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. ? ? ?
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