| P53 is a tumour suppressor gene found mutated in over half of all human cancers. Centred at the core of a complex signaling pathway, in response to various cellular stressors p53 functions as a transcription factor, and is able to transactivate dozens of target genes involved in cell cycle arrest, apoptosis, and others. How p53 is able to discriminate between these different cellular outcomes genes is complex, and remains an area of intense study. In this thesis, I describe aspects of stimulus-specific regulation of a particular p53 target, p21, as well novel mutations of p53 that predispose the protein to induce a particular cellular outcome.;First, previous work in our lab has shown that p53 activates distinct transcriptional programs in response to replication stress induced by hydroxyurea (HU) and DNA damage induced by daunorubicin. This phenomenon occurs despite the equal induction of p53 protein after both treatments. Specifically, in response to HU treatment, p21 mRNA production is blocked at the level of transcription elongation. In this thesis, I provide data supporting a direct role for the checkpoint kinase Chk1, but not Chk2, in effecting this block to p21 transcription after replication stress. Using high-resolution quantitative chromatin immunoprecipitation, I show that downregulating the ATR/Chk1 axis is able to rescue transcription elongation at p21 after S-phase block, and that this correlates with the increased recruitment of various transcription elongation and 3' mRNA processing factors to the locus.;Second, I show that two novel mutations within the tetramerization domain of p53, K351 and K357Q (2KQ), severely impair the protein's ability to bind and transactivate its target genes when expressed at physiological levels. Curiously, 2KQ is nonetheless able to effect apoptosis when stimulated by 5-FU. Furthermore, when massively overexpressed, 2KQ is able to induce higher amounts of p21 protein than WT p53, yet surprisingly is still unable to arrest cells in G1. This may be due to the fact that even when highly expressed, 2KQ cannot transactivate miR-34a or CCNG1. Finally, I show that these tetramerization lysines can be acetylated in vivo by p300/CBP, perhaps elucidating a mechanism by which p53 can be shunted away from its cell cycle arrest program in certain scenarios.;In summary, this thesis describes a novel mechanism by which a checkpoint kinase can influence the stimulus-specific regulation of p53 target genes, as well as mutations which impact upon the promoter selectivity of p53. The data presented herein contribute to the growing body of knowledge concerning the regulation of p53 target gene selectivity. |
