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

p53 C 末端结构域在组织稳态、肿瘤抑制和肿瘤发生中的作用

• 批准号: 9056104
• 负责人: James J Manfredi
• 金额: $ 61.67万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-15 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9056104
• 关键词: Acute; Affect; Autocrine Communication; Basic Amino Acids; Binding; Binding Proteins; Birth; Breast Cancer Cell; C-terminal; Cells; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Complex; Consensus; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Defect; Engineering; Epigenetic Process; Failure; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Hematopoietic; Homeostasis; Human; Human Cell Line; In Vitro; KDR gene; Knock-in; Knock-in Mouse; Knowledge; Laboratories; Lighting; Link; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; Nucleic Acids; Nucleosomes; Oncogenic; Pathology; Pathway interactions; Play; Protein Binding Domain; Protein p53; Proteins; Proteomics; Protocols documentation; Publishing; Reporting; Research; Role; Site; TP53 gene; Tissues; Tumor Angiogenesis; Tumor Suppression; Tumor Suppressor Proteins; Tumor-Derived; Work; base; crosslink; diagnosis design; gain of function; human disease; improved; in vivo; insight; mutant; new therapeutic target; postnatal; promoter; public health relevance; transcription factor; transcriptome sequencing; tumor; tumorigenesis; unpublished works

 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.

 描述（由申请人提供）：肿瘤抑制因子p53在人类癌症中起关键作用。p53作为调节基因表达的转录因子发挥其大部分活性。其C-末端结构域（CTD）高度富集碱性氨基酸，并被广泛地后修饰。已经广泛研究了C-末端在与DNA和蛋白质的相互作用中起作用，从而调节p53在细胞中的活性。 基因表达。然而，对CTD如何促进p53功能的充分理解仍然是难以捉摸的。这项拟议的研究将建立在Manfredi和Prives实验室的初步和已发表的工作基础上，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示。目的1建立在来自Manfredi产生的基因敲入小鼠的数据基础上，其中CTD的缺失导致出生后致死（Hamard et al. 2013. Genes Dev 27，1868）。Manfredi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，并将鉴定以组织特异性方式结合和调节CTD的细胞蛋白。目标2基于Prives实验室的最新发现（Laptenko等人，2015年）。Mol Cell 57，1034），显示CTD是p53结合至所选靶基因内的位点所需的，并且CTD引起稳定p53-DNA复合物形成的中央核心结构域中的结构变化。Prives实验室将获得有关需要CTD以实现最佳结合的DNA序列库的数据，并将确定需要CTD的DNA内核心结构域碱基接触。核心结构域突变体p53（称为RE）可以在体外挽救CTD缺失的缺陷，Prives将使用基因编辑来改变人类细胞系，以从内源性p53基因座表达p53 ΔCTD或RE/ΔCTD。Manfredi小组将产生一只RE/ΔCTD小鼠，并确定这是否会影响他们先前报道的病理学和基因表达。目的3将提供CTD如何影响肿瘤衍生的突变体p53以促进肿瘤发生（功能获得活性）的见解，建立在Prives组的先前研究的基础上（Freed-Pastor et al. 2012.单元148、244）。Prives小组最近未发表的工作表明，突变型p53可以诱导VEGFR 2的表达，VEGFR 2是肿瘤血管生成所需的VEGF受体。他们发现突变型p53与VEGFR 2启动子处的核小体结合，并且是VEGFR 2启动子处的核小体丢失所必需的，VEGFR 2启动子需要SWI/SNF染色质重塑复合物。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用于VEGFR 2表达，CTD是否需要重塑VEGR 2启动子，以及是否需要与SWI/SNF合作。Prives还将识别由突变型p53全局调控的基因，这些基因需要CTD以及哪些途径是CTD依赖的。Manfredi将在有或没有CTD的情况下产生敲入突变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。