we demonstrate that DjPiwiB-piRNA complexes are required for the proper expression of Djmcm2 and Djhistone h4 in neoblasts and that DjPiwiB-piRNA complexes regulate the transient expression of Djcalu during neoblast differentiation. Thus, DjPiwiB-piRNA complexes regulate the correct expression patterns during neoblast self-renewal and differentiation. © 2018 Japanese Society of Developmental Biologists.

SR proteins in Vertical Integration of Gene Expression from Transcription to RNA Processing to Translation

PubMed Central

Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G.; Fu, Xiang-Dong

2009-01-01

Summary SR proteins have been studied extensively as a family of RNA binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and co-localize with genes that are engaged in specific intra- and inter-chromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings therefore highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell cycle progression in higher eukaryotic cells. PMID:19595711

SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

PubMed

Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G; Fu, Xiang-Dong

2009-07-10

SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription

PubMed Central

Wozniak, Glenn G.

2014-01-01

Monoubiquitylation of histone H2B on Lys123 (H2BK123ub1) plays a multifaceted role in diverse DNA-templated processes, yet the mechanistic details by which this modification is regulated are not fully elucidated. Here we show in yeast that H2BK123ub1 is regulated in part through the protein stability of the E3 ubiquitin ligase Bre1. We found that Bre1 stability is controlled by the Rtf1 subunit of the polymerase-associated factor (PAF) complex and through the ability of Bre1 to catalyze H2BK123ub1. Using a domain in Rtf1 that stabilizes Bre1, we show that inappropriate Bre1 levels lead to defects in gene regulation. Collectively, these data uncover a novel quality control mechanism used by the cell to maintain proper Bre1 and H2BK123ub1 levels, thereby ensuring proper control of gene expression. PMID:25085417

MicroRNA-146a constrains multiple parameters of intestinal immunity and increases susceptibility to DSS colitis.

PubMed

Runtsch, Marah C; Hu, Ruozhen; Alexander, Margaret; Wallace, Jared; Kagele, Dominique; Petersen, Charisse; Valentine, John F; Welker, Noah C; Bronner, Mary P; Chen, Xinjian; Smith, Daniel P; Ajami, Nadim J; Petrosino, Joseph F; Round, June L; O'Connell, Ryan M

2015-10-06

Host-microbial interactions within the mammalian intestines must be properly regulated in order to promote host health and limit disease. Because the microbiota provide constant immunological signals to intestinal tissues, a variety of regulatory mechanisms have evolved to ensure proper immune responses to maintain homeostasis. However, many of the genes that comprise these regulatory pathways, including immune-modulating microRNAs (miRNAs), have not yet been identified or studied in the context of intestinal homeostasis. Here, we investigated the role of microRNA-146a (miR-146a) in regulating intestinal immunity and barrier function and found that this miRNA is expressed in a variety of gut tissues in adult mice. By comparing intestinal gene expression in WT and miR-146a-/- mice, we demonstrate that miR-146a represses a subset of gut barrier and inflammatory genes all within a network of immune-related signaling pathways. We also found that miR-146a restricts the expansion of intestinal T cell populations, including Th17, Tregs, and Tfh cells. GC B cells, Tfh ICOS expression, and the production of luminal IgA were also reduced by miR-146a in the gut. Consistent with an enhanced intestinal barrier, we found that miR-146a-/- mice are resistant to DSS-induced colitis, a model of Ulcerative Colitis (UC), and this correlated with elevated colonic miR-146a expression in human UC patients. Taken together, our data describe a role for miR-146a in constraining intestinal barrier function, a process that alters gut homeostasis and enhances at least some forms of intestinal disease in mice.

MicroRNA-146a constrains multiple parameters of intestinal immunity and increases susceptibility to DSS colitis

PubMed Central

Runtsch, Marah C.; Hu, Ruozhen; Alexander, Margaret; Wallace, Jared; Kagele, Dominique; Petersen, Charisse; Valentine, John F.; Welker, Noah C.; Bronner, Mary P.; Chen, Xinjian; Smith, Daniel P.; Ajami, Nadim J.; Petrosino, Joseph F.; Round, June L.; O'Connell, Ryan M.

2015-01-01

Host-microbial interactions within the mammalian intestines must be properly regulated in order to promote host health and limit disease. Because the microbiota provide constant immunological signals to intestinal tissues, a variety of regulatory mechanisms have evolved to ensure proper immune responses to maintain homeostasis. However, many of the genes that comprise these regulatory pathways, including immune-modulating microRNAs (miRNAs), have not yet been identified or studied in the context of intestinal homeostasis. Here, we investigated the role of microRNA-146a (miR-146a) in regulating intestinal immunity and barrier function and found that this miRNA is expressed in a variety of gut tissues in adult mice. By comparing intestinal gene expression in WT and miR-146a−/− mice, we demonstrate that miR-146a represses a subset of gut barrier and inflammatory genes all within a network of immune-related signaling pathways. We also found that miR-146a restricts the expansion of intestinal T cell populations, including Th17, Tregs, and Tfh cells. GC B cells, Tfh ICOS expression, and the production of luminal IgA were also reduced by miR-146a in the gut. Consistent with an enhanced intestinal barrier, we found that miR-146a−/− mice are resistant to DSS-induced colitis, a model of Ulcerative Colitis (UC), and this correlated with elevated colonic miR-146a expression in human UC patients. Taken together, our data describe a role for miR-146a in constraining intestinal barrier function, a process that alters gut homeostasis and enhances at least some forms of intestinal disease in mice. PMID:26456940

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

PubMed

Huang, Daosheng; Guo, Guoji; Yuan, Ping; Ralston, Amy; Sun, Lingang; Huss, Mikael; Mistri, Tapan; Pinello, Luca; Ng, Huck Hui; Yuan, Guocheng; Ji, Junfeng; Rossant, Janet; Robson, Paul; Han, Xiaoping

2017-12-07

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

Selection of Reference Genes for Expression Studies in Diaphorina citri (Hemiptera: Liviidae).

PubMed

Bassan, Meire Menezes; Angelotti-Mendonc A, Je Ssika; Alves, Gustavo Rodrigues; Yamamoto, Pedro Takao; Moura O Filho, Francisco de Assis Alves

2017-12-05

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is considered the main vector of the bacteria associated with huanglongbing, a very serious disease that has threatened the world citrus industry. The absence of efficient control management protocols, including a lack of resistant cultivars, has led to the development of different approaches to study this pathosystem. The production of resistant genotypes relies on D. citri gene expression analyses by RT-qPCR to assess control of the vector population. High-quality, reliable RT-qPCR analyses depend upon proper reference gene selection and validation. However, adequate D. citri reference genes have not yet been identified. In the present study, we evaluated the genes EF 1-α, ACT, GAPDH, RPL7, RPL17, and TUB as candidate reference genes for this insect. Gene expression stability was evaluated using the mathematical algorithms deltaCt, NormFinder, BestKeeper, and geNorm, at five insect developmental stages, grown on two different plant hosts [Citrus sinensis (L.) Osbeck (Sapindales: Rutaceae) and Murraya paniculata (L.) Jack (Sapindales: Rutaceae)]. The final gene ranking was calculated using RefFinder software, and the V-ATPase-A gene was selected for validation. According to our results, two reference genes are recommended when different plant hosts and developmental stages are considered. Considering gene expression studies in D. citri grown on M. paniculata, regardless of the insect developmental stage, GAPDH and RPL7 have the best fit as reference genes in RT-qPCR analyses, whereas GAPDH and EF 1-α are recommended as reference genes in insect studies using C. sinensis. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

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

Al-Dosari, Mohammed; Zhang Guisheng; Knapp, Joseph E.

2006-01-13

Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), {alpha}-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken {beta} actin (ACT), nuclear factor {kappa} B (NF{kappa}B), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promotermore » exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within First 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.« less

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity

PubMed Central

Masoudi, Neda; Tavazoie, Saeed; Glenwinkel, Lori; Ryu, Leesun; Kim, Kyuhyung

2018-01-01

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor–encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an “ur-” bHLH gene. PMID:29672507

Gene networks specific for innate immunity define post-traumatic stress disorder.

PubMed

Breen, M S; Maihofer, A X; Glatt, S J; Tylee, D S; Chandler, S D; Tsuang, M T; Risbrough, V B; Baker, D G; O'Connor, D T; Nievergelt, C M; Woelk, C H

2015-12-01

The molecular factors involved in the development of Post-Traumatic Stress Disorder (PTSD) remain poorly understood. Previous transcriptomic studies investigating the mechanisms of PTSD apply targeted approaches to identify individual genes under a cross-sectional framework lack a holistic view of the behaviours and properties of these genes at the system-level. Here we sought to apply an unsupervised gene-network based approach to a prospective experimental design using whole-transcriptome RNA-Seq gene expression from peripheral blood leukocytes of U.S. Marines (N=188), obtained both pre- and post-deployment to conflict zones. We identified discrete groups of co-regulated genes (i.e., co-expression modules) and tested them for association to PTSD. We identified one module at both pre- and post-deployment containing putative causal signatures for PTSD development displaying an over-expression of genes enriched for functions of innate-immune response and interferon signalling (Type-I and Type-II). Importantly, these results were replicated in a second non-overlapping independent dataset of U.S. Marines (N=96), further outlining the role of innate immune and interferon signalling genes within co-expression modules to explain at least part of the causal pathophysiology for PTSD development. A second module, consequential of trauma exposure, contained PTSD resiliency signatures and an over-expression of genes involved in hemostasis and wound responsiveness suggesting that chronic levels of stress impair proper wound healing during/after exposure to the battlefield while highlighting the role of the hemostatic system as a clinical indicator of chronic-based stress. These findings provide novel insights for early preventative measures and advanced PTSD detection, which may lead to interventions that delay or perhaps abrogate the development of PTSD.

Neurofibromin Deficiency-Associated Transcriptional Dysregulation Suggests a Novel Therapy for Tibial Pseudoarthrosis in NF1

PubMed Central

Paria, Nandina; Cho, Tae-Joon; Choi, In Ho; Kamiya, Nobuhiro; Kayembe, Kay; Mao, Rong; Margraf, Rebecca L.; Obermosser, Gerlinde; Oxendine, Ila; Sant, David W.; Song, Mi Hyun; Stevenson, David A.; Viskochil, David H.; Wise, Carol A.; Kim, Harry K.W.; Rios, Jonathan J

2014-01-01

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic bi-allelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinosital-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant over-expression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing. PMID:24932921

Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map.

PubMed

Smith, Ian; Greenside, Peyton G; Natoli, Ted; Lahr, David L; Wadden, David; Tirosh, Itay; Narayan, Rajiv; Root, David E; Golub, Todd R; Subramanian, Aravind; Doench, John G

2017-11-01

The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss-of-function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that microRNA (miRNA)-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a consensus gene signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 single guide RNAs (sgRNAs) in 6 cells lines and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.

The imidazopyridine derivative JK184 reveals dual roles for microtubules in Hedgehog signaling.

PubMed

Cupido, Tommaso; Rack, Paul G; Firestone, Ari J; Hyman, Joel M; Han, Kyuho; Sinha, Surajit; Ocasio, Cory A; Chen, James K

2009-01-01

Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

Mammary gland morphology and gene expression signature of prepubertal male and female rats following exposure to exogenous estradiol

USDA-ARS?s Scientific Manuscript database

In order to properly screen environmental chemicals for potential toxic effects such as increased cancer risk and risk of infertility resulting from actions similar to those of female sex steroids such as estrogens, it is essential to understand the effects of treatment with the most important femal...

Activation of the Arabidopsis B class homeotic genes by APETALA1.

PubMed

Ng, M; Yanofsky, M F

2001-04-01

Proper development of petals and stamens in Arabidopsis flowers requires the activities of APETALA3 (AP3) and PISTILLATA (PI), whose transcripts can be detected in the petal and stamen primordia. Localized expression of AP3 and PI requires the activities of at least three genes: APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO). It has been proposed that UFO provides spatial cues and that LFY specifies competence for AP3 and PI expression in the developing flower. To understand the epistatic relationship among AP1, LFY, and UFO in regulating AP3 and PI expression, we generated two versions of AP1 that have strong transcriptional activation potential. Genetic and molecular analyses of transgenic plants expressing these activated AP1 proteins show that the endogenous AP1 protein acts largely as a transcriptional activator in vivo and that AP1 specifies petals by regulating the spatial domains of AP3 and PI expression through UFO.

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity.

PubMed

Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

2017-04-10

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors.

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene.

PubMed

Wang, Guo-Kun; Zhang, Meng; Gong, Jiang-Feng; Guo, Qi-Fang; Feng, Ya-Nan; Wang, Wei

2012-12-01

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin. The ubiquitin-26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA(3) conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.

Molecular insight in gastric cancer induction: an overview of cancer stemness genes.

PubMed

Akhavan-Niaki, Haleh; Samadani, Ali Akbar

2014-04-01

Gastric cancer is one of the most outgoing human cancers in the world. Two main functional types were described: Intestinal adenocarcinoma and diffuse one. The most important purpose of this review is to analyze and investigate the main genetic factors involved in tumorogenesis of stomach and the molecular mechanism of their expression regulation alongside with the importance of cancer stem cells and their relationship with gastric cancer. It is evident that proper diagnosis of molecular case of cancer may lead to absolute treatment and at least reduction in the disease severity. However, stemness factors such as Sox2, Oct3/4, and Nanog were related with induced pluripotent stem cells, proposing a correlation between these stemness factors and cancer stem cells. Moreover, aberrant induction by Helicobacter pylori of the intestinal-specific homeobox transcription factors, CDX1 and CDX2, also plays an important role in this modification. There are some genes which are directly activated by CDX1 in gastric cancer and distinguished stemness-related reprogramming factors like SALL4 and KLF5. Correspondingly, we also aimed to present the main important epigenetic changes such as DNA methylation, histone modification, and chromatin modeling of stemness genes in disease development. Remarkably, a better understanding of molecular bases of cancer may lead to novel diagnostic, therapeutic, and preventive approaches by some genetic and epigenetic changes such as gene amplifications, gene silencing by DNA methylation, losses of imprinting, LOH, and mutations. Consequently, genome-wide searches of gene expression are widely important for surveying the proper mechanisms of cancer emergence and development. Conspicuously, this review explains an outline of the molecular mechanism and new approaches in gastric cancer.

Zebrafish hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch.

PubMed

Hunter, Michael P; Prince, Victoria E

2002-07-15

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated "knock-down" of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as "selector genes." 2002 Elsevier Science (USA).

Node and midline defects are associated with left-right development in Delta1 mutant embryos.

PubMed

Przemeck, Gerhard K H; Heinzmann, Ulrich; Beckers, Johannes; Hrabé de Angelis, Martin

2003-01-01

Axes formation is a fundamental process of early embryonic development. In addition to the anteroposterior and dorsoventral axes, the determination of the left-right axis is crucial for the proper morphogenesis of internal organs and is evolutionarily conserved in vertebrates. Genes known to be required for the normal establishment and/or maintenance of left-right asymmetry in vertebrates include, for example, components of the TGF-beta family of intercellular signalling molecules and genes required for node and midline function. We report that Notch signalling, which previously had not been implicated in this morphogenetic process, is required for normal left-right determination in mice. We show, that the loss-of-function of the delta 1 (Dll1) gene causes a situs ambiguous phenotype, including randomisation of the direction of heart looping and embryonic turning. The most probable cause for this left-right defect in Dll1 mutant embryos is a failure in the development of proper midline structures. These originate from the node, which is disrupted and deformed in Dll1 mutant embryos. Based on expression analysis in wild-type and mutant embryos, we suggest a model, in which Notch signalling is required for the proper differentiation of node cells and node morphology.

Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development

PubMed Central

José-Edwards, Diana S.; Oda-Ishii, Izumi; Nibu, Yutaka; Di Gregorio, Anna

2013-01-01

T-box genes are potent regulators of mesoderm development in many metazoans. In chordate embryos, the T-box transcription factor Brachyury (Bra) is required for specification and differentiation of the notochord. In some chordates, including the ascidian Ciona, members of the Tbx2 subfamily of T-box genes are also expressed in this tissue; however, their regulatory relationships with Bra and their contributions to the development of the notochord remain uncharacterized. We determined that the notochord expression of Ciona Tbx2/3 (Ci-Tbx2/3) requires Ci-Bra, and identified a Ci-Tbx2/3 notochord CRM that necessitates multiple Ci-Bra binding sites for its activity. Expression of mutant forms of Ci-Tbx2/3 in the developing notochord revealed a role for this transcription factor primarily in convergent extension. Through microarray screens, we uncovered numerous Ci-Tbx2/3 targets, some of which overlap with known Ci-Bra-downstream notochord genes. Among the Ci-Tbx2/3 notochord targets are evolutionarily conserved genes, including caspases, lineage-specific genes, such as Noto4, and newly identified genes, such as MLKL. This work sheds light on a large section of the notochord regulatory circuitry controlled by T-box factors, and reveals new components of the complement of genes required for the proper formation of this structure. PMID:23674602

Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development.

PubMed

José-Edwards, Diana S; Oda-Ishii, Izumi; Nibu, Yutaka; Di Gregorio, Anna

2013-06-01

T-box genes are potent regulators of mesoderm development in many metazoans. In chordate embryos, the T-box transcription factor Brachyury (Bra) is required for specification and differentiation of the notochord. In some chordates, including the ascidian Ciona, members of the Tbx2 subfamily of T-box genes are also expressed in this tissue; however, their regulatory relationships with Bra and their contributions to the development of the notochord remain uncharacterized. We determined that the notochord expression of Ciona Tbx2/3 (Ci-Tbx2/3) requires Ci-Bra, and identified a Ci-Tbx2/3 notochord CRM that necessitates multiple Ci-Bra binding sites for its activity. Expression of mutant forms of Ci-Tbx2/3 in the developing notochord revealed a role for this transcription factor primarily in convergent extension. Through microarray screens, we uncovered numerous Ci-Tbx2/3 targets, some of which overlap with known Ci-Bra-downstream notochord genes. Among the Ci-Tbx2/3 notochord targets are evolutionarily conserved genes, including caspases, lineage-specific genes, such as Noto4, and newly identified genes, such as MLKL. This work sheds light on a large section of the notochord regulatory circuitry controlled by T-box factors, and reveals new components of the complement of genes required for the proper formation of this structure.

Orgyia pseudotsugata baculovirus p10 and polyhedron envelope protein genes: analysis of their relative expression levels and role in polyhedron structure.

PubMed

Gross, C H; Russell, R L; Rohrmann, G F

1994-05-01

To investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the beta-glucuronidase (GUS) or beta-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10- viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10-GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra.

Monitoring the regulation of gene expression in a growing organ using a fluid mechanics formalism

PubMed Central

2010-01-01

Background Technological advances have enabled the accurate quantification of gene expression, even within single cell types. While transcriptome analyses are routinely performed, most experimental designs only provide snapshots of gene expression. Molecular mechanisms underlying cell fate or positional signalling have been revealed through these discontinuous datasets. However, in developing multicellular structures, temporal and spatial cues, known to directly influence transcriptional networks, get entangled as the cells are displaced and expand. Access to an unbiased view of the spatiotemporal regulation of gene expression occurring during development requires a specific framework that properly quantifies the rate of change of a property in a moving and expanding element, such as a cell or an organ segment. Results We show how the rate of change in gene expression can be quantified by combining kinematics and real-time polymerase chain reaction data in a mechanistic model which considers any organ as a continuum. This framework was applied in order to assess the developmental regulation of the two reference genes Actin11 and Elongation Factor 1-β in the apex of poplar root. The growth field was determined by time-lapse photography and transcript density was obtained at high spatial resolution. The net accumulation rates of the transcripts of the two genes were found to display highly contrasted developmental profiles. Actin11 showed pulses of up and down regulation in the accelerating and decelerating parts of the growth zone while the dynamic of EF1β were much slower. This framework provides key information about gene regulation in a developing organ, such as the location, the duration and the intensity of gene induction/repression. Conclusions We demonstrated that gene expression patterns can be monitored using the continuity equation without using mutants or reporter constructions. Given the rise of imaging technologies, this framework in our view opens a new way to dissect the molecular basis of growth regulation, even in non-model species or complex structures. PMID:20202192

The Genetic Program of Pancreatic β-Cell Replication In Vivo.

PubMed

Klochendler, Agnes; Caspi, Inbal; Corem, Noa; Moran, Maya; Friedlich, Oriel; Elgavish, Sharona; Nevo, Yuval; Helman, Aharon; Glaser, Benjamin; Eden, Amir; Itzkovitz, Shalev; Dor, Yuval

2016-07-01

The molecular program underlying infrequent replication of pancreatic β-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating β-cells and determined their transcriptome. Replicating β-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in β-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating β-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in β-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are "left behind" and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating β-cells in vivo. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation

PubMed Central

Masè, Michela; Grasso, Margherita; Avogaro, Laura; D’Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

2017-01-01

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-Cq, GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions. PMID:28117343

Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break

PubMed Central

Lindgren, Emma; Hägg, Sara; Giordano, Fosco; Björkegren, Johan; Ström, Lena

2014-01-01

Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding. PMID:25483075

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

PubMed

Bottardi, Stefania; Mavoungou, Lionel; Pak, Helen; Daou, Salima; Bourgoin, Vincent; Lakehal, Yahia A; Affar, El Bachir; Milot, Eric

2014-12-01

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

The IKAROS Interaction with a Complex Including Chromatin Remodeling and Transcription Elongation Activities Is Required for Hematopoiesis

PubMed Central

Bottardi, Stefania; Mavoungou, Lionel; Pak, Helen; Daou, Salima; Bourgoin, Vincent; Lakehal, Yahia A.; Affar, El Bachir; Milot, Eric

2014-01-01

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation. PMID:25474253

Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold

PubMed Central

Hasegawa, Tomoya; Hall, Christopher J; Crosier, Philip S; Abe, Gembu; Kawakami, Koichi; Kudo, Akira; Kawakami, Atsushi

2017-01-01

Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta (il1b). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration. DOI: http://dx.doi.org/10.7554/eLife.22716.001 PMID:28229859

Modeling gene expression measurement error: a quasi-likelihood approach

PubMed Central

Strimmer, Korbinian

2003-01-01

Background Using suitable error models for gene expression measurements is essential in the statistical analysis of microarray data. However, the true probabilistic model underlying gene expression intensity readings is generally not known. Instead, in currently used approaches some simple parametric model is assumed (usually a transformed normal distribution) or the empirical distribution is estimated. However, both these strategies may not be optimal for gene expression data, as the non-parametric approach ignores known structural information whereas the fully parametric models run the risk of misspecification. A further related problem is the choice of a suitable scale for the model (e.g. observed vs. log-scale). Results Here a simple semi-parametric model for gene expression measurement error is presented. In this approach inference is based an approximate likelihood function (the extended quasi-likelihood). Only partial knowledge about the unknown true distribution is required to construct this function. In case of gene expression this information is available in the form of the postulated (e.g. quadratic) variance structure of the data. As the quasi-likelihood behaves (almost) like a proper likelihood, it allows for the estimation of calibration and variance parameters, and it is also straightforward to obtain corresponding approximate confidence intervals. Unlike most other frameworks, it also allows analysis on any preferred scale, i.e. both on the original linear scale as well as on a transformed scale. It can also be employed in regression approaches to model systematic (e.g. array or dye) effects. Conclusions The quasi-likelihood framework provides a simple and versatile approach to analyze gene expression data that does not make any strong distributional assumptions about the underlying error model. For several simulated as well as real data sets it provides a better fit to the data than competing models. In an example it also improved the power of tests to identify differential expression. PMID:12659637

Chromatin organization and global regulation of Hox gene clusters

PubMed Central

Montavon, Thomas; Duboule, Denis

2013-01-01

During development, a properly coordinated expression of Hox genes, within their different genomic clusters is critical for patterning the body plans of many animals with a bilateral symmetry. The fascinating correspondence between the topological organization of Hox clusters and their transcriptional activation in space and time has served as a paradigm for understanding the relationships between genome structure and function. Here, we review some recent observations, which revealed highly dynamic changes in the structure of chromatin at Hox clusters, in parallel with their activation during embryonic development. We discuss the relevance of these findings for our understanding of large-scale gene regulation. PMID:23650639

Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila.

PubMed

Zmojdzian, Monika; de Joussineau, Svetlana; Da Ponte, Jean Philippe; Jagla, Krzysztof

2018-01-17

The Drosophila heart, composed of discrete subsets of cardioblasts and pericardial cells, undergoes Hox-triggered anterior-posterior morphogenesis, leading to a functional subdivision into heart proper and aorta, with its most anterior part forming a funnel-shaped cardiac outflow. Cardioblasts differentiate into Tin-positive 'working myocytes' and Svp-expressing ostial cells. However, developmental fates and functions of heart-associated pericardial cells remain elusive. Here, we show that the pericardial cells that express the transcription factor Even Skipped adopt distinct fates along the anterior-posterior axis. Among them, the most anterior Antp-Ubx-AbdA - negative cells form a novel cardiac outflow component we call the outflow hanging structure, whereas the Antp-expressing cells differentiate into wing heart precursors. Interestingly, Hox gene expression in the Even Skipped-positive cells not only underlies their antero-posterior diversification, but also influences heart morphogenesis in a non-cell-autonomous way. In brief, we identify a new cardiac outflow component derived from a subset of Even Skipped-expressing cells that stabilises the anterior heart tip, and demonstrate non-cell-autonomous effects of Hox gene expression in the Even Skipped-positive cells on heart morphogenesis. © 2018. Published by The Company of Biologists Ltd.

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

Foxl2 function in ovarian development.

PubMed

Uhlenhaut, Nina Henriette; Treier, Mathias

2006-07-01

Foxl2 is a forkhead transcription factor essential for proper reproductive function in females. Human patients carrying mutations in the FOXL2 gene display blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), an autosomal dominant disease associated with eyelid defects and premature ovarian failure in females. Recently, animal models for BPES have been developed that in combination with a catalogue of human FOXL2 mutations provide further insight into its molecular function. Mice homozygous mutant for Foxl2 display craniofacial malformations and female infertility. The analysis of the murine phenotype has revealed that Foxl2 is required for granulosa cell function. These ovarian somatic cells surround and nourish the oocyte and play an important role in follicle formation and activation. Mutations upstream of FOXL2 in humans, not affecting the coding sequence itself, have also been shown to cause BPES, which points to the existence of a distant regulatory element necessary for proper gene expression. The same regulatory sequences may be deleted in the goat polled intersex syndrome (PIS), in which FoxL2 expression is severely reduced. Sequence comparison of FoxL2 from several vertebrate species has shown that it is a highly conserved gene involved in ovary development. Thus, the detailed understanding of Foxl2 function and regulation and the identification of its transcriptional targets may open new avenues for the treatment of female infertility in the future.

Chromatin landscape and circadian dynamics: Spatial and temporal organization of clock transcription

PubMed Central

Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

2015-01-01

Circadian rhythms drive the temporal organization of a wide variety of physiological and behavioral functions in ∼24-h cycles. This control is achieved through a complex program of gene expression. In mammals, the molecular clock machinery consists of interconnected transcriptional–translational feedback loops that ultimately ensure the proper oscillation of thousands of genes in a tissue-specific manner. To achieve circadian transcriptional control, chromatin remodelers serve the clock machinery by providing appropriate oscillations to the epigenome. Recent findings have revealed the presence of circadian interactomes, nuclear “hubs” of genome topology where coordinately expressed circadian genes physically interact in a spatial and temporal-specific manner. Thus, a circadian nuclear landscape seems to exist, whose interplay with metabolic pathways and clock regulators translates into specific transcriptional programs. Deciphering the molecular mechanisms that connect the circadian clock machinery with the nuclear landscape will reveal yet unexplored pathways that link cellular metabolism to epigenetic control. PMID:25378702

Gene Expression Profiling Identifies FKBP39 as an Inhibitor of Autophagy in Larval Drosophila Fat Body

PubMed Central

Juhász, Gábor; Puskás, László G.; Komonyi, Orbán; Érdi, Balázs; Maróy, Péter; Neufeld, Thomas P.; Sass, Miklós

2007-01-01

In Drosophila, the fat body undergoes a massive burst of autophagy at the end of larval development in preparation for the pupal transition. To identify genes involved in this process, we carried out a microarray analysis. We found that mRNA levels of the homologs of Atg8, the coat protein of early autophagic structures, and lysosomal hydrolases were upregulated, consistent with previous results. Genes encoding mitochondrial proteins and many chaperones were downregulated, including the inhibitor of eIF2alpha kinases and the peptidyl-prolyl cis-trans isomerase (PPiase) FKBP39. Genetic manipulation of FKBP39 expression had a significant effect on autophagy, potentially through modulation of the transcription factor Foxo. Accordingly, we found that Foxo mutants can not properly undergo autophagy in response to starvation, and that overexpression of Foxo induces autophagy. PMID:17363962

Posttranscriptional control of neuronal development by microRNA networks.

PubMed

Gao, Fen-Biao

2008-01-01

The proper development of the nervous system requires precise spatial and temporal control of gene expression at both the transcriptional and translational levels. In different experimental model systems, microRNAs (miRNAs) - a class of small, endogenous, noncoding RNAs that control the translation and stability of many mRNAs - are emerging as important regulators of various aspects of neuronal development. Further dissection of the in vivo physiological functions of individual miRNAs promises to offer novel mechanistic insights into the gene regulatory networks that ensure the precise assembly of a functional nervous system.

Malignant pleural mesothelioma and mesothelial hyperplasia: A new molecular tool for the differential diagnosis.

PubMed

Bruno, Rossella; Alì, Greta; Giannini, Riccardo; Proietti, Agnese; Lucchi, Marco; Chella, Antonio; Melfi, Franca; Mussi, Alfredo; Fontanini, Gabriella

2017-01-10

Malignant pleural mesothelioma (MPM) is a rare asbestos related cancer, aggressive and unresponsive to therapies. Histological examination of pleural lesions is the gold standard of MPM diagnosis, although it is sometimes hard to discriminate the epithelioid type of MPM from benign mesothelial hyperplasia (MH).This work aims to define a new molecular tool for the differential diagnosis of MPM, using the expression profile of 117 genes deregulated in this tumour.The gene expression analysis was performed by nanoString System on tumour tissues from 36 epithelioid MPM and 17 MH patients, and on 14 mesothelial pleural samples analysed in a blind way. Data analysis included raw nanoString data normalization, unsupervised cluster analysis by Pearson correlation, non-parametric Mann Whitney U-test and molecular classification by the Uncorrelated Shrunken Centroid (USC) Algorithm.The Mann-Whitney U-test found 35 genes upregulated and 31 downregulated in MPM. The unsupervised cluster analysis revealed two clusters, one composed only of MPM and one only of MH samples, thus revealing class-specific gene profiles. The Uncorrelated Shrunken Centroid algorithm identified two classifiers, one including 22 genes and the other 40 genes, able to properly classify all the samples as benign or malignant using gene expression data; both classifiers were also able to correctly determine, in a blind analysis, the diagnostic categories of all the 14 unknown samples.In conclusion we delineated a diagnostic tool combining molecular data (gene expression) and computational analysis (USC algorithm), which can be applied in the clinical practice for the differential diagnosis of MPM.

Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains.

PubMed Central

Vázquez, M; Dávalos, A; de las Peñas, A; Sánchez, F; Quinto, C

1991-01-01

Nodulation by Rhizobium, Bradyrhizobium, and Azorhizobium species in the roots of legumes and nonlegumes requires the proper expression of plant genes and of both common and specific bacterial nodulation genes. The common nodABC genes form an operon or are physically mapped together in all species studied thus far. Rhizobium leguminosarum bv. phaseoli strains are classified in two groups. The type I group has reiterated nifHDK genes and a narrow host range of nodulation. The type II group has a single copy of the nifHDK genes and a wide host range of nodulation. We have found by genetic and nucleotide sequence analysis that in type I strain CE-3, the functional common nodA gene is separated from the nodBC genes by 20 kb and thus is transcriptionally separated from the latter genes. This novel organization could be the result of a complex rearrangement, as we found zones of identity between the two separated nodA and nodBC regions. Moreover, this novel organization of the common nodABC genes seems to be a general characteristic of R. leguminosarum bv. phaseoli type I strains. Despite the separation, the coordination of the expression of these genes seems not to be altered. PMID:1991718

Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

Cancer.gov

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic

Tissue Architecture and Microenvironment Sustain Hormone Signaling | Center for Cancer Research

Cancer.gov

Cells interact with their environments in part through protein receptors embedded in the cell membrane. Activation of a receptor by external signaling molecules sets off a complex chain of events within the cell that can result in alterations in protein structure and function and/or changes in gene expression. Proper integration of these signals is crucial for normal cell

Inferring nonlinear gene regulatory networks from gene expression data based on distance correlation.

PubMed

Guo, Xiaobo; Zhang, Ye; Hu, Wenhao; Tan, Haizhu; Wang, Xueqin

2014-01-01

Nonlinear dependence is general in regulation mechanism of gene regulatory networks (GRNs). It is vital to properly measure or test nonlinear dependence from real data for reconstructing GRNs and understanding the complex regulatory mechanisms within the cellular system. A recently developed measurement called the distance correlation (DC) has been shown powerful and computationally effective in nonlinear dependence for many situations. In this work, we incorporate the DC into inferring GRNs from the gene expression data without any underling distribution assumptions. We propose three DC-based GRNs inference algorithms: CLR-DC, MRNET-DC and REL-DC, and then compare them with the mutual information (MI)-based algorithms by analyzing two simulated data: benchmark GRNs from the DREAM challenge and GRNs generated by SynTReN network generator, and an experimentally determined SOS DNA repair network in Escherichia coli. According to both the receiver operator characteristic (ROC) curve and the precision-recall (PR) curve, our proposed algorithms significantly outperform the MI-based algorithms in GRNs inference.

Inferring Nonlinear Gene Regulatory Networks from Gene Expression Data Based on Distance Correlation

PubMed Central

Guo, Xiaobo; Zhang, Ye; Hu, Wenhao; Tan, Haizhu; Wang, Xueqin

2014-01-01

Nonlinear dependence is general in regulation mechanism of gene regulatory networks (GRNs). It is vital to properly measure or test nonlinear dependence from real data for reconstructing GRNs and understanding the complex regulatory mechanisms within the cellular system. A recently developed measurement called the distance correlation (DC) has been shown powerful and computationally effective in nonlinear dependence for many situations. In this work, we incorporate the DC into inferring GRNs from the gene expression data without any underling distribution assumptions. We propose three DC-based GRNs inference algorithms: CLR-DC, MRNET-DC and REL-DC, and then compare them with the mutual information (MI)-based algorithms by analyzing two simulated data: benchmark GRNs from the DREAM challenge and GRNs generated by SynTReN network generator, and an experimentally determined SOS DNA repair network in Escherichia coli. According to both the receiver operator characteristic (ROC) curve and the precision-recall (PR) curve, our proposed algorithms significantly outperform the MI-based algorithms in GRNs inference. PMID:24551058

Insights into inner ear-specific gene regulation: epigenetics and non-coding RNAs in inner ear development and regeneration

PubMed Central

Avraham, Karen B.

2016-01-01

The vertebrate inner ear houses highly specialized sensory organs, tuned to detect and encode sound, head motion and gravity. Gene expression programs under the control of transcription factors orchestrate the formation and specialization of the non-sensory inner ear labyrinth and its sensory constituents. More recently, epigenetic factors and non-coding RNAs emerged as an additional layer of gene regulation, both in inner ear development and disease. In this review, we provide an overview on how epigenetic modifications and non-coding RNAs, in particular microRNAs (miRNAs), influence gene expression and summarize recent discoveries that highlight their critical role in the proper formation of the inner ear labyrinth and its sensory organs. In contrast to non-mammalian vertebrates, adult mammals lack the ability to regenerate inner ear mechano-sensory hair cells. Finally, we discuss recent insights into how epigenetic factors and miRNAs may facilitate, or in the case of mammals, restrict sensory hair cell regeneration. PMID:27836639

The ins and outs of editing and splicing of plastid RNAs: lessons from parasitic plants.

PubMed

Tillich, Michael; Krause, Kirsten

2010-07-31

In chloroplasts of higher plants, editing and splicing of transcripts is a prerequisite for the proper expression of the plastid genetic information and thereby for photosynthesis. Holoparasitic plants differ from photosynthetic plants in that they have abandoned a photoautotrophic life style, which has led to a reduction or loss of photosynthetic activity. The analysis of several parasitic plant plastid genomes revealed that coding capacities were reduced to different extent, encompassing genes that regulate plastid gene expression as well as photosynthesis genes. The reorganization of the plastid genome is also reflected in overall increases in point mutation rates that parallel the vanishing of RNA editing sites. Unprecedented in land plants is the parallel loss of the plastid gene coding for an intron maturase and all but one group IIa introns in two parasitic species. These observations highlight the plastome-wide effects that are associated with a relaxed evolutionary pressure in plants living a heterotrophic life style. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Definition of Cis-Acting Elements Regulating Expression of the Drosophila Melanogaster Ninae Opsin Gene by Oligonucleotide-Directed Mutagenesis

PubMed Central

Mismer, D.; Rubin, G. M.

1989-01-01

We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter. PMID:2521839

Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, Bombyx mori.

PubMed

Cheng, Yang; Wang, Xue-yang; Du, Chang; Gao, Juan; Xu, Jia-ping

2014-05-30

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a highly pathogenic virus in the sericultural industry, often causing severe damage leading to large economic losses. The immune mechanisms of B. mori against this virus remain obscure. Previous studies had demonstrated Bmlipase-1, BmNox and Bmserine protease-2 showing antiviral activity in vitro, but data on the transcription levels of these proteins in different resistant strains were not reported. In order to determine the resistance level of the four different strains (P50, A35, A40, A53) and gain a better understanding of the mechanism of resistance to BmNPV in B. mori, the relative expression level of the genes coding the three antiviral proteins in larval haemolymph and midgut of different B. mori strains resistant to BmNPV was determined. The results showed that these genes expressed significantly higher in the resistant strains compared to the susceptible strain, and the differential expression levels were consistent with the LC50 values in different strains. The transcription level of the target genes almost all up-regulated in the larvae midgut and down-regulated in the haemolymph. The results indicate the correlation of these genes to BmNPV resistance in B. mori. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

Genome-wide profiling of chromosome interactions in Plasmodium falciparum characterizes nuclear architecture and reconfigurations associated with antigenic variation

PubMed Central

Lemieux, Jacob E; Kyes, Sue A; Otto, Thomas D; Feller, Avi I; Eastman, Richard T; Pinches, Robert A; Berriman, Matthew; Su, Xin-zhuan; Newbold, Chris I

2013-01-01

Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites. PMID:23980881

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants[OPEN

PubMed Central

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Havaux, Michel; Schmülling, Thomas

2016-01-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. PMID:27354555

Whole blood genome-wide gene expression profile in males after prolonged wakefulness and sleep recovery.

