Genome-Wide Mapping of in Vivo Protein-DNA Interactions

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305-5120, USA.
Science (Impact Factor: 31.48). 07/2007; 316(5830):1497-502. DOI: 10.1126/science.1141319
Source: PubMed

ABSTRACT In vivo protein-DNA interactions connect each transcription factor with its direct targets to form a gene network scaffold.
To map these protein-DNA interactions comprehensively across entire mammalian genomes, we developed a large-scale chromatin
immunoprecipitation assay (ChIPSeq) based on direct ultrahigh-throughput DNA sequencing. This sequence census method was then
used to map in vivo binding of the neuron-restrictive silencer factor (NRSF; also known as REST, for repressor element–1 silencing
transcription factor) to 1946 locations in the human genome. The data display sharp resolution of binding position [±50 base
pairs (bp)], which facilitated our finding motifs and allowed us to identify noncanonical NRSF-binding motifs. These ChIPSeq
data also have high sensitivity and specificity [ROC (receiver operator characteristic) area ≥ 0.96] and statistical confidence
(P <10–4), properties that were important for inferring new candidate interactions. These include key transcription factors in the
gene network that regulates pancreatic islet cell development.

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Available from: Richard M Myers, Jul 28, 2015
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    • "As specified for a subset of other genes (Pullen et al., 2010; Quintens et al., 2008), REST is thus " disallowed " in beta cells, as it is in neurons (Atouf et al., 1997). The observation made by ChIP seq analysis that REST binds to the chromatin of drivers of islet cell development (Johnson et al., 2007), together with the fact that REST clearance in neural progenitors has been evoked as a trigger for neural differentiation (Ballas et al., 2005), prompted us to assess the role of REST in the developing pancreas. "
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    ABSTRACT: To contribute to devise successful beta-cell differentiation strategies for the cure of Type1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program. Copyright © 2015. Published by Elsevier Inc.
    Developmental Biology 07/2015; DOI:10.1016/j.ydbio.2015.07.002 · 3.64 Impact Factor
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    • "CarD modulates transcription through its direct interaction with RNAP [6] [7]. To determine at which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used ChIP-seq [2] to survey the distribution of CarD throughout the M. smegmatis chromosome . Our data shows that CarD is localized to promoters throughout the M. smegmatis genome, indicating that CarD functions during transcription initiation. "
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    ABSTRACT: CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis (6). We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σA were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σA would be predominantly enriched at promoters based on work in Escherichia coli (3), however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σA. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 (5) as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, (accession no. GSE48164).
    12/2014; 2. DOI:10.1016/j.gdata.2014.05.012
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    • "Regulation of PDYN gene expression is a complex phenomenon that may implicate several transcription factors including avian myelocytomatosis viral oncogene homolog (c-Myc), neuron restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) [36] [52] [53], USF1/2, AP-1 family protein, FBJ murine osteosarcoma viral oncogene homolog B (FosB), cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), downstream regulatory element (DRE) antagonist modulator (DREAM), yin-yang1 (YY1) and NF-kB [54] [55] [56] [57]. c-Myc may directly interact with its binding motif in PDYN gene to regulate its function while AP-1 may either interact directly or recruit other proteins to regulate PDYN expression. "
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    ABSTRACT: Single nucleotide polymorphisms (SNPs) both in coding and non-coding regions govern gene functions prompting differential vulnerability to diseases, heterogeneous response to pharmaceutical regimes and environmental anomalies. These genetic variations, SNPs, may alter an individual's susceptibility for alcohol dependence by remodeling DNA-protein interaction patterns in prodynorphin (PDYN) and the κ-opioid receptor (OPRK1) genes. In order to elaborate the underlying molecular mechanism behind these susceptibility differences we used bioinformatics tools to retrieve differential DNA-protein interactions at PDYN and OPRK1 SNPs significantly associated with alcohol dependence. Our results show allele-specific DNA-protein interactions depicting allele-specific mechanisms implicated in differential regulation of gene expression. Several transcription factors, for instance, VDR, RXR-alpha, NFYA, CTF family, USF-1, USF2, ER, AR and predominantly SP family show an allele-specific binding affinity with PDYN gene; likewise, GATA, TBP, AP-1, USF-2, C/EBPbeta, Cart-1 and ER interact with OPRK1 SNPs on intron 2 in an allele-specific manner. In a nutshell, transition of a single nucleotide may modify differential DNA-protein interactions at OPRK1 and PDYN's SNPs, significantly associated with pathology that may lead to altered individual vulnerability for alcohol dependence.
    Computers in Biology and Medicine 10/2014; 53. DOI:10.1016/j.compbiomed.2014.07.021 · 1.46 Impact Factor
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