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Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [ChIP-seq]

ABSTRACT: The highly conserved histone chaperone FACT (Facilitates Chromatin Transcription) is thought to contribute to the disassembly and reassembly of nucleosomes in the wake of RNA Polymerase II (Pol II) passage through chromatin. However, FACT’s roles in chromatin biology and transcriptional regulation in vivo in higher eukaryotes are not well understood. Here, we report that depletion of FACT leads to a reduction in the duration of promoter-proximal pausing by Pol II in Drosophila S2 cells. In addition, FACT depletion leads to a modest decrease in the occupancy of histone H3, and to decrease in occupancy of histone H2A.v in promoter-proximal nucleosomes. Finally, we observed a dramatic 5’ to 3’ repositioning of the co-transcriptionally deposited histone modifications H3K4me3 and H3K36me3 in the FACT depleted cells. Taken together, our findings are consistent with the model that FACT contributes to the interplay between chromatin architecture and control of Pol II promoter-proximal pausing.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE113470 | GEO | 2019/06/18

REPOSITORIES: GEO

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Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [RNA-seq]

Project description:The highly conserved histone chaperone FACT (Facilitates Chromatin Transcription) is thought to contribute to the disassembly and reassembly of nucleosomes in the wake of RNA Polymerase II (Pol II) passage through chromatin. However, FACT’s roles in chromatin biology and transcriptional regulation in vivo in higher eukaryotes are not well understood. Here, we report that depletion of FACT leads to a reduction in the duration of promoter-proximal pausing by Pol II in Drosophila S2 cells. In addition, FACT depletion leads to a modest decrease in the occupancy of histone H3, and to decrease in occupancy of histone H2A.v in promoter-proximal nucleosomes. Finally, we observed a dramatic 5’ to 3’ repositioning of the co-transcriptionally deposited histone modifications H3K4me3 and H3K36me3 in the FACT depleted cells. Taken together, our findings are consistent with the model that FACT contributes to the interplay between chromatin architecture and control of Pol II promoter-proximal pausing.

2019-06-18 | GSE129235 | GEO

A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing

Project description:This SuperSeries is composed of the SubSeries listed below. FACT (Facilitates Chromatin Transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including H3K4 and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.

2019-06-14 | GSE129236 | GEO

A Unified View of the Sequence and Functional Organization of the Human RNA Polymerase II Promoter

Project description:To expand our understanding of the interplay of human RNA polymerase II (Pol II) promoter elements, promoter-proximal pausing, local chromatin structure, and co-transcriptional RNA processing, the 5′ and 3′ ends of nascent, capped transcripts and the locations of nearby nucleosomes were accurately identified. High-quality visualization tools revealed sequence elements defining the core promoter, sites of pausing, and nucleosome positioning across over 177,000 transcription start regions (TSRs). We found that cap methylation begins as transcripts attain a length of about 30 nt. A nuclear run-off assay was developed that utilizes the unique properties of the DNA fragmentation factor (DFF) to demonstrate the relative location of nucleosomes and paused Pol II. Our data support the conclusion that human Pol II promoters are functional TFIID binding sites with built-in downstream information directing ubiquitous promoter proximal pausing and the downstream nucleosome location.

2020-04-29 | GSE139237 | GEO

Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [ChIP-seq]

Project description:Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [ChIP-seq]

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Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [RNA-seq]

Project description:Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [RNA-seq]

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Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation: ChIP-chip data

Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the âdefaultâ chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy. All experiments were done using two channels per chip, comparing DNA immunoprecipitated by the indicated antibody to matching input chromatin used for affinity purification. Where appropriate, replicate data sets were averaged.

2010-10-18 | E-GEOD-20471 | biostudies-arrayexpress

Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation: ChIP-chip data

Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the “default” chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy.

2010-10-18 | GSE20471 | GEO

Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation: MNase-seq data

Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the "default" chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy.

2010-10-18 | GSE22119 | GEO

Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation: Expression data

Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the “default” chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy.

2010-10-18 | GSE24133 | GEO

The histone deacetylase SIRT6 controls transcription elongation via promoter-proximal pausing (PRO-Seq)

Project description:Transcriptional regulation in eukaryotes commonly occurs at promoter-proximal regions wherein transcriptionally engaged RNA Polymerase II (Pol II) pauses before proceeding towards productive elongation. The roles of chromatin in this process remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 regulates transcription elongation by binding to Pol II and anchoring the Negative ELongation Factor NELF, thereby preventing the release of Pol II towards elongation. Absence of SIRT6, or conditions of glucose deprivation, lead to increased levels of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of the CDK9 kinase, phosphorylation of NELF and Pol II, and recruitment of the transcription factors MYC and BRD4, the PAF1 Complex, as well as several positive elongation factors, in turn releasing Pol II into productive elongation. Collectively, we identified SIRT6 as the first pausing-dedicated histone deacetylase, regulating intragenic H3K9ac and H3K56ac as critical chromatin modifications modulating transcriptional pausing and elongation.

2019-10-31 | GSE130691 | GEO

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