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Vishwanath R Iyer

Publications and source records attributed to Vishwanath R Iyer.

14 recordsLinked to original sources
PubMed2006

FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.

DNA segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes from chromatin and are experimentally identified by their hypersensitivity to nucleases. Here we demonstrate a simple procedure for the isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type-specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays.

Cells, Cultured↗
PubMed2006

PI3K signaling and miRNA expression during the response of quiescent human fibroblasts to distinct proliferative stimuli.

BACKGROUND: Serum treatment of quiescent human dermal fibroblasts induces proliferation, coupled with a complex physiological response that is indicative of their normal role in wound-healing. However, it is not known to what extent such complex transcriptional events are specific to a given cell type and signal, and how these global changes are coordinately regulated. We have profiled the global transcriptional program of human fibroblasts from two different tissue sources to distinct growth stimuli, and identified a striking conservation in their gene-expression signatures. RESULTS: We found that the wound-healing program of gene expression was not specific to the response of dermal fibroblasts to serum but was regulated more broadly. However, there were specific differences among different stimuli with regard to signaling pathways that mediate these transcriptional programs. Our data suggest that the PI3-kinase pathway is differentially involved in mediating the responses of cells to serum as compared with individual peptide growth factors. Expression profiling indicated that let7 and other miRNAs with similar expression profiles may be involved in regulating the transcriptional program in response to proliferative signals. CONCLUSION: This study provides insights into how different stimuli use distinct as well as conserved signaling and regulatory mechanisms to mediate genome-wide transcriptional reprogramming during cell proliferation. Our results indicate that conservation of transcriptional programs and their regulation among different cell types may be much broader than previously appreciated.

Cell Line↗
PubMed2006

Toward understanding the genetics of alcohol drinking through transcriptome meta-analysis.

Much evidence from studies in humans and animals supports the hypothesis that alcohol addiction is a complex disease with both hereditary and environmental influences. Molecular determinants of excessive alcohol consumption are difficult to study in humans. However, several rodent models show a high or low degree of alcohol preference, which provides a unique opportunity to approach the molecular complexities underlying the genetic predisposition to drink alcohol. Microarray analyses of brain gene expression in three selected lines, and six isogenic strains of mice known to differ markedly in voluntary alcohol consumption provided >4.5 million data points for a meta-analysis. A total of 107 arrays were obtained and arranged into six experimental data sets, allowing the identification of 3,800 unique genes significantly and consistently changed between all models of high or low amounts of alcohol consumption. Several functional groups, including mitogen-activated protein kinase signaling and transcription regulation pathways, were found to be significantly overrepresented and may play an important role in establishing a high level of voluntary alcohol drinking in these mouse models. Data from the general meta-analysis was further filtered by a congenic strain microarray set, from which cis-regulated candidate genes for an alcohol preference quantitative trait locus on chromosome 9 were identified: Arhgef12, Carm1, Cryab, Cox5a, Dlat, Fxyd6, Limd1, Nicn1, Nmnat3, Pknox2, Rbp1, Sc5d, Scn4b, Tcf12, Vps11, and Zfp291 and four ESTs. The present study demonstrates the use of (i) a microarray meta-analysis to analyze a behavioral phenotype (in this case, alcohol preference) and (ii) a congenic strain for identification of cis regulation.

Alcohol Drinking↗
PubMed2006

Systematic profiling of cellular phenotypes with spotted cell microarrays reveals mating-pheromone response genes.

We have developed spotted cell microarrays for measuring cellular phenotypes on a large scale. Collections of cells are printed, stained for subcellular features, then imaged via automated, high-throughput microscopy, allowing systematic phenotypic characterization. We used this technology to identify genes involved in the response of yeast to mating pheromone. Besides morphology assays, cell microarrays should be valuable for high-throughput in situ hybridization and immunoassays, enabling new classes of genetic assays based on cell imaging.

Gene Expression Profiling↗
PubMed2005

Patterns of gene expression in the frontal cortex discriminate alcoholic from nonalcoholic individuals.

