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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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE62171 | GEO | 2015/12/11

SECONDARY ACCESSION(S): PRJNA263366

REPOSITORIES: GEO

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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

SPT6 functions in transcriptional pause/release via PAF1C recruitment

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.

2022-08-09 | PXD033771 | Pride

CDK12 and Integrator-PP2A complex Modulates LEO1 Phosphorylation for Processive Transcription Elongation

Project description:Cyclin-dependent kinase 12 (CDK12) interacts with Cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD). To gain a broader understanding of CDK12 cellular function, we used chemical-genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the PAF1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. We also found that LEO1 interacts with, and is dephosphorylated by, the Integrator-PP2A complex (INTAC) and that INTAC promotes the association of PAF1C with Pol II. Together, this study reveals a previously unknown role for CDK12 and INTAC in regulating LEO1 phosphorylation for transcriptional regulation, providing important insights into gene transcription and its regulation.

2023-06-08 | PXD038558 | Pride

SPT6 functions in transcriptional pause-release via PAF1C recruitment [RNA-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202189 | GEO

SPT6 functions in transcriptional pause-release via PAF1C recruitment [PRO-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202187 | GEO

SPT6 functions in transcriptional pause-release via PAF1C recruitment [ChIP-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202184 | GEO

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Project description:Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

2020-06-03 | GSE115290 | GEO

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

2020-06-18 | GSE133156 | GEO

PAF1 regulation of promoter-proximal pause release via enhancer activation

Project description:Gene expression in metazoans is regulated by RNA Polymerase II (Pol II) promoter-proximal pausing and its release. Previously, we identified that Pol II-associated factor 1 (PAF1) modulates the release of paused Pol II into productive elongation. Here, we find that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of these enhancers. Enhancer activation as the result of Paf1 loss releases Pol II from paused promoters of nearby PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner.

2017-09-01 | GSE97527 | GEO

The CDK12-INTAC Axis Modulates LEO1 Phosphorylation for Processive Transcription Elongation

Project description:Cyclin-dependent kinase 12 (CDK12) interacts with Cyclin K to form a a functional nuclear kinase complex, which has been reported to phosphorylate the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) for transcriptional regulation and co-transcriptional RNA processing. However, the precise mechanisms and targets of CDK12 action remain largely unknown. Here, we combined a chemical genetic screen and phosphoproteomic strategies and identified a landscape of nuclear CDK12 substrates, which included proteins that regulate transcription, chromatin organization, and RNA splicing. Next, we confirmed that the LEO1 subunit of the transcription elongation factor PAF1 complex (PAF1C) is a bona fide substrate of CDK12. Acute depletion of LEO1 reduces Pol II occupancy on the chromatin, while mutations of LEO1 phosphorylation sites to non-phosphorylatable alanine residues attenuated the association of PAF1C with elongating Pol II and chromatin, resulting in impaired processive transcription elongation. Furthermore, LEO1 C-terminus could interact with and be dephosphorylated by the Integrator-PP2A complex (INTAC), while acute depletion of INTAC in cells promotes the association between PAF1C and elongating Pol II on the chromatin. Together, this study provides a novel transcriptional regulatory mechanism that the CDK12-INTAC axis fine-tunes LEO1 phosphorylation for processive transcription elongation.

2023-05-19 | GSE217544 | GEO

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