Interactions of p53 and p73 with human promoters

The p53 family of transcription factors, p53, p63, and p73, mediates both common and differential biological functions. While p53 has its crucial role in tumor suppression, p63 and p73 have essential functions in embryonic development and differentiation control. This dissertation describes the use of chromatin immunoprecipitation coupled with microarray analysis (ChIP -chip) to study the promoter specificity of p53 and p73. A direct comparison of the promoter occupancy profiles of p53 and p73 was performed in the same cellular context, both at steady state and following hydroxyurea (HU) treatment. As expected, we found that p53 and p73 have overlapping and distinct promoter occupancy and target gene expression profiles under both conditions. Interestingly, HU appears to alter the promoter occupancy profile of p73 more than that of p53. Moreover, our results demonstrated that p53 and p73 are likely to associate with the same 250 bp genomic region in their overlapping promoters. Expression profiling in cells depleted for both p53 and p73 suggests that ̃10% of the p53 and p73 binding sites show p53 and p73- dependent transcriptional effects. Together with our motif analysis, our results suggest that p53 and p73 can also interact indirectly with the promoter-proximal region possibly via protein-protein interactions with other transcription factors. Chapter 1 introduces the common and differential functions of the p53 family members and summarizes the current high-throughput technologies employed to identify protein-DNA interactions. Chapter 2 describes a small- scale comparison of p53 and p73 promoter occupancy profiles using the ChIP-DSL technology. Chapter 3 describes a detailed comparison of p53 and p73 interactions with the promoter-proximal region using ChIP- coupled with the NimbleGen 1.5 kb human promoter arrays. It also introduces a model-based algorithm and other bioinformatics tools for the identification and characterization of the in vivo binding sites of p53 and p73. Finally, integration of ChIP-chip data with target gene expression analysis led to the identification of potential direct transcriptional targets of p53 and p73. Chapter 4 discusses the possible mechanisms by which the p53 family members mediate differential target gene specificity and presents some of the future challenges for ChIP-chip studie