Mechanisms of Gene-Specific Transcriptional Regulation Within the p53 Network

描述（由申请人提供）：p53网络是人类癌症中最常见的失调基因回路。超过一半的肿瘤在TP 53基因中携带突变，并且在剩余部分中，p53可能通过阻遏物如MDM 2的过度活化而减弱。激活的p53参与多种细胞应激反应，包括细胞周期阻滞和细胞凋亡。据估计，全世界有1100万患者携带具有正常版本的p53蛋白的肿瘤，其可能被重新激活以进行选择性消除。 癌细胞。因此，利用p53的肿瘤抑制功能的治疗设计是现代医学的首要任务。然而，这些努力受到p53是一种高度多效性因子的事实的阻碍，在这个意义上，它仅在某些情况下提供细胞死亡。是什么决定了细胞对p53激活的反应是死亡还是存活？p53是一种与DNA结合并激活数百个靶基因表达的转录因子，其中只有一些参与凋亡。根据上下文，一些p53靶基因比其他基因表达更强，这种变异性影响细胞命运。我们的研究项目的目标是阐明p53网络中驱动基因特异性调控的分子机制。在这项资助计划中，我们报告了三个新的分子机制介导的p53靶基因的选择性调节。这些机制中的每一个都将通过制定以下具体目标来进一步研究：1.明确CTCF、Polycomb复合物和lncRNA如何调控CTCF A。我们发现，有效的凋亡p53靶基因cDNAA是由一种新的机制，涉及绝缘子蛋白CTCF，染色质调控复合物PRC 1 -2和两个长的非编码RNA的调节。我们将使用创新的实验方法来剖析这些因素之间的相互作用。这些努力可能使策略选择性地增加这种凋亡基因在癌细胞中的表达。2.阐明基因特异性阻遏的机制？Np63？我们发现癌蛋白？Np63通过涉及各种抑制性表观遗传事件的新机制，如组蛋白变体的沉积和组蛋白脱乙酰化/脱甲基化，关闭了大部分p53靶基因。我们将调查各种作用？Np 63辅阻遏物和？Np 63蛋白结构域在这种新的基因调控模式中的作用，这可能揭示了靶向这种有效的癌基因用于治疗目的的策略。3.为了表征通过全基因组shRNA筛选鉴定的p21：p21 A表达比率的新型调节剂。我们在癌细胞中完成了一项遗传筛选，以确定调节p53依赖性细胞周期阻滞的关键介质p21和p53依赖性细胞周期阻滞剂之间平衡的因素。我们现在将研究这些辅助调节因子的作用机制，这些辅助调节因子可以靶向确定p53激活后的细胞命运。这些研究将显著推进我们对p53靶基因如何差异调节的理解，并且还有助于设计用于治疗目的的操纵p53程序的策略。 公共卫生相关性：癌细胞中最常见的遗传改变之一是肿瘤抑制蛋白p53的功能失活。该研究项目的目标是破译调节p53下游基因的分子机制，这可能使新的治疗策略的设计成为可能。

DESCRIPTION (provided by applicant): The p53 network is the most commonly deregulated gene circuitry in human cancer. More than half of tumors carry mutations in the TP53 gene, and in the remaining fraction p53 is likely attenuated by hyperactivation of repressors such as MDM2. Activated p53 participates in various cellular responses to stress including cell cycle arrest and apoptosis. It is estimated that 11 million patients worldwide carry tumors with a normal version of the p53 protein, which could potentially be reactivated for selective elimination of cancer cells. Thus, the design of therapies exploiting the tumor suppressive function of p53 is a top priority in modern medicine. However, these efforts are hampered by the fact that p53 is a highly pleiotropic factor, in the sense that it delivers cell death only in some scenarios. What determines whether cells die or survive in response to p53 activation? p53 is a transcription factor that binds to DNA and activates the expression of hundreds of target genes, only some of which are involved in apoptosis. Depending on the context, some p53 target genes are expressed more strongly than others, and this variability affects cell fate. The goal of our research program is to elucidate the molecular mechanisms driving gene-specific regulation within the p53 network. In this grant proposal we report three novel molecular mechanisms mediating the selective regulation of p53 target genes. Each of these mechanisms will be further investigated by developing the following Specific Aims: 1. To define how CTCF, Polycomb complexes and lncRNAs regulate PUMA. We discovered that the potent apoptotic p53 target gene PUMA is regulated by a novel mechanism involving the insulator protein CTCF, the chromatin regulatory complexes PRC1-2 and two long non-coding RNAs. We will dissect the functional interplay between these factors at the PUMA locus using innovative experimental approaches. These efforts may enable strategies to selectively increase expression of this apoptotic gene in cancer cells. 2. To elucidate the mechanism of gene-specific repression by ?Np63¿. We discovered that the oncoprotein ?Np63¿ shuts down a large subset of p53 target genes by a novel mechanism involving various repressive epigenetic events, such as deposition of histone variants and histone deacetylation /demethylation. We will investigate the role of various ?Np63¿ corepressors and ?Np63¿ protein domains in this novel mode of gene regulation, which may reveal strategies to target this potent oncogene for therapeutic purposes. 3. To characterize novel regulators of the p21: PUMA expression ratio identified by a genome-wide shRNA screen. We completed a genetic screen in cancer cells to identify factors regulating the balance between p21, the key mediator of p53-dependent cell cycle arrest, and PUMA. We will now investigate the mechanism of action of these coregulators, which could be targeted to define cell fate upon p53 activation. These studies will significantly advance our understanding of how p53 target genes are differentially regulated, and also contribute toward the design of strategies manipulating the p53 program for therapeutic purposes. PUBLIC HEALTH RELEVANCE: One of the most common genetic alterations in cancer cells is the functional inactivation of the tumor suppressor protein p53. The goal of this research project is to decipher the molecular mechanisms regulating the genes acting downstream of p53, which may enable the design of novel therapeutic strategies.