CleaveRec8 - Molecular Mechanisms Underlying Meiotic Cohesin Removal

在有丝分裂中，遗传物质的正确分离取决于一种环状的、包裹DNA的蛋白质复合物（称为黏连蛋白）的及时去除。虽然有丝分裂前期的磷酸化从染色体臂上去除了粘连蛋白，但只有当着丝粒处的蛋白磷酸酶2A保护的粘连蛋白被分离酶蛋白水解切割时，后期才开始。在减数分裂中，两个特化的细胞分裂没有S期的介入，产生单倍体配子。首先同源染色体的分离和随后姐妹染色单体的分离需要在减数分裂I中从染色体臂和在减数分裂II中从着丝粒逐步置换粘着蛋白。令人惊讶的是，这两种分裂都需要Rec8的Separase依赖性切割，Rec8在功能上取代了减数分裂中的Scc1，并且需要磷酸化以作为Separase的底物。而单细胞真菌S.在这项合作研究中，我们将1）定位和功能分析磷酸化，这使得哺乳动物Rec8对Separase切割敏感，2）鉴定激酶，其对于使Rec8对蛋白水解敏感是必要且足够的，并且表征其失活的表型结果。在实验上更易处理的有丝分裂细胞中的功能性Rec8测定与评估体外和体内Rec8切割的能力配对，使我们处于实现这些目标的独特位置。与我们最近的其他发现一起，这也使我们能够3）解开两次减数分裂之间快速Separase失活的分子细节，4）澄清为什么姐妹染色单体的分离在减数分裂II中依赖于CyclinA，并且过早地被这种Cyclin的不可降解形式而不是其他形式触发，以及5）解决减数分裂粘着蛋白是否对蛋白水解无关的解离敏感，如果是，我们相信，我们的共同努力将产生重要的见解，分子机制，确保正确的染色体分离在哺乳动物减数分裂。在人类中，女性减数分裂容易出错，并且错误率随着年龄的增长呈指数级增加。减数分裂中的错误分离导致非整倍体配子的产生，并在受精后产生非整倍体胚胎。大多数非整倍体是不能存活的，并导致胎儿自然流产，除了某些三体，如21三体，这是由于在90%的情况下，女性减数分裂错误。研究表明，在接近绝经期的妇女中，粘附素的过早丢失是卵母细胞非整倍体发生率高的原因之一。因此，我们需要了解凝聚素是如何在减数分裂中被去除的，以深入了解与年龄相关的女性生殖问题。

In mitosis the correct segregation of the genetic material depends on the timely removal of a ring-shaped, DNA-embracing protein complex, named Cohesin. While phosphorylations in mitotic prophase remove Cohesin from chromosome arms, anaphase only commences when protein phosphatase 2A-protected Cohesin at centromeres is proteolytically cleaved by Separase. In meiosis, two specialized cell divisions without an intervening S-phase generate haploid gametes. The separation of homologous chromosomes first and sister chromatids later requires the stepwise displacement of Cohesin from chromosome arms in meiosis I and from centromeres in meiosis II. Surprisingly, both these divisions require the Separase-dependent cleavage of Rec8, which functionally replaces Scc1 in meiotic Cohesin and needs to be phosphorylated to serve as a substrate for Separase. Whereas the molecular details of stepwise Rec8 removal in the single cell fungus S. cerevisiae are starting to emerge, it remains largely unknown how the localization- and time-specific cleavage of Rec8 is controlled in mammalian meiosis and whether the prophase pathway may contribute to displacement of meiotic Cohesin.In this collaborative study we will 1) map and functionally analyse the phosphorylations, which render mammalian Rec8 susceptible to cleavage by Separase, and 2) identify the kinases, which are necessary and sufficient to sensitise Rec8 to proteolysis, and characterize phenotypic consequences of their inactivation. A functional Rec8 assay in experimentally more tractable mitotic cells paired with the ability to assess Rec8-cleavage in vitro and in vivo put us in a unique position to achieve these goals. Together with other recent discoveries of ours, this also enables us to 3) unravel the molecular details of rapid Separase inactivation between the two meiotic divisions, 4) clarify why separation of sister chromatids depends on CyclinA in meiosis II and is prematurely triggered by non-degradable forms of this Cyclin but not others, and 5) address whether meiotic cohesin is susceptible to proteolysis-independent dissociation and, if yes, at which position the ring is opened.We are confident that our mutual efforts will yield important insights into molecular mechanisms ensuring correct chromosome segregation in mammalian meiosis. In humans, female meiosis is error-prone and the error rate increases exponentially with age. Missegregations in meiosis lead to the generation of aneuploid gametes and, upon fertilization, to aneuploid embryos. Most aneuploidies are not viable and lead to spontaneous fetal abortions, except certain trisomies, such as trisomy 21, which is due to errors in female meiotic divisions in 90% of cases. It has been shown that precocious loss of Cohesin is one reason for the high incidence of oocyte aneuploidies in women closer to menopause. Therefore, we need to understand how Cohesin is removed in meiosis to gain insights into age-related reproductive problems in women.