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CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Taras Velychko

LAB HEAD: Henning Urlaub

PROVIDER: PXD037842 | Pride | 2024-05-31

REPOSITORIES: Pride

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			Action	DRS
	200303_homo_sapiens_swiss_prot.fasta	Fasta
	I_Parfentev_T_Velychko_250220_010320_CDK7_NMPP1_30min_TMT_bRP_1.raw	Raw
	I_Parfentev_T_Velychko_250220_010320_CDK7_NMPP1_30min_TMT_bRP_10.raw	Raw
	I_Parfentev_T_Velychko_250220_010320_CDK7_NMPP1_30min_TMT_bRP_10b.raw	Raw
	I_Parfentev_T_Velychko_250220_010320_CDK7_NMPP1_30min_TMT_bRP_10c.raw	Raw

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CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

Project description:Cyclin-dependent kinase 7 (CDK7), part of general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (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 Pol II retention at promoters, leading to decreased Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although Pol II, initiation factors, and Mediator accumulate at promoters, Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, Pol II phosphorylation increases, initiation factors and Mediator are released, allowing recruitment of elongation factors and increase in Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from Pol II, facilitating Pol II escape from the promoter.

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2021-09-08 | PXD017611 | Pride

BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation

Project description:Understanding the precise functions and relationship of BRD2 with other bromodomain and extraterminal motif (BET) proteins is central for the application of BET-specific and pan inhibitors. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at low levels of H3K4me3-modified promoters and Pol II elongation by suppressing R-loops. Single and double depletion revealed that BRD2 and BRD3, but not BRD4, redundantly and independently function in Pol II transcription at different promoters and cooperatively occupy enhancers. Interestingly, we found that depletion of BRD2 affects the expression of different genes during differentiation processes, priming with promoter regulation in ES cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state.

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Identification of CDK9 targets using DRB and quantitative phosphoproteomics

Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

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

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Project description:After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. Here we used chromatin immunoprecipitation (ChIP) to elucidate the general initiation-elongation transition of Pol II in yeast. We extended our ChIP-chip occupancy profiling to capping enzymes and the cap-binding complex (CBC). We found that the first two capping enzymes, Cet1 and Ceg1, are recruited near the TSS, whereas the third enzyme, the methyltransferase Abd1, is recruited about 110 nt downstream of the TSS, and CBC is recruited further downstream. ChIP was performed using TAP-tagged S. cerevisiae strains (see protocols).

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Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

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