Analysis of the COP9 signalosome for Retinoblastoma function

COP9信号体对视网膜母细胞瘤功能的分析

• 批准号: 7589728
• 负责人: David N Arnosti
• 金额: $ 27.93万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589728
• 关键词: Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Biology; COP9 protein; Cell Cycle Regulation; Chromosomes; Complex; Data; Development; Developmental Gene; Drosophila Rbf protein; Drosophila genus; Elements; Embryo; Family; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Human; In Vitro; Individual; Malignant Neoplasms; Mediating; Molecular Genetics; Mutate; Pathway interactions; Pattern; Physiological; Play; Process; Property; Proteins; Recruitment Activity; Regulation; Repression; Research; Retinoblastoma; Retinoblastoma Protein; Role; Staining method; Stains; System; Testing; Therapeutic Intervention; Transcriptional Regulation; base; chromatin immunoprecipitation; cofactor; design; fly; gene repression; in vivo; insight; mutant; novel; overexpression; promoter; tool; tumor

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.

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，在发育过程中发挥着关键作用（尽管人们对此知之甚少）。 Rbf 蛋白在发育过程中的部署存在差异，并且被认为对基因表达具有不同的功能和影响。 Rbf 蛋白的功能特性由不同辅因子的募集决定，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 复合物调节发育胚胎中 Rbf 蛋白的稳定性。其次，COP9 复合物可以作为转录共阻遏物，通过 Rbf 蛋白制定转录抑制模式。该项目将利用果蝇中可用的强大生化和遗传工具来全面了解果蝇 Rbf 蛋白靶基因调控中的 COP9 信号体。首先，我们将对定义 COP9 信号体和 Rbf 蛋白之间关联的机制进行详细检查，作为我们后续功能测定的重要先决条件。其次，我们将使用染色质免疫沉淀检查 Rbf 相关 COP9 复合物的生理作用，以确定该假定的辅助因子何时何地与靶基因启动子处的 Rbf 蛋白相关。第三，将检查 Rbf 蛋白不稳定性的重要性以及 COP9 复合物在发育控制基因抑制模式期间在此过程中的作用。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，该测定在发育过程中测试Rbf转录调节活性，并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起着关键作用，并且是在高比例的人类肿瘤中发生突变的关键调节因子。阐明先前未识别的 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，从而有助于设计针对广泛人类癌症的治疗干预措施。