Stress- and Promoter-Specific Mechanisms of Transcritpional Activation by p53

描述(申请人提供)：肿瘤抑制蛋白P53调节基因的表达，控制细胞对各种形式的压力的反应。在没有p53的情况下，细胞无法启动转录程序，导致细胞周期停滞或凋亡，从而导致癌症监视机制的缺陷。我们研究的长期目标是了解这些基因是如何受P53和其他因素转录调控的，以产生最合适的细胞对压力的反应，并防止肿瘤生长。基于我们以前的工作，我们认为P53利用的一组转录辅助因子在不同类型的压力和不同的靶基因之间是不同的。这项建议的目的是阐明这种组合调控的分子基础。我们的具体目标是：1.阐明P53依赖的p21/Waf1/Cip1基因转录激活的应激特异性机制。P21介导细胞周期停滞反应，其表达水平因应激源的不同而有很大差异。我们将确定影响其激活的应激特异性辅助因素。2.确定在没有应激依赖信号的情况下，p53如何在Nutlin激活时激活转录。Nutlin是一种小分子，能够在不对细胞施加压力的情况下激活P53，从而使我们能够剖析应激依赖信号在P53转录反应中的作用。3.确定P53基因启动子特异的辅助因子，以激活PUMA和Fas/APO1基因。参与不同细胞通路的P53靶基因受到差异调控。这表明不同的辅因子可能对不同的基因亚集的激活很重要。为了实现我们的目标，我们将使用高通量染色质免疫沉淀分析来检测不同形式的应激激活过程中P53及其几个辅助因子与P53靶基因的相互作用。通过使用RNAi技术干扰其表达，将测试假定的辅助因子在细胞对应激反应中的作用。由于大多数抗癌治疗依赖于P53活性来触发肿瘤细胞的死亡，我们的研究可能为P53的作用模式提供新的见解，并为人类癌症的药物干预提供新的靶点。

DESCRIPTION (provided by applicant): The tumor supressor protein p53 regulates the expression of genes that control the cellular response to various forms of stress. In the absence of p53, cells fail to initiate transcriptional programs that result in cell cycle arrest or apoptosis, thus generating a defect in the cancer surveillance mechanism. The long-term goal of our research is to understand how these genes are transcriptionally regulated by p53 and other factors in order to produce the most appropriate cellular response to stress and prevent tumor growth. Based on our previous work, we propose that the set of transcriptional co-factors utilized by p53 varies among distinct types of stress and different target genes. The goal of this proposal is to elucidate the molecular basis of this combinatorial regulation. Our specific aims are: 1. To elucidate the stress-specific mechanism of p53-dependent transcriptional activation of the p21/Waf1/Cip1 gene. p21 mediates the cell cycle arrest response and its levels of expression vary greatly depending on the source of stress. We will identify stress-specific co-factors that influence its activation. 2. To determine how p53 activates transcription in the absence of stress-dependent signaling upon activation by nutlin. Nutlin is a small molecule capable of activating p53 without stressing the cells, thus allowing us to dissect the role of stress-dependent signaling in the p53 transcriptional response. 3. To identify promoter-specific co-factors used by p53 to activate the apoptotic genes PUMA and Fas/APO1. p53 target genes involved in diverse cellular pathways are differentially regulated. This suggest that different co-factors may be important for activation of distinct subsets of genes. To achieve our aims we will use high-throughput Chromatin Immunoprecipitation assays to examine the interaction of p53 and several co-factors with p53 target genes during activation by different forms of stress. The role of putative co-factors in the cellular response to stress will be tested by interfering with its expression using RNAi technology. Because most anti-cancer therapies rely on p53 activity to trigger the death of tumor cells, our research may provide new insights in the mode of p53 action and yield new targets for pharmacological intervention in human cancer.