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
