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

描述（由申请人提供）：交换对于减数分裂期间染色体的准确分离至关重要。在酿酒酵母中，交换由ScZip 3特异性促进，ScZip 3是SUMO的推定E3连接酶。人类ScZip 3同源物RNF 212的等位基因变体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ZIP 3/RNF 212的作用，我们构建了Zip 3-/-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交换中的作用。假设是ZIP 3促进的重组和/或染色体蛋白的SUMO化促进减数分裂交换。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测试这一假设。具体目标是：1。分析鼠标ZIP 3的功能。初步的免疫荧光细胞学显示，小鼠ZIP 3（MmZIP 3）专门定位于染色体突触的区域。定位可以独立于重组发生，并且在联会复合体蛋白SYCP 1不存在的情况下被解除调节。免疫荧光共染色实验将用于测试MmZIP 3通常定位于重组位点的想法。将分析额外的突变株系以进一步确定MmZIP 3定位的遗传要求。将使用组织学和免疫荧光细胞学方法详细分析Zip 3-/-突变小鼠。2.鉴定酵母和小鼠ZIP 3蛋白的减数分裂SUMO结合物和底物。酵母zip 3突变体中SUMO-蛋白缀合物的谱发生了显著改变。ScZip 3-依赖性SUMO-缀合物将通过质谱法鉴定。同时，将检查候选靶标的ScZip 3依赖性SUMO化。在酵母靶标保守的情况下，这些实验将为MmZIP 3的伴侣和潜在底物的鉴定提供信息。我们还将利用酵母双杂交筛选和睾丸提取物的免疫共沉淀来鉴定MmZIP 3伴侣和底物。ScZip 3和MmZIP 3依赖性体外SUMO化试验将使用纯化组分复溶，并用于确认候选底物。3.分析SUMO化对特异性目的2中鉴定的蛋白质的作用。将使用定点诱变来减少酵母中已鉴定底物的SUMO化，并将使用遗传分析、专门的DNA物理测定和免疫荧光细胞学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育、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。 公共卫生相关性：有性生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育、流产和遗传疾病，特别是癌症有关。更好地了解它们的机制和调节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.