Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Fred Dick

Abstract

This thesis investigates mechanistic links between genome integrity and the recruitment of chromatin organizing proteins to repetitive DNA sequences mediated by the retinoblastoma tumor suppressor protein (pRB). I demonstrate that a CDK-resistant interaction between the pRB C-terminus and the E2F1 coiled-coil marked box domain establishes a scaffold that facilitates recruitment of multiple chromatin-organizing proteins to repetitive sequences across the genome throughout the cell cycle. Specifically, pRB recruits the enhancer-of-zeste-homologue 2 (EZH2) histone methyltransferase to establish repressive facultative heterochromatin at repetitive sequences, and the Condensin II complex to ensure proper DNA replication and mitotic progression. To disrupt the CDK-resistant pRB-E2F1 interaction in vivo, a gene-targeted mutant mouse strain bearing a germline F832A substitution (Rb1S) is generated. Viable homozygous mutants permit exploration of CDK-resistant pRB-E2F1 functions in cell culture and in vivo. Rb1S/S MEFs and adult splenocytes exhibit pronounced misregulation of endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs) and major satellites (MaSats). Misexpression is associated with reduced co-occupancy of pRB and EZH2, along with reduced H3K27me3 at repetitive genomic regions but not developmental H3K27me3 targets. Furthermore, Rb1S/S MEFs exhibit increased γH2AX, aneuploidy, ppRPA32, and chromosome segregation errors. γH2AX accumulates specifically at major satellites that exhibit reduced co-occupancy of pRB and Condensin II. Collectively, the consequences of perturbed EZH2 and Condensin II recruitment contribute to a state of genomic instability in Rb1S/S cells that likely underlie the onset of spontaneous lymphomas that arise from the spleen or mesenteric lymph nodes of aged homozygous mutant mice. Finally, I explore whether the pRB-E2F1 scaffold provides an opportunity for therapeutic exploitation, and whether these properties direct alter tumor phenotypes in combination with p53 inactivation. Overall, this work suggests that chromatin-organization mediated through the CDK-resistant pRB-E2F1 complex underscores a previously unknown facet of pRB-mediated tumor suppression.

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