The CDK12-INTAC Axis Modulates LEO1 Phosphorylation for Processive Transcription Elongation
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ABSTRACT:
PROVIDER: PRJNA899509 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22230504_1.fastq.gz | Fastqsanger.gz | |||
| SRR22230504_2.fastq.gz | Fastqsanger.gz | |||
| SRR22230505_1.fastq.gz | Fastqsanger.gz | |||
| SRR22230505_2.fastq.gz | Fastqsanger.gz | |||
| SRR22230506_1.fastq.gz | Fastqsanger.gz |
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Project description:Cyclin-dependent kinase 12 (CDK12) interacts with Cyclin K to form a a functional nuclear kinase complex, which has been reported to phosphorylate the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) for transcriptional regulation and co-transcriptional RNA processing. However, the precise mechanisms and targets of CDK12 action remain largely unknown. Here, we combined a chemical genetic screen and phosphoproteomic strategies and identified a landscape of nuclear CDK12 substrates, which included proteins that regulate transcription, chromatin organization, and RNA splicing. Next, we confirmed that the LEO1 subunit of the transcription elongation factor PAF1 complex (PAF1C) is a bona fide substrate of CDK12. Acute depletion of LEO1 reduces Pol II occupancy on the chromatin, while mutations of LEO1 phosphorylation sites to non-phosphorylatable alanine residues attenuated the association of PAF1C with elongating Pol II and chromatin, resulting in impaired processive transcription elongation. Furthermore, LEO1 C-terminus could interact with and be dephosphorylated by the Integrator-PP2A complex (INTAC), while acute depletion of INTAC in cells promotes the association between PAF1C and elongating Pol II on the chromatin. Together, this study provides a novel transcriptional regulatory mechanism that the CDK12-INTAC axis fine-tunes LEO1 phosphorylation for processive transcription elongation.
2023-05-19 | GSE217544 | GEO
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
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 C-terminal domain of RNA polymerase II (Pol II). To gain a comprehensive understanding of CDK12's 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 polymerase-associated factor 1 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. Moreover, we discovered that LEO1 interacts with and is dephosphorylated by the Integrator-PP2A complex (INTAC) and that INTAC depletion promotes the association of PAF1C with Pol II. Together, this study reveals an uncharacterized role for CDK12 and INTAC in regulating LEO1 phosphorylation, providing important insights into gene transcription and its regulation.
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Project description:Domains of heterochromatin play important roles in the maintenance and regulation of eukaryotic genomes. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates the existence of robust mechanisms that limit heterochromatin spreading and thereby avoid silencing of expressed genes. A number of specific sequence elements have been found to serve as barriers to heterochromatin spreading; however, the mechanisms by which spreading is curtailed are generally not well understood. Here we uncover a role for PAF complex component Leo1 in regulating heterochromatin cis-spreading. A genetic screen revealed that loss of Leo1 results in spreading of heterochromatin across a centromeric (IRC) boundary element in fission yeast. Similar heterochromatin spreading was seen upon deletion of other components of the PAF complex, but not other factors involved in transcription-coupled chromatin modification, indicating a specific role for the PAF complex in heterochromatin regulation. Loss of Leo1 is associated with reduced levels of H4K16 acetylation at the boundary, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1? cells, suggesting that Leo1 antagonises heterochromatin spreading by facilitating H4K16 acetylation. Interestingly, Leo1 also regulates heterochromatin spreading independently of boundaries, and loss of Leo1 causes redistribution of heterochromatin, in particular resulting in substantial expansion of telomeric heterochromatin domains. The PAF complex is known to be an important regulator of transcription-related chromatin modifications; our findings reveal a previously undescribed role for this complex in global regulation of heterochromatin spreading in cis. 8 samples: input (whole cell extract) and IP from H3K9me2 ChIP in wild-type and leo1? cells, in duplicate
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