Doctor of Philosophy
Dr Fred Dick
The Retinoblastoma protein (pRB) is a key regulator of the cell cycle and is functionally inactivated in most cancers. pRB has been proposed to utilize simultaneous interactions with E2F transcription factors and chromatin regulatory proteins to repress transcription and block cell cycle progression. The goal of this study is to characterize the physiological role of pRB interactions with chromatin regulatory proteins. I used gene targeted mice carrying point mutations in the murine Rb1 gene (Rb1∆L) that specifically disrupt pRB’s LXCXE binding cleft, and thereby its ability to interact with chromatin regulatory proteins while leaving its ability to bind E2Fs intact. Embryonic fibroblasts from Rb1∆L/∆L mice fail to properly arrest DNA synthesis in response to senescence inducing signals such as oncogene expression and g Irradiation. Failure to incorporate repressive heterochromatin marks like H3K9me3 results in de-repression of key cell cycle genes during senescence. However, this function of pRB is dispensable during normal differentiation and development, suggesting a specific role during stress responsive cell cycle arrest. Furthermore, during cellular senescence, pRB uses LXCXE binding cleft dependent interactions to recruit Promyelocytic leukemia protein (PML) to E2F target gene promoters. This function of pRB is important for establishment of heterochromatin marks and stable silencing of these genes thereby creating a permanent cell cycle arrest. Disruption of this function of pRB by the ∆L mutation confers susceptibility to escape from senescence and allows transformation in vitro. However, the same mutation does not enhance tumorigenesis in tumor models with activated ras mutations. Rb1∆L/∆L mice expressing oncogenic KrasG12D show delayed lung tumor formation compared to controls, which correlate with increased apoptosis in the early lesions following ras activation. Furthermore, DMBA treatment to induce ras mutations also fail to reveal greater susceptibility to cancer in Rb1∆L/∆L mice suggesting that loss of chromatin regulation by pRB has context dependent outcomes and does not universally enhance tumorigenesis in vivo.
Overall, this thesis enhances our current understanding of the unique role of pRB among the pocket proteins in cell cycle regulation by showing how pRB utilizes LXCXE binding cleft mediated interactions to stably block cell cycle in response to oncogenic stress signals.
Talluri, Srikanth, "Characterizing the role of the Retinoblastoma protein LXCXE binding cleft in cellular senescence and tumor suppression" (2013). Electronic Thesis and Dissertation Repository. 1377.