Spp1蛋白质作为接头分子联系组蛋白修饰与减数分裂重组起始的三维结构基础

During meiosis, homologous chromosome recombination is needed for the formation of genetic diversity. Meiotic recombination is initiated by the production of programmed DNA double-strand breaks (DSBs). Histone H3 lysine 4 trimethylation (H3K4me3) marks meiotic recombination hotspots in yeast and mammals, but how histone methylation relates to meiotic DSB formation remained unclear for a long time. Spp1 is recently shown in Saccharomyces cerevisiae to tether potential DSB hotspots to chromosome axes, by interacting with both H3K4me3 and Mer2, and then promote meiotic DSB formation. Mer2 is a protein required for DSB formation. However, relatively little is known about the structural basis and interactional mechanism of the Spp1 protein. Our program aims to obtain the three-dimensional complex structures of Spp1 N-terminal region with H3 tail peptide and Spp1 C-terminal region with Mer2, and then elucidate the structural basis of Spp1 recognizing specific histone post-translational modifications and binding with Mer2. Our study will describe the functional mechanism of Spp1, playing a pivotal role in linking histone modification to initiation of meiotic recombination, at atomic-level resolution.

减数分裂同源染色体重组是遗传多样性形成所必须的，其过程起始于程序性DNA双链断裂（DNA double-strand breaks, DSBs）发生。在酵母和哺乳动物细胞中，组蛋白H3第4位赖氨酸的三甲基化（H3K4me3）标记减数分裂重组热点区域。可是一直都不清楚组蛋白甲基化如何与减数分裂DSBs生成相联系。直到最近报道了酿酒酵母蛋白质Spp1通过其N端区域结合H3K4me3和C端区域结合DSBs生成所需的蛋白质Mer2，将染色质袢环上的潜在DSB热点绑定到染色体轴附近，从而利于DSBs发生。然而相关的结构基础与结合机理尚不清楚。本项目拟解析Spp1 N端区域与H3小肽以及Spp1 C端区域与Mer2的复合物三维结构，揭示Spp1识别特定的组蛋白翻译后修饰以及结合Mer2的结构基础。通过本项目的研究将在原子分辨率水平阐明Spp1作为关键接头分子联系组蛋白修饰与减数分裂重组起始的作用机理。

减数分裂同源染色体重组是遗传多样性形成所必需的，其过程起始于程序性DNA双链断裂（double-strand breaks，DSBs）的产生。在酵母和哺乳动物细胞中，H3K4me3标记减数分裂重组热点区域。酿酒酵母Sc_Spp1蛋白通过其N端区域结合H3K4me3和C端区域结合Mer2蛋白，从而将染色质袢环上潜在DSB热点绑定到染色体轴上，利于DSBs产生。但是，Spp1作为联系组蛋白修饰和减数分裂重组起始的关键桥梁蛋白，其相关的相互作用机理还不清楚。本研究解析了酿酒酵母Spp1的N端区域Sc_Spp1NTD和组蛋白H3(1-7)K4me3小肽的复合物晶体结构，从原子分辨率水平揭示了Sc_Spp1识别组蛋白翻译后修饰的结构基础。Sc_Spp1NTD由紧密结合的PHD结构域和C3H锌指模块组成。PHD负责识别H3K4me3，锌指模块负责稳定PHD结构的完整性。H3上其它翻译后修饰，包括R2对称和非对称二甲基化（R2me2s/me2a）、T3和T6磷酸化（T3ph和T6ph）不同程度的影响Sc_Spp1NTD对H3K4me3的识别。等温滴定量热实验表明Sc_Spp1NTD在裂殖酵母和哺乳动物中的同源区域同样识别H3K4me3，且R2me2s/me2a、T3ph和T6ph对H3K4me3的识别有不同程度的影响。另外，减数分裂特异性激酶Mek1通过抑制以姐妹染色单体为模板的DSBs修复，促进减数分裂同源染色体重组的偏好性。本研究解析了酿酒酵母Sc_Mek1的N端FHA结构域单体及其和Sc_Hop1上含磷酸化修饰Thr318的小肽Sc_Hop1(313-322)pT318的复合物晶体结构，揭示了Mek1的FHA结构域对磷酸化小肽的特异性识别机理。本项目研究结果为进一步探究减数分裂程序性DSBs产生和修复调控机理提供指导。