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Time course of localization of histone modifications, insulators, RNA polymerase II on TNF alpha responsive genes

ABSTRACT: The many modifications involved in transcription are extensively coupled, and levels of histone modifications, insulators, and RNA polymerase II could be measured using genomic tiling microarrays. After 0, 30, 60 min of stimulation of HUVEC with TNF alpha, we crosslinked cells and fixed, then immunoprecipitated to prepare DNA using antibodies for modified histone, insulators and RNA polymerase. We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course

ORGANISM(S): Homo sapiens

PROVIDER: GSE15157 | GEO | 2010/03/23

SECONDARY ACCESSION(S): PRJNA114927

REPOSITORIES: GEO

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Time course of localization of histone modifications, insulators, RNA polymerase II on TNF alpha responsive genes

Project description:The many modifications involved in transcription are extensively coupled, and levels of histone modifications, insulators, and RNA polymerase II could be measured using genomic tiling microarrays. After 0, 30, 60 min of stimulation of HUVEC with TNF alpha, we crosslinked cells and fixed, then immunoprecipitated to prepare DNA using antibodies for modified histone, insulators and RNA polymerase. We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course Using tumor necrosis factor a (TNF-alpha) to switch on transcription of selected human genes rapidly and synchronously, we apply samples collected every 30 min to arrays

2010-03-23 | E-GEOD-15157 | biostudies-arrayexpress

Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- ORF enrichment

Project description:Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states. Keywords: Genomewide mapping of regulatory elements through differential fractionation of crosslinked chromatin based on nucleosome occupancy. ORF enrichment- Cells were crosslinked and nuclei isolated to prepare solubilized chromatin. Crosslinks were then reversed by incubation at 65°C, and DNA was prepared. In initial experiments, immunoprecipitation using antimethyl-lysine histone H3 antibody was performed before the crosslinks were reversed. However, the IPs were not required for ORF enrichment.

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Homo sapiens

Project description:Time course of localization of histone modifications, insulators, RNA polymerase II on TNF alpha responsive genes

| PRJNA114927 | ENA

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Epigenome editing strategies for the functional annotation of topological insulators

Project description:The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs), within which the majority of gene-enhancer interactions occur. Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries3,4. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, mitotically-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing with dCas9-KRAB and dCas9-DNMT3A3L. Finally, we apply these strategies to activate PDGFRA expression in glioblastoma stem cells, thus simulating an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

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The Open Chromatin Landscape of Kaposi's Sarcoma-Associated Herpesvirus

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Project description:By CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation in silico and genetic deletions in vivo revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.

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