Molecular Analysis of Kinetochore Function

着丝粒功能的分子分析

• 批准号: 10400840
• 负责人: Iain McPherson Cheeseman
• 金额: $ 72.15万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400840
• 关键词: Area; Binding; Biochemical; Biological; Biological Assay; CRISPR/Cas technology; Cell Cycle; Cell division; Cells; Centromere; Chromatin; Chromosome Segregation; Chromosomes; Complement; Complex; DNA; Defect; Development; Diagnosis; Ensure; Environment; Epigenetic Process; Failure; Gene Targeting; Generations; Genes; Genetic; Genetic Screening; Goals; Histones; Human; Individual; Kinetochores; Laboratories; Meiosis; Microtubule Depolymerization; Microtubules; Mitosis; Modeling; Molecular; Molecular Analysis; Molecular Machines; Molecular Structure; Movement; Phenotype; Polymers; Process; Proteins; Proteomics; Regulation; Rod; Site; Structure; Variant; Vertebrates; Work; base; cancer therapy; centromere protein A; experimental study; genetic approach; genetic information; genome editing; genome-wide; loss of function; programs; tumor progression

Project Summary/Abstract The goal of my laboratory is to define the molecular mechanisms by which accurate cell division occurs. Our efforts focus on the kinetochore, the central player in directing chromosome segregation. The kinetochore is a macromolecular structure that connects chromosomes to the microtubule polymers that power their movement. Our goal is to generate a coherent model for how the kinetochore functions as an integrated molecular machine. To direct faithful chromosome segregation, kinetochores must form two key interaction interfaces. First, kinetochores must associate with a single site on each chromosome to direct the assembly of a stable kinetochore structure. In vertebrates, this site is defined epigenetically by the presence of a specialized histone variant termed CENP-A, and through contributions of a 16-subunit Constitutive Centromere-Associated Network (CCAN). Together, these proteins form the interface with centromeric chromatin. Despite the identification of these molecules, it remains unclear how the CCAN is established and reorganized during the cell cycle, and also how these processes are modulated during different cell division programs, such as in the context of meiosis and early development. In addition, centromeres must have a specific open chromatin environment to facilitate proper kinetochore function, but the relationship between the CCAN and centromere chromatin is poorly defined. Second, kinetochores must form robust interactions with dynamic microtubule polymers and harness the force generated by depolymerizing microtubules to direct chromosome segregation. To understand this elegant interface, it is critical to define the individual contributions of key outer kinetochore microtubule-binding complexes and also assess their integrated activities. The kinetochore must also sense and correct microtubule attachments to ensure high fidelity chromosome segregation, requiring the functions from the spindle assembly checkpoint components. To understand these critical kinetochore activities and the functional requirements for chromosome segregation, it is also important to define the complete complement of human genes that are required for chromosome segregation. The advent of CRISPR/Cas9-based genome editing has transformed the capability to conduct functional genetics experiments in human cells. This includes the ability to systematically screen gene targets for their loss of function phenotypes using cell biological assays and genome-wide functional genetics screening to analyze context-dependent essentiality to define synthetic lethality relationships. For the work in this proposal, our lab will investigate the fundamental mechanisms of chromosome segregation and kinetochore function, focusing on three related areas: 1) Specification and formation of the centromere- DNA interface, 2) Generation and regulation of dynamic kinetochore-microtubule interactions, 3) Functional genetic approaches to analyze chromosome segregation. We will analyze key open questions in these important areas using combined cell biological, biochemical, proteomic, and functional genetics approaches.

项目总结/摘要 我的实验室的目标是确定精确的细胞分裂发生的分子机制。我们 努力集中在着丝粒上，着丝粒是指导染色体分离的中心角色。动粒是一种 连接染色体和微管聚合物的大分子结构，微管聚合物为染色体的运动提供动力。 我们的目标是产生一个连贯的模型，如何动粒功能作为一个完整的分子 机为了指导忠实的染色体分离，动粒必须形成两个关键的相互作用界面。 首先，动粒必须与每条染色体上的单个位点相关联，以指导染色体的组装。 稳定的动粒结构。在脊椎动物中，该位点在表观遗传学上由一个专门的 称为CENP-A的组蛋白变体，并通过16个亚基组成型着丝粒相关的贡献 网络（CCAN）。这些蛋白质共同形成与着丝粒染色质的界面。尽管 尽管这些分子的鉴定，但仍不清楚CCAN是如何在细胞周期中建立和重组的。 细胞周期，以及这些过程在不同的细胞分裂程序中是如何调节的，例如在细胞周期中， 减数分裂和早期发育的背景。此外，着丝粒必须有一个特定的开放染色质 环境，以促进适当的动粒功能，但CCAN和着丝粒之间的关系， 染色质界限不清。第二，动粒必须与动力学形成强大的相互作用， 微管聚合物和利用解聚微管产生的力量， 染色体分离要理解这个优雅的接口，定义个人 关键外动粒微管结合复合物的贡献，并评估其综合 活动动粒还必须感知和纠正微管附着以确保高保真度 染色体分离，需要来自纺锤体组装检查点组件的功能。到 了解这些关键的动粒活动和染色体分离的功能要求， 同样重要的是要确定人类基因的完整补充， 染色体分离基于CRISPR/Cas9的基因组编辑的出现改变了 能够在人类细胞中进行功能遗传学实验。这包括系统地 使用细胞生物学测定和全基因组筛选功能丧失表型的基因靶标 功能遗传学筛选，分析背景依赖的必要性，以确定合成致死率 关系。 本实验室将对染色体分离的基本机制进行研究 和动粒功能，集中在三个相关领域：1）着丝粒的规范和形成- DNA界面，2）动态运动-微管相互作用的产生和调节，3）功能 遗传学方法来分析染色体分离。我们将分析这些关键的开放性问题， 利用组合的细胞生物学、生物化学、蛋白质组学和功能遗传学方法的重要领域。