Transcriptional repression in response to p53

描述(由申请人提供)DNA损伤导致细胞周期的G2期停滞，这是一种从酵母到人类的保守反应。在动物细胞中，P53确保稳定地维持这种停滞，细胞不会因DNA受损而进入有丝分裂，这可能会损害基因组的完整性，并有助于肿瘤的发生。P53有助于稳定停滞的一种方式是通过触发正常情况下有丝分裂所需蛋白质的下调。这种作用需要RB家族蛋白与E2F蛋白形成复合体，抑制细胞周期调控基因的转录。我们对cdc2和plk1启动子的分析表明，p53的抑制需要一个先前发现的称为cde/chr的DMA元件。CDE部分类似于E2F位点，而CHR没有相似之处。在我们的第一个特定目标中，我们建议使用染色质免疫沉淀(ChlP)来确定当P53表达被诱导时，Rb和E2F蛋白是否与体内cdc2和plk1启动子的CDE/CHR区域相关。Cdc2和plk1启动子的CDE/CHR元件被发现非常接近转录的起始点，这增加了Rb/E2F与这些元件结合的可能性，从而可能在植物上阻止预起始复合体的成分的结合。这一假设将在我们的第二个目标中使用ChlP和启动子修饰进行验证。 其他研究小组认为，包括cdc2和plk1在内的几个启动子中的CCAAT元件对p53的抑制很重要。在我们的第三个目标中，我们建议使用ChlP和凝胶位移分析来分析过度表达p53的细胞中的cdc2和plk1启动子，以检验这一模型。这些研究将提供关于p53用来抑制有丝分裂所需的两个基因的详细机制的信息，并可能揭示Rb/E2F用来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错乱提供新的见解。

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.