MOLECULAR GENETICS OF HUMAