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基因特异性功能的分子基础 项目总结 P53是一种肿瘤抑制基因，其基因是人类癌症中最常见的突变基因。作为“守护者” 基因组“，p53被各种遗传毒性、致癌和代谢应激稳定和激活(包括 其他)。作为一种传统的位点特异性DNA结合转录激活剂，p53激活了一系列广泛的 靶基因反过来调节许多细胞过程，包括细胞周期停滞、DNA修复、细胞凋亡、 自动调节和新陈代谢。像其他转录激活子一样，增强子/启动子结合的p53相互作用 具有包括染色质重塑/组蛋白修饰因子和其他因素的多种共激活剂(例如， 介体和TAFs)，介导与RNA聚合酶II的直接通信和一般转录 核心启动子的启动因子(GTF)。作为对压力的反应，P53的功能受到大量的后 翻译修饰，包括磷酸化、乙酰化和甲基化。两个主要成果 DNA损伤介导的P53激活主要是细胞周期停滞和细胞凋亡。做出选择的基础是 对应激反应中的细胞周期停滞和细胞凋亡知之甚少，但这在一定程度上取决于 压力和细胞环境。几项研究已经报道了选择性地参与P53的辅因子- 促凋亡基因的依赖转录，尽管其基因选择性的潜在机制 功能不明确。两个研究小组已经证明，DNA损伤诱导的P53 K120乙酰化对 关键促凋亡基因(PUMA和Bax)的转录，而不是提前停滞(P21)或p53自身调节(Hdm2) 基因。进一步观察到PUMA和Bax与PUMA和Bax结合不依赖K120乙酰化导致 假设p53K120ac通过与特定辅因子(S)的相互作用发挥作用，该辅因子可能是 以前已确定的促凋亡因素或尚未确定的因素。拟议的项目将寻求确定 并阐明了直接介导促凋亡基因特异性功能的辅因子的作用机制 P53K120ac。在目标1中，对DNA受损的HCT116细胞进行生化和基于细胞的方法将是 用于识别和验证(I)依赖于p53 K120乙酰化的促凋亡基因相关因子 (通过芯片分析)和(Ii)p53K120ac相互作用/相关蛋白(通过蛋白质组分析)。在目标2中， 将使用基于细胞的综合方法(基因编辑/蛋白质敲除)和生化方法来建立(I) P53K120ac辅因子在细胞内促凋亡基因的特异性功能，(II)辅因子与 彪马和bax与p21和mdm2基因的比较，(Iii)p53K120ac辅因子依赖的基因特异性转录 以及(Iv)p53K120ac及其相关辅因子通过多种途径的作用机制。 蛋白质之间的相互作用。这些研究将为肿瘤中P53的关键促凋亡功能提供新的线索。 压制。

“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.