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.
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.
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.
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.
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.
Project description:When CDK19 was initially discovered, it was assumed that CDK19 played a redundant role to CDK8 because they share 84% amino acid sequence similarity. However, biological clues such as CDK19s different chromosomal location and its more limited tissue distribution suggested a role distinct from CDK8 To investigate whether CDK19 and CDK8 had distinct biological functions in Triple-Negative breast cancer, we knocked down CDK19 and CDK8 in MDA-MB231 and examined differences in the resulting gene expression.