The NELF pausing checkpoint mediates the functional divergence of Cdk9
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ABSTRACT:
PROVIDER: PRJNA870098 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21091505.fastq.gz | Fastqsanger.gz | |||
| SRR21091506.fastq.gz | Fastqsanger.gz | |||
| SRR21091507.fastq.gz | Fastqsanger.gz | |||
| SRR21091508.fastq.gz | Fastqsanger.gz | |||
| SRR21091509.fastq.gz | Fastqsanger.gz |
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Project description:Cdk9 is an essential transcriptional kinase that is conserved across distant eukaryotes, but we previously found that acute inhibition of Cdk9 causes different transcriptional phenotypes in NELF-containing higher eukaryotes (like mammals and Drosophila) vs. the NELF-lacking fission yeast S. pombe. NELF is known to participate in promoter-proximal pausing of RNA Polymerase II, and using NELF-depleted Drosophila cells, we find that this NELF-mediated pause is necessary to prevent gene body entry by Pol II in the absence of Cdk9. Without NELF, the abnormal gene body entry that occurs following Cdk9 inhibition is strikingly similar to that seen in S. pombe, and in either case, these Pol II complexes have elongation defects and are unable to complete gene transcription. These results show that NELF enforces an early checkpoint for Cdk9 and efficiently shuts down gene transcription in its absence. We propose this would have been critical for the emergence of gene regulatory strategies acting via Cdk9 to modulate transcription of specific genes.
2023-03-22 | GSE211397 | GEO
Project description:Promoter-proximal pausing by RNA Pol II is a rate-determining step in gene transcription that is hypothesized to be a prominent point at which regulatory factors act. The pausing factor NELF is known to induce and stabilize pausing, but not all kinds of pausing are NELF-mediated. Here, we find that NELF-depleted Drosophila melanogaster cells functionally recapitulate the NELF-independent pausing we previously observed in fission yeast (which lack NELF). Critically, only NELF-mediated pausing establishes a strict requirement for Cdk9 kinase activity for the release of paused Pol II into productive elongation. Upon inhibition of Cdk9, cells with NELF efficiently shutdown gene transcription, while in NELF-depleted cells, defective, non-productive transcription continues unabated. By introducing a strict checkpoint for Cdk9, the evolution of NELF was likely critical to enable increased regulation of Cdk9 in higher eukaryotes, as Cdk9 availability can be restricted to limit gene transcription without inducing wasteful, non-productive transcription.
| S-EPMC10182999 | biostudies-literature
Project description:Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown. mES cells were infected with a shRNA hairpin to knockdown Spt5, Spt4, NelfE or control. The resulting cells were then treated with flavopiridol (1uM for 60 minutes). DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total RNA Pol2 (Rpb1 N-terminus, Santa Cruz sc-899).
2011-01-19 | E-GEOD-26658 | biostudies-arrayexpress
Project description:Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown.
2011-01-19 | GSE26658 | GEO
Project description:Controlled gene expression is achieved through the intricate regulation of Pol II progression through transcription-cycle checkpoints. While the contribution of CDK9 for Pol II pause-release is well established, the requirement for other CDKs has not been fully elucidated. Here we propose a new role for CDK11 in the Pol II pausing-to-elongation transition at a checkpoint that precedes and is independent from CDK9. Selective CDK11 inhibition or degradation results in acute ablation of RNA synthesis near the beginning of transcriptional units and genome-wide stalling of Pol II at TSS-proximal-regions. High-resolution chromatin-immunoprecipitation and precision-nuclear-run-on assays reveals spatial differences between CDK11- and CDK9-dependent Pol II pause sites, with CDK11 regulating Pol II upstream of the CDK9 checkpoint within the pausing zone. Cancer cells exhibit profound reliance on CDK11, with CDK11 inhibition reducing tumour burden in in vivo models of blood cancer, demonstrating the importance of CDK11-dependent Pol II regulation for aggressive haematological malignancies.
2025-09-09 | PXD066230 | Pride
Project description:The NELF complex is a metazoan-specific factor essential for establishing transcription pausing. Although NELF has been implicated in cell fate regulation, the cellular regulation of NELF and its intrinsic role in specific lineage differentiation remains largely unknown. Using mammalian hematopoietic differentiation as a model system, here we identified a dynamic change of NELF-mediated transcription pausing as a novel mechanism regulating hematopoietic differentiation. We found a sharp decrease of NELF protein abundance during granulocytic differentiation with a consequent genome-wide reduction of transcription pausing. This loss of pausing coincides with activation of granulocyte-affiliated genes and diminished expression of progenitor markers. Functional studies revealed that sustained expression of NELF inhibits granulocytic differentiation, whereas NELF depletion in progenitor cells leads to premature differentiation towards the granulocytic lineage. Our results thus uncover a previously unrecognized regulation of transcription pausing by modulating NELF protein abundance to control cellular differentiation.
2019-05-31 | GSE98555 | GEO
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:NELF-A phosphorylation by P-TEFb is a key event in the pausing release. We conduct IMAC-LC-MS-MS to identified NELF-A phosphorylation site by activation P-TEFb kinase.
2016-03-23 | PXD003433 | 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.
2017-10-09 | GSE96056 | 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.
2021-05-23 | GSE163803 | GEO