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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE115290 | GEO | 2020/06/03

REPOSITORIES: GEO

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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.

2020-06-18 | GSE133156 | GEO

Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modification on the C-terminal domain (CTD) of the largest subunit , generating a sophisticated PTM landscape for the spatiotemporal recruitment to transcriptional regulators. To delineate the protein interactomes recruited to Pol II at different stages of transcription, we in vitro reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 positions, the hallmark phosphorylation at the initation and productive elongation stages of transcription, respectively. Distinctive protein interactomes indicates different proteins are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) specifically binds to the Ser2 of the heptad. The loss of the interaction between CHERP and Pol II results in broad alternative splicing events. Our method points to a new method to distinguish the PTM codes that coordinate the transcription process.

2023-02-06 | MSV000091213 | MassIVE

Threonine-4 of Mammalian RNA Polymerase II CTD is Targeted by Polo-like Kinase-3 and Required for Transcriptional Elongation

Project description:Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5’ and 3’ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3’ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.

2012-06-04 | GSE37519 | GEO

Genome-wide occupanies of Ssu72, Pol II, pSer2, pSer5, pSer7 and pThr4 of Ssu72 Wild Type and Knock-out MEFs and hepatocytes

Project description:We report the genome-wide occupanies for high-throughput profiling of Pol II and Pol II CTD phosphorylation modifications in mammalian cells and tissues. By obtaining over three billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, embryonic fibroblasts and primary hepatocytes. Loss of mammalian Ssu72 was found to be related to defect of transcriptional elongation though the defects of Pol II CTD phophorylational dynamics. In addition, depletion of Ssu72 resulted in defects of Pol II elongation by reducing CTD Ser2 and Thr4 phosphorylation which induced Pol II accumulation at the proximal promoter region by increasing CTD Ser5 and Ser7 phosphorylation of transcriptionally active genes in a tissue-specific manner. Our results revealed a fundamental role of mammalian Ssu72 in regulating RNA Pol II-mediated transcriptional plasticity for cell-type-specific homeostatic maintenance.

2020-12-28 | GSE122102 | GEO

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

CTD tyrosine phosphorylation impairs termination factor recruitment to transcribing RNA polymerase II

Project description:ChIP-chip was performed to identify the genomic binding locations for the termination factors Nrd1, and Rtt103, and for RNA polymerase (Pol) II phosphorylated at the tyrosine 1 and threonine 4 position of its C-terminal domain (CTD). In different phases of the transcription cycle, Pol II recruits different factors via its CTD, which consists of heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Here we show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, and that this impairs recruitment of termination factors. Tyr1 phosphorylation levels rise downstream of the transcription start site (TSS), and decrease before the polyadenylation (pA) site. Tyr1-phosphorylated gene bodies are depleted of CTD-binding termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation blocks CTD binding by these termination factors, but stimulates binding of elongation factor Spt6. These results show that CTD modifications can not only stimulate but also block factor recruitment, and lead to an extended CTD code for transcription cycle coordination.

2012-06-29 | E-MTAB-1060 | biostudies-arrayexpress

Role of RNA polymerase II "pausing" in Drosophila development

Project description:According to previous studies, during Drosophila embryogenesis, RNA polymerase II is recruited to promoters at developmental stages preceding the stages of active transcription of genes. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. We performed ChIP-Seq analysis using antibodies to various modifications of RNA polymerase II (total, Pol II CTD Ser5P and Pol II CTD Ser2P), as well as to subunits of NELF, DSIF, PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that like in mid-embryogenesis during metamorphosis, promoters bind RNA polymerase II in the "paused" state preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in "pause" is phosphorylated at Ser5 and Ser2 of Rpb1 CTD and binds NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex includes Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex is phosphorylated at Ser5 at Rpb1 CTD, but not at Ser2.

2022-09-10 | GSE210971 | GEO

Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:We have discovered distinctive protein interactomes for Ser2 and Ser5 phosphorylation of the C-terminal domain (CTD) of RNA Pol II. Calcium Homeostasis Endoplasmic reticulum (CHERP) was identified in our pulldown to directly interact with Ser2 phosphomarks on the CTD of Pol II. As Pol II is a chromatin-associated protein, we sought to identify genomic regions that CHERP, a Pol II binding protein, occupy through ChIP-seq analysis.

2023-08-17 | GSE226908 | GEO

HDACs and Phosphorylated Pol II CTD recruit Spt6 for cotranscriptional histone reassembly

Project description:Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C-terminus that recognizes Pol II CTD peptides phosphorylated on Ser2, Ser5 or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' end of genes, where phosphorylated Ser2 reaches its maximum level. Additionally, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' end of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo.

2014-09-25 | GSE61713 | GEO

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

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