Alcohol dependence is characterized by tolerance, physical dependence, and craving. The neuroadaptations underlying these effects of chronic alcohol abuse are likely due to altered gene expression. Previous gene expression studies using human post-mortem brain demonstrated that several gene families were altered by alcohol abuse. However, most of these changes in gene expression were small. It is not clear if gene expression profiles have sufficient power to discriminate control from alcoholic individuals and how consistent gene expression changes are when a relatively large sample size is examined. In the present study, microarray analysis (approximately 47,000 elements) was performed on the superior frontal cortex of 27 individual human cases (14 well characterized alcoholics and 13 matched controls). A partial least squares statistical procedure was applied to identify genes with altered expression levels in alcoholics. We found that genes involved in myelination, ubiquitination, apoptosis, cell adhesion, neurogenesis, and neural disease showed altered expression levels. Importantly, genes involved in neurodegenerative diseases such as Alzheimer's disease were significantly altered suggesting a link between alcoholism and other neurodegenerative conditions. A total of 27 genes identified in this study were previously shown to be changed by alcohol abuse in previous studies of human post-mortem brain. These results revealed a consistent re-programming of gene expression in alcohol abusers that reliably discriminates alcoholic from non-alcoholic individuals.

Adult↗
PubMed2005

The heme activator protein Hap1 represses transcription by a heme-independent mechanism in Saccharomyces cerevisiae.

The yeast heme activator protein Hap1 binds to DNA and activates transcription of genes encoding functions required for respiration and for controlling oxidative damage, in response to heme. Hap1 contains a DNA-binding domain with a C6 zinc cluster motif, a coiled-coil dimerization element, typical of the members of the yeast Gal4 family, and an acidic activation domain. The regulation of Hap1 transcription-activating activity is controlled by two classes of Hap1 elements, repression modules (RPM1-3) and heme-responsive motifs (HRM1-7). Previous indirect evidence indicates that Hap1 may repress transcription directly. Here we show, by promoter analysis, by chromatin immunoprecipitation, and by electrophoretic mobility shift assay, that Hap1 binds directly to DNA and represses transcription of its own gene by at least 20-fold. We found that Hap1 repression of the HAP1 gene occurs independently of heme concentrations. While DNA binding is required for transcriptional repression by Hap1, deletion of Hap1 activation domain and heme-regulatory elements has varying effects on repression. Further, we found that repression by Hap1 requires the function of Hsp70 (Ssa), but not Hsp90. These results show that Hap1 binds to its own promoter and represses transcription in a heme-independent but Hsp70-dependent manner.

Chromatin↗
PubMed2005

Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP.

Shigella flexneri requires iron for survival, and the genes for iron uptake and homeostasis are regulated by the Fur protein. Microarrays were used to identify genes regulated by Fur and to study the physiological effects of iron availability in S. flexneri. These assays showed that the expression of genes involved in iron acquisition and acid response was induced by low-iron availability and by inactivation of fur. A fur null mutant was acid sensitive in media at pH 2.5, and acid sensitivity was also observed in the wild-type strain grown under iron-limiting conditions. Acid resistance of the fur mutant in minimal medium was restored by addition of glutamate during acid challenge, indicating that the glutamate-dependent acid resistance system was not defective. Inactivation of ryhB, a small regulatory RNA whose expression is repressed by Fur, restored acid resistance in the fur mutant, while overexpressing ryhB increased acid sensitivity in the wild-type strain. RyhB-regulated genes were identified by microarray analysis. The expression of one of the RyhB-repressed genes, ydeP, which encodes a putative oxidoreductase, suppressed acid sensitivity in the fur mutant. Furthermore, an S. flexneri ydeP mutant was defective for both glutamate-independent and glutamate-dependent acid resistance. The repression of ydeP by RyhB may be indirect, as real time polymerase chain reaction (PCR) experiments indicated that RyhB negatively regulates evgA, which encodes an activator of ydeP. These results demonstrate that the acid sensitivity defect of the S. flexneri fur mutant is due to repression of ydeP by RyhB, most likely via repression of evgA.

Bacterial Proteins↗
PubMed2004

Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment.

Identifying the chromosomal targets of transcription factors is important for reconstructing the transcriptional regulatory networks underlying global gene expression programs. We have developed an unbiased genomic method called sequence tag analysis of genomic enrichment (STAGE) to identify the direct binding targets of transcription factors in vivo. STAGE is based on high-throughput sequencing of concatemerized tags derived from target DNA enriched by chromatin immunoprecipitation. We first used STAGE in yeast to confirm that RNA polymerase III genes are the most prominent targets of the TATA-box binding protein. We optimized the STAGE protocol and developed analysis methods to allow the identification of transcription factor targets in human cells. We used STAGE to identify several previously unknown binding targets of human transcription factor E2F4 that we independently validated by promoter-specific PCR and microarray hybridization. STAGE provides a means of identifying the chromosomal targets of DNA-associated proteins in any sequenced genome.

