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

有丝分裂中姐妹染色单体凝聚力的磷酸化和脱乙酰化驱动的溶解

• 批准号: 450806808
• 负责人: Professor Dr. Olaf Stemmann
• 金额: --
• 依托单位: Lehrstuhl für Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/450806808?language=en
• 关键词: Phosphorylation; deacetylation; driven; dissolution; sister

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 Does this mean that the prophase pathway is no longer active in prometaphase, and - if yes - how is it switched off at the right time?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.

细胞分裂对于人类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，每条染色体由两个相同的姐妹染色单体组成，它们在拓扑上被粘连蛋白（一种环形蛋白质复合物）包围。姐妹染色单体的内聚力是通过黏连蛋白 Smc3 亚基的乙酰化来增强的，对于遗传物质的无差错分配减半至关重要。然而，在染色体分离发生之前，需要解决内聚力问题。在后生动物中，这分两次发生。首先，蛋白激酶和Wapl的协同作用以非蛋白水解方式打开粘连蛋白环。虽然这种所谓的前期途径从染色体臂上去除了粘连蛋白，但着丝粒处的粘连蛋白受到 Sgo1 依赖性去磷酸化的保护。分离酶的激活导致剩余粘连蛋白的 Rad21 亚基裂解，正是这种依赖于蛋白水解的第二波开环波触发了后期。在酵母中，不会发生粘连蛋白释放活性的早期有丝分裂上调，所有与染色体结合的粘连蛋白都会被 Rad21 裂解去除。虽然一项研究声称粘连蛋白从染色体上解离是后期 Smc3 脱乙酰化的先决条件，但当脱乙酰化被阻止时，粘连蛋白（甚至裂解的）染色质结合的延长对这一观点提出了挑战。我们最近发现，分离酶一旦去除其抑制剂，就会与 HDAC8（人类 Smc3 脱乙酰酶）形成复合物。使用染色质结合粘连蛋白进行酶测定，我们将阐明 Rad21 裂解和 Smc3 脱乙酰化的时间顺序和（相互）依赖性。Sgo1 在中期留下着丝粒粘连蛋白，这提出了尚未解决的重要问题：为什么脱保护的着丝粒粘连蛋白没有通过磷酸化和 Wapl 依赖性开环去除？这是否意味着前期途径在前中期不再活跃，如果是的话，它是如何在正确的时间关闭的？ Sgo1 如何在机制水平上靶向着丝粒粘连蛋白（然后从中去除）？为什么 Sgo1 完全离开着丝粒？这是必需的，因为否则粘连蛋白的分离酶依赖性切割会受到损害？在这里，我们提出结合生化重建、基因组编辑和细胞生物学实验来回答所有这些问题，并全面了解人类染色体分离。