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Fission yeast Cdk7 controls gene expression through both its CAK and CTD kinase activities

ABSTRACT: Cyclin dependent kinases activation and RNA polymerase II transcription are linked by the Cdk7 kinase that phosphorylates Cdks as a trimeric CAK complex, and serine 5 within the PolII C-terminal domain (CTD) as TFIIH bound CAK. However, the physiological importance of integrating these processes is not understood. Beside the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. Both enzymes were proposed to act redundantly to activate Cdc2. Using an improved analogue sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20 amino acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through both its well-described RNA Polymerase II CTD kinase activity, but also through the Cdc2 activating kinase activity.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE27425 | GEO | 2012/02/19

SECONDARY ACCESSION(S): PRJNA137229

REPOSITORIES: GEO

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The Cdk-activating kinase (CAK) couples cell growth to cell division

Project description:We report that inactivation of the fission yeast Cdk7 kinase affects gene expression through both its RNA Polymerase II CTD kinase activity and its Cdc2-activating kinase activity. The ribosome biogenesis cluster is specifically downregulated when Cdc2 T167 phosphorylation is abolished, which results is slow growth. We propose that Cdc2 activation by CAK defines the rate change point observed in mid G2 and that CAK therefore couples cell growth to cell division. Total RNA was isolated from two biological replicates for all conditions, and each biological replicate was hybridized in duplicate on Agilent arrays (dye-swap).

2012-02-19 | E-GEOD-27425 | biostudies-arrayexpress

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription

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

Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast

2016-06-16 | GSE83416 | GEO

Pol II and CTD Phosphorylation ChIP-chip of ATP-analog sensitive Kin28 budding yeast

2016-06-16 | GSE83412 | GEO

Schizosaccharomyces pombe

Project description:Fission yeast Cdk7 controls gene expression through both its CAK and CTD kinase activities

| PRJNA137229 | ENA

Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast

Project description:During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a â??checkpointâ?? during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo. Total RNA was collected from wild-type and analog-sensitive Kin28 strains treated with reversible inhibitor 1-NAPP-1, irreversible inhibitor CMK, and solvent control DMSO. Equivalent ratios of S. pombe : S. cerevisiae cells were added to each sample before RNA extraction for normalization of read counts after sequencing. Nascent RNA was purified from total RNA by 4-thiouracil labeling, biotinylation, and streptavidin-pulldown. As a negative control, nascent RNA was also extracted from total RNA from cells that had not been treated with 4-thiouracil.

2016-06-16 | E-GEOD-83416 | biostudies-arrayexpress

Structure of TFIIK for phosphorylation of CTD of RNA polymerase II

Project description:TFIIH is a 10-protein complex that is conserved through out eukaryotes. TFIIH has two primary cellular functions: transcription initiation and nucleotide excision repair (NER). In transcription initiation, TFIIH acts as a structural scaffold, phosphorylates the RNA polymerase II (pol II) C-terminal domain (CTD) and translocates promoter DNA through the pol II active site to facilitate start site selection. In NER, again is a structural scaffold, opens a bubble around damaged DNA and scans the damaged strand for bulky lesions. In yeast (Saccharomyces cerevisiae), TFIIH is composed of the two helicases Ssl2 and Rad3, the scaffolding subunits Tfb1, Tfb2, Tfb4 and Ssl1 and the kinase subunits Kin28, Ccl1 and Tfb3.

2021-07-07 | PXD021211 | Pride

Human TFIIH kinase CDK7 regulates transcription-associated epigenetic modification

Project description:CDK7 phosphorylates the RNA polymerase II (pol II) CTD and activates the P-TEFb- associated kinase, CDK9, but its regulatory roles remain obscure. Using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3'-ends upon CDK7 inhibition. We also found that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5'-ends and H3K36me3 was displaced toward gene 3'-ends in CDK7as cells. Together, these results implicate a CDK7-dependent "CTD code" that regulates epigenetic marks in addition to RNA processing and pol II pausing.

2017-08-01 | GSE100040 | GEO

Kin28 Regulates the Transient Association of Mediator with Core Promoters

Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter.

2014-03-24 | GSE55402 | GEO

Pol II Ser7phospho ChIP-seq in mES cells

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

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