Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [RNA-seq]
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PROVIDER: PRJNA530570 | ENA |
REPOSITORIES: ENA
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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 | GSE113470 | GEO
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
Project description:Drosophila FACT Regulates Promoter Proximal Pol II Pausing and Chromatin Architecture [ChIP-seq]
| PRJNA451204 | ENA
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
Project description:RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.
2020-05-28 | GSE132764 | GEO
Project description:A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing
| PRJNA530563 | ENA
Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans. ChIP-seq of Pol II of different forms, SEC subunits, PAFc subunits and H2Bub in human cell lines targeted by PAF1 or scramble shRNA. ChIP-seq of total Pol II in HCT116 cells targeted by BRE1A or scramble shRNA. ChIP-seq of total Pol II in S2 cells targeted by Paf1 or LacZ RNAi. Total RNA-seq, nascent RNA-seq and GRO-seq in HCT116 cells targeted by PAF1 or scramble shRNA.
2015-08-13 | E-GEOD-70408 | biostudies-arrayexpress
Project description:m6A RNA methylation regulates promoter proximal pausing of RNA Pol II
| PRJNA603113 | ENA
Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans.
2015-08-13 | GSE70408 | GEO
Project description:Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread in higher eukaryotes. Previous studies have shown that GAF is enriched at paused genes, but the role of GAF in pausing has not been well characterized on a genome-wide level. To investigate the role of GAF in pausing, we RNAi-depleted GAF from Drosophila S2 cells, and examined the effects on promoter-proximal polymerase. We confirmed the importance of GAF for pausing on the classic pause model gene Hsp70. To determine the dependence of pausing on GAF genome-wide, we assayed the levels of transcriptionally-engaged polymerase genome-wide using GRO-seq in control and GAF-RNAi cells. We found that promoter-proximal polymerase was significantly reduced on a subset of paused genes with GAF-bound promoters. There is a dramatic change in nucleosome distribution at genes with reduction in pausing upon GAF depletion and intergenic GAF binding sites in GAF knock-down, suggesting that GAF allows the establishment of pausing at these genes by directing nucleosome displacement off of the promoter. In addition, the insulator factor BEAF, BEAF-interacting protein Chriz, and transcription M1BP enrichment on unaffected genes suggests that redundant transcription factors or insulators protect other GAF-bound paused genes from GAF knock-down effects. Three biological replicates of MNase digested chromatin from LacZ-RNAi and GAGA factor-RNAi cells.
2015-04-04 | E-GEOD-58956 | biostudies-arrayexpress