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

基本信息

批准号：
7571311
负责人：
PETER E WARBURTON
金额：
$ 29.38万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
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/7571311
关键词：
Arabidopsis Artificial Chromosomes Biological Assay Cell Line Cell division Cell hybridization 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 Human Chromosomes Hybrid Cells Immunofluorescence Immunologic Kinetochores Lead Length Lysine Maintenance Malignant Neoplasms Metaphase Methylation Mitotic Modeling Modification Mus Nature Patients Play Positioning Attribute Production Public Health RNA RNA Interference Reverse Transcriptase Polymerase Chain Reaction Role Satellite DNA Shotguns Signal Transduction Sister Sister Chromatid Small Interfering RNA Specificity Testing Thinking Transcript Transfection Upper arm Variant base centromere protein A cohesion design heterochromatin-specific nonhistone chromosomal protein HP-1 human DICER1 protein insight novel promoter segregation vector

项目摘要

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）：人类丝粒形成的表观遗传学中的非编码RNA。我们将研究人类丝omles的形成和功能的表观遗传学，该丝粒的形成和功能是负责在细胞分裂过程中适当染色体隔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性，这代表了先天缺陷和癌症的主要原因。人的中心粒含有高度重复的α卫星DNA。将α卫星DNA转染到细胞中可以导致新的丝粒和人工染色体（HAC）形成，尽管以低频为单位。二含染色体含有灭活的中心粒，但保留了α卫星的阵列。 Neopertromeres是在低拷贝的非竞争性DNA上形成的，没有α卫星DNA。因此，无活跃的丝粒和新中心粒表明，α卫星DNA既不足够，甚至既不是必要的，也不必进行丝粒的形成和功能。取而代之的是，丝粒形成是一个很大程度上独立的表观遗传过程，取决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。动力学染色质包含Centromere蛋白A（CENP-A），这是一种丝粒特异性组蛋白H3变体，散布在赖氨酸4（H3K4DIME）处的组蛋白H3二甲基化。 CENPA结构域周围是丝状异染色质，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，它已经在S. pombe，果蝇，拟南芥和小鼠中显示出来，丝状异染色质的组装实际上需要将丝粒DNA的双向顺式转录到DSRNA中，该转录指导了正确的表观遗传修饰，以实现异染料素形成和cenp-a染料蛋白的异降解素形成和建立。但是，这些丝粒转录本的确切性质尚不清楚，尤其是在哺乳动物和人类细胞中。因此，我们建议对新型非编码中心RNA的表观遗传学标记进行发现和执行功能分析，这些表观遗传标记负责人的中心粒形成和功能，如以下三个特定目的所述。 1）我们将使用RT-PCR发现从HT1080细胞中的人内源性centress抄录的非编码RNA，从而将17染色体的Alpha卫星作为模型。对特定的中心α卫星DNA转录物的检测和详细表征将提供有关其在人类细胞中丝粒异染色质表观遗传沉默中的作用的见解。 2）我们将确定RNAi在使用新型的HAC矢量建立和维持新的HAC矢量中的作用，以提供适当的Alpha卫星特异性RNAi信号来启动中心粒细胞异染色质组装。将研究迪切尔耗竭对HAC和内源性染色体有丝分裂稳定性的影响。 3）我们将使用我们独特的带有新中心染色体的患者衍生细胞系的RNAi和异染色质以及姐妹染色体内聚的模式的作用。公共卫生相关性：中心粒是负责细胞分裂过程中适当姐妹染色单体隔离的关键染色体成分。内唑centromeres是表观遗传确定的，涉及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尚不清楚，它们的发现将为人们对丝粒形成和功能的需求提供深入的了解。