Maintaining the integrity of a genome

项目总结/摘要 为了完全掌握基因组不稳定性的分子起源，该领域必须在分子水平上理解 着丝粒如何促进染色体的稳定传递。基因组不稳定性是 各种人类病理学，包括癌症和生殖老化。我们的长期目标是确定 进化上保守的粘着蛋白复合体通过其在染色体中的作用维持基因组的完整性， 分离、染色体组织和双链断裂修复。姐妹染色单体凝聚力的丧失是 推测是染色体不稳定的主要原因。本申请的目的是产生一种 粘附素如何作用于单个人类着丝粒以实现着丝粒粘附的分子模型 染色体分离准确。核心假设是，粘连蛋白和DNA连接在一起， 创建姐妹染色单体凝聚的着丝粒独特景观，以防止染色体不稳定性。的 因此，人类着丝粒的变异和着丝粒的凝聚力可能会影响每种病毒的传播。 染色体。我们将测试的想法，着丝粒特异性凝聚力必须被视为一个遗传 姐妹染色单体凝聚和分离的决定因素，以便有一个完整的模型， 染色体不稳定性通过两个特定的目的发生：1）发现着丝粒内聚的景观， 个体人类着丝粒和2）检查染色体着丝粒的凝聚力如何维持整倍性。 在第一个目标下，校准的配对末端ChIP seq将用于相对于动粒的粘着蛋白结合的图谱 蛋白质和人组织培养细胞中的人着丝粒阵列。这一做法将得到以下方面的补充： 细胞中相同的三种成分（着丝粒、动粒和粘着蛋白）的超分辨率成像，以及 将包括基于成像的着丝粒特异性内聚的测定。这些方法将共同 产生一个线性和三维地图内和周围的个别人类着丝粒的凝聚力。第二个目标 我们将研究着丝粒特异性的着丝粒凝聚模式如何阻止染色体 培养细胞和异种移植肿瘤组织中的错误分离事件。结果将是根本性的 基于着丝粒阵列的内聚疲劳的原理和由此产生的染色体不稳定性模式。的 这项研究是创新的，因为它结合了人类着丝粒DNA阵列的最新信息， 着丝粒DNA的凝聚力组织的工作模型，和新的定量分子，基因组， 和成像工具，以探讨如何着丝粒凝聚力强制准确的姐妹染色单体分离。的 拟议的研究是重要的，因为着丝粒阵列可能是未被识别的遗传决定因素， 染色体不稳定性许多类型的癌症都与看似随机的不稳定模式有关， 可能具有独特的着丝粒内聚特征的分子起源。此外，许多癌症 与影响染色体分离机制的突变相关，如粘着蛋白。的结果 该项目将更全面地了解染色体不稳定性的机制。

Project Summary/Abstract In order to fully grasp the molecular origins of genome instability, the field must understand at a molecular level how centromeres work to promote the stable transmission of chromosomes. Genome instability underlies a variety of human pathologies, including cancer and reproductive aging. Our long-term goal is to determine how the evolutionarily conserved cohesin complex maintains genome integrity through its roles in chromosome segregation, chromosome organization, and double-strand break repair. Loss of sister chromatid cohesion is speculated to be a major contributor to chromosome instability. The objective of this application is to produce a molecular model for how cohesin operates at individual human centromeres to achieve centromeric cohesion and accurate chromosome segregation. The central hypothesis is that cohesin and DNA catenation together create centromere-unique landscapes of sister chromatid cohesion to prevent chromosome instability. The variation in human centromeres and centromeric cohesion may therefore impact the transmission of each chromosome. We will test the idea that centromere-specific cohesion must be considered as a genetic determinant of sister chromatid cohesion and segregation in order to have a complete model for how chromosomal instability occurs through two specific aims: 1) discover the landscape of centromeric cohesion at individual human centromeres and 2) examine how chromosome centromeric cohesion maintains euploidy. Under the first aim, calibrated paired-end ChIP seq will be used to map cohesin binding relative to kinetochore proteins and human centromeric arrays in human tissue culture cells. This approach will be complemented by superresolution imaging of the same three components (centromeres, kinetochores, and cohesin) in cells, and will include imaging-based determination of centromere-specific cohesion. Together these approaches will produce a linear and 3D map of cohesion within and around individual human centromeres. In the second aim we will examine how centromere-specific patterns of centromeric cohesion prevent chromosome missegregation events in cultured cells and in xenograft tumor tissue. The outcome will be fundamental principles of centromeric array-based cohesion fatigue and resulting patterns of chromosome instability. The research is innovative because it incorporates the latest information on human centromeric DNA arrays, a new working model for the organization of centromeric DNA by cohesion, and new quantitative molecular, genomic, and imaging tools to probe how centromeric cohesion enforces accurate sister chromatid segregation. The proposed research is significant because centromeric arrays may be unrecognized genetic determinants of chromosome instability. Many types of cancer are associated with seemingly random patterns of instability that may have molecular origins in unique centromeric cohesion profiles. Furthermore, many cancers are associated with mutations that impact chromosome segregation machinery, such as cohesin. The outcome of this project will be a more complete picture of the mechanisms underlying chromosomal instability.