Role of lysine deacetylase Hst2 in mitotic chromosome condensation

有丝分裂染色体是细胞分裂的特有标志。然而，到目前为止，它们的分子结构、组成和导致它们形成的机制在很大程度上仍然是个谜。染色质的粘性吸引了大量非特异性结合蛋白，其结构随着环境条件的变化而发生动态变化，这阻碍了生化分析。使用体内用遗传安装的非天然氨基酸进行交联的方法，我的实验室已经确定了一系列组蛋白修饰，在有丝分裂开始时启动染色质的缩合。这揭示了有丝分裂染色体凝聚的一个全新的机制方面，它独立于并平行于典型的凝集素机制。在这个级联中的一个关键角色是赖氨酸脱乙酰酶HST2，它通过组蛋白H3尾巴的磷酸化被招募到染色质中。它介导组蛋白H4尾部乙酰化的去除，以促进其与邻近核小体的相互作用。这增加了染色体凝聚的驱动力。在这项提案中，我们希望通过寻找酶的进一步相互作用伙伴来分析HST2招募的细节。我们还希望为这些酶找到更多的脱乙酰基底物，因为我们的初步结果表明，H4脱乙酰基并不是它在缩合过程中唯一的任务。最后，我们希望重建体外缩合过程的各个方面，以分析其动力学特性。从这个项目中，我们想要对HST2如何协调染色体凝聚有一个详细的机制了解。这一机制可能在所有真核生物中都是保守的，并最终可能成为设计新的癌症治疗策略的目标。

Mitotic chromosomes are characteristic hallmarks of cell division. However, till date have their molecular structure, their composition and the mechanisms leading to their formation remained largely mysterious. Biochemical analyses are hampered by the stickiness of chromatin, attracting large numbers of unspecific binding proteins, and the dynamic changes in its structure in response to changing environmental conditions. Using an in vivo crosslinking approach with genetically installed unnatural amino acids my lab has identified a cascade of histone modifications that initiates the condensation of chromatin at the onset of mitosis. This revealed an entirely new mechanistic aspect of mitotic chromosome condensation independent and parallel to the canonical condensin machinery. A key player in this cascade is lysine deacetylase Hst2, which is recruited to chromatin by phosphorylation of the histone H3 tail. It mediates the removal of an acetylation from histone H4 tail to facilitate its interaction with neighbouring nucleosomes. This adds a driving force to chromosome condensation. In this proposal we want to analyse the details of the recruitment of Hst2 by searching for further interaction partners of the enzyme. We also expect to find additional deacetylation substrates for the enzymes because our preliminary results indicate the H4 deacetylation is not its only task in the condensation process. Finally, we want to reconstitute aspects of the condensation process in vitro to analyse its kinetic properties. From this project we want to get a detailed mechanistic understanding of how Hst2 coordinates chromosome condensation. This mechanism is likely conserved across all eukaryotes and may eventually become a target for the design of new therapeutic strategies in the treatment of cancer.