PubMed

Pellegrino, R; Sunaga, D Y; Guindalini, C; Martins, R C S; Mazzotti, D R; Wei, Z; Daye, Z J; Andersen, M L; Tufik, S

2012-11-01

Although the specific functions of sleep have not been completely elucidated, the literature has suggested that sleep is essential for proper homeostasis. Sleep loss is associated with changes in behavioral, neurochemical, cellular, and metabolic function as well as impaired immune response. Using high-resolution microarrays we evaluated the gene expression profiles of healthy male volunteers who underwent 60 h of prolonged wakefulness (PW) followed by 12 h of sleep recovery (SR). Peripheral whole blood was collected at 8 am in the morning before the initiation of PW (Baseline), after the second night of PW, and one night after SR. We identified over 500 genes that were differentially expressed. Notably, these genes were related to DNA damage and repair and stress response, as well as diverse immune system responses, such as natural killer pathways including killer cell lectin-like receptors family, as well as granzymes and T-cell receptors, which play important roles in host defense. These results support the idea that sleep loss can lead to alterations in molecular processes that result in perturbation of cellular immunity, induction of inflammatory responses, and homeostatic imbalance. Moreover, expression of multiple genes was downregulated following PW and upregulated after SR compared with PW, suggesting an attempt of the body to re-establish internal homeostasis. In silico validation of alterations in the expression of CETN3, DNAJC, and CEACAM genes confirmed previous findings related to the molecular effects of sleep deprivation. Thus, the present findings confirm that the effects of sleep loss are not restricted to the brain and can occur intensely in peripheral tissues.

A statistical approach to identify, monitor, and manage incomplete curated data sets.

PubMed

Howe, Douglas G

2018-04-02

Many biological knowledge bases gather data through expert curation of published literature. High data volume, selective partial curation, delays in access, and publication of data prior to the ability to curate it can result in incomplete curation of published data. Knowing which data sets are incomplete and how incomplete they are remains a challenge. Awareness that a data set may be incomplete is important for proper interpretation, to avoiding flawed hypothesis generation, and can justify further exploration of published literature for additional relevant data. Computational methods to assess data set completeness are needed. One such method is presented here. In this work, a multivariate linear regression model was used to identify genes in the Zebrafish Information Network (ZFIN) Database having incomplete curated gene expression data sets. Starting with 36,655 gene records from ZFIN, data aggregation, cleansing, and filtering reduced the set to 9870 gene records suitable for training and testing the model to predict the number of expression experiments per gene. Feature engineering and selection identified the following predictive variables: the number of journal publications; the number of journal publications already attributed for gene expression annotation; the percent of journal publications already attributed for expression data; the gene symbol; and the number of transgenic constructs associated with each gene. Twenty-five percent of the gene records (2483 genes) were used to train the model. The remaining 7387 genes were used to test the model. One hundred and twenty-two and 165 of the 7387 tested genes were identified as missing expression annotations based on their residuals being outside the model lower or upper 95% confidence interval respectively. The model had precision of 0.97 and recall of 0.71 at the negative 95% confidence interval and precision of 0.76 and recall of 0.73 at the positive 95% confidence interval. This method can be used to identify data sets that are incompletely curated, as demonstrated using the gene expression data set from ZFIN. This information can help both database resources and data consumers gauge when it may be useful to look further for published data to augment the existing expertly curated information.

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

PubMed

Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

2015-09-01

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum

PubMed Central

Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

2015-01-01

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a “seesaw model” in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors. PMID:26360497

Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A.

PubMed

Sánchez, Marta; Prim, Núria; Rández-Gil, Francisca; Pastor, F I Javier; Diaz, Pilar

2002-05-05

Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive. Copyright 2002 Wiley Periodicals, Inc.

Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

PubMed

Fu, Lijuan; Shi, Zhimin; Luo, Guanzheng; Tu, Weihong; Wang, XiuJie; Fang, Zhide; Li, XiaoChing

2014-10-01

Mutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3' untranslated region (3' UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs). We identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3' UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed. Our results reveal novel regulatory functions of the human FOXP2 3' UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.

HSF and Msn2/4p can exclusively or cooperatively activate the yeast HSP104 gene.

PubMed

Grably, Melanie R; Stanhill, Ariel; Tell, Osnat; Engelberg, David

2002-04-01

In an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5' deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Deltamsn4Delta cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2Delta cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Deltamsn2Deltamsn4Delta cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.

Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation.

PubMed

Nambu-Nishida, Yumiko; Sakihama, Yuri; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

2018-01-01

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, P TDH3 , P FBA1 , and P TDH1 were favorable for high expression, and P SED1 , P HXT7 , P PDC1 , P TEF1 , P TPI1 , and P PGK1 were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. P TEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. P ZWF1 and P SOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. P ALD3 and P TKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Positron emission tomography and gene therapy: basic concepts and experimental approaches for in vivo gene expression imaging.

PubMed

Peñuelas, Iván; Boán, JoséF; Martí-Climent, Josep M; Sangro, Bruno; Mazzolini, Guillermo; Prieto, Jesús; Richter, José A

2004-01-01

More than two decades of intense research have allowed gene therapy to move from the laboratory to the clinical setting, where its use for the treatment of human pathologies has been considerably increased in the last years. However, many crucial questions remain to be solved in this challenging field. In vivo imaging with positron emission tomography (PET) by combination of the appropriate PET reporter gene and PET reporter probe could provide invaluable qualitative and quantitative information to answer multiple unsolved questions about gene therapy. PET imaging could be used to define parameters not available by other techniques that are of substantial interest not only for the proper understanding of the gene therapy process, but also for its future development and clinical application in humans. This review focuses on the molecular biology basis of gene therapy and molecular imaging, describing the fundamentals of in vivo gene expression imaging by PET, and the application of PET to gene therapy, as a technology that can be used in many different ways. It could be applied to avoid invasive procedures for gene therapy monitoring; accurately diagnose the pathology for better planning of the most adequate therapeutic approach; as treatment evaluation to image the functional effects of gene therapy at the biochemical level; as a quantitative noninvasive way to monitor the location, magnitude and persistence of gene expression over time; and would also help to a better understanding of vector biology and pharmacology devoted to the development of safer and more efficient vectors.

Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

PubMed

Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

2016-01-01

An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg. © 2015 FEBS.

Differential expression of antimicrobial peptides in active and latent tuberculosis and its relationship with diabetes mellitus.

PubMed

Gonzalez-Curiel, Irma; Castañeda-Delgado, Julio; Lopez-Lopez, Nallely; Araujo, Zaida; Hernandez-Pando, Rogelio; Gandara-Jasso, Benjamin; Macias-Segura, Noe; Enciso-Moreno, Antonio; Rivas-Santiago, Bruno

2011-08-01

Tuberculosis (TB) is one of the most important infectious diseases, causing 1.8 million deaths annually worldwide. This problem has increased because of the association with human immmunodeficiency virus and diabetes mellitus type 2, mainly in developing countries. In the past few years it has been highlighted the significance of antimicrobial peptides in the immunopathogenesis of TB ex vivo and in experimental models studies. In this study we analyzed the expression of CAMP, DEFA1, DEFB4, and DEFB103A in patients with latent TB and progressive TB with and without comorbidity with diabetes mellitus type 2. Antimicrobial peptide gene expression increased during progressive TB, which could be used as a biomarker for reactivation. By contrast, patients with diabetes mellitus type 2 have lower antimicrobial peptides gene expression, suggesting that the lack of its proper production in these patients contribute to enhance the risk for TB reactivation. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development.

PubMed

Ma, Jingqun; Brennan, Kaelan J; D'Aloia, Mitch R; Pascuzzi, Pete E; Weake, Vikki M

2016-08-09

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B): Nonstop, Sgf11, E(y)2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7). Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp) is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia. Copyright © 2016 Ma et al.

High density DNA microarrays: algorithms and biomedical applications.

PubMed

Liu, Wei-Min

2004-08-01

DNA microarrays are devices capable of detecting the identity and abundance of numerous DNA or RNA segments in samples. They are used for analyzing gene expressions, identifying genetic markers and detecting mutations on a genomic scale. The fundamental chemical mechanism of DNA microarrays is the hybridization between probes and targets due to the hydrogen bonds of nucleotide base pairing. Since the cross hybridization is inevitable, and probes or targets may form undesirable secondary or tertiary structures, the microarray data contain noise and depend on experimental conditions. It is crucial to apply proper statistical algorithms to obtain useful signals from noisy data. After we obtained the signals of a large amount of probes, we need to derive the biomedical information such as the existence of a transcript in a cell, the difference of expression levels of a gene in multiple samples, and the type of a genetic marker. Furthermore, after the expression levels of thousands of genes or the genotypes of thousands of single nucleotide polymorphisms are determined, it is usually important to find a small number of genes or markers that are related to a disease, individual reactions to drugs, or other phenotypes. All these applications need careful data analyses and reliable algorithms.

LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome

PubMed Central

Ariel, Federico; Latrasse, David; Mariappan, Kiruthiga Gayathri; Kim, Soon-Kap; Crespi, Martin; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2016-01-01

Precise expression patterns of genes in time and space are essential for proper development of multicellular organisms. Dynamic chromatin conformation and spatial organization of the genome constitute a major step in this regulation to modulate developmental outputs. Polycomb repressive complexes (PRCs) mediate stable or flexible gene repression in response to internal and environmental cues. In Arabidopsis thaliana, LHP1 co-localizes with H3K27me3 epigenetic marks throughout the genome and interacts with PRC1 and PRC2 members as well as with a long noncoding RNA. Here, we show that LHP1 is responsible for the spreading of H3K27me3 towards the 3’ end of the gene body. We also identified a subset of LHP1-activated genes and demonstrated that LHP1 shapes local chromatin topology in order to control transcriptional co-regulation. Our work reveals a general role of LHP1 from local to higher conformation levels of chromatin configuration to determine its accessibility to define gene expression patterns. PMID:27410265

CHD1 regulates cell fate determination by activation of differentiation-induced genes

PubMed Central

Baumgart, Simon J.; Najafova, Zeynab; Hossan, Tareq; Xie, Wanhua; Nagarajan, Sankari; Kari, Vijayalakshmi; Ditzel, Nicholas; Kassem, Moustapha

2017-01-01

Abstract The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination. PMID:28475736

CHD1 regulates cell fate determination by activation of differentiation-induced genes.

PubMed

Baumgart, Simon J; Najafova, Zeynab; Hossan, Tareq; Xie, Wanhua; Nagarajan, Sankari; Kari, Vijayalakshmi; Ditzel, Nicholas; Kassem, Moustapha; Johnsen, Steven A

2017-07-27

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

PubMed

Chen, B; Han, B H; Sun, X H; Lim, R W

1997-01-15

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

PubMed Central

Chen, B; Han, B H; Sun, X H; Lim, R W

1997-01-01

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected. PMID:9016574

Developmental expression of the Notch signaling pathway genes during mouse preimplantation development.

PubMed

Cormier, Sarah; Vandormael-Pournin, Sandrine; Babinet, Charles; Cohen-Tannoudji, Michel

2004-10-01

Notch signaling is an evolutionary conserved pathway involved in intercellular signaling and essential for proper cell fate choices during development. Thus, it could be involved in mouse preimplantation development where intercellular signaling plays a crucial role, particularly between the inner cell mass and the trophectoderm of the blastocyst. At their face value, the phenotypes observed when disrupting each of the four Notch genes known in the mouse do not support this view as none of them involves perturbation of preimplantation development. However this could be due to functional redundancy and/or maternal expression. As a first step to address this issue, we decided to examine the expression in early development of various genes known to participate in Notch signaling. Here, we report on the expression pattern of Notch1-4, Jagged1 (Jag1), Jag2, Delta-like1 (Dll-1), Dll-3, Dll-4, Rbpsuh, Deltex1(Dtx1)and Dtx2 genes during preimplantation development from unfertilized eggs until late blastocyst stage using a RT-PCR strategy. We show that Notch1, 2, Jag1-2, Dll-3, Rbpsuh and Dtx2 transcripts are expressed at all stages. Notch4 and Dll-4 mRNAs are synthesized from the 2-cell through to the hatched blastocyst stage. Notch3, Dll-1 and Dtx1exhibit a stage dependent expression as their mRNAs are detected in 2-cell embryos and in hatched blastocysts, but are absent or weakly detected at the morula stage. Finally, we show that all the above genes are expressed both in Embryonic and Trophoblast Stem cells (ES and TS cells, respectively). Our results suggest that the Notch pathway may be active during mouse preimplantation development.

Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation.

PubMed

Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie; Courtin, Christophe M; Verstrepen, Kevin J

2013-12-01

The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate pathways associated with starvation and stress responses. Further analysis shows that genes regulated by the high-osmolarity glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces the fermentation capacity. Together, our results demonstrate that cells embedded in a solid matrix such as bread dough suffer severe osmotic stress and that a proper induction of the HOG pathway is critical for optimal fermentation.

EGO-1, a C. elegans RdRP, Modulates Gene Expression via Production of mRNA-Templated Short Antisense RNAs

PubMed Central

Maniar, Jay M.; Fire, Andrew Z.

2011-01-01

SUMMARY Background The development of the germline in Caenorhabditis elegans is a complex process involving the regulation of thousands of genes in a coordinated manner. Several genes required for small RNA biogenesis and function are among those required for the proper organization of the germline. EGO-1 is a putative RNA-directed RNA polymerase (RdRP) that is required for multiple aspects of C. elegans germline development and efficient RNAi of germline-expressed genes. RdRPs have been proposed to act through a variety of mechanisms including the post-transcriptional targeting of specific mRNAs as well as through a direct interaction with chromatin. Despite extensive investigation, the molecular role of EGO-1 has remained enigmatic. Results Here we use high-throughput small RNA and messenger RNA sequencing to investigate EGO-1 function. We found that EGO-1 is required to produce a distinct pool of small RNAs antisense to a number of germline-expressed mRNAs through several developmental stages. These potential mRNA targets fall into distinct classes, including genes required for kinetochore and nuclear pore assembly, histone-modifying activities and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong association between the loss of small RNAs and the rise of mRNA levels in ego-1(−) animals. Conclusions Our data support the conclusion that EGO-1 produces triphosphorylated small RNAs derived from mRNA templates and that these small RNAs modulate gene expression through the targeting of their cognate mRNAs. PMID:21396820

CHESS (CgHExpreSS): a comprehensive analysis tool for the analysis of genomic alterations and their effects on the expression profile of the genome.

PubMed

Lee, Mikyung; Kim, Yangseok

2009-12-16

Genomic alterations frequently occur in many cancer patients and play important mechanistic roles in the pathogenesis of cancer. Furthermore, they can modify the expression level of genes due to altered copy number in the corresponding region of the chromosome. An accumulating body of evidence supports the possibility that strong genome-wide correlation exists between DNA content and gene expression. Therefore, more comprehensive analysis is needed to quantify the relationship between genomic alteration and gene expression. A well-designed bioinformatics tool is essential to perform this kind of integrative analysis. A few programs have already been introduced for integrative analysis. However, there are many limitations in their performance of comprehensive integrated analysis using published software because of limitations in implemented algorithms and visualization modules. To address this issue, we have implemented the Java-based program CHESS to allow integrative analysis of two experimental data sets: genomic alteration and genome-wide expression profile. CHESS is composed of a genomic alteration analysis module and an integrative analysis module. The genomic alteration analysis module detects genomic alteration by applying a threshold based method or SW-ARRAY algorithm and investigates whether the detected alteration is phenotype specific or not. On the other hand, the integrative analysis module measures the genomic alteration's influence on gene expression. It is divided into two separate parts. The first part calculates overall correlation between comparative genomic hybridization ratio and gene expression level by applying following three statistical methods: simple linear regression, Spearman rank correlation and Pearson's correlation. In the second part, CHESS detects the genes that are differentially expressed according to the genomic alteration pattern with three alternative statistical approaches: Student's t-test, Fisher's exact test and Chi square test. By successive operations of two modules, users can clarify how gene expression levels are affected by the phenotype specific genomic alterations. As CHESS was developed in both Java application and web environments, it can be run on a web browser or a local machine. It also supports all experimental platforms if a properly formatted text file is provided to include the chromosomal position of probes and their gene identifiers. CHESS is a user-friendly tool for investigating disease specific genomic alterations and quantitative relationships between those genomic alterations and genome-wide gene expression profiling.

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

Accurate and fast multiple-testing correction in eQTL studies.

PubMed

Sul, Jae Hoon; Raj, Towfique; de Jong, Simone; de Bakker, Paul I W; Raychaudhuri, Soumya; Ophoff, Roel A; Stranger, Barbara E; Eskin, Eleazar; Han, Buhm

2015-06-04

In studies of expression quantitative trait loci (eQTLs), it is of increasing interest to identify eGenes, the genes whose expression levels are associated with variation at a particular genetic variant. Detecting eGenes is important for follow-up analyses and prioritization because genes are the main entities in biological processes. To detect eGenes, one typically focuses on the genetic variant with the minimum p value among all variants in cis with a gene and corrects for multiple testing to obtain a gene-level p value. For performing multiple-testing correction, a permutation test is widely used. Because of growing sample sizes of eQTL studies, however, the permutation test has become a computational bottleneck in eQTL studies. In this paper, we propose an efficient approach for correcting for multiple testing and assess eGene p values by utilizing a multivariate normal distribution. Our approach properly takes into account the linkage-disequilibrium structure among variants, and its time complexity is independent of sample size. By applying our small-sample correction techniques, our method achieves high accuracy in both small and large studies. We have shown that our method consistently produces extremely accurate p values (accuracy > 98%) for three human eQTL datasets with different sample sizes and SNP densities: the Genotype-Tissue Expression pilot dataset, the multi-region brain dataset, and the HapMap 3 dataset. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Domain-swapped T cell receptors improve the safety of TCR gene therapy

PubMed Central

Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

2016-01-01

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

Transcript and protein environmental biomarkers in fish--a review.

PubMed

Tom, Moshe; Auslander, Meirav

2005-04-01

The levels of contaminant-affected gene products (transcripts and proteins) are increasingly utilized as environmental biomarkers, and their appropriate implementation as diagnostic tools is discussed. The required characteristics of a gene product biomarker are accurate evaluation using properly normalized absolute units, aiming at long-term comparability of biomarker levels over a wide geographical range and among many laboratories. Quantitative RT-PCR and competitive ELISA are suggested as preferred evaluation methods for transcript and protein, respectively. Constitutively expressed RNAs or proteins which are part of the examined homogenate are suggested as normalizing agents, compensating for variable processing efficiency. Essential characterization of expression patterns is suggested, providing reference values to be compared to the monitored levels. This comparison would enable estimation of the intensity of biological effects of contaminants. Contaminant-independent reference expression patterns should include natural fluctuations of the biomarker level. Contaminant-dependent patterns should include dose response to model contaminants chronically administered in two environmentally-realistic routes, reaching extreme sub-lethal affected levels. Recent studies using fish as environmental sentinel species, applying gene products as environmental biomarkers, and implementing at least part of the depicted methodologies are reviewed.

Bmp2 Deletion Causes an Amelogenesis Imperfecta Phenotype Via Regulating Enamel Gene Expression

PubMed Central

GUO, FENG; FENG, JUNSHENG; WANG, FENG; LI, WENTONG; GAO, QINGPING; CHEN, ZHUO; SHOFF, LISA; DONLY, KEVIN J.; GLUHAK-HEINRICH, JELICA; CHUN, YONG HEE PATRICIA; HARRIS, STEPHEN E.; MACDOUGALL, MARY; CHEN, SHUO

2015-01-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. PMID:25545831

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

PubMed

Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

2015-08-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. © 2015 Wiley Periodicals, Inc.

The mammalian homologue of mago nashi encodes a serum-inducible protein.

PubMed

Zhao, X F; Colaizzo-Anas, T; Nowak, N J; Shows, T B; Elliott, R W; Aplan, P D

1998-01-15

The products of at least 11 maternal effect genes have been shown to be essential for proper germ plasm assembly in Drosophila melanogaster embryos. Here we report the isolation and characterization of the mammalian counterpart for one of these genes (named MAGOH for mago nashi homologue). The predicted amino acid sequence of mouse and human MAGOH are completely identical; MAGOH homologues from the nematode Caenorhabditis elegans and rice grain Oryza sativa also show a remarkable degree of amino acid conservation. MAGOH was mapped to chromosome 1p33-p34 in the human and a syntenic region of chromosome 4 in the mouse. Of note, MAGOH mRNA expression is not limited to germ plasm, but is expressed ubiquitously in adult tissues and can be induced by serum stimulation of quiescent fibroblasts.

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

PubMed Central

Wakabayashi, Shunichi; Soma, Atsumi; Sato, Saeko; Nakatake, Yuhki; Oda, Mayumi; Murakami, Miyako; Sakota, Miki; Chikazawa-Nohtomi, Nana

2016-01-01

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings. PMID:27802135

Functional Neuroprotection and Efficient Regulation of GDNF Using Destabilizing Domains in a Rodent Model of Parkinson's Disease

PubMed Central

Quintino, Luis; Manfré, Giuseppe; Wettergren, Erika Elgstrand; Namislo, Angrit; Isaksson, Christina; Lundberg, Cecilia

2013-01-01

Glial cell line–derived neurotrophic factor (GDNF) has great potential to treat Parkinson's disease (PD). However, constitutive expression of GDNF can over time lead to side effects. Therefore, it would be useful to regulate GDNF expression. Recently, a new gene inducible system using destabilizing domains (DD) from E. coli dihydrofolate reductase (DHFR) has been developed and characterized. The advantage of this novel DD is that it is regulated by trimethoprim (TMP), a well-characterized drug that crosses the blood–brain barrier and can therefore be used to regulate gene expression in the brain. We have adapted this system to regulate expression of GDNF. A C-terminal fusion of GDNF and a DD with an additional furin cleavage site was able to be efficiently regulated in vitro, properly processed and was able to bind to canonical GDNF receptors, inducing a signaling cascade response in target cells. In vivo characterization of the protein showed that it could be efficiently induced by TMP and it was only functional when gene expression was turned on. Further characterization in a rodent model of PD showed that the regulated GDNF protected neurons, improved motor behavior of animals and was efficiently regulated in a pathological setting. PMID:23881415

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

PubMed Central

Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

2017-01-01

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors. DOI: http://dx.doi.org/10.7554/eLife.26287.001 PMID:28394252

Localization of male-specifically expressed MROS genes of Silene latifolia by PCR on flow-sorted sex chromosomes and autosomes.

PubMed

Kejnovský, E; Vrána, J; Matsunaga, S; Soucek, P; Siroký, J; Dolezel, J; Vyskot, B

2001-07-01

The dioecious white campion Silene latifolia (syn. Melandrium album) has heteromorphic sex chromosomes, XX in females and XY in males, that are larger than the autosomes and enable their separation by flow sorting. The group of MROS genes, the first male-specifically expressed genes in dioecious plants, was recently identified in S. latifolia. To localize the MROS genes, we used the flow-sorted X chromosomes and autosomes as a template for PCR with internal primers. Our results indicate that the MROS3 gene is located in at least two copies tandemly arranged on the X chromosome with additional copy(ies) on the autosome(s), while MROS1, MROS2, and MROS4 are exclusively autosomal. The specificity of PCR products was checked by digestion with a restriction enzyme or reamplification using nested primers. Homology search of databases has shown the presence of five MROS3 homologues in A. thaliana, four of them arranged in two tandems, each consisting of two copies. We conclude that MROS3 is a low-copy gene family, connected with the proper pollen development, which is present not only in dioecious but also in other dicot plant species.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Epigenetic regulation of intragenic transposable elements impacts gene transcription in Arabidopsis thaliana

PubMed Central

Le, Tu N.; Miyazaki, Yuji; Takuno, Shohei; Saze, Hidetoshi

2015-01-01

Genomes of higher eukaryotes, including plants, contain numerous transposable elements (TEs), that are often silenced by epigenetic mechanisms, such as histone modifications and DNA methylation. Although TE silencing adversely affects expression of nearby genes, recent studies reveal the presence of intragenic TEs marked by repressive heterochromatic epigenetic marks within transcribed genes. However, even for the well-studied plant model Arabidopsis thaliana, the abundance of intragenic TEs, how they are epigenetically regulated, and their potential impacts on host gene expression, remain unexplored. In this study, we comprehensively analyzed genome-wide distribution and epigenetic regulation of intragenic TEs in A. thaliana. Our analysis revealed that about 3% of TEs are located within gene bodies, dominantly at intronic regions. Most of them are shorter and less methylated than intergenic TEs, but they are still targeted by RNA-directed DNA methylation-dependent and independent pathways. Surprisingly, the heterochromatic epigenetic marks at TEs are maintained within actively transcribed genes. Moreover, the heterochromatic state of intronic TEs is critical for proper transcription of associated genes. Our study provides the first insight into how intragenic TEs affect the transcriptional landscape of the A. thaliana genome, and suggests the importance of epigenetic mechanisms for regulation of TEs within transcriptional gene units. PMID:25813042

COE loss-of-function analysis reveals a genetic program underlying maintenance and regeneration of the nervous system in planarians.

PubMed

Cowles, Martis W; Omuro, Kerilyn C; Stanley, Brianna N; Quintanilla, Carlo G; Zayas, Ricardo M

2014-10-01

Members of the COE family of transcription factors are required for central nervous system (CNS) development. However, the function of COE in the post-embryonic CNS remains largely unknown. An excellent model for investigating gene function in the adult CNS is the freshwater planarian. This animal is capable of regenerating neurons from an adult pluripotent stem cell population and regaining normal function. We previously showed that planarian coe is expressed in differentiating and mature neurons and that its function is required for proper CNS regeneration. Here, we show that coe is essential to maintain nervous system architecture and patterning in intact (uninjured) planarians. We took advantage of the robust phenotype in intact animals to investigate the genetic programs coe regulates in the CNS. We compared the transcriptional profiles of control and coe RNAi planarians using RNA sequencing and identified approximately 900 differentially expressed genes in coe knockdown animals, including 397 downregulated genes that were enriched for nervous system functional annotations. Next, we validated a subset of the downregulated transcripts by analyzing their expression in coe-deficient planarians and testing if the mRNAs could be detected in coe+ cells. These experiments revealed novel candidate targets of coe in the CNS such as ion channel, neuropeptide, and neurotransmitter genes. Finally, to determine if loss of any of the validated transcripts underscores the coe knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of coe-regulated genes implicated in CNS regeneration and patterning, including orthologs of sodium channel alpha-subunit and pou4. Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

PubMed Central

Blazie, Stephen M.; Geissel, Heather C.; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-01-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. PMID:28348061

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues.

PubMed

Blazie, Stephen M; Geissel, Heather C; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-06-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. Copyright © 2017 Blazie et al.

Bayesian inference of the number of factors in gene-expression analysis: application to human virus challenge studies.

PubMed

Chen, Bo; Chen, Minhua; Paisley, John; Zaas, Aimee; Woods, Christopher; Ginsburg, Geoffrey S; Hero, Alfred; Lucas, Joseph; Dunson, David; Carin, Lawrence

2010-11-09

Nonparametric Bayesian techniques have been developed recently to extend the sophistication of factor models, allowing one to infer the number of appropriate factors from the observed data. We consider such techniques for sparse factor analysis, with application to gene-expression data from three virus challenge studies. Particular attention is placed on employing the Beta Process (BP), the Indian Buffet Process (IBP), and related sparseness-promoting techniques to infer a proper number of factors. The posterior density function on the model parameters is computed using Gibbs sampling and variational Bayesian (VB) analysis. Time-evolving gene-expression data are considered for respiratory syncytial virus (RSV), Rhino virus, and influenza, using blood samples from healthy human subjects. These data were acquired in three challenge studies, each executed after receiving institutional review board (IRB) approval from Duke University. Comparisons are made between several alternative means of per-forming nonparametric factor analysis on these data, with comparisons as well to sparse-PCA and Penalized Matrix Decomposition (PMD), closely related non-Bayesian approaches. Applying the Beta Process to the factor scores, or to the singular values of a pseudo-SVD construction, the proposed algorithms infer the number of factors in gene-expression data. For real data the "true" number of factors is unknown; in our simulations we consider a range of noise variances, and the proposed Bayesian models inferred the number of factors accurately relative to other methods in the literature, such as sparse-PCA and PMD. We have also identified a "pan-viral" factor of importance for each of the three viruses considered in this study. We have identified a set of genes associated with this pan-viral factor, of interest for early detection of such viruses based upon the host response, as quantified via gene-expression data.

Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

PubMed

Hoffmann, H M; Catron, K M; van Wijnen, A J; McCabe, L R; Lian, J B; Stein, G S; Stein, J L

1994-12-20

The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

Separable roles of UFO during floral development revealed by conditional restoration of gene function.

PubMed

Laufs, Patrick; Coen, Enrico; Kronenberger, Jocelyne; Traas, Jan; Doonan, John

2003-02-01

The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.

A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis

PubMed Central

Platre, Matthieu Pierre; Barberon, Marie; Caillieux, Erwann; Colot, Vincent

2016-01-01

Summary Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis. PMID:26662936

Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR.

PubMed

Phillips, Brittany L; Banerjee, Ayan; Sanchez, Brenda J; Di Marco, Sergio; Gallouzi, Imed-Eddine; Pavlath, Grace K; Corbett, Anita H

2018-06-25

RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the Pabpn1 transcript is unstable, contributing to low steady-state Pabpn1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate Pabpn1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate Pabpn1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases Pabpn1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates Pabpn1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.

H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

PubMed Central

Subramanian, Vidya; Mazumder, Aprotim; Surface, Lauren E.; Butty, Vincent L.; Fields, Paul A.; Alwan, Allison; Torrey, Lillian; Thai, Kevin K.; Levine, Stuart S.; Bathe, Mark; Boyer, Laurie A.

2013-01-01

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.ZAP3) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.ZAP3 interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.ZAP3 was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.ZAP3 ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.ZAP3 ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.ZAP3 displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.ZAP3 mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment. PMID:23990805

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins

PubMed Central

Chen, Yunjia; Qiu, Shihong; Luan, Chi-Hao; Luo, Ming

2007-01-01

Background Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics. Results With bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies. Conclusion The DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics. PMID:17663785

microRNA-133: expression, function and therapeutic potential in muscle diseases and cancer.

PubMed

Yu, Hao; Lu, Yinhui; Li, Zhaofa; Wang, Qizhao

2014-01-01

microRNAs (miRNAs) are a class of small non-coding RNAs that are 18-25 nucleotides (nt) in length and negatively regulate gene expression post-transcriptionally. miRNAs are known to mediate myriad processes and pathways. While many miRNAs are expressed ubiquitously, some are expressed in a tissue specific manner. miR-133 is one of the most studied and best characterized miRNAs to date. Specifically expressed in muscles, it has been classified as myomiRNAs and is necessary for proper skeletal and cardiac muscle development and function. Genes encoding miR-133 (miR-133a-1, miR-133a-2 and miR-133b) are transcribed as bicistronic transcripts together with miR-1-2, miR-1-1, and miR-206, respectively. However, they exhibit opposing impacts on muscle development. miR-133 gets involved in muscle development by targeting a lot of genes, including SFR, HDAC4, cyclin D2 and so on. Its aberrant expression has been linked to many diseases in skeletal muscle and cardiac muscle such as cardiac hypertrophy, muscular dystrophy, heart failure, cardiac arrhythmia. Beyond the study in muscle, miR-133 has been implicated in cancer and identified as a key factor in cancer development, including bladder cancer, prostate cancer and so on. Much more attention has been drawn to the versatile molecular functions of miR-133, making it a truly valuable therapeutic gene in miRNA-based gene therapy. In this review, we identified and summarized the results of studies of miR-133 with emphasis on its function in human diseases in muscle and cancer, and highlighted its therapeutic value. It might provide researchers a new insight into the biological significance of miR-133.

Concerted action of two dlx paralogs in sensory placode formation.

PubMed

Solomon, Keely S; Fritz, Andreas

2002-07-01

Sensory placodes are ectodermal thickenings that give rise to elements of the vertebrate cranial sensory nervous system, including the inner ear and nose. Although mutations have been described in humans, mice and zebrafish that perturb ear and nose development, no mutation is known to prevent sensory placode formation. Thus, it has been postulated that a functional redundancy exists in the genetic mechanisms that govern sensory placode development. We describe a zebrafish deletion mutation, b380, which results in a lack of both otic and olfactory placodes. The b380 deletion removes several known genes and expressed sequence tags, including dlx3 and dlx7, two transcription factors that share a homoeobox domain similar in sequence to the Drosophila Distal-less gene. dlx3 and dlx7 are expressed in an overlapping pattern in the regions that produce the otic and olfactory placodes in zebrafish. We present evidence suggesting that it is specifically the removal of these two genes that leads to the otic and olfactory phenotype of b380 mutants. Using morpholinos, antisense oligonucleotides that effectively block translation of target genes, we find that functional reduction of both dlx genes contributes to placode loss. Expression patterns of the otic marker pax2.1, olfactory marker anxV and eya1, a marker of both placodes, in morpholino-injected embryos recapitulate the reduced expression of these genes seen in b380 mutants. We also examine expression of dlx3 and dlx7 in the morpholino-injected embryos and present evidence for existence of auto- and cross-regulatory control of expression among these genes. We demonstrate that dlx3 is necessary and sufficient for proper otic and olfactory placode development. However, our results indicate that dlx3 and dlx7 act in concert and their importance in placode formation is only revealed by inactivating both paralogs.

Genome-wide coexpression dynamics: Theory and application

PubMed Central

Li, Ker-Chau

2002-01-01

High-throughput expression profiling enables the global study of gene activities. Genes with positively correlated expression profiles are likely to encode functionally related proteins. However, all biological processes are interlocked, and each protein may play multiple cellular roles. Thus the coexpression of any two functionally related genes may depend on the constantly varying, yet often-unknown cellular state. To initiate a systematic study on this issue, a theory of coexpression dynamics is presented. This theory is used to rationalize a strategy of conducting a genome-wide search for the most critical cellular players that may affect the coexpression pattern of any two genes. In one example, using a yeast data set, our method reveals how the enzymes associated with the urea cycle are expressed to ensure proper mass flow of the involved metabolites. The correlation between ARG2 and CAR2 is found to change from positive to negative as the expression level of CPA2 increases. This delicate interplay in correlation signifies a remarkable control on the influx and efflux of ornithine and reflects well the intrinsic cellular demand for arginine. In addition to the urea cycle, our examples include SCH9 and CYR1 (both implicated in a recent longevity study), cytochrome c1 (mitochondrial electron transport), calmodulin (main calcium-binding protein), PFK1 and PFK2 (glycolysis), and two genes, ECM1 and YNL101W, the functions of which are newly revealed. The complexity in computation is eased by a new result from mathematical statistics. PMID:12486219

ArraySolver: an algorithm for colour-coded graphical display and Wilcoxon signed-rank statistics for comparing microarray gene expression data.

PubMed

Khan, Haseeb Ahmad

2004-01-01

The massive surge in the production of microarray data poses a great challenge for proper analysis and interpretation. In recent years numerous computational tools have been developed to extract meaningful interpretation of microarray gene expression data. However, a convenient tool for two-groups comparison of microarray data is still lacking and users have to rely on commercial statistical packages that might be costly and require special skills, in addition to extra time and effort for transferring data from one platform to other. Various statistical methods, including the t-test, analysis of variance, Pearson test and Mann-Whitney U test, have been reported for comparing microarray data, whereas the utilization of the Wilcoxon signed-rank test, which is an appropriate test for two-groups comparison of gene expression data, has largely been neglected in microarray studies. The aim of this investigation was to build an integrated tool, ArraySolver, for colour-coded graphical display and comparison of gene expression data using the Wilcoxon signed-rank test. The results of software validation showed similar outputs with ArraySolver and SPSS for large datasets. Whereas the former program appeared to be more accurate for 25 or fewer pairs (n < or = 25), suggesting its potential application in analysing molecular signatures that usually contain small numbers of genes. The main advantages of ArraySolver are easy data selection, convenient report format, accurate statistics and the familiar Excel platform.

ArraySolver: An Algorithm for Colour-Coded Graphical Display and Wilcoxon Signed-Rank Statistics for Comparing Microarray Gene Expression Data

PubMed Central

2004-01-01

The massive surge in the production of microarray data poses a great challenge for proper analysis and interpretation. In recent years numerous computational tools have been developed to extract meaningful interpretation of microarray gene expression data. However, a convenient tool for two-groups comparison of microarray data is still lacking and users have to rely on commercial statistical packages that might be costly and require special skills, in addition to extra time and effort for transferring data from one platform to other. Various statistical methods, including the t-test, analysis of variance, Pearson test and Mann–Whitney U test, have been reported for comparing microarray data, whereas the utilization of the Wilcoxon signed-rank test, which is an appropriate test for two-groups comparison of gene expression data, has largely been neglected in microarray studies. The aim of this investigation was to build an integrated tool, ArraySolver, for colour-coded graphical display and comparison of gene expression data using the Wilcoxon signed-rank test. The results of software validation showed similar outputs with ArraySolver and SPSS for large datasets. Whereas the former program appeared to be more accurate for 25 or fewer pairs (n ≤ 25), suggesting its potential application in analysing molecular signatures that usually contain small numbers of genes. The main advantages of ArraySolver are easy data selection, convenient report format, accurate statistics and the familiar Excel platform. PMID:18629036

TEG-1 CD2BP2 controls miRNA levels by regulating miRISC stability in C. elegans and human cells

PubMed Central

Wang, Chris; Gupta, Pratyush; Fressigne, Lucile; Bossé, Gabriel D.; Wang, Xin; Simard, Martin J.

2017-01-01

Abstract MiRNAs post-transcriptionally regulate gene expression by recruiting the miRNA-induced silencing complex (miRISC) to target mRNAs. However, the mechanisms by which miRISC components are maintained at appropriate levels for proper function are largely unknown. Here, we demonstrate that Caenorhabditis elegans TEG-1 regulates the stability of two miRISC effectors, VIG-1 and ALG-1, which in turn affects the abundance of miRNAs in various families. We demonstrate that TEG-1 physically interacts with VIG-1, and complexes with mature let-7 miRNA. Also, loss of teg-1 in vivo phenocopies heterochronic defects observed in let-7 mutants, suggesting the association of TEG-1 with miRISC is necessary for let-7 to function properly during development. Loss of TEG-1 function also affects the abundance and function of other microRNAs, suggesting that TEG-1's role is not specific to let-7. We further demonstrate that the human orthologs of TEG-1, VIG-1 and ALG-1 (CD2BP2, SERBP1/PAI-RBP1 and AGO2) are found in a complex in HeLa cells, and knockdown of CD2BP2 results in reduced miRNA levels; therefore, TEG-1's role in affecting miRNA levels and function is likely conserved. Together, these data demonstrate that TEG-1 CD2BP2 stabilizes miRISC and mature miRNAs, maintaining them at levels necessary to properly regulate target gene expression. PMID:28180320

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

PubMed

Mohammadzadeh, Sara; Roohvand, Farzin; Memarnejadian, Arash; Jafari, Anis; Ajdary, Soheila; Salmanian, Ali-Hatef; Ehsani, Parastoo

2016-01-01

Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

PubMed Central

Stibitz, S; Weiss, A A; Falkow, S

1988-01-01

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA. Images PMID:2898470

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study[W][OA

PubMed Central

Graeber, Kai; Linkies, Ada; Wood, Andrew T.A.; Leubner-Metzger, Gerhard

2011-01-01

Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR. PMID:21666000

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

PubMed

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu; Makaroff, Christopher; Ma, Hong; Wang, Yingxiang

2016-08-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. © 2016 American Society of Plant Biologists. All rights reserved.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3[OPEN

PubMed Central

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu

2016-01-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. PMID:27385818

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

PubMed

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Feussner, Ivo; Havaux, Michel; Riefler, Michael; Schmülling, Thomas

2016-07-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. © 2016 American Society of Plant Biologists. All rights reserved.

