Analysis of the COP9 signalosome for Retinoblastoma function

描述（由申请人提供）：该项目的长期目标是阐明果蝇视网膜母细胞瘤家族（Rbf）蛋白和相关辅因子在发育环境中的活性。rbf蛋白是细胞周期控制的关键调节因子，在发育过程中起着关键作用，尽管人们对此了解甚少。rbf蛋白质在发育过程中有不同的分布，被认为对基因表达有不同的功能和影响。Rbf蛋白的功能特性取决于不同辅因子的招募，我们已经确定了COP9信号体复合物和Rbf2之间的新关联。我们的初步数据表明，COP9信号体可能以两种重要的方式影响Rbf功能。首先，COP9复合物调节发育胚胎中Rbf蛋白的稳定性。第二，COP9复合物可以作为转录辅助抑制因子，通过Rbf蛋白来制定转录抑制模式。该项目将采用果蝇中可用的强大的生物化学和遗传工具来开发果蝇Rbf蛋白在靶基因调控中的COP9信号体的全面图片。首先，我们将对COP9信号体和Rbf蛋白之间的关联机制进行详细研究，这是我们后续功能测定的重要先决条件。其次，我们将研究Rbf相关的COP9复合物的生理作用，使用染色质免疫沉淀，以确定何时何地这种推定的辅因子与Rbf蛋白在靶基因启动子。第三，将研究Rbf蛋白不稳定性的重要性以及COP 9复合物在发育控制的基因抑制模式期间在这一过程中的作用。第四，我们将确定COP9信号体作为Rbf介导的基因抑制的转录共调节因子的生理作用。COP9复合物作为共阻遏物的功能将通过遗传测定来检查，所述遗传测定在发育期间测试Rbf转录调节活性，并且通过转录测定来确定单个COP9亚基在果蝇中特定基因的Rbf调节中的重要性。视网膜母细胞瘤蛋白在发育中具有关键作用，并且是在高比例的人类肿瘤中突变的关键调节因子。阐明以前未鉴定的COP9对Rbf基因调控的功能、其作用模式及其在发育基因调控中的作用将为RB作用提供重要的见解，这将有助于在广泛的人类癌症中设计治疗干预措施。

DESCRIPTION (provided by applicant): The long-term aim of this project is to elucidate the activity of Drosophila Retinoblastoma family (Rbf) proteins and associated cofactors in a developmental setting. Rbf proteins are key regulators of cell cycle control and play pivotal, although poorly understood, roles in development. Rbf proteins are differentially deployed during development and are thought to have distinct functions and effects on gene expression. The functional properties of Rbf proteins are dictated by the recruiting of distinct cofactors, and we have identified a novel association between the COP9 signalosome complex and Rbf2. Our preliminary data indicates that the COP9 signalosome may function in two important ways to influence Rbf function. First, the COP9 complex regulates Rbf protein stability in the developing embryo. Second, the COP9 complex may serve as a transcriptional co-repressor to enact patterns of transcriptional repression by Rbf proteins. This project will employ powerful biochemical and genetic tools that are available in Drosophila to develop a comprehensive picture of the COP9 signalosome in target gene regulation by Drosophila Rbf proteins. First, we will perform a detailed examination of the mechanism defining the association between the COP9 signalosome and Rbf proteins, as a vital pre-requisite to our subsequent functional assays. Second, we will examine the physiological role of the Rbf-associated COP9 complex using chromatin immunoprecipitation to determine when and where this putative cofactor is associated with Rbf proteins at target gene promoters. Third, the significance of Rbf protein instability and the role for the COP9 complex in this process during developmentally controlled gene repression patterns will be examined. Fourth, we will determine the physiological role of the COP9 signalosome as transcriptional co-regulatory factor for Rbf- mediated gene repression. The function of the COP9 complex as a co-repressor will be examined by means of genetic assays that test Rbf transcriptional regulatory activity during development and by transcription assays to determine the importance of individual COP9 subunits in Rbf regulation of specific genes in the fly. Retinoblastoma proteins have critical roles in development and are key regulators mutated in a high percentage of human tumors. Elucidation of the previously unidentified COP9 function for Rbf gene regulation, its mode of action, and its role in developmental gene regulation will provide important insight into RB action that will facilitate the design of therapeutic interventions in a wide spectrum of human cancers.