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

“促凋亡基因转录中 p53K120 乙酰化的基因特异性功能的分子基础” 项目概要 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”中，p53 被各种遗传毒性、致癌性和代谢应激（其中包括 其他）。作为一种传统的位点特异性 DNA 结合转录激活剂，p53 可激活多种 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、 自我调节和新陈代谢。与其他转录激活因子一样，增强子/启动子结合的 p53 相互作用 具有多种共激活剂，包括染色质重塑/组蛋白修饰因子和其他因子（例如， 介导与 RNA 聚合酶 II 和一般转录的直接通讯的介体和 TAF 核心启动子的起始因子（GTF）。为了应对压力，p53 的功能受到许多后处理的影响。 翻译修饰包括磷酸化、乙酰化和甲基化。两大成果 DNA 损伤介导的 p53 激活是细胞周期停滞和细胞凋亡。选择的依据 应激反应中的细胞周期停滞和细胞凋亡尚不清楚，但部分取决于应激反应的程度 压力和细胞环境。一些研究报告了选择性参与 p53- 的辅因子 促凋亡基因的依赖转录，尽管其基因选择性的机制 功能不明确。两个研究小组表明 DNA 损伤诱导的 p53 K120 乙酰化对于 关键促凋亡基因（PUMA 和 BAX）的转录，但不促凋亡基因 (p21) 或 p53 自动调节基因 (HDM2) 的转录 基因。进一步观察发现，p53 与 PUMA 和 BAX 的结合不依赖于 K120 乙酰化，导致 p53K120ac 通过与特定辅因子相互作用发挥作用的假设，该辅因子可能是 先前确定的促凋亡因素或尚未确定的因素。拟议项目将寻求确定 并阐明直接介导促凋亡基因特异性功能的辅助因子的作用机制 p53K120ac。在目标 1 中，将采用基于 DNA 损伤的 HCT116 细胞的生化和细胞方法 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 ChIP 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 辅因子的细胞内促凋亡基因特异性功能，(ii) 与辅因子关联的动力学 PUMA 和 BAX 与 p21 和 MDM2 基因，(iii) p53K120ac 辅因子依赖的基因特异性转录 PUMA 体外实验； (iv) p53K120ac 和相关辅因子通过各种途径的作用机制 蛋白质-蛋白质相互作用。这些研究将为 p53 在肿瘤中的关键促凋亡功能提供新的线索 镇压。

“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.