Meiosis, SUMOylation and the ZIP3 Protein: Parallel Studies in Mouse and Yeast.

描述(由申请人提供)：交叉对于减数分裂过程中准确的染色体分离是必不可少的。在酿酒酵母中，交换是由ScZip3特异性地促进的，ScZip3是相扑的E3-连接酶。人类ScZip3同源基因RNF212的等位基因变异与全基因组减数分裂重组率的变化有关。为了研究哺乳动物ZIP3/RNF212的作用，我们构建了一只Zip3/-敲除小鼠。我们的长期目标是了解翻译后蛋白质修饰在减数分裂互换中的作用。假设是ZIP3促进的重组和/或染色体蛋白的SUMO化促进了减数分裂的交叉。我们将使用遗传学、细胞学和分子方法的组合在小鼠和酵母中验证这一假说。具体目的是：1.分析小鼠ZIP3的功能。初步的免疫荧光细胞学研究表明，小鼠ZIP3(MmZIP3)特异定位于染色体突触区域。定位可以独立于重组发生，并且在没有联会复合体蛋白SYCP1的情况下被解除调控。免疫荧光共染色实验将被用来测试MmZIP3通常定位于重组位点的想法。将分析更多的突变系，以进一步确定MmZIP3本地化的遗传要求。Zip3/-突变小鼠将使用组织学和免疫荧光细胞学方法进行详细分析。2.鉴定酵母和小鼠ZIP3蛋白的减数分裂相扑偶联物和底物。在酵母zip3突变体中，相扑蛋白结合物的光谱发生了显著变化。依赖于ScZip3的相扑结合物将通过质谱学进行鉴定。同时，将对候选靶标进行ScZip3依赖的SUMO化检测。在酵母靶标保守的情况下，这些实验将有助于确定MmZIP3的合作伙伴和潜在底物。我们还将利用酵母双杂交筛选和免疫共沉淀从睾丸提取液中鉴定MmZIP3伴侣和底物。依赖于ScZip3和MmZIP3的体外SUMO化分析将使用纯化的组分进行重组，并用于确定候选底物。3.分析特定目的蛋白的糖基化作用2.利用定点突变技术减少酵母菌中已鉴定底物的糖基化，并通过遗传分析、特殊的DNA物理分析和免疫荧光细胞学研究其对减数分裂重组和染色体行为的影响。相关性：重组和翻译后修饰的缺陷与人类不孕不育、流产和遗传病，特别是癌症有关。因此，了解重组的机制和调控有助于我们更好地了解这些疾病的病因。 公共卫生相关性：有性繁殖和染色体修复需要染色体配对和同源重组。这些过程中的缺陷与人类不孕、流产和遗传病，特别是癌症有关。更好地了解它们的机制和调控将有助于我们更好地了解这些疾病的病因。

DESCRIPTION (provided by applicant): Crossing-over is essential for accurate chromosome segregation during meiosis. In Saccharomyces cerevisiae, crossing-over is specifically promoted by ScZip3, a putative E3-ligase for SUMO. Allelic variants of the human ScZip3 homolog, RNF212, have been linked to changes in genome-wide meiotic recombination rates. To examine the role of mammalian ZIP3/RNF212, we have constructed a Zip3-/- knock-out mouse. Our long-term goal is to understand the roles of post-translational protein modification in meiotic crossing-over. The hypothesis is that ZIP3-promoted SUMOylation of recombination and/or chromosomal proteins promotes meiotic crossing-over. We will test this hypothesis using a combination of genetic, cytological and molecular approaches in both mouse and yeast. The Specific Aims are: 1. To analyze the function of mouse ZIP3. Preliminary immunofluorescence cytology shows that mouse ZIP3 (MmZIP3) localizes specifically to regions of chromosome synapsis. Localization can occur independently of recombination and is deregulated in the absence of synaptonemal complex protein, SYCP1. Immunofluorescence co-staining experiments will be used to test the idea that MmZIP3 normally localizes to sites of recombination. Additional mutant lines will be analyzed to further define the genetic requirements for MmZIP3 localization. Zip3-/- mutant mice will be analyzed in detail using histological and immunofluorescence cytology approaches. 2. To identify meiotic SUMO-conjugates and substrates of yeast and mouse ZIP3 proteins. The spectrum of SUMO-protein conjugates is dramatically altered in yeast zip3 mutants. ScZip3-dependent SUMO- conjugates will be identified by mass spectrometry. In parallel, candidate targets will be examined for ScZip3- dependent SUMOylation. In cases where yeast targets are conserved, these experiments will inform the identification of partners and potential substrates of MmZIP3. We will also utilize yeast 2-hybrid screening and co-immunoprecipitation from testis extracts to identify MmZIP3 partners and substrates. ScZip3- and MmZIP3- dependent in vitro SUMOylation assays will be reconstituted using purified components and used to confirm candidate substrates. 3. To analyze the role of SUMOylation for proteins identified in Specific Aim 2. Site-directed mutagenesis will be used to diminish SUMOylation of identified substrates in yeast and the effects on meiotic recombination and chromosome behavior will be examined using genetic analysis, specialized DNA physical assays, and immunofluorescence cytology. Relevance: Defects in recombination and post-translational modification have been linked to human infertility, miscarriage and genetic diseases, particularly cancer. An understanding of the mechanism and regulation of recombination will therefore help us better understand the etiology of these diseases. PUBLIC HEALTH RELEVANCE: Chromosome pairing and homologous recombination are required for sexual reproduction and chromosome repair. Defects in these processes are linked to human infertility, miscarriage and genetic diseases, particularly cancer. A greater understanding of their mechanism and regulation will help us better understand the etiology of these diseases.