The meiotic role of dtopors in male Drosophila

描述(申请人提供)：肿瘤抑制因子Topors是一种E3型泛素和相扑连接酶，可与许多重要的细胞周期调节蛋白结合，包括P53、拓扑异构酶I和DJ-1。它通过泛素结合蛋白小体降解的靶标，或通过总甲基化改变靶标的结合性质或细胞定位来调节蛋白质的功能。此外，Topors与早幼粒细胞白血病小体有关，这些核结构与肿瘤发生有关，参与了多种功能，包括总和、转录调节、细胞周期控制和抗病毒防御。TOPORS在癌细胞系中表达下调，这表明其活性对于抑制肿瘤发生具有重要意义。果蝇已经证明了一个信息模型来表征保守的Topors同源基因Dtopors的功能。Dtopors蛋白起泛素连接酶的作用，通过指导毛发转录因子的蛋白分解来调节早期胚胎模式。此外，它还通过在绝缘体元件上的组装在基因调控中发挥第二个作用。绝缘体元件被认为是通过建立染色质结构域来限制顺式作用的转录调节元件的活动来发挥作用的。Dtopors蛋白与绝缘体蛋白和层蛋白相关，并被认为在将绝缘体复合体与核层连接的过程中发挥着重要的功能作用。Dtopors蛋白对雄性生殖系中适当的染色体分离也是必不可少的，在雄性生殖系中，它是染色体及时凝聚、正常核层形成和中心体复制调节所必需的。在减数分裂前期细胞中，Dtopors聚集成类似于PML小体的核内病灶。Dtopors中的突变导致这些焦点的破坏，生殖系基因组的不稳定性和非整倍体配子的产生。这些发现表明，Dtopors在果蝇雄性生殖系中的功能特征可能揭示出重要的保守的肿瘤抑制机制。这类研究将使用细胞学、分子和遗传学相结合的方法进行。雄性生殖系对Dtopors的需求将通过检查野生型和突变型Dtopors的时间和空间调节表达的结果来确定。形成病灶和挽救生殖系缺陷的个别方面的能力将从细胞学和遗传学两方面进行检测。将在减数分裂过程中研究与Dtopors的已知有丝分裂伙伴Mod(MDG4)的潜在相互作用。将进行修饰筛选，以识别在减数分裂中与dtopors相互作用的新基因，基于对dtopors等位基因特异性雄性不育的抑制和dtopors诱导的细胞学缺陷的改变。这些研究将表征dTopors在减数分裂细胞分裂中的作用，重要的是将有助于识别dtopors途径中的基因，这些基因可能对人类Topors在肿瘤抑制中的活动具有重要意义。Topors是一种多功能的人类肿瘤抑制基因，已知可以改变参与细胞周期进程和DNA损伤修复的蛋白质的活性。对果蝇dtopors的研究揭示了对其作用机制的重要见解。这项建议旨在表征dtopors在男性减数分裂中的功能，这是减数分裂染色体分离所必需的，以了解人类Topors在肿瘤抑制中的类似作用。

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.