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

描述（由申请人提供）：在减数分裂过程中，交叉是准确的染色体分离所必需的。在酿酒酵母中，交叉是由ScZip3特异性促进的，ScZip3是一种被认为是SUMO的e3连接酶。人类ScZip3同源基因RNF212的等位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修饰在减数分裂交叉中的作用。假设是zip3促进重组和/或染色体蛋白的sumo化促进减数分裂交叉。我们将在小鼠和酵母中使用遗传学、细胞学和分子方法的组合来验证这一假设。具体目标是：1。分析鼠标ZIP3的功能。初步免疫荧光细胞学显示小鼠ZIP3 （MmZIP3）特异性定位于染色体突触区域。定位可以独立于重组发生，并且在突触复合体蛋白SYCP1缺失的情况下不受调节。免疫荧光共染色实验将用于验证MmZIP3通常定位于重组位点的想法。将分析其他突变系，以进一步确定MmZIP3定位的遗传要求。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sumo偶联物和底物。sumo蛋白偶联物的光谱在酵母zip3突变体中发生了显著改变。依赖于sczip3的SUMO-共轭物将通过质谱鉴定。与此同时，候选靶点将被检查是否依赖于ScZip3的SUMOylation。在酵母菌靶点保守的情况下，这些实验将为确定MmZIP3的合作伙伴和潜在底物提供信息。我们还将利用酵母2杂交筛选和睾丸提取物的共免疫沉淀来鉴定MmZIP3伴侣和底物。ScZip3和MmZIP3依赖的体外SUMOylation实验将使用纯化的组分重建，并用于确定候选底物。3. 分析SUMOylation在特异性靶蛋白2中鉴定的蛋白中的作用。位点定向诱变将用于减少酵母菌中已识别底物的SUMOylation，并将使用遗传分析，专门的DNA物理分析和免疫荧光细胞学来检查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症、流产和遗传疾病，特别是癌症有关。因此，了解重组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.