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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE217544 | GEO | 2023/05/19

REPOSITORIES: GEO

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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

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

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.

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

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

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The PAF1 complex promotes 3‘ processing of pervasive transcripts

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2022-03-03 | PXD031531 | Pride

THOC5 complexes with other proteins (DDX5, DDX17 and CDK12) essential in primitive cell survival to regulate R loop structures and RNA elongation rate.

Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites except those at the promotor regions suggesting that THOC5 is involved in transcriptional elongation. Indeed, THOC5 independent genes in cells expressing fast Pol II became THOC5 dependent in cells expressing slow Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells

2022-12-12 | PXD035092 | Pride

Spt6 directly interacts with Cdc73 and is required for Paf1C occupancy at active genes in Saccharomyces cerevisiae

Project description:The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.

2023-02-24 | GSE201436 | GEO

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

THOC5 complexes with DDX5, DDX17 and CDK12 to regulate R loop structures and transcription elongation rate

Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50-60% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence at the start site, on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites suggesting that THOC5 is involved in transcriptional elongation. Indeed, DRB/TTchem-seq that measures elongation rates in vivo revealed an accumulation of released Pol II near to TSS and a decrease of elongation rates in THOC5 depleted cells. Furthermore, THOC5 is more recruited to its target genes in cells expressing slow Pol II than those expressing fast Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. 3’ cleavage of 50% of THOC5 target genes is also regulated by CDK12 and THOC6. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells.

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