Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dick, Fred A.

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, Rb1L, 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.

Summary for Lay Audience

In human cells, maintaining the integrity of DNA is critical to preserve proper function and health. If genomic instability does occur, it can lead to cancer. It has recently been discovered that the retinoblastoma protein (pRB), typically known for its role in regulating cellular growth rates to provide protection against cancer, also aids in the maintenance of genome stability. Intriguingly, mutations to pRB can arise in cancer cells where cellular growth is already compromised. This suggests that pRB’s functions in genome stability could underlie cancer relevant characteristics that are independent of its ability to regulate cellular growth rates. The overall aim of this thesis is to characterize the different means through which pRB contributes to the preservation of genome integrity. To investigate this, pRB mutations were created in a number of cancer cell lines. Cells with pRB mutations have increased basal levels of DNA damage and increased errors when cells are dividing. When transplanted into immune compromised mice, cells with pRB mutation have an increased capacity to form new tumors in the lungs. This thesis also uses a mouse model with a targeted mutation to investigate the genome stability functions of pRB when it is in a complex with another protein, condensin II, which is known to fold DNA to package it tightly. I found that condensin II and another protein known to alter DNA packaging, TFIIIC, are both reliant on pRB to be recruited to the beginning of genes facing opposite directions. Recruitment of condensin II and TFIIIC at these locations is required for proper DNA packaging and gene expression. In addition, pRB deletion in cancer causes similar misexpression of genes at these locations, indicating that loss of pRB and hence condensin II and TFIIIC localization impacts gene expression in cancer cells. Overall, this work demonstrates that beyond altered cellular growth rates, loss of pRB can also contribute to cancer progression through enhanced DNA damage and altered DNA packaging. In the future, these recently discovered characteristics could be used to select the best therapeutic tools for patients with pRB loss.

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