Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA378674 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5331794_1.fastq.gz | Fastqsanger.gz | |||
| SRR5331794_2.fastq.gz | Fastqsanger.gz | |||
| SRR5331795_1.fastq.gz | Fastqsanger.gz | |||
| SRR5331795_2.fastq.gz | Fastqsanger.gz | |||
| SRR5331796_1.fastq.gz | Fastqsanger.gz |
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Project description:Gene expression by RNA Polymerase II (RNAPII) is tightly controlled by Cyclindependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The RNAPII pausing checkpoint, engaged after transcription initiation, is controlled by CDK9 to regulate transcription in metazoans. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPIICTD, and is recruited to transcription pausing sites by the Integrator complex subunit INTS6. INTS6 depletion confers resistance to CDK9 inhibition in a variety of normal and tumor cell lines. Loss of INTS6 abolishes the Integrator-PP2A association leading to unrestrained CDK9 activity, which amplifies transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill MLLrearranged leukemias and solid tumors and provide therapeutic benefit in vivo. These data demonstrate that finely-tuned gene expression relies on the balance of kinase and phosphatase activity at the pausing checkpoint.
2022-08-12 | PXD022962 | Pride
Project description:Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a ‘multi-omics’ approach to show that the duration of polymerase pausing generally limits the frequency of transcription initiation in human cells (‘pause-initiation limit’). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. We show that CDK9 activity decreases the pause duration but also increases the initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.
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Project description:Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region. Pausing Pol II limits the frequency of transcription initiation (‘pause-initiation limit’), predicting that the pause duration must be decreased for transcriptional activation. To test this prediction, we conducted a genome-wide kinetic analysis of the heat shock response in human cells. We show that the pause-initiation limit restricts transcriptional activation at most genes. Gene activation generally requires the activity of the P-TEFb kinase CDK9, which decreases the duration of Pol II pausing and thereby enables an increase in the productive initiation frequency. The transcription of enhancer elements is generally not pause-limited and can be activated without CDK9 activity. Our results define the kinetics of Pol II transcriptional regulation in human cells at all gene classes during a natural transcription response.
2019-07-10 | GSE123980 | GEO
Project description:Gene expression by RNA Polymerase II (RNAPII) is tightly controlled by Cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The RNAPII pausing checkpoint, engaged after transcription initiation, is controlled by CDK9 to regulate transcription in metazoans. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPII-CTD, and is recruited to transcription pausing sites by the Integrator complex subunit INTS6. INTS6 depletion confers resistance to CDK9 inhibition in a variety of normal and tumor cell lines. Loss of INTS6 abolishes the Integrator-PP2A association leading to unrestrained CDK9 activity, which amplifies transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill MLL-rearranged leukemias and solid tumors and provide therapeutic benefit in vivo. These data demonstrate that f inely-tuned gene expression relies on the balance of kinase and phosphatase activity at the pausing checkpoint.
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2021-05-23 | GSE163803 | GEO
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.
2022-08-22 | PXD026720 | Pride
Project description:Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA Polymerase II and associated factors to control transitions between these steps and act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, though their roles remain less well understood. Here we discover that the PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, this reveals an essential new role for a phosphatase complex in transcription pause release and shows that the PNUTS complex is essential for RNA Poll II-dependent transcription.
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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:RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.
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