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Genome-wide reductions in active chromatin, paused RNA Polymerase II and nucleosome turnover during heat shock

ABSTRACT: Heat shock rapidly induces expression of a small set of genes while globally repressing transcription, making it an attractive system for studying alterations in the chromatin landscape that accompany changes in gene regulation. We have characterized these changes using low-salt extraction of intact micrococcal nuclease (MNase)-treated Drosophila S2 cell nuclei to determine the active nucleosomal and subnucleosomal chromatin landscapes. The low-salt-soluble fraction corresponds to classical "active" chromatin and includes distinct size fractions of MNase-protected particles that can be precisely mapped by paired-end sequencing. After heat shock, the distribution of low-salt-soluble nucleosomes showed an overall reduction over gene bodies, consistent with down-regulation of transcription. No global changes were detected in the subnucleosomal landscape upstream of transcriptional start sites, however, we observed a genome-wide reduction of paused RNA Polymerase II from the active chromatin fraction. Furthermore, nucleosome turnover decreased within gene bodies in a pattern similar to that observed when transcription elongation was artificially inhibited. These observations suggest that reduced Pol II affinity and processivity is the dominant nuclear mechanism for genome-wide repression during heat shock. Our ability to precisely map both nucleosomal and subnucleosomal particles directly from classical active chromatin extracts to assay changes in the chromatin landscape provides a simple general strategy for epigenome characterization.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE30755 | GEO | 2011/11/15

SECONDARY ACCESSION(S): PRJNA144131

REPOSITORIES: GEO

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Genome-wide reductions in active chromatin, paused RNA Polymerase II and nucleosome turnover during heat shock

Project description:Heat shock rapidly induces expression of a small set of genes while globally repressing transcription, making it an attractive system for studying alterations in the chromatin landscape that accompany changes in gene regulation. We have characterized these changes using low-salt extraction of intact micrococcal nuclease (MNase)-treated Drosophila S2 cell nuclei to determine the active nucleosomal and subnucleosomal chromatin landscapes. The low-salt-soluble fraction corresponds to classical "active" chromatin and includes distinct size fractions of MNase-protected particles that can be precisely mapped by paired-end sequencing. After heat shock, the distribution of low-salt-soluble nucleosomes showed an overall reduction over gene bodies, consistent with down-regulation of transcription. No global changes were detected in the subnucleosomal landscape upstream of transcriptional start sites, however, we observed a genome-wide reduction of paused RNA Polymerase II from the active chromatin fraction. Furthermore, nucleosome turnover decreased within gene bodies in a pattern similar to that observed when transcription elongation was artificially inhibited. These observations suggest that reduced Pol II affinity and processivity is the dominant nuclear mechanism for genome-wide repression during heat shock. Our ability to precisely map both nucleosomal and subnucleosomal particles directly from classical active chromatin extracts to assay changes in the chromatin landscape provides a simple general strategy for epigenome characterization. High-throughput sequencing (Illumina HiSeq 2000) We have characterized changes to the active nucleosomal and subnucleosomal landscape during the heat shock response in Drosophila cells by genome-wide profiling of low-salt extracted micrococcal nuclease-treated nuclei, paused RNA Polymerase II and CATCH-IT nucleosome turnover.

2011-11-15 | E-GEOD-30755 | biostudies-arrayexpress

Nucleosomal and subnucleosomal organization of transcription cis-regulatory elements in mouse embryonic stem cells (chip-seq gradient)

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2024-06-03 | GSE255089 | GEO

Nucleosomal and subnucleosomal organization of transcription cis-regulatory elements in mouse embryonic stem cells

Project description:We report a comprehensive analysis of nucleosomal and subnucleosomal organization at cis-regulatory elements (CREs) in mouse embryonic stem (ES) cells. We used chromatin fragmented by a moderate dose of micrococcal nuclease (MNase) as input for ChIP-seq with antibodies against histones and transcription factors and high-coverage deep-sequencing. Centrifugation of chromatin through sucrose gradients, followed by ChIP-seq, allowed the characterization of different subclasses of subnucleosomal particles having distribution patterns specific to enhancers, gene promoters and CTCF binding sites. We also provide datasets showing that this particular chromatin organization of CREs is conserved in the human melanoma 501Mel cell line.

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Nucleosomal and subnucleosomal organization of transcription cis-regulatory elements in mouse embryonic stem cells

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Nucleosomal and subnucleosomal organization of transcription cis-regulatory elements in mouse embryonic stem cells

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Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis

Project description:Background: Analysis of the effect that chromatin structure has on the expression patterns of eukaryotic genes has recently expanded knowledge of the complex influence genome accessibility has on genome function. Interlaced with regular nucleosomal patterning are other mobile and labile sub-nucleosomal-sized protein structures bound to the genome such as transcription factors (TF), initiation complexes, and modified nucleosomes. Results: We present chromatin structure maps of the A. thaliana Col-0 wild-type (in vitro cell culture) combining differential micrococcal nuclease (MNase) digestion and analysis by paired-end next-generation sequencing with RNASeq data to provide insight into the sensitive genomic regions and variable DNA-bound particle size related to transcriptional activity. The landscape of sub-nucleosomal sized particles (subNSPs) has been investigated, revealing the DNA-binding complex positioning genome-wide. We observe the dynamic changes in the distribution of these complexes surrounding genomic features, particularly at transcription start sites (TSS). Differential digestion reveals the presence of MNase-sensitive smaller particles and a labile -1 nucleosome upstream of the TSS of active genes. These changes correlate with gene expression differences resulting from different environmental conditions of light- or dark growth both globally and locally, and a complex profile of bound particles is directly visualised. Conclusions: Analysis of the A. thaliana genome reveals that resolution of chromatin particle size by differential MNase digestion allows detection of the sensitive features in chromatin structure that have hereto been obfuscated. The concomitant analysis of transcript levels reveals the impact of transient extrinsic factors in modifying the sub- nucleosomal landscape in association with transcriptional changes, giving insight into the binding of transcription-associated factors.

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2014-10-28 | E-GEOD-54524 | biostudies-arrayexpress

Asymmetric nucleosomes flank promoters in the budding yeast genome

Project description:Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that nucleosomes at 5% of budding yeast nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling. We have analyzed the chromatin landscape of the yeast genome using paired-end MNase-seq and the chromatin binding of yeast remodelers Swr1, Ino80 and RSC at base-pair resolution using native chromatin immunoprecipitation followed by sequencing (N-ChIP-seq).

2014-12-04 | E-GEOD-59523 | biostudies-arrayexpress

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