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Effects of Inhibition of CDK8/19 Mediator Kinase on serum-regulated gene expression in 293 cells

ABSTRACT: Effects of Inhibition of CDK8/19 Mediator Kinase on serum-regulated gene expression in 293 cells

PROVIDER: PRJNA819404 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18463519_1.fastq.gz	Fastqsanger.gz
	SRR18463519_2.fastq.gz	Fastqsanger.gz
	SRR18463520_1.fastq.gz	Fastqsanger.gz
	SRR18463520_2.fastq.gz	Fastqsanger.gz
	SRR18463521_1.fastq.gz	Fastqsanger.gz

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Effects of Inhibition of CDK8/19 Mediator Kinase on serum-regulated gene expression in 293 cells

Project description:We have investigated the effects of CDK8/19 inhibitor Senexin B on the gene expression regulated by serum stimulation in 293 cells

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The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

Project description:The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex Mediator that regulates RNA polymerase II (Pol II transcription. The CKM has negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. Here, we prepare recombinant CKM from the yeast Saccharomyces cerevisiae and show that it binds core Mediator (cMed) to sterically inhibit cMed binding to the Pol II preinitiation complex (PIC) in vitro. We further show that the Cdk8 kinase activity of CKM counteracts CKM-cMed interaction,thereby releasing CKM and enabling Mediator to bind the PIC. Finally, we report that the kinase activity of Cdk8 is required for gene activation during heat shock in vivo, but not under steady state growth conditions. These results converge with previous literature on a model for CKM function. In this model, CKM negatively regulates Mediator function at upstream activating sequences by preventing Mediator binding to the PIC at the promoter. During gene activation, Cdk8 kinase activity may release Mediator and allow its binding to the PIC, thereby stimulating transcription initiation and accounting for the positive function of CKM.

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Project description:The Mediator complex allows communication between transcription factors and RNA polymerase II (RNAPII). CDK8, the kinase found in some variants of Mediator, has been characterized mostly as a transcriptional repressor. Recently, CDK8 was demonstrated to be a potent oncoprotein. Here we show that CDK8 is predominantly a positive regulator of gene expression within the serum response network, as it is required for expression of several members of the AP-1 and EGR family of oncogenic transcription factors (e.g. FOS, JUN, EGR1-3). Mechanistic studies demonstrate that CDK8 is not required for recruitment of RNAPII and promoter escape at these loci. Instead, CDK8 depletion leads to the appearance of slower elongation complexes carrying hypophosphorylated RNAPII. We show that CDK8-Mediator regulates precise steps in the assembly of a functional elongation complex, including the recruitment of P-TEFb and BRD4, but is dispensable for recruitment of SPT5 and FACT. Furthermore, CDK8-Mediator specifically interacts with P-TEFb. Thus, we uncovered a novel role for CDK8 in transcriptional regulation that may contribute to its oncogenic effects. We used microarrays to analyze the expression of immediate early genes in response to serum in cells with and without the Mediator associated kinase, CDK8.

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Project description:We use biochemically reconstituted transcription initiation components including the Mediator core and Cdk8 kinase modules (CKM) to investigate the function of the CKM in regulating the Mediator-RNA polymerase II interaction in a highly purified system. We use cross-linking coupled to mass spectrometry to map the interaction of the CKM and core Mediator, and in vitro phosphorylation assays followed by phosphopeptide enrichment coupled to mass spectrometry to identify Cdk8 phosphorylation targets in this context and biochemically dissect their functions. Finally, we investigate the function of phosphorylation by Cdk8 on transcription in vivo. Based on that, we propose a model to integrate the phosphorylation-dependent and -independent functions of the CKM in transcription initiation.

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Project description:We have investigated the effects of CDK8/19 inhibitor Senexin B on the gene expression regulated by PMA 24hr-treatment in 293 cells.

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Project description:RNA-Seq analysis was carried out to investigate the effects of selective CDK8/19 inhibitor Senexin B on gene expression in HEK293 cells (293-WT) or the CDK8/19 double-knockout (293-dKO) derivatives.

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Project description:RNA-Seq analysis was carried out to investigate the effects of CDK8/19 inhibitor Senexin B on the gene expression regulated by 2hr-treatment with PMA in 293 cells.

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