组蛋白二聚体的动态结构及其生物学功能研究

Nucleosome is the fundamental unit of chromatin. Intrinsic dynamic properties of nucleosome structure, as well as nucleosome assembly process, confer nucleosome dynamics which are strongly associated with gene misregulation and human diseases. Histone dimeric structure, which mainly exists in H2A-H2B and H3-H4 form, involves in constitution of nucleosome and various nucleosome subunits. In addition, histone dimeric structure plays important roles in nucleosome assembly process. Therefore, Histone dimeric structure is the key for better understanding nuclesosome assembly and the dynamic properties of nuclesome structure. Nuclear Magnetic Resonance (NMR) spectroscopy will be used to investigate histone dimeric structures and their important functions. Solution structure determination and structure dynamics are our major foci. First, we will determine the solution structure of H2A.Z-H2B and Cse4-H4 and identify their dynamic properties. Second, because histone dimeric structures are sensitive to change of salt concentration or protein denaturant, the dynamic equilibrium of various conformations will be altered if we challenge histone dimeric structure with salt or denatured. Hydrogen deuterium exchange approach will be applied to identify the equilibrium movement. Last, histone variant and histone chaperone are coupled with histone dimeric structure, either structurally or functionally. The way they used to interrupt histone dimeric structure will highlight their prominent roles in regulating nucleosome dynamic.

核小体是染色质的基本结构单位，核小体的动态变化与基因表达调控异常导致的各种疾病的发生机制有密切联系。核小体动态结构和核小体组装是核小体动态变化的必要基础和调控手段。组蛋白二聚体是组成核小体亚单位的基本结构，也是核小体组装过程中的关键蛋白分子，因此组蛋白二聚体的研究对深入理解核小体动态结构和核小体组装机制具有重要意义。本课题将利用核磁共振方法研究组蛋白二聚体的动态结构，并揭示相关的生物学功能。通过测定组蛋白二聚体H2A.Z-H2B和Cse4-H4的溶液三维结构阐明其溶液构象的动态特点。通过氢氘交换实验研究组蛋白二聚体的溶液构象如何在离子强度和蛋白质变性剂的影响下重新分布和相互转变。通过研究组蛋白变体和组蛋白伴侣对组蛋白二聚体动态结构的影响,揭示组蛋白二聚体的动态结构与其功能的联系。

核小体是染色体的基本单位，其核心区域由组蛋白H2A,H2B,H3,H4和其周围的DNA组成。组蛋白单体在溶液状态下呈无规则构象，然而，组蛋白形成二聚体后形成稳定的高级结构并在核小体组装过程中发挥重要作用。对组蛋白变体和组蛋白伴侣与组蛋白二聚体的相互作用研究较多。H2A与H2A.Z分别与H2B形成的组蛋白二聚体具有非常相似的结构,但二者具备明显不同的生物学功能,但其分子机制还不清楚。本项目通过X-射线晶体学和核磁共振结合的实验方法分别解析了组蛋白二聚体的晶体结构及其相关动力学数据，并以此为基础解释了组蛋白二聚体在溶液状态下的特性，为了解组蛋白二聚体在核小体动态组装等方面的生物功能研究奠定了基础。本项目通过核磁氢氘交换实验和圆二色谱实验分析发现两个组蛋白二聚体都可以形成稳定的疏水核心，但是H2A.Z-H2B的交换速率要明显慢于H2A-H2B，首次证明了H2A.Z-H2B在慢速时间尺度上的稳定性要明显高于H2A-H2B，并通过其他生化实验揭示造成这种稳定性差异的主要结构元件位于α2-helix上。本项目还进一步通过单分子磁镊技术证明H2A.Z核小体比H2A核小体更稳定，并且这种稳定性的差异也是由α2-helix作为稳定元件主导造成的。本项目的研究结果为最终理解H2A核小体作为染色质重塑复合物SWR1的特异性底物提供了动力学依据。