Human TFIIH kinase CDK7 regulates transcription-associated epigenetic modification
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE100040 | GEO | 2017/08/01
SECONDARY ACCESSION(S): PRJNA390543
REPOSITORIES: GEO
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Project description:Affinity Purification and Mass Spectrometry (AP-MS) using the phosphorylated RNAPII C-terminal domain (CTD) identified the capping enzyme and the H3K4 methyltransferase complex SETD1A/B. Subsequent inhibition of CDK7 revealed displaced H3K4me3 marks at the 3'-end of genes.
2017-05-10 | MSV000081069 | MassIVE
Project description: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.
2024-05-31 | PXD037842 | Pride
Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged.
2015-04-23 | GSE60358 | GEO
Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
2015-04-23 | E-GEOD-60358 | biostudies-arrayexpress
Project description:Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways. ChIP-Seq of total/Ser5P Pol II in HCT116 cells treated with DMSO or TPL in serum starvation/activation or normal conditions. Nascent RNA-seq in HCT116 cells treated with DMSO or TPL in starved condition.
2015-01-05 | E-GEOD-63314 | biostudies-arrayexpress
Project description:CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e. distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a “master regulator” role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g. SPT5, SF3B1) are phosphorylated by the 3-subunit CDK-activating kinase (CAK; CCNH, MAT1, CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
2020-10-31 | GSE151699 | GEO
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
Project description:Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways.
2015-01-05 | GSE63314 | GEO
Project description:RNA Polymerase II transcribes protein-coding and many non-coding RNA genes in eukaryotes. The largest subunit of RNA Polymerase II, Rpb1, contains a hepta-peptide repeat on its C-terminal tail with three potential phosphorylation sites (Serine 2, Serine 5 and Serine 7). Mammalian Rpb1 contains 52 repeats. The phosphorylation events are catalyzed by specific protein kinases where the phosphorylation of specific residues is coupled to the transcription cycle. For example, the Cdk7 subunit of TFIIH phosphorylates both Serine 5 and Serine 7 during intiation and the Cdk9 subunit of P-TEFb phosphorylates Serine 2 during the transition into productive elongation. The dataset presented here is the genome-wide distribution of RNA Pol II with Serine 7 of the CTD phosphorylated in murine embryonic stem cells. This data, in addition to phospho-specific datasets generated in the same cell type in Rahl et al. Cell 2010 and Seila et al. Science 2008, represents the genome-wide distribution of multiple RNA Pol II isoforms in murine embryonic stem cells: total Pol II, hypophosphorylated CTD Pol II, Serine 2 phosphorylated CTD Pol II, Serine 5 phosphorylated CTD Pol II and Serine 7 phosphorylated CTD Pol II. An antibody specific to RNA Pol II Serine 7 phosphorylated CTD (gift of Dirk Eick; Chapman et al. Science 2008) was used to enrich for DNA fragments associated with this Pol II isoform in murine embryonic stem cells. DNA was purified and prepared for Illumina/Solexa sequencing following their standard protocol. This is a single dataset but together with datasets from Rahl et al. Cell 2010 and Seila et al. Science 2008, these datasets represent the genome-wide distribution of multiple RNA Pol II isoforms in murine embryonic stem cells: total Pol II, hypophosphorylated CTD Pol II, Serine 2 phosphorylated CTD Pol II, Serine 5 phosphorylated CTD Pol II and Serine 7 phosphorylated CTD Pol II.
2010-05-20 | E-GEOD-21917 | biostudies-arrayexpress
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.
2015-12-11 | GSE62171 | GEO