Article

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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    • "Interestingly, in the K562 cell genome, 46% of the p300 enhancer marks (Heintzman et al., 2007) have at least one CBS located within 2 kb (Figure S4B). On the other hand, 54% of the marks of the silencer factor REST/NRSF (Johnson et al., 2007) have at least one CBS located within 2 kb (Figure S4C). These observations suggest that CTCF/cohesin-mediated DNA-looping interaction may enhance or inhibit gene expression, depending on the proximity of the CBS to p300 or REST/NRSF. "
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    ABSTRACT: Graphical Abstract Highlights d The orientation of Pcdh CBSs determines the direction of topological DNA looping d Directional CTCF binding to CBSs is crucial for loop topology and gene expression d The CTCF binding orientation functions similarly in b-globin and the whole genome d CTCF/cohesin-mediated directional DNA-looping determines chromosome architecture
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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.
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    • "To determine where histone modifications or transcription factors are located in the DNA , chromatin immunoprecipita - tion combined with next generation sequencing ( ChIP - seq ) protocols were developed several years ago ( Barski et al . , 2007 ; Johnson et al . , 2007 ; Mikkelsen et al . , 2007 ) . In a typi - cal ChIP experiment , a chromatin fragment is precipitated using antibodies directed against a specific histone modifica - tion or DNA - binding protein . The DNA is isolated from the precipitated chromatin and sequenced using Next Generation Sequencing . A genome - wide profile is generated th"
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