Role of the p53 C-terminal domain in tissue homeostasis, tumor suppression, and oncogenesis

 描述(申请人提供)：肿瘤抑制基因P53被认为在人类癌症中起关键作用。P53作为一种转录因子在很大程度上发挥着调节基因表达的作用。它的C末端结构域(CTD)富含碱性氨基酸，并被广泛的翻译后修饰。人们广泛研究C末端在与DNA和蛋白质相互作用中的作用，从而调节P53的活性。 基因表达。然而，对于CTD如何参与P53功能的充分理解仍然是个未知数。这项拟议的研究将建立在Manfredi和Prives实验室的初步工作和已发表的工作的基础上，以澄清CTD在生物体内平衡、肿瘤抑制和肿瘤发生中的作用。这三个特定的目标将为CTD在体外、细胞和小鼠中的作用提供前所未有的启发。目标1建立在Manfredi产生的敲入小鼠的数据基础上，其中CTD缺失会导致出生后死亡(Hamard等人。2013年。基因发展史27,1868)。曼弗雷迪实验室将分析P53 CTD的缺失如何影响不同组织中的基因表达，并将识别以组织特有的方式结合和调节CTD的细胞蛋白质。AIM 2基于Prives实验室的最新发现(Laptenko等人。2015年。Mol Cell 57,1034)表明，CTD是p53与选定靶基因内的位点结合所必需的，并且CTD导致中央核心域的结构变化，从而稳定p53-DNA复合体的形成。Prives实验室将获得有关需要CTD才能最佳结合的DNA序列的数据，并将确定DNA中需要CTD的核心结构域碱基接触。核心区突变体P53(简称RE)在体外可以弥补CTD缺失的缺陷，Prives将利用基因编辑来改变人类细胞系，使其表达P53ΔCTD或来自内源性P53基因的RE/ΔCTD。曼弗雷迪研究小组将培育一只RE/Δ慢性结缔组织病小鼠，并确定这是否会影响他们之前报告的病理和基因表达。目的3将在Prives小组(Freed-Pastor等人)先前研究的基础上，深入了解CTD如何影响肿瘤衍生突变体p53以促进肿瘤发生(功能活性获得)。2012年。牢房148,244)。Prives小组最近未发表的工作表明，突变的p53可以诱导VEGFR2的表达，VEGFR2是肿瘤血管生成所需的血管内皮生长因子受体。他们发现，突变的p53与VEGFR2启动子上的核小体丢失有关，并且是丢失所需的，这需要SWI/SNF染色质重塑复合体。为了确定CTD是否以及如何在突变型p53获得致癌功能中发挥作用，Prives将确定突变型p53是否需要CTD来表达VEGFR2，是否需要CTD来重塑VEGR2启动子，以及是否需要与SWI/SNF协同作用。PRIVES还将识别由突变型p53全球调控的需要CTD的基因，以及哪些途径是CTD依赖的。Manfredi将产生带有或不带有CTD的敲入突变的p53小鼠，并探索其在体内肿瘤发生中的作用。一个长期目标是将这些发现与癌症联系起来。

 DESCRIPTION (provided by applicant): The tumor suppressor p53 is implicated as playing a key role in human cancer. p53 exerts much of its activity as a transcription factor that regulates gene expression. Its C-terminus domain (CTD) is highly enriched in basic amino acids and is extensively post-translationally modified. The C-terminus has been studied extensively as playing a role in interactions with both DNA and protein, thereby regulating the activity of p53 in gene expression. Nevertheless, a full understanding of how the CTD contributes to p53 function has remained elusive. The proposed research will build on preliminary and published work from the Manfredi and Prives laboratories to clarify the roles of the CTD in organismal homeostasis, tumor suppression, and oncogenesis. The three Specific Aims will provide unprecedented illumination of the roles of the CTD in vitro, in cells and in mice. Aim 1 builds on data from a knock-in mouse generated by Manfredi where deletion of the CTD results in postnatal lethality (Hamard et al. 2013. Genes Dev 27, 1868). The Manfredi lab will analyze how loss of the p53 CTD affects gene expression in different tissues and will identify cellular proteins that bind and regulate the CTD in a tissue specific manner. Aim 2 is based on recent findings from the Prives lab (Laptenko et al. 2015. Mol Cell 57, 1034) showing that the CTD is required for p53 to bind to sites within select target genes and that the CTD causes structural changes in the central core domain that stabilize p53-DNA complex formation. The Prives lab will obtain data about the repertoire of DNA sequences that require the CTD for optimal binding, and will determine the core-domain base contacts within DNA that require the CTD. A core domain mutant p53 (termed RE) can rescue the defects of deletion of CTD in vitro and Prives will use gene editing to alter human cell lines to express either p53 ΔCTD or RE/ΔCTD from the endogenous p53 locus. The Manfredi group will generate a RE/ΔCTD mouse and determine whether this affects the pathology and gene expression that they previously reported. Aim 3 will provide insight into how the CTD impacts tumor-derived mutant p53 to promote oncogenesis (gain-of-function activity), building on previous studies of the Prives group (Freed-Pastor et al. 2012. Cell 148, 244). Recent unpublished work from the Prives group showed that mutant p53 can induce expression of VEGFR2, the VEGF receptor that is needed for tumor angiogenesis. They found that mutant p53 binds to and is needed for loss of nucleosomes at the VEGFR2 promoter that requires the SWI/SNF chromatin remodeling complex. To ascertain whether and how the CTD plays a role in mutant p53 gain of oncogenic function, Prives will determine whether mutant p53 requires the CTD for VEGFR2 expression, whether the CTD is needed for remodeling the VEGR2 promoter, and whether it is needed to cooperate with SWI/SNF. Prives will also identify genes globally regulated by mutant p53 that require the CTD and which pathways are CTD-dependent. Manfredi will generate knock-in mutant p53 mice with or without the CTD and explore its role in tumorigenesis in vivo. A long term goal would be to link such discoveries to cancer.