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

PubMed

Huang, Jinjin; Xia, Ji; Yang, Zhen; Guan, Feifei; Cui, Di; Guan, Guohua; Jiang, Wei; Li, Ying

2014-01-01

We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%. Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

Interaction of Electromagnetic Fields with Chondrocytes in Gel Culture

DTIC Science & Technology

1990-02-01

biosynthesis due to applied electromagnetic fields. The results suggest that both normal chondrocytes and swarm rat chondrosarcoma cells in agarose Culture...and Swarm rat chondrosarcoma cells in agarose iii culture can, under proper culture conditions, continue to synthesize ma- trix macromolecules at a...cartilagc, and in rat chondrosarcoma cells (a continuous cell line). The overt gene expression of chondrocytes results in the synthesis and deposition of a

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

PubMed Central

Spike, Caroline A.; Bader, Jason; Reinke, Valerie; Strome, Susan

2008-01-01

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). PMID:18234720

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells.

PubMed

Spike, Caroline A; Bader, Jason; Reinke, Valerie; Strome, Susan

2008-03-01

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs).

Synthetic data sets for the identification of key ingredients for RNA-seq differential analysis.

PubMed

Rigaill, Guillem; Balzergue, Sandrine; Brunaud, Véronique; Blondet, Eddy; Rau, Andrea; Rogier, Odile; Caius, José; Maugis-Rabusseau, Cathy; Soubigou-Taconnat, Ludivine; Aubourg, Sébastien; Lurin, Claire; Martin-Magniette, Marie-Laure; Delannoy, Etienne

2018-01-01

Numerous statistical pipelines are now available for the differential analysis of gene expression measured with RNA-sequencing technology. Most of them are based on similar statistical frameworks after normalization, differing primarily in the choice of data distribution, mean and variance estimation strategy and data filtering. We propose an evaluation of the impact of these choices when few biological replicates are available through the use of synthetic data sets. This framework is based on real data sets and allows the exploration of various scenarios differing in the proportion of non-differentially expressed genes. Hence, it provides an evaluation of the key ingredients of the differential analysis, free of the biases associated with the simulation of data using parametric models. Our results show the relevance of a proper modeling of the mean by using linear or generalized linear modeling. Once the mean is properly modeled, the impact of the other parameters on the performance of the test is much less important. Finally, we propose to use the simple visualization of the raw P-value histogram as a practical evaluation criterion of the performance of differential analysis methods on real data sets. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The primary role of zebrafish nanog is in extra-embryonic tissue.

PubMed

Gagnon, James A; Obbad, Kamal; Schier, Alexander F

2018-01-09

The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZ nanog ) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZ nanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation. © 2018. Published by The Company of Biologists Ltd.

Robustness of equations that define molecular subtypes of glioblastoma tumors based on five transcripts measured by RT-PCR.

PubMed

Castells, Xavier; Acebes, Juan José; Majós, Carles; Boluda, Susana; Julià-Sapé, Margarida; Candiota, Ana Paula; Ariño, Joaquín; Barceló, Anna; Arús, Carles

2015-01-01

Glioblastoma (Gb) is one of the most deadly tumors. Its molecular subtypes are yet to be fully characterized while the attendant efforts for personalized medicine need to be intensified in relation to glioblastoma diagnosis, treatment, and prognosis. Several molecular signatures based on gene expression microarrays were reported, but the use of microarrays for routine clinical practice is challenged by attendant economic costs. Several authors have proposed discriminant equations based on RT-PCR. Still, the discriminant threshold is often incompletely described, which makes proper validation difficult. In a previous work, we have reported two Gb subtypes based on the expression levels of four genes: CHI3L1, LDHA, LGALS1, and IGFBP3. One Gb subtype presented with low expression of the four genes mentioned, and of MGMT in a large portion of the patients (with anticipated high methylation of its promoter), and mutated IDH1. Here, we evaluate the robustness of the equations fitted with these genes using RT-PCR values in a set of 64 cases and importantly, define an unequivocal discriminant threshold with a view to prognostic implications. We developed two approaches to generate the discriminant equations: 1) using the expression level of the four genes mentioned above, and 2) using those genes displaying the highest correlation with survival among the aforementioned four ones, plus MGMT, as an attempt to further reduce the number of genes. The ease of equations' applicability, reduction in cost for raw data, and robustness in terms of resampling-based classification accuracy warrant further evaluation of these equations to discern Gb tumor biopsy heterogeneity at molecular level, diagnose potential malignancy, and prognosis of individual patients with glioblastomas.

Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6.

PubMed

Liu, Wan-Ju; Reece-Hoyes, John S; Walhout, Albertha J M; Eisenmann, David M

2014-05-13

Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo. We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status.

PubMed

Dai, Weijun; Li, Wencheng; Hoque, Mainul; Li, Zhuyun; Tian, Bin; Makeyev, Eugene V

2015-07-06

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons.

Hyperinnervation improves Xenopus laevis limb regeneration.

PubMed

Mitogawa, Kazumasa; Makanae, Aki; Satoh, Akira

2018-01-15

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamics of the Saccharomyces cerevisiae Transcriptome during Bread Dough Fermentation

PubMed Central

Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie

2013-01-01

The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate pathways associated with starvation and stress responses. Further analysis shows that genes regulated by the high-osmolarity glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces the fermentation capacity. Together, our results demonstrate that cells embedded in a solid matrix such as bread dough suffer severe osmotic stress and that a proper induction of the HOG pathway is critical for optimal fermentation. PMID:24056467

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

PubMed

Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Tena, Juan J; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F; Roy, Anna R; Galjart, Niels; Delgado-Olguin, Paul; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis; Manzanares, Miguel

2017-08-01

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart

PubMed Central

Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F.; Roy, Anna R.; Galjart, Niels; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis

2017-01-01

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development. PMID:28846746

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

PubMed

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

PubMed Central

Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

2016-01-01

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

Repression of Middle Sporulation Genes in Saccharomyces cerevisiae by the Sum1-Rfm1-Hst1 Complex Is Maintained by Set1 and H3K4 Methylation

PubMed Central

Jaiswal, Deepika; Jezek, Meagan; Quijote, Jeremiah; Lum, Joanna; Choi, Grace; Kulkarni, Rushmie; Park, DoHwan; Green, Erin M.

2017-01-01

The conserved yeast histone methyltransferase Set1 targets H3 lysine 4 (H3K4) for mono, di, and trimethylation and is linked to active transcription due to the euchromatic distribution of these methyl marks and the recruitment of Set1 during transcription. However, loss of Set1 results in increased expression of multiple classes of genes, including genes adjacent to telomeres and middle sporulation genes, which are repressed under normal growth conditions because they function in meiotic progression and spore formation. The mechanisms underlying Set1-mediated gene repression are varied, and still unclear in some cases, although repression has been linked to both direct and indirect action of Set1, associated with noncoding transcription, and is often dependent on the H3K4me2 mark. We show that Set1, and particularly the H3K4me2 mark, are implicated in repression of a subset of middle sporulation genes during vegetative growth. In the absence of Set1, there is loss of the DNA-binding transcriptional regulator Sum1 and the associated histone deacetylase Hst1 from chromatin in a locus-specific manner. This is linked to increased H4K5ac at these loci and aberrant middle gene expression. These data indicate that, in addition to DNA sequence, histone modification status also contributes to proper localization of Sum1. Our results also show that the role for Set1 in middle gene expression control diverges as cells receive signals to undergo meiosis. Overall, this work dissects an unexplored role for Set1 in gene-specific repression, and provides important insights into a new mechanism associated with the control of gene expression linked to meiotic differentiation. PMID:29066473

Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822).

PubMed

López-Landavery, Edgar A; Portillo-López, Amelia; Gallardo-Escárate, Cristian; Del Río-Portilla, Miguel A

2014-10-10

The red abalone Haliotis rufescens is one of the most important species for aquaculture in Baja California, México, and despite this, few gene expression studies have been done in tissues such as gill, head and gonad. For this purpose, reverse transcription and quantitative real time PCR (RT-qPCR) is a powerful tool for gene expression evaluation. For a reliable analysis, however, it is necessary to select and validate housekeeping genes that allow proper transcription quantification. Stability of nine housekeeping genes (ACTB, BGLU, TUBB, CY, GAPDH, HPRTI, RPL5, SDHA and UBC) was evaluated in different tissues of red abalone (gill, head and gonad/digestive gland). Four-fold serial dilutions of cDNA (from 25 ngμL(-1) to 0.39 ngμL(-1)) were used to prepare the standard curve, and it showed gene efficiencies between 0.95 and 0.99, with R(2)=0.99. geNorm and NormFinder analysis showed that RPL5 and CY were the most stable genes considering all tissues, whereas in gill HPRTI and BGLU were most stable. In gonad/digestive gland, RPL5 and TUBB were the most stable genes with geNorm, while SDHA and HPRTI were the best using NormFinder. Similarly, in head the best genes were RPL5 and UBC with geNorm, and GAPDH and CY with NormFinder. The technical variability analysis with RPL5 and abalone gonad/digestive gland tissue indicated a high repeatability with a variation coefficient within groups ≤ 0.56% and between groups ≤ 1.89%. These results will help us for further research in reproduction, thermoregulation and endocrinology in red abalone. Copyright © 2014 Elsevier B.V. All rights reserved.

Adaptations to High Salt in a Halophilic Protist: Differential Expression and Gene Acquisitions through Duplications and Gene Transfers

PubMed Central

Harding, Tommy; Roger, Andrew J.; Simpson, Alastair G. B.

2017-01-01

The capacity of halophiles to thrive in extreme hypersaline habitats derives partly from the tight regulation of ion homeostasis, the salt-dependent adjustment of plasma membrane fluidity, and the increased capability to manage oxidative stress. Halophilic bacteria, and archaea have been intensively studied, and substantial research has been conducted on halophilic fungi, and the green alga Dunaliella. By contrast, there have been very few investigations of halophiles that are phagotrophic protists, i.e., protozoa. To gather fundamental knowledge about salt adaptation in these organisms, we studied the transcriptome-level response of Halocafeteria seosinensis (Stramenopiles) grown under contrasting salinities. We provided further evolutionary context to our analysis by identifying genes that underwent recent duplications. Genes that were highly responsive to salinity variations were involved in stress response (e.g., chaperones), ion homeostasis (e.g., Na+/H+ transporter), metabolism and transport of lipids (e.g., sterol biosynthetic genes), carbohydrate metabolism (e.g., glycosidases), and signal transduction pathways (e.g., transcription factors). A significantly high proportion (43%) of duplicated genes were also differentially expressed, accentuating the importance of gene expansion in adaptation by H. seosinensis to high salt environments. Furthermore, we found two genes that were lateral acquisitions from bacteria, and were also highly up-regulated and highly expressed at high salt, suggesting that this evolutionary mechanism could also have facilitated adaptation to high salt. We propose that a transition toward high-salt adaptation in the ancestors of H. seosinensis required the acquisition of new genes via duplication, and some lateral gene transfers (LGTs), as well as the alteration of transcriptional programs, leading to increased stress resistance, proper establishment of ion gradients, and modification of cell structure properties like membrane fluidity. PMID:28611746

Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function.

PubMed

Rayon-Estrada, Violeta; Harjanto, Dewi; Hamilton, Claire E; Berchiche, Yamina A; Gantman, Emily Conn; Sakmar, Thomas P; Bulloch, Karen; Gagnidze, Khatuna; Harroch, Sheila; McEwen, Bruce S; Papavasiliou, F Nina

2017-12-12

Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells. Copyright © 2017 the Author(s). Published by PNAS.

ABA-insensitive3, ABA-insensitive5, and DELLAs Interact to activate the expression of SOMNUS and other high-temperature-inducible genes in imbibed seeds in Arabidopsis.

PubMed

Lim, Soohwan; Park, Jeongmoo; Lee, Nayoung; Jeong, Jinkil; Toh, Shigeo; Watanabe, Asuka; Kim, Junghyun; Kang, Hyojin; Kim, Dong Hwan; Kawakami, Naoto; Choi, Giltsu

2013-12-01

Seeds monitor the environment to germinate at the proper time, but different species respond differently to environmental conditions, particularly light and temperature. In Arabidopsis thaliana, light promotes germination but high temperature suppresses germination. We previously reported that light promotes germination by repressing SOMNUS (SOM). Here, we examined whether high temperature also regulates germination through SOM and found that high temperature activates SOM expression. Consistent with this, som mutants germinated more frequently than the wild type at high temperature. The induction of SOM mRNA at high temperature required abscisic acid (ABA) and gibberellic acid biosynthesis, and ABA-insensitive3 (ABI3), ABI5, and DELLAs positively regulated SOM expression. Chromatin immunoprecipitation assays indicated that ABI3, ABI5, and DELLAs all target the SOM promoter. At the protein level, ABI3, ABI5, and DELLAs all interact with each other, suggesting that they form a complex on the SOM promoter to activate SOM expression at high temperature. We found that high-temperature-inducible genes frequently have RY motifs and ABA-responsive elements in their promoters, some of which are targeted by ABI3, ABI5, and DELLAs in vivo. Taken together, our data indicate that ABI3, ABI5, and DELLAs mediate high-temperature signaling to activate the expression of SOM and other high-temperature-inducible genes, thereby inhibiting seed germination.

Gene expression levels of gamma-glutamyl hydrolase in tumor tissues may be a useful biomarker for the proper use of S-1 and tegafur-uracil/leucovorin in preoperative chemoradiotherapy for patients with rectal cancer.

PubMed

Sadahiro, Sotaro; Suzuki, T; Tanaka, A; Okada, K; Saito, G; Miyakita, H; Ogimi, T; Nagase, H

2017-06-01

Preoperative chemoradiotherapy (CRT) using 5-fluorouracil (5-FU)-based chemotherapy is the standard of care for rectal cancer. The effect of additional chemotherapy during the period between the completion of radiotherapy and surgery remains unclear. Predictive factors for CRT may differ between combination chemotherapy with S-1 and with tegafur-uracil/leucovorin (UFT/LV). The subjects were 54 patients with locally advanced rectal cancer who received preoperative CRT with S-1 or UFT/LV. The pathological tumor response was assessed according to the tumor regression grade (TRG). The expression levels of 18 CRT-related genes were determined using RT-PCR assay. A pathological response (TRG 1-2) was observed in 23 patients (42.6%). In a multivariate logistic regression analysis for pathological response, the overall expression levels of four genes, HIF1A, MTHFD1, GGH and TYMS, were significant, and the accuracy rate of the predictive model was 83.3%. The effects of the gene expression levels of GGH on the response differed significantly according to the treatment regimen. The total pathological response rate of both high-GGH patients in the S-1 group and low-GGH patients in the UFT/LV group was 58.3%. Additional treatment with 5-FU-based chemotherapy during the interval between radiotherapy and surgery is not beneficial in patients who have received 5-FU-based CRT. The expression levels of four genes, HIF1A, MTHFD1, GGH and TYMS, in tumor tissues can predict the response to preoperative CRT including either S-1 or UFT/LV. In particular, the gene expression level of GGH in tumor tissues may be a useful biomarker for the appropriate use of S-1 and UFT/LV in CRT.

Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

PubMed Central

Calvanese, Vincenzo; Mallya, Meera; Campbell, R Duncan; Aguado, Begoña

2008-01-01

Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own. PMID:18817541

Grouping and characterization of putative glycosyltransferase genes from Panax ginseng Meyer.

PubMed

Khorolragchaa, Altanzul; Kim, Yu-Jin; Rahimi, Shadi; Sukweenadhi, Johan; Jang, Moon-Gi; Yang, Deok-Chun

2014-02-15

Glycosyltransferases are members of the multigene family of plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and few have been functionally characterized in detail. Korean ginseng (Panax ginseng Meyer), belonging to Araliaceae, has been well known as a popular mysterious medicinal herb in East Asia for over 2,000 years. A total of 704 glycosyltransferase unique sequences have been found from a ginseng expressed sequence tag (EST) library, and these sequences encode enzymes responsible for the secondary metabolite biosynthesis. Finally, twelve UDP glycosyltransferases (UGTs) were selected as the candidates most likely to be involved in triterpenoid synthesis. In this study, we classified the candidate P. ginseng UGTs (PgUGTs) into proper families and groups, which resulted in eight UGT families and six UGT groups. We also investigated those gene candidates encoding for glycosyltransferases by analysis of gene expression in methyl jasmonate (MeJA)-treated ginseng adventitious roots and different tissues from four-year-old ginseng using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). For organ-specific expression, most of PgUGT transcription levels were higher in leaves and roots compared with flower buds and stems. The transcription of PgUGTs in adventitious roots treated with MeJA increased as compared with the control. PgUGT1 and PgUGT2, which belong to the UGT71 family genes expressed in MeJA-treated adventitious roots, were especially sensitive, showing 33.32 and 38.88-fold expression increases upon 24h post-treatments, respectively. © 2013 Elsevier B.V. All rights reserved.

Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of a GATA1 Protein Lacking the N-terminal Domain*

PubMed Central

Kobayashi, Eri; Shimizu, Ritsuko; Kikuchi, Yuko; Takahashi, Satoru; Yamamoto, Masayuki

2010-01-01

GATA1 is essential for the differentiation of erythroid cells and megakaryocytes. The Gata1 gene is composed of multiple untranslated first exons and five common coding exons. The erythroid first exon (IE exon) is important for Gata1 gene expression in hematopoietic lineages. Because previous IE exon knockdown analyses resulted in embryonic lethality, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the floxed IE exon in adulthood using an inducible Cre expression system. In this conditional knock-out mouse line, the Gata1 mRNA level was significantly down-regulated in the megakaryocyte lineage, resulting in thrombocytopenia with a marked proliferation of megakaryocytes. By contrast, in the erythroid lineage, Gata1 mRNA was expressed abundantly utilizing alternative first exons. Especially, the IEb/c and newly identified IEd exons were transcribed at a level comparable with that of the IE exon in control mice. Surprisingly, in the IE-null mouse, these transcripts failed to produce full-length GATA1 protein, but instead yielded GATA1 lacking the N-terminal domain inefficiently. With low level expression of the short form of GATA1, IE-null mice showed severe anemia with skewed erythroid maturation. Notably, the hematological phenotypes of adult IE-null mice substantially differ from those observed in mice harboring conditional ablation of the entire Gata1 gene. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and selecting the correct transcription start site of the Gata1 gene. PMID:19854837

Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

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

Springer, Patricia S

2010-07-12

The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes thatmore » encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.« less

The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens.

PubMed

Hind, Sarah R; Pulliam, Sarah E; Veronese, Paola; Shantharaj, Deepak; Nazir, Azka; Jacobs, Nekaiya S; Stratmann, Johannes W

2011-02-01

The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

PubMed Central

O'Connell, K F; Leys, C M; White, J G

1998-01-01

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle. PMID:9649522

Discordant timing between antennae disrupts sun compass orientation in migratory monarch butterflies

PubMed Central

Guerra, Patrick A; Merlin, Christine; Gegear, Robert J; Reppert, Steven M

2014-01-01

To navigate during their long-distance migration, monarch butterflies (Danaus plexippus) use a time-compensated sun compass. The sun compass timing elements reside in light-entrained circadian clocks in the antennae. Here we show that either antenna is sufficient for proper time compensation. However, migrants with either antenna painted black (to block light entrainment) and the other painted clear (to permit light entrainment) display disoriented group flight. Remarkably, when the black-painted antenna is removed, re-flown migrants with a single, clear-painted antenna exhibit proper orientation behaviour. Molecular correlates of clock function reveal that period and timeless expression is highly rhythmic in brains and clear-painted antennae, while rhythmic clock gene expression is disrupted in black-painted antennae. Our work shows that clock outputs from each antenna are processed and integrated together in the monarch time-compensated sun compass circuit. This dual timing system is a novel example of the regulation of a brain-driven behaviour by paired organs. PMID:22805565

The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.

PubMed

Claycomb, Julie M; Batista, Pedro J; Pang, Ka Ming; Gu, Weifeng; Vasale, Jessica J; van Wolfswinkel, Josien C; Chaves, Daniel A; Shirayama, Masaki; Mitani, Shohei; Ketting, René F; Conte, Darryl; Mello, Craig C

2009-10-02

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq.

PubMed

Whitmore, S Scott; Wagner, Alex H; DeLuca, Adam P; Drack, Arlene V; Stone, Edwin M; Tucker, Budd A; Zeng, Shemin; Braun, Terry A; Mullins, Robert F; Scheetz, Todd E

2014-12-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq

PubMed Central

Whitmore, S. Scott; Wagner, Alex H.; DeLuca, Adam P.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Zeng, Shemin; Braun, Terry A.; Mullins, Robert F.; Scheetz, Todd E.

2014-01-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell-types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. PMID:25446321

Holoprosencephaly: from Homer to Hedgehog.

PubMed

Ming, J E; Muenke, M

1998-03-01

Holoprosencephaly (HPE), a common developmental defect affecting the forebrain and face, is etiologically heterogeneous and exhibits wide phenotypic variation. Graded degrees of severity of the brain malformation are also reflected in the highly variable craniofacial malformations associated with HPE. In addition, individuals with microforms of HPE, who usually have normal cognition and normal brain imaging, are at risk for having children with HPE. Some obligate carriers for HPE may not have any phenotypic abnormalities. Recurrent chromosomal rearrangements in individuals with HPE suggest loci containing genes important for brain development, and abnormalities in these genes may result in HPE. Recently, Sonic Hedgehog (SHH) was the first gene identified as causing HPE in humans. Proper function of SHH depends on cholesterol modification. Other candidate genes that may be involved in HPE include components of the SHH pathway, elements involved in cholesterol metabolism, and genes expressed in the developing forebrain.

The Role of c-FLIP(L) in Regulating Apoptotic Pathways in Prostate Cancer

DTIC Science & Technology

2006-12-01

which regulates gene expression 3. c-Fos has been shown to play an important role in development, inflammation and oncogenic processes. For example...important role in development, inflammation and oncogenic processes. For example, TNF-family induction of c-Fos plays an important role in proper bone c...identifying the down-stream targets of c-Fos has significant implications in understanding of normal development, inflammation and oncogenesis (10). In

Genome expression analyses revealing the modulation of the Salmonella Rcs regulon by the attenuator IgaA.

PubMed

Mariscotti, Javier F; García-del Portillo, Francisco

2009-03-01

Intracellular growth attenuator A (IgaA) was identified as a Salmonella enterica regulator limiting bacterial growth inside fibroblasts. Genetic evidence further linked IgaA to repression of the RcsCDB regulatory system, which responds to envelope stress. How IgaA attenuates this system is unknown. Here, we present genome expression profiling data of S. enterica serovar Typhimurium igaA mutants grown at high osmolarity and displaying exacerbated Rcs responses. Transcriptome data revealed that IgaA attenuates gene expression changes requiring phosphorylated RcsB (RcsB~P) activity. Some RcsB-regulated genes, yciGFE and STM1862 (pagO)-STM1863-STM1864, were equally expressed in wild-type and igaA strains, suggesting a maximal expression at low levels of RcsB ~P. Other genes, such as metB, ypeC, ygaC, glnK, glnP, napA, glpA, and nirB, were shown for the first time and by independent methods to be regulated by the RcsCDB system. Interestingly, IgaA-deficient strains with reduced RcsC or RcsD levels exhibited different Rcs responses and distinct virulence properties. spv virulence genes were differentially expressed in most of the analyzed strains. spvA expression required RcsB and IgaA but, unexpectedly, was also impaired upon stimulation of the RcsC-->RcsD-->RcsB phosphorelay. Overproduction of either RcsB(+) or a nonphosphorylatable RcsB(D56Q) variant in strains displaying low spvA expression unveiled that both dephosphorylated RcsB and RcsB~P are required for optimal spvA expression. Taken together, our data support a model with IgaA attenuating the RcsCDB system by favoring the switch of RcsB~P to the dephosphorylated state. This role of IgaA in constantly fine-tuning the RcsB~P/RcsB ratio may ensure the proper expression of important virulence factors, such as the Spv proteins.

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

PubMed Central

Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

2015-01-01

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

PubMed

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

An Arabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperatures.

PubMed

Guo, Yan; Xiong, Liming; Ishitani, Manabu; Zhu, Jian-Kang

2002-05-28

Low temperature regulates gene expression in bacteria, yeast, and animals as well as in plants. However, the signal transduction cascades mediating the low temperature responses are not well understood in any organism. To identify components in low temperature signaling genetically, we isolated Arabidopsis thaliana mutants in which cold-responsive genes are no longer induced by low temperatures. One of these mutations, los1-1, specifically blocks low temperature-induced transcription of cold-responsive genes. Surprisingly, cold-induced expression of the early response transcriptional activators, C-repeat/dehydration responsive element binding factors (CBF/DREB1s), is enhanced by the los1-1 mutation. The los1-1 mutation also reduces the capacity of plants to develop freezing tolerance but does not impair the vernalization response. Genetic analysis indicated that los1-1 is a recessive mutation in a single nuclear gene. The LOS1 gene encodes a translation elongation factor 2-like protein. Protein labeling studies show that new protein synthesis is blocked in los1-1 mutant plants specifically in the cold. These results reveal a critical role of new protein synthesis in the proper transduction of low temperature signals. Our results also suggest that cold-induced transcription of CBF/DREB1s is feedback inhibited by their gene products or by products of their downstream target genes.

The canonical Wnt signaling activator, R-spondin2, regulates craniofacial patterning and morphogenesis within the branchial arch through ectodermal-mesenchymal interaction

PubMed Central

Jin, Yong-Ri; Turcotte, Taryn J.; Crocker, Alison L.; Han, Xiang Hua; Yoon, Jeong Kyo

2011-01-01

R-spondins are a recently characterized family of secreted proteins that activate Wnt/β-catenin signaling. Herein, we determine R-spondin2 (Rspo2) function in craniofacial development in mice. Mice lacking a functional Rspo2 gene exhibit craniofacial abnormalities such as mandibular hypoplasia, maxillary and mandibular skeletal deformation, and cleft palate. We found that loss of the mouse Rspo2 gene significantly disrupted Wnt/β-catenin signaling and gene expression within the first branchial arch (BA1). Rspo2, which is normally expressed in BA1 mesenchymal cells, regulates gene expression through a unique ectoderm-mesenchyme interaction loop. The Rspo2 protein, potentially in combination with ectoderm-derived Wnt ligands, up-regulates Msx1 and Msx2 expression within mesenchymal cells. In contrast, Rspo2 regulates expression of the Dlx5, Dlx6, and Hand2 genes in mesenchymal cells via inducing expression of their upstream activator, Endothelin1 (Edn1), within ectodermal cells. Loss of Rspo2 also causes increased cell apoptosis, especially within the aboral (or caudal) domain of the BA1, resulting in hypoplasia of the BA1. Severely reduced expression of Fgf8, a survival factor for mesenchymal cells, in the ectoderm of Rspo2−/− embryos is likely responsible for increased cell apoptosis. Additionally, we found that cleft palate in Rspo2−/− mice is not associated with defects intrinsic to the palatal shelves. A possible cause of cleft palate is a delay of proper palatal shelf elevation that may result from the small mandible and a failure of lowering the tongue. Thus, our study identifies Rspo2 as a mesenchyme-derived factor that plays critical roles in regulating BA1 patterning and morphogenesis through ectodermal-mesenchymal interaction and a novel genetic factor for cleft palate. PMID:21237142

A comprehensive catalogue of the coding and non-coding transcripts of the human inner ear

PubMed Central

Corneveaux, Jason J.; Ohmen, Jeffrey; White, Cory; Allen, April N.; Lusis, Aldons J.; Van Camp, Guy; Huentelman, Matthew J.; Friedman, Rick A.

2015-01-01

The mammalian inner ear consists of the cochlea and the vestibular labyrinth (utricle, saccule, and semicircular canals), which participate in both hearing and balance. Proper development and life-long function of these structures involves a highly complex coordinated system of spatial and temporal gene expression. The characterization of the inner ear transcriptome is likely important for the functional study of auditory and vestibular components, yet, primarily due to tissue unavailability, detailed expression catalogues of the human inner ear remain largely incomplete. We report here, for the first time, comprehensive transcriptome characterization of the adult human cochlea, ampulla, saccule and utricle of the vestibule obtained from patients without hearing abnormalities. Using RNA-Seq, we measured the expression of >50,000 predicted genes corresponding to approximately 200,000 transcripts, in the adult inner ear and compared it to 32 other human tissues. First, we identified genes preferentially expressed in the inner ear, and unique either to the vestibule or cochlea. Next, we examined expression levels of specific groups of potentially interesting RNAs, such as genes implicated in hearing loss, long non-coding RNAs, pseudogenes and transcripts subject to nonsense mediated decay (NMD). We uncover the spatial specificity of expression of these RNAs in the hearing/balance system, and reveal evidence of tissue specific NMD. Lastly, we investigated the non-syndromic deafness loci to which no gene has been mapped, and narrow the list of potential candidates for each locus. These data represent the first high-resolution transcriptome catalogue of the adult human inner ear. A comprehensive identification of coding and non-coding RNAs in the inner ear will enable pathways of auditory and vestibular function to be further defined in the study of hearing and balance. Expression data are freely accessible at https://www.tgen.org/home/research/research-divisions/neurogenomics/supplementary-data/inner-ear-transcriptome.aspx PMID:26341477

MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

PubMed

Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A M

2013-01-01

Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

Genome-Wide Transcriptome Analysis During Anthesis Reveals New Insights into the Molecular Basis of Heat Stress Responses in Tolerant and Sensitive Rice Varieties.

PubMed

González-Schain, Nahuel; Dreni, Ludovico; Lawas, Lovely M F; Galbiati, Massimo; Colombo, Lucia; Heuer, Sigrid; Jagadish, Krishna S V; Kater, Martin M

2016-01-01

Rice is one of the main food crops in the world. In the near future, yield is expected to be under pressure due to unfavorable climatic conditions, such as increasing temperatures. Therefore, improving rice germplasm in order to guarantee rice production under harsh environmental conditions is of top priority. Although many physiological studies have contributed to understanding heat responses during anthesis, the most heat-sensitive stage, molecular data are still largely lacking. In this study, an RNA-sequencing approach of heat- and control-treated reproductive tissues during anthesis was carried out using N22, one of the most heat-tolerant rice cultivars known to date. This analysis revealed that expression of genes encoding a number of transcription factor families, together with signal transduction and metabolic pathway genes, is repressed. On the other hand, expression of genes encoding heat shock factors and heat shock proteins was highly activated. Many of these genes are predominantly expressed at late stages of anther development. Further physiological experiments using heat-tolerant N22 and two sensitive cultivars suggest that reduced yield in heat-sensitive plants may be associated with poor pollen development or production in anthers prior to anthesis. In parallel, induction levels of a set of heat-responsive genes in these tissues correlated well with heat tolerance. Altogether, these findings suggest that proper expression of protective chaperones in anthers is needed before anthesis to overcome stress damage and to ensure fertilization. Genes putatively controlling this process were identified and are valuable candidates to consider for molecular breeding of highly productive heat-tolerant cultivars. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Epigallocatechin gallate promotes the development of mouse 2-cell embryos in vitro by regulating mitochondrial activity and expression of genes related to p53 signalling pathway.

PubMed

Zhang, Weiyu; Lv, Junjie; Zhang, Yanqin; Jiang, Yufei; Chu, Chenfeng; Wang, Shie

2014-11-01

Preliminary studies have found that the epigallocatechin gallate (EGCG) at proper concentration could promote development of pre-implantation mouse embryos in vitro. However, the underlying mechanisms have not been well understood. In this study, we collected 1-cell embryos from Kunming (KM) mice, cultured them in M16 medium or M16 medium supplemented with 10 μg/mL EGCG and investigated the effects of EGCG on mitochondrial activity and reactive oxygen species (ROS) level of 2-cell embryos. Furthermore, we explored expression differences of genes related to p53 signalling pathway in 2-cell embryos using a PCR array. The results showed that ROS level and mitochondrial membrane potential were significantly lower in embryos cultured in the EGCG group than in the M16 group (p < 0.05), while the adenosine triphosphate content was slightly lower than in the M16 group (p > 0.05). PCR array test results showed that 18 genes were differentially expressed, among which eight genes involving cell growth, cell cycle regulation and mRNA transcription were up-regulated and 10 genes involving apoptosis, cell cycle arrest and DNA repair were down-regulated in the EGCG groups. It is concluded that EGCG could promote the development of 1-cell embryos in vitro possibly due to its ability to scavenge ROS and regulate mitochondrial activity. In addition, EGCG could influence expression of genes related to p53 signalling pathway in 2-cell embryos and promote cell cycle progression. © 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

PubMed

Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M

2013-01-01

N-MYC down-regulated-like (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

PubMed Central

Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

2013-01-01

Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Methodology/Principal Findings Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. Conclusion/Significance NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients. PMID:24223735

Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

PubMed Central

O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

2015-01-01

Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

PubMed

Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

PubMed

Rha, Jennifer; Jones, Stephanie K; Fidler, Jonathan; Banerjee, Ayan; Leung, Sara W; Morris, Kevin J; Wong, Jennifer C; Inglis, George Andrew S; Shapiro, Lindsey; Deng, Qiudong; Cutler, Alicia A; Hanif, Adam M; Pardue, Machelle T; Schaffer, Ashleigh; Seyfried, Nicholas T; Moberg, Kenneth H; Bassell, Gary J; Escayg, Andrew; García, Paul S; Corbett, Anita H

2017-10-01

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

microRNA regulation of T-cell differentiation and function

PubMed Central

Jeker, Lukas T.; Bluestone, Jeffrey A.

2013-01-01

Summary microRNAs (miRNAs) are emerging as key controllers of T-cell differentiation and function. Their expression is dynamically regulated by extracellular signals such as costimulation and cytokine signals. miRNAs set thresholds for gene expression and optimize protein concentrations of genetic networks. Absence of individual miRNAs can lead to severe immune dysfunction. Here we review emerging principles and provide examples of important functions exerted by miRNAs. Although our understanding of miRNA function in T-cell differentiation is still rudimentary, the available evidence leaves no doubt that these small posttranscriptional regulators are indispensable for proper functioning of the immune system. PMID:23550639

ACR4, a putative receptor kinase gene of Arabidopsis thaliana, that is expressed in the outer cell layers of embryos and plants, is involved in proper embryogenesis.

PubMed

Tanaka, Hirokazu; Watanabe, Masaru; Watanabe, Daisuke; Tanaka, Toshihiro; Machida, Chiyoko; Machida, Yasunori

2002-04-01

The surfaces of higher plants are characterized by epidermis, which usually consists of a single layer of cells. The epidermis is derived from the outer cell layer of the embryo or protoderm, which arises as a result of periclinal cell division. After seed germination, most of the epidermal cells of the aerial parts of plants are derived from the outer cell layer of the shoot apical meristem (the L1 layer). Thus, knowledge of how the protoderm and/or L1 layer is established is fundamental to understanding the morphogenesis of higher plants. Here, we report the isolation of a gene encoding an Arabidopsis homologue (ACR4) of the maize putative receptor kinase CRINKLY4 (CR4), which is involved in epidermal differentiation. The domain organization of the predicted amino acid sequence of ACR4 is essentially identical to that of CR4. ACR4-GFP fusion protein localized to the cell surface when expressed in tobacco cell (BY-2) culture. ACR4 transcripts were detected in all the organs of the Arabidopsis plant. In developing embryos and shoot apices, ACR4 transcripts accumulated in protoderm and epidermis at relatively higher levels than in the inner tissues. Over-expression of antisense ACR4 in Arabidopsis plants resulted in malformation of embryos to varying degrees. These results suggest that ACR4 is, at a minimum, involved in the normal morphogenesis of embryos, most likely through properly differentiating protoderm cells.

Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses.

PubMed

Vandelle, Elodie; Vannozzi, Alessandro; Wong, Darren; Danzi, Davide; Digby, Anne-Marie; Dal Santo, Silvia; Astegno, Alessandra

2018-06-04

Calcium (Ca 2+ ) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca 2+ binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca 2+ signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes. We assigned gene family nomenclature, analyzed gene structure, chromosomal location and gene duplication, as well as protein motif organization. The phylogenetic clustering revealed a total of eight subgroups, including one unique clade of VviCaMs distinct from VviCMLs. VviCaMs were found to contain four EF-hand motifs whereas VviCML proteins have one to five. Most of grapevine CML genes were intronless, while VviCaMs were intron rich. All the genes were well spread among the 19 grapevine chromosomes and displayed a high level of duplication. The expression profiling of VviCaM/VviCML genes revealed a broad expression pattern across all grape organs and tissues at various developmental stages, and a significant modulation in biotic stress-related responses. Our results highlight the complexity of CaM/CML protein family also in grapevine, supporting the versatile role of its different members in modulating cellular responses to various stimuli, in particular to biotic stresses. This work lays the foundation for further functional and structural studies on specific grapevine CaMs/CMLs in order to better understand the role of Ca 2+ -binding proteins in grapevine and to explore their potential for further biotechnological applications. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.

PubMed

Pérez-Rico, A; Crespo, F; Sanmartín, M L; De Santiago, A; Vega-Pla, J L

2014-10-01

Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specificity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous concentration gradient. RNA purification followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective amplification of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Specificities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that five out of the nine candidate genes amplified properly (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which β-Actin and the L32 Ribosomal protein showed the highest stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B. Copyright © 2014 Elsevier B.V. All rights reserved.

Epigenetic Deregulation of MicroRNAs in Rhabdomyosarcoma and Neuroblastoma and Translational Perspectives

PubMed Central

Romania, Paolo; Bertaina, Alice; Bracaglia, Giorgia; Locatelli, Franco; Fruci, Doriana; Rota, Rossella

2012-01-01

Gene expression control mediated by microRNAs and epigenetic remodeling of chromatin are interconnected processes often involved in feedback regulatory loops, which strictly guide proper tissue differentiation during embryonal development. Altered expression of microRNAs is one of the mechanisms leading to pathologic conditions, such as cancer. Several lines of evidence pointed to epigenetic alterations as responsible for aberrant microRNA expression in human cancers. Rhabdomyosarcoma and neuroblastoma are pediatric cancers derived from cells presenting features of skeletal muscle and neuronal precursors, respectively, blocked at different stages of differentiation. Consistently, tumor cells express tissue markers of origin but are unable to terminally differentiate. Several microRNAs playing a key role during tissue differentiation are often epigenetically downregulated in rhabdomyosarcoma and neuroblastoma and behave as tumor suppressors when re-expressed. Recently, inhibition of epigenetic modulators in adult tumors has provided encouraging results causing re-expression of anti-tumor master gene pathways. Thus, a similar approach could be used to correct the aberrant epigenetic regulation of microRNAs in rhabdomyosarcoma and neuroblastoma. The present review highlights the current insights on epigenetically deregulated microRNAs in rhabdomyosarcoma and neuroblastoma and their role in tumorigenesis and developmental pathways. The translational clinical implications and challenges regarding modulation of epigenetic chromatin remodeling/microRNAs interconnections are also discussed. PMID:23443118

Gene Chips: A New Tool for Biology

NASA Astrophysics Data System (ADS)

Botstein, David

2005-03-01

The knowledge of many complete genomic sequences has led to a ``grand unification of biology,'' consisting of direct evidence that most of the basic cellular functions of all organisms are carried out by genes and proteins whose primary sequences are directly related by descent (i.e. orthologs). Further, genome sequences have made it possible to study all the genes of a single organism simultaneously. We have been using DNA microarrays (sometime referred to as ``gene chips'') to study patterns of gene expression and genome rearrangement in yeast and human cells under a variety of conditions and in human tumors and normal tissues. These experiments produce huge volumes of data; new computational and statistical methods are required to analyze them properly. Examples from this work will be presented to illustrate how genome-scale experiments and analysis can result in new biological insights not obtainable by traditional analyses of genes and proteins one by one. For lymphomas, breast tumors, lung tumors, liver tumors, gastric tumors, brain tumors and soft tissue tumors we have been able, by the application of clustering algorithms, to subclassify tumors of similar anatomical origin on the basis of their gene expression patterns. These subclassifications appear to be reproducible and clinically as well as biologically meaningful. By studying synchronized cells growing in culture, we have identified many hundreds of yeast and human genes that are expressed periodically, at characteristically different points in the cell division cycle. In humans, it turns out that most of these genes are the same genes that comprise the ``proliferation cluster,'' i.e. the genes whose expression is specifically associated with the proliferativeness of tumors and tumor cell lines. Finally, we have been applying a variant of our DNA microarray technology (which we call ``array comparative hybridization'') to follow the DNA copy number of genes, both in tumors and in yeast cells undergoing adaptive evolution during hundreds of generations of growth in continuous culture. These studies suggest a basic similarity in mechanism between adaptive evolution in yeast and tumor progression in humans.

Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.).

PubMed

Lipka, A; Paukszto, L; Majewska, M; Jastrzebski, J P; Myszczynski, K; Panasiewicz, G; Szafranska, B

2017-09-01

The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein β2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α 7 (Itga7), downregulated in twins, and Nrk, gap junction protein β6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α 1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber subplacental transcriptome. Enhancement of transcriptomic resources for C. fiber will improve understanding of the pathways relevant to proper placental development and successful reproduction.

Paradigm Shift in Thyroid Hormone Mechanism of Action | Center for Cancer Research

Cancer.gov

Thyroid hormone (TH) is one of the primary endocrine regulators of human metabolism and homeostasis. Acting through three forms of the thyroid hormone receptor (THR; alpha-1, beta-1, and beta-2), TH regulates target gene expression in nearly every cell in the body, modulating fundamental processes, such as basal metabolic rate, long bone growth, and neural maturation. TH is also essential for proper development and differentiation of all cells of the human body.

Transcription and translation of the sigG gene is tuned for proper execution of the switch from early to late gene expression in the developing Bacillus subtilis spore

PubMed Central

Mearls, Elizabeth B.; Jackter, Jacquelin; Colquhoun, Jennifer M.; Matthews, Allison J.; Fenton, Colleen

2018-01-01

A cascade of alternative sigma factors directs developmental gene expression during spore formation by the bacterium Bacillus subtilis. As the spore develops, a tightly regulated switch occurs in which the early-acting sigma factor σF is replaced by the late-acting sigma factor σG. The gene encoding σG (sigG) is transcribed by σF and by σG itself in an autoregulatory loop; yet σG activity is not detected until σF-dependent gene expression is complete. This separation in σF and σG activities has been suggested to be due at least in part to a poorly understood intercellular checkpoint pathway that delays sigG expression by σF. Here we report the results of a careful examination of sigG expression during sporulation. Unexpectedly, our findings argue against the existence of a regulatory mechanism to delay sigG transcription by σF and instead support a model in which sigG is transcribed by σF with normal timing, but at levels that are very low. This low-level expression of sigG is the consequence of several intrinsic features of the sigG regulatory and coding sequence—promoter spacing, secondary structure potential of the mRNA, and start codon identity—that dampen its transcription and translation. Especially notable is the presence of a conserved hairpin in the 5’ leader sequence of the sigG mRNA that occludes the ribosome-binding site, reducing translation by up to 4-fold. Finally, we demonstrate that misexpression of sigG from regulatory and coding sequences lacking these features triggers premature σG activity in the forespore during sporulation, as well as inappropriate σG activity during vegetative growth. Altogether, these data indicate that transcription and translation of the sigG gene is tuned to prevent vegetative expression of σG and to ensure the precise timing of the switch from σF to σG in the developing spore. PMID:29702640

Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

PubMed Central

2012-01-01

Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the regulatory sequences of these genes could not be confirmed. Conclusions In the thalamus, β-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of β-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies. PMID:23157480

A Protocol for Epigenetic Imprinting Analysis with RNA-Seq Data.

PubMed

Zou, Jinfeng; Xiang, Daoquan; Datla, Raju; Wang, Edwin

2018-01-01

Genomic imprinting is an epigenetic regulatory mechanism that operates through expression of certain genes from maternal or paternal in a parent-of-origin-specific manner. Imprinted genes have been identified in diverse biological systems that are implicated in some human diseases and in embryonic and seed developmental programs in plants. The molecular underpinning programs and mechanisms involved in imprinting are yet to be explored in depth in plants. The recent advances in RNA-Seq-based methods and technologies offer an opportunity to systematically analyze epigenetic imprinting that operates at the whole genome level in the model and crop plants. We are interested using Arabidopsis model system, to investigate gene expression patterns associated with parent of origin and their implications to imprinting during embryo and seed development. Toward this, we have generated early embryo development RNA-Seq-based transcriptome datasets in F1s from a genetic cross between two diverse Arabidopsis thaliana ecotypes Col-0 and Tsu-1. With the data, we developed a protocol for evaluating the maternal and paternal contributions of genes during the early stages of embryo development after fertilization. This protocol is also designed to consider the contamination from other potential seed tissues, sequencing quality, proper processing of sequenced reads and variant calling, and appropriate inference of the parental contributions based on the parent-of-origin-specific single-nucleotide polymorphisms within the expressed genes. The approach, methods and the protocol developed in this study can be used for evaluating the effects of epigenetic imprinting in plants.

Bayesian inference of the number of factors in gene-expression analysis: application to human virus challenge studies

PubMed Central

2010-01-01

Background Nonparametric Bayesian techniques have been developed recently to extend the sophistication of factor models, allowing one to infer the number of appropriate factors from the observed data. We consider such techniques for sparse factor analysis, with application to gene-expression data from three virus challenge studies. Particular attention is placed on employing the Beta Process (BP), the Indian Buffet Process (IBP), and related sparseness-promoting techniques to infer a proper number of factors. The posterior density function on the model parameters is computed using Gibbs sampling and variational Bayesian (VB) analysis. Results Time-evolving gene-expression data are considered for respiratory syncytial virus (RSV), Rhino virus, and influenza, using blood samples from healthy human subjects. These data were acquired in three challenge studies, each executed after receiving institutional review board (IRB) approval from Duke University. Comparisons are made between several alternative means of per-forming nonparametric factor analysis on these data, with comparisons as well to sparse-PCA and Penalized Matrix Decomposition (PMD), closely related non-Bayesian approaches. Conclusions Applying the Beta Process to the factor scores, or to the singular values of a pseudo-SVD construction, the proposed algorithms infer the number of factors in gene-expression data. For real data the "true" number of factors is unknown; in our simulations we consider a range of noise variances, and the proposed Bayesian models inferred the number of factors accurately relative to other methods in the literature, such as sparse-PCA and PMD. We have also identified a "pan-viral" factor of importance for each of the three viruses considered in this study. We have identified a set of genes associated with this pan-viral factor, of interest for early detection of such viruses based upon the host response, as quantified via gene-expression data. PMID:21062443

Epigenetics of sex determination and gonadogenesis.

PubMed

Piferrer, Francesc

2013-04-01

Epigenetics is commonly defined as the study of heritable changes in gene function that cannot be explained by changes in DNA sequence. The three major epigenetic mechanisms for gene expression regulation include DNA methylation, histone modifications, and non-coding RNAs. Epigenetic mechanisms provide organisms with the ability to integrate genomic and environmental information to modify the activity of their genes for generating a particular phenotype. During development, cells differentiate, acquire, and maintain identity through changes in gene expression. This is crucial for sex determination and differentiation, which are among the most important developmental processes for the proper functioning and perpetuation of species. This review summarizes studies showing how epigenetic regulatory mechanisms contribute to sex determination and reproductive organ formation in plants, invertebrates, and vertebrates. Further progress will be made by integrating several approaches, including genomics and Next Generation Sequencing to create epigenetic maps related to different aspects of sex determination and gonadogenesis. Epigenetics will also contribute to understand the etiology of several disorders of sexual development. It also might play a significant role in the control of reproduction in animal farm production and will aid in recognizing the environmental versus genetic influences on sex determination of sensitive species in a global change scenario. Copyright © 2013 Wiley Periodicals, Inc.

The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation[C][W

PubMed Central

Catalá, Rafael; López-Cobollo, Rosa; Mar Castellano, M.; Angosto, Trinidad; Alonso, José M.; Ecker, Joseph R.; Salinas, Julio

2014-01-01

In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis. PMID:25122152

Repeat-mediated epigenetic dysregulation of the FMR1 gene in the fragile X-related disorders.

PubMed

Usdin, Karen; Kumari, Daman

2015-01-01

The fragile X-related disorders are members of the Repeat Expansion Diseases, a group of genetic conditions resulting from an expansion in the size of a tandem repeat tract at a specific genetic locus. The repeat responsible for disease pathology in the fragile X-related disorders is CGG/CCG and the repeat tract is located in the 5' UTR of the FMR1 gene, whose protein product FMRP, is important for the proper translation of dendritic mRNAs in response to synaptic activation. There are two different pathological FMR1 allele classes that are distinguished only by the number of repeats. Premutation alleles have 55-200 repeats and confer risk of fragile X-associated tremor/ataxia syndrome and fragile X-associated primary ovarian insufficiency. Full mutation alleles on the other hand have >200 repeats and result in fragile X syndrome, a disorder that affects learning and behavior. Different symptoms are seen in carriers of premutation and full mutation alleles because the repeat number has paradoxical effects on gene expression: Epigenetic changes increase transcription from premutation alleles and decrease transcription from full mutation alleles. This review will cover what is currently known about the mechanisms responsible for these changes in FMR1 expression and how they may relate to other Repeat Expansion Diseases that also show repeat-mediated changes in gene expression.

Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity

PubMed Central

Li, Bo; Jiang, Shan; Yu, Xiao; Cheng, Cheng; Chen, Sixue; Cheng, Yanbing; Yuan, Joshua S.; Jiang, Daohong; He, Ping; Shan, Libo

2015-01-01

Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression. PMID:25770109

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

PubMed

Maconochie, M K; Nonchev, S; Studer, M; Chan, S K; Pöpperl, H; Sham, M H; Mann, R S; Krumlauf, R

1997-07-15

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.

PubMed

Couture, Camille; Zaniolo, Karine; Carrier, Patrick; Lake, Jennifer; Patenaude, Julien; Germain, Lucie; Guérin, Sylvain L

2016-02-01

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.

ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs Interact to Activate the Expression of SOMNUS and Other High-Temperature-Inducible Genes in Imbibed Seeds in Arabidopsis[W

PubMed Central

Lim, Soohwan; Park, Jeongmoo; Lee, Nayoung; Jeong, Jinkil; Toh, Shigeo; Watanabe, Asuka; Kim, Junghyun; Kang, Hyojin; Kim, Dong Hwan; Kawakami, Naoto; Choi, Giltsu

2013-01-01

Seeds monitor the environment to germinate at the proper time, but different species respond differently to environmental conditions, particularly light and temperature. In Arabidopsis thaliana, light promotes germination but high temperature suppresses germination. We previously reported that light promotes germination by repressing SOMNUS (SOM). Here, we examined whether high temperature also regulates germination through SOM and found that high temperature activates SOM expression. Consistent with this, som mutants germinated more frequently than the wild type at high temperature. The induction of SOM mRNA at high temperature required abscisic acid (ABA) and gibberellic acid biosynthesis, and ABA-INSENSITIVE3 (ABI3), ABI5, and DELLAs positively regulated SOM expression. Chromatin immunoprecipitation assays indicated that ABI3, ABI5, and DELLAs all target the SOM promoter. At the protein level, ABI3, ABI5, and DELLAs all interact with each other, suggesting that they form a complex on the SOM promoter to activate SOM expression at high temperature. We found that high-temperature-inducible genes frequently have RY motifs and ABA-responsive elements in their promoters, some of which are targeted by ABI3, ABI5, and DELLAs in vivo. Taken together, our data indicate that ABI3, ABI5, and DELLAs mediate high-temperature signaling to activate the expression of SOM and other high-temperature-inducible genes, thereby inhibiting seed germination. PMID:24326588

JAK and STAT members in channel catfish: Identification, phylogenetic analysis and expression profiling after Edwardsiella ictaluri infection.

PubMed

Jin, Yulin; Zhou, Tao; Li, Ning; Liu, Shikai; Xu, Xiaoyan; Pan, Ying; Tan, Suxu; Shi, Huitong; Yang, Yujia; Yuan, Zihao; Wang, Wenwen; Luo, Jian; Gao, Dongya; Dunham, Rex; Liu, Zhanjiang

2018-04-01

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is one of the main pleiotropic cascades used to transmit information from extracellular receptors to the nucleus, which results in DNA transcription and expression of genes involved in immunity, proliferation, differentiation, migration, apoptosis, and cell survival. Members of JAK family and STAT family have been extensively studied in different mammalian species because of their important roles in innate and adaptive immune responses. However, they have not been systematically studied among teleost fish species. In this study, five JAK family members and eight STAT family members were identified and characterized from channel catfish. Phylogenetic analysis was conducted to properly annotate these genes. Syntenic analysis was also conducted to establish orthology, and confirm the results from phylogenetic analysis. Compared to mammals, more members of the JAK and STAT family were identified in channel catfish genome. Expression of JAK and STAT family members was detected in healthy catfish tissues, but was induced in gill, liver, and intestine after bacterial challenge. Notably, the significant upregulation of STAT1b gene in catfish liver, gill and intestine after Edwardsiella ictaluri infection supported the notion that high STAT1 expression are involved in defense against pathogens. Collectively, the increased expression of JAK and STAT members in tested tissues suggested their crucial function in defending the host against pathogen invasion. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulatory link between DNA methylation and active demethylation in Arabidopsis

PubMed Central

Lei, Mingguang; Zhang, Huiming; Julian, Russell; Tang, Kai; Xie, Shaojun; Zhu, Jian-Kang

2015-01-01

De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression. PMID:25733903

Epigenetic mechanisms: critical contributors to long-term memory formation.

PubMed

Lubin, Farah D; Gupta, Swati; Parrish, R Ryley; Grissom, Nicola M; Davis, Robin L

2011-12-01

Recent advances in chromatin biology have identified a role for epigenetic mechanisms in the regulation of neuronal gene expression changes, a necessary process for proper synaptic plasticity and memory formation. Experimental evidence for dynamic chromatin remodeling influencing gene transcription in postmitotic neurons grew from initial reports describing posttranslational modifications of histones, including phosphorylation and acetylation occurring in various brain regions during memory consolidation. An accumulation of recent studies, however, has also highlighted the importance of other epigenetic modifications, such as DNA methylation and histone methylation, as playing a role in memory formation. This present review examines learning-induced gene transcription by chromatin remodeling underlying long-lasting changes in neurons, with direct implications for the study of epigenetic mechanisms in long-term memory formation and behavior. Furthermore, the study of epigenetic gene regulation, in conjunction with transcription factor activation, can provide complementary lines of evidence to further understanding transcriptional mechanisms subserving memory storage.

Overexpression of maize anthocyanin regulatory gene Lc affects rice fertility.

PubMed

Li, Yuan; Zhang, Tao; Shen, Zhong-Wei; Xu, Yu; Li, Jian-Yue

2013-01-01

Seventeen independent transgenic rice plants with the maize anthocyanin regulatory gene Lc under control of the CaMV 35S promoter were obtained and verified by molecular identification. Ten plants showed red spikelets during early development of florets, and the degenerate florets were still red after heading. Additionally, these plants exhibited intense pigmentation on the surface of the anther and the bottom of the ovary. They were unable to properly bloom and were completely sterile. Following pollination with normal pollen, these plants yielded red caryopses but did not mature normally. QRT-PCR analysis indicated that mRNA accumulation of the CHS-like gene encoding a chalcone synthase-related protein was increased significantly in the sterile plant. This is the first report to suggest that upregulation of the CHS gene expression may result in rice sterility and affect the normal development of rice seeds.

GLI1, a master regulator of the hallmark of pancreatic cancer.

PubMed

Kasai, Kenji

2016-12-01

Hedgehog signaling is highly conserved across species and governs proper embryonic development. Germline gene mutations that reduce this signaling activity cause a variety of developmental abnormalities such as holoprosencephaly, while those that enhance Hedgehog signaling activity induce a tumor-predisposition condition Nevoid basal cell carcinoma syndrome. Furthermore, dysregulated activation of Hedgehog signaling has been recognized in various sporadic malignancies, including pancreatic adenocarcinoma. Pancreatic adenocarcinoma develops through a multistep carcinogenesis starting with oncogenic mutation of the KRAS gene. During this process, precancerous or cancer cells secrete Hedgehog ligand proteins to promote characteristic desmoplastic stroma around the cells, which in turn activates the expression of the downstream transcription factor GLI1 inside the cells. The quantitative and spatiotemporal dysregulation of GLI1 subsequently leads to the expression of transcriptional target genes of GLI1 that govern the hallmark of malignant properties. Here, after a brief introductory outline, a perspective is offered of Hedgehog signaling with a special focus on the role of GLI1 in pancreatic carcinogenesis. © 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Nucleoplasmic Nup98 controls gene expression by regulating a DExH/D-box protein.

PubMed

Capitanio, Juliana S; Montpetit, Ben; Wozniak, Richard W

2018-01-01

The nucleoporin Nup98 has been linked to the regulation of transcription and RNA metabolism, 1-3 but the mechanisms by which Nup98 contributes to these processes remains largely undefined. Recently, we uncovered interactions between Nup98 and several DExH/D-box proteins (DBPs), a protein family well-known for modulating gene expression and RNA metabolism. 4-6 Analysis of Nup98 and one of these DBPs, DHX9, showed that they directly interact, their association is facilitated by RNA, and Nup98 binding stimulates DHX9 ATPase activity. 7 Furthermore, these proteins were dependent on one another for their proper association with a subset of gene loci to control transcription and modulate mRNA splicing. 7 On the basis of these observations, we proposed that Nup98 functions to regulate DHX9 activity within the nucleoplasm. 7 Since Nup98 is associated with several DBPs, regulation of DHX9 by Nup98 may represent a paradigm for understanding how Nup98, and possibly other FG-Nup proteins, could direct the diverse cellular activities of multiple DBPs.

Differential Expression of Programmed Cell Death on the Follicular Development in Normal and Miniature Pig Ovary

PubMed Central

Kim, Sang Hwan; Min, Kwan Sik; Kim, Nam Hyung; Yoon, Jong Taek

2012-01-01

Follicles are important in oocyte maturation. Successful estrous cycle requires remodeling of follicular cells, and proper execution of programmed cell death is crucial for normal follicular development. The objectives of the present study were to understand programmed cell death during follicle development, to analyze the differential follicle development patterns, and to assess the patterns of apoptosis and autophagy expression during follicle development in normal and miniature pigs. Through the analysis of differential patterns of programmed cell death during follicular development in porcine, MAP1LC3A, B and other autophagy-associated genes (ATG5, mTOR, Beclin-1) were found to increase in normal pigs, while it decreased in miniature pigs. However, for the apoptosis-associated genes, progression of genes during follicular development increased in miniature pigs, while it decreased in normal pigs. Thus, results show that normal and miniature pigs showed distinct patterns of follicular remodeling manifesting that programmed cell death largely depends on the types of pathway during follicular development (Type II or autophagy for normal pigs and Type I or apoptosis for miniature pigs). PMID:23056260

A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status

PubMed Central

Dai, Weijun; Li, Wencheng; Hoque, Mainul; Li, Zhuyun; Tian, Bin; Makeyev, Eugene V.

2015-01-01

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3′-untranslated regions (3′ UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. PMID:26144867

Molecular anatomy of the developing limb in the coquí frog, Eleutherodactylus coqui.

PubMed

Gross, Joshua B; Kerney, Ryan; Hanken, James; Tabin, Clifford J

2011-01-01

The vertebrate limb demonstrates remarkable similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this morphological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size-reduced embryos of the Puerto Rican coquí frog, Eleutherodactylus coqui. This animal demonstrates direct development, a life-history strategy marked by rapid progression from egg to adult and absence of a free-living, aquatic larva. Nonetheless, coquí exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We investigated the extent to which coquí limb bud development conforms to the model of limb development derived from amniote studies. Toward this end, we characterized dynamic patterns of expression for 13 critical patterning genes across three principle stages of limb development. As expected, most genes demonstrate expression patterns that are essentially unchanged compared to amniote species. For example, we identified an EcFgf8-expression domain within the apical ectodermal ridge (AER). This expression pattern defines a putatively functional AER signaling domain, despite the absence of a morphological ridge in coquí embryos. However, two genes, EcMeis2 and EcAlx4, demonstrate altered domains of expression, which imply a potential shift in gene function between coquí frogs and amniote model systems. Unexpectedly, several genes thought to be critical for limb patterning in other systems, including EcFgf4, EcWnt3a, EcWnt7a, and EcGremlin, demonstrated no evident expression pattern in the limb at the three stages we analyzed. The absence of EcFgf4 and EcWnt3a expression during limb patterning is perhaps not surprising, given that neither gene is critical for proper limb development in the mouse, based on knockout and expression analyses. In contrast, absence of EcWnt7a and EcGremlin is surprising, given that expression of these molecules appears to be absolutely essential in all other model systems so far examined. Although this analysis substantiates the existence of a core set of ancient limb-patterning molecules, which likely mediate identical functions across highly diverse vertebrate forms, it also reveals remarkable evolutionary flexibility in the genetic control of a conserved morphological pattern across evolutionary time. © 2011 Wiley Periodicals, Inc.

The ArcB Leucine Zipper Domain Is Required for Proper ArcB Signaling

PubMed Central

Nuñez Oreza, Luis Alberto; Alvarez, Adrián F.; Arias-Olguín, Imilla I.; Torres Larios, Alfredo; Georgellis, Dimitris

2012-01-01

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70–121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling. PMID:22666479

In Vivo Exposure to Inorganic Arsenic Alters Differentiation-Specific Gene Expression of Adipose-Derived Mesenchymal Stem/Stromal Cells in C57BL/6J Mouse Model

PubMed Central

Shearer, Joseph J.; Figueiredo Neto, Manoel; Umbaugh, C. Samuel; Figueiredo, Marxa L.

2017-01-01

Abstract The number of mesenchymal stem cell (MSC) therapeutic modalities has grown in recent years. Adipose-derived mesenchymal stem/stromal cells (ASCs) can be isolated and expanded relatively easily as compared with their bone-marrow counterparts, making them a particularly promising source of MSCs. And although the biological mechanisms surrounding ASCs are actively being investigated, little is known about the effects that in vivo environmental exposures might have on their ability to properly differentiate. Therefore, we hypothesized that ASCs isolated from mice exposed to inorganic arsenic (iAs) would have an altered response towards adipogenic, osteogenic, and/or chondrogenic differentiation. To test this hypothesis, C57BL/6J male mice were provided drinking water containing 0, 300, or 1000 ppb iAs. ASCs were then isolated and differentiated, which was assessed by immunocytochemistry and real-time quantitative PCR (RT-qPCR). Our results showed that total urinary arsenic equilibrated within 1 week of exposure to iAs and was maintained throughout the study. ASCs isolated from each exposure group maintained differentiation capabilities for each lineage. The magnitude of differentiation-specific gene expression, however, appeared to be concentration dependent. For osteogenesis and chondrogenesis, differentiation-specific gene expression decreased, whereas adipogenesis showed a biphasic response with an initial decrease followed by an increase in adipogenic-related gene expression following iAs exposure. These results suggest that the level in which differentiation-specific genes are induced within these stromal cells might be sensitive to environmental contaminants. These findings highlight the need to take into account potential environmental exposures prior to selecting stromal cell donors, so ASCs can achieve optimal efficiency in regenerative therapy applications. PMID:28206643

MCM-BP Is Required for Repression of Life-Cycle Specific Genes Transcribed by RNA Polymerase I in the Mammalian Infectious Form of Trypanosoma brucei

PubMed Central

Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A. M.

2013-01-01

Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei. PMID:23451133

An Arabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperatures

PubMed Central

Guo, Yan; Xiong, Liming; Ishitani, Manabu; Zhu, Jian-Kang

2002-01-01

Low temperature regulates gene expression in bacteria, yeast, and animals as well as in plants. However, the signal transduction cascades mediating the low temperature responses are not well understood in any organism. To identify components in low temperature signaling genetically, we isolated Arabidopsis thaliana mutants in which cold-responsive genes are no longer induced by low temperatures. One of these mutations, los1–1, specifically blocks low temperature-induced transcription of cold-responsive genes. Surprisingly, cold-induced expression of the early response transcriptional activators, C-repeat/dehydration responsive element binding factors (CBF/DREB1s), is enhanced by the los1–1 mutation. The los1–1 mutation also reduces the capacity of plants to develop freezing tolerance but does not impair the vernalization response. Genetic analysis indicated that los1–1 is a recessive mutation in a single nuclear gene. The LOS1 gene encodes a translation elongation factor 2-like protein. Protein labeling studies show that new protein synthesis is blocked in los1–1 mutant plants specifically in the cold. These results reveal a critical role of new protein synthesis in the proper transduction of low temperature signals. Our results also suggest that cold-induced transcription of CBF/DREB1s is feedback inhibited by their gene products or by products of their downstream target genes. PMID:12032361

Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

PubMed

Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

2015-10-01

Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

PubMed Central

Carpio, Lomeli R.; Bradley, Elizabeth W.; McGee-Lawrence, Meghan E.; Weivoda, Megan M.; Poston, Daniel D.; Dudakovic, Amel; Xu, Ming; Tchkonia, Tamar; Kirkland, James L.; van Wijnen, Andre J.; Oursler, Merry Jo; Westendorf, Jennifer J.

2017-01-01

Histone deacetylase (HDAC) inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, each of these drugs inhibits multiple HDACs and has detrimental effects on the skeleton. To better understand how HDAC inhibitors affect endochondral bone formation, we conditionally deleted one of their targets, Hdac3, pre- and postnatally in type II collagen α1 (Col2α1)–expressing chondrocytes. Embryonic deletion was lethal, but postnatal deletion of Hdac3 delayed secondary ossification center formation, altered maturation of growth plate chondrocytes, and increased osteoclast activity in the primary spongiosa. HDAC3-deficient chondrocytes exhibited increased expression of cytokine and matrix-degrading genes (Il-6, Mmp3, Mmp13, and Saa3) and a reduced abundance of genes related to extracellular matrix production, bone development, and ossification (Acan, Col2a1, Ihh, and Col10a1). Histone acetylation increased at and near genes that had increased expression. The acetylation and activation of nuclear factor κB (NF-κB) were also increased in HDAC3-deficient chondrocytes. Increased cytokine signaling promoted autocrine activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and NF-κB pathways to suppress chondrocyte maturation, as well as paracrine activation of osteoclasts and bone resorption. Blockade of interleukin-6 (IL-6)–JAK–STAT signaling, NF-κB signaling, and bromodomain extraterminal proteins, which recognize acetylated lysines and promote transcriptional elongation, significantly reduced Il-6 and Mmp13 expression in HDAC3-deficient chondrocytes and secondary activation in osteoclasts. The JAK inhibitor ruxolitinib also reduced osteoclast activity in Hdac3 conditional knockout mice. Thus, HDAC3 controls the temporal and spatial expression of tissue-remodeling genes and inflammatory responses in chondrocytes to ensure proper endochondral ossification during development. PMID:27507649

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification

PubMed Central

Schaub, Christoph; Nagaso, Hideyuki; Jin, Hong; Frasch, Manfred

2012-01-01

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1–Slou and Org-1–Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila. PMID:22318630

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification.

PubMed

Schaub, Christoph; Nagaso, Hideyuki; Jin, Hong; Frasch, Manfred

2012-03-01

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.

RNAi Functions in Adaptive Reprogramming of the Genome | Center for Cancer Research

Cancer.gov

The regulation of transcribing DNA into RNA, including the production, processing, and degradation of RNA transcripts, affects the expression and the regulation of the genome in ways that are just beginning to be unraveled. A surprising discovery in recent years is that the vast majority of the genome is transcribed to yield an abundance of RNA transcripts. Many transcripts are regulated by the exosome, a multi-protein complex that degrades RNAs, and may also be targeted, under certain conditions, by the RNA interference (RNAi) pathway. These RNA degrading activities can recruit factors to silence certain regions of the genome by condensing the DNA into tightly-packed heterochromatin. For some chromosomal regions, such as centromeres and telomeres, which lie at the center and ends of chromosomes, respectively, silencing must be stably enforced through each cell generation. For other regions, silencing mechanisms must be easily reversible to activate gene expression in response to changing environmental or developmental conditions. Thus, the regulation of gene silencing is key to maintaining the integrity of the genome and proper cellular expression patterns, which, when disrupted can underlie many diseases, including cancer.

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation.

PubMed

Naro, Chiara; Jolly, Ariane; Di Persio, Sara; Bielli, Pamela; Setterblad, Niclas; Alberdi, Antonio J; Vicini, Elena; Geremia, Raffaele; De la Grange, Pierre; Sette, Claudio

2017-04-10

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Concerted transcription of auxin and carbohydrate homeostasis-related genes underlies improved adventitious rooting of microcuttings derived from far-red treated Eucalyptus globulus Labill mother plants.

PubMed

Ruedell, Carolina Michels; de Almeida, Márcia Rodrigues; Fett-Neto, Arthur Germano

2015-12-01

Economically important plant species, such as Eucalyptus globulus, are often rooting recalcitrant. We have previously shown that far-red light enrichment applied to E. globulus donor-plants improved microcutting rooting competence and increased rooting zone/shoot carbohydrate ratio. To better understand this developmental response, the relative expression profiles of genes involved in auxin signaling (ARF6, ARF8, AGO1), biosynthesis (YUC3) and transport (AUX1, PIN1, PIN2); sucrose cleavage (SUS1, CWINV1), transport (SUC5), hexose phosphorylation (HXK1, FLN1) and starch biosynthesis (SS3) were quantified during adventitious rooting of E. globulus microcuttings derived from donor plants exposed to far-red or white light. Expression of auxin transport-related genes increased in the first days of root induction. Far-red enrichment of donor plants induced ARF6, ARF8 and AGO1 in microcuttings. The first two gene products could activate GH3 and other rooting related genes, whereas AGO1 deregulation of the repressor ARF17 may relief rooting inhibition. Increased sink strength at the basal stem with sucrose unloading in root tissue mediated by SUC and subsequent hydrolysis by SUS1 were also supported by gene expression profile. Fructose phosphorylation and starch biosynthesis could also contribute to proper carbon allocation at the site of rooting, as evidenced by increased expression of related genes. These data are in good agreement with increased contents of hexoses and starch at the cutting base severed from far-red exposed donor plants. To sum up, pathways integrating auxin and carbohydrate metabolism were activated in microcuttings derived from donor plants exposed to far red light enrichment, thereby improving rooting response in E. globulus. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Identification and characterization of calcium transporter gene family in finger millet in relation to grain calcium content.

PubMed

Singh, Uma M; Metwal, Mamta; Singh, Manoj; Taj, Gohar; Kumar, Anil

2015-07-15

Calcium (Ca) is an essential mineral for proper growth and development of plants as well as animals. In plants including cereals, calcium is deposited in seed during its development which is mediated by specialized Ca transporters. Common cereal seeds contain very low amounts of Ca while the finger millet (Eleusine coracana) contains exceptionally high amounts of Ca in seed. In order to understand the role of Ca transporters in grain Ca accumulation, developing seed transcriptome of two finger millet genotypes (GP-1, low Ca and GP-45 high Ca) differing in seed Ca content was sequenced using Illumina paired-end sequencing technology and members of Ca transporter gene family were identified. Out of 109,218 and 120,130 contigs, 86 and 81 contigs encoding Ca transporters were identified in GP-1 and GP-45, respectively. After removal of redundant sequences, a total of 19 sequences were confirmed as Ca transporter genes, which includes 11 Ca(2+) ATPases, 07 Ca(2+)/cation exchangers and 01 Ca(2+) channel. The differential expressions of all genes were analyzed from transcriptome data and it was observed that 9 and 3 genes were highly expressed in GP-45 and GP-1 genotypes respectively. Validation of transcriptome expression data of selected Ca transporter genes was performed on different stages of developing spikes of both genotypes grown under different concentrations of exogenous Ca. In both genotypes, significant correlation was observed between the expression of these genes, especially EcCaX3, and on the amount of Ca accumulated in seed. The positive correlation of seed mass with the amount of Ca concentration was also observed. The efficient Ca transport property and responsiveness of EcCAX3 towards exogenous Ca could be utilized in future biofortification program. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome-wide investigation and expression analyses of WD40 protein family in the model plant foxtail millet (Setaria italica L.).

PubMed

Mishra, Awdhesh Kumar; Muthamilarasan, Mehanathan; Khan, Yusuf; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I-V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

PubMed Central

Mishra, Awdhesh Kumar; Muthamilarasan, Mehanathan; Khan, Yusuf; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses. PMID:24466268

Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

PubMed

Tramontana, S; Bionaz, M; Sharma, A; Graugnard, D E; Cutler, E A; Ajmone-Marsan, P; Hurley, W L; Loor, J J

2008-08-01

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.

Molecular response to water stress in two contrasting Mediterranean pines (Pinus pinaster and Pinus pinea).

PubMed

Perdiguero, Pedro; Barbero, María Del Carmen; Cervera, María Teresa; Collada, Carmen; Soto, Alvaro

2013-06-01

Adaptation to water stress has determined the evolution and diversification of vascular plants. Water stress is forecasted to increase drastically in the next decades in certain regions, such as in the Mediterranean basin. Consequently, a proper knowledge of the response and adaptations to drought stress is essential for the correct management of plant genetic resources. However, most of the advances in the understanding of the molecular response to water stress have been attained in angiosperms, and are not always applicable to gymnosperms. In this work we analyse the transcriptional response of two emblematic Mediterranean pines, Pinus pinaster and Pinus pinea, which show noticeable differences in their performance under water stress. Using microarray analysis, up to 113 genes have been detected as significantly induced by drought in both species. Reliability of expression patterns has been confirmed by RT-PCR. While induced genes with similar profiles in both species can be considered as general candidate genes for the study of drought response in conifers, genes with diverging expression patterns can underpin the differences displayed by these species under water stress. Most promising candidate genes for drought stress response include genes related to carbohydrate metabolism, such as glycosyltransferases or galactosidases, sugar transporters, dehydrins and transcription factors. Additionally, differences in the molecular response to drought and polyethylene-glycol-induced water stress are also discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Reference genes for real-time PCR quantification of messenger RNAs and microRNAs in mouse model of obesity.

PubMed

Matoušková, Petra; Bártíková, Hana; Boušová, Iva; Hanušová, Veronika; Szotáková, Barbora; Skálová, Lenka

2014-01-01

Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.

Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos.

PubMed

Yamada, Shigehiro; Hotta, Kohji; Yamamoto, Takamasa S; Ueno, Naoto; Satoh, Nori; Takahashi, Hiroki

2009-04-01

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.

[Companion diagnostics in the era of personalized medicine--chairmen's introductory remarks].

PubMed

Fukutsuka, Katsuhiro; Takubo, Takayuki

2014-04-01

Personalized medicine is a medical model that proposes the customization of treatment for individual patients. In this model, diagnostic tests are essential for selecting safer and more efficacious treatments. The term "companion diagnostics" has been used to describe these tests, whereby molecular assays that measure the levels of proteins or specific gene mutations are used to provide a specific therapy for an individual by stratifying the disease status, selecting the proper medication, and tailoring dosages. Examples of companion diagnostics in the field of cancer medicine for molecular targeted therapy include tests for the ALK-fusion gene in non-small cell lung cancer and expression of CCR4 in adult T-cell leukemia. For breast cancer, the expression of HER2 protein is evaluated by immunohistochemistry (IHC), and gene amplification of HER2 is tested by fluorescence in situ hybridization (FISH); both tests consist of pre-analysis, analysis, and post-analysis processes that require quality control to ensure the reliability of the results. This symposium includes: 1) future aspects of companion diagnostics addressing many of the problems that must be overcome, 2) companion diagnostics using FISH focusing on HER2 amplification and ALK alteration, 3) newly developed diagnostic tests using tumor specimens and cell-free DNA in serum, and 4) CCR4 expression detected by IHC and flow cytometry.

Different cucumber CsYUC genes regulate response to abiotic stresses and flower development.

PubMed

Yan, Shuangshuang; Che, Gen; Ding, Lian; Chen, Zijing; Liu, Xiaofeng; Wang, Hongyin; Zhao, Wensheng; Ning, Kang; Zhao, Jianyu; Tesfamichael, Kiflom; Wang, Qian; Zhang, Xiaolan

2016-02-09

The phytohormone auxin is essential for plant growth and development, and YUCCA (YUC) proteins catalyze a rate-limiting step for endogenous auxin biosynthesis. Despite YUC family genes have been isolated from several species, systematic expression analyses of YUCs in response to abiotic stress are lacking, and little is known about the function of YUC homologs in agricultural crops. Cucumber (Cucumis sativus L.) is a world cultivated vegetable crop with great economical and nutritional value. In this study, we isolated 10 YUC family genes (CsYUCs) from cucumber and explored their expression pattern under four types of stress treatments. Our data showed that CsYUC8 and CsYUC9 were specifically upregulated to elevate the auxin level under high temperature. CsYUC10b was dramatically increased but CsYUC4 was repressed in response to low temperature. CsYUC10a and CsYUC11 act against the upregulation of CsYUC10b under salinity stress, suggesting that distinct YUC members participate in different stress response, and may even antagonize each other to maintain the proper auxin levels in cucumber. Further, CsYUC11 was specifically expressed in the male flower in cucumber, and enhanced tolerance to salinity stress and regulated pedicel and stamen development through auxin biosynthesis in Arabidopsis.

Tissue Architecture and Microenvironment Sustain Hormone Signaling | Center for Cancer Research

Cancer.gov

Cells interact with their environments in part through protein receptors embedded in the cell membrane. Activation of a receptor by external signaling molecules sets off a complex chain of events within the cell that can result in alterations in protein structure and function and/or changes in gene expression. Proper integration of these signals is crucial for normal cell growth and development. A more complete understanding of these normal processes will help elucidate how aberrant signaling results in diseases such as cancer.  

The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect[C][W][OA

PubMed Central

Ngo, Quy A.; Baroux, Celia; Guthörl, Daniela; Mozerov, Peter; Collinge, Margaret A.; Sundaresan, Venkatesan; Grossniklaus, Ueli

2012-01-01

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin–dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of MINISEED3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways. PMID:23064319

contamDE: differential expression analysis of RNA-seq data for contaminated tumor samples.

