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）蛋白和相关辅助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，在发育过程中发挥着关键的作用，尽管人们对其知之甚少。Rbf蛋白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蛋白的功能特性是由不同的辅助因子的募集决定的，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信号体可能通过两种重要方式影响Rbf功能。首先，COP9复合物调节胚胎发育中Rbf蛋白的稳定性。其次，COP9复合体可能作为转录共抑制因子，以制定Rbf蛋白的转录抑制模式。该项目将利用强大的果蝇生物化学和遗传工具，全面研究果蝇Rbf蛋白调控靶基因的COP9信号体。首先，我们将详细研究COP9信号体和Rbf蛋白之间关联的机制，这是我们后续功能分析的重要先决条件。其次，我们将使用染色质免疫沉淀法检查Rbf相关的COP9复合体的生理作用，以确定这种假定的辅助因子何时何地与靶基因启动子上的Rbf蛋白相关。第三，在发育控制的基因抑制模式中，Rbf蛋白不稳定性的意义以及COP9复合物在这一过程中的作用将被检查。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复合体作为共抑制因子的功能将通过基因分析来检测Rbf在发育过程中的转录调控活性，并通过转录分析来确定单个COP9亚基在果蝇中特定基因的Rbf调控中的重要性。视网膜母细胞瘤蛋白在发育过程中起着关键作用，是人类肿瘤中高比例突变的关键调节因子。阐明先前未知的COP9在Rbf基因调控中的功能、作用模式及其在发育基因调控中的作用，将为了解RB的作用提供重要的见解，这将有助于设计针对多种人类癌症的治疗干预措施。

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.