Transcriptional repression in response to p53

描述（由申请人提供）DNA损伤诱导细胞周期的G2期停滞，这是从酵母到人类的保守反应。在动物细胞中，p53确保稳定地维持停滞，并且细胞不会进入具有受损DMA的有丝分裂，这可能损害基因组完整性并有助于肿瘤发生。p53促进稳定停滞的一种方式是触发有丝分裂通常所需的蛋白质的下调。这种作用需要Rb家族蛋白与E2 F蛋白形成复合物并抑制细胞周期调控基因的转录。我们对cdc 2和plkl启动子的分析表明，p53的抑制需要一个先前鉴定的称为CDE/CHR. CDE部分类似于E2 F位点的DMA元件，而CD 2F没有相似性。在我们的第一个具体的目的，我们建议使用染色质免疫沉淀（ChIP），以确定是否Rb和E2 F蛋白与CDE/CDE区域的cdc 2和plkl启动子在体内p53表达诱导时。发现cdc 2和plkl启动子的CDE/E2 F元件非常接近转录的起始位点，这提高了Rb/E2 F与这些元件的结合可能在物理上阻断前起始复合物的组分的缔合的可能性。该假设将在我们的第二个目标中使用ChIP和启动子修饰进行测试。 其他研究小组已经提出，在几个启动子中的CCAAT元件，包括cdc 2和plkl，对于p53的抑制是重要的。在我们的第三个目标中，我们提出使用ChIP和凝胶位移分析来分析过表达p53的细胞中的cdc 2和plkl启动子，以检验该模型。这些研究将提供有关p53抑制有丝分裂所需的两个基因的机制的详细信息，并可能揭示Rb/E2 F调节基因表达的新机制。由于p53在癌症中经常发生突变，我们的研究应该为肿瘤发生过程中基因表达如何紊乱提供新的见解。

DESCRIPTION (provided by applicant) DNA damage induces arrest in the G2 phase of the cell cycle, a response conserved from yeast to humans. In animal cells, p53 ensures that the arrest is stably maintained and cells do not enter mitosis with damaged DMA, which could compromise genomic integrity and contribute to tumorigenesis. One way that p53 contributes to the stable arrest is by triggering the down regulation of proteins normally needed for mitosis. This effect requires Rb family proteins which form a complex with E2F proteins and repress the transcription of cell cycle-regulated genes. Our analysis of the cdc2 and plkl promoters shows that repression by p53 requires a previously identified DMA element called the CDE/CHR. The CDE partially resembles an E2F site, while the CHR has no similarity. In our first Specific Aim we propose to use chromatin immunoprecipitation (ChlP) to determine if Rb and E2F proteins are associated with the CDE/CHR regions of the cdc2 and plkl promoters in vivo when p53 expression is induced. The CDE/CHR elements of the cdc2 and plkl promoters are found very close to the start sites of transcription raising the possibility that binding of Rb/E2F to these elements may physcially block the association of components of the preinitiation complex. This hypothesis will be tested in our second Aim using ChlP and promoter modification. Other groups have suggested that CCAAT elements in several promoters, including cdc2 and plkl are important for repression by p53. In our third Aim, we propose to use ChlP and gel shift analysis to analyze the cdc2 and plkl promoters in cells overexpressing p53 to examine this model. These studies will provide detailed information about the mechanisms used by p53 to repress two genes required for mitosis, and may uncover new mechanisms used by Rb/E2F to regulate gene expression. Since p53 is frequently mutated in cancer, our studies should provide new insight into how gene expression is deranged during tumorigenesis.