The meiotic role of dtopors in male Drosophila

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DESCRIPTION (provided by applicant): The tumor suppressor Topors is an E3-type ubiquitin and SUMO ligase that binds to a number of important cell cycle regulatory proteins, including p53, topoisomerase I, and DJ-1. It regulates protein function by ubiquitin-conjugating targets for proteosome degradation, or by altering binding properties or cellular localizations of targets by sumoylation. Additionally, Topors associates with Promyeloleucytic Leukemia Bodies, nuclear structures implicated in oncogenesis that are involved in a diversity of functions including sumolyation, transcriptional regulation, cell cycle control and anti-viral defense. Topors is downregulated in cancer cell lines, suggesting its activities are important for suppressing oncogenesis. Drosophila has proven an informative model for characterizing functions of the conserved Topors homolog, Dtopors. The Dtopors protein acts as a ubiquitin ligase, and regulates early embryonic pattern by directing proteolysis of the Hairy transcription factor. Additionally, it plays a second role in gene regulation via assembly at insulator elements. Insulator elements are proposed to function by establishing chromatin domains that restrict the activities of cis-acting transcriptional regulatory elements. The Dtopors protein associates with insulator proteins and lamin and has been hypothesized to play an important functional role in tethering insulator complexes to the nuclear lamina. The Dtopors protein is also essential for proper chromosome segregation in the male germ line, where it is required for timely chromosome condensation, normal nuclear lamina formation, and the regulation of centrosome duplication. In meiotic prophase cells, Dtopors assembles into intranuclear foci reminiscent of PML bodies. Mutants in dtopors lead to disruption of these foci, germline genomic instability and the production of aneuploid gametes. These findings suggest that characterization of Dtopors function in the Drosophila male germ line may reveal important conserved mechanisms of tumor suppression. Such studies will be pursued using a combined cytological, molecular and genetic approach. Requirements for Dtopors in the male germ line will be defined by examining the outcome of temporally and spatially regulated expression of wild type and mutant forms of Dtopors. The ability to form foci and to rescue individual aspects of the germ line defects will be examined both cytologically and genetically. A potential interaction with Mod(mdg4), a known mitotic partner of Dtopors, will be investigated in meiosis. Modifier screens will be performed to identify new genes that interact with dtopors in meiosis, based on suppression of a dtopors allele-specific male sterility and on alteration of dtopors-induced cytological defects. These studies will characterize the involvement of dTopors in meiotic cell division, and importantly will lead to the identification of genes in the dtopors pathway that may have important implications for the activities of human Topors in tumor suppression. Topors is a multifunctional human tumor suppressor gene known to alter the activities of proteins involved in cell cycle progression and DNA damage repair. Studies of dtopors, the functional homolog in the fruit fly Drosophila melanogaster, have revealed important insights on its mechanism of action. This proposal aims to characterize dtopors functions in male meiosis, where it is particularly critical and is required for meiotic chromosome segregation, towards understanding of analogous roles of human Topors in tumor suppression.