Mechanism of Gene Silencing by DNA Methylation

DNA甲基化导致基因沉默的机制

基本信息

批准号：
6877201
负责人：
Yi Zhang
金额：
$ 24万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-05-01 至 2006-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (Verbatim from the applicant's abstract): Methylation of cytosine at CpG dinucleotides is a common feature of many higher eukaryotic genomes. DNA methylation plays important roles in diverse biological processes including embryogenesis, genomic imprinting, X-chromosome inactivation, and cancer. A major biological consequence of DNA methylation is gene silencing. However, the mechanism underlying methylated DNA silencing is not known. In an effort to understand how core histone acetylation and deacetylation regulate transcription, we previously purified and characterized two histone deacetylase complexes, the Sin3 and the NuRD complexes. Both complexes have been shown to be involved in methylated DNA silencing. To understand the relationship between nucleosome remodeling, histone deacetylation and methylated DNA silencing, we have purified the methyl-CpG specific binding protein complex MeCP1. Our preliminary studies revealed that in addition to the methyl-CpG binding protein MBD2, the MeCP1 complex contains all the known NuRD components indicating NuRD can be targeted to methylated DNA. We propose to extend this study with the following specific aims: 1. Cloning and characterization of the p66 and p68 components of the MeCP1 complex. We will isolate the cDNAs encoding p66 and p68. Their roles in methyl-CpG binding, nucleosome remodeling, and histone deacetylation will be analyzed. 2. Functional characterization of the MeCP1 complex. We will investigate chromosomal localization of the MeCP1 complex in response to DNA demethylation. We will also characterize the ATPase, nucleosome remodeling and histone deacetylase activities of the MeCP1 complex. 3. Identification of MeCP1 targeted genes. We will identify genes that are targeted by the MeCP1 complex using chromatin immunoprecipitation (CHIP) approach. The identified candidate genes will be verified by Northern blot analysis using the MTA2 null cells. 4. Understanding the mechanism of transcriptional repression by MeCP1. We will investigate the transcription repression mechanism of MeCP1 using a reconstituted in vitro chromatin transcription system. The proposed studies will provide a thorough characterization of the MeCP1 complex. They will also reveal the potential targets and repression mechanism of the MeCP1 complex.

描述（逐字摘自申请人的摘要）：胞嘧啶的甲基化 CpG二核苷酸是许多高等真核生物基因组的共同特征。脱氧核糖核酸甲基化在多种生物过程中发挥着重要作用，包括胚胎发生、基因组印记、X 染色体失活和癌症。一个 DNA甲基化的主要生物学后果是基因沉默。然而，甲基化 DNA 沉默的机制尚不清楚。努力了解核心组蛋白乙酰化和脱乙酰化如何调节转录，我们之前纯化并表征了两种组蛋白脱乙酰酶复合物、Sin3 和 NuRD 复合物。两种复合物均已被证明参与甲基化 DNA 沉默。要了解之间的关系核小体重塑、组蛋白脱乙酰化和甲基化 DNA 沉默，我们纯化了甲基-CpG 特异性结合蛋白复合物 MeCP1。我们的初步研究表明，除了甲基-CpG 结合蛋白 MBD2，MeCP1 复合体包含所有已知的 NuRD 成分，表明 NuRD 可以靶向甲基化 DNA。我们建议扩大这项研究具体目标如下： 1. p66 和 p68 的克隆和表征 MeCP1 复合物的组成部分。我们将分离编码 p66 的 cDNA 和第 68 页。它们在甲基 CpG 结合、核小体重塑和组蛋白中的作用将分析脱乙酰化。 2. MeCP1 的功能表征复杂的。我们将研究 MeCP1 复合物的染色体定位对 DNA 去甲基化的反应。我们还将表征 ATP 酶、核小体 MeCP1 复合物的重塑和组蛋白脱乙酰酶活性。 3. MeCP1 靶基因的鉴定。我们将确定基因使用染色质免疫沉淀 (CHIP) 以 MeCP1 复合物为目标方法。确定的候选基因将通过 Northern blot 进行验证使用 MTA2 无效细胞进行分析。 4. 理解机制 MeCP1 的转录抑制。我们将调查转录使用重构的体外染色质抑制 MeCP1 的机制转录系统。拟议的研究将提供全面的 MeCP1 复合物的表征。他们还将揭示潜力 MeCP1复合物的靶点和抑制机制。