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: 33.61). 07/2007; 316(5830):1497-502. DOI: 10.1126/science.1141319
Source: PubMed


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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    • "The number of REST target genes is still debated. The first ∼2000 genes were proposed because of their positivity for RE-1, a DNA sequence of possible REST binding (Bruce et al., 2004; Wu and Xie, 2006; Johnson et al., 2007). More recently, thousands of additional genes, possibly RESTdependent but RE-1-negative, have been identified based on integrated computational analyses of available ChIP-Seq datasets, carried out mostly in non-neural cells (Otto et al., 2007; ENCODE Project Consortium et al., 2012; Johnson et al., 2012). "
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    ABSTRACT: The role of REST changes in neurons, including the rapid decrease of its level during differentiation and its fluctuations during many mature functions and diseases, is well established. However, identification of many thousand possible REST-target genes, mostly based on indirect criteria, and demonstration of their operative dependence on the repressor have been established for only a relatively small fraction. In the present study, starting from our recently published work, we have expanded the identification of REST-dependent genes, investigated in two clones of the PC12 line, a recognized neuronal cell model, spontaneously expressing different levels of REST: very low as in neurons and much higher as in most non-neural cells. The molecular, structural and functional differences of the two PC12 clones were shown to depend largely on their different REST level and the ensuing variable expression of some dependent genes. Comprehensive RNA-Seq analyses of the 13,700 genes expressed, validated by parallel RT-PCR and western analyses of mRNAs and encoded proteins, identified in the high-REST clone two groups of almost 900 repressed and up-regulated genes. Repression is often due to direct binding of REST to target genes; up-regulation to indirect mechanism(s) mostly mediated by REST repression of repressive transcription factors. Most, but not all, genes governing neurosecretion, excitability, and receptor channel signaling were repressed in the high REST clone. The genes governing expression of non-channel receptors (G protein-coupled and others), although variably affected, were often up-regulated together with the genes of intracellular kinases, small G proteins, cytoskeleton, cell adhesion, and extracellular matrix proteins. Expression of REST-dependent genes governing functions other than those mentioned so far were also identified. The results obtained by the parallel investigation of the two PC12 clones revealed the complexity of the REST molecular and functional role, deciphering new aspects of its participation in neuronal functions. The new findings could be relevant for further investigation and interpretation of physiological processes typical of neurons. Moreover, they could be employed as tools in the study of neuronal diseases recently shown to depend on REST for their development.
    Frontiers in Cellular Neuroscience 11/2015; 9(8). DOI:10.3389/fncel.2015.00438 · 4.29 Impact Factor
    • "The development of high-throughput techniques lead to an ever-increasing number of experimental data sets that can be used for inferring binding motifs computationally. Techniques like ChIP-chip (Ren et al., 2000), ChIP-seq (Johnson et al., 2007), ChIP-exo (Rhee and Pugh, 2011), or ORGANIC (Kasinathan et al., 2014) allow for studying the in-vivo binding regions of a target TF on a genomic scale. One extensive resource of TF ChIP-seq data in human is the ENCODE project (The ENCODE Project Consortium, 2012). "

    German Conference on Bioinformatics 2015, University Alliance Ruhr, Dortmund; 09/2015
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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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