动粒亚基CENP-H/I/K对着丝粒特异识别与动粒组装新机制的研究

In mitosis, the precise alignment and separation of sister chromosomes depend on the kinetochore, a protein complex on both sides of centromere. Kinetochore is a macromolecular complex assembled by dozens of core proteins (CENPs), which connects chromosomes and spindles. CENP-A nucleosome is the epigenetic marker of centromere localization as a starting position for kinetochore assembly. Our group has revealed the molecular assembly mechanism of core subunit CENP-H/I/K complex, but the mechanism of kinetochore assembly and centromere recognition is unclear.Previous studies believed that CENP-C depends on direct interaction with CENP-A and recruits other components to complete assembly of the whole kinetochore, but this model faces many questions. In this project, Our latest study found that CENP-I can interact directly with centromeric DNA and CENP-A nucleosomes, which means that CENP-I may play a more important role in centromere recognition and kinetochore assembly. In this project,We will use biochemical and biophysical methods to determine the CENP-H/I/K-NCPs-CENP-A structure, combined with cell biology and super resolution microscopy to clarify the dependence and assembly sequence among kinetochore subunits, and reveal the molecular mechanism of CENP-H/I/K involved in centromere recognition and kinetochore assembly initiation. elucidate the mechanism of CENP-H/I/K complex in centromere recognition and kinetochore assembly.

真核生物有丝分裂中，姐妹染色单体的准确分离与平均分配依赖于着丝粒及其两侧的动粒复合体。CENP-A核小体是着丝粒的表观遗传标志，也是动粒组装的起始位点。动粒内层由多种着丝粒蛋白（CENPs）组装而成，本课题组曾揭示核心亚基CENP-H/I/K分子组装机制，但着丝粒识别与动粒组装机制尚不清晰，以往研究认为CENP-C与CENP-A的相互作用是动粒组装的起始。我们最新研究发现CENP-I与着丝粒DNA及CENP-A核小体存在直接相互作用，暗示CENP-I可能在着丝粒识别与动粒组装中发挥更重要的功能。本项目拟利用生物化学与生物物理学等方法，解析CENP-H/I/K与着丝粒DNA及CENP-A核小体的互作方式与结构基础，建立以CENP-H/I/K识别着丝粒新模型；联合细胞生物学及超分辨显微技术厘清动粒亚基间依赖关系与组装顺序，揭示CENP-H/I/K启动动粒组装的新机制。

细胞的有丝分裂过程中，中期染色体的正确排列和分离是遗传物质平均分配给子代细胞的前提。纺锤体微管通过连接染色体上特化结构-着丝粒并牵引染色体向两级运动完成姐妹染色单体的分离，动粒附着在着丝粒区域，是一个超大蛋白复合体，负责微管的连接，是微管识别着丝粒的重要平台。组蛋白H3的变体CENP-A作为着丝粒的表观遗传性标志，特异性定位于着丝粒并启动动粒蛋白的招募和组装。内层动粒包含16个亚基（CENPs），其中CENP-C和CENP-N亚基与CENP-A核小体存在直接相互作用并启动动粒的完整组装。.本研究课题通过体外纯化真菌和人源内层动粒蛋白复合物CENP-H/I/K/M蛋白复合物并进行生化分析，发现CENP-I亚基除维持动粒组装外能直接与着丝粒DNA相互作用。鉴定出CENP-I N端 HEAT REPEAT 结构域负责着丝粒DNA互作，并对着丝粒DNA的AT富集元件的识别更具偏好性。利用结构分析和点突变策略筛选出CENP-I互作位点，基于CENP-I突变体的胞生物学实验表明，打破CENP-I与DNA的相互作用将直接阻碍CENP-I在着丝粒上的有效定位，并造成中期细胞染色体的错误排列。.进一步的核小体消化实验表明CENP-I通过识别CENP-A核小体DNA并维持了后者地结构稳定性。在细胞水平，CENP-I识别着丝粒DNA并促进了G1期CENP-A地有效定位，从而在动粒的整体组装中发挥重要作用。.由此，本研究课题成功鉴定动粒亚基CENP-I为DNA结合蛋白，促进其自身功能定位的同时也为CENP-A的着丝粒定位机制提供了全新的分子视角，有助于理解动粒的动态组装和着丝粒的维持。