PubMed

Shen, Qi; Hu, Jiyuan; Jiang, Ning; Hu, Xiaohua; Luo, Zewei; Zhang, Hong

2016-03-01

Accurate detection of differentially expressed genes between tumor and normal samples is a primary approach of cancer-related biomarker identification. Due to the infiltration of tumor surrounding normal cells, the expression data derived from tumor samples would always be contaminated with normal cells. Ignoring such cellular contamination would deflate the power of detecting DE genes and further confound the biological interpretation of the analysis results. For the time being, there does not exists any differential expression analysis approach for RNA-seq data in literature that can properly account for the contamination of tumor samples. Without appealing to any extra information, we develop a new method 'contamDE' based on a novel statistical model that associates RNA-seq expression levels with cell types. It is demonstrated through simulation studies that contamDE could be much more powerful than the existing methods that ignore the contamination. In the application to two cancer studies, contamDE uniquely found several potential therapy and prognostic biomarkers of prostate cancer and non-small cell lung cancer. An R package contamDE is freely available at http://homepage.fudan.edu.cn/zhangh/softwares/ zhanghfd@fudan.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability

PubMed Central

Ruiz, Oscar N.; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

2015-01-01

Summary Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183 000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioniens in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation. PMID:21518240

Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein.

PubMed

Garrity, P A; Rao, Y; Salecker, I; McGlade, J; Pawson, T; Zipursky, S L

1996-05-31

Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney.

PubMed

Drake, Keri A; Adam, Mike; Mahoney, Robert; Potter, S Steven

2018-04-20

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

Characterization of axon formation in the embryonic stem cell-derived motoneuron.

PubMed

Pan, Hung-Chuan; Wu, Ya-Ting; Shen, Shih-Cheng; Wang, Chi-Chung; Tsai, Ming-Shiun; Cheng, Fu-Chou; Lin, Shinn-Zong; Chen, Ching-Wen; Liu, Ching-San; Su, Hong-Lin

2011-01-01

The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

RNAi and heterochromatin repress centromeric meiotic recombination

PubMed Central

Ellermeier, Chad; Higuchi, Emily C.; Phadnis, Naina; Holm, Laerke; Geelhood, Jennifer L.; Thon, Genevieve; Smith, Gerald R.

2010-01-01

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis. PMID:20421495

Multiclass classification for skin cancer profiling based on the integration of heterogeneous gene expression series.

PubMed

Gálvez, Juan Manuel; Castillo, Daniel; Herrera, Luis Javier; San Román, Belén; Valenzuela, Olga; Ortuño, Francisco Manuel; Rojas, Ignacio

2018-01-01

Most of the research studies developed applying microarray technology to the characterization of different pathological states of any disease may fail in reaching statistically significant results. This is largely due to the small repertoire of analysed samples, and to the limitation in the number of states or pathologies usually addressed. Moreover, the influence of potential deviations on the gene expression quantification is usually disregarded. In spite of the continuous changes in omic sciences, reflected for instance in the emergence of new Next-Generation Sequencing-related technologies, the existing availability of a vast amount of gene expression microarray datasets should be properly exploited. Therefore, this work proposes a novel methodological approach involving the integration of several heterogeneous skin cancer series, and a later multiclass classifier design. This approach is thus a way to provide the clinicians with an intelligent diagnosis support tool based on the use of a robust set of selected biomarkers, which simultaneously distinguishes among different cancer-related skin states. To achieve this, a multi-platform combination of microarray datasets from Affymetrix and Illumina manufacturers was carried out. This integration is expected to strengthen the statistical robustness of the study as well as the finding of highly-reliable skin cancer biomarkers. Specifically, the designed operation pipeline has allowed the identification of a small subset of 17 differentially expressed genes (DEGs) from which to distinguish among 7 involved skin states. These genes were obtained from the assessment of a number of potential batch effects on the gene expression data. The biological interpretation of these genes was inspected in the specific literature to understand their underlying information in relation to skin cancer. Finally, in order to assess their possible effectiveness in cancer diagnosis, a cross-validation Support Vector Machines (SVM)-based classification including feature ranking was performed. The accuracy attained exceeded the 92% in overall recognition of the 7 different cancer-related skin states. The proposed integration scheme is expected to allow the co-integration with other state-of-the-art technologies such as RNA-seq.

Immunohistochemistry as a surrogate for molecular testing: a review.

PubMed

Swanson, Paul E

2015-02-01

Despite the myriad of genetic and epigenetic alterations in human neoplasms that seem to demand specific molecular probes for their identification and practical application to diagnostic pathology, immunohistochemistry (IHC) remains a vital component of laboratory testing in the emerging molecular era. The development and proper application of sensitive and specific antibodies raised against cryptic proteins only expressed in quantity after gene translocation, translocation-specific chimeric fusion peptides, and gene products overexpressed because of gene amplification demonstrate that IHC is a legitimate surrogate for traditional cytogenetic and in situ hybridization-based identification of chromosomal abnormalities, if not a viable molecular technique in its own right. Similarly, the detection of mutational events, through the reliable demonstration of protein loss, the identification of proteins overexpressed because of activating mutations, the specific visualization of mutant gene products, and the localization of splice variant gene products emphasizes the potential value of IHC as a surrogate for mutational analyses of genes important to both diagnosis and prediction of therapeutic response. In the latter setting IHC also provides a means of approximating gene expression profiles in the molecular classification and risk stratification of human neoplasms. For time being, the application of appropriately targeted sensitive and specific antibodies provides a cost-effective screening modality, if not replacement, for selected molecular techniques, but IHC will lose its value if the development of companion tests for emerging novel biomarkers does not keep pace with molecular techniques, particularly as the costs and time constraints of genomic sequencing diminish over time.

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

PubMed

Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

2013-11-01

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

Statins cause profound effects on gene expression in human cancer cells in vitro: the role of membrane microdomains.

PubMed

Garnett, David John; Greenhough, Trevor James

2012-01-01

There is increasing evidence that statin treatment can be beneficial in certain cancer patients. To determine if these benefits are a direct result of the cholesterol-lowering effects of statins or a result of secondary, protein transcription effects, the impacts of pravastatin and a cholesterol sequestrating agent methyl-beta-cyclodextrin (MbetaCD) on mRNA expression in the breast cancer cell MDA-MB-231 and the lung carcinoma cell Calu-1 have been compared by microarray techniques. The effects of these agents on cholesterol-rich rafts and caveolae, which have significance in cancer signaling, have also been examined. Both treatments caused a general downregulation of not only signal transduction including cancer pathway proteins, but also apoptosis and chemokine pathways, with statins impacting 35 genes by twofold or greater in MDA-MB-231 and > 300 genes in Calu-1. These manifold dysregulations could also explain the various side effects reportedly caused by statins. MbetaCD produced far fewer statistical events than pravastatin in the breast cancer line but many more in the lung cell line. Pravastatin increased expression of CAV1 but caveolae density decreased and overall raft density was unaffected. MbetaCD also caused an increase in CAV1 expression and reduced the prevalence of both rafts and caveolae. It is proposed that sequestration of cholesterol from the membrane by MbetaCD is not equivalent to blockade of the cholesterol pathway and causes different effects on microdomain-mediated signal transduction dependant on the cell line. The profound effects of statins on mRNA expression can be explained by the failure of caveolin-1 to properly complex with cholesterol in an altered sterol environment, with caveolae acting as the main loci for signaling directed towards those transcription processes unaffected by MbetaCD. Targeted inhibition of the postmevalonate pathway could offer an opportunity to specifically reduce caveolae-based signaling in cancer cells. The observed impact of pravastatin on gene expression may explain the pleiotropic effects of statins when they are used as adjuvants in chemotherapy and suggests impact on gene expression as a possible cause of side effects from statin use.

MYB10 and MYB72 are required for growth under iron-limiting conditions.

PubMed

Palmer, Christine M; Hindt, Maria N; Schmidt, Holger; Clemens, Stephan; Guerinot, Mary Lou

2013-11-01

Iron is essential for photosynthesis and is often a limiting nutrient for plant productivity. Plants respond to conditions of iron deficiency by increasing transcript abundance of key genes involved in iron homeostasis, but only a few regulators of these genes have been identified. Using genome-wide expression analysis, we searched for transcription factors that are induced within 24 hours after transferring plants to iron-deficient growth conditions. Out of nearly 100 transcription factors shown to be up-regulated, we identified MYB10 and MYB72 as the most highly induced transcription factors. Here, we show that MYB10 and MYB72 are functionally redundant and are required for plant survival in alkaline soil where iron availability is greatly restricted. myb10myb72 double mutants fail to induce transcript accumulation of the nicotianamine synthase gene NAS4. Both myb10myb72 mutants and nas4-1 mutants have reduced iron concentrations, chlorophyll levels, and shoot mass under iron-limiting conditions, indicating that these genes are essential for proper plant growth. The double myb10myb72 mutant also showed nickel and zinc sensitivity, similar to the nas4 mutant. Ectopic expression of NAS4 rescues myb10myb72 plants, suggesting that loss of NAS4 is the primary defect in these plants and emphasizes the importance of nicotianamine, an iron chelator, in iron homeostasis. Overall, our results provide evidence that MYB10 and MYB72 act early in the iron-deficiency regulatory cascade to drive gene expression of NAS4 and are essential for plant survival under iron deficiency.

Topographical mapping of α- and β-keratins on developing chicken skin integuments: Functional interaction and evolutionary perspectives

PubMed Central

Wu, Ping; Ng, Chen Siang; Yan, Jie; Lai, Yung-Chih; Chen, Chih-Kuan; Lai, Yu-Ting; Wu, Siao-Man; Chen, Jiun-Jie; Luo, Weiqi; Widelitz, Randall B.; Li, Wen-Hsiung; Chuong, Cheng-Ming

2015-01-01

Avian integumentary organs include feathers, scales, claws, and beaks. They cover the body surface and play various functions to help adapt birds to diverse environments. These keratinized structures are mainly composed of corneous materials made of α-keratins, which exist in all vertebrates, and β-keratins, which only exist in birds and reptiles. Here, members of the keratin gene families were used to study how gene family evolution contributes to novelty and adaptation, focusing on tissue morphogenesis. Using chicken as a model, we applied RNA-seq and in situ hybridization to map α- and β-keratin genes in various skin appendages at embryonic developmental stages. The data demonstrate that temporal and spatial α- and β-keratin expression is involved in establishing the diversity of skin appendage phenotypes. Embryonic feathers express a higher proportion of β-keratin genes than other skin regions. In feather filament morphogenesis, β-keratins show intricate complexity in diverse substructures of feather branches. To explore functional interactions, we used a retrovirus transgenic system to ectopically express mutant α- or antisense β-keratin forms. α- and β-keratins show mutual dependence and mutations in either keratin type results in disrupted keratin networks and failure to form proper feather branches. Our data suggest that combinations of α- and β-keratin genes contribute to the morphological and structural diversity of different avian skin appendages, with feather-β-keratins conferring more possible composites in building intrafeather architecture complexity, setting up a platform of morphological evolution of functional forms in feathers. PMID:26598683

Morphological restoration of gonadotrope population by thymulin gene therapy in nude mice

PubMed Central

Reggiani, Paula; Martines, Eliana; Ferese, Celia; Goya, Rodolfo; Cónsole, Gloria

2009-01-01

Summary The integrity of the thymus during the first week of life is necessary for a proper maturation of the pituitary-gonadal axis as revealed by the significantly reduced levels of circulating gonadotropins in congenitally athymic (nude) mice. In the present work we studied the impact of athymia and the effect of neonatal thymulin gene therapy on the pituitaries of adult nude mice. Also circulating thymulin and gonadotropin levels were evaluated. We used an adenoviral vector expressing a synthetic gene for the thymic peptide thymulin (metFTS) termed RAd-FTS. On postnatal day 1, each experimental heterozygous (nu/+) and homozygous (nu/nu) pup of both sexes received a single bilateral i.m. injection of RAd-FTS or RAd-GFP/TK, a control vector expressing green fluorescent protein. On postnatal days 51-52, mice were bled and sacrificed, their pituitaries were immediately dissected, fixed and immunostained. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density (VD: cell area/reference area), cell density (CD: number of cells/reference area), and cell size (expressed in μm2). Serum thymulin levels were measured by a bioassay and gonadotropin levels were assayed by RIA. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels. The histometrical analysis revealed that the treatment prevented the reduction in gonadotrope CD and the VD in athymic mice. Our data suggest that thymulin gene therapy may be an effective strategy to approach reproductive deficits associated with endocrine thymus dysfunction. PMID:19337971

Analysis of gene network robustness based on saturated fixed point attractors

PubMed Central

2014-01-01

The analysis of gene network robustness to noise and mutation is important for fundamental and practical reasons. Robustness refers to the stability of the equilibrium expression state of a gene network to variations of the initial expression state and network topology. Numerical simulation of these variations is commonly used for the assessment of robustness. Since there exists a great number of possible gene network topologies and initial states, even millions of simulations may be still too small to give reliable results. When the initial and equilibrium expression states are restricted to being saturated (i.e., their elements can only take values 1 or −1 corresponding to maximum activation and maximum repression of genes), an analytical gene network robustness assessment is possible. We present this analytical treatment based on determination of the saturated fixed point attractors for sigmoidal function models. The analysis can determine (a) for a given network, which and how many saturated equilibrium states exist and which and how many saturated initial states converge to each of these saturated equilibrium states and (b) for a given saturated equilibrium state or a given pair of saturated equilibrium and initial states, which and how many gene networks, referred to as viable, share this saturated equilibrium state or the pair of saturated equilibrium and initial states. We also show that the viable networks sharing a given saturated equilibrium state must follow certain patterns. These capabilities of the analytical treatment make it possible to properly define and accurately determine robustness to noise and mutation for gene networks. Previous network research conclusions drawn from performing millions of simulations follow directly from the results of our analytical treatment. Furthermore, the analytical results provide criteria for the identification of model validity and suggest modified models of gene network dynamics. The yeast cell-cycle network is used as an illustration of the practical application of this analytical treatment. PMID:24650364

Transcriptomic Response of Drosophila Melanogaster Pupae Developed in Hypergravity

NASA Technical Reports Server (NTRS)

Hosamani, Ravikumar; Hateley, Shannon; Bhardwaj, Shilpa R.; Pachter, Lior; Bhattacharya, Sharmila

2016-01-01

The metamorphosis of Drosophila is evolutionarily adapted to Earth's gravity, and is a tightly regulated process. Deviation from 1g to microgravity or hypergravity can influence metamorphosis, and alter associated gene expression. Understanding the relationship between an altered gravity environment and developmental processes is important for NASA's space travel goals. In the present study, 20 female and 20 male synchronized (Canton S, 2 to 3day old) flies were allowed to lay eggs while being maintained in a hypergravity environment (3g). Centrifugation was briefly stopped to discard the parent flies after 24hrs of egg laying, and then immediately continued until the eggs developed into P6-staged pupae (25 - 43 hours after pupation initiation). Post hypergravity exposure, P6-staged pupae were collected, total RNA was extracted using Qiagen RNeasy mini kits. We used RNA-Seq and qRT-PCR techniques to profile global transcriptomic changes in early pupae exposed to chronic hypergravity. During the pupal stage, Drosophila relies upon gravitational cues for proper development. Assessing gene expression changes in the pupa under altered gravity conditions helps highlight gravity dependent genetic pathways. A robust transcriptional response was observed in hypergravity-exposed pupae compared to controls, with 1,513 genes showing a significant (q < 0.05) difference in gene expression. Five major biological processes were affected: ion transport, redox homeostasis, immune response, proteolysis, and cuticle development. This outlines the underlying molecular changes occurring in Drosophila pupae in response to hypergravity.

Configurable pattern-based evolutionary biclustering of gene expression data

PubMed Central

2013-01-01

Background Biclustering algorithms for microarray data aim at discovering functionally related gene sets under different subsets of experimental conditions. Due to the problem complexity and the characteristics of microarray datasets, heuristic searches are usually used instead of exhaustive algorithms. Also, the comparison among different techniques is still a challenge. The obtained results vary in relevant features such as the number of genes or conditions, which makes it difficult to carry out a fair comparison. Moreover, existing approaches do not allow the user to specify any preferences on these properties. Results Here, we present the first biclustering algorithm in which it is possible to particularize several biclusters features in terms of different objectives. This can be done by tuning the specified features in the algorithm or also by incorporating new objectives into the search. Furthermore, our approach bases the bicluster evaluation in the use of expression patterns, being able to recognize both shifting and scaling patterns either simultaneously or not. Evolutionary computation has been chosen as the search strategy, naming thus our proposal Evo-Bexpa (Evolutionary Biclustering based in Expression Patterns). Conclusions We have conducted experiments on both synthetic and real datasets demonstrating Evo-Bexpa abilities to obtain meaningful biclusters. Synthetic experiments have been designed in order to compare Evo-Bexpa performance with other approaches when looking for perfect patterns. Experiments with four different real datasets also confirm the proper performing of our algorithm, whose results have been biologically validated through Gene Ontology. PMID:23433178

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

PubMed

Beckwith, Esteban J; Hernando, Carlos E; Polcowñuk, Sofía; Bertolin, Agustina P; Mancini, Estefania; Ceriani, M Fernanda; Yanovsky, Marcelo J

2017-10-01

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period ( per ) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model. Copyright © 2017 by the Genetics Society of America.

Iron-heme-Bach1 axis is involved in erythroblast adaptation to iron deficiency.

PubMed

Kobayashi, Masahiro; Kato, Hiroki; Hada, Hiroshi; Itoh-Nakadai, Ari; Fujiwara, Tohru; Muto, Akihiko; Inoguchi, Yukihiro; Ichiyanagi, Kenji; Hojo, Wataru; Tomosugi, Naohisa; Sasaki, Hiroyuki; Harigae, Hideo; Igarashi, Kazuhiko

2017-03-01

Iron plays the central role in oxygen transport by erythrocytes as a constituent of heme and hemoglobin. The importance of iron and heme is also to be found in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is deactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1 -/- mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses these genes in erythroblasts under iron deficiency to balance the levels of heme and globin. Moreover, an increase in genome-wide DNA methylation was observed in erythroblasts of Bach1 -/- mice under iron deficiency. These findings reveal the principle role of iron as a regulator of gene expression in erythroblast maturation and suggest that the iron-heme-Bach1 axis is important for a proper adaptation of erythroblast to iron deficiency to avoid toxic aggregates of non-heme globin. Copyright© Ferrata Storti Foundation.

Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation.

PubMed

Kozlova, Liudmila V; Gorshkov, Oleg V; Mokshina, Natalia E; Gorshkova, Tatyana A

2015-05-01

Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.

Molecular control of gut formation in the spider Parasteatoda tepidariorum.

PubMed

Feitosa, Natália Martins; Pechmann, Matthias; Schwager, Evelyn E; Tobias-Santos, Vitória; McGregor, Alistair P; Damen, Wim G M; Nunes da Fonseca, Rodrigo

2017-05-01

The development of a digestive system is an essential feature of bilaterians. Studies of the molecular control of gut formation in arthropods have been studied in detail in the fruit fly Drosophila melanogaster. However, little is known in other arthropods, especially in noninsect arthropods. To better understand the evolution of arthropod alimentary system, we investigate the molecular control of gut development in the spider Parasteatoda tepidariorum (Pt), the primary chelicerate model species for developmental studies. Orthologs of the ectodermal genes Pt-wingless (Pt-wg) and Pt-hedgehog (Pt-hh), of the endodermal genes, Pt-serpent (Pt-srp) and Pt-hepatocyte-nuclear factor-4 (Pt-hnf4) and of the mesodermal gene Pt-twist (Pt-twi) are expressed in the same germ layers during spider gut development as in D. melanogaster. Thus, our expression data suggest that the downstream molecular components involved in gut development in arthropods are conserved. However, Pt-forkhead (Pt-fkh) expression and function in spiders is considerably different from its D. melanogaster ortholog. Pt-fkh is expressed before gastrulation in a cell population that gives rise to endodermal and mesodermal precursors, suggesting a possible role for this factor in specification of both germ layers. To test this hypothesis, we knocked down Pt-fkh via RNA interference. Pt-fkh RNAi embryos not only fail to develop a proper gut, but also lack the mesodermal Pt-twi expressing cells. Thus, in spiders Pt-fkh specifies endodermal and mesodermal germ layers. We discuss the implications of these findings for the evolution and development of gut formation in Ecdysozoans. © 2017 Wiley Periodicals, Inc.

In vivo retroviral gene transfer into human bronchial epithelia of xenografts.

PubMed

Engelhardt, J F; Yankaskas, J R; Wilson, J M

1992-12-01

Cystic fibrosis (CF) is the most common lethal inherited disease in the Caucasian population with an incidence of approximately 1 in 2,500 live births. Pulmonary complications of CF, which are the most morbid aspects of the disease, are caused by primary abnormalities in epithelial cells that lead to impaired mucociliary clearance. One potential therapeutic strategy is to reconstitute expression of the CF gene in airway epithelia by somatic gene transfer. To this end, we have developed an animal model of the human airway using bronchial xenografts and have tested the efficiency of in vivo retroviral gene transfer. Using the LacZ reporter gene, we find the efficiency of in vivo retroviral gene transfer to be dramatically dependent on the regenerative and mitotic state of the epithelium. Within an undifferentiated regenerating epithelium in which 40% of nuclei labeled with BrdU, 5-10% retroviral gene transfer was obtained. In contrast, no gene transfer was noted in a fully differentiated epithelium in which 1% of nuclei labeled with BrdU. These findings suggest that retroviral mediated gene transfer to the airway in vivo may be feasible if the proper regenerative state can be induced.

CellTree: an R/bioconductor package to infer the hierarchical structure of cell populations from single-cell RNA-seq data.

PubMed

duVerle, David A; Yotsukura, Sohiya; Nomura, Seitaro; Aburatani, Hiroyuki; Tsuda, Koji

2016-09-13

Single-cell RNA sequencing is fast becoming one the standard method for gene expression measurement, providing unique insights into cellular processes. A number of methods, based on general dimensionality reduction techniques, have been suggested to help infer and visualise the underlying structure of cell populations from single-cell expression levels, yet their models generally lack proper biological grounding and struggle at identifying complex differentiation paths. Here we introduce cellTree: an R/Bioconductor package that uses a novel statistical approach, based on document analysis techniques, to produce tree structures outlining the hierarchical relationship between single-cell samples, while identifying latent groups of genes that can provide biological insights. With cellTree, we provide experimentalists with an easy-to-use tool, based on statistically and biologically-sound algorithms, to efficiently explore and visualise single-cell RNA data. The cellTree package is publicly available in the online Bionconductor repository at: http://bioconductor.org/packages/cellTree/ .

Tomato Male sterile 1035 is essential for pollen development and meiosis in anthers

PubMed Central

Jeong, Hee-Jin; Kang, Jin-Ho; Zhao, Meiai; Kwon, Jin-Kyung; Choi, Hak-Soon; Bae, Jung Hwan; Lee, Hyun-ah; Joung, Young-Hee; Choi, Doil; Kang, Byoung-Cheorl

2014-01-01

Male fertility in flowering plants depends on proper cellular differentiation in anthers. Meiosis and tapetum development are particularly important processes in pollen production. In this study, we showed that the tomato male sterile (ms10 35) mutant of cultivated tomato (Solanum lycopersicum) exhibited dysfunctional meiosis and an abnormal tapetum during anther development, resulting in no pollen production. We demonstrated that Ms10 35 encodes a basic helix–loop–helix transcription factor that is specifically expressed in meiocyte and tapetal tissue from pre-meiotic to tetrad stages. Transgenic expression of the Ms10 35 gene from its native promoter complemented the male sterility of the ms10 35 mutant. In addition, RNA-sequencing-based transcriptome analysis revealed that Ms10 35 regulates 246 genes involved in anther development processes such as meiosis, tapetum development, cell-wall degradation, pollen wall formation, transport, and lipid metabolism. Our results indicate that Ms10 35 plays key roles in regulating both meiosis and programmed cell death of the tapetum during microsporogenesis. PMID:25262227

von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

PubMed Central

Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

2007-01-01

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

Extracellular small RNAs: what, where, why?

PubMed Central

Hoy, Anna M.; Buck, Amy H.

2012-01-01

miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753

A Novel Nondevelopmental Role of the SAX-7/L1CAM Cell Adhesion Molecule in Synaptic Regulation in Caenorhabditis elegans

PubMed Central

Opperman, Karla; Moseley-Alldredge, Melinda; Yochem, John; Bell, Leslie; Kanayinkal, Tony; Chen, Lihsia

2015-01-01

The L1CAM family of cell adhesion molecules is a conserved set of single-pass transmembrane proteins that play diverse roles required for proper nervous system development and function. Mutations in L1CAMs can cause the neurological L1 syndrome and are associated with autism and neuropsychiatric disorders. L1CAM expression in the mature nervous system suggests additional functions besides the well-characterized developmental roles. In this study, we demonstrate that the gene encoding the Caenorhabditis elegans L1CAM, sax-7, genetically interacts with gtl-2, as well as with unc-13 and rab-3, genes that function in neurotransmission. These sax-7 genetic interactions result in synthetic phenotypes that are consistent with abnormal synaptic function. Using an inducible sax-7 expression system and pharmacological reagents that interfere with cholinergic transmission, we uncovered a previously uncharacterized nondevelopmental role for sax-7 that impinges on synaptic function. PMID:25488979

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients

PubMed Central

Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the “neurotrophin-MAPK signaling pathway” was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment. PMID:28900628

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients.

PubMed

Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Ji, Lin-Dan

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the "neurotrophin-MAPK signaling pathway" was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment.

Exploring miRNA based approaches in cancer diagnostics and therapeutics.

PubMed

Mishra, Shivangi; Yadav, Tanuja; Rani, Vibha

2016-02-01

MicroRNAs (miRNAs), a highly conserved class of tissue specific, small non-protein coding RNAs maintain cell homeostasis by negative gene regulation. Proper controlling of miRNA expression is required for a balanced physiological environment, as these small molecules influence almost every genetic pathway from cell cycle checkpoint, cell proliferation to apoptosis, with a wide range of target genes. Deregulation in miRNAs expression correlates with various cancers by acting as tumor suppressors and oncogenes. Although promising therapies exist to control tumor development and progression, there is a lack of efficient diagnostic and therapeutic approaches for delineating various types of cancer. The molecularly different tumors can be differentiated by specific miRNA profiling as their phenotypic signatures, which can hence be exploited to surmount the diagnostic and therapeutic challenges. Present review discusses the involvement of miRNAs in oncogenesis with the analysis of patented research available on miRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation

PubMed Central

Nakaki, Ryo; Shimamura, Teppei; Matsunaga, Taichi; Yamamizu, Kohei; Katayama, Shiori; Suehiro, Jun-ichi; Osawa, Tsuyoshi; Aburatani, Hiroyuki; Kodama, Tatsuhiko; Wada, Youichiro; Yamashita, Jun K.

2017-01-01

Abstract Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine. PMID:28334937

Effect of recipient breed on delivery rate of cloned miniature pig.

PubMed

Koo, Ok Jae; Park, Hee Jung; Kwon, Dae Kee; Kang, Jung Taek; Jang, Goo; Lee, Byeong Chun

2009-08-01

The miniature pig is regarded as a better organ donor breed for xenotransplantation than other pig breeds because the size of their organs is similar to that of humans. To improve efficiency of cloned miniature pig production, we analysed the effect of breed difference between donor cells and embryo recipients on pregnancy rate and delivery rate. Cloned porcine embryos derived from domestic or miniature pig donor cells were transferred to domestic or miniature recipient pigs. Delivery rate was significantly higher when embryos reconstructed with miniature pig donor cells were transferred to miniature pig recipients as compared with that of embryos transferred to domestic pig recipients. However, pregnancy rates were similar between the two groups. The breed of donor cells, but not of embryo recipients, seems likely to affect litter size. From a 13 610 gene cDNA microarray, 1551 (11.7%) genes showed significantly different levels of expression between the fetuses of the two breeds. Vascular endothelial growth factor and c-kit ligand genes related to implantation and maintenance of pregnancy were significantly down-regulated in miniature pigs. In conclusion, the differential gene expression in fetuses interferes with proper fetal/maternal interactions, and results in late-stage pregnancy loss. Our results indicate that the miniature pig is the preferred embryo recipient breed than domestic pig for producing cloned miniature piglets.

Zebrafish bcl2l is a survival factor in thyroid development.

PubMed

Porreca, Immacolata; De Felice, Elena; Fagman, Henrik; Di Lauro, Roberto; Sordino, Paolo

2012-06-15

Regulated cell death, defined in morphological terms as apoptosis, is crucial for organ morphogenesis. While differentiation of the thyroid gland has been extensively studied, nothing is yet known about the survival mechanisms involved in the development of this endocrine gland. Using the zebrafish model system, we aim to understand whether genes belonging to the Bcl-2 family that control apoptosis are implicated in regulation of cell survival during thyroid development. Evidence of strong Bcl-2 gene expression in mouse thyroid precursors prompted us to investigate the functions played by its zebrafish homologs during thyroid development. We show that the bcl2-like (bcl2l) gene is expressed in the zebrafish thyroid primordium. Morpholino-mediated knockdown and mutant analyses revealed that bcl2l is crucial for thyroid cell survival and that this function is tightly modulated by the transcription factors pax2a, nk2.1a and hhex. Also, the bcl2l gene appears to control a caspase-3-dependent apoptotic mechanism during thyroid development. Thyroid precursor cells require an actively maintained survival mechanism to properly proceed through development. The bcl2l gene operates in the inhibition of cell death under direct regulation of a thyroid specific set of transcription factors. This is the first demonstration of an active mechanism to ensure survival of the thyroid primordium during morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Nuclear receptors and metabolism: from feast to famine.

PubMed

Hong, Suk-Hyun; Ahmadian, Maryam; Yu, Ruth T; Atkins, Annette R; Downes, Michael; Evans, Ronald M

2014-05-01

The ability to adapt to cycles of feast and famine is critical for survival. Communication between multiple metabolic organs must be integrated to properly metabolise nutrients. By controlling networks of genes in major metabolic organs, nuclear hormone receptors (NHRs) play central roles in regulating metabolism in a tissue-specific manner. NHRs also establish daily rhythmicity by controlling the expression of core clock genes both centrally and peripherally. Recent findings show that many of the metabolic effects of NHRs are mediated through certain members of the fibroblast growth factor (FGF) family. This review focuses on the roles of NHRs in critical metabolic organs, including adipose tissue, liver and muscle, during the fed and fasted states, as well as their roles in circadian metabolism and downstream regulation of FGFs.

Axial level-specific regulation of neuronal development: lessons from PITX2.

PubMed

Waite, Mindy R; Martin, Donna M

2015-02-01

Transcriptional regulation of gene expression is vital for proper control of proliferation, migration, differentiation, and survival of developing neurons. Pitx2 encodes a homeodomain transcription factor that is highly expressed in the developing and adult mammalian brain. In humans, mutations in PITX2 result in Rieger syndrome, characterized by defects in the development of the eyes, umbilicus, and teeth and variable abnormalities in the brain, including hydrocephalus and cerebellar hypoplasia. Alternative splicing of Pitx2 in the mouse results in three isoforms, Pitx2a, Pitx2b, and Pitx2c, each of which is expressed symmetrically along the left-right axis of the brain throughout development. Here, we review recent evidence for axial and brain region-specific requirements for Pitx2 during neuronal migration and differentiation, highlighting known isoform contributions. © 2014 Wiley Periodicals, Inc.

BMP, Wnt and FGF signals are integrated through evolutionarily conserved enhancers to achieve robust expression of Pax3 and Zic genes at the zebrafish neural plate border

PubMed Central

Garnett, Aaron T.; Square, Tyler A.; Medeiros, Daniel M.

2012-01-01

Neural crest cells generate a range of cells and tissues in the vertebrate head and trunk, including peripheral neurons, pigment cells, and cartilage. Neural crest cells arise from the edges of the nascent central nervous system, a domain called the neural plate border (NPB). NPB induction is known to involve the BMP, Wnt and FGF signaling pathways. However, little is known about how these signals are integrated to achieve temporally and spatially specific expression of genes in NPB cells. Furthermore, the timing and relative importance of these signals in NPB formation appears to differ between vertebrate species. Here, we use heat-shock overexpression and chemical inhibitors to determine whether, and when, BMP, Wnt and FGF signaling are needed for expression of the NPB specifiers pax3a and zic3 in zebrafish. We then identify four evolutionarily conserved enhancers from the pax3a and zic3 loci and test their response to BMP, Wnt and FGF perturbations. We find that all three signaling pathways are required during gastrulation for the proper expression of pax3a and zic3 in the zebrafish NPB. We also find that, although the expression patterns driven by the pax3a and zic3 enhancers largely overlap, they respond to different combinations of BMP, Wnt and FGF signals. Finally, we show that the combination of the two pax3a enhancers is less susceptible to signaling perturbations than either enhancer alone. Taken together, our results reveal how BMPs, FGFs and Wnts act cooperatively and redundantly through partially redundant enhancers to achieve robust, specific gene expression in the zebrafish NPB. PMID:23034628

Function, dynamics and evolution of network motif modules in integrated gene regulatory networks of worm and plant.

PubMed

Defoort, Jonas; Van de Peer, Yves; Vermeirssen, Vanessa

2018-06-05

Gene regulatory networks (GRNs) consist of different molecular interactions that closely work together to establish proper gene expression in time and space. Especially in higher eukaryotes, many questions remain on how these interactions collectively coordinate gene regulation. We study high quality GRNs consisting of undirected protein-protein, genetic and homologous interactions, and directed protein-DNA, regulatory and miRNA-mRNA interactions in the worm Caenorhabditis elegans and the plant Arabidopsis thaliana. Our data-integration framework integrates interactions in composite network motifs, clusters these in biologically relevant, higher-order topological network motif modules, overlays these with gene expression profiles and discovers novel connections between modules and regulators. Similar modules exist in the integrated GRNs of worm and plant. We show how experimental or computational methodologies underlying a certain data type impact network topology. Through phylogenetic decomposition, we found that proteins of worm and plant tend to functionally interact with proteins of a similar age, while at the regulatory level TFs favor same age, but also older target genes. Despite some influence of the duplication mode difference, we also observe at the motif and module level for both species a preference for age homogeneity for undirected and age heterogeneity for directed interactions. This leads to a model where novel genes are added together to the GRNs in a specific biological functional context, regulated by one or more TFs that also target older genes in the GRNs. Overall, we detected topological, functional and evolutionary properties of GRNs that are potentially universal in all species.

Transcriptome Profiles of Isolated Murine Achilles Tendon Proper- and Peritenon- Derived Progenitor Cells.

PubMed

Mienaltowski, Michael J; Cánovas, Angela; Fates, Valerie A; Hampton, Angela R; Pechanec, Monica Y; Islas-Trejo, Alma; Medrano, Juan F

2018-06-21

Progenitor cells of the tendon proper and peritenon have unique properties that could impact their utilization in tendon repair strategies. While a few markers have been found to aid in distinguishing progenitors cells from each region, there is great value in identifying more markers. In this study, we hypothesized that RNAseq could be used to improve our understanding of those markers that define these cell types. Transcriptome profiles were generated for pools of mouse Achilles tendon progenitor cells from both regions and catalogues of potential markers were generated. Moreover, common (e.g., glycoprotein, signaling, and proteinaceous extracellular matrix) and unique (e.g., cartilage development versus angiogenesis and muscle contraction) biological processes and molecular functions were described for progenitors from each region. Real-time quantitative PCR of a subset of genes was used to gain insight into the heterogeneity amongst individual progenitor colonies from each region. Markers like Scx, Mkx, Thbs4, and Wnt10a were consistently able to distinguish tendon proper progenitors from peritenon progenitors; expression variability for other genes suggested greater cell type complexity for potential peritenon progenitor markers. This is the first effort to define Achilles tendon progenitor markers by region. Further efforts to investigate the value of these catalogued markers are required by screening more individual colonies of progenitors for more markers. Findings from this study advance efforts in the discernment of cell type specific markers for tendon proper and peritenon progenitor cells; insight into marker sets could improve tracking and sorting strategies for these cells for future therapeutic strategies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Glucose Alters Per2 Rhythmicity Independent of AMPK, Whereas AMPK Inhibitor Compound C Causes Profound Repression of Clock Genes and AgRP in mHypoE-37 Hypothalamic Neurons.

PubMed

Oosterman, Johanneke E; Belsham, Denise D

2016-01-01

Specific neurons in the hypothalamus are regulated by peripheral hormones and nutrients to maintain proper metabolic control. It is unclear if nutrients can directly control clock gene expression. We have therefore utilized the immortalized, hypothalamic cell line mHypoE-37, which exhibits robust circadian rhythms of core clock genes. mHypoE-37 neurons were exposed to 0.5 or 5.5 mM glucose, comparable to physiological levels in the brain. Per2 and Bmal1 mRNAs were assessed every 3 hours over 36 hours. Incubation with 5.5 mM glucose significantly shortened the period and delayed the phase of Per2 mRNA levels, but had no effect on Bmal1. Glucose had no significant effect on phospho-GSK3β, whereas AMPK phosphorylation was altered. Thus, the AMPK inhibitor Compound C was utilized, and mRNA levels of Per2, Bmal1, Cryptochrome1 (Cry1), agouti-related peptide (AgRP), carnitine palmitoyltransferase 1C (Cpt1c), and O-linked N-acetylglucosamine transferase (Ogt) were measured. Remarkably, Compound C dramatically reduced transcript levels of Per2, Bmal1, Cry1, and AgRP, but not Cpt1c or Ogt. Because AMPK was not inhibited at the same time or concentrations as the clock genes, we suggest that the effect of Compound C on gene expression occurs through an AMPK-independent mechanism. The consequences of inhibition of the rhythmic expression of clock genes, and in turn downstream metabolic mediators, such as AgRP, could have detrimental effects on overall metabolic processes. Importantly, the effects of the most commonly used AMPK inhibitor Compound C should be interpreted with caution, considering its role in AMPK-independent repression of specific genes, and especially clock gene rhythm dysregulation.

Glucose Alters Per2 Rhythmicity Independent of AMPK, Whereas AMPK Inhibitor Compound C Causes Profound Repression of Clock Genes and AgRP in mHypoE-37 Hypothalamic Neurons

PubMed Central

Oosterman, Johanneke E.; Belsham, Denise D.

2016-01-01

Specific neurons in the hypothalamus are regulated by peripheral hormones and nutrients to maintain proper metabolic control. It is unclear if nutrients can directly control clock gene expression. We have therefore utilized the immortalized, hypothalamic cell line mHypoE-37, which exhibits robust circadian rhythms of core clock genes. mHypoE-37 neurons were exposed to 0.5 or 5.5 mM glucose, comparable to physiological levels in the brain. Per2 and Bmal1 mRNAs were assessed every 3 hours over 36 hours. Incubation with 5.5 mM glucose significantly shortened the period and delayed the phase of Per2 mRNA levels, but had no effect on Bmal1. Glucose had no significant effect on phospho-GSK3β, whereas AMPK phosphorylation was altered. Thus, the AMPK inhibitor Compound C was utilized, and mRNA levels of Per2, Bmal1, Cryptochrome1 (Cry1), agouti-related peptide (AgRP), carnitine palmitoyltransferase 1C (Cpt1c), and O-linked N-acetylglucosamine transferase (Ogt) were measured. Remarkably, Compound C dramatically reduced transcript levels of Per2, Bmal1, Cry1, and AgRP, but not Cpt1c or Ogt. Because AMPK was not inhibited at the same time or concentrations as the clock genes, we suggest that the effect of Compound C on gene expression occurs through an AMPK-independent mechanism. The consequences of inhibition of the rhythmic expression of clock genes, and in turn downstream metabolic mediators, such as AgRP, could have detrimental effects on overall metabolic processes. Importantly, the effects of the most commonly used AMPK inhibitor Compound C should be interpreted with caution, considering its role in AMPK-independent repression of specific genes, and especially clock gene rhythm dysregulation. PMID:26784927

Transcriptional response of skeletal muscle to a low-protein gestation diet in porcine offspring accumulates in growth- and cell cycle-regulating pathways.

