Mechanisms of Gene-Specific Transcriptional Regulation Within the p53 Network

描述(申请人提供)：P53网络是人类癌症中最常见的非调控基因电路。超过一半的肿瘤携带TP53基因的突变，在剩余的部分中，p53可能被MDM2等阻滞物的过度激活而减弱。激活的P53参与了细胞对应激的各种反应，包括细胞周期停滞和细胞凋亡。据估计，全世界有1100万名患者携带有正常版本的p53蛋白的肿瘤，这种蛋白可能会被重新激活以选择性消除 癌细胞的数量。因此，设计利用p53的肿瘤抑制功能的治疗方法是现代医学的首要任务。然而，这些努力受到这样一个事实的阻碍，即P53是一个高度多效性的因素，从某种意义上说，它只在某些情况下导致细胞死亡。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结合的转录因子，可以激活数百个靶基因的表达，其中只有一部分参与了细胞凋亡。根据上下文的不同，某些p53靶基因的表达比其他基因更强，这种可变性影响细胞的命运。我们研究计划的目标是阐明在p53网络中驱动基因特异性调控的分子机制。在这项拨款提案中，我们报告了三种新的分子机制，它们介导了对p53靶基因的选择性调控。这些机制将通过制定以下具体目标来进一步研究：1.确定CTCF、Polycomb复合体和lncRNAs如何调节PUMA。我们发现，强大的凋亡靶基因PUMA受到一种新的机制的调控，涉及绝缘蛋白CTCF、染色质调节复合体PRC1-2和两个长的非编码RNA。我们将使用创新的实验方法剖析这些因素在PUMA基因座上的功能相互作用。这些努力可能使有选择地增加这种凋亡基因在癌细胞中表达的策略成为可能。2.阐明Np63基因特异性抑制的机制。我们发现，癌蛋白Np63通过一种新的机制关闭了P53靶基因的一大部分，该机制涉及各种抑制性表观遗传事件，如组蛋白变体的沉积和组蛋白去乙酰化/去甲基化。我们将研究不同的Np63辅抑制子和Np63蛋白结构域在这种新的基因调控模式中的作用，这可能揭示针对这种有效的癌基因的治疗目的的策略。3.通过全基因组shRNA筛选鉴定p21：彪马表达比率的新调节因子。我们完成了对癌细胞的基因筛查，以确定调节p21和PUMA之间平衡的因素。p21是P53依赖的细胞周期停滞的关键中介。我们现在将研究这些协同调节因子的作用机制，这可能是为了定义细胞在P53激活时的命运。这些研究将极大地促进我们对p53靶基因是如何差异调控的理解，并有助于为治疗目的而操纵p53程序的策略的设计。

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.