Molecular Analysis of Kinetochore Function

着丝粒功能的分子分析

• 批准号: 9812941
• 负责人: Iain McPherson Cheeseman
• 金额: $ 8.21万
• 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/9812941
• 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; 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; retinal rods; 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 我实验室的目的是定义出现准确细胞分裂的分子机制。我们的 努力专注于指导染色体隔离的核心参与者Kinetochore。动力学是 大分子结构将染色体连接到动力运动的微管聚合物。 我们的目标是生成一个连贯的模型，以使动力学如何充当综合分子 机器。要指导忠实的染色体隔离，动力学必须形成两个关键的相互作用界面。 首先，动力学必须与每个染色体上的一个位点相关联，以指导A组装 稳定的动力学结构。在脊椎动物中，该站点是通过专业的存在在表观遗传上定义的 组蛋白变体称为CENP-A，并通过16个亚基构型粒子相关的贡献 网络（CCAN）。这些蛋白质一起与丝粒染色质形成界面。尽管有 鉴定这些分子，尚不清楚如何在期间建立和重组CCAN 细胞周期，以及如何在不同的细胞分裂程序中​​调制这些过程，例如 减数分裂和早期发展的背景。此外，中心粒必须具有特定的开放染色质 促进适当的动力学功能的环境，但是CCAN与Centromere之间的关系 染色质的定义很差。其次，动力学必须与动态形成强大的相互作用 微管聚合物和利用通过去聚合微管产生的力直接产生的力 染色体分离。要了解这个优雅的界面，定义个人至关重要 关键的外部动力学微管结合复合物的贡献，还评估其综合 活动。动力学还必须感知并纠正微管附件，以确保高保真度 染色体隔离，需要纺锤体组件检查点组件的功能。到 了解这些关键的动力学活动以及染色体隔离的功能要求 定义人类基因的完整补体也很重要 染色体分离。基于CRISPR/CAS9的基因组编辑的到来改变了 在人类细胞中进行功能遗传学实验的能力。这包括系统的能力 筛选基因靶标，用于使用细胞生物学测定和全基因组的功能表型丧失功能表型 功能遗传学筛选以分析上下文依赖性的本质以定义合成杀伤力 关系。 对于本提案中的工作，我们的实验室将研究染色体隔离的基本机制 和动力学功能，重点关注三个相关领域：1）丝粒的规范和形成 DNA界面，2）动态运动学微管相互作用的生成和调节，3）功能 分析染色体分离的遗传方法。我们将在其中分析关键的开放问题 使用联合细胞生物学，生化，蛋白质组学和功能遗传学方法的重要区域。