Genomic Analysis of Centromere Assembly and Function

着丝粒组装和功能的基因组分析

• 批准号: 9753267
• 负责人: BETH A SULLIVAN
• 金额: $ 33.38万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753267
• 关键词: Address; Affect; Aneuploidy; Architecture; Area; Biological Assay; CRISPR/Cas technology; Centromere; Chimeric Proteins; Chromatin; Chromatin Fiber; Chromosomal Stability; Chromosomes; Chromosomes, Artificial, Human; Chromosomes, Human, Pair 17; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Abnormality; DNA; Defect; Dicentric chromosome; Disease; Engineering; Epigenetic Process; Genetic Transcription; Genome; Genome Stability; Genome engineering; Genomics; Goals; Histones; Homo sapiens; Human; Human Chromosomes; Individual; Infertility; Kinetochores; Knowledge; Label; Link; Location; Maintenance; Malignant Neoplasms; Maps; Mediating; Modeling; Molecular; Phenotype; Population; Process; Proteins; RNA; Research; Satellite DNA; Satellite RNA; Site; Stretching; Testing; Transcript; Variant; Work; centromere protein A; chromosome movement; experimental study; gene therapy; genomic variation; negative affect; neglect; recruit; repaired; single molecule

Centromeres are essential to genome inheritance. Abnormal centromere function is associated with birth defects, infertility, and cancer. Human centromeric (CEN) chromatin typically forms on alpha satellite DNA, a 171bp monomeric sequence that is tandemly organized into large multi-megabase arrays. More than half of endogenous human chromosomes have two distinct alpha satellite arrays, either of which can be the site of centromere assembly. Using Homo sapiens chromosome 17 (HSA17) as a model, we demonstrated that in most individuals, the centromere is formed at the primary alpha satellite array. Less frequently (30%), the centromere assembles at the alternative array. We showed that centromere location is dictated by genomic variation within the primary alpha satellite array. When the array contains extensive size and sequence variation, the centromere is usually assembled at the alternative array nearby. However, if the centromere forms at a variant array, the kinetochore is architecturally flawed, resulting in chromosome aneuploidy. The molecular basis for how genomic variation affects centromere assembly and maintenance is not clear. Most human chromosomes must choose between two (or more) locations at which to build a stable centromere, so our work will address a fundamental gap in the knowledge of basic processes governing centromere choice and chromosome stability. In this proposal, we will define molecular links between human centromere assembly and genomic variation in alpha satellite DNA. The proposed work will test the hypothesis that alpha satellite arrays containing variant higher order repeat (HORs) are sub-optimally organized for proper kinetochore assembly. We also postulate that variant HORs produce unstable transcripts that cannot interact appropriately with centromere proteins (CENPs). The experiments in this proposal will define the altered molecular relationship of alpha satellite variation and centromere protein assembly by: 1) mapping alpha satellite long-range organization of HORs and CENPs and characterizing RNA-CENP interactions at normal and defective centromeres, 2) distinguishing if variant arrays are unable to recruit versus retain CENPs, 3) using human artificial chromosome assembly assays to test the ability of variant arrays to assemble centromeres de novo, and 4) repairing defective centromeres using CRISPR engineering. This proposal will address mechanisms of centromere choice and assembly by focusing on the largely unexplored area of genomic variation within highly repetitive DNA.

着丝粒对基因组遗传是必不可少的。着丝粒功能异常与出生有关 缺陷、不孕不育和癌症。人着丝粒（CEN）染色质通常形成在α卫星DNA上， 一个171 bp的单体序列，串联组成大的多兆碱基阵列。一半以上 内源性人类染色体有两个不同的α卫星阵列，其中任何一个都可以是位点， 着丝粒组装使用智人17号染色体（HSA 17）作为模型，我们证明，在 大多数个体的着丝粒形成于初级α随体阵列。较少（30%）， 着丝粒在交替排列处组装。我们发现，着丝粒的位置是由基因组决定的， 主要阿尔法卫星阵列内的变化。当数组包含大量的大小和序列时， 在一种变型中，着丝粒通常在附近的交替阵列处组装。然而，如果着丝粒 当染色体在一个变体阵列上形成时，动粒在结构上是有缺陷的，导致染色体非整倍性。的 基因组变异如何影响着丝粒装配和维持的分子基础尚不清楚。最 人类染色体必须在两个（或多个）位置之间进行选择，以建立稳定的着丝粒， 我们的工作将解决一个根本性的差距，在知识的基本过程，支配着丝粒的选择 和染色体稳定性。在这个建议中，我们将定义人类着丝粒之间的分子联系 α卫星DNA的组装和基因组变异。这项拟议中的工作将测试阿尔法的假设， 包含变异高阶重复序列（HOR）的卫星阵列被次优地组织以进行适当的 动粒装配我们还假设变异的HORs产生不稳定的转录本， 适当地与着丝粒蛋白（CENP）结合。本提案中的实验将定义 α卫星变异与着丝粒蛋白组装的分子关系：1）定位α HORs和CENP的卫星远程组织，并在正常条件下表征RNA-CENP相互作用 和有缺陷的着丝粒，2）区分变体阵列是否不能募集相对于保留CENP，3） 使用人类人工染色体组装测定来测试变体阵列组装的能力 着丝粒从头修复，和4）使用CRISPR工程修复有缺陷的着丝粒。这项建议会 解决机制的着丝粒的选择和大会，重点放在很大程度上未开发的领域， 高度重复DNA中的基因组变异。