Molecular control of centromere specification and kinetochore assembly

 描述(申请人提供)：本提案的目的是确定着丝粒-DNA界面的性质，并确定着丝粒特定的染色质如何指导人类细胞中功能着丝粒结构的组装。在每一次细胞分裂过程中，遗传物质的整个组合必须准确地分割到子细胞。即使是一个单一的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，对发育和疾病造成严重后果，包括肿瘤形成。在指导染色体分离的中心角色是着丝粒，这是一种大的大分子结构，调节纺锤体微管和每条染色体上称为着丝粒的区域之间的附着。确定动粒功能的分子基础对于了解导致肿瘤细胞生长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， 着丝粒是由与序列无关的表观遗传机制指定的，这种机制涉及含有组蛋白H3变体CENP-A的核小体的靶向沉积。一个根本的悬而未决的问题是，动粒的其余部分是如何在CENP-A下游组装的。我们以前的工作表明，CENP-A不足以直接在人类细胞中组装完整的、有功能的动粒结构。我们已经确定由组蛋白折叠蛋白CENP-T、-W、-S和-X组成的异四聚体复合体是动粒-DNA界面的关键附加成分。CENP-T-W-S-X具有与核小体相似的结构，并具有序列无关的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，指导动粒组装。因此，CENP-T-W-S-X复合体在连接运动中心的DNA和微管界面上起着关键作用。然而，目前尚不清楚CENP-T-W-S-X复合体是如何专门针对着丝粒在每条染色体上产生一个单一的、功能性的微管附着位点的。这项拟议的工作将通过评估以下方面来确定指导CENP-T-W-S-X复合体着丝粒定位的机制：1)其定位所需的CENP-T-W-S-X复合体的内在序列特征；2)与CENP-T-W-S-X复合体相关的外部因素使其沉积或维持在着丝粒上；以及3)控制CENP-T-W-S-X复合体定位的调控修饰和染色质特征。

 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.