Date of Award
Doctor of Philosophy (PhD)
Dylan J. Taatjes
Corrella S. Detweiler
Norman R. Pace
Robin D. Dowell
The Mediator complex is a required co-activator of RNA Polymerase II (Pol II), the enzyme responsible for the transcription of all protein coding genes in eukaryotes. Mediator facilitates transcription factor-dependent gene expression by directly interacting with gene specific transcription factors and Pol II and the general transcription machinery. Mediator consists of up to 29 subunits; a four-subunit “CDK module” reversibly associates with Core-Mediator and alters its structure and activity. Most of the work on the CDK module has been done in yeast, and has focused on the CDK module subunits CDK8, MED12, and MED13. These three subunits have paralog proteins CDK19, MED12L, and MED13L that are present only in vertebrates. Information about these CDK module paralog subunits is limited. CDK19, the paralog of CDK8, associates with all other CDK module subunits and with Core-Mediator. As one of the only two enzymatic subunits of Mediator, the CDK19 kinase likely has important roles in transcription, but these roles remain uncharacterized.
Here I initiated experiments to define a role for CDK19 in human cancer cells. Using osteosarcoma cells (SJSA) that naturally lack endogenous CDK8 protein, I have identified a role for CDK19 in cellular proliferation under standard growth conditions, and I have demonstrated that this function is not dependent upon its kinase activity. Furthermore, using a combination of RNA-Seq and qRT-PCR in SJSA cells with stable CDK19 knockdown, I have established evidence of a role for CDK19 in the transcriptional response to 5-fluorouracil (5-FU), an inducer of genotoxic and metabolic stress. These experiments also revealed a specific requirement for the CDK19 protein in the activation of p53 target genes during 5-FU treatment. To further probe a potential role for CDK19 in the p53 response, I completed studies in control vs. CDK19 knockdown cells treated with Nutlin, a highly specific, well-tested activator of p53. Remarkably, I observed that the presence of CDK19 enhanced survival in Nutlin-treated cells and was required for SJSA cells to return to a proliferative state following Nutlin treatment. As with the general proliferative defect under normal growth conditions, this effect was observed to be kinase-independent. Taken together, the results summarized herein implicate CDK19 as a key regulator of the p53 stress response in SJSA cells. These data also suggest that in cancers that are sensitive to Nutlin, concomitant targeting of CDK19 might represent an effective therapeutic strategy.
Audetat, Katherine Audrey, "Regulation of Gene Expression by the Mediator Kinase Cdk19" (2016). Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations. 76.