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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Knockdown effect of CDK8 and CDK19 in HeLa S3 cell

ABSTRACT: Mediator complex has been known as pivotal regulator of RNA polymerase II. Mediator complex has two CDK subunits in vertebrates, named CDK8 and CDK19. To elucidate functional difference between CDK8 and CDK19 in human cell, we employ siRNA mediate knockdown assay using HeLa S3 cell line. According to this assay these CDKs possess highly redundancy in HeLa S3 cell transcription regulation mechanism but in several genes, each CDK shows gene specific regulatory function. HeLa S3 cells were seeded on 6-well plate at 4 x 104 cells per well in 2ml antibiotic free DMEM medium before 24hr transfection. We transfect siRNA using Lipofectamine 2000 (invitrogen) according to its manual and siRNA final concentration in the medium was 20nM. After culturing for 48hr, transfection medium was changed to fresh antibiotic containing medium. Then the cells were incubated for additional 12hr and then RNA was prepared using RNeazy column (QIAGEN). Five μg of prepared RNA was subjected to the gene expression analysis using Human Genome U133 Plus 2.0 (Affymetrix) followed by characterizations with GeneSpring software (Agilent) and Pathway Analysis software (Ingenuity). In this analysis we employ two individual siRNA against each CDK and three replicates in each condition.

ORGANISM(S): Homo sapiens

SUBMITTER: Taiki Tsutsui

PROVIDER: E-GEOD-45210 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Knockdown effect of CDK8 or CDK19 in HeLa S3 cell

Project description:Mediator complex has been known as pivotal regulator of RNA polymerase II. Mediator complex has two CDK subunits in vertebrates, named CDK8 and CDK19. To elucidate functional difference between CDK8 and CDK19 in human cell, we employ siRNA mediate knockdown assay using HeLa S3 cell line. According to this assay these CDKs possess highly redundancy in HeLa S3 cell transcription regulation mechanism but in several genes, each CDK shows gene specific regulatory function. HeLa S3 cells were seeded on 6-well plate at 4 x 104 cells per well in 2ml antibiotic free DMEM medium before 24hr transfection. We transfect siRNA using Lipofectamine 2000 (invitrogen) according to its manual and siRNA final concentration in the medium was 20nM. After culturing for 48hr, transfection medium was changed to fresh antibiotic containing medium. Then the cells were incubated for additional 12hr and then RNA was prepared using RNeazy column (QIAGEN). Five μg of prepared RNA was subjected to the gene expression analysis using Human Genome U133 Plus 2.0 (Affymetrix) followed by characterizations with GeneSpring software (Agilent) and Pathway Analysis software (Ingenuity). In this analysis we employ two individual siRNA against each CDK and three replicates in each condition.

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12hr 5-FU treatment vs. DMSO in SJSA cells (from 'A kinase independent role for CDK19 in p53 response')

Project description:The human Mediator complex regulates RNA Polymerase II transcription genomewide. A general factor that regulates Mediator function is the four-subunit Mediator kinase module, which contains either CDK8 or CDK19. Whereas CDK8 has been linked to specific signaling cascades and oncogenesis, the cellular roles of its paralog, CDK19, are poorly studied. To define cellular roles for CDK19, we used osteosarcoma cells (SJSA) that naturally lack endogenous CDK8 protein. Although stable CDK19 knockdown was tolerated in SJSA cells, it caused reduced proliferation vs. control shRNA cells. Global gene expression analyses (RNA-Seq) suggested that defects in cholesterol biosynthesis contributed to reduced proliferation in CDK19 knockdown cells (vs. shCTRL). Notably, proliferation defects were rescued with transient expression of wild-type or kinase-dead CDK19. Using RNA-Seq and other assays, we established a general role for CDK19 in the transcriptional response to 5-fluorouracil (5-FU), an inducer of genotoxic and metabolic stress. These experiments also implicated CDK19 in activation of p53 target genes during 5-FU treatment. To further probe a potential role for CDK19 in the p53 response, SJSA cells (shCDK19 vs. shCTRL) were treated with the p53 activator Nutlin-3. Remarkably, CDK19 was required for SJSA cells to return to a proliferative state following Nutlin-3 treatment, and this effect was kinase-independent. These results implicate CDK19 as a regulator of p53 stress responses and suggest a manifestation of CDK19 knockdown—potentially reduced levels of cholesterol metabolites—blocks cellular resistance to Nutlin-3.

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24hr CA treatment vs. DMSO in HCT116 cells (from 'Identification of CDK8 and CDK19 substrates in human cells using cortistatin A and quantitative phosphoproteomics')

Project description:Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we report here the first large-scale identification of Mediator kinase substrates in human cells (HCT116). Among over 16,000 quantified phosphosites, we identified 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-Seq data correlated with Mediator kinase targets, CA effects on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, which tracked about 7,000 proteins across six time points (0 â?? 24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation; contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest cellular roles beyond transcription, including metabolism and DNA repair. HCT116 cells were treated with either 100nM CA or DMSO in biological triplicate for each population (6 samples total). Treatment was for 24h for compound and vehicle.

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Identification of CDK8 and CDK19 substrates in human cells using cortistatin A and quantitative phosphoproteomics

Project description:Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we report here the first large-scale identification of Mediator kinase substrates in human cells (HCT116). Among over 16,000 quantified sites, we identified 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, CA effects on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, which tracked about 7,000 proteins across six time points (0-24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation; contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest roles beyond transcription, including metabolism and DNA repair.

2018-10-24 | PXD003698 | Pride

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