The centromeric nucleosome in yeast: Interaction with the inner kinetochore and assembly by the chaperone Yta7/ ATAD2

在有丝分裂过程中，遗传信息从母细胞到子细胞的忠实传递要求每条染色体的姐妹染色单体正确地分离到子细胞。为此，着丝点一边附着在着丝染色质上，另一边附着在微管上，从而提供了一个物理连接，使染色单体在染色体分离期间被拉向细胞的相反两极。在上一期的研究中，我们描述了酵母中内部着丝点与着丝粒核小体的附着。具体来说，我们确定了异源二聚体Okp1 CENP Q/ Ame1 CENP- u与酵母CENP- a同源物Cse4的延伸氨基端之间的物理相互作用，并且我们发现这种相互作用通过Cse4 CENP- an端翻译后修饰来调节。Okp1 CENP Q/ Ame1 CENP- u是内着丝点CTF19复合体的一部分，相当于人类的CCAN。此外，我们已经鉴定出组蛋白伴侣Yta7 ATAD2是酵母中着丝粒Cse4 CENP-A水平的新调节剂。我们假设Yta7是一种含有溴域的AAA+ atp酶，与展开酶具有同源性，其功能是展开Cse4/ H4，并将其交给CENP-A伴侣Scm3 HJURP，将其整合到着丝粒核小体中。目前的项目建议在这项工作的基础上，获得关于Okp1 CENP Q/ Ame1 CENP- u如何与Cse4 n端相互作用的分子见解。具体来说，我们将使用双正交氨基酸和交联/质谱以及结构分析获得的接近信息来确定酵母的着丝粒核小体与内部着丝点的Okp1/ Ame1之间相互作用的分子细节。这对于理解着丝点与染色质的物理连接是如何实现的很重要。此外，我们将详细阐明Yta7 ATAD2调控着丝粒染色质的机制。我们将利用生化和结构方法表征其与Cse4/ H4和Cse4伴侣Scm3的相互作用及其在染色质组装中的作用。我们将结合体内分析来确定Yta7的功能相关残基和结构域。总之，该项目将提供对着丝粒调控的机制见解，这对于理解这一过程中的缺陷如何导致染色体错分离非常重要。

The faithful transmission of genetic information from mother to daughter cells during mitosis requires that the sister chromatids of each chromosome are properly segregated to the daughter cells. For this, the kinetochore attaches on one side to the centromeric chromatin and on the other side to the microtubule and thus provides a physical link that allows the chromatids to be pulled towards opposite poles of the cell during chromosome segregation. In the last grant period, we have characterized the attachment of the inner kinetochore to the centromeric nucleosome in the yeast Saccharomyces cerevisiae. Specifically, we identified a physical interaction between the heterodimer Okp1 CENP Q/ Ame1 CENP-U and the extended amino-terminus of the yeast CENP-A homolog Cse4, and we found this interaction to be modulated by posttranslational modifications on the Cse4 CENP-A N-terminus. Okp1 CENP Q/ Ame1 CENP-U is part of the CTF19 complex of the inner kinetochore, the yeast equivalent of human CCAN. Furthermore, we have identified the histone chaperone Yta7 ATAD2 as a novel regulator of centromeric Cse4 CENP-A levels in yeast. We postulate that Yta7, a bromodomain-containing AAA+ ATPase with homology to unfoldases, functions to unfold Cse4/ H4 and to hand it to the CENP-A chaperone Scm3 HJURP for incorporation into the centromeric nucleosome. The current project proposes to build on this work and to obtain molecular insights into how Okp1 CENP Q/ Ame1 CENP-U interacts with the Cse4 N-terminus. Specifically, we will use proximity information obtained using biorthogonal amino acids and cross-linking/ mass spectrometry as well as structural analysis to determine the molecular details of the interaction between the centromeric nucleosome of yeast and Okp1/ Ame1 of the inner kinetochore. This is important in order to understand how physical attachment of the kinetochore to chromatin is achieved. Furthermore, we will elucidate in mechanistic detail how Yta7 ATAD2 regulates centromeric chromatin. We will characterize its interaction with Cse4/ H4 and the Cse4 chaperone Scm3 and its role in chromatin assembly using biochemical and structural approaches. We will combine this with an in vivo analysis to determine functionally relevant residues and domains of Yta7. Altogether, this project will provide mechanistic insights into regulation at the centromere that are important to understand how defects in this process contribute to chromosome missegregation.