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RNA polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II.

ABSTRACT: Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.

SUBMITTER: Yu M

PROVIDER: S-EPMC8729149 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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RNA polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II.

Yu Ming M Yang Wenjing W Ni Ting T Tang Zhanyun Z Nakadai Tomoyoshi T Zhu Jun J Roeder Robert G RG

Science (New York, N.Y.) 20151201 6266

Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positi ...[more]

PMID: 26659056

Similar Datasets

RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.

2015-12-11 | E-GEOD-62171 | biostudies-arrayexpress

RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

2015-12-11 | GSE62171 | GEO

JMJD5 couples with CDK9 to release the paused RNA polymerase II.

Project description:More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

| S-EPMC7443887 | biostudies-literature

Multiple P-TEFbs cooperatively regulate the release of promoter-proximally paused RNA polymerase II.

Project description:The association of DSIF and NELF with initiated RNA Polymerase II (Pol II) is the general mechanism for inducing promoter-proximal pausing of Pol II. However, it remains largely unclear how the paused Pol II is released in response to stimulation. Here, we show that the release of the paused Pol II is cooperatively regulated by multiple P-TEFbs which are recruited by bromodomain-containing protein Brd4 and super elongation complex (SEC) via different recruitment mechanisms. Upon stimulation, Brd4 recruits P-TEFb to Spt5/DSIF via a recruitment pathway consisting of Med1, Med23 and Tat-SF1, whereas SEC recruits P-TEFb to NELF-A and NELF-E via Paf1c and Med26, respectively. P-TEFb-mediated phosphorylation of Spt5, NELF-A and NELF-E results in the dissociation of NELF from Pol II, thereby transiting transcription from pausing to elongation. Additionally, we demonstrate that P-TEFb-mediated Ser2 phosphorylation of Pol II is dispensable for pause release. Therefore, our studies reveal a co-regulatory mechanism of Brd4 and SEC in modulating the transcriptional pause release by recruiting multiple P-TEFbs via a Mediator- and Paf1c-coordinated recruitment network.

| S-EPMC5001612 | biostudies-literature

SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase.

Project description:RNAP II is frequently paused near gene promoters in mammals, and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR-splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes.

| S-EPMC4103662 | biostudies-literature

STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.

Project description:Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.

| S-EPMC9020433 | biostudies-literature

The histone variant macroH2A1.1 regulates RNA polymerase II-paused genes within defined chromatin interaction landscapes.

Project description:The histone variant macroH2A1.1 plays a role in cancer development and metastasis. To determine the underlying molecular mechanisms, we mapped the genome-wide localization of endogenous macroH2A1.1 in the human breast cancer cell line MDA-MB-231. We demonstrate that macroH2A1.1 specifically binds to active promoters and enhancers in addition to facultative heterochromatin. Selective knock down of macroH2A1.1 deregulates the expression of hundreds of highly active genes. Depending on the chromatin landscape, macroH2A1.1 acts through two distinct molecular mechanisms. The first mitigates excessive transcription by binding over domains including the promoter and the gene body. The second stimulates expression of RNA polymerase II (Pol II)-paused genes, including genes regulating mammary tumor cell migration. In contrast to the first mechanism, macroH2A1.1 specifically associates with the transcription start site of Pol II-paused genes. These processes occur in a predefined local 3D genome landscape, but do not require rewiring of enhancer-promoter contacts. We thus propose that macroH2A1.1 serves as a transcriptional modulator with a potential role in assisting the conversion of promoter-locked Pol II into a productive, elongating Pol II.

| S-EPMC9016624 | biostudies-literature

Cohesin selectively binds and regulates genes with paused RNA polymerase.

Project description:BackgroundThe cohesin complex mediates sister chromatid cohesion and regulates gene transcription. Prior studies show that cohesin preferentially binds and regulates genes that control growth and differentiation and that even mild disruption of cohesin function alters development. Here we investigate how cohesin specifically recognizes and regulates genes that control development in Drosophila.ResultsGenome-wide analyses show that cohesin selectively binds genes in which RNA polymerase II (Pol II) pauses just downstream of the transcription start site. These genes often have GAGA factor (GAF) binding sites 100 base pairs (bp) upstream of the start site, and GT dinucleotide repeats 50 to 800 bp downstream in the plus strand. They have low levels of histone H3 lysine 36 trimethylation (H3K36me3) associated with transcriptional elongation, even when highly transcribed. Cohesin depletion does not reduce polymerase pausing, in contrast to depletion of the NELF (negative elongation factor) pausing complex. Cohesin, NELF, and Spt5 pausing and elongation factor knockdown experiments indicate that cohesin does not inhibit binding of polymerase to promoters or physically block transcriptional elongation, but at genes that it strongly represses, it hinders transition of paused polymerase to elongation at a step distinct from those controlled by Spt5 and NELF.ConclusionsOur findings argue that cohesin and pausing factors are recruited independently to the same genes, perhaps by GAF and the GT repeats, and that their combined action determines the level of actively elongating RNA polymerase.

| S-EPMC3193539 | biostudies-literature

JMJD5 couples with CDK9 to release the paused RNA polymerase II

Project description:More than 30% of genes in higher eukaryotes are regulated by RNA Polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS) thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxy-terminal domain of Pol II together with phosphorylation of Ser5 within the second repeat, HR-Ser2p(1,2)-Ser5p(2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

2020-06-27 | GSE153322 | GEO

H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

Project description:Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and proposed to regulate transcription initiation. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate elucidation of the specific role of H3K4me3 in transcriptional regulation. Here, by using mouse embryonic stem cells (mESCs) as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to complete loss of all H3K4 methylation. H3K4me3 turnover occurs more rapidly than H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Surprisingly, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. Notably, we show that H3K4me3 is required for the recruitment of the Integrator Complex Subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.

2023-02-10 | PXD039176 | Pride

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