CleaveRec8 - Molecular Mechanisms Underlying Meiotic Cohesin Removal

在有丝分裂中，遗传物质的正确分离依赖于一种环状的、包含dna的蛋白质复合体的及时移除，这种复合体被称为内聚蛋白。虽然有丝分裂前期的磷酸化会使染色体臂上的内聚蛋白脱落，但只有当蛋白磷酸酶2a保护的着丝粒上的内聚蛋白被分离酶蛋白水解裂解时，后期才会开始。在减数分裂中，两个特殊的细胞分裂没有中间的s期产生单倍体配子。同源染色体先分离，姐妹染色单体后分离，需要在减数分裂I时从染色体臂和减数分裂II时从着丝粒中逐步移出内聚蛋白。令人惊讶的是，这两种分裂都需要Rec8的分离依赖裂解，Rec8在功能上取代了减数分裂内聚蛋白中的Scc1，并且需要磷酸化以作为分离酶的底物。虽然Rec8在单细胞真菌cerevisiae中逐步去除的分子细节开始出现，但在哺乳动物减数分裂中，Rec8的定位和时间特异性切割是如何被控制的，以及前期途径是否有助于减数分裂内聚蛋白的位移，在很大程度上仍然未知。在这项合作研究中，我们将1)绘制和功能分析使哺乳动物Rec8易于被分离酶切割的磷酸化，2)鉴定使Rec8对蛋白质水解敏感的必要和充分的激酶，并表征其失活的表型后果。在实验中更易于处理的有丝分裂细胞中进行功能性Rec8检测，并在体外和体内评估Rec8切割的能力，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)揭示两个减数分裂分裂之间快速分离酶失活的分子细节，4)阐明为什么姐妹染色单体的分离依赖于减数分裂II中的周期蛋白a，并且由该周期蛋白的不可降解形式而不是其他形式过早触发，5)解决减数分裂内聚蛋白是否易受蛋白质水解独立解离的影响，如果是，在哪个位置打开环。我们相信，我们的共同努力将对哺乳动物减数分裂中确保正确染色体分离的分子机制产生重要的见解。在人类中，女性减数分裂容易出错，错误率随着年龄的增长呈指数增长。减数分裂过程中的错离导致非整倍体配子的产生，受精后产生非整倍体胚胎。大多数非整倍体不能存活并导致自然流产，除了某些三体，如21三体，90%的病例是由于女性减数分裂错误造成的。已有研究表明，内聚蛋白的过早丧失是接近绝经期妇女卵母细胞非整倍体高发的原因之一。因此，我们需要了解内聚蛋白是如何在减数分裂中被移除的，以深入了解女性年龄相关的生殖问题。

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.