Mechanisms of epigenetic assembly, maintenance and propagation of human centromeres

项目摘要 在细胞分裂过程中，染色体（遗传的基本单位）向每个子细胞的传递是由 着丝粒我们的实验室有兴趣了解人类着丝粒是如何组装，维持和 在整个细胞周期中传播。哺乳动物的着丝粒不是由DNA序列决定的， 通过表观遗传获得一种叫做CENP-A的组蛋白H3变体。我们之前已经表明，大多数 含CENP-A的染色质由同型八聚体核小体组成， 不仅复制DNA，而且纠正异位CENP-A沉积中的错误，导致异位CENP-A沉积的去除。 CENP-A和仅将CENP-A限制于着丝粒。与此同时，同样的DNA复制机制 为了精确地将CENP-A再循环到子着丝粒上， 序列的CENP-A从染色体上去除的DNA复制机制的联合作用 着丝粒上CENP-A的保留和再循环导致人类染色体的表观遗传维持， 每个染色体上的单个位点上的着丝粒。我们未来五年的目标是利用端粒- to-Telomere基因组组装，首次描述了完全组装的人类着丝粒基因组 地图和DNA甲基化数据，以建立CENCODE，人类着丝粒的表观基因组景观。的 CENCODE将使用CENP-A、CENP-B和CENP-C的现有和新的ChIP测序和Cut & Run数据集构建。 C和CENP-T/W/S/X核小体样复合物在每个细胞周期点，以及可用的DNA甲基化 数据，映射到端粒到端粒基因组组装内的着丝粒，以创建一个新的基因组 和人类着丝粒的表观基因组图谱。我们将确定CENP-A结合与 使用长读纳米孔在新着丝粒和CENP-A沉积的异位位点处的DNA甲基化 测序和着丝粒DNA甲基化的功能重要性。这个项目将奠定基础 用于将来研究衰老和癌症中的着丝粒表观遗传学变化。接下来，我们将确定 人类着丝粒的位置稳定性和CENP-A是否能够指定着丝粒位置 在单个细胞周期和整个细胞增殖过程中以碱基对分辨率精确和稳定， 通过使用携带新着丝粒的患者来源的细胞系。过度表达的贡献 CENP-A和/或HJURP，两者已知在几种类型的癌症中升高，对人着丝粒漂移和/或 扩张将被确定。我们将探讨异位负载着丝粒的错误纠正机制 目的是防止染色体分离失败以及新着丝粒形成的蛋白质。最后， 创新的单分子方法将用于定义组蛋白的组成和组合， 在位于整个细胞的单个含CENP-A的核小体内的表观遗传翻译后修饰 基因组：在重复的人类着丝粒，在非着丝粒异位位点，和在新着丝粒。

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.