The Role of Retroelements in Centromere Function

着丝粒在细胞分裂过程中染色体分离中的基本功能需要一个 涉及染色质特性和动粒变化的复杂的表观遗传事件级联 集合。有丝分裂过程中着丝粒的形成是一个循环的顶峰，这个循环被“加载”(fiNed)所驱动， 或将新合成的CENP-A，一种变异组蛋白，沉积到着丝粒染色质中。CENP-ATIS 组蛋白促进了细胞周期末期晚期/G1期早期的忠实组装 HJURP的监护人。这种组装级联对遗传、表观遗传和 环境侮辱，对基因组/细胞稳定性造成灾难性后果，但基因组 指导CENP-A核小体准确组装的元件还不是很清楚。一个中环 理解CENP-A核小体组装的基因组特征是 观察到已建立的着丝粒充满了卫星DNA，而从头开始的着丝粒 (例如，新温差)没有卫星，但受到逆行元件的影响，如LINE-1s。fi-1s当它 似乎CENP-A核小体的占据可能不需要特定的fic DNA序列，越来越多的证据表明 证明RNA是导致忠实CENP-A的表观遗传级联反应的关键组成部分 核小体组装。然而，序列规格fi城市，时空要求和 这些着丝粒RNA的转录调控目前尚不清楚。在初步数据中，我们 表明着丝粒逆转录元件(CENTE)是参与的RNA聚合酶的位置并参与 在人类着丝粒的CENP-A组装级联中，连接了一个共同的转录基因组特征 fi首次在人类天然着丝粒和从头开始着丝粒上对CENP-A进行组装。利用我们的 在着丝粒组装、非编码RNA、染色体工程和基因组学方面的专业知识，我们拥有 制定了三个目标，每个目标都有创新的方法，使我们能够提供不偏不倚的 着丝粒转录何时在细胞周期转录物中启动的评估 起始和延伸发生，以及这些转录本如何介导着丝粒核小体组装。我们 将使用染色体工程来直接测试CENTE及其转录活性 有足够的ffi促进着丝粒在染色体上的从头组装。这些经过改造的染色体 提供了一个新的模型来研究引导着丝粒组装和着丝粒组装的过程 稳定，并检查着丝粒组蛋白的错误整合。其结果是 这项研究将使我们目前对着丝粒组装和维护的理解出现很大差距 在正常细胞中，并为染色体异常呈现的潜在事件提供了有价值的见解 在人类疾病、不孕不育和高转移潜能的癌症中。

The essential function of centromeres in chromosome segregation during cell division requires a complex cascade of epigenetic events involving changes to chromatin character and kinetochore assembly. Kinetochore formation during mitosis is the culmination of a cycle defined by the “loading”, or deposition, of newly synthesized CENP-A, a variant histone, into centromeric chromatin. CENP-Atis faithful assembly during late telophase/early G1 of the cell cycle is facilitated by its histone chaperone, HJURP. This assembly cascade is sensitive to perturbation by genetic, epigenetic and environmental insults, with catastrophic consequences for genome/cell stability, but the genomic elements that guide accurate CENP-A nucleosome assembly are not well understood. A central conundrum in understanding the genomic features that aQract CENP-A nucleosome assembly is the observation that established centromeres are replete with satellite DNA while de novo centromeres (e.g. neocentromeres) lack satellites, yet are defined by retroelements, such as LINE-1s. While it appears CENP-A nucleosome occupancy may not require specific DNA sequences, mounting evidence demonstrates that RNA is a critical component of the epigenetic cascade leading to faithful CENP-A nucleosome assembly. However, the sequence specificity, spatiotemporal requirements for and transcriptional regulation of these centromeric RNAs are currently unknown. In Preliminary Data, we show that centromeric retroelements (cenTEs) are sites of engaged RNA polymerase and are involved in the CENP-A assembly cascade at human centromeres, linking a common transcribed genomic feature to CENP-A assembly at both native and de novo centromeres in humans for the first time. Leveraging our expertise in centromere assembly, noncoding RNAs, chromosome engineering, and genomics, we have formulated three aims, each with an innovative approach that will allow us to provide an unbiased assessment of where within centromeres transcription initiates, when during the cell cycle transcript initiation and elongation occur, and how these transcripts mediate centromere nucleosome assembly. We will use chromosome engineering to directly test whether cenTEs and their transcriptional activity are sufficient to facilitate de novo centromere assembly on chromosomes. These engineered chromosomes provide a new model to study the processes guiding cenTE-mediated centromere assembly and stabilization, and to examine misregulated incorporation of centromeric histones. The outcomes of this study will fill a large gap in our current understanding of centromere assembly and maintenance in normal cells, and provide valuable insight into events underlying chromosome aberration presenting in human diseases, infertility, and cancers of high metastatic potential.