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Kin28 Regulates the Transient Association of Mediator with Core Promoters

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE55402 | GEO | 2014/03/24

SECONDARY ACCESSION(S): PRJNA239629

REPOSITORIES: GEO

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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. We took a systematic approach to examine the genome-wide distribution (using ChIP-chip) of the various Mediator subunits. Mediator occupancy was also assayed in mutants for most of the CTD kinases and in strains where some CTD serines had been replaced by alanines. Mediator ChIPs were performed with Myc-tagged subunits, except in some preliminary experiments where polyclonal antibodies were used. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In some of the preliminary experiments, non immunoprecipitated DNA (input) was used as the control. In addition to Mediator ChIPs, the project includes TFIIB and RNAPII (Rpb3) ChIP-chip datasets. All ChIP-chip experiments were done in duplicates except for the preliminary experiments that were done in monoplicat for the most part. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

2014-03-24 | E-GEOD-55402 | biostudies-arrayexpress

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

2016-06-16 | GSE83412 | GEO

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

2016-06-16 | GSE83416 | GEO

Chromatin immunoprecipitation of yeast to assess changes in RNA Polymerase II, Ser5-P, and Ser7-P occupancy upon chemical inhibition of Kin28

Project description:Chromatin Immunoprecipitation to assess changes in RNA Polymerase II, Ser5-P, and Ser7-P occupancy in S. cerevisiae upon chemical inhibition of Kin28

2009-07-01 | E-TABM-689 | biostudies-arrayexpress

Transient transcriptome sequencing (TT-Seq) and 5'P-Seq of ATP-analog sensitive Kin28 budding yeast

Project description:Inhibition of Kin28/CDK7, the kinase subunit of TFIIH, leads to defects in transcription of protein-coding genes. Despite a severe reduction in nascent RNA synthesis, the majority of mRNAs retain their steady-state level upon inhibition. In this study, we examined the determinants of mRNA stability in cells experiencing transcriptional crisis via irreversible chemical inhibition of Kin28. We discovered that the inhibited Kin28 transcriptome resembles the transcriptome of cells treated with an inhibitor of protein synthesis. Indeed, inhibition of Kin28 induces a coordinated decrease in translation and an increase in P-body formation. Unexpectedly, integrated stress response effectors do not trigger the observed proteostasis and ribostasis. Rather, mRNAs that are buffered from degradation display a preference for Ski2 over Nab2 and are differentially sensitive to the 5′-exonuclease Xrn1. These findings reveal that a nuclear kinase, well-known for its role in early stages of RNA synthesis, orchestrates multiple molecular processes in different cellular compartments.

2022-01-18 | GSE125409 | GEO

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

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

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

ChIP-seq of Mediator subunits in Mediator mutants and wild type yeast

Project description:Mediator subunits Med17 (Srb4) and Med15 (Gal11) were subjected to ChIP in wild type yeast, med18 (srb5), med20 (srb2), and med2 med3 med15 (triple mutant) yeast, all in kin28-anchor away yeast after 1 hr rapamycin treatment to evict Kin28 from the nucleus. This results in inactivation of Kin28, which reduces Mediator turnover at promoters and allows stronger Mediator ChIP signal at promoters. <br></br>Both Med17 and Med15 were tagged with myc and precipitated using the monoclonal antibody against c-myc. Please note that a specific control for rapamycin treatment was not performed in this experiment, as that has been well documented in the literature for this experiment, using what is called the anchor away method. The untagged sample is a control for non-specific immunoprecipitation.

2017-12-01 | E-MTAB-5824 | biostudies-arrayexpress

Saccharomyces cerevisiae

Project description:Kin28 Regulates the Transient Association of Mediator with Core Promoters

| PRJNA239629 | ENA

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

Project description:Cyclin-dependent kinase 7 (CDK7), part of general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (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 Pol II retention at promoters, leading to decreased Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although Pol II, initiation factors, and Mediator accumulate at promoters, Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, Pol II phosphorylation increases, initiation factors and Mediator are released, allowing recruitment of elongation factors and increase in Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from Pol II, facilitating Pol II escape from the promoter.

2024-06-01 | GSE218269 | GEO

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