Ectopic centromere assembly in humans

人类异位着丝粒组装

• 批准号: 10059192
• 负责人: BETH A SULLIVAN
• 金额: $ 22.58万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10059192
• 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

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.

中心粒对于基因组遗传至关重要，作为动力学组装和协调的位点 细胞分裂期间的染色体分离。异常的中心粒功能与先天缺陷有关， 不育和癌症。大多数人的中心粒始终在包含高度重复α的区域形成 卫星DNA。但是，在癌细胞中，新的，异位的丝粒（新中心粒）经常出现完整的 在缺乏α卫星DNA的基因组区域的染色体或小的超级标记染色体（SSMC）。 模型生物（果蝇，酵母，鸡细胞）的研究表明，基因组的某些区域， 包括周围粒细胞，异染色质和开放染色质或高转录的区域尤其是 适合新中心粒激活。关于人类新中心粒形成的机制或 新中心瘤在肿瘤中的作用。这主要是由于缺乏研究异位的实验系统 人类细胞中的丝粒形成。在此提案中，我们建议建立基于CRISPR的测定法以诱导 对特定人类染色体的新中心粒子比较其（EPI）基因组组织和功能效率， 并在形成部位定义DNA和染色质特征。在AIM 1中，我们将使用基于CRISPR的方法来 在去除或抑制后，独立触发新中质膜形成在单个人类染色体上 本地的中心。我们的理由是，通过删除或丧失了最大的CENP-A浓度， Neopertromeres将出现在具有低浓度CENP-A和/或容易受到CENP-A的异位部位。 入侵。在AIM 2中，我们将通过绘制染色质来定义新中心粒形成的功能后果 异位丝粒区域的结构，并测量新中心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 改变我们创建和研究人类染色体异常并扩大我们对基因组功能的看法的能力 和可塑性。我们提出的实验将建立新的方法，以诱导异位中心粒组装 特定的人类染色体可以更好地了解新中心粒形成的临床后果。

项目成果

A genetic memory initiates the epigenetic loop necessary to preserve centromere position.

• DOI: 10.15252/embj.2020105505
• 发表时间: 2020-10-15
• 期刊: The EMBO journal
• 作者: Hoffmann S;Izquierdo HM;Gamba R;Chardon F;Dumont M;Keizer V;Hervé S;McNulty SM;Sullivan BA;Manel N;Fachinetti D
• 通讯作者: Fachinetti D