Genomic Analysis of Centromere Assembly and Function

着丝粒组装和功能的基因组分析

基本信息

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
项目类别：
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

项目摘要

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.

着丝粒对于基因组遗传至关重要。着丝粒功能异常与出生有关缺陷、不孕症和癌症。人类着丝粒 (CEN) 染色质通常形成于 α 卫星 DNA 上，串联组织成大型多兆碱基阵列的 171bp 单体序列。超过一半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着丝粒组装。使用智人 17 号染色体（HSA17）作为模型，我们证明了大多数个体的着丝粒是在初级阿尔法卫星阵列处形成的。不太常见（30%），着丝粒在替代阵列处组装。我们发现着丝粒的位置是由基因组决定的主阿尔法卫星阵列内的变化。当数组包含大量大小和序列时变异时，着丝粒通常组装在附近的替代阵列上。然而，如果着丝粒在变异阵列中形成，动粒在结构上有缺陷，导致染色体非整倍体。这基因组变异如何影响着丝粒组装和维持的分子基础尚不清楚。最多人类染色体必须在两个（或更多）位置之间进行选择来构建稳定的着丝粒，因此我们的工作将解决控制着丝粒选择的基本过程知识方面的根本差距和染色体稳定性。在本提案中，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。拟议的工作将检验阿尔法的假设包含变体高阶重复序列 (HOR) 的卫星阵列的组织方式不是最理想的着丝粒组装。我们还假设变异 HOR 产生不能相互作用的不稳定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改变的 α卫星变异和着丝粒蛋白组装的分子关系：1）绘制α图谱 HOR 和 CENP 的卫星远程组织并表征正常情况下的 RNA-CENP 相互作用和有缺陷的着丝粒，2) 区分变体阵列是否无法招募和保留 CENP，3) 使用人类人工染色体组装检测来测试变体阵列的组装能力从头开始着丝粒，4) 使用 CRISPR 工程修复有缺陷的着丝粒。该提案将通过关注很大程度上尚未探索的领域来解决着丝粒选择和组装的机制高度重复的DNA中的基因组变异。