Molecular control of centromere specification and kinetochore assembly

 描述（由申请人提供）：本提案的目标是定义着丝粒-DNA界面的性质，并确定着丝粒特异性染色质如何指导人类细胞中功能性着丝粒结构的组装。在每次细胞分裂过程中，整个遗传物质必须准确地分配给子细胞。即使是单个染色体错误分离事件也可能是灾难性的，导致数百个基因的丢失或获得，对发育和疾病（包括肿瘤发生）造成严重后果。在指导染色体分离的中心球员是动粒，一个大的大分子结构，介导的附件之间的纺锤体微管和一个区域的每个染色体称为着丝粒。确定动粒功能的分子基础对于理解可能产生肿瘤细胞的缺陷过程以及评估疾病诊断和治疗的最佳靶点至关重要。在脊椎动物中， 着丝粒由序列独立的表观遗传机制指定，该机制涉及含有组蛋白H3变体CENP-A的核小体的靶向沉积。一个基本的未回答的问题是，动粒的其余部分如何在CENP-A下游组装。我们以前的工作表明，CENP-A不足以指导人类细胞中完整的功能性动粒结构的组装。我们已经确定了一个异源四聚体复合物组成的组蛋白折叠蛋白CENP-T，-W，-S，和-X作为一个关键的附加组件的kinetochore-DNA接口。CENP-T-W-S-X复合物显示出与核小体的结构相似性，并具有序列非依赖性DNA结合活性。此外，CENP-T直接与外部动粒微管结合蛋白相互作用以指导动粒组装。因此，CENP-T-W-S-X复合物在连接着丝粒处的DNA和微管界面中起关键作用。然而，CENP-T-W-S-X复合物如何专门靶向着丝粒以在每条染色体上产生单个功能性微管附着位点仍然是未知的。这项拟议的工作将通过评估来定义指导CENP-T-W-S-X复合物的着丝粒定位的机制：1）CENP-T-W-S-X复合物定位所需的内在序列特征，2）与CENP-T-W-S-X复合物相关以将其存款或维持在着丝粒的外在因子，以及3）控制CENP-T-W-S-X复合物定位的调节修饰和染色质特征。

 DESCRIPTION (provided by applicant): The goal of this proposal is to define the nature of the kinetochore-DNA interface and determine how centromere-specific chromatin directs the assembly of a functional kinetochore structure in human cells. During each cell division, the entire complement of genetic material must be accurately partitioned to the daughter cells. Even a single chromosome mis-segregation event can be catastrophic, resulting in the loss or gain of hundreds of genes, with severe consequences for development and disease, including tumorigenesis. The central player in directing chromosome segregation is the kinetochore, a large macromolecular structure that mediates attachments between spindle microtubules and a region of each chromosome termed the centromere. Determining the molecular basis for kinetochore function is crucial to understand the defective processes that can give rise to tumor cells, and to evaluate the best targets for the diagnosis and treatment of disease. In vertebrates, centromeres are specified by sequence-independent epigenetic mechanisms that involve the targeted deposition of nucleosomes containing the histone H3-variant, CENP-A. A fundamental unanswered question is how the remainder of the kinetochore is assembled downstream of CENP-A. Our previous work demonstrated that CENP-A is not sufficient to direct assembly of a complete, functional kinetochore structure in human cells. We have identified a heterotetrameric complex comprised of the histone fold proteins CENP-T, -W, -S, and -X as a critical additional component of the kinetochore-DNA interface. The CENP-T-W-S-X complex displays structural similarity to a nucleosome and possesses sequence-independent DNA binding activity. In addition, CENP-T interacts directly with outer kinetochore microtubule-binding proteins to direct kinetochore assembly. Thus, the CENP-T-W-S-X complex plays a pivotal role in connecting the DNA and microtubule interfaces at kinetochores. However, it remains unknown how the CENP-T-W-S-X complex is targeted exclusively to centromeres to generate a single, functional microtubule attachment site on each chromosome. This proposed work will define the mechanisms that direct the centromere localization of the CENP-T-W-S-X complex by assessing: 1) The intrinsic sequence features of the CENP-T-W-S-X complex that are required for its localization, 2) The extrinsic factors that associate with the CENP-T-W-S-X complex to deposit or maintain it at centromeres, and 3) The regulatory modifications and chromatin features that control CENP-T-W-S-X complex localization.