Genomic Analysis of Centromere Assembly and Function

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。人类着丝粒(CEN)染色质通常形成在阿尔法卫星DNA上， 一个171bp的单体序列，按序组织成大的多兆碱基阵列。超过一半的 内源性人类染色体有两个不同的阿尔法卫星阵列，其中任何一个都可以是 着丝粒集合。以智人17号染色体(HSA17)为模型，我们在 大多数个体的着丝粒是在初级阿尔法卫星阵列上形成的。不太常见的(30%)， 着丝粒聚集在另一个阵列上。我们发现着丝粒的定位是由基因组决定的 主要的阿尔法卫星阵列内的变化。当数组包含大量大小和序列时 对于变异，着丝粒通常组装在附近的替代阵列上。然而，如果着丝粒 在变异阵列上形成，着丝粒在结构上有缺陷，导致染色体非整倍体。这个 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。多数 人类染色体必须在建立稳定着丝粒的两个(或更多)位置之间进行选择，因此 我们的工作将解决在控制着丝粒选择的基本过程方面的知识的根本差距 和染色体的稳定性。在这项提案中，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。拟议中的工作将检验阿尔法的假设 包含可变高阶重复(HORS)的卫星阵列被次优地组织为适当的 动粒组件。我们还假设，变异的hors产生不稳定的转录本，不能相互作用。 与着丝粒蛋白(CENPs)相适应。本提案中的实验将定义更改后的 α卫星变异与着丝粒蛋白组装的分子关系：1)定位阿尔法 正常情况下HORS和CENPs的卫星远程组织和RNA-CENP相互作用的特征 和有缺陷的着丝粒，2)区分变异阵列是否无法招募和保留CENP，3) 使用人类人工染色体组装试验来测试变异阵列的组装能力 着丝粒从头开始，以及4)使用CRISPR工程修复有缺陷的着丝粒。这项提议将 通过重点关注在很大程度上未被探索的区域，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。

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.