Functional investigation of the role of the retinoblastoma protein in genome stability

Aren E. Marshall, The University of Western Ontario

Abstract

Genome instability is an enabling characteristic of cancerous cells. It has recently been discovered that the retinoblastoma protein (pRB), typically known for its role in cell cycle regulation, also aids in the maintenance of genome stability. Intriguingly, mutations to the pRB gene, RB1, can arise late in tumorigenesis in cancer cells whose cell cycle regulation is already compromised by another mutation. This suggests that pRB’s functions in genome stability could underlie cancer relevant characteristics that are independent of its ability to negatively regulate proliferation. The overall aim of this thesis is to characterize the different means through which pRB contributes to the preservation of genome integrity. Using CRISPR/Cas9, isogenic RB1 mutant genotypes were created in a number of cancer cell lines. Cells with at least one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. When the underlying origins of these phenotypes were investigated further, I discovered elevated levels of reactive oxygen species as well as impaired homologous recombination repair in cells with RB1 mutations. When xenografted into immune compromised mice, RB1 mutation also results in an increased capacity to seed new tumors in the lungs. This thesis also investigates the functions of the pRB-condensin II complex in maintaining genome stability, specifically in interphase cells. Using a gene-targeted mouse model that disrupts the ability of pRB to recruit condensin II, Rb1^L, locations of pRB-dependent condensin II recruitment were investigated. I found that both condensin II and another architectural protein complex, TFIIIC, are recruited to promoters between bidirectional genes by a mechanism that is reliant on pRB. Recruitment of these architectural proteins at bidirectional promoters is required to establish long-range chromosome interactions and transcriptional insulation between gene pairs. In addition, pRB deletion in cancer causes similar misregulation at divergent promoters, indicating that loss of insulation impacts the transcriptome of cancer cells. Overall, this work demonstrates that beyond altered proliferative control, loss of pRB can also contribute to cancer progression through enhanced DNA damage and altered chromosome topology.