PubMed

Oster, Michael; Murani, Eduard; Metges, Cornelia C; Ponsuksili, Siriluck; Wimmers, Klaus

2012-08-17

Inadequate maternal protein supply during gestation represents an environmental factor that affects physiological signaling pathways with long-term consequences for growth, function, and structure of various tissues. Hypothesizing that the offspring's transcriptome is persistently altered by maternal diets, we used a porcine model to monitor the longitudinal expression changes in muscle to identify pathways relevant to fetal initiation of postnatal growth and development. German Landrace gilts were fed isoenergetic gestational diets containing 6.5% (LP) or 12.1% protein. The longissimus dorsi samples were collected from offspring at 94 days postconception (dpc) and 1, 28, and 188 days postnatum (dpn) for expression profiling. At 94 dpc, 1 dpn, and 28 dpn relatively few transcripts (<130) showed an altered abundance between the dietary groups. In fact, at 94 dpc genes of G2/M checkpoint regulation and mitotic roles of Polo-like kinases showed lowered transcript abundance in LP. At 188 dpn 677 transcripts were altered including those related to oxidative phosphorylation, citrate cycle, fatty acid metabolism (higher abundance in LP) and cell cycle regulation (lower abundance in LP). Correspondingly, transcriptional alterations during pre and postnatal development differed considerably among dietary groups, particularly for genes related to cell cycle regulation (G1/S and G2/M checkpoint regulation; cyclines), growth factor signaling (GH, IGF1, mTOR, RAN, VEGF, INSR), lipid metabolism, energy metabolism, and nucleic acid metabolism. In skeletal muscle, fetal programming related to maternal LP diets disturbed gene expression in growth-related pathways into adulthood. Diet-dependent gene expression may hamper proper development, thereby affecting signaling pathways related to energy utilization.

Partial loss of Smad7 function impairs bone remodeling, osteogenesis and enhances osteoclastogenesis in mice.

PubMed

Li, Nan; Lee, Wayne Yuk-Wai; Lin, Si-En; Ni, Ming; Zhang, Ting; Huang, Xiao-Ru; Lan, Hui-Yao; Li, Gang

2014-10-01

Smad7 is well demonstrated as a negative regulator of TGF-β signaling. Its alteration in expression often results in diseases such as cancer and fibrosis. However, the exact role of Smad7 in regulating bone remodeling during mammalian development has not been properly delineated. In this study we performed experiments to clarify the involvement of Smad7 in regulating osteogenesis and osteoclastogenesis both invivo and invitro. Genetically engineered Smad7(ΔE1) (KO) mice were used, whereby partial functional of Smad7 is lost by deleting exon I of the Smad7 gene and the truncated proteins cause a hypomorphic allele. Analysis with μCT imagery and bone histomorphometry showed that the KO mice had lower TbN, TbTh, higher TbSp in the metaphysic region of the femurs at 6, 12, 24weeks from birth, as well as decreased MAR and increased osteoclast surface compared with the WT mice. In vitro BM-MSC multi-lineage differentiation evaluation showed that the KO group had reduced osteogenic potential, fewer mineralized nodules, lower ALP activity, and reduced gene expression of Col1A1, Runx2 and OCN. The adipogenic potential was elevated in the KO group with more formation of lipid droplets, and increased gene expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was elevate, with emergence of more osteoclasts, larger resorptive areas, and increased gene expression of TRAP and CTR. Our results indicate that partial loss of Smad7 function in mice leads to compromised bone formation and enhanced bone resorption. Thus, Smad7 is acknowledged as a novel key regulator between osteogenesis and osteoclastogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Immediate-early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c-fos mRNA to adaptation.

PubMed

Ons, Sheila; Rotllant, David; Marín-Blasco, Ignacio J; Armario, Antonio

2010-06-01

Stress exposure resulted in brain induction of immediate-early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c-fos and arc (activity-regulated cytoskeleton-associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c-fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos-like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO-induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double-labelling in the LS of IMO-exposed rats revealed that arc was expressed in only one-third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c-fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

PubMed

Ruiz, Oscar N; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

2011-06-01

Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Plasticity of the myelination genomic fabric.

PubMed

Iacobas, Sanda; Thomas, Neil M; Iacobas, Dumitru A

2012-03-01

This study aimed to quantify the influence of the astrocyte proximity on myelination genomic fabric (MYE) of oligodendrocytes, defined as the most interconnected and stably expressed gene web responsible for myelination. Such quantitation is important to evaluate whether astrocyte signaling may contribute to demyelination when impaired and remyelination when properly restored. For this, we compared changes in the gene expression profiles of immortalized precursor oligodendrocytes (Oli-neu), stimulated to differentiate by the proximity of nontouching astrocytes or treatment with db-cAMP. In a previous paper, we reported that the astrocyte proximity upregulated or turned-on a large number of myelination genes and substantially enriched the Ca(2+)-signaling and cytokine receptor regulatory networks of MYE in Oli-neu cells. Here, we introduce the "transcriptomic distance" to evaluate fabric remodeling and "pair-wise relevance" to identify the most influential gene pairs. Together with the prominence gene analysis used to select and rank the fabric genes, these novel analytical tools provide a comprehensively quantitative view of the physio/pathological transformations of the transcriptomic programs of myelinating cells. Applied to our data, the analyses revealed not only that the astrocyte neighborhood is a substantially more powerful regulator of myelination than the differentiating treatment but also the molecular mechanisms of the two differentiating paradigms are different. By inducing a profound remodeling of MYE and regulatory transcriptomic networks, the astrocyte-oligodendrocyte intercommunication may be considered as a major player in both pathophysiology and therapy of neurodegenerative diseases related to myelination.

Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2005-04-01

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

PubMed Central

Yan, Xu; Bishop, David J.

2018-01-01

Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content. PMID:29746477

The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

PubMed Central

Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

2016-01-01

DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

Genome-Wide Analysis of SREBP1 Activity around the Clock Reveals Its Combined Dependency on Nutrient and Circadian Signals

PubMed Central

Naldi, Aurélien; Baruchet, Michaël; Canella, Donatella; Le Martelot, Gwendal; Guex, Nicolas; Desvergne, Béatrice; Delorenzi, Mauro; Deplancke, Bart; Desvergne, Béatrice; Guex, Nicolas; Herr, Winship; Naef, Felix; Rougemont, Jacques; Schibler, Ueli; Deplancke, Bart; Guex, Nicolas; Herr, Winship; Guex, Nicolas; Andersin, Teemu; Cousin, Pascal; Gilardi, Federica; Gos, Pascal; Martelot, Gwendal Le; Lammers, Fabienne; Canella, Donatella; Gilardi, Federica; Raghav, Sunil; Fabbretti, Roberto; Fortier, Arnaud; Long, Li; Vlegel, Volker; Xenarios, Ioannis; Migliavacca, Eugenia; Praz, Viviane; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; David, Fabrice; Jarosz, Yohan; Kuznetsov, Dmitry; Liechti, Robin; Martin, Olivier; Delafontaine, Julien; Sinclair, Lucas; Cajan, Julia; Krier, Irina; Leleu, Marion; Migliavacca, Eugenia; Molina, Nacho; Naldi, Aurélien; Rey, Guillaume; Symul, Laura; Guex, Nicolas; Naef, Felix; Rougemont, Jacques; Bernasconi, David; Delorenzi, Mauro; Andersin, Teemu; Canella, Donatella; Gilardi, Federica; Martelot, Gwendal Le; Lammers, Fabienne; Baruchet, Michaël; Raghav, Sunil

2014-01-01

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1 −/− mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved. PMID:24603613

Genome Editing in Human Pluripotent Stem Cells.

PubMed

Carlson-Stevermer, Jared; Saha, Krishanu

2017-01-01

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication

PubMed Central

Olsen, Michelle E.; Filone, Claire Marie; Rozelle, Dan; Mire, Chad E.; Agans, Krystle N.; Hensley, Lisa

2016-01-01

ABSTRACT Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates. EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein. PMID:27460797

Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PubMed Central

Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo; Hans, Wolfgang; Wuelling, Manuela; Mentz, Bettina; Multhaupt, Hinke Arnolda; Fog, Cathrine Kolster; Jensen, Klaus Thorleif; Rappsilber, Juri; Vortkamp, Andrea; Coulton, Les; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Calogero, Raffaele Adolfo; Couchman, John Robert; Lund, Anders Henrik

2012-01-01

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix. PMID:22589746

CORECLUST: identification of the conserved CRM grammar together with prediction of gene regulation.

PubMed

Nikulova, Anna A; Favorov, Alexander V; Sutormin, Roman A; Makeev, Vsevolod J; Mironov, Andrey A

2012-07-01

Identification of transcriptional regulatory regions and tracing their internal organization are important for understanding the eukaryotic cell machinery. Cis-regulatory modules (CRMs) of higher eukaryotes are believed to possess a regulatory 'grammar', or preferred arrangement of binding sites, that is crucial for proper regulation and thus tends to be evolutionarily conserved. Here, we present a method CORECLUST (COnservative REgulatory CLUster STructure) that predicts CRMs based on a set of positional weight matrices. Given regulatory regions of orthologous and/or co-regulated genes, CORECLUST constructs a CRM model by revealing the conserved rules that describe the relative location of binding sites. The constructed model may be consequently used for the genome-wide prediction of similar CRMs, and thus detection of co-regulated genes, and for the investigation of the regulatory grammar of the system. Compared with related methods, CORECLUST shows better performance at identification of CRMs conferring muscle-specific gene expression in vertebrates and early-developmental CRMs in Drosophila.

Editing the Neuronal Genome: a CRISPR View of Chromatin Regulation in Neuronal Development, Function, and Plasticity.

PubMed

Yang, Marty G; West, Anne E

2016-12-01

The dynamic orchestration of gene expression is crucial for the proper differentiation, function, and adaptation of cells. In the brain, transcriptional regulation underlies the incredible diversity of neuronal cell types and contributes to the ability of neurons to adapt their function to the environment. Recently, novel methods for genome and epigenome editing have begun to revolutionize our understanding of gene regulatory mechanisms. In particular, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be a particularly accessible and adaptable technique for genome engineering. Here, we review the use of CRISPR/Cas9 in neurobiology and discuss how these studies have advanced understanding of nervous system development and plasticity. We cover four especially salient applications of CRISPR/Cas9: testing the consequences of enhancer mutations, tagging genes and gene products for visualization in live cells, directly activating or repressing enhancers in vivo , and manipulating the epigenome. In each case, we summarize findings from recent studies and discuss evolving adaptations of the method.

MicroRNAs in genetic disease: rethinking the dosage.

PubMed

Henrion-Caude, Alexandra; Girard, Muriel; Amiel, Jeanne

2012-08-01

To date, the general assumption was that most mutations interested protein-coding genes only. Thus, only few illustrations have mentioned here that mutations may occur in non-protein coding genes such as microRNAs (miRNAs). We thus report progress in delineating their contribution as phenotypic modulators, genetic switches and fine-tuners of gene expression. We reasoned that browsing their contribution to genetic disease may provide a framework for understanding the proper requirements to devise miRNA-based therapy strategies, in particular the relief of an appropriate dosage. Gain and loss of function of miRNA enforce the need to respectively antagonize or supply the miRNAs. We further categorized human disease according to the different ways in which the miRNA was altered arising either de novo, or inherited whether as a mendelian or as an epistatic trait, uncovering its role in epigenetics. We discuss how improving our knowledge on the contribution of miRNAs to genetic disease may be beneficial to devise appropriate gene therapy strategies.

MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type

PubMed Central

Wu, Hai; Naya, Francisco J.; McKinsey, Timothy A.; Mercer, Brian; Shelton, John M.; Chin, Eva R.; Simard, Alain R.; Michel, Robin N.; Bassel-Duby, Rhonda; Olson, Eric N.; Williams, R. Sanders

2000-01-01

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained. PMID:10790363

DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

PubMed Central

Hua, Brian L.; Orr-Weaver, Terry L.

2017-01-01

Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. PMID:28874453

Molecular cloning of low-temperature-inducible ribosomal proteins from soybean.

PubMed

Kim, Kee-Young; Park, Seong-Whan; Chung, Young-Soo; Chung, Chung-Han; Kim, Jung-In; Lee, Jai-Heon

2004-05-01

Three ribosomal protein genes induced by low-temperature treatment were isolated from soybean. GmRPS13 (742 bp) encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974). GmRPS6 (925 bp) encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533). GmRPL37 (494 bp) encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216). The expression of these ribosomal protein genes started to increase 3 d after low-temperature treatment, whereas the cold-stress protein src1 was highly induced from the first day. Such late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The induction of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under low-temperature conditions.

Core labeling of adenovirus with EGFP

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

Le, Long P.; Le, Helen N.; Nelson, Amy R.

2006-08-01

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expressionmore » vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.« less

Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data.

PubMed

Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong; Li, Mingyao; Zhang, Nancy R

2017-11-02

Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data

PubMed Central

Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong

2017-01-01

Abstract Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. PMID:29036714

Development and characterization of a eukaryotic expression system for human type II procollagen.

PubMed

Wieczorek, Andrew; Rezaei, Naghmeh; Chan, Clara K; Xu, Chuan; Panwar, Preety; Brömme, Dieter; Merschrod S, Erika F; Forde, Nancy R

2015-12-15

Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Transcriptional atlas of cardiogenesis maps congenital heart disease interactome.

PubMed

Li, Xing; Martinez-Fernandez, Almudena; Hartjes, Katherine A; Kocher, Jean-Pierre A; Olson, Timothy M; Terzic, Andre; Nelson, Timothy J

2014-07-01

Mammalian heart development is built on highly conserved molecular mechanisms with polygenetic perturbations resulting in a spectrum of congenital heart diseases (CHD). However, knowledge of cardiogenic ontogeny that regulates proper cardiogenesis remains largely based on candidate-gene approaches. Mapping the dynamic transcriptional landscape of cardiogenesis from a genomic perspective is essential to integrate the knowledge of heart development into translational applications that accelerate disease discovery efforts toward mechanistic-based treatment strategies. Herein, we designed a time-course transcriptome analysis to investigate the genome-wide dynamic expression landscape of innate murine cardiogenesis ranging from embryonic stem cells to adult cardiac structures. This comprehensive analysis generated temporal and spatial expression profiles, revealed stage-specific gene functions, and mapped the dynamic transcriptome of cardiogenesis to curated pathways. Reconciling known genetic underpinnings of CHD, we deconstructed a disease-centric dynamic interactome encoded within this cardiogenic atlas to identify stage-specific developmental disturbances clustered on regulation of epithelial-to-mesenchymal transition (EMT), BMP signaling, NF-AT signaling, TGFb-dependent EMT, and Notch signaling. Collectively, this cardiogenic transcriptional landscape defines the time-dependent expression of cardiac ontogeny and prioritizes regulatory networks at the interface between health and disease. Copyright © 2014 the American Physiological Society.

c-Maf is required for the development of dorsal horn laminae III/IV neurons and mechanoreceptive DRG axon projections.

PubMed

Hu, Jia; Huang, Tianwen; Li, Tingting; Guo, Zhen; Cheng, Leping

2012-04-18

Establishment of proper connectivity between peripheral sensory neurons and their central targets is required for an animal to sense and respond to various external stimuli. Dorsal root ganglion (DRG) neurons convey sensory signals of different modalities via their axon projections to distinct laminae in the dorsal horn of the spinal cord. In this study, we found that c-Maf was expressed predominantly in the interneurons of laminae III/IV, which primarily receive inputs from mechanoreceptive DRG neurons. In the DRG, c-Maf⁺ neurons also coexpressed neurofilament-200, a marker for the medium- and large-diameter myelinated afferents that transmit non-noxious information. Furthermore, mouse embryos deficient in c-Maf displayed abnormal development of dorsal horn laminae III/IV neurons, as revealed by the marked reduction in the expression of several marker genes for these neurons, including those for transcription factors MafA and Rora, GABA(A) receptor subunit α5, and neuropeptide cholecystokinin. In addition, among the four major subpopulations of DRG neurons marked by expression of TrkA, TrkB, TrkC, and MafA/GFRα2/Ret, c-Maf was required selectively for the proper differentiation of MafA⁺/Ret⁺/GFRα2⁺ low-threshold mechanoreceptors (LTMs). Last, we found that the central and peripheral projections of mechanoreceptive DRG neurons were compromised in c-Maf deletion mice. Together, our results indicate that c-Maf is required for the proper development of MafA⁺/Ret⁺/GFRα2⁺ LTMs in the DRG, their afferent projections in the dorsal horn and Pacinian corpuscles, as well as neurons in laminae III/IV of the spinal cord.

Developing molecular tools for Chlamydomonas reinhardtii

NASA Astrophysics Data System (ADS)

Noor-Mohammadi, Samaneh

Microalgae have garnered increasing interest over the years for their ability to produce compounds ranging from biofuels to neutraceuticals. A main focus of researchers has been to use microalgae as a natural bioreactor for the production of valuable and complex compounds. Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. To take full advantage of these organisms' natural abilities, sophisticated molecular tools are needed to be able to introduce and functionally express multiple gene biosynthetic pathways in its genome. To achieve the above objective, we have sought to establish a method to construct, integrate and express multigene operons in the chloroplast and nuclear genome of the model microalgae Chlamydomonas reinhardtii. Here we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast, or by random integration in the nuclear genome of C. reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii and up to three reporter proteins (Ble, AphVIII, and GFP) in its nuclear genome. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated-region selection when constructing a target pathway. In addition, this work focuses on expressing the cofactor regeneration enzyme phosphite dehydrogenase (PTDH) in the chloroplast and nuclear genomes of C. reinhardtii. The PTDH enzyme converts phosphite into phosphate and NAD(P)+ into NAD(P)H. The reduced nicotinamide cofactor NAD(P)H plays a pivotal role in many biochemical oxidation and reduction reactions, thus this enzyme would allow regeneration of NAD(P)H in a microalgae strain over-expressing a NAD(P)H-dependent oxidoreductase. A phosphite dehydrogenase gene was introduced into the chloroplast genome (codon optimized) and nuclear genome of C. reinhardtii by biolistic transformation and electroporation in separate events, respectively. Successful expression of the heterologous protein was confirmed by transcript analysis and protein analysis. In conclusion, this new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast or nuclear genome of microalgae, and this should aid current efforts to engineer algae for recombinant protein expression, biofuels production and production of other desirable natural products.

Molecular characterization and expression analysis of a heat shock protein 90 gene from disk abalone (Haliotis discus).

PubMed

Wang, Ning; Whang, Ilson; Lee, Jae-Seong; Lee, Jehee

2011-06-01

Heat shock protein 90s (hsp90s) are chaperones that contribute to the proper folding of cellular proteins and help animals cope with the cellular protein damages in stress conditions. In this study, an hsp90 gene was isolated from disc abalone (Haliotis discus). The complete nucleotide sequence of the hsp90 gene contains an open reading frame of 2,184 base pairs, encoding an 84 kDa protein. Disk abalone hsp90 shares high sequence similarity with other hsp90 family proteins. Although the phylogenetic analysis did not classify it into the hsp90α group, the inductivity of this gene was confirmed by heat shock and lipopolysaccharide (LPS) challenge test. Disk abalone hsp90 gene displayed a rapid and reversible induction response to both an exposure of typical heat shock and the LPS challenge. Once given the sublethal heat shock treatment, the transcription of disk abalone hsp90 gene was significantly up-regulated. With a recovery of 12 h, the transcription of disk abalone hsp90 gene gradually attenuated to the control level. These observations reflected the feedback regulation of abalone heat shock responses faithfully. In response to LPS challenge, the transcription of disk abalone hsp90 gene was significantly increased within 2 h and it approached maximum induction at 4 h later and recovered finally the reference level in 24 h. Take all together, the cloning and expression analysis of disk abalone hsp90 gene provided useful molecular information of abalone responses in stress conditions and potential ways to monitor the chronic stressors in abalone culture environments and diagnose the animal health status.

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

NASA Technical Reports Server (NTRS)

Halfon, M. S.; Keshishian, H.

1998-01-01

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Zinc Transport Differs in Rat Spermatogenic Cell Types and Is Affected by Treatment with Cyclophosphamide1

PubMed Central

Downey, Anne Marie; Hales, Barbara F.; Robaire, Bernard

2016-01-01

Adequate zinc levels are required for proper cellular functions and for male germ cell development. Zinc transport is accomplished by two families of zinc transporters, the ZIPs and the ZnTs, that increase and decrease cytosolic zinc levels, respectively. However, very little is known about zinc transport in the testis. Furthermore, whether cytotoxic agents such as cyclophosphamide (CPA), a known male germ cell toxicant, can affect zinc transport and homeostasis is unknown. We examined zinc transporter expression and zinc transport in pachytene spermatocytes (PS) and round spermatids (RS) in a normal state and after exposure to CPA. We observed differences in the expression of members of the ZnT and ZIP families in purified populations of PS and RS. We also observed that RS accumulate more zinc over time than PS. The expression of many zinc binding genes was altered after CPA treatment. Interestingly, we found that the expression levels of ZIP5 and ZIP14 were increased in PS from animals treated daily with 6 mg/kg CPA for 4 wk but not in RS. This up-regulation led to an increase in zinc uptake in PS but not in RS from treated animals compared to controls. These data suggest that CPA treatment may alter zinc homeostasis in male germ cells leading to an increased need for zinc. Altered zinc homeostasis may disrupt proper germ cell development and contribute to infertility and effects on progeny. PMID:27281708

Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol.

PubMed

Suzuki, Tadashi; Yano, Keiichi; Sugimoto, Seiji; Kitajima, Ken; Lennarz, William J; Inoue, Sadako; Inoue, Yasuo; Emori, Yasufumi

2002-07-23

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

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

Zhang, Pengpeng; Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; Liang, Xinrong

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7{sup CreER} and Mtor{sup flox/flox} mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number andmore » size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7{sup CreER} was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes.« less

The lipodystrophic hotspot lamin A p.R482W mutation deregulates the mesodermal inducer T/Brachyury and early vascular differentiation gene networks.

PubMed

Briand, Nolwenn; Guénantin, Anne-Claire; Jeziorowska, Dorota; Shah, Akshay; Mantecon, Matthieu; Capel, Emilie; Garcia, Marie; Oldenburg, Anja; Paulsen, Jonas; Hulot, Jean-Sebastien; Vigouroux, Corinne; Collas, Philippe

2018-04-15

The p.R482W hotspot mutation in A-type nuclear lamins causes familial partial lipodystrophy of Dunnigan-type (FPLD2), a lipodystrophic syndrome complicated by early onset atherosclerosis. Molecular mechanisms underlying endothelial cell dysfunction conferred by the lamin A mutation remain elusive. However, lamin A regulates epigenetic developmental pathways and mutations could perturb these functions. Here, we demonstrate that lamin A R482W elicits endothelial differentiation defects in a developmental model of FPLD2. Genome modeling in fibroblasts from patients with FPLD2 caused by the lamin A R482W mutation reveals repositioning of the mesodermal regulator T/Brachyury locus towards the nuclear center relative to normal fibroblasts, suggesting enhanced activation propensity of the locus in a developmental model of FPLD2. Addressing this issue, we report phenotypic and transcriptional alterations in mesodermal and endothelial differentiation of induced pluripotent stem cells we generated from a patient with R482W-associated FPLD2. Correction of the LMNA mutation ameliorates R482W-associated phenotypes and gene expression. Transcriptomics links endothelial differentiation defects to decreased Polycomb-mediated repression of the T/Brachyury locus and over-activation of T target genes. Binding of the Polycomb repressor complex 2 to T/Brachyury is impaired by the mutated lamin A network, which is unable to properly associate with the locus. This leads to a deregulation of vascular gene expression over time. By connecting a lipodystrophic hotspot lamin A mutation to a disruption of early mesodermal gene expression and defective endothelial differentiation, we propose that the mutation rewires the fate of several lineages, resulting in multi-tissue pathogenic phenotypes.

Role of adipokinetic hormone and adenosine in the anti-stress response in Drosophila melanogaster.

PubMed

Zemanová, Milada; Stašková, Tereza; Kodrík, Dalibor

2016-01-01

The role of adipokinetic hormone (AKH) and adenosine in the anti-stress response was studied in Drosophila melanogaster larvae and adults carrying a mutation in the Akh gene (Akh(1)), the adenosine receptor gene (AdoR(1)), or in both of these genes (Akh(1) AdoR(1) double mutant). Stress was induced by starvation or by the addition of an oxidative stressor paraquat (PQ) to food. Mortality tests revealed that the Akh(1) mutant was the most resistant to starvation, while the AdoR(1) mutant was the most sensitive. Conversely, the Akh(1) AdoR(1) double mutant was more sensitive to PQ toxicity than either of the single mutants. Administration of PQ significantly increased the Drome-AKH level in w(1118) and AdoR(1) larvae; however, this was not accompanied by a simultaneous increase in Akh gene expression. In contrast, PQ significantly increased the expression of the glutathione S-transferase D1 (GstD1) gene. The presence of both a functional adenosine receptor and AKH seem to be important for the proper control of GstD1 gene expression under oxidative stress, however, the latter appears to play more dominant role. On the other hand, differences in glutathione S-transferase (GST) activity among the strains, and between untreated and PQ-treated groups were minimal. In addition, the glutathione level was significantly lower in all untreated AKH- or AdoR-deficient mutant flies as compared with the untreated control w(1118) flies and further declined following treatment with PQ. All oxidative stress characteristics modified by mutations in Akh gene were restored or even improved by 'rescue' mutation in flies which ectopically express Akh. Thus, the results of the present study demonstrate the important roles of AKH and adenosine in the anti-stress response elicited by PQ in a D. melanogaster model, and provide the first evidence for the involvement of adenosine in the anti-oxidative stress response in insects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification, expression, and comparative genomic analysis of the IPT and CKX gene families in Chinese cabbage (Brassica rapa ssp. pekinensis)

PubMed Central

2013-01-01

Background Cytokinins (CKs) have significant roles in various aspects of plant growth and development, and they are also involved in plant stress adaptations. The fine-tuning of the controlled CK levels in individual tissues, cells, and organelles is properly maintained by isopentenyl transferases (IPTs) and cytokinin oxidase/dehydrogenases (CKXs). Chinese cabbage is one of the most economically important vegetable crops worldwide. The whole genome sequencing of Brassica rapa enables us to perform the genome-wide identification and functional analysis of the IPT and CKX gene families. Results In this study, a total of 13 BrIPT genes and 12 BrCKX genes were identified. The gene structures, conserved domains and phylogenetic relationships were analyzed. The isoelectric point, subcellular localization and glycosylation sites of the proteins were predicted. Segmental duplicates were found in both BrIPT and BrCKX gene families. We also analyzed evolutionary patterns and divergence of the IPT and CKX genes in the Cruciferae family. The transcription levels of BrIPT and BrCKX genes were analyzed to obtain an initial picture of the functions of these genes. Abiotic stress elements related to adverse environmental stimuli were found in the promoter regions of BrIPT and BrCKX genes and they were confirmed to respond to drought and high salinity conditions. The effects of 6-BA and ABA on the expressions of BrIPT and BrCKX genes were also investigated. Conclusions The expansion of BrIPT and BrCKX genes after speciation from Arabidopsis thaliana is mainly attributed to segmental duplication events during the whole genome triplication (WGT) and substantial duplicated genes are lost during the long evolutionary history. Genes produced by segmental duplication events have changed their expression patterns or may adopted new functions and thus are obtained. BrIPT and BrCKX genes respond well to drought and high salinity stresses, and their transcripts are affected by exogenous hormones, such as 6-BA and ABA, suggesting their potential roles in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. The appropriate modulation of endogenous CKs levels by IPT and CKX genes is a promising approach for developing economically important high-yielding and high-quality stress-tolerant crops in agriculture. PMID:24001366

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

PubMed Central

Tondeleir, Davina; Noelanders, Rivka; Bakkali, Karima; Ampe, Christophe

2014-01-01

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11. PMID:24409333

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression

PubMed Central

Garcia, Gloria R.; Goodale, Britton C.; Wiley, Michelle W.; La Du, Jane K.; Hendrix, David A.

2017-01-01

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway. PMID:28385905

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

PubMed

Garcia, Gloria R; Goodale, Britton C; Wiley, Michelle W; La Du, Jane K; Hendrix, David A; Tanguay, Robert L

2017-06-01

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA ( slincR ) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway. Copyright © 2017 by The Author(s).

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

PubMed Central

Videau, Patrick; Rivers, Orion S.; Ushijima, Blake; Oshiro, Reid T.; Kim, Min Joo; Philmus, Benjamin

2016-01-01

ABSTRACT To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:l-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. IMPORTANCE Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program. PMID:26811320

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120.

PubMed

Videau, Patrick; Rivers, Orion S; Ushijima, Blake; Oshiro, Reid T; Kim, Min Joo; Philmus, Benjamin; Cozy, Loralyn M

2016-04-01

To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Treacher Collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation.

PubMed

Gonzales, Bianca; Henning, Dale; So, Rolando B; Dixon, Jill; Dixon, Michael J; Valdez, Benigno C

2005-07-15

Treacher Collins syndrome (TCS) is characterized by defects in craniofacial development, which results from mutations in the TCOF1 gene. TCOF1 encodes the nucleolar phosphoprotein treacle, which interacts with upstream binding factor (UBF) and affects transcription of the ribosomal DNA gene. The present study shows participation of treacle in the 2'-O-methylation of pre-rRNA. Antisense-mediated down-regulation of treacle expression in Xenopus laevis oocytes reduced 2'-O-methylation of pre-rRNA. Analysis of RNA isolated from wild-type and Tcof1+/- heterozygous mice embryos from strains that exhibit a lethal phenotype showed significant reduction in 2'-O-methylation at nucleotide C463 of 18S rRNA. The level of pseudouridylation of U1642 of 18S rRNA from the same RNA samples was not affected suggesting specificity. There is no significant difference in rRNA methylation between wild-type and heterozygous embryos of DBA x BALB/c mice, which have no obvious craniofacial phenotype. The function of treacle in pre-rRNA methylation is most likely mediated by its direct physical interaction with NOP56, a component of the ribonucleoprotein methylation complex. Although treacle co-localizes with UBF throughout mitosis, it co-localizes with NOP56 and fibrillarin, a putative methyl transferase, only during telophase when rDNA gene transcription and pre-rRNA methylation are known to commence. These observations suggest that treacle might link RNA polymerase I-catalyzed transcription and post-transcriptional modification of pre-rRNA. We hypothesize that haploinsufficiency of treacle in TCS patients results in inhibition of production of properly modified mature rRNA in addition to inhibition of rDNA gene transcription, which consequently affects proliferation and proper differentiation of specific embryonic cells during development.

Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia.

PubMed

Novakovic, Boris; Evain-Brion, Danièle; Murthi, Padma; Fournier, Thiery; Saffery, Richard

2017-06-01

Placental functioning relies on the appropriate differentiation of progenitor villous cytotrophoblasts (CTBs) into extravillous cytotrophoblasts (EVCTs), including invasive EVCTs, and the multinucleated syncytiotrophoblast (ST) layer. This is accompanied by a general move away from a proliferative, immature phenotype. Genome-scale expression studies have provided valuable insight into genes that are associated with the shift to both an invasive EVCT and ST phenotype, whereas genome-scale DNA methylation analysis has shown that differentiation to ST involves widespread methylation shifts, which are counteracted by low oxygen. In the current study, we sought to identify DNA methylation variation that is associated with transition from CTB to ST in vitro and from a noninvasive to invasive EVCT phenotype after culture on Matrigel. Of the several hundred differentially methylated regions that were identified in each comparison, the majority showed a loss of methylation with differentiation. This included a large differentially methylated region (DMR) in the gene body of death domain-associated protein 6 ( DAXX ), which lost methylation during both CTB syncytialization to ST and EVCT differentiation to invasive EVCT. Comparison to publicly available methylation array data identified the same DMR as among the most consistently differentially methylated genes in placental samples from preeclampsia pregnancies. Of interest, in vitro culture of CTB or ST in low oxygen increases methylation in the same region, which correlates with delayed differentiation. Analysis of combined epigenomics signatures confirmed DAXX DMR as a likely regulatory element, and direct gene expression analysis identified a positive association between methylation at this site and DAXX expression levels. The widespread dynamic nature of DAXX methylation in association with trophoblast differentiation and placenta-associated pathologies is consistent with an important role for this gene in proper placental development and function.-Novakovic, B., Evain-Brion, D., Murthi, P., Fournier, T., Saffery, R. Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia. © FASEB.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

PubMed Central

Perkins, Archibald S.; Kirschmeier, Paul T.; Gattoni-Celli, Sebastiano; Weinstein, I. Bernard

1983-01-01

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt+ colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt+ phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells. Images PMID:6308426

Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity

PubMed Central

2010-01-01

Background The European sea bass (Dicentrarchus labrax) is a marine fish of great importance for fisheries and aquaculture. Functional genomics offers the possibility to discover the molecular mechanisms underlying productive traits in farmed fish, and a step towards the application of marker assisted selection methods in this species. To this end, we report here on the development of an oligo DNA microarray for D. labrax. Results A database consisting of 19,048 unique transcripts was constructed, of which 12,008 (63%) could be annotated by similarity and 4,692 received a GO functional annotation. Two non-overlapping 60mer probes were designed for each unique transcript and in-situ synthesized on glass slides using Agilent SurePrint™ technology. Probe design was positively completed for 19,035 target clusters; the oligo microarray was then applied to profile gene expression in mandibles and whole-heads of fish affected by prognathism, a skeletal malformation that strongly affects sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish, with a significant enrichment in genes related to nervous system development and functioning. A set of genes spanning a wide dynamic range in gene expression level were selected for quantitative RT-PCR validation. Fold change correlation between microarray and qPCR data was always significant. Conclusions The microarray platform developed for the European sea bass has a high level of flexibility, reliability, and reproducibility. Despite the well known limitations in achieving a proper functional annotation in non-model species, sufficient information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on putative mechanisms involved on mandibular prognathism, suggesting that bone/nervous system development might play a role in this phenomenon. PMID:20525278

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

PubMed Central

Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

2014-01-01

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

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.

Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1.

PubMed

Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

2017-06-01

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

Maintenance of the marginal zone B cell compartment specifically requires the RNA-binding protein ZFP36L1

PubMed Central

Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S.; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

2017-01-01

RNA binding proteins (RBP) of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU rich elements in the 3’UTR and promoting mRNA decay. Here we show an indispensible role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal zone (MZ) B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene expression program related to signaling, cell-adhesion and locomotion, in part by limiting the expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RBP for maintaining cellular identity between closely related cell types. PMID:28394372

A robust two-way semi-linear model for normalization of cDNA microarray data

PubMed Central

Wang, Deli; Huang, Jian; Xie, Hehuang; Manzella, Liliana; Soares, Marcelo Bento

2005-01-01

Background Normalization is a basic step in microarray data analysis. A proper normalization procedure ensures that the intensity ratios provide meaningful measures of relative expression values. Methods We propose a robust semiparametric method in a two-way semi-linear model (TW-SLM) for normalization of cDNA microarray data. This method does not make the usual assumptions underlying some of the existing methods. For example, it does not assume that: (i) the percentage of differentially expressed genes is small; or (ii) the numbers of up- and down-regulated genes are about the same, as required in the LOWESS normalization method. We conduct simulation studies to evaluate the proposed method and use a real data set from a specially designed microarray experiment to compare the performance of the proposed method with that of the LOWESS normalization approach. Results The simulation results show that the proposed method performs better than the LOWESS normalization method in terms of mean square errors for estimated gene effects. The results of analysis of the real data set also show that the proposed method yields more consistent results between the direct and the indirect comparisons and also can detect more differentially expressed genes than the LOWESS method. Conclusions Our simulation studies and the real data example indicate that the proposed robust TW-SLM method works at least as well as the LOWESS method and works better when the underlying assumptions for the LOWESS method are not satisfied. Therefore, it is a powerful alternative to the existing normalization methods. PMID:15663789

The Growth-Suppressive Function of the Polycomb Group Protein Polyhomeotic Is Mediated by Polymerization of Its Sterile Alpha Motif (SAM) Domain*

PubMed Central

Robinson, Angela K.; Leal, Belinda Z.; Chadwell, Linda V.; Wang, Renjing; Ilangovan, Udayar; Kaur, Yogeet; Junco, Sarah E.; Schirf, Virgil; Osmulski, Pawel A.; Gaczynska, Maria; Hinck, Andrew P.; Demeler, Borries; McEwen, Donald G.; Kim, Chongwoo A.

2012-01-01

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions. PMID:22275371

The TORMOZ Gene Encodes a Nucleolar Protein Required for Regulated Division Planes and Embryo Development in Arabidopsis[W

PubMed Central

Griffith, Megan E.; Mayer, Ulrike; Capron, Arnaud; Ngo, Quy A.; Surendrarao, Anandkumar; McClinton, Regina; Jürgens, Gerd; Sundaresan, Venkatesan

2007-01-01

Embryogenesis in Arabidopsis thaliana is marked by a predictable sequence of oriented cell divisions, which precede cell fate determination. We show that mutation of the TORMOZ (TOZ) gene yields embryos with aberrant cell division planes and arrested embryos that appear not to have established normal patterning. The defects in toz mutants differ from previously described mutations that affect embryonic cell division patterns. Longitudinal division planes of the proembryo are frequently replaced by transverse divisions and less frequently by oblique divisions, while divisions of the suspensor cells, which divide only transversely, appear generally unaffected. Expression patterns of selected embryo patterning genes are altered in the mutant embryos, implying that the positional cues required for their proper expression are perturbed by the misoriented divisions. The TOZ gene encodes a nucleolar protein containing WD repeats. Putative TOZ orthologs exist in other eukaryotes including Saccharomyces cerevisiae, where the protein is predicted to function in 18S rRNA biogenesis. We find that disruption of the Sp TOZ gene results in cell division defects in Schizosaccharomyces pombe. Previous studies in yeast and animal cells have identified nucleolar proteins that regulate the exit from M phase and cytokinesis, including factors involved in pre-rRNA processing. Our study suggests that in plant cells, nucleolar functions might interact with the processes of regulated cell divisions and influence the selection of longitudinal division planes during embryogenesis. PMID:17616738

Homeostasis of Hyaluronic Acid in Normal and Scarred Vocal Folds

PubMed Central

Tateya, Ichiro; Tateya, Tomoko; Watanuki, Makoto; Bless, Diane M.

