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

描述（由申请人提供）：肿瘤抑制蛋白p53调节控制细胞对各种形式应激反应的基因的表达。在缺乏p53的情况下，细胞不能启动导致细胞周期停滞或凋亡的转录程序，从而在癌症监视机制中产生缺陷。我们研究的长期目标是了解这些基因是如何通过p53和其他因子进行转录调控的，以产生最适当的细胞应激反应并防止肿瘤生长。基于我们以前的工作，我们提出p53利用的转录辅因子的集合在不同类型的应激和不同的靶基因之间变化。这个建议的目标是阐明这种组合调节的分子基础。我们的具体目标是：1.阐明p21/Waf 1/Cip 1基因的p53依赖性转录激活的胁迫特异性机制。p21介导细胞周期停滞反应，其表达水平根据应激源而变化很大。我们将确定影响其激活的压力特异性辅助因子。2.确定p53如何在nutlin激活后，在缺乏应激依赖信号的情况下激活转录。Nutlin是一种能够激活p53而不对细胞施加压力的小分子，从而使我们能够剖析压力依赖性信号在p53转录反应中的作用。3.鉴定p53启动子特异性辅因子，以激活凋亡基因p53 A和Fas/APO 1。参与不同细胞途径的p53靶基因受到不同的调节。这表明不同的辅因子可能对不同基因子集的激活很重要。为了实现我们的目标，我们将使用高通量染色质免疫沉淀法来检查p53和几个辅因子与p53靶基因在不同形式的应激激活过程中的相互作用。假定的辅因子在细胞应激反应中的作用将通过使用RNAi技术干扰其表达来测试。由于大多数抗癌疗法依赖于p53活性来触发肿瘤细胞的死亡，因此我们的研究可能会为p53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.