Mechanisms of epigenetic assembly, maintenance and propagation of human centromeres

人类着丝粒的表观遗传组装、维持和繁殖机制

• 批准号: 10626793
• 负责人: Yael Nechemia-Arbely
• 金额: $ 39.75万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-19 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626793
• 关键词: Aging; Base Pairing; Binding; Cell Cycle; Cell Line; Cell Proliferation; Cell division; Centromere; ChIP-seq; Chromatin; Chromosome Arm; Chromosome Segregation; Chromosomes; Complex; Congenital Abnormality; DNA; DNA Methylation; DNA Sequence; DNA biosynthesis; Data; Data Set; Daughter; Deposition; Development; Epigenetic Process; Excision; Failure; Foundations; Future; Genome; Genomic Instability; Genomics; Goals; Histone H3; Histones; Human; Infertility; Maintenance; Malignant Neoplasms; Maps; Mediating; Medical; Molecular; Nucleosomes; Patients; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Recycling; Resolution; Running; Site; Specific qualifier value; Variant; Vision; cancer type; centromere protein A; centromere protein C; chromosome replication; daughter cell; epigenomics; innovation; interest; nanopore; novel; overexpression; prevent; protein complex; single molecule; telomere; tumor

PROJECT SUMMARY Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Our lab is interested in understanding how human centromeres are assembled, maintained and propagated across the cell cycle. Mammalian centromeres are determined not by DNA sequence but rather through epigenetic acquisition of a histone H3 variant called CENP-A. We have previously shown that most CENP-A-containing chromatin consist of homotypic octameric nucleosomes and that DNA replication functions not only to replicate DNA but also to correct errors in ectopic CENP-A deposition, leading to removal of ectopic CENP-A and restricting CENP-A to centromeres only. In parallel, the same DNA replication machinery is capable to precisely recycle CENP-A onto the daughter centromeres to re-assemble CENP-A onto the same centromeric sequences. The combined actions of the DNA replication machinery of CENP-A removal from the chromosome arms and CENP-A retention and recycling at the centromere results in the epigenetic maintenance of human centromeres at a single locus on each chromosome. Our vision for the next five years is to harness the Telomere- to-Telomere genome assembly, that contains the first description of fully assembled human centromeric genomic maps and DNA methylation data, to build the CENCODE, an epigenomic landscape of human centromeres. The CENCODE will be built using available and new ChIP-sequencing and Cut & Run datasets of CENP-A, CENP- C and CENP-T/W/S/X nucleosome-like complex at each cell cycle point, as well as available DNA methylation data, mapped to the centromeres within the Telomere-to-Telomere genome assembly to create a novel genomic and epigenomic map for human centromeres. We will determine the relationship between CENP-A binding and DNA methylation at neocentromeres and at ectopic sites of CENP-A deposition using long-read nanopore sequencing, and the functional importance of centromeric DNA methylation. This project will lay the foundation for future examination of centromeric epigenetic changes in aging and cancer. Next we will determine the positional stability of human centromeres and whether CENP-A is capable to specify centromere position precisely and stably across a single cell cycle and throughout cellular proliferation at base-pair resolution, through the use of patient derived cell lines that harbor a neocentromere. The contribution of overexpressed CENP-A and/or HJURP, both known to be elevated in several types of cancer, to human centromere drift and/or expansion will be determined. We will explore error corrections mechanisms of ectopically loaded centromeric proteins that aim to prevent failure of chromosome segregation as well as neocentromere formation. Finally, innovative single molecule approaches will be used to define histone compositions and combinations of epigenetic posttranslational modifications within single CENP-A-containing nucleosomes located across the genome: at repetitive human centromeres, at non-centromeric ectopic sites, and at neocentromeres.

项目摘要 在细胞分裂过程中，染色体的赋予每个子细胞的基本单位是由细胞分裂介导的 中心。我们的实验室有兴趣了解人类的中心粒如何组装，维护和 在整个细胞周期中传播。哺乳动物的共粒不是由DNA序列确定的，而是确定 通过表观遗传学获取称为CENP-A的组蛋白H3变体。我们以前表明大多数 含CENP-A的染色质由同型八聚体核小体和DNA复制功能组成 不仅要复制DNA，还要纠正异位CENP-A沉积中的错误，导致异位去除 CENP-A并将CENP-A限制为centromeres。同时，相同的DNA复制机械能够 精确地将CENP-A回收到女儿的centromeres中，以将CENP-A重新组装到同一中心粒上 序列。从染色体中去除CENP-A的DNA复制机制的组合作用 武器和CENP-A保留和回收丝粒会导致人类的表观维持 每个染色体上的一个基因座的centromeres。我们接下来五年的愿景是利用端粒 - To-Telomere基因组组件，其中包含完全组装的人丝粒基因组的首次描述 地图和DNA甲基化数据，以构建柱面，这是人类centromeres的表观基因组景观。这 Cencode将使用可用的和新的芯片序列和剪切和运行数据集构建 C和CENP-T/W/S/X在每个细胞周期点上的核小体样复合物以及可用的DNA甲基化 数据，映射到端粒到核粒基因组组件内的丝粒，以创建一种新型的基因组 和人类centromeres的表观基因组图。我们将确定CENP-A结合与 使用长阅读纳米孔的新中心粒和CENP-A沉积的异位部位的DNA甲基化 测序以及丝粒DNA甲基化的功能重要性。这个项目将奠定基础 为了将来检查衰老和癌症的丝粒表观遗传变化。接下来我们将确定 人类丝粒的位置稳定性以及CENP-A是否能够指定中心粒位置 精确，稳定在单个细胞周期以及以基本对分辨率下的整个细胞增殖， 通过使用带有新中心粒的患者衍生细胞系。过表达的贡献 CENP-A和/或HJURP（已知在几种类型的癌症中都升高到人类的丝粒漂移和/或 将确定扩展。我们将探索异位加载的中心质体的错误校正机制 旨在防止染色体分离以及新中质粒形成的蛋白质。最后， 创新的单分子方法将用于定义组蛋白组成和组合 位于单个CENP-A的核小体内的表观遗传学后修饰 基因组：在重复的人centromeres，在非中心异位位点和新中心粒。