2015-01-01

Summary Objectives/Hypothesis Vocal fold scarring is one of the most challenging laryngeal disorders to treat. Hyaluronic acid (HA) is the main component of lamina propria, and it plays an important role in proper vocal fold vibration and is also thought to be important in fetal wound healing without scarring. Although several animal models of vocal fold scarring have been reported, little is known about the way in which HA is maintained in vocal folds. The purpose of this study was to clarify the homeostasis of HA by examining the expression of hyaluronan synthase (Has) and hyaluronidase (Hyal), which produce and digest HA, respectively. Study Design Experimental prospective animal study. Methods Vocal fold stripping was performed on 38 Sprague-Dawley rats. Vocal fold tissue was collected at five time points (3 days–2 months). Expression of HA was examined by immunohistochemistry, and messenger RNA (mRNA) expression of Has and Hyal was examined by real-time polymerase chain reaction and in-situ hybridization. Results In scarred vocal folds, expression of Has1 and Has2 increased at day 3 together with expression of HA and returned to normal at 2 weeks. At 2 months, Has3 and Hyal3 mRNA showed higher expressions than normal. Conclusions Expression patterns of Has and Hyal genes differed between normal, acute-scarred, and chronic-scarred vocal folds, indicating the distinct roles of each enzyme in maintaining HA. Continuous upregulation of Has genes in the acute phase may be necessary to achieve scarless healing of vocal folds. PMID:25499520

Stable expression of calpain 3 from a muscle transgene in vivo: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation

PubMed Central

Spencer, M. J.; Guyon, J. R.; Sorimachi, H.; Potts, A.; Richard, I.; Herasse, M.; Chamberlain, J.; Dalkilic, I.; Kunkel, L. M.; Beckmann, J. S.

2002-01-01

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development. PMID:12084932

Premature terminator analysis sheds light on a hidden world of bacterial transcriptional attenuation.

PubMed

Naville, Magali; Gautheret, Daniel

2010-01-01

Bacterial transcription attenuation occurs through a variety of cis-regulatory elements that control gene expression in response to a wide range of signals. The signal-sensing structures in attenuators are so diverse and rapidly evolving that only a small fraction have been properly annotated and characterized to date. Here we apply a broad-spectrum detection tool in order to achieve a more complete view of the transcriptional attenuation complement of key bacterial species. Our protocol seeks gene families with an unusual frequency of 5' terminators found across multiple species. Many of the detected attenuators are part of annotated elements, such as riboswitches or T-boxes, which often operate through transcriptional attenuation. However, a significant fraction of candidates were not previously characterized in spite of their unmistakable footprint. We further characterized some of these new elements using sequence and secondary structure analysis. We also present elements that may control the expression of several non-homologous genes, suggesting co-transcription and response to common signals. An important class of such elements, which we called mobile attenuators, is provided by 3' terminators of insertion sequences or prophages that may be exapted as 5' regulators when inserted directly upstream of a cellular gene. We show here that attenuators involve a complex landscape of signal-detection structures spanning the entire bacterial domain. We discuss possible scenarios through which these diverse 5' regulatory structures may arise or evolve.

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor.

PubMed

Willemse, Joost; Mommaas, A Mieke; van Wezel, Gilles P

2012-03-01

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants-which fail to properly localise FtsZ-could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades.

humpty dumpty is required for developmental DNA amplification and cell proliferation in Drosophila.

PubMed

Bandura, Jennifer L; Beall, Eileen L; Bell, Maren; Silver, Hannah R; Botchan, Michael R; Calvi, Brian R

2005-04-26

The full complement of proteins required for the proper regulation of genome duplication are yet to be described. We employ a genetic DNA-replication model system based on developmental amplification of Drosophila eggshell (chorion) genes [1]. Hypomorphic mutations in essential DNA replication genes result in a distinct thin-eggshell phenotype owing to reduced amplification [2]. Here, we molecularly identify the gene, which we have named humpty dumpty (hd), corresponding to the thin-eggshell mutant fs(3)272-9 [3]. We confirm that hd is essential for DNA amplification in the ovary and show that it also is required for cell proliferation during development. Mosaic analysis of hd mutant cells during development and RNAi in Kc cells reveal that depletion of Hd protein results in severe defects in genomic replication and DNA damage. Most Hd protein is found in nuclear foci, and some may traverse the nuclear envelope. Consistent with a role in DNA replication, expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Hd protein sequence is conserved from plants to humans, and published microarrays indicate that expression of its putative human ortholog also peaks at G1/S [4]. Our data suggest that hd defines a new gene family likely required for cell proliferation in all multicellular eukaryotes.

Putrescine Is Involved in Arabidopsis Freezing Tolerance and Cold Acclimation by Regulating Abscisic Acid Levels in Response to Low Temperature1

PubMed Central

Cuevas, Juan C.; López-Cobollo, Rosa; Alcázar, Rubén; Zarza, Xavier; Koncz, Csaba; Altabella, Teresa; Salinas, Julio; Tiburcio, Antonio F.; Ferrando, Alejandro

2008-01-01

The levels of endogenous polyamines have been shown to increase in plant cells challenged with low temperature; however, the functions of polyamines in the regulation of cold stress responses are unknown. Here, we show that the accumulation of putrescine under cold stress is essential for proper cold acclimation and survival at freezing temperatures because Arabidopsis (Arabidopsis thaliana) mutants defective in putrescine biosynthesis (adc1, adc2) display reduced freezing tolerance compared to wild-type plants. Genes ADC1 and ADC2 show different transcriptional profiles upon cold treatment; however, they show similar and redundant contributions to cold responses in terms of putrescine accumulation kinetics and freezing sensitivity. Our data also demonstrate that detrimental consequences of putrescine depletion during cold stress are due, at least in part, to alterations in the levels of abscisic acid (ABA). Reduced expression of NCED3, a key gene involved in ABA biosynthesis, and down-regulation of ABA-regulated genes are detected in both adc1 and adc2 mutant plants under cold stress. Complementation analysis of adc mutants with ABA and reciprocal complementation tests of the aba2-3 mutant with putrescine support the conclusion that putrescine controls the levels of ABA in response to low temperature by modulating ABA biosynthesis and gene expression. PMID:18701673

Transcriptional regulation of the tad locus in Aggregatibacter actinomycetemcomitans: a termination cascade.

PubMed

Kram, Karin E; Hovel-Miner, Galadriel A; Tomich, Mladen; Figurski, David H

2008-06-01

The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.

Comparison of trophic factors' expression between paralyzed and recovering muscles after facial nerve injury. A quantitative analysis in time course.

PubMed

Grosheva, Maria; Nohroudi, Klaus; Schwarz, Alisa; Rink, Svenja; Bendella, Habib; Sarikcioglu, Levent; Klimaschewski, Lars; Gordon, Tessa; Angelov, Doychin N

2016-05-01

After peripheral nerve injury, recovery of motor performance negatively correlates with the poly-innervation of neuromuscular junctions (NMJ) due to excessive sprouting of the terminal Schwann cells. Denervated muscles produce short-range diffusible sprouting stimuli, of which some are neurotrophic factors. Based on recent data that vibrissal whisking is restored perfectly during facial nerve regeneration in blind rats from the Sprague Dawley (SD)/RCS strain, we compared the expression of brain derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), insulin growth factors 1 and 2 (IGF1, IGF2) and nerve growth factor (NGF) between SD/RCS and SD-rats with normal vision but poor recovery of whisking function after facial nerve injury. To establish which trophic factors might be responsible for proper NMJ-reinnervation, the transected facial nerve was surgically repaired (facial-facial anastomosis, FFA) for subsequent analysis of mRNA and proteins expressed in the levator labii superioris muscle. A complicated time course of expression included (1) a late rise in BDNF protein that followed earlier elevated gene expression, (2) an early increase in FGF2 and IGF2 protein after 2 days with sustained gene expression, (3) reduced IGF1 protein at 28 days coincident with decline of raised mRNA levels to baseline, and (4) reduced NGF protein between 2 and 14 days with maintained gene expression found in blind rats but not the rats with normal vision. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of lesion-associated neurotrophic factors and cytokines in denervated muscles. The increase of FGF-2 protein and concomittant decrease of NGF (with no significant changes in BDNF or IGF levels) during the first week following FFA in SD/RCS blind rats possibly prevents the distal branching of regenerating axons resulting in reduced poly-innervation of motor endplates. Copyright © 2016 Elsevier Inc. All rights reserved.

Gene Expression Measurement Module (GEMM) - a fully automated, miniaturized instrument for measuring gene expression in space

NASA Astrophysics Data System (ADS)

Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh

2012-07-01

The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions, under light and dark cycles exposed to polar orbit for a period of 6 months. The integration and end-to-end technology validation of this instrument will be discussed. In particular, preliminary results demonstrating that the instrument properly carries out cellular lysis, nucleic acid extraction and its purification is being assessed by reverse transcription polymerase chain reaction (PCR) and real time PCR, in addition to microarray analysis of selected genes. Once developed, the system can be used with minor modifications for multiple experiments on different platforms in space, including extensions to higher organisms and microbial monitoring. A proposed version of GEMM that is capable of handing both microbial and tissue samples on the International Space Station will be briefly reviewed.

Excitation-transcription coupling in parvalbumin-positive interneurons employs a novel CaM Kinase-dependent pathway distinct from excitatory neurons

PubMed Central

Cohen, Samuel M.; Ma, Huan; Kuchibhotla, Kishore V.; Watson, Brendon O.; Buzsáki, György; Froemke, Robert C.; Tsien, Richard W.

2016-01-01

Properly functional CNS circuits depend on inhibitory interneurons that in turn rely upon activity-dependent gene expression for morphological development, connectivity and excitatory-inhibitory coordination. Despite its importance, excitation-transcription coupling in inhibitory interneurons is poorly understood. Here, we report that PV+ interneurons employ a novel CaMK-dependent pathway to trigger CREB phosphorylation and gene expression. As in excitatory neurons, voltage-gated Ca2+ influx through CaV1 channels triggers CaM nuclear translocation via local Ca2+ signaling. However, PV+ interneurons are distinct in that nuclear signaling is mediated by γCaMKI, not γCaMKII. CREB phosphorylation also proceeds with slow, sigmoid kinetics, rate-limited by paucity of CaMKIV, protecting against saturation of phospho-CREB in the face of higher firing rates and bigger Ca2+ transients. Our findings support the generality of CaM shuttling to drive nuclear CaMK activity, and are relevant to disease pathophysiology, insofar as dysfunction of PV+ interneurons and molecules underpinning their excitation-transcription coupling both relate to neuropsychiatric disease. PMID:27041500

Sumoylation Dynamics During Keratinocyte Differentiation

PubMed Central

Deyrieux, Adeline F.; Rosas-Acosta, Germán; Ozbun, Michelle A.; Wilson, Van G.

2012-01-01

Summary SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several critical keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), calcium-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding critical sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASxβ), SUMO2, and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similar to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Lastly, abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process. PMID:17164289

Regulation of inflorescence architecture by intertissue layer ligand-receptor communication between endodermis and phloem.

PubMed

Uchida, Naoyuki; Lee, Jin Suk; Horst, Robin J; Lai, Hung-Hsueh; Kajita, Ryoko; Kakimoto, Tatsuo; Tasaka, Masao; Torii, Keiko U

2012-04-17

Multicellular organisms achieve final body shape and size by coordinating cell proliferation, expansion, and differentiation. Loss of function in the Arabidopsis ERECTA (ER) receptor-kinase gene confers characteristic compact inflorescence architecture, but its underlying signaling pathways remain unknown. Here we report that the expression of ER in the phloem is sufficient to rescue compact er inflorescences. We further identified two Epidermal Patterning Factor-like (EPFL) secreted peptide genes, EPFL4 and EPFL6/CHALLAH (CHAL), as redundant, upstream components of ER-mediated inflorescence growth. The expression of EPFL4 or EPFL6 in the endodermis, a layer adjacent to phloem, is sufficient to rescue the er-like inflorescence of epfl4 epfl6 plants. EPFL4 and EPFL6 physically associate with ER in planta. Finally, transcriptome analysis of er and epfl4 epfl6 revealed a potential downstream component as well as a role for plant hormones in EPFL4/6- and ER-mediated inflorescence growth. Our results suggest that intercell layer communication between the endodermis and phloem mediated by peptide ligands and a receptor kinase coordinates proper inflorescence architecture in Arabidopsis.

Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome.

PubMed

Rodrigo, Isabel; Hill, Robert E; Balling, Rudi; Münsterberg, Andrea; Imai, Kenji

2003-02-01

We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and that they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.

Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta

PubMed Central

Gerlach, Gary F.; Wingert, Rebecca A.

2014-01-01

The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by inhibiting pax2a expression. These studies provide a valuable foundation for further analysis of MET during nephrogenesis, and have implications for understanding the pathways that affect nephron epithelial cells during kidney disease and regeneration. PMID:25446529

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

PubMed

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe; Bustin, Michael

2017-04-07

An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation

PubMed Central

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M.; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe

2017-01-01

Abstract An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. PMID:27923998

ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice

PubMed Central

Zhao, Hongyu; Ruan, Wenyuan; Deng, Minjuan; Wang, Fang; Peng, Jinrong; Luo, Jie; Chen, Zhixiang

2017-01-01

Root meristem activity determines root growth and root architecture and consequently affects water and nutrient uptake in plants. However, our knowledge about the regulation of root meristem activity in crop plants is very limited. Here, we report the isolation and characterization of a short root mutant in rice (Oryza sativa) with reduced root meristem activity. This root growth defect is caused by a mutation in ABNORMAL INFLORESCENCE MERISTEM1 (AIM1), which encodes a 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in β-oxidation. The reduced root meristem activity of aim1 results from reduced salicylic acid (SA) levels and can be rescued by SA application. Furthermore, reduced SA levels are associated with reduced levels of reactive oxygen species (ROS) in aim1, likely due to increased expression of redox and ROS-scavenging-related genes, whose increased expression is (at least in part) caused by reduced expression of the SA-inducible transcriptional repressors WRKY62 and WRKY76. Like SA, ROS application substantially increased root length and root meristem activity in aim1. These results suggest that AIM1 is required for root growth in rice due to its critical role in SA biosynthesis: SA maintains root meristem activity through promoting ROS accumulation by inducing the activity of WRKY transcriptional repressors, which repress the expression of redox and ROS-scavenging genes. PMID:28298519

Drosophila gene tao-1 encodes proteins with and without a Ste20 kinase domain that affect cytoskeletal architecture and cell migration differently

PubMed Central

Pflanz, Ralf; Voigt, Aaron; Yakulov, Toma; Jäckle, Herbert

2015-01-01

Tao-1, the single representative of the Sterile 20 kinase subfamily in Drosophila, is best known for destabilizing microtubules at the actin-rich cortex, regulating the cytoskeletal architecture of cells. More recently, Tao-1 was shown to act in the Salvador–Warts–Hippo pathway by phosphorylating Hippo, regulating cell growth as well as cell polarity. Here, we show that tao-1 encodes two proteins, one with the Sterile 20 kinase domain (Tao-L) and one without it (Tao-S), and that they act in an antagonistic manner. Tao-L expression causes lamellipodia-like cell protrusions, whereas Tao-S expression results in filopodia-like structures that make cells stick to the surface they attach to. Ectopic Tao-1 expression in the anterior region of Drosophila embryos results in pole cell formation as normally observed at the posterior end. Tao-S expression causes primordial germ cells (PGCs) to adhere to the inner wall of the gut primordia and prevents proper transepithelial migration to the gonads. Conversely, RNAi knockdowns of Tao-1 cause disordered migration of PGCs out of the gut epithelium, their dispersal within the embryo and cell death. The results reveal a novel function of Tao-1 in cell migration, which is based on antagonistic activities of two proteins encoded by a single gene. PMID:25589578

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

2012-03-01

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development

PubMed Central

Schertel, Claus; Albarca, Monica; Rockel-Bauer, Claudia; Kelley, Nicholas W.; Bischof, Johannes; Hens, Korneel

2015-01-01

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such “bivalent” chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue. PMID:25568052

Nuclear ribosome biogenesis mediated by the DIM1A rRNA dimethylase is required for organized root growth and epidermal patterning in Arabidopsis.

PubMed

Wieckowski, Yana; Schiefelbein, John

2012-07-01

Position-dependent patterning of hair and non-hair cells in the Arabidopsis thaliana root epidermis is a powerful system to study the molecular basis of cell fate specification. Here, we report an epidermal patterning mutant affecting the ADENOSINE DIMETHYL TRANSFERASE 1A (DIM1A) rRNA dimethylase gene, predicted to participate in rRNA posttranscriptional processing and base modification. Consistent with a role in ribosome biogenesis, DIM1A is preferentially expressed in regions of rapid growth, and its product is nuclear localized with nucleolus enrichment. Furthermore, DIM1A preferentially accumulates in the developing hair cells, and the dim1A point mutant alters the cell-specific expression of the transcriptional regulators GLABRA2, CAPRICE, and WEREWOLF. Together, these findings suggest that establishment of cell-specific gene expression during root epidermis development is dependent upon proper ribosome biogenesis, possibly due to the sensitivity of the cell fate decision to relatively small differences in gene regulatory activities. Consistent with its effect on the predicted S-adenosyl-l-Met binding site, dim1A plants lack the two 18S rRNA base modifications but exhibit normal pre-rRNA processing. In addition to root epidermal defects, the dim1A mutant exhibits abnormal root meristem division, leaf development, and trichome branching. Together, these findings provide new insights into the importance of rRNA base modifications and translation regulation for plant growth and development.

Nuclear Ribosome Biogenesis Mediated by the DIM1A rRNA Dimethylase Is Required for Organized Root Growth and Epidermal Patterning in Arabidopsis[C][W

PubMed Central

Wieckowski, Yana; Schiefelbein, John

2012-01-01

Position-dependent patterning of hair and non-hair cells in the Arabidopsis thaliana root epidermis is a powerful system to study the molecular basis of cell fate specification. Here, we report an epidermal patterning mutant affecting the ADENOSINE DIMETHYL TRANSFERASE 1A (DIM1A) rRNA dimethylase gene, predicted to participate in rRNA posttranscriptional processing and base modification. Consistent with a role in ribosome biogenesis, DIM1A is preferentially expressed in regions of rapid growth, and its product is nuclear localized with nucleolus enrichment. Furthermore, DIM1A preferentially accumulates in the developing hair cells, and the dim1A point mutant alters the cell-specific expression of the transcriptional regulators GLABRA2, CAPRICE, and WEREWOLF. Together, these findings suggest that establishment of cell-specific gene expression during root epidermis development is dependent upon proper ribosome biogenesis, possibly due to the sensitivity of the cell fate decision to relatively small differences in gene regulatory activities. Consistent with its effect on the predicted S-adenosyl-l-Met binding site, dim1A plants lack the two 18S rRNA base modifications but exhibit normal pre-rRNA processing. In addition to root epidermal defects, the dim1A mutant exhibits abnormal root meristem division, leaf development, and trichome branching. Together, these findings provide new insights into the importance of rRNA base modifications and translation regulation for plant growth and development. PMID:22829145

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

PubMed

Ochiai, Kyoko; Muto, Akihiko; Tanaka, Hiromu; Takahashi, Shinichiro; Igarashi, Kazuhiko

2008-03-01

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

Program Specificity for Ptf1a in Pancreas versus Neural Tube Development Correlates with Distinct Collaborating Cofactors and Chromatin Accessibility

PubMed Central

Meredith, David M.; Borromeo, Mark D.; Deering, Tye G.; Casey, Bradford H.; Savage, Trisha K.; Mayer, Paul R.; Hoang, Chinh; Tung, Kuang-Chi; Kumar, Manonmani; Shen, Chengcheng; Swift, Galvin H.

2013-01-01

The lineage-specific basic helix-loop-helix transcription factor Ptf1a is a critical driver for development of both the pancreas and nervous system. How one transcription factor controls diverse programs of gene expression is a fundamental question in developmental biology. To uncover molecular strategies for the program-specific functions of Ptf1a, we identified bound genomic regions in vivo during development of both tissues. Most regions bound by Ptf1a are specific to each tissue, lie near genes needed for proper formation of each tissue, and coincide with regions of open chromatin. The specificity of Ptf1a binding is encoded in the DNA surrounding the Ptf1a-bound sites, because these regions are sufficient to direct tissue-restricted reporter expression in transgenic mice. Fox and Sox factors were identified as potential lineage-specific modifiers of Ptf1a binding, since binding motifs for these factors are enriched in Ptf1a-bound regions in pancreas and neural tube, respectively. Of the Fox factors expressed during pancreatic development, Foxa2 plays a major role. Indeed, Ptf1a and Foxa2 colocalize in embryonic pancreatic chromatin and can act synergistically in cell transfection assays. Together, these findings indicate that lineage-specific chromatin landscapes likely constrain the DNA binding of Ptf1a, and they identify Fox and Sox gene families as part of this process. PMID:23754747

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

PubMed

Philip, Naomi H; DeLaney, Alexandra; Peterson, Lance W; Santos-Marrero, Melanie; Grier, Jennifer T; Sun, Yan; Wynosky-Dolfi, Meghan A; Zwack, Erin E; Hu, Baofeng; Olsen, Tayla M; Rongvaux, Anthony; Pope, Scott D; López, Carolina B; Oberst, Andrew; Beiting, Daniel P; Henao-Mejia, Jorge; Brodsky, Igor E

2016-10-01

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense.

Many human accelerated regions are developmental enhancers

PubMed Central

Capra, John A.; Erwin, Genevieve D.; McKinsey, Gabriel; Rubenstein, John L. R.; Pollard, Katherine S.

2013-01-01

The genetic changes underlying the dramatic differences in form and function between humans and other primates are largely unknown, although it is clear that gene regulatory changes play an important role. To identify regulatory sequences with potentially human-specific functions, we and others used comparative genomics to find non-coding regions conserved across mammals that have acquired many sequence changes in humans since divergence from chimpanzees. These regions are good candidates for performing human-specific regulatory functions. Here, we analysed the DNA sequence, evolutionary history, histone modifications, chromatin state and transcription factor (TF) binding sites of a combined set of 2649 non-coding human accelerated regions (ncHARs) and predicted that at least 30% of them function as developmental enhancers. We prioritized the predicted ncHAR enhancers using analysis of TF binding site gain and loss, along with the functional annotations and expression patterns of nearby genes. We then tested both the human and chimpanzee sequence for 29 ncHARs in transgenic mice, and found 24 novel developmental enhancers active in both species, 17 of which had very consistent patterns of activity in specific embryonic tissues. Of these ncHAR enhancers, five drove expression patterns suggestive of different activity for the human and chimpanzee sequence at embryonic day 11.5. The changes to human non-coding DNA in these ncHAR enhancers may modify the complex patterns of gene expression necessary for proper development in a human-specific manner and are thus promising candidates for understanding the genetic basis of human-specific biology. PMID:24218637

Deployment of a Prototype Plant GFP Imager at the Arthur Clarke Mars Greenhouse of the Haughton Mars Project.

PubMed

Paul, Anna-Lisa; Bamsey, Matthew; Berinstain, Alain; Braham, Stephen; Neron, Philip; Murdoch, Trevor; Graham, Thomas; Ferl, Robert J

2008-04-18

The use of engineered plants as biosensors has made elegant strides in the past decades, providing keen insights into the health of plants in general and particularly in the nature and cellular location of stress responses. However, most of the analytical procedures involve laboratory examination of the biosensor plants. With the advent of the green fluorescence protein (GFP) as a biosensor molecule, it became at least theoretically possible for analyses of gene expression to occur telemetrically, with the gene expression information of the plant delivered to the investigator over large distances simply as properly processed fluorescence images. Spaceflight and other extraterrestrial environments provide unique challenges to plant life, challenges that often require changes at the gene expression level to accommodate adaptation and survival. Having previously deployed transgenic plant biosensors to evaluate responses to orbital spaceflight, we wished to develop the plants and especially the imaging devices required to conduct such experiments robotically, without operator intervention, within extraterrestrial environments. This requires the development of an autonomous and remotely operated plant GFP imaging system and concomitant development of the communications infrastructure to manage dataflow from the imaging device. Here we report the results of deploying a prototype GFP imaging system within the Arthur Clarke Mars Greenhouse (ACMG) an autonomously operated greenhouse located within the Haughton Mars Project in the Canadian High Arctic. Results both demonstrate the applicability of the fundamental GFP biosensor technology and highlight the difficulties in collecting and managing telemetric data from challenging deployment environments.

Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor.

PubMed

Deppe, Janina; Popp, Tanja; Egea, Virginia; Steinritz, Dirk; Schmidt, Annette; Thiermann, Horst; Weber, Christian; Ries, Christian

2016-05-01

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

Novel isoforms of Dlg are fundamental for neuronal development in Drosophila.

PubMed

Mendoza, Carolina; Olguín, Patricio; Lafferte, Gabriela; Thomas, Ulrich; Ebitsch, Susanne; Gundelfinger, Eckart D; Kukuljan, Manuel; Sierralta, Jimena

2003-03-15

Drosophila discs-large (dlg) mutants exhibit multiple developmental abnormalities, including severe defects in neuronal differentiation and synaptic structure and function. These defects have been ascribed to the loss of a single gene product, Dlg-A, a scaffold protein thought to be expressed in many cell types. Here, we describe that additional isoforms arise as a consequence of different transcription start points and alternative splicing of dlg. At least five different dlg gene products are predicted. We identified a subset of dlg-derived cDNAs that include novel exons encoding a peptide homologous to the N terminus of the mammalian protein SAP97/hDLG (S97N). Dlg isoforms containing the S97N domain are expressed at larval neuromuscular junctions and within the CNS of both embryos and larvae but are not detectable in epithelial tissues. Strong hypomorphic dlg alleles exhibit decreased expression of S97N, which may account for neural-specific aspects of the pleiomorphic dlg mutant phenotype. Selective inhibition of the expression of S97N-containing proteins in embryos by double-strand RNA leads to severe defects in neuronal differentiation and axon guidance, without overt perturbations in epithelia. These results indicate that the differential expression of dlg products correlates with distinct functions in non-neural and neural cells. During embryonic development, proteins that include the S97N domain are essential for proper neuronal differentiation and organization, acting through mechanisms that may include the adequate localization of cell fate determinants.

Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture.

PubMed

Xie, Pingyuan; Sun, Yi; Ouyang, Qi; Hu, Liang; Tan, Yueqiu; Zhou, Xiaoying; Xiong, Bo; Zhang, Qianjun; Yuan, Ding; Pan, Yi; Liu, Tiancheng; Liang, Ping; Lu, Guangxiu; Lin, Ge

2014-02-01

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. © AlphaMed Press.

Essential oil from Cymbopogon flexuosus as the potential inhibitor for HSP90.

PubMed

Gaonkar, Roopa; Shiralgi, Yallappa; Lakkappa, Dhananjaya B; Hegde, Gurumurthy

2018-01-01

The essential oil of Cymbopogon flexuosus or lemongrass oil (LO) is reported to have antibacterial, antifungal and anticancerous effects. HSP90 is one of the major chaperones responsible for the proper folding of cancer proteins. In this paper we show that the essential oil of C. flexuosus significantly suppresses the HSP90 gene expression. The cytotoxicity of the compounds was tested by MTT assay and the gene expression studies were carried out using HEK-293 and MCF-7 cells. Also we tested the efficacy of the major component of this essential oil viz. citral and geraniol in inhibiting the HSP90 expression. The oil was found to be more cytotoxic to MCF-7 cells with different IC 50 values for the oil (69.33 μg/mL), citral (140.7 μg/mL) and geraniol (117 μg/mL). The fold change of expression was calculated by RT-qPCR using ΔΔCt (2 ^-ΔΔCt ) method and it was 0.1 and 0.03 in MCF-7 cells at 80 μg/mL and 160 μg/mL of LO. Western blot results showed suppression of HSP90 protein expression and HSP90 - ATPase activity inhibition was also observed using LO. This study shows the anticancer mechanism exhibited by the essential oil of C. flexuosus is by the inhibition of the important chaperone protein HSP90.

No control genes required: Bayesian analysis of qRT-PCR data.

PubMed

Matz, Mikhail V; Wright, Rachel M; Scott, James G

2013-01-01

Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR) is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC) algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts). Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization) but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.

Gene Families of Cuticular Proteins Analogous to Peritrophins (CPAPs) in Tribolium castaneum Have Diverse Functions

PubMed Central

Jasrapuria, Sinu; Specht, Charles A.; Kramer, Karl J.; Beeman, Richard W.; Muthukrishnan, Subbaratnam

2012-01-01

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal ‘stiff-jointed’ gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species. PMID:23185457

Gene families of cuticular proteins analogous to peritrophins (CPAPs) in Tribolium castaneum have diverse functions.

PubMed

Jasrapuria, Sinu; Specht, Charles A; Kramer, Karl J; Beeman, Richard W; Muthukrishnan, Subbaratnam

2012-01-01

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

Identification and Functional Analysis of Healing Regulators in Drosophila

PubMed Central

Álvarez-Fernández, Carmen; Tamirisa, Srividya; Prada, Federico; Chernomoretz, Ariel; Podhajcer, Osvaldo; Blanco, Enrique; Martín-Blanco, Enrique

2015-01-01

Wound healing is an essential homeostatic mechanism that maintains the epithelial barrier integrity after tissue damage. Although we know the overall steps in wound healing, many of the underlying molecular mechanisms remain unclear. Genetically amenable systems, such as wound healing in Drosophila imaginal discs, do not model all aspects of the repair process. However, they do allow the less understood aspects of the healing response to be explored, e.g., which signal(s) are responsible for initiating tissue remodeling? How is sealing of the epithelia achieved? Or, what inhibitory cues cancel the healing machinery upon completion? Answering these and other questions first requires the identification and functional analysis of wound specific genes. A variety of different microarray analyses of murine and humans have identified characteristic profiles of gene expression at the wound site, however, very few functional studies in healing regulation have been carried out. We developed an experimentally controlled method that is healing-permissive and that allows live imaging and biochemical analysis of cultured imaginal discs. We performed comparative genome-wide profiling between Drosophila imaginal cells actively involved in healing versus their non-engaged siblings. Sets of potential wound-specific genes were subsequently identified. Importantly, besides identifying and categorizing new genes, we functionally tested many of their gene products by genetic interference and overexpression in healing assays. This non-saturated analysis defines a relevant set of genes whose changes in expression level are functionally significant for proper tissue repair. Amongst these we identified the TCP1 chaperonin complex as a key regulator of the actin cytoskeleton essential for the wound healing response. There is promise that our newly identified wound-healing genes will guide future work in the more complex mammalian wound healing response. PMID:25647511

The Populus class III HD ZIP, popREVOLUTA, influences cambium initiation and patterning of woody stems.

PubMed

Robischon, Marcel; Du, Juan; Miura, Eriko; Groover, Andrew

2011-03-01

The secondary growth of a woody stem requires the formation of a vascular cambium at an appropriate position and proper patterning of the vascular tissues derived from the cambium. Class III homeodomain-leucine zipper (HD ZIP) transcription factors have been implicated in polarity determination and patterning in lateral organs and primary vascular tissues and in the initiation and function of shoot apical meristems. We report here the functional characterization of a Populus class III HD ZIP gene, popREVOLUTA (PRE), that demonstrates another role for class III HD ZIPs in regulating the development of cambia and secondary vascular tissues. PRE is orthologous to Arabidopsis (Arabidopsis thaliana) REVOLUTA and is expressed in both the shoot apical meristem and in the cambial zone and secondary vascular tissues. Transgenic Populus expressing a microRNA-resistant form of PRE presents unstable phenotypic abnormalities affecting both primary and secondary growth. Surprisingly, phenotypic changes include abnormal formation of cambia within cortical parenchyma that can produce secondary vascular tissues in reverse polarity. Genes misexpressed in PRE mutants include transcription factors and auxin-related genes previously implicated in class III HD ZIP functions during primary growth. Together, these results suggest that PRE plays a fundamental role in the initiation of the cambium and in regulating the patterning of secondary vascular tissues.

Hypoxia and fetal heart development.

PubMed

Patterson, A J; Zhang, L

2010-10-01

Fetal hearts show a remarkable ability to develop under hypoxic conditions. The metabolic flexibility of fetal hearts allows sustained development under low oxygen conditions. In fact, hypoxia is critical for proper myocardial formation. Particularly, hypoxia inducible factor 1 (HIF-1) and vascular endothelial growth factor play central roles in hypoxia-dependent signaling in fetal heart formation, impacting embryonic outflow track remodeling and coronary vessel growth. Although HIF is not the only gene involved in adaptation to hypoxia, its role places it as a central figure in orchestrating events needed for adaptation to hypoxic stress. Although "normal" hypoxia (lower oxygen tension in the fetus as compared with the adult) is essential in heart formation, further abnormal hypoxia in utero adversely affects cardiogenesis. Prenatal hypoxia alters myocardial structure and causes a decline in cardiac performance. Not only are the effects of hypoxia apparent during the perinatal period, but prolonged hypoxia in utero also causes fetal programming of abnormality in the heart's development. The altered expression pattern of cardioprotective genes such as protein kinase c epsilon, heat shock protein 70, and endothelial nitric oxide synthase, likely predispose the developing heart to increased vulnerability to ischemia and reperfusion injury later in life. The events underlying the long-term changes in gene expression are not clear, but likely involve variation in epigenetic regulation.

Detection of Pathological Changes in the Aorta during Thoracic Aortic Aneurysm Progression on Molecular Level

PubMed Central

Rabajdová, Miroslava; Špaková, Ivana; Panagiotis, Artemiou; Ferenčáková, Michaela; Rybár, Dušan; Bobrov, Nikita; Sabol, František

2017-01-01

The progression of thoracic aortic aneurysm depends on regulation of aortic wall homeostasis and on changes in the structural components of the extracellular matrix, which are affected by multiple molecular signalling pathways. We decided to correlate the diameter of ascending thoracic aneurysm with gene expression of inflammation markers (IL-6, CRP), cytokine receptors (IL-6R, TNFR1, and TNFR2), and extracellular matrix components (Emilin-1, MMP9, and TIMP) for detection of the degree of pathological process of TAA formation. The experimental group was divided into three groups according to the diameter of the aortic aneurysm. Whole blood and tissue samples were properly collected and used for nucleic acid, chromatin, and protein isolation. The mRNA levels were detected by qRT-PCR. For the detection of protein levels a Cytokine Array IV assay kit was used in combination with a biochip analyzer. In aortic tissue, significant positive correlations were found between increased mRNA levels of inflammatory cytokines (CRP and IL-6) on both mRNA levels in tissue and protein from the blood with maximum in stage 3. Changes of gene expression of selected genes can be used for the experimental study of the inflammatory receptor inhibitors during trials targeted on slowing down the progress of aortic wall aneurysm. PMID:29158612

A comparative study of covariance selection models for the inference of gene regulatory networks.

PubMed

Stifanelli, Patrizia F; Creanza, Teresa M; Anglani, Roberto; Liuzzi, Vania C; Mukherjee, Sayan; Schena, Francesco P; Ancona, Nicola

2013-10-01

The inference, or 'reverse-engineering', of gene regulatory networks from expression data and the description of the complex dependency structures among genes are open issues in modern molecular biology. In this paper we compared three regularized methods of covariance selection for the inference of gene regulatory networks, developed to circumvent the problems raising when the number of observations n is smaller than the number of genes p. The examined approaches provided three alternative estimates of the inverse covariance matrix: (a) the 'PINV' method is based on the Moore-Penrose pseudoinverse, (b) the 'RCM' method performs correlation between regression residuals and (c) 'ℓ(2C)' method maximizes a properly regularized log-likelihood function. Our extensive simulation studies showed that ℓ(2C) outperformed the other two methods having the most predictive partial correlation estimates and the highest values of sensitivity to infer conditional dependencies between genes even when a few number of observations was available. The application of this method for inferring gene networks of the isoprenoid biosynthesis pathways in Arabidopsis thaliana allowed to enlighten a negative partial correlation coefficient between the two hubs in the two isoprenoid pathways and, more importantly, provided an evidence of cross-talk between genes in the plastidial and the cytosolic pathways. When applied to gene expression data relative to a signature of HRAS oncogene in human cell cultures, the method revealed 9 genes (p-value<0.0005) directly interacting with HRAS, sharing the same Ras-responsive binding site for the transcription factor RREB1. This result suggests that the transcriptional activation of these genes is mediated by a common transcription factor downstream of Ras signaling. Software implementing the methods in the form of Matlab scripts are available at: http://users.ba.cnr.it/issia/iesina18/CovSelModelsCodes.zip. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Transcriptomics of aged Drosophila motor neurons reveals a matrix metalloproteinase that impairs motor function.

PubMed

Azpurua, Jorge; Mahoney, Rebekah E; Eaton, Benjamin A

2018-04-01

The neuromuscular junction (NMJ) is responsible for transforming nervous system signals into motor behavior and locomotion. In the fruit fly Drosophila melanogaster, an age-dependent decline in motor function occurs, analogous to the decline experienced in mice, humans, and other mammals. The molecular and cellular underpinnings of this decline are still poorly understood. By specifically profiling the transcriptome of Drosophila motor neurons across age using custom microarrays, we found that the expression of the matrix metalloproteinase 1 (dMMP1) gene reproducibly increased in motor neurons in an age-dependent manner. Modulation of physiological aging also altered the rate of dMMP1 expression, validating dMMP1 expression as a bona fide aging biomarker for motor neurons. Temporally controlled overexpression of dMMP1 specifically in motor neurons was sufficient to induce deficits in climbing behavior and cause a decrease in neurotransmitter release at neuromuscular synapses. These deficits were reversible if the dMMP1 expression was shut off again immediately after the onset of motor dysfunction. Additionally, repression of dMMP1 enzymatic activity via overexpression of a tissue inhibitor of metalloproteinases delayed the onset of age-dependent motor dysfunction. MMPs are required for proper tissue architecture during development. Our results support the idea that matrix metalloproteinase 1 is acting as a downstream effector of antagonistic pleiotropy in motor neurons and is necessary for proper development, but deleterious when reactivated at an advanced age. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Xcat, a novel mouse model for Nance-Horan syndrome inhibits expression of the cytoplasmic-targeted Nhs1 isoform.

PubMed

Huang, Kristen M; Wu, Junhua; Duncan, Melinda K; Moy, Chris; Dutra, Amalia; Favor, Jack; Da, Tong; Stambolian, Dwight

2006-01-15

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X-chromosome. Sequence and FISH analysis of the X-chromosome region containing the Xcat mutation reveal a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT-PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wild-type neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1-GFP fusion proteins were done to determine whether the amino acids encoded by exon 1 were critical for protein localization. We found the presence of Nhs1 exon 1 critical for localization of the fusion protein to the cytoplasm, whereas fusion proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1 containing isoform results in the abnormal phenotype of Xcat.

Differential effects of social isolation in adolescent and adult mice on behavior and cortical gene expression.

PubMed

Lander, Sharon S; Linder-Shacham, Donna; Gaisler-Salomon, Inna

2017-01-01

Intact function of the medial prefrontal cortex (mPFC) function relies on proper development of excitatory and inhibitory neuronal populations and on integral myelination processes. Social isolation (SI) affects behavior and brain circuitry in adulthood, but previous rodent studies typically induced prolonged (post-weaning) exposure and failed to directly compare between the effects of SI in adolescent and adulthood. Here, we assessed the impact of a 3-week SI period, starting in mid-adolescence (around the onset of puberty) or adulthood, on a wide range of behaviors in adult male mice. Additionally, we asked whether adolescent SI would differentially affect the expression of excitatory and inhibitory neuronal markers and myelin-related genes in mPFC. Our findings indicate that mid-adolescent or adult SI increase anxiogenic behavior and locomotor activity. However, SI in adolescence uniquely affects the response to the psychotomimetic drug amphetamine, social and novelty exploration and performance in reversal and attentional set shifting tasks. Furthermore, adolescent but not adult SI increased the expression of glutamate markers in the adult mPFC. Our results imply that adolescent social deprivation is detrimental for normal development and may be particularly relevant to the investigation of developmental psychopathology. Copyright © 2016 Elsevier B.V. All rights reserved.

