Date Approved

5-2021

Document Type

Dissertation

Degree Name

PhD in Cell and Molecular Biology

Department

Molecular Biology

College

Graduate School of Biomedical Sciences

First Advisor

Katrina Cooper, PhD

Committee Member 1

Dmitriy Markov, PhD

Committee Member 2

Hristo Houbaviy, PhD

Committee Member 3

Natalia Shcherbik, PhD

Committee Member 4

Michael Law, PhD

Subject(s)

Cyclin-Dependent Kinase 8, Oxidative Stress, Cell Death, Cyclin C, Transcription, Genetic, Cell Physiological Phenomena, Proteasome Inhibition

Disciplines

Biological Phenomena, Cell Phenomena, and Immunity | Cell Biology | Laboratory and Basic Science Research | Medical Molecular Biology | Medicine and Health Sciences | Molecular Biology

Abstract

In response to various sources of cellular stress, the coordination of intracellular events is necessary to elicit the appropriate molecular response. In particular, the reprogramming of gene expression by stress-specific transcription factors drives the activation of signaling pathways, triggering either cell survival or regulated cell death pathways. The Cdk8 kinase module (CKM) is a highly conserved transcriptional regulatory complex with a role in this decision. The CKM is composed of Cdk8, its activating partner cyclin C, and two scaffold proteins, Med12 and Med13. The CKM is a detachable subunit of the Mediator complex, which interacts with RNA polymerase II to control transcription. While the CKM in yeast is predominantly a repressor of transcription, it plays a dual role in transcription as both an activator and repressor in mammalian cells. A more in-depth analysis of the role of the cyclin C in controlling transcription, particularly how the CKM itself is regulated, encompasses the focus of this thesis.

In addition to its nuclear role in transcription, cyclin C independently relocalizes to the cytoplasm in response to oxidative stress to induce mitochondrial fission and intrinsic programmed cell death (iPCD). Our laboratory revealed that that in yeast, the degradation of Med13 is important for cyclin C nuclear release in response to oxidative stress. The ubiquitin ligase Grr1 is required for the degradation of Med13, as shown here. This study also revealed a complex set of phosphorylation events required for Med13 degradation and nuclear release of cyclin C. Unlike yeast, which release all cyclin C into the cytoplasm, only about 20% of total cyclin C is translocated in mammalian cells. This suggest that cyclin C continues to regulate transcription following oxidative stress. To determine the transcriptional role of cyclin C, RNA sequencing (RNA-seq) studies were performed on Ccnc+/+ and Ccnc-/- mouse embryonic fibroblasts (MEFs) before and after oxidative stress induced by H2O2 treatment. These analyses revealed that the CKM acts equally as both an activator and a repressor of transcription of nearly 5,000 genes. The CKM both positively and negatively controls genes required for cell growth or cell death. These results indicate that oxidative stress either enhances or diminishes the transcriptional impact of cyclin C, in a locus-specific manner. To determine if the CKM regulates transcription through direct interaction with DNA, chromatin immunoprecipitation (ChIP) experiments were utilized. The results shown here revealed the CKM regulates transcription directly through promoter occupancy. In response to oxidative stress, the CKM is released from promoters where its activity in repression is diminished, while simultaneously recruited to other loci requiring additional activation by cyclin C. Using the Cdk8 inhibitor, Senexin A, we found that CKM-dependent activation required kinase activity whereas repression did not. Thus, revealing the CKM controls transcription as an activator or a repressor by separate mechanisms.

The results from our RNA-seq analyses indicated that cyclin C may alter transcription in response to other forms of cellular stress. To determine if the role of cyclin C is stress-specific, we tested the cellular response to autophagy inducing agents, mTOR inhibitor Torin1, or the proteasome inhibitor, MG132. ChIP studies revealed a different pattern of promoter occupancy changes by the CKM in response to Torin1, compared to H2O2 stress. Of particular interest, the CKM activates genes essential for autophagosome-lysosome fusion in response to MG132. Accordingly, Ccnc-/- MEFs or Ccnc-/- pancreatic tumor cells show a significantly increased sensitivity to MG132-induced iPCD. The combination of the autophagosome-lysosome fusion inhibitor chloroquine (CQ) with MG132 resulted in an increase in iPCD. This is suggestive that the increased sensitivity to MG132-induced iPCD in Ccnc-/- MEFs could be due to the observed deficiency of the transcription of genes involved in autophagosome-lysosome fusion. In conclusion, cyclin C is shown here to be a dynamic stress-specific transcriptional regulator with essential roles in the cellular response to both oxidative stress and autophagy induction, and accordingly, the determination of cell fate.

Download

Share

COinS