Non-coding RNAs in the epigenetics of human centromere formation

非编码RNA在人类着丝粒形成的表观遗传学中的作用

• 批准号: 7935567
• 负责人: PETER E WARBURTON
• 金额: $ 14.83万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935567
• 关键词: Arabidopsis; Biological Assay; Cell Line; Cell division; Cells; Centromere; Chromatin; Chromosome Cohesion; Chromosome Segregation; Chromosomes; Chromosomes, Artificial, Human; Chromosomes, Human, Pair 17; Collection; Congenital Abnormality; DNA; Defect; Detection; Dicentric chromosome; Double-Stranded RNA; Drosophila genus; Engineering; Epigenetic Process; Fission Yeast; Frequencies; Functional RNA; Genetic Transcription; Genomic Instability; Heterochromatin; Histone H3; Human; Hybrid Cells; Immunofluorescence Immunologic; Kinetochores; Lead; Length; Lysine; Maintenance; Malignant Neoplasms; Metaphase; Methylation; Mitotic; Modeling; Modification; Mus; Nature; Patients; Play; Positioning Attribute; Production; RNA; RNA Interference; Reverse Transcriptase Polymerase Chain Reaction; Role; Satellite DNA; Shotguns; Signal Transduction; Sister; Sister Chromatid; Small Interfering RNA; Specificity; Testing; Transcript; Transfection; Upper arm; Variant; base; centromere protein A; cohesion; design; heterochromatin-specific nonhistone chromosomal protein HP-1; human DICER1 protein; insight; novel; promoter; public health relevance; segregation; vector

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.

描述(由申请人提供)： 人类着丝粒形成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，着丝粒是负责细胞分裂过程中适当的染色体分离的基本染色体成分。染色体分离的缺陷导致基因组不稳定，这是导致出生缺陷和癌症的主要原因。人类着丝粒含有高度重复的阿尔法卫星DNA。将α卫星DNA导入细胞可导致从头开始的着丝粒和人类人工染色体(HAC)的形成，尽管频率很低。双着丝粒染色体含有失活的着丝粒，但保留了阿尔法卫星阵列。新着丝粒是在无α卫星DNA的低拷贝非重复DNA上形成的功能齐全的人类着丝粒。因此，不活跃的着丝粒和新着丝粒分别表明，α卫星DNA对着丝粒的形成和功能既不充分，也不必要。相反，着丝粒的形成是一个在很大程度上独立于序列的表观遗传过程，依赖于以已知表观遗传标记为特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)，着丝粒特异的组蛋白H3变异体，其间夹杂着赖氨酸4位二甲基化的组蛋白H3(H3K4diMe)。围绕CENPA结构域的是着丝粒异染色质，其特征是组蛋白H3赖氨酸9甲基化(H3K9me)和异染色质蛋白1(HP1)。值得注意的是，在S.pombe、果蝇、拟南芥和小鼠中已经表明，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染色质建立的正确的表观遗传修饰。然而，这些着丝粒转录本的确切性质尚不清楚，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基于着丝粒RNA的新型非编码着丝粒遗传标记的功能分析，这些标记负责人类着丝粒的形成和功能，具体描述如下三个目标。1)以17号染色体α卫星为模型，利用RT-PCR技术在HT1080细胞中发现人内源性着丝粒转录的非编码RNA。对特定着丝粒α卫星DNA转录本的检测和详细鉴定将提供对它们在人类细胞着丝粒异染色质表观遗传沉默中的作用的深入了解。2)我们将通过使用新型HAC载体来确定RNAi在从头开始着丝粒的建立和维持中的作用，该载体旨在提供适当的α卫星特异性RNAi信号来启动着丝粒异染色质组装。我们将研究Dector耗竭对HAC和内源染色体有丝分裂稳定性的影响。3)我们将利用我们独特的具有新着丝粒染色体的患者来源的细胞系，研究RNAi和异染色质的作用以及姐妹染色体在人类新着丝粒中的聚集方式。公共卫生相关性：着丝粒是负责在细胞分裂过程中适当分离姐妹染色单体的关键染色体成分。后生动物着丝粒是表观遗传决定的，涉及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巨大的洞察力。