Investigations into the Role of TAF1-mediated Phosphorylation in Gene Regulation

In response to cellular stresses, the transcription factor p53, a tumor suppressor, controls cell cycle arrest, DNA repair, or senescence by modulating the expression of target genes. Phosphorylation of p53 at threonine-residue 55 (p53-Thr55) by the TBP-associated factor 1 (TAF1), a subunit of the general transcription factor TFIID, suppresses the interaction of p53 with target genes and promotes nuclear export of p53, resulting in degradation of p53 and G1 cell cycle progression. TAF1-mediated p53-Thr55 phosphorylation in response to UV damage or high glucose (HG) treatment terminates the transcription of p21, an important regulator of G1 progression. In Chapter 2, as a first step to identify additional p53 target genes regulated by TAF1-mediated p53-Thr55 phosphorylation through genome-wide transcription and protein-DNA interaction assays, I recapitulated the HG-induced, TAF1-, and p53-dependent repression of p21 and confirmed the conditions under which maximal repression of p21 can be observed. Furthermore, my preliminary results suggest that HG can alter p53 protein levels in response to UV damage and provide a model for how HG increases the cancer risk of diabetics.In Chapter 3, I collected lists of p53- and TAF1- target genes, and HG-regulated genes via bioinformatics. I identified a set of genes commonly activated in response to different stress conditions: 12 p53-activated and 11 p53-repressed TAF1 target genes differentially expressed by HG, suggesting that these genes are regulated via TAF1-mediated p53-Thr55 phosphorylation. Furthermore, I describe a number of transcription factors representing putative targets for regulation via TAF-mediated phosphorylation.In Chapter 4, I describe tools to further investigate the relationship between TAF1 and p53. A 2x3'BS p21-LUC construct shall allow discernment of the role of different p53 DNA binding sites in the differential recruitment of TAF1 by DNA bound p53. Two TAF1 kinase mutant expression constructs shall facilitate the future elucidation of the TAF1 kinase domain responsible for p53-Thr55 phosphorylation in vivo. Finally, my preliminary results suggest that the p53 target gene NOXA is regulated via TAF1-mediated p53-Thr55 phosphorylation in response to UV, indicating that TAF1-mediated phosphorylation of p53-Thr55 may be involved in regulating the expression of a subset of p53 target genes