Non-coding RNAs in the epigenetics of human centromere formation

描述（由申请人提供）： 人类着丝粒形成的表观遗传学中的非编码RNA。我们将研究人类着丝粒的形成和功能的表观遗传学，着丝粒是细胞分裂过程中染色体分离的基本组成部分。染色体分离缺陷导致基因组不稳定，这是出生缺陷和癌症的主要原因。人类着丝粒含有高度重复的α卫星DNA。将α卫星DNA转染到细胞中可以导致从头着丝粒和人人工染色体（HAC）形成，尽管频率较低。双着丝粒染色体含有失活的着丝粒，但保留α随体阵列。新着丝粒是在没有α卫星DNA的低拷贝非重复DNA上形成的全功能人类着丝粒。因此，不活跃的着丝粒和新着丝粒表明，α卫星DNA是既不充分，也不是必要的，分别为着丝粒的形成和功能。相反，着丝粒形成是一个很大程度上序列独立的表观遗传过程，依赖于由已知表观遗传标记表征的多个不同染色质结构域的形成。着丝粒染色质含有着丝粒蛋白A（CENP-A），一种着丝粒特异性组蛋白H3变体，散布有赖氨酸4处二甲基化的组蛋白H3（H3 K4 diMe）。围绕CENPA结构域的是着丝粒异染色质，其特征在于组蛋白H3赖氨酸9甲基化（H3 K9 me）和异染色质蛋白1（HP 1）。值得注意的是，它已被证明在S。粟酒属、果蝇属、拟南芥属和小鼠的研究表明，着丝粒异染色质的组装实际上需要着丝粒DNA双向顺式转录成dsRNA，这指导了异染色质形成和CENP-A染色质建立的正确表观遗传修饰。然而，这些着丝粒转录本的确切性质是未知的，特别是在哺乳动物和人类细胞中。因此，我们建议发现并进行新的非编码着丝粒RNA为基础的表观遗传标记，负责人类着丝粒的形成和功能的功能分析，如以下三个具体目标所述。1)我们将以17号染色体α随体为模型，利用RT-PCR技术在HT 1080细胞中发现人内源性着丝粒转录的非编码RNA。特异性着丝粒α卫星DNA转录物的检测和详细表征将提供对它们在人类细胞中着丝粒异染色质的表观遗传沉默中的作用的见解。2)我们将确定RNAi在从头着丝粒的建立和维持中的作用，使用新的HAC载体工程化以提供适当的α卫星特异性RNAi信号来启动着丝粒异染色质组装。将研究Dicer消耗对HAC和内源染色体的有丝分裂稳定性的影响。3)我们将研究RNAi和异染色质的作用和模式的姐妹染色体凝聚在人类neocentromeres，使用我们独特的收集患者来源的细胞系与neocentromeres。公共卫生相关性：着丝粒是细胞分裂过程中负责姐妹染色单体分离的关键染色体成分。后生动物的着丝粒是由表观遗传学决定的，包括着丝粒重复序列的转录和基于RNAi的异染色质的建立。这些表观遗传标记目前在人类细胞中是未知的，它们的发现将为着丝粒形成和功能的要求提供很大的见解。

DESCRIPTION (provided by applicant): Non-coding RNAs in the epigenetics of human centromere formation. We will examine the epigenetics of the formation and function of human centromeres, the fundamental chromosomal component responsible for proper chromosome segregation during cell division. Defects in chromosome segregation leads to genome instability, which represents a major cause of birth defects and cancer. Human centromeres contain highly repetitive alpha satellite DNA. Transfection of alpha satellite DNA into cells can result in de novo centromere and human artificial chromosome (HAC) formation, albeit at low frequency. Dicentric chromosomes contain inactivated centromeres but retain the array of alpha satellite. Neocentromeres are fully functional human centromeres that have formed on low copy non-repetitive DNA with no alpha satellite DNA. Thus, inactive centromeres and neocentromeres demonstrate that alpha satellite DNA is neither sufficient nor even necessary, respectively, for centromere formation and function. Instead, centromere formation is a largely sequence independent epigenetic process dependent on the formation of multiple distinct chromatin domains characterized by known epigenetic marks. The kinetochore chromatin contains CENtromere Protein A (CENP-A), a centromere-specific histone H3 variant, interspersed with Histone H3 dimethylated at lysine 4 (H3K4diMe). Surrounding the CENPA domain is the centromeric heterochromatin, characterized by histone H3 lysine 9 methylation (H3K9me) and heterochromatin protein 1 (HP1). Remarkably, it has been shown in S. pombe, Drosophila, Arabidopsis and mouse that assembly of centromeric heterochromatin actually requires bidirectional cis transcription of centromeric DNA into dsRNA, which directs the correct epigenetic modifications for heterochromatin formation and establishment of CENP-A chromatin. However, the exact nature of these centromeric transcripts are unknown, especially in mammalian and human cells. Thus, we propose to discover and perform functional analysis of the novel non-coding centromeric RNA-based epigenetic marks that are responsible for human centromere formation and function, as described in the following three specific aims. 1) We will discover the non-coding RNA transcribed from human endogenous centromeres in HT1080 cells using RT-PCR, examining chromosome 17 alpha satellite as a model. Detection and detailed characterization of specific centromeric alpha satellite DNA transcripts will provide insights into their role in epigenetic silencing of centromeric heterochromatin in human cells. 2) We will determine the role of RNAi in establishment and maintenance of de novo centromeres in using novel HAC vectors engineered to provide the proper alpha satellite-specific RNAi signals to initiate centromeric heterochromatin assembly. The effect of Dicer depletion on mitotic stability of HACs and endogenous chromosomes will be investigated. 3) We will investigate the role of RNAi and heterochromatin and mode of sister chromosome cohesion at human neocentromeres, using our unique collection of patient-derived cell lines with neocentric chromosomes. PUBLIC HEALTH RELEVANCE: Centromeres are the critical chromosomal component responsible for proper sister chromatid segregation during cell division. Metazoan centromeres are epigenetically determined, involving transcription of centromeric repeats and RNAi-based establishment of heterochromatin. These epigenetic marks are currently unknown in human cells, and their discovery will provide great insight into the requirements for centromere formation and function.