Ectopic centromere assembly in humans

人类异位着丝粒组装

• 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
• 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

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.

着丝粒是基因组遗传所必需的，作为动粒组装和协调的场所 细胞分裂时染色体分离。异常的着丝粒功能与出生缺陷有关， 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α的区域形成的 卫星DNA然而，在癌细胞中，新的异位着丝粒（新着丝粒）经常出现在完整的染色体上。 染色体或小的额外标记染色体（sSMC）在缺乏α卫星DNA的基因组区域。 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， 易受新着丝粒激活的影响。关于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。这在很大程度上是由于缺乏实验系统来研究异位 人类细胞中的着丝粒形成。在这项提案中，我们建议建立基于CRISPR的检测方法， 在特定的人类染色体上的新着丝粒，比较它们的（epi）基因组组织和功能效率， 并定义DNA和染色质在形成部位的特征。在目标1中，我们将使用基于CRISPR的方法， 在去除或抑制后独立地触发个体人染色体上的新着丝粒形成， 原始的着丝粒我们的理由是，通过去除或使最大浓度的CENP-A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影响的异位部位 入侵在目标2中，我们将通过绘制染色质图谱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 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新的方法来诱导异位着丝粒组装在 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。