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target postnatal promoter public health relevance transcription factor transcriptome sequencing tumor tumorigenesis unpublished works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（由适用提供）：肿瘤抑制p53隐含在人类癌症中起关键作用。 p53将其大部分活性作为调节基因表达的转录因子。它的C末端结构域（CTD）在碱性氨基酸中高度富集，并且经过广泛的翻译后修饰。 C-末端已被广泛研究，因为在与DNA和蛋白质的相互作用中发挥了作用，从而调节了p53的活性基因表达。然而，对CTD如何对p53功能的贡献的充分理解仍然难以捉摸。拟议的研究将基于曼弗雷迪的初步和发表的工作，并赞扬实验室，以阐明CTD在有机稳态，肿瘤抑制和肿瘤发生中的作用。这三个具体目标将为CTD在细胞和小鼠中的角色提供前所未有的照明。 AIM 1建立在Manfredi产生的敲入小鼠中的数据，其中CTD的删除导致产后致死性（Hamard等人，2013。GenesDev 27，1868）。 Manfredi实验室将分析p53 CTD的丧失如何影响不同组织中的基因表达，并将鉴定以组织特定方式结合和调节CTD的细胞蛋白。 AIM 2基于Prives Lab的最新发现（Laptenko等人，2015。MolCell 57，1034），表明CTD是P53需要与所选靶基因内的位点结合所必需的，并且CTD在中心核心域中导致稳定p53-DNA复合物形成的中心核心结构域中的结构变化。 Prives Lab将获得有关需要CTD才能获得最佳结合的DNA序列曲目的数据，并将确定需要CTD的DNA中的核心域基础接触。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，而Prives将使用基因编辑来改变人类细胞系以从内源性p53基因座中表达p53ΔCTD或RE/ΔCTD。 Manfredi组将产生RE/ΔCTD小鼠，并确定这是否会影响他们先前报道的病理和基因表达。 AIM 3将提供有关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）的洞察力，这是基于Prives组的先前研究（Freed-Pastor等，2012。Cell 148，244）。 Prives组的最近未发表的工作表明，突变体p53可以影响VEGFR2的表达，VEGFR2是肿瘤血管生成所需的VEGF受体。他们发现，突变体p53与需要SWI/SNF染色质重塑复合物的VEGFR2启动子处的核体损失结合，并且是必需的。为了确定CTD是否以及如何在致癌功能的突变体p53增益中发挥作用，Prives将确定突变体p53是否需要CTD的VEGFR2表达，是否需要CTD来重塑VEGR2启动子，以及是否需要与SWI/SNF合作。 PRIVE还将确定需要CTD和哪些途径CTD依赖性的突变体p53全球调节的基因。曼弗雷迪（Manfredi）将产生有或没有CTD的敲门突变体P53小鼠，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。