Mechanisms of epigenetic assembly, maintenance and propagation of human centromeres

项目总结 染色体是遗传的基本单位，在细胞分裂过程中，染色体传递到每个子细胞是由 着丝粒。我们的实验室有兴趣了解人类着丝粒是如何组装、维护和 在整个细胞周期中传播。哺乳动物着丝粒不是由DNA序列决定的，而是由DNA序列决定的 通过表观遗传获得称为CENP-A的组蛋白H3变异体。我们之前已经表明，大多数 含CENP-A染色质由同型八聚体核小体组成，DNA复制功能 不仅复制DNA，而且纠正异位CENP-A沉积中的错误，导致异位移除 CENP-A和将CENP-A仅限于着丝粒。同时，同样的DNA复制机制也能够 精确地将CENP-A循环到子着丝粒上，将CENP-A重新组装到相同的着丝粒上 序列。CENP-A从染色体上移除的DNA复制机制的联合作用 ARMS和CENP-A在着丝粒的保留和再循环导致人类表观遗传的维持 着丝粒位于每条染色体上的单个位置。我们对未来五年的愿景是利用端粒- TO-端粒基因组组装，包含对完全组装的人类着丝粒基因组的第一个描述 图和DNA甲基化数据，以建立CENCODE，人类着丝粒的表观基因组图谱。这个 CENCODE将使用CENP-A、CENP-A和CENP-A的现有和新的芯片测序和切割和运行数据集来构建 C和CENP-T/W/S/X核小体复合体在每个细胞周期点，以及有效的DNA甲基化 数据，映射到端粒到端粒基因组组装中的着丝粒以创建新的基因组 和人类着丝粒的表观基因组图谱。我们将确定CENP-A结合和 长读纳米孔在CENP-A沉积的新着丝粒和异位位置上的DNA甲基化 测序，以及着丝粒DNA甲基化的功能重要性。这一项目将奠定基础 用于未来对衰老和癌症中着丝粒表观遗传学变化的检查。接下来我们将确定 人类着丝粒的位置稳定性和CENP-A是否能够确定着丝粒位置 以碱基对分辨率精确而稳定地跨越单个细胞周期和整个细胞增殖过程， 通过使用含有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，均已知在几种类型的癌症中升高，导致人类着丝粒漂移和/或 扩张将是确定的。我们将探索异位负载着丝粒的纠错机制 旨在防止染色体分离失败和新着丝粒形成的蛋白质。最后， 创新的单分子方法将被用来定义组蛋白的组成和组合 单个含CENP-A的核小体中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.