Ectopic centromere assembly in humans

人类异位着丝粒组装

基本信息

批准号：
9898095
负责人：
BETH A SULLIVAN
金额：
$ 17.85万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9898095
关键词：
Address Animal Model Biological Biological Assay Biological Models Biology Cell division Cells Centromere Chicken Cells Chromatin Chromatin Structure Chromosome Arm Chromosome Breakage Chromosome Markers Chromosome Segregation Chromosome abnormality Chromosomes Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collection Congenital Abnormality DNA DNA Damage Disease Drosophila genus Engineering Environment Euchromatin Event Excision Gene Expression Genetic Transcription Genome Genome Stability Genome engineering Genomic Instability Genomic Segment Genomics Heterochromatin Histones Human Human Chromosomes Human Engineering Human Genome Image Individual Infertility Kinetochores Location Lysine Malignant Neoplasms Maps Measures Mediating Methods Molecular Neoplasms Normal Cell Outcome Outcome Study Patients Recovery Replication Origin Reporting Research Proposals Role Satellite DNA Site System Testing Validation Variant Work Yeasts base cancer cell cancer genome centromere protein A chromosome movement chromothripsis experimental study functional plasticity patient population

项目摘要

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.

着丝粒对于基因组遗传至关重要，是着丝粒组装和协调的场所细胞分裂过程中染色体分离。着丝粒功能异常与出生缺陷有关，不孕症和癌症。大多数人类着丝粒始终在含有高度重复 α 的区域形成卫星DNA。然而，在癌细胞中，新的异位着丝粒（新着丝粒）经常出现在完整的细胞上。缺乏α卫星DNA的基因组区域的染色体或小型额外标记染色体（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，包括着丝粒周围、异染色质和开放染色质或高转录区域，特别是适合新着丝粒激活。人们对人类新着丝粒形成的机制知之甚少。新着丝粒在肿瘤形成中的作用。这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。在本提案中，我们建议建立基于 CRISPR 的检测来诱导特定人类染色体上的新着丝粒，比较它们的（表观）基因组组织和功能效率，并定义形成位点的 DNA 和染色质特征。在目标 1 中，我们将使用基于 CRISPR 的方法去除或抑制后独立触发个体人类染色体上新着丝粒的形成天然着丝粒。我们的理由是，通过去除最大浓度的 CENP-A 或使其丧失能力，新着丝粒将出现在含有低浓度 CENP-A 和/或易受 CENP-A 影响的异位位点入侵。在目标 2 中，我们将通过绘制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并测量新着丝粒形成对基因表达的影响周围的染色质环境。这项研究的技术和智力成果将从根本上转变我们创造和研究人类染色体异常的能力并扩展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新方法来诱导异位着丝粒组装特定的人类染色体，以更好地了解新着丝粒形成的临床后果。