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

着丝粒是染色体上着丝粒组装的区域，它们在着丝粒染色质和微管之间建立物理联系，用于有丝分裂中的染色体分离。在酿酒酵母中，着丝粒染色质由含有组蛋白H3变体CENP-A Cse 4而不是典型H3的单个核小体定义。Cse 4的N端延伸与Okp 1/Ame 1相互作用（S.人CENP-Q/ CENP-U的酿酒酵母同源物），其是Ctf 19/CCAN复合物的组分，并将内部动粒蛋白募集到着丝粒染色质。在本研究中，我们将研究CENP-A Cse 4核小体核心区在S.啤酒。具体来说，我们将研究Cse 4 α N-螺旋中的翻译后修饰，该修饰靠近DNA进入着丝粒核小体的入口/出口位点。这将通过分析邻近Cse 4残基中的选定突变来补充，所述突变在突变时引起明显的着丝粒缺陷。我们将分离Cse 4核心区缺陷的抑制突变，并评估动粒突变背景下致病突变的影响。将使用分子遗传学和生物化学方法研究相应的因素，以剖析它们在着丝粒和动粒调节中的功能。由于Cse 4修饰的位点，预期这些因子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而起作用。因此，他们将确定新的调控着丝粒，并将提供分子的见解着丝粒核小体的作用，动粒组装和功能。

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.