Molecular basis of gene-specific function of p53K120 acetylation in proapoptotic gene transcription - Resubmission - 1

p53K120乙酰化在促凋亡基因转录中的基因特异性功能的分子基础

“Molecular basis of gene-specific function of p53K120 acetylation in proapoptotic gene transcription” PROJECT SUMMARY p53 is a tumor suppressor whose gene is the most commonly mutated gene in human cancer. As the “guardian of the genome”, p53 is stabilized and activated by various genotoxic, oncogenic and metabolic stresses (amongst others). As a conventional site-specific DNA-binding transcriptional activator, p53 activates a broad array of target genes that in turn regulate many cellular processes that include cell cycle arrest, DNA repair, apoptosis, auto-regulation, and metabolism. Like other transcriptional activators, enhancer/promoter-bound p53 interacts with diverse co-activators that include chromatin remodeling/histone modifying factors and other factors (e.g., Mediator and TAFs) that mediate direct communication with RNA polymerase II and the general transcription initiation factors (GTFs) at core promoters. In response to stress, the function of p53 is subject to numerous post- translational modifications that include phosphorylation, acetylation and methylation. Two major outcomes of DNA damage-mediated activation of p53 are cell-cycle arrest and apoptosis. The basis for the choice between cell-cycle arrest and apoptosis in a stress response is poorly understood, but dependent in part on the extent of the stress and the cellular context. Several studies have reported cofactors that are selectively involved in p53- dependent transcription of proapoptotic genes, although the mechanisms underlying their gene-selective functions are not clear. Two groups have shown that DNA damage-induced p53 K120 acetylation is critical for transcription of key proapoptotic genes (PUMA and BAX) but not proarrest (p21) or p53 autoregulatory (HDM2) genes. The further observation that p53 binding to PUMA and BAX is independent of K120 acetylation leads to the hypothesis that p53K120ac acts through interactions with a specific cofactor(s), potentially one of the previously identified proapoptotic factors or an as yet unidentified factor. The proposed project will seek to identify and elucidate the mechanism of action of cofactors that directly mediate the pro-apoptotic gene-specific functions of p53K120ac. In Aim 1, biochemical and cell-based approaches with DNA-damaged HCT116 cells will be employed to identify and validate (i) proapoptotic gene-associated factors dependent upon p53 K120 acetylation (by ChIP analysis) and (ii) p53K120ac-interacting/associated proteins (by proteomic analyses). In Aim 2, integrated cell-based (gene editing/protein knockdown) and biochemical approaches will be used to establish (i) intracellular proapoptotic gene-specific functions of p53K120ac cofactors, (ii) kinetics of cofactor association with PUMA and BAX versus p21 and MDM2 genes, (iii) p53K120ac cofactor-dependent, gene-specific transcription of PUMA in vitro; and (iv) the mechanism of action of p53K120ac and associated cofactors through various protein-protein interactions. These studies will shed new light on a key proapoptotic function of p53 in tumor suppression.