Ectopic centromere assembly in humans

着丝粒是基因组遗传所必需的，是着丝粒组装和协调的场所。 细胞分裂过程中的染色体分离。着丝粒功能异常与出生缺陷有关， 不孕不育和癌症。大多数人类着丝粒都是在含有高度重复的阿尔法的区域一致形成的 卫星DNA。然而，在癌细胞中，新的异位着丝粒(新着丝粒)经常出现在完整的 位于缺乏α卫星DNA的基因组区域的染色体或小的额外标记染色体(SSMC)。 对模式生物(果蝇、酵母、鸡细胞)的研究表明，基因组的某些区域， 包括着丝粒、异染色质和开放染色质或高转录区域 易受新着丝粒激活的。关于人类新着丝点的形成或形成的机制知之甚少。 新着丝粒在肿瘤形成中的作用。这在很大程度上是由于缺乏研究异位的实验系统。 人类细胞中着丝粒的形成。在这项建议中，我们建议建立基于CRISPR的检测方法来诱导 人类特定染色体上的新着丝粒，比较它们(Epi)的基因组组织和功能效率， 并确定形成部位的DNA和染色质特征。在目标1中，我们将使用基于CRISPR的方法来 在单个人类染色体上独立触发新着丝粒的形成后，移除或抑制 原生着丝粒。我们的理论是，通过去除或使最大浓度的CENP-A丧失能力， 新着丝点将出现在含有低浓度CENP-A和/或易受CENP-A影响的异位部位 入侵。在目标2中，我们将通过定位染色质来定义新着丝粒形成的功能后果 异位着丝粒区域的结构和测量新着丝粒形成对基因表达和 周围的染色质环境。这项研究的技术和智力成果将从根本上 改变我们创造和研究人类染色体异常的能力，扩大我们对基因组功能的看法 和可塑性。我们提出的实验将建立诱导异位着丝粒组装的新方法 为了更好地了解新着丝粒形成的临床后果，我们对人类特定的染色体进行了研究。

Centromeres are essential to genome inheritance, serving as the site of kinetochore assembly and coordinating chromosome segregation during cell division. Abnormal centromere function is associated with birth defects, infertility, and cancer. Most human centromeres are consistently formed at regions containing highly repetitive alpha satellite DNA. However, in cancer cells, new, ectopic centromeres (neocentromeres) frequently arise on intact chromosomes or small supernumerary marker chromosomes (sSMCs) at genomic regions that lack alpha satellite DNA. Studies in model organisms (Drosophila, yeast, chicken cells) have suggested that certain regions of the genome, including pericentromeres, heterochromatin, and regions of open chromatin or high transcription, are particularly amenable to neocentromere activation. Little is known about the mechanisms of human neocentromere formation or the role of neocentromeres in neoplasia. This is largely due to the lack of experimental systems to study ectopic centromere formation in human cells. In this proposal, we propose to establish CRISPR-based assays to induce neocentromeres on specific human chromosomes, compare their (epi)genomic organization and functional efficiency, and define DNA and chromatin features at sites of formation. In Aim 1, we will use CRISPR-based approaches to independently trigger neocentromere formation on individual human chromosomes after removal or suppression of the native centromere. Our rationale is that by removing or incapacitating the largest concentration of CENP-A, neocentromeres will arise at ectopic sites that harbor low concentrations of CENP-A and/or are vulnerable to CENP-A invasion. In Aim 2, we will define the functional consequences of neocentromere formation by mapping chromatin structure at ectopic centromere regions and measuring the effect of neocentromere formation on gene expression and the surrounding chromatin environment. The technological and intellectual outcomes of this study will fundamentally transform our ability to create and study human chromosome abnormalities and expand our view of genome function and plasticity. Our proposed experiments will establish new methods to induce ectopic centromere assembly on specific human chromosomes to better understand the clinical consequences of neocentromere formation.