Plant twitter: ligands under 140 amino acids enforcing stomatal patterning.

PubMed

Rychel, Amanda L; Peterson, Kylee M; Torii, Keiko U

2010-05-01

Stomata are an essential land plant innovation whose patterning and density are under genetic and environmental control. Recently, several putative ligands have been discovered that influence stomatal density, and they all belong to the epidermal patterning factor-like family of secreted cysteine-rich peptides. Two of these putative ligands, EPF1 and EPF2, are expressed exclusively in the stomatal lineage cells and negatively regulate stomatal density. A third, EPFL6 or CHALLAH, is also a negative regulator of density, but is expressed subepidermally in the hypocotyl. A fourth, EPFL9 or STOMAGEN, is expressed in the mesophyll tissues and is a positive regulator of density. Genetic evidence suggests that these ligands may compete for the same receptor complex. Proper stomatal patterning is likely to be an intricate process involving ligand competition, regional specificity, and communication between tissue layers. EPFL-family genes exist in the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, and rice, Oryza sativa, and their sequence analysis yields several genes some of which are related to EPF1, EPF2, EPFL6, and EPFL9. Presence of these EPFL family members in the basal land plants suggests an exciting hypothesis that the genetic components for stomatal patterning originated early in land plant evolution.

Integration of multimodal RNA-seq data for prediction of kidney cancer survival

PubMed Central

Schwartzi, Matt; Parkl, Martin; Phanl, John H.; Wang., May D.

2016-01-01

Kidney cancer is of prominent concern in modern medicine. Predicting patient survival is critical to patient awareness and developing a proper treatment regimens. Previous prediction models built upon molecular feature analysis are limited to just gene expression data. In this study we investigate the difference in predicting five year survival between unimodal and multimodal analysis of RNA-seq data from gene, exon, junction, and isoform modalities. Our preliminary findings report higher predictive accuracy-as measured by area under the ROC curve (AUC)-for multimodal learning when compared to unimodal learning with both support vector machine (SVM) and k-nearest neighbor (KNN) methods. The results of this study justify further research on the use of multimodal RNA-seq data to predict survival for other cancer types using a larger sample size and additional machine learning methods. PMID:27532026

Evolution of robustness to damage in artificial 3-dimensional development.

PubMed

Joachimczak, Michał; Wróbel, Borys

2012-09-01

GReaNs is an Artificial Life platform we have built to investigate the general principles that guide evolution of multicellular development and evolution of artificial gene regulatory networks. The embryos develop in GReaNs in a continuous 3-dimensional (3D) space with simple physics. The developmental trajectories are indirectly encoded in linear genomes. The genomes are not limited in size and determine the topology of gene regulatory networks that are not limited in the number of nodes. The expression of the genes is continuous and can be modified by adding environmental noise. In this paper we evolved development of structures with a specific shape (an ellipsoid) and asymmetrical pattering (a 3D pattern inspired by the French flag problem), and investigated emergence of the robustness to damage in development and the emergence of the robustness to noise. Our results indicate that both types of robustness are related, and that including noise during evolution promotes higher robustness to damage. Interestingly, we have observed that some evolved gene regulatory networks rely on noise for proper behaviour. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The DEAD-box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish.

PubMed

Zhang, Linlin; Yang, Yuxi; Li, Beibei; Scott, Ian C; Lou, Xin

2018-04-23

RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism, including RNA synthesis, processing and degradation. The function and mechanism of action of most of these helicases in animal development and human disease remain largely unexplored. In a zebrafish mutagenesis screen to identify genes essential for heart development we identified a mutant that disrupts the gene encoding the RNA helicase DEAD-box 39ab ( ddx39ab ). Homozygous ddx39ab mutant embryos exhibit profound cardiac and trunk muscle dystrophy, along with lens abnormalities, caused by abrupt terminal differentiation of cardiomyocyte, myoblast and lens fiber cells. Loss of ddx39ab hindered splicing of mRNAs encoding epigenetic regulatory factors, including members of the KMT2 gene family, leading to misregulation of structural gene expression in cardiomyocyte, myoblast and lens fiber cells. Taken together, these results show that Ddx39ab plays an essential role in establishment of the proper epigenetic status during differentiation of multiple cell lineages. © 2018. Published by The Company of Biologists Ltd.

Editing the Neuronal Genome: a CRISPR View of Chromatin Regulation in Neuronal Development, Function, and Plasticity

PubMed Central

Yang, Marty G.; West, Anne E.

2016-01-01

The dynamic orchestration of gene expression is crucial for the proper differentiation, function, and adaptation of cells. In the brain, transcriptional regulation underlies the incredible diversity of neuronal cell types and contributes to the ability of neurons to adapt their function to the environment. Recently, novel methods for genome and epigenome editing have begun to revolutionize our understanding of gene regulatory mechanisms. In particular, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be a particularly accessible and adaptable technique for genome engineering. Here, we review the use of CRISPR/Cas9 in neurobiology and discuss how these studies have advanced understanding of nervous system development and plasticity. We cover four especially salient applications of CRISPR/Cas9: testing the consequences of enhancer mutations, tagging genes and gene products for visualization in live cells, directly activating or repressing enhancers in vivo, and manipulating the epigenome. In each case, we summarize findings from recent studies and discuss evolving adaptations of the method. PMID:28018138

Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development

PubMed Central

Sehra, Bhupinder; Franks, Robert G.

2017-01-01

In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve. PMID:29085379

[Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].

PubMed

Wang, Yue-qi; Zhou, Yan; Cheng, Na; Chen, Mu-xin; Ai, Lin; Liu, Yu-hua; Zhang, Jian-guo; Luo, Jia-jun; Xu, Xue-nian

2015-04-01

To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA. The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P < 0.01). The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.

Characterization of antennal sensilla, larvae morphology and olfactory genes of Melipona scutellaris stingless bee

PubMed Central

de Carvalho, Washington João; Fujimura, Patrícia Tieme; Bonetti, Ana Maria; Goulart, Luiz Ricardo; Cloonan, Kevin; da Silva, Neide Maria; Araújo, Ester Cristina Borges; Ueira-Vieira, Carlos; Leal, Walter S.

2017-01-01

There is growing evidence in the literature suggesting that caste differentiation in the stingless bee, Melipona scutellaris, and other bees in the genus Melipona, is triggered by environmental signals, particularly a primer pheromone. With the proper amount of food and a chemical stimulus, 25% of females emerge as queens, in agreement with a long-standing “two loci/two alleles model” proposed in the 1950s. We surmised that these larvae must be equipped with an olfactory system for reception of these chemical signals. Here we describe for the first time the diversity of antennal sensilla in adults and the morphology of larvae of M. scutellaris. Having found evidence for putative olfactory sensilla in larvae, we next asked whether olfactory proteins were expressed in larvae. Since the molecular basis of M. scutellaris is still unknown, we cloned olfactory genes encoding chemosensory proteins (CSP) and odorant-binding proteins (OBPs) using M. scutellaris cDNA template and primers designed on the basis CSPs and OBPs previously reported from the European honeybee, Apis mellifera. We cloned two CSP and two OBP genes and then attempted to express the proteins encoded by these genes. With a recombinant OBP, MscuOBP8, and a combinatorial single-chain variable fragment antibody library, we generated anti-MscuOBP8 monoclonal antibody. By immunohistochemistry we demonstrated that the anti-MscuOBP8 binds specifically to the MscuOBP8. Next, we found evidence that MscuOBP8 is expressed in M. scutellaris larvae and it is located in the mandibular region, thus further supporting the hypothesis of olfactory function in immature stages. Lastly, molecular modeling suggests that MscuOBP8 may function as a carrier of primer pheromones or other ligands. PMID:28423045

Characterization of antennal sensilla, larvae morphology and olfactory genes of Melipona scutellaris stingless bee.

PubMed

Carvalho, Washington João de; Fujimura, Patrícia Tieme; Bonetti, Ana Maria; Goulart, Luiz Ricardo; Cloonan, Kevin; da Silva, Neide Maria; Araújo, Ester Cristina Borges; Ueira-Vieira, Carlos; Leal, Walter S

2017-01-01

There is growing evidence in the literature suggesting that caste differentiation in the stingless bee, Melipona scutellaris, and other bees in the genus Melipona, is triggered by environmental signals, particularly a primer pheromone. With the proper amount of food and a chemical stimulus, 25% of females emerge as queens, in agreement with a long-standing "two loci/two alleles model" proposed in the 1950s. We surmised that these larvae must be equipped with an olfactory system for reception of these chemical signals. Here we describe for the first time the diversity of antennal sensilla in adults and the morphology of larvae of M. scutellaris. Having found evidence for putative olfactory sensilla in larvae, we next asked whether olfactory proteins were expressed in larvae. Since the molecular basis of M. scutellaris is still unknown, we cloned olfactory genes encoding chemosensory proteins (CSP) and odorant-binding proteins (OBPs) using M. scutellaris cDNA template and primers designed on the basis CSPs and OBPs previously reported from the European honeybee, Apis mellifera. We cloned two CSP and two OBP genes and then attempted to express the proteins encoded by these genes. With a recombinant OBP, MscuOBP8, and a combinatorial single-chain variable fragment antibody library, we generated anti-MscuOBP8 monoclonal antibody. By immunohistochemistry we demonstrated that the anti-MscuOBP8 binds specifically to the MscuOBP8. Next, we found evidence that MscuOBP8 is expressed in M. scutellaris larvae and it is located in the mandibular region, thus further supporting the hypothesis of olfactory function in immature stages. Lastly, molecular modeling suggests that MscuOBP8 may function as a carrier of primer pheromones or other ligands.

CSP41b, a protein identified via FOX hunting using Eutrema salsugineum cDNAs, improves heat and salinity stress tolerance in transgenic Arabidopsis thaliana.

PubMed

Ariga, Hirotaka; Tanaka, Tomoko; Ono, Hirokazu; Sakata, Yoichi; Hayashi, Takahisa; Taji, Teruaki

2015-08-14

Eutrema salsugineum (also known as Thellungiella salsuginea and formerly Thellungiella halophila), a species closely related to Arabidopsis thaliana, shows tolerance not only to salt stress, but also to chilling, freezing, and high temperatures. To identify genes responsible for stress tolerance, we conducted Full-length cDNA Over-eXpressing gene (FOX) hunting among a collection of E. salsugineum cDNAs that were stress-induced according to gene ontology analysis or over-expressed in E. salsugineum compared with A. thaliana. We identified E. salsugineum CSP41b (chloroplast stem-loop-binding protein of 41 kDa; also known as CRB, chloroplast RNA binding; named here as EsCSP41b) as a gene that can confer heat and salinity stress tolerance on A. thaliana. A. thaliana CSP41b is reported to play an important role in the proper functioning of the chloroplast: the atcsp41b mutant is smaller and paler than wild-type plants and shows altered chloroplast morphology and photosynthetic performance. We observed that AtCSP41b-overexpressing transgenic A. thaliana lines also exhibited marked heat tolerance and significant salinity stress tolerance. The EsCSP41b-overexpressing transgenic A. thaliana lines showed significantly higher photosynthesis activity than wild-type plants not only under normal growth conditions but also under heat stress. In wild-type plants, the expression levels of both EsCSP41b and AtCSP41b were significantly reduced under heat or salinity stress. We conclude that maintenance of CSP41b expression under abiotic stresses may alleviate photoinhibition and improve survival under such stresses. Copyright © 2015 Elsevier Inc. All rights reserved.

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance.

PubMed

Gruner, Katrin; Zeier, Tatyana; Aretz, Christina; Zeier, Jürgen

2018-04-16

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR-defective mlo2 mutants were still competent in systemically increasing the levels of the SAR-activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR-related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA- or Pip-inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

The dlx5a/dlx6a Genes Play Essential Roles in the Early Development of Zebrafish Median Fin and Pectoral Structures

PubMed Central

Heude, Églantine; Shaikho, Sarah; Ekker, Marc

2014-01-01

The Dlx5 and Dlx6 genes encode homeodomain transcription factors essential for the proper development of limbs in mammalian species. However, the role of their teleost counterparts in fin development has received little attention. Here, we show that dlx5a is an early marker of apical ectodermal cells of the pectoral fin buds and of the median fin fold, but also of cleithrum precursor cells during pectoral girdle development. We propose that early median fin fold establishment results from the medial convergence of dlx5a-expressing cells at the lateral edges of the neural keel. Expression analysis also shows involvement of dlx5a during appendage skeletogenesis. Using morpholino-mediated knock down, we demonstrate that disrupted dlx5a/6a function results in pectoral fin agenesis associated with misexpression of bmp4, fgf8a, and1 and msx genes. In contrast, the median fin fold presents defects in mesenchymal cell migration and actinotrichia formation, whereas the initial specification seems to occur normally. Our results demonstrate that the dlx5a/6a genes are essential for the induction of pectoral fin outgrowth, but are not required during median fin fold specification. The dlx5a/6a knock down also causes a failure of cleithrum formation associated with a drastic loss of runx2b and col10a1 expression. The data indicate distinct requirements for dlx5a/6a during median and pectoral fin development suggesting that initiation of unpaired and paired fin formation are not directed through the same molecular mechanisms. Our results refocus arguments on the mechanistic basis of paired appendage genesis during vertebrate evolution. PMID:24858471

The Ornithine Decarboxylase Gene Is Essential for Cell Survival during Early Murine Development

PubMed Central

Pendeville, Hélène; Carpino, Nick; Marine, Jean-Christophe; Takahashi, Yutaka; Muller, Marc; Martial, Joseph A.; Cleveland, John L.

2001-01-01

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation. PMID:11533243

Phenotype comparison confirms ZMYND11 as a critical gene for 10p15.3 microdeletion syndrome.

PubMed

Tumiene, Birute; Čiuladaitė, Ž; Preikšaitienė, E; Mameniškienė, R; Utkus, A; Kučinskas, V

2017-11-01

Proper epigenetic regulation processes are crucial in the normal development of the human brain. An ever-increasing group of neurodevelopmental disorders due to derangements of epigenetic regulation involve both microdeletion and monogenic syndromes. Some of these syndromes have overlapping clinical phenotypes due to haploinsufficiency-sensitive genes involved in microdeletions. It was shown recently that the ZMYND11 gene has important functions in epigenetic regulation as an unconventional transcription co-repressor of highly expressed genes, possibly acting in the repression of cryptic transcription from gene bodies. The aim of our study was to compare the clinical phenotypes of patients with 10p15.3 deletions with the phenotypes of patients with loss-of-function ZMYND11 mutations. The results of our study further confirm that the ZMYND11 gene is the critical gene for the clinical phenotype of 10p15.3 microdeletion involving the terminal ~4 Mb of chromosome 10p. In addition, accumulating clinical data allow for further characterisation of this syndrome, including neurodevelopmental disorder, characteristic dysmorphic features and some other more frequent symptoms, such as behavioural disturbances, hypotonia, seizures, low birth weight, short stature in those older than 10 years of age, genitourinary malformations and recurrent infections.

Zinc deficiency induces vascular pro-inflammatory parameters associated with NF-kappaB and PPAR signaling.

PubMed

Shen, Huiyun; Oesterling, Elizabeth; Stromberg, Arnold; Toborek, Michal; MacDonald, Ruth; Hennig, Bernhard

2008-10-01

Marginal intake of dietary zinc can be associated with increased risk of cardiovascular diseases. In the current study we hypothesized that vascular dysfunction and associated inflammatory events are activated during a zinc deficient state. We tested this hypothesis using both vascular endothelial cells and mice lacking the functional LDL-receptor gene. Zinc deficiency increased oxidative stress and NF-kappaB DNA binding activity, and induced COX-2 and E-selectin gene expression, as well as monocyte adhesion in cultured endothelial cells. The NF-kappaB inhibitor CAPE significantly reduced the zinc deficiency-induced COX-2 expression, suggesting regulation through NF-kappaB signaling. PPAR can inhibit NF-kappaB signaling, and our previous data have shown that PPAR transactivation activity requires adequate zinc. Zinc deficiency down-regulated PPARalpha expression in cultured endothelial cells. Furthermore, the PPARgamma agonist rosiglitazone was unable to inhibit the adhesion of monocytes to endothelial cells during zinc deficiency, an event which could be reversed by zinc supplementation. Our in vivo data support the importance of PPAR dysregulation during zinc deficiency. For example, rosiglitazone induced inflammatory genes (e.g., MCP-1) only during zinc deficiency, and adequate zinc was required for rosiglitazone to down-regulate pro-inflammatory markers such as iNOS. In addition, rosiglitazone increased IkappaBalpha protein expression only in zinc adequate mice. Finally, plasma data from LDL-R-deficient mice suggest an overall pro-inflammatory environment during zinc deficiency and support the concept that zinc is required for proper anti-inflammatory or protective functions of PPAR. These studies suggest that zinc nutrition can markedly modulate mechanisms of the pathology of inflammatory diseases such as atherosclerosis.

Epigenetic regulation and chromatin remodeling in learning and memory.

PubMed

Kim, Somi; Kaang, Bong-Kiun

2017-01-13

Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms.

Islet 1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood

PubMed Central

Nasif, Sofia; de Souza, Flavio S. J.; González, Laura E.; Yamashita, Miho; Orquera, Daniela P.; Rubinstein, Marcelo

2015-01-01

Food intake and body weight regulation depend on proper expression of the proopiomelanocortin gene (Pomc) in a group of neurons located in the mediobasal hypothalamus of all vertebrates. These neurons release POMC-encoded melanocortins, which are potent anorexigenic neuropeptides, and their absence from mice or humans leads to hyperphagia and severe obesity. Although the pathophysiology of hypothalamic POMC neurons is well understood, the genetic program that establishes the neuronal melanocortinergic phenotype and maintains a fully functional neuronal POMC phenotype throughout adulthood remains unknown. Here, we report that the early expression of the LIM-homeodomain transcription factor Islet 1 (ISL1) in the developing hypothalamus promotes the terminal differentiation of melanocortinergic neurons and is essential for hypothalamic Pomc expression since its initial onset and throughout the entire lifetime. We detected ISL1 in the prospective hypothalamus just before the onset of Pomc expression and, from then on, Pomc and Isl1 coexpress. ISL1 binds in vitro and in vivo to critical homeodomain binding DNA motifs present in the neuronal Pomc enhancers nPE1 and nPE2, and mutations of these sites completely disrupt the ability of these enhancers to drive reporter gene expression to hypothalamic POMC neurons in transgenic mice and zebrafish. ISL1 is necessary for hypothalamic Pomc expression during mouse and zebrafish embryogenesis. Furthermore, conditional Isl1 inactivation from POMC neurons impairs Pomc expression, leading to hyperphagia and obesity. Our results demonstrate that ISL1 specifies the identity of hypothalamic melanocortin neurons and is required for melanocortin-induced satiety and normal adiposity throughout the entire lifespan. PMID:25825735

JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

PubMed Central

Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

2006-01-01

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

Interactive knowledge discovery and data mining on genomic expression data with numeric formal concept analysis.

PubMed

González-Calabozo, Jose M; Valverde-Albacete, Francisco J; Peláez-Moreno, Carmen

2016-09-15

Gene Expression Data (GED) analysis poses a great challenge to the scientific community that can be framed into the Knowledge Discovery in Databases (KDD) and Data Mining (DM) paradigm. Biclustering has emerged as the machine learning method of choice to solve this task, but its unsupervised nature makes result assessment problematic. This is often addressed by means of Gene Set Enrichment Analysis (GSEA). We put forward a framework in which GED analysis is understood as an Exploratory Data Analysis (EDA) process where we provide support for continuous human interaction with data aiming at improving the step of hypothesis abduction and assessment. We focus on the adaptation to human cognition of data interpretation and visualization of the output of EDA. First, we give a proper theoretical background to bi-clustering using Lattice Theory and provide a set of analysis tools revolving around [Formula: see text]-Formal Concept Analysis ([Formula: see text]-FCA), a lattice-theoretic unsupervised learning technique for real-valued matrices. By using different kinds of cost structures to quantify expression we obtain different sequences of hierarchical bi-clusterings for gene under- and over-expression using thresholds. Consequently, we provide a method with interleaved analysis steps and visualization devices so that the sequences of lattices for a particular experiment summarize the researcher's vision of the data. This also allows us to define measures of persistence and robustness of biclusters to assess them. Second, the resulting biclusters are used to index external omics databases-for instance, Gene Ontology (GO)-thus offering a new way of accessing publicly available resources. This provides different flavors of gene set enrichment against which to assess the biclusters, by obtaining their p-values according to the terminology of those resources. We illustrate the exploration procedure on a real data example confirming results previously published. The GED analysis problem gets transformed into the exploration of a sequence of lattices enabling the visualization of the hierarchical structure of the biclusters with a certain degree of granularity. The ability of FCA-based bi-clustering methods to index external databases such as GO allows us to obtain a quality measure of the biclusters, to observe the evolution of a gene throughout the different biclusters it appears in, to look for relevant biclusters-by observing their genes and what their persistence is-to infer, for instance, hypotheses on their function.

Cdk5 Regulates Activity-Dependent Gene Expression and Dendrite Development.

PubMed

Liang, Zhuoyi; Ye, Tao; Zhou, Xiaopu; Lai, Kwok-On; Fu, Amy K Y; Ip, Nancy Y

2015-11-11

The proper growth and arborization of dendrites in response to sensory experience are essential for neural connectivity and information processing in the brain. Although neuronal activity is important for sculpting dendrite morphology, the underlying molecular mechanisms are not well understood. Here, we report that cyclin-dependent kinase 5 (Cdk5)-mediated transcriptional regulation is a key mechanism that controls activity-dependent dendrite development in cultured rat neurons. During membrane depolarization, Cdk5 accumulates in the nucleus to regulate the expression of a subset of genes, including that of the neurotrophin brain-derived neurotrophic factor, for subsequent dendritic growth. Furthermore, Cdk5 function is mediated through the phosphorylation of methyl-CpG-binding protein 2, a key transcriptional repressor that is mutated in the mental disorder Rett syndrome. These findings collectively suggest that the nuclear import of Cdk5 is crucial for activity-dependent dendrite development by regulating neuronal gene transcription during neural development. Neural activity directs dendrite development through the regulation of gene transcription. However, how molecular signals link extracellular stimuli to the transcriptional program in the nucleus remains unclear. Here, we demonstrate that neuronal activity stimulates the translocation of the kinase Cdk5 from the cytoplasmic compartment into the nucleus; furthermore, the nuclear localization of Cdk5 is required for dendrite development in cultured neurons. Genome-wide transcriptome analysis shows that Cdk5 deficiency specifically disrupts activity-dependent gene transcription of bdnf. The action of Cdk5 is mediated through the modulation of the transcriptional repressor methyl-CpG-binding protein 2. Therefore, this study elucidates the role of nuclear Cdk5 in the regulation of activity-dependent gene transcription and dendritic growth. Copyright © 2015 the authors 0270-6474/15/3515127-08$15.00/0.

Comparative MicroRNA Expression Patterns in Fibroblasts after Low and High Doses of Low-LET Radiation Exposure

NASA Technical Reports Server (NTRS)

Maes, Olivier C.; Xu, Suying; Hada, Megumi; Wu, Honglu; Wang, Eugenia

2007-01-01

Exposure to ionizing radiation causes DNA damage to cells, and provokes a plethora of cellular responses controlled by unique gene-directed signaling pathways. MicroRNAs (miRNAs) are small (22-nucleotide), non-coding RNAs which functionally silence gene expression by either degrading the messages or inhibiting translation. Here we investigate radiation-dependent changes in these negative regulators by comparing the expression patterns of all 462 known human miRNAs in fibroblasts, after exposure to low (0.1 Gy) or high (2 Gy) doses of X-rays at 30 min, 2, 6 and 24 hrs post-treatment. The expression patterns of microRNAs after low and high doses of radiation show a similar qualitative down-regulation trend at early (0.5 hr) and late (24 hr) time points, with a quantitatively steeper slope following the 2 Gy exposures. Interestingly, an interruption of this downward trend is observed after the 2 Gy exposure, i.e. a significant up-regulation of microRNAs at 2 hrs, then reverting to the downward trend by 6 hrs; this interruption at the intermediate time point was not observed with the 0.1 Gy exposure. At the early time point (0.5 hr), candidate gene targets of selected down-regulated microRNAs, common to both 0.1 and 2 Gy exposures, were those functioning in chromatin remodeling. Candidate target genes of unique up-regulated microRNAs seen at a 2 hr intermediate time point, after the 2 Gy exposure only, are those involved in cell death signaling. Finally, putative target genes of down-regulated microRNAs seen at the late (24 hr) time point after either doses of radiation are those involved in the up-regulation of DNA repair, cell signaling and homeostasis. Thus we hypothesize that after radiation exposure, microRNAs acting as hub negative regulators for unique signaling pathways needed to be down-regulated so as to de-repress their target genes for the proper cellular responses, including DNA repair and cell maintenance. The unique microRNAs up-regulated at 2 hr after 2 Gy suggest the cellular response to functionally suppress the apoptotic death signaling reflex after exposure to high dose radiation. Further analyses with transcriptome and global proteomic profiling will validate the reciprocal expression of signature microRNAs selected in our radiation-exposed cells, and their candidate target gene families, and test our hypothesis that unique radiation-specific microRNAs are keys in governing signaling responses for damage control of this environmental hazard.

Gene Cloning and Expression of Cellulase of Bacillus amyloliquefaciens Isolated from the Cecum of Goose.

PubMed

Sun, Linghong; Cao, Jiangyan; Liu, Ying; Wang, Junjie; Guo, Panpan; Wang, Zaigui

2017-01-02

A kind of bacteria secreting cellulase and showing probiotic attributes was isolated from the cecum of goose and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens S1. In vitro assays, the enzymatic activity of the strain was determined by the reducing-sugar method, and the proper culture conditions of producing cellulase and some properties of the cellulase were investigated. The cultural mixture of the bacteria had a high cellulase activity of 1.25 U/mL. In order to improve the utilization rate of the cellulase, some properties of the cellulase were studied. The best reaction pH of the enzymes was 7.0 and the optimum reaction temperature was 60°C. The enzyme was a kind of neutral cellulase that possessing strong resistance against heat and acidity. It showed high activity to absorbent cotton, soybean meal, and filter paper. Meanwhile, a gene encoding a kind of cellulase was cloned and prokaryotic expressed in Escherichia coli. The gene had 1500 bp in length, encoding a protein of 55 kDa, which was confirmed by SDS-PAGE and Western blotting. This study explored the possibility of degrading ability of bacteria with its probiotic attributes to enhance digestibility of the feed and gut health of animal. It also provided some basis for its further functional analysis and practical application as a microbial preparation for the breeding.

EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

PubMed

Zjablovskaja, Polina; Kardosova, Miroslava; Danek, Petr; Angelisova, Pavla; Benoukraf, Touati; Wurm, Alexander A; Kalina, Tomas; Sian, Stephanie; Balastik, Martin; Delwel, Ruud; Brdicka, Tomas; Tenen, Daniel G; Behre, Gerhard; Fiore, Fréderic; Malissen, Bernard; Horejsi, Vaclav; Alberich-Jorda, Meritxell

2017-04-01

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli

PubMed Central

Easton, Donna M.; Totsika, Makrina; Allsopp, Luke P.; Phan, Minh-Duy; Idris, Adi; Wurpel, Daniël J.; Sherlock, Orla; Zhang, Bing; Venturini, Carola; Beatson, Scott A.; Mahony, Timothy J.; Cobbold, Rowland N.; Schembri, Mark A.

2011-01-01

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence. PMID:21687429

Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis

PubMed Central

Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Chris; Wu, Yan; Costa, Mike; de Sauvage, Frederic J.; Klein, Ophir D.

2015-01-01

Summary Proper organ homeostasis requires tight control of adult stem cells and differentiation through integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a de-repression of the Wnt signaling pathway, leading to mis-expression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

PubMed Central

Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A. A.; Baumann, Marcus E. M.; Selmer, Thorsten

2013-01-01

Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the presented cloning platform, the system might represent an important step towards new routes in synthetic biology. PMID:23861944

Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm.

PubMed

Lu, Junjie; Baccei, Anna; Lummertz da Rocha, Edroaldo; Guillermier, Christelle; McManus, Sean; Finney, Lydia A; Zhang, Cheng; Steinhauser, Matthew L; Li, Hu; Lerou, Paul H

2018-04-01

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Role of H1 Linker Histones in Mammalian Development and Stem Cell Differentiation

PubMed Central

Pan, Chenyi; Fan, Yuhong

2016-01-01

H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. PMID:26689747

[Molecular and immunohistochemical diagnostics in melanoma].

PubMed

Schilling, B; Griewank, K G

2016-07-01

To provide appropriate therapy and follow-up to patients with malignant melanoma, proper diagnostics are of critical importance. Targeted therapy of advanced melanoma is based on the molecular genetic analyses of tumor tissue. In addition, sequencing of genes and other genetic approaches can provide insight into the origin of melanocytic tumors and can aid in distinguishing benign from malignant lesions. In this regard, spizoid neoplasms remain a challenging entity. Aside from genetic analyses of tumor tissue, immunohistochemistry remains an essential tool in melanoma diagnostics and TNM classification. With new immunotherapies being approved for advanced melanoma, immunohistochemistry to determine PD-L1 expression has gained clinical interest. While PD-L1 expression is associated with response to PD-1 blockade, a substantial number of patients without PD-L1 expression can still experience tumor remission upon treatment. In this review, current and future developments in melanoma diagnostics with regard to molecular genetics and immunohistochemistry are summarized. The utilization of such analyses in clinical decision making is also discussed.

Regulation of mRNA abundance by polypyrimidine tract-binding protein-controlled alternate 5' splice site choice.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-11-01

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

PubMed

Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

2014-11-01

Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

PubMed

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana

PubMed Central

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts. PMID:26451798

Cell wall invertase as a regulator in determining sequential development of endosperm and embryo through glucose signaling early in seed development.

PubMed

Wang, Lu; Liao, Shengjin; Ruan, Yong-Ling

2013-01-01

Seed development depends on coordination among embryo, endosperm and seed coat. Endosperm undergoes nuclear division soon after fertilization, whereas embryo remains quiescent for a while. Such a developmental sequence is of great importance for proper seed development. However, the underlying mechanism remains unclear. Recent results on the cellular domain- and stage-specific expression of invertase genes in cotton and Arabidopsis revealed that cell wall invertase may positively and specifically regulate nuclear division of endosperm after fertilization, thereby playing a role in determining the sequential development of endosperm and embryo, probably through glucose signaling.

Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells.

PubMed

Ebeid, Michael; Sripal, Prashanth; Pecka, Jason; Beisel, Kirk W; Kwan, Kelvin; Soukup, Garrett A

2017-01-01

Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.

[Correlation of codon biases and potential secondary structures with mRNA translation efficiency in unicellular organisms].

PubMed

Vladimirov, N V; Likhoshvaĭ, V A; Matushkin, Iu G

2007-01-01

Gene expression is known to correlate with degree of codon bias in many unicellular organisms. However, such correlation is absent in some organisms. Recently we demonstrated that inverted complementary repeats within coding DNA sequence must be considered for proper estimation of translation efficiency, since they may form secondary structures that obstruct ribosome movement. We have developed a program for estimation of potential coding DNA sequence expression in defined unicellular organism using its genome sequence. The program computes elongation efficiency index. Computation is based on estimation of coding DNA sequence elongation efficiency, taking into account three key factors: codon bias, average number of inverted complementary repeats, and free energy of potential stem-loop structures formed by the repeats. The influence of these factors on translation is numerically estimated. An optimal proportion of these factors is computed for each organism individually. Quantitative translational characteristics of 384 unicellular organisms (351 bacteria, 28 archaea, 5 eukaryota) have been computed using their annotated genomes from NCBI GenBank. Five potential evolutionary strategies of translational optimization have been determined among studied organisms. A considerable difference of preferred translational strategies between Bacteria and Archaea has been revealed. Significant correlations between elongation efficiency index and gene expression levels have been shown for two organisms (S. cerevisiae and H. pylori) using available microarray data. The proposed method allows to estimate numerically the coding DNA sequence translation efficiency and to optimize nucleotide composition of heterologous genes in unicellular organisms. http://www.mgs.bionet.nsc.ru/mgs/programs/eei-calculator/.

Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae.

PubMed

Klauser, Benedikt; Rehm, Charlotte; Summerer, Daniel; Hartig, Jörg S

2015-01-01

Synthetic RNA-based switches are a growing class of genetic controllers applied in synthetic biology to engineer cellular functions. In this chapter, we detail a protocol for the selection of posttranscriptional controllers of gene expression in yeast using the Schistosoma mansoni hammerhead ribozyme as a central catalytic unit. Incorporation of a small molecule-sensing aptamer domain into the ribozyme renders its activity ligand-dependent. Aptazymes display numerous advantages over conventional protein-based transcriptional controllers, namely, the use of little genomic space for encryption, their modular architecture allowing for easy reprogramming to new inputs, the physical linkage to the message to be controlled, and the ability to function without protein cofactors. Herein, we describe the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. We also highlight how to adapt the protocol to screen for switches responsive to other ligands. Reprogramming of the sensor unit and incorporation into any RNA of interest enables the fulfillment of a variety of regulatory functions. However, proper functioning of the aptazyme is largely dependent on optimal connection between the aptamer and the catalytic core. We obtained functional switches from a pool of variants carrying randomized connection sequences by an in vivo selection in MaV203 yeast cells that allows screening of a large sequence space of up to 1×10(9) variants. The protocol given explains how to construct aptazyme libraries, carry out the in vivo selection and characterize novel ON- and OFF-switches. © 2015 Elsevier Inc. All rights reserved.

Golgi Localized Barley MTP8 Proteins Facilitate Mn Transport

PubMed Central

Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

2014-01-01

Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species. PMID:25486417

ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice.

PubMed

Xu, Lei; Zhao, Hongyu; Ruan, Wenyuan; Deng, Minjuan; Wang, Fang; Peng, Jinrong; Luo, Jie; Chen, Zhixiang; Yi, Keke

2017-03-01

Root meristem activity determines root growth and root architecture and consequently affects water and nutrient uptake in plants. However, our knowledge about the regulation of root meristem activity in crop plants is very limited. Here, we report the isolation and characterization of a short root mutant in rice ( Oryza sativa ) with reduced root meristem activity. This root growth defect is caused by a mutation in ABNORMAL INFLORESCENCE MERISTEM1 ( AIM1 ), which encodes a 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in β-oxidation. The reduced root meristem activity of aim1 results from reduced salicylic acid (SA) levels and can be rescued by SA application. Furthermore, reduced SA levels are associated with reduced levels of reactive oxygen species (ROS) in aim1 , likely due to increased expression of redox and ROS-scavenging-related genes, whose increased expression is (at least in part) caused by reduced expression of the SA-inducible transcriptional repressors WRKY62 and WRKY76. Like SA, ROS application substantially increased root length and root meristem activity in aim1 These results suggest that AIM1 is required for root growth in rice due to its critical role in SA biosynthesis: SA maintains root meristem activity through promoting ROS accumulation by inducing the activity of WRKY transcriptional repressors, which repress the expression of redox and ROS-scavenging genes. © 2017 American Society of Plant Biologists. All rights reserved.

Neuropathological and transcriptomic characteristics of the aged brain

PubMed Central

Miller, Jeremy A; Guillozet-Bongaarts, Angela; Gibbons, Laura E; Postupna, Nadia; Renz, Anne; Beller, Allison E; Sunkin, Susan M; Ng, Lydia; Rose, Shannon E; Smith, Kimberly A; Szafer, Aaron; Barber, Chris; Bertagnolli, Darren; Bickley, Kristopher; Brouner, Krissy; Caldejon, Shiella; Chapin, Mike; Chua, Mindy L; Coleman, Natalie M; Cudaback, Eiron; Cuhaciyan, Christine; Dalley, Rachel A; Dee, Nick; Desta, Tsega; Dolbeare, Tim A; Dotson, Nadezhda I; Fisher, Michael; Gaudreault, Nathalie; Gee, Garrett; Gilbert, Terri L; Goldy, Jeff; Griffin, Fiona; Habel, Caroline; Haradon, Zeb; Hejazinia, Nika; Hellstern, Leanne L; Horvath, Steve; Howard, Kim; Howard, Robert; Johal, Justin; Jorstad, Nikolas L; Josephsen, Samuel R; Kuan, Chihchau L; Lai, Florence; Lee, Eric; Lee, Felix; Lemon, Tracy; Li, Xianwu; Marshall, Desiree A; Melchor, Jose; Mukherjee, Shubhabrata; Nyhus, Julie; Pendergraft, Julie; Potekhina, Lydia; Rha, Elizabeth Y; Rice, Samantha; Rosen, David; Sapru, Abharika; Schantz, Aimee; Shen, Elaine; Sherfield, Emily; Shi, Shu; Sodt, Andy J; Thatra, Nivretta; Tieu, Michael; Wilson, Angela M; Montine, Thomas J; Larson, Eric B; Bernard, Amy; Crane, Paul K; Ellenbogen, Richard G

2017-01-01

As more people live longer, age-related neurodegenerative diseases are an increasingly important societal health issue. Treatments targeting specific pathologies such as amyloid beta in Alzheimer’s disease (AD) have not led to effective treatments, and there is increasing evidence of a disconnect between traditional pathology and cognitive abilities with advancing age, indicative of individual variation in resilience to pathology. Here, we generated a comprehensive neuropathological, molecular, and transcriptomic characterization of hippocampus and two regions cortex in 107 aged donors (median = 90) from the Adult Changes in Thought (ACT) study as a freely-available resource (http://aging.brain-map.org/). We confirm established associations between AD pathology and dementia, albeit with increased, presumably aging-related variability, and identify sets of co-expressed genes correlated with pathological tau and inflammation markers. Finally, we demonstrate a relationship between dementia and RNA quality, and find common gene signatures, highlighting the importance of properly controlling for RNA quality when studying dementia. PMID:29120328

Rho-associated kinase is a therapeutic target in neuroblastoma.

PubMed

Dyberg, Cecilia; Fransson, Susanne; Andonova, Teodora; Sveinbjörnsson, Baldur; Lännerholm-Palm, Jessika; Olsen, Thale K; Forsberg, David; Herlenius, Eric; Martinsson, Tommy; Brodin, Bertha; Kogner, Per; Johnsen, John Inge; Wickström, Malin

2017-08-08

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN -driven neuroblastoma growth in TH- MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

Ncam1a and Ncam1b: two carriers of polysialic acid with different functions in the developing zebrafish nervous system.

PubMed

Langhauser, Melanie; Ustinova, Jana; Rivera-Milla, Eric; Ivannikov, Darja; Seidl, Carmen; Slomka, Christin; Finne, Jukka; Yoshihara, Yoshihiro; Bastmeyer, Martin; Bentrop, Joachim

2012-02-01

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.