Molecular Analysis of Kinetochore Function

着丝粒功能的分子分析

• 批准号: 10152611
• 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/10152611
• 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.

项目摘要/摘要 我的实验室的目标是确定准确的细胞分裂发生的分子机制。我们的 努力的重点是着丝粒，这是指导染色体分离的核心角色。动毛虫是一种 将染色体连接到为其运动提供动力的微管聚合物的大分子结构。 我们的目标是生成一个连贯的模型，说明动粒如何作为一个完整的分子发挥作用 机器。为了指导正确的染色体分离，着丝点必须形成两个关键的相互作用界面。 首先，动点必须与每条染色体上的单个位置相关联，以指导 稳定的动粒结构。在脊椎动物中，这个位置在表观遗传学上是由特殊的 称为CENP-A的组蛋白变异体，并通过16个亚单位组成着丝粒相关的贡献 网络(CCAN)。这些蛋白质共同形成与着丝粒染色质的界面。尽管 对于这些分子的鉴定，目前尚不清楚CCAN是如何在 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 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。 环境有助于正常的着丝粒功能，但CCAN和着丝粒之间的关系 染色质的定义不明确。其次，Kintochore必须与Dynamic 微管聚合物和利用解聚微管产生的力来定向 染色体分离。要理解这种优雅的界面，关键是要定义个人 关键的外部动粒微管结合复合体的贡献及其整合性评估 活动。动粒还必须感知和纠正微管连接，以确保高保真 染色体分离，需要来自纺锤体组件检查点的功能。至 了解这些关键的动粒活动和染色体分离的功能要求，它 定义人类基因的完整补充也很重要，这些基因是 染色体分离。基于CRISPR/Cas9的基因组编辑的出现改变了 在人类细胞中进行功能遗传学实验的能力。这包括系统地 应用细胞生物学方法和全基因组技术筛选功能缺失的基因靶点 功能遗传学筛查以分析上下文相关的重要性以确定合成致死性 两性关系。 在这项建议中，我们的实验室将研究染色体分离的基本机制。 和着丝粒功能，集中在三个相关领域：1)着丝粒的指定和形成-- DNA界面，2)动态动粒-微管相互作用的产生和调节，3)功能 分析染色体分离的遗传学方法。我们将分析这些问题中的关键未解决问题 使用细胞生物学、生化、蛋白质组学和功能遗传学相结合的方法的重要领域。