Mechanisms of Delta-N-p63-alpha transcriptional repression and its role in squamo

描述（由申请人提供）：p53相关蛋白Np 631是一种驱动各种来源的鳞状细胞癌（SCC）发生和进展的强效癌基因。虽然该因子的生理水平是维持上皮干细胞所必需的，但Np 631过表达促进癌细胞增殖和存活，并抑制细胞周期停滞和凋亡。该项目的总体目标是确定Np 631影响基因转录和细胞行为的机制。我们的初步研究结果表明，在H226 SCC细胞中，Np 631是一种有效的基因特异性抑制剂的促凋亡因子NOXA，其机制独立于其能力，结合染色质在p53反应元件，并在监管步骤，以下p53介导的组蛋白乙酰化。然而，在不同的SCC细胞系中，其他研究人员已经表明，Np 631耗尽去抑制其他p53/p73应答基因。该项目的第一个具体目标将确定其他家族成员p53和p73对H226细胞和其他细胞系中Np 631缺失的转录和细胞效应的相对贡献。将使用稳定表达的shRNA构建体产生一组缺乏p53和/或p73的同基因H226细胞系，并且将确定Np 631消耗对细胞存活、增殖、凋亡和>30个p53/p73靶基因的表达的影响。此外，NOXA和Np 631将在H226细胞中同时被敲低，以确定NOXA上调是否对Np 631敲低后细胞增殖的丧失至关重要。这些研究将使我们能够确定p53/p73，Np 631和NOXA之间的上位关系。第二个特异性目的将确定Np 631抑制特异性p53/p73靶基因表达的分子机制。通过在基础和激活的p53条件下对Np 631敲低之前和之后在NOXA和p21位点发生的转录事件进行详尽的染色质免疫沉淀（ChIP）分析，将鉴定受Np 631影响的精确调节步骤。最终的具体目标将确定生物相关的Np 631突变对基因特异性抑制的影响。将通过用野生型Np 631或在DNA结合结构域、SAM结构域和其它区域中含有天然存在的突变的构建体拯救Np 631敲低细胞来进行体内结构-功能分析。这些实验将确定Np 631的关键功能区域，并进一步阐明Np 631的作用机制。预计在该项目期间产生的知识将极大地提高我们对Np 631的理解，其作用机制及其在正常上皮维持和肿瘤发生中的作用，以及帮助实现由Np 631过表达驱动的各种癌症的治疗控制策略。

DESCRIPTION (provided by applicant): The p53-related protein, Np631, is a potent oncogene driving the development and progression of squamous cell carcinomas (SCC) of various origins. While physiologic levels of this factor are required for epithelial stem cell maintenance, Np631 overexpression promotes cancer cel proliferation and survival, and inhibits cel cycle arrest and apoptosis. The overall goal of this project is to define the mechanisms by which Np631 impacts gene transcription and cell behavior. Our initial findings indicate that in H226 SCC cells, Np631 is a potent gene-specific repressor of the pro-apoptotic factor NOXA, by mechanisms independent of its ability to bind chromatin at p53 responsive elements, and at regulatory steps that follow p53-mediated histone acetylation. Yet in different SCC cell lines, other investigators have shown that Np631 depletion de-represses other p53/p73-responsive genes. The first Specific Aim of this project will define the relative contributions of the other family members, p53 and p73, to the transcriptional and celular efects of Np631 depletion in H226 cells and other cell lines. A panel of isogenic H226 cell lines lacking p53 and/or p73 will be created using stably-expressed shRNA constructs, and the effect of Np631 depletion on cel survival, proliferation, apoptosis, and the expression of >30 p53/p73 target genes will be determined. Furthermore, NOXA and Np631 will be concomitantly knocked down in H226 cells to determine whether NOXA upregulation is crucial for the loss of cell proliferation upon Np631 knockdown. These studies will enable us to define the epistatic relationship between p53/p73, Np631, and NOXA. The second Specific Aim will identify the molecular mechanism by which Np631 represses the expression of specific p53/p73 target genes. By performing an exhaustive chromatin immunoprecipitation (ChIP) analysis of the transcriptional events taking place at the NOXA and p21 loci before and after Np631 knockdown under conditions of both basal and activated p53, the precise regulatory steps affected by Np631 will be identified. The final Specific Aim will determine the impact of biologically-relevant Np631 mutations on gene-specific repression. An in vivo structure-function analysis will be performed by rescuing Np631 knockdown cells with either wild type Np631 or constructs containing naturally occurring mutations in the DNA binding domain, SAM domain, and other regions. These experiments will identify crucial functional regions of Np631 and will further illuminate the Np631 mechanism of action. It is anticipated that the knowledge generated during this project will vastly improve our understanding of Np631, its mechanism of action, and its role in normal epithelial maintenance and oncogenesis, as well as to help enable strategies for the therapeutic control of the various cancers driven by Np631 overexpression.