X CHROMOSOME INACTIVATION AND DNA METHYLATION

表观遗传现象，即可遗传的变化，不 不可逆转地改变DNA碱基序列，越来越多地成为 被认为对哺乳动物的正常发育、疾病和 衰老。最近的研究已经确凿地证明，在小鼠和 人类某些基因的功能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。 哺乳动物最典型的表观遗传机制是DNA 通过形成5-甲基胞嘧啶进行修饰。X染色体 失活(XCI)很早就知道会显示父母印迹， 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和印记基因作为实验 我们将努力更好地了解分子机制 潜在的表观遗传机制。更具体而言，我们建议：(I) 末端转移酶依赖的聚合酶链式反应的改进及应用研究 DNA和RNA分析的新程序(TDPCR)；(Ii)继续 DNA甲基化和DNA甲基化的动态随机模型的研究 定量测定甲基化参数，如体内速率 在几个选定的CpG位点上发生从头甲基化和去甲基化。 基于TDPCR的检测有望促进实现 这些研究；(3)确定核酸酶可及性差异 利用一种新的方法在活性和非活性X-连锁和印记基因之间进行比较 LMPCR/Snupe方法；以及(Iv)使用质谱学方法 DNA结合蛋白的鉴定、鉴定及应用 这种方法论用于研究与表观遗传有关的蛋白质 机械装置。 X染色体失活和双亲印记在几个 遗传性疾病，包括可遗传精神疾病的主要形式 发育迟缓(脆性X综合征)和一些家族性肿瘤。脱氧核糖核酸 甲基化日益被认为是一个重要的因素 治疗多种癌症。

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.