Cells, Cultured↗
PubMed2004

Exploring the post-transcriptional RNA world with DNA microarrays.

Genomic approaches are valuable for understanding the complex layer of gene regulation that involves the control of RNA processing, localization and stability. Recent work provides a prime example of the power of unbiased microarray-based methods to discover unexpected functions for proteins in the RNA world. The challenges ahead relate to extending such approaches to larger genomes and to integrating this type of information with that generated by standard expression profiling.

Autoantigens↗
PubMed2004

Genome-wide analysis of the biology of stress responses through heat shock transcription factor.

Heat shock transcription factor (HSF) and the promoter heat shock element (HSE) are among the most highly conserved transcriptional regulatory elements in nature. HSF mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. While HSF is essential for cell viability in Saccharomyces cerevisiae, oogenesis and early development in Drosophila melanogaster, extended life span in Caenorhabditis elegans, and extraembryonic development and stress resistance in mammals, little is known about its full range of biological target genes. We used whole-genome analyses to identify virtually all of the direct transcriptional targets of yeast HSF, representing nearly 3% of the genomic loci. The majority of the identified loci are heat-inducibly bound by yeast HSF, and the target genes encode proteins that have a broad range of biological functions including protein folding and degradation, energy generation, protein trafficking, maintenance of cell integrity, small molecule transport, cell signaling, and transcription. This genome-wide identification of HSF target genes provides novel insights into the role of HSF in growth, development, disease, and aging and in the complex metabolic reprogramming that occurs in all cells in response to stress.

Animals↗
PubMed2004

Global role of TATA box-binding protein recruitment to promoters in mediating gene expression profiles.

The recruitment of TATA box-binding protein (TBP) to promoters is one of the rate-limiting steps during transcription initiation. However, the global importance of TBP recruitment in determining the absolute and changing levels of transcription across the genome is not known. We used a genomic approach to explore the relationship between TBP recruitment to promoters and global gene expression profiles in Saccharomyces cerevisiae. Our data indicate that first, RNA polymerase III promoters are the most prominent binding targets of TBP in vivo. Second, the steady-state transcript levels of genes throughout the genome are proportional to the occupancy of their promoters by TBP, and changes in the expression levels of these genes are closely correlated with changes in TBP recruitment to their promoters. Third, a consensus TATA element does not appear to be a major determinant of either TBP binding or gene expression throughout the genome. Our results indicate that the recruitment of TBP to promoters in vivo is of universal importance in determining gene expression levels in yeast, regardless of the nature of the core promoter or the type of activator or repressor that may mediate changes in transcription. The primary data reported here are available at http://www.iyerlab.org/tbp.

DNA, Fungal↗
PubMed2003

The Longhorn Array Database (LAD): an open-source, MIAME compliant implementation of the Stanford Microarray Database (SMD).

BACKGROUND: The power of microarray analysis can be realized only if data is systematically archived and linked to biological annotations as well as analysis algorithms. DESCRIPTION: The Longhorn Array Database (LAD) is a MIAME compliant microarray database that operates on PostgreSQL and Linux. It is a fully open source version of the Stanford Microarray Database (SMD), one of the largest microarray databases. LAD is available at http://www.longhornarraydatabase.org CONCLUSIONS: Our development of LAD provides a simple, free, open, reliable and proven solution for storage and analysis of two-color microarray data.

Database Management Systems↗
PubMed2002

Characterizing the physical genome.

The genome of an organism is a dynamic physical entity, comprising genomic DNA bound to many different proteins and organized into chromosomes. A thorough characterization of the physical genome is relevant to our understanding of processes such as the regulation of gene expression, DNA replication and repair, recombination, chromosome segregation, epigenetic inheritance and genomic instability. Methods based on microarrays are beginning to provide a detailed picture of this physical genome, and they complement the genome-wide studies of mRNA expression profiling that have previously been so successful.

Animals↗