Specification of meiotic cohesin function by divergent alpha-kleisin subunits

 描述（由申请人提供）：减数分裂开始时，复制的姐妹染色单体通过由粘着蛋白复合物介导的姐妹染色单体粘着（SCC）结合在一起。随后，同源染色体通过交换重组而连接。基因组拷贝数（倍性）减少时，一个单一的凝聚素亚基，α-kleisin逐步蛋白水解，触发两轮染色体分离：同源分离第一，然后姐妹篇。在芽殖酵母中的开创性工作表明，同源物和姐妹篇的连续分离需要有丝分裂Kleisin Scc 1被减数分裂特异性Kleisin Rec 8取代;只有Rec 8可以在两个步骤中被切割。然而，Rec 8不是所有生物中唯一的减数分裂Kleisin。我展示了C。除了REC-8外，线虫减数分裂还需要两个几乎相同且功能完全冗余的α-Kleisin，称为COH-3和COH-4（以下简称COH-3/4）。值得注意的是，REC-8和COH-3/4粘着蛋白在几乎每一个性质上都不同，包括它们如何与减数分裂染色体缔合，它们一旦结合到染色体上如何变得粘着，以及它们在减数分裂I的前期何时、何地以及如何在上述Kleisin的蛋白水解切割之前与染色体解离。最近的研究表明，植物和哺乳动物利用的减数分裂Kleisin组功能上与我们在线虫中鉴定的相似，并且植物和哺乳动物Kleisin赋予减数分裂粘附素与线虫中发生的不同性质。由于减数分裂SCC的缺陷被认为是导致人类流产、出生缺陷和不育的主要因素，我们建议确定使用不同kleisins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是任何功能差异的基础。我们的初步证据表明，参与细胞周期进程的许多激酶，包括ATM、ATL、CHK-2、波罗和Aurora B，差异调节REC-8和COH-3/4粘附素，我们建议鉴定Kleisin特异性翻译后修饰，并确定它们在SCC建立和蛋白水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，除了经典的WAPL依赖性途径之外，分离酶独立的粘附素去除途径是否起作用，如我们的初步数据所示。最后，我们将进行大规模的遗传筛查，以确定破坏REC-8粘附素介导的SCC而不是COH-3/4粘附素介导的突变。该筛选可以鉴定区分克莱辛以及克莱辛内有助于其独特性质的序列的粘附素调节剂。由于在许多类型的癌症中发现了内聚蛋白亚基和内聚蛋白调节剂的突变，并且与科尔内利亚德兰格和罗伯茨-SC短肢综合征等内聚蛋白病有关，因此拟议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.