FACT orchestrates the interplay between chromatin structure and transcription [ChIP-Seq]
Ontology highlight
ABSTRACT: Transcription by RNA polymerase II (RNA Pol II) depends on transcription factors and chromatin factors. Here we use rapid factor depletion and multiomics analysis to investigate how a histone chaperone, FAcilitates Chromatin Transcription (FACT), influence nascent transcription by RNA PolII in human cells. Depletion of a FACT subunit, SSRP1, led to rapid changes in chromatin structure and concomitantly strongly compromised RNA synthesis. FACT depletion led to a multilayered transcriptional defect, including loss of promoter proximal pausing, deregulated release into elongation and drop-off of RNA Pol II in promoter-distant gene regions. We combined these analyses with biochemical dissection of transcription of a chromatinized template to show that FACT supports both elongation and pausing of RNA Pol II. Our study also provides new evidence how the position of promoter proximal pausing is defined by the +1 nucleosome in human cells.
Project description:Transcription by RNA polymerase II (RNA Pol II) depends on transcription factors and chromatin factors. Here we use rapid factor depletion and multiomics analysis to investigate how a histone chaperone, FAcilitates Chromatin Transcription (FACT), influence nascent transcription by RNA PolII in human cells. Depletion of a FACT subunit, SSRP1, led to rapid changes in chromatin structure and concomitantly strongly compromised RNA synthesis. FACT depletion led to a multilayered transcriptional defect, including loss of promoter proximal pausing, deregulated release into elongation and drop-off of RNA Pol II in promoter-distant gene regions. We combined these analyses with biochemical dissection of transcription of a chromatinized template to show that FACT supports both elongation and pausing of RNA Pol II. Our study also provides new evidence how the position of promoter proximal pausing is defined by the +1 nucleosome in human cells.
Project description:Transcription by RNA polymerase II (RNA Pol II) depends on transcription factors and chromatin factors. Here we use rapid factor depletion and multiomics analysis to investigate how a histone chaperone, FAcilitates Chromatin Transcription (FACT), influence nascent transcription by RNA PolII in human cells. Depletion of a FACT subunit, SSRP1, led to rapid changes in chromatin structure and concomitantly strongly compromised RNA synthesis. FACT depletion led to a multilayered transcriptional defect, including loss of promoter proximal pausing, deregulated release into elongation and drop-off of RNA Pol II in promoter-distant gene regions. We combined these analyses with biochemical dissection of transcription of a chromatinized template to show that FACT supports both elongation and pausing of RNA Pol II. Our study also provides new evidence how the position of promoter proximal pausing is defined by the +1 nucleosome in human cells.
Project description:Transcription by RNA polymerase II (RNA Pol II) depends on transcription factors and chromatin factors. Here we use rapid factor depletion and multiomics analysis to investigate how a histone chaperone, FAcilitates Chromatin Transcription (FACT), influence nascent transcription by RNA PolII in human cells. Depletion of a FACT subunit, SSRP1, led to rapid changes in chromatin structure and concomitantly strongly compromised RNA synthesis. FACT depletion led to a multilayered transcriptional defect, including loss of promoter proximal pausing, deregulated release into elongation and drop-off of RNA Pol II in promoter-distant gene regions. We combined these analyses with biochemical dissection of transcription of a chromatinized template to show that FACT supports both elongation and pausing of RNA Pol II. Our study also provides new evidence how the position of promoter proximal pausing is defined by the +1 nucleosome in human cells.
Project description:Transcription by RNA polymerase II (RNA Pol II) depends on transcription factors and chromatin factors. Here we use rapid factor depletion and multiomics analysis to investigate how a histone chaperone, FAcilitates Chromatin Transcription (FACT), influence nascent transcription by RNA PolII in human cells. Depletion of a FACT subunit, SSRP1, led to rapid changes in chromatin structure and concomitantly strongly compromised RNA synthesis. FACT depletion led to a multilayered transcriptional defect, including loss of promoter proximal pausing, deregulated release into elongation and drop-off of RNA Pol II in promoter-distant gene regions. We combined these analyses with biochemical dissection of transcription of a chromatinized template to show that FACT supports both elongation and pausing of RNA Pol II. Our study also provides new evidence how the position of promoter proximal pausing is defined by the +1 nucleosome in human cells.
Project description:FACT has been identified as a histone chaperone that enables transcription through chromatin in vitro, but its role in regulating chromatin structure and transcription in vivo remains unclear. In this study, we have investigated the function and molecular mechanism of FACT in unprecedented detail, by using a rapid depletion system in combination with high-resolution genomic analyses. We show that acute depletion of FACT leads to changes in 3D chromatin structure and a concomitant multilayered transcriptional defect, including loss of promoter-proximal pausing, deregulated elongation and increased drop-off of RNA Pol II. Integration of in vitro transcription assays shows that FACT stimulates RNA Pol II pausing by stabilizing nucleosomal architecture, while simultaneously promoting transcription through the +1 nucleosome. In addition to providing detailed mechanistic insight into the seemingly contradictory functions of FACT and the regulation of promoter-proximal pausing, our study indicates a direct coupling of chromatin structure and transcription.
Project description:Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
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.
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.
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.
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.