Phosphorylation- and deacetylation-driven dissolution of sister chromatid cohesion in mitosis

细胞分裂对人类的生长和繁殖至关重要。在细胞开始分裂之前，储存在染色体上的遗传信息会被复制。复制后，每个染色体由两个相同的姐妹染色单体组成，它们在拓扑上被环状蛋白质复合体粘附素所拥抱。姐妹染色单体的凝聚力是通过粘附素的Smc3亚单位的乙酰化而增强的，对于遗传物质的无错误分配减半是必不可少的。然而，在染色体分离发生之前，需要解决凝聚力问题。在后生动物中，这种情况分两波发生。首先，蛋白激酶和WAPL的协同作用以一种非蛋白水解性的方式打开粘附素环。虽然这一所谓的前期途径将粘附素从染色体臂上移除，但着丝粒上的粘附素受到依赖于Sgo1的去磷酸化的保护。分离酶的激活导致剩余粘附素的Rad21亚基的裂解，正是这种依赖于蛋白分解的第二波开环触发了后期。在酵母中，粘附素释放活性的早期有丝分裂上调不会发生，所有与染色体结合的粘附素都会被Rad21切割去除。虽然一项研究声称，粘附素从染色体上解离是Smc3在后期去乙酰化的先决条件，但当脱乙酰化被阻止时，粘附素的染色质结合时间延长(甚至被切割)，这一观点受到了挑战。我们最近发现，分离酶一旦去除它的抑制剂，就会与人类Smc3脱乙酰基酶HDAC8形成复合体。使用染色质结合的粘附素进行酶分析，我们将澄清Rad21裂解和Smc3去乙酰化的时间顺序和(相互)依赖。Sgo1在着丝粒粘附素的前中期留下着丝粒粘附素，这提出了重要的尚未解决的问题：为什么去保护的着丝粒粘附素不能通过磷酸化和WAPL依赖的环打开来移除？这是否意味着前期途径在前中期不再活跃，如果是的话，它如何在正确的时间被关闭？在机械学水平上，Sgo1是如何针对着丝粒粘附素(后来从着丝粒粘附素中移除)的？为什么Sgo1要离开着丝点呢？这是因为依赖于分离酶的粘附素的切割否则会受到损害吗？在这里，我们提出了生化重组、基因组编辑和细胞生物学实验相结合的方法来回答所有这些问题，并对人类染色体分离有一个全面的了解。

Cell division is essential for human growth and reproduction. Before a cell begins to divide, the genetic information stored on the chromosomes is duplicated. After replication, each chromosome consists of two identical sister chromatids, which are topologically embraced by cohesin, a ring-shaped protein complex. Sister chromatid cohesion is enforced by acetylation of cohesin's Smc3 subunit and essential for error-free distributive halving of the genetic material. However, before chromosome segregation can take place, cohesion needs to be resolved. In metazoans, this occurs in two waves. Firstly, the concerted actions of protein kinases and Wapl open cohesin rings in a non-proteolytic manner. While this so-called prophase pathway removes cohesin from chromosome arms, cohesin at centromeres is protected by Sgo1-dependent dephosphorylation. Activation of separase results in cleavage of the Rad21 subunit of remaining cohesin, and it is this proteolysis-dependent, second wave of ring opening which triggers anaphase. In yeast, where the early mitotic upregulation of the cohesin release activity does not occur, all chromosomally bound cohesin is removed by Rad21 cleavage. While one study claims that dissociation of cohesin from chromosomes is pre-requisite for deacetylation of Smc3 in anaphase, this view is challenged by the prolonged chromatin association of (even cleaved) cohesin when deacetylation is prevented. We recently discovered that separase, once stripped off its inhibitors, forms a complex with HDAC8, the human Smc3 deacetylase. Using chromatin bound cohesin for enzymatic assays, we will clarify the temporal order and (inter)dependence of Rad21 cleavage and Smc3 deacetylation.Sgo1 leaves centromeric cohesin in prometaphase, which raises important as yet unresolved questions: Why is de-protected centromeric cohesin not removed by phosphorylation- and Wapl-dependent ring opening? Does this mean that the prophase pathway is no longer active in prometaphase, and - if yes - how is it switched off at the right time? How is Sgo1 targeted to centromeric cohesin (and later removed from it) on a mechanistic level? And why does Sgo1 leave the centromeres at all? Is this required because separase-dependent cleavage of cohesin would otherwise be impaired? Here, we propose a combination of biochemical reconstitutions, genome editing and cell biological experiments to answer all of these questions and obtain a comprehensive understanding of human chromosome segregation.