X CHROMOSOME INACTIVATION AND DNA METHYLATION

X 染色体失活和 DNA 甲基化

• 批准号: 2761808
• 负责人: ARTHUR D RIGGS
• 金额: $ 43.16万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2761808
• 关键词: 5 methylcytosine; DNA binding protein; DNA footprinting; DNA methylation; DNA replication; affinity chromatography; cell differentiation; developmental genetics; gene expression; gene induction /repression; genetic transcription; genomic imprinting; laboratory mouse; mass spectrometry; methyltransferase; oligonucleotides; polymerase chain reaction; protein purification; sex chromosomes; tissue /cell culture; transcription factor; transposon /insertion element

Epigenetic phenomena, which are heritable changes that do not irreversibly alter DNA base sequence, are increasingly becoming recognized as important for normal mammalian development, disease, and aging. Recent studies have firmly established that both in mouse and man some genes function differently depending on whether they come from the mother or father; by definition this is due to parental imprinting. The best characterized epigenetic mechanism in mammals is DNA modification by the formation of 5-methylcytosine. X chromosome inactivation (XCI) has long been known to display parental imprinting, stable somatic inheritance of chromatin activity states, and DNA methylation changes. Using XCI and imprinted genes as our experimental system, we will try to better understand the molecular mechanisms underlying epigenetic mechanisms. More specifically we propose to: (i) Improve and study application of Terminal transferase-Dependent PCR (TDPCR), a new procedure, to the analysis of DNA and RNA; (ii) continue investigation of a dynamic, stochastic model for DNA methylation and quantitatively determine methylation parameters such as the in vivo rate of de novo methylation and demethylation at several selected CpG sites. An assay based on TDPCR is expected to facilitate accomplishment of these studies; (iii) determine nuclease accessibility differences between active and inactive X-linked and imprinted genes using a novel LMPCR/SNuPE approach; and (iv) use mass spectrometry for the identification and characterization of DNA binding proteins and apply this methodology to the study of proteins involved in epigenetic mechanisms. X chromosome inactivation and parental imprinting play a role in several genetic diseases, including the major form of heritable mental retardation (fragile X syndrome) and some familial tumors. DNA methylation is becoming increasingly recognized as a significant factor for many cancers.

表观遗传现象是可遗传的变化，但不 不可逆地改变 DNA 碱基序列，正日益成为 被认为对哺乳动物的正常发育、疾病和 老化。 最近的研究已经证实，无论是在小鼠还是 有些基因的功能不同，取决于它们是否来自 母亲或父亲；根据定义，这是由于父母的印记。 哺乳动物中最有特征的表观遗传机制是 DNA 通过形成 5-甲基胞嘧啶进行修饰。 X染色体 失活（XCI）长期以来一直被认为可以显示亲代印记， 染色质活性状态和 DNA 的稳定体细胞遗传 甲基化改变。 使用 XCI 和印记基因作为我们的实验 系统，我们将尝试更好地理解分子机制 潜在的表观遗传机制。 更具体地说，我们建议：（i） 末端转移酶依赖性PCR的改进和应用研究 (TDPCR)，一种用于分析 DNA 和 RNA 的新程序； (二) 继续 DNA 甲基化动态随机模型的研究 定量测定甲基化参数，例如体内速率 几个选定的 CpG 位点的从头甲基化和去甲基化。 基于 TDPCR 的检测预计将有助于实现 这些研究； (iii) 确定核酸酶可及性差异 使用一种新颖的方法来区分活性和非活性的 X 连锁基因和印记基因 LMPCR/SNuPE 方法； (iv) 使用质谱分析法 DNA结合蛋白的鉴定和表征并应用 这种方法用于研究参与表观遗传的蛋白质 机制。 X 染色体失活和亲代印记在多种因素中发挥作用 遗传疾病，包括主要形式的遗传性精神疾病 发育迟缓（脆性 X 综合征）和一些家族性肿瘤。脱氧核糖核酸 甲基化越来越被认为是一个重要因素 对于许多癌症。