组蛋白乙酰转移酶MOF与核小体以及长非编码RNA roX1/2的结构生物学研究

Acetylation on histone H4 lys16 is a prevalent epigenetic mark associated with the actively transcribed chromatins. This histone mark is mainly established by the histone acetyltransferase MOF in mammals and flies. MOF is essential for mouse embryonic stem cells pluripotency and early development, and it also mediates dosage compensation of the male X chromosome genes in flies. Biochemical and functional analyses indicate that MOF primarily resides in two different multi-subunit complexes, the MSL complex and the NSL complex, that have distinct cellular functions. In both complexes, the catalytic activities of MOF on its substrate nucleosome are tightly regulated by its associated partners (the MSL1 and MSL3 proteins in the MSL complex and the NSL1 protein in the NSL complex). In addition, the recruitment of MOF on specific genomic loci is also directed by other components in the complexes. For example, in the male Drosophila, the targeting and the assembly of the MSL complex on X chromosome is mediated by a long non-coding RNA roX1/2. However, till now, little is known at the structural level about how the MOF complexes recognize and acetylate nucleosome, and how MOF is targeted on specific chromatin regions by its partners. To address these questions, we set out to study the atomic structures of the two MOF complexes as well as their complexes with nucleosome and with the lncRNA roX1/2. These studies will reveal the structural basis for the recognition between the MOF complexes and nucleosome, and also provide the regulatory mechanisms on the acetylation activity of MOF. In addition, our research will provide structural insights into the regulations of the roX1/2 RNA on the recruitment and the assembly of the Drosophila MSL complex on X chromosome. Generally speaking, this project will elucidate how histone modifications are established at the nucleosomal level. It can also enrich our knowledge on the structural and functional mechanisms of lncRNAs in epigenetic regulation.

组蛋白乙酰转移酶MOF是一种重要的表观遗传调控因子，它负责在染色质上建立组蛋白H4K16乙酰化标记，参与哺乳动物胚胎发育、雄性果蝇X染色体基因剂量补偿等重要的生物学过程。MOF主要存在于MSL和NSL两类复合物中，复合物中的其它组分对于MOF的核小体修饰活性及其在染色质上的定位等起到十分重要的调控作用。目前，关于MOF识别、修饰核小体的分子机理尚不清楚，对于MOF定位到染色质特定区域的结构基础也仍不了解。针对这两方面的研究问题，本项目拟结合生物化学和结构生物学方法，致力于在三维结构水平上解析MSL和NSL复合物识别、修饰核小体的结构基础，并揭示长非编码RNA roX1/2参与果蝇MSL复合物在X染色体定位和组装的调控机理。本项目的完成，一方面将在核小体水平上解析组蛋白修饰酶的催化和调控机制，另一方面将揭示lncRNA参与表观遗传调控的结构机理，促进对lncRNA的三维结构和功能机理的了解。

组蛋白修饰是染色质表观遗传调控的重要方式，其通过对核小体组蛋白的可逆性共价修饰来动态调节染色质结构与功能。组蛋白修饰通常发生在延伸于核小体核心结构之外的组蛋白尾端区域。以往对组蛋白修饰的结构生物学研究主要以组蛋白尾端区域的合成多肽作为底物来研究组蛋白修饰酶对组蛋白尾端序列的特异性识别和修饰机制，相对缺少在核小体水平上对组蛋白修饰的结构和调控机制研究。为了研究组蛋白修饰酶对核小体的底物识别和活性调控机制，我们建立了重组核小体的体外制备和体外化学修饰体系，并通过冷冻电镜单颗粒重构技术对多种组蛋白修饰酶识别、修饰核小体的结构机制进行解析。对组蛋白H4K16位点乙酰转移酶MSL复合物与核小体的电镜结构研究表明，MSL复合物结合于核小体核心结构表面，与多种组蛋白发生相互作用，但MSL复合物与核小体的结合具有较高的动态性，可能需要其它核小体结合蛋白或组蛋白修饰标记来稳定其与核小体的相互作用。关于组蛋白H3K79位点甲基转移酶DOT1L与H2BK120位点泛素化修饰的核小体的复合物电镜结构解析，揭示了DOT1L特异性识别和修饰组蛋白H3K79位点的结构机制，并且阐明了H2BK120位点泛素化修饰是通过增强DOT1L与核小体的相互作用来实现H2BK120ub1对H3K79me的组蛋白修饰串扰调控。我们还解析了组蛋白H3K4位点甲基转移酶MLL1、MLL3复合物与H2BK120ub1或无修饰核小体的多个复合物电镜结构。该研究首次揭示了核小体结构对MLL复合物的酶活调控及其分子机制，阐明了H2BK120ub1对MLL甲基化活性的串扰调控机制，并发现了MLL复合物关键组分WDR5蛋白对MLL家族成员活性调控的迥异的分子机理及底物特异性调控机制。此外，我们还研究了MSL复合物中lncRNA roX2与蛋白亚基MLE和MSL2的相互作用机制，揭示了三者相互作用的关键区域及可能的分子识别机制。本项目的研究不仅揭示了组蛋白修饰酶对核小体的精确识别和特异修饰的结构机制，也为相关人类疾病的病理机制研究和靶向药物开发提供了高分辨率结构信息。