Characterization of novel modifiers of dE2F/RBF function

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DESCRIPTION (provided by applicant): The retinoblastoma protein (pRB) is an important tumor suppressor that functions as a negative regulator of cell proliferation by restraining the activity of the E2F transcription factor. The E2F transcription factor is a critical regulator of the G1 to S transition and its activity is rate-limiting for S phase entry. In most tumor cells, the pRB pathway is believed to be functionally inactivated; this highlights the significance of pRB and E2F in the regulation of mammalian cell proliferation. While the critical role of pRB and E2F is well established, how precisely these proteins regulate cell cycle progression, what these proteins do, and why their activities are " so important, is not completely understood. Adding to the problem, there is an increasing number of proteins which have been shown to interact with pRB. Currently, the field faces a major challenge to identify the interactions which are functionally significant for pRB function. The proposed research aims to take advantage of Drosophila as a model organism to identify important regulators of endogenous E2F/RBF represser activity. In Drosophila, there are two E2F genes. dE2F1 is a strong activator of transcription and positive regulator of cell proliferation. During larval development, its activity is counterbalanced by the represser dE2F2. Our approach is based on our previous finding, that the strong block in cell proliferation in de2f1 mutants is due to the unchecked activity of the dE2F2/RBF represser complex. We propose to perform a genetic screen for mutations which suppress the de2f1 mutant phenotype. These mutations may be in genes whose products are functionally important for dE2F2/RBF to block cell cycle in vivo. Two specific aims are proposed: (1) To perform a full scale screen for suppressors of the de2f1 mutant phenotype on the right arm of chromosome 3, and to develop strategies for and to initiate screening of, other autosomal arms. (2) To carry out a detailed phenotypic analysis and mapping of suppressors of the de2f1 mutant phenotype. Our goal is to identify functional partners of the dE2F2/RBF represser complex which are critical for dE2F2/RBF- dependent block in cell proliferation in vivo. Given the pivotal role of pRB in tumor suppression, an understanding of how the function of the Drosophila homologues dE2F2/RBF is regulated will provide crucial clues in understanding the regulation of mammalian cell growth and the ways in which such controls may go wrong in human cancer.