Role of lysine deacetylase Hst2 in mitotic chromosome condensation

有丝分裂染色体是细胞分裂的特征性标志。然而，到目前为止，它们的分子结构，组成和形成机制仍然很神秘。生化分析受到染色质粘性的阻碍，吸引大量非特异性结合蛋白，以及其结构随环境条件变化而发生的动态变化。使用体内交联方法与基因安装的非天然氨基酸，我的实验室已经确定了一个级联组蛋白修饰，启动在有丝分裂开始时的染色质的凝聚。这揭示了一个全新的机制方面的有丝分裂染色体凝聚独立和平行的经典凝聚素机制。该级联反应中的关键参与者是赖氨酸脱乙酰酶Hst 2，其通过组蛋白H3尾部的磷酸化被募集到染色质。它介导从组蛋白H4尾部去除乙酰化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。在这个提议中，我们希望通过寻找酶的进一步相互作用伙伴来分析Hst 2募集的细节。我们还希望找到其他的酶的脱乙酰基底物，因为我们的初步结果表明，H4脱乙酰基不是它在缩合过程中的唯一任务。最后，我们希望在体外重构缩合过程的各个方面，以分析其动力学性质。从这个项目中，我们希望获得Hst 2如何协调染色体浓缩的详细机制理解。这种机制可能在所有真核生物中都是保守的，并可能最终成为癌症治疗中新治疗策略设计的目标。

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.