Regulation of kinetochore function in yeast by modification of the core region of the centromeric nucleosome

着丝粒是染色体上组装着丝粒的区域，它们在着丝粒染色质和微管之间建立了物理联系，以便在有丝分裂中进行染色体分离。在酿酒酵母中，着丝粒染色质是由单个核小体定义的，该核小体含有组蛋白H3变体CENP-A Cse4而不是典型的H3。Cse4延伸的N端与Ctf19/Ccan复合体的组成部分Okp1/Ame1(人CENP-Q/CENP-U的酿酒酵母同源物)相互作用，并将内部动粒蛋白招募到着丝粒染色质。在这项研究中，我们将研究CENP-A Cse4核小体的核心区在酿酒酵母着丝粒组装和着丝粒功能中的功能。具体地说，我们将研究CSE4DNA N-螺旋的翻译后修饰，该修饰位于α进入着丝粒核小体的入口/出口位置附近。此外，还将分析邻近Cse4残基中的选定突变，这些突变在突变时会导致明显的着丝粒缺陷。我们将分离Cse4核心区缺陷的抑制突变，并将评估致病突变在动粒突变背景下的影响。将使用分子遗传学和生物化学方法研究各自的因素，以剖析它们在着丝粒和着丝粒调节中的功能。由于Cse4修饰的位置，这些因子有望通过着丝粒核小体的定位和滑动，或通过内部动粒蛋白与核心核小体的相互作用发挥作用。因此，他们将确定着丝粒的新调节器，并将提供对着丝粒核小体在着丝粒组装和功能中所起作用的分子洞察。

Centromeres are the regions of the chromosome where the kinetochore is assembled, and they establish a physical link between the centromeric chromatin and the microtubule for chromosome segregation in mitosis. In Saccharomyces cerevisiae, centromeric chromatin is defined by a single nucleosome that contains the histone H3 variant CENP-A Cse4 instead of canonical H3. The extended N-terminus of Cse4 interacts with Okp1/ Ame1 (the S. cerevisiae homologs of human CENP-Q/ CENP-U), which are components of the Ctf19 /CCAN complex, and recruits the inner kinetochore proteins to centromeric chromatin. In this proposal, we will investigate the function of the core region of the CENP-A Cse4 nucleosome in kinetochore assembly and centromere function in S. cerevisiae. Specifically, we will investigate a posttranslational modification in the αN-helix of Cse4 that lies close to the entry/ exit site of the DNA into the centromeric nucleosome. This will be complemented by the analysis of selected mutations in neighbouring Cse4 residues, which cause a distinct centromeric defect when mutated. We will isolate suppressor mutations of defects in the Cse4 core region, and the effect of the causative mutation in the context of kinetochore mutations will be evaluated. The respective factors will be investigated using molecular genetic and biochemical approaches in order to dissect their function in the regulation of the centromere and kinetochore. Due to the site of Cse4 modification, such factors are expected to act through positioning and sliding of the centromeric nucleosome, or through interaction of inner kinetochore proteins with the core nucleosome. They will thus identify novel regulators of the centromere and will provide molecular insights into the role of the centromeric nucleosome in kinetochore assembly and function.