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

酵母中的着丝粒核小体：与内部动粒的相互作用以及分子伴侣 Yta7/ ATAD2 的组装

• 批准号: 254324152
• 负责人: Professorin Dr. Ann Elizabeth Ehrenhofer-Murray
• 金额: --
• 依托单位: Arbeitsgruppe Molekulare Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/254324152?language=en
• 关键词: centromeric; nucleosome; yeast; Interaction; inner

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.

在有丝分裂期间，遗传信息从母细胞向子细胞的忠实传递需要每条染色体的姐妹染色单体被适当地分离到子细胞。为此，着丝粒一边附着在着丝粒染色质上，另一边附着在微管上，从而提供了一种物理联系，允许染色单体在染色体分离期间被拉向细胞的相反两极。在上一次授权期，我们描述了酿酒酵母的内部着丝粒与着丝粒核小体的连接。具体地说，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的扩展氨基末端之间的物理相互作用，并且我们发现这种相互作用受到Cse4 CENP-A N端的翻译后修饰的调节。Okp1 CENP Q/Ame1 CENP-U是内部着丝粒CTF19复合体的一部分，CTF19是相当于人类CCAN的酵母。此外，我们还发现组蛋白伴侣Yta7 ATAD2是一种新的酵母着丝粒Cse4 CENP-A水平的调节因子。我们推测，Yta7是一个含有溴结构域的AAA+ATPase，与Unfoldase同源，其功能是解开Cse4/H4，并将其交给CENP-A伴侣Scm3 HJURP，以整合到着丝粒核小体中。目前的项目建议在这一工作的基础上，对Okp1CENP Q/Ame1CENP-U如何与Cse4N末端相互作用进行分子研究。具体地说，我们将使用双正交氨基酸和交联/质谱学获得的邻近信息以及结构分析来确定酵母着丝粒核小体与内部着丝粒的Okp1/Ame1相互作用的分子细节。这一点对于了解着丝粒与染色质的物理附着是如何实现的是重要的。此外，我们将详细阐明Yta7ATAD2是如何调节着丝粒染色质的。我们将利用生化和结构分析的方法研究它与Cse4/H4和Cse4分子伴侣Scm3的相互作用以及它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，这个项目将提供对着丝粒调控的机械性见解，这对于理解这一过程中的缺陷如何导致染色体错误分离非常重要。