Specification of meiotic cohesin function by divergent alpha-kleisin subunits

 描述(申请人提供)：当减数分裂开始时，复制的姐妹染色单体由粘附素复合体介导的姐妹染色单体凝聚(SCC)结合在一起。随后，同源染色体通过交叉重组联系在一起。当单个粘附素亚基α-kleisin的逐步蛋白分解触发两轮染色体分离时，基因组拷贝数(倍体)会减少：同源基因首先分离，然后是姐妹。发芽酵母的开创性工作表明，同系物和姐妹蛋白的连续分离需要有丝分裂的kleisin Scc1被减数分裂特异的kleisin Rec8取代；只有Rec8可以分两步切割。然而，Rec8并不是所有生物中唯一的减数分裂Kleisin。我指出，线虫的减数分裂需要两个几乎相同且在功能上完全冗余的α-kleisin，称为Coh-3和Coh-4(以下称为Coh-3/4)，此外还需要Rec-8。值得注意的是，REC-8和COH-3/4粘附素几乎在每种性质上都不同，包括它们如何与减数分裂染色体结合，它们如何与染色体结合，以及它们在减数分裂I的前期，在上述kleisin蛋白水解性切割之前，何时、何地以及如何从染色体分离。最近的研究表明，植物和哺乳动物利用的减数分裂kleisin组似乎在功能上类似于我们在线虫中鉴定的那些kleisin，并且植物和哺乳动物的kleisins赋予减数分裂粘连蛋白不同的特性，就像线虫一样。由于减数分裂SCC中的缺陷被认为是导致人类流产、出生缺陷和不孕的主要因素，我们建议确定使用不同kleisin的减数分裂粘附素之间功能差异的分子机制。Rec-8和COH-3/4在不同的时间表达，我们将确定表达时间是否存在任何功能差异。我们的初步证据表明，许多参与细胞周期进程的激酶，包括ATM、ATL、CHK-2、Polo和Aurora B，对REC-8和COH-3/4粘附素有不同的调节作用，我们建议识别Kleisin特异的翻译后修饰，并确定它们在鳞状细胞癌建立和蛋白降解非依赖性粘附素去除中的功能重要性。我们还将确定在减数分裂过程中，除了典型的依赖于WAPL的途径外，是否有其他途径可以去除分离酶非依赖性粘附素，正如我们的初步数据所表明的那样。最后，我们将进行大规模的基因筛查，以确定由REC-8粘附素而不是COH-3/4粘附素介导的破坏SCC的突变。这一筛选可以识别区别于Kleisin的粘附素调节因子以及Kleisin中影响其独特性质的序列。由于粘附素亚单位和粘附素调节器的突变在多种类型的癌症中被发现，并与科妮莉亚·德朗格和罗伯茨-斯卡伯茨青光眼综合征等粘附素疾病有关，因此这项拟议的研究的相关性超出了生殖健康。

 DESCRIPTION (provided by applicant): When meiosis begins, replicated sister chromatids are held together by sister chromatid cohesion (SCC) mediated by the cohesin complex. Subsequently, homologous chromosomes become linked through crossover recombination. Genome copy number (ploidy) is reduced when stepwise proteolysis of a single cohesin subunit, the α-kleisin, triggers two rounds of chromosome segregation: homologs separate first, then sisters. Pioneering work in budding yeast showed that the successive separation of homologs and sisters requires that the mitotic kleisin Scc1 be replaced by the meiosis-specific kleisin Rec8; only Rec8 can be cleaved in two steps. However, Rec8 is not the sole meiotic kleisin in all organisms. I showed that C. elegans meiosis requires two nearly identical and completely functionally redundant α-kleisins, called COH-3 and COH-4 (henceforth, COH-3/4), in addition to REC-8. Remarkably, REC-8 and COH-3/4 cohesins differ in nearly every property, including how they associate with meiotic chromosomes, how they become cohesive once bound to chromosomes, and when, where, and how they dissociate from chromosomes in prophase of meiosis I, prior to the proteolytic cleavage of the kleisin described above. It has recently been shown that plants and mammals utilize sets of meiotic kleisins that appear functionally similar to those we identified in nematodes, and that the plant and mammalian kleisins endow meiotic cohesins with divergent properties as occurs in nematodes. Because defects in meiotic SCC are thought to be a major factor contributing to human miscarriage, birth defects, and infertility, we propose to identify the molecular mechanisms that underlie the functional differences between meiotic cohesins that use different kleisins. REC-8 and COH-3/4 are expressed at different times, and we will determine whether expression timing underlies any functional differences. Our preliminary evidence suggests that numerous kinases involved in cell cycle progression, including ATM, ATL, CHK-2, Polo, and Aurora B, differentially regulate REC-8 and COH-3/4 cohesin, and we propose to identify kleisin-specific post- translational modifications and determine their functional importance in SCC establishment and proteolysis- independent cohesin removal. We will also determine whether pathways other than the canonical WAPL- dependent pathway for separase-independent cohesin removal function during meiosis, as suggested by our preliminary data. Finally, we will conduct a large-scale genetic screen to identify mutations that disrupt SCC mediated by REC-8 cohesin but not COH-3/4 cohesin. This screen can identify cohesin regulators that differentiate between kleisins as well as sequences within kleisins that contribute to their unique properties. Because mutations in cohesin subunits and cohesin regulators are found in numerous types of cancer and are linked to cohesinopathies like Cornelia de Lange and Roberts-SC phocomelia syndromes, the relevance of the proposed research extends beyond reproductive health.