Knockdown effect of CDK8 or CDK19 in HeLa S3 cell
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE32108 | GEO | 2011/09/15
SECONDARY ACCESSION(S): PRJNA147465
REPOSITORIES: GEO
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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.
2013-03-16 | GSE45210 | GEO
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.
2011-09-14 | E-GEOD-32108 | biostudies-arrayexpress
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.
2013-03-16 | E-GEOD-45210 | biostudies-arrayexpress
Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
2015-10-26 | E-GEOD-67847 | biostudies-arrayexpress
Project description:Transcriptional responses to interferon require Mediator kinase-dependent pause release and mechanistically distinct functions of CDK8 and CDK19.
2019-07-15 | GSE129501 | GEO
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.
2017-05-09 | GSE89807 | GEO
Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Colo205 colon cancer cells were treated with 350 nM of Compound 1 or with vehicle (DMSO) for 2 or 6h. Four samples with four biological repeats each. Samples were hybridized against human reference. Compound 1 is a close analogue of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. It was shown to be potent and selective inhibitor of the Mediator complex-associated protein kinases CDK8 and CDK19. Its is ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
2015-10-26 | E-GEOD-67845 | biostudies-arrayexpress
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
Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. LS-174-T colon cancer cells were treated with 715 nM of Compound 6 (IC90 of TCF dependent luciferase) for 0, 4 or 24h. Three samples with three biological repeats each. Sample were hybridized against human reference. Compound 6 is a close analogue of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. It was shown to be potent and selective inhibitor of the Mediator complex-associated protein kinases CDK8 and CDK19. Its is ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
2015-10-26 | E-GEOD-67846 | biostudies-arrayexpress
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.
2016-02-26 | E-GEOD-78506 | biostudies-arrayexpress