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

基本信息

批准号：
9195074
负责人：
James J Manfredi
金额：
$ 55.97万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
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/9195074
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 Expression Profiling 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target postnatal promoter public health relevance transcription factor transcriptome sequencing tumor tumorigenesis unpublished works

项目摘要

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 在人类癌症中发挥着关键作用，其大部分活性是作为调节基因表达的转录因子，其 C 末端结构域 (CTD) 在碱性中高度富集。 C 末端通常被研究为在与 DNA 和蛋白质的相互作用中发挥作用，从而调节 p53 的活性。然而，对 CTD 如何促进 p53 功能的全面了解仍然难以捉摸。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的基础上，以阐明 CTD 在机体稳态、肿瘤抑制、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阐明。目标 1 建立在敲入小鼠的数据基础上。 Manfredi 认为删除 CTD 会导致出生后致死（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，并将鉴定结合和调节的细胞蛋白。以组织特异性方式进行 CTD 目标 2 基于 Prives 实验室的最新发现（Laptenko 等人，2015 年。Mol Cell 57， 1034）表明 p53 需要 CTD 才能与选定的靶基因内的位点结合，并且 CTD 会导致中央核心结构域发生结构变化，从而稳定 p53-DNA 复合物的形成。 Prives 实验室将获得有关 DNA 序列库的数据。需要 CTD 才能实现最佳结合，并将确定 DNA 内需要 CTD 的核心域碱基接触。核心域突变体 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 小组将生成 RE/ΔCTD 小鼠，并确定这是否会影响他们之前报道的病理学和基因表达。目标 3 将基于 Prives 小组之前的研究，深入了解 CTD 如何影响肿瘤衍生突变体 p53 以促进肿瘤发生（功能获得活性） (Freed-Pastor et al. 2012. Cell 148, 244) Prives 小组最近未发表的研究表明，突变型 p53 可以诱导 VEGFR2 的表达，VEGFR2 是肿瘤血管生成所需的 VEGF 受体。 VEGFR2 启动子处的核小体丢失需要 SWI/SNF 染色质重塑复合物来确定是否和。 CTD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配合。 Prives 还将鉴定需要 CTD 的突变 p53 全局调控的基因，以及哪些途径依赖于 CTD，Manfredi 将产生带有或不带有 CTD 的敲入突变 p53 小鼠。 CTD 并探索其在体内肿瘤发生中的作用，长期目标是将这些发现与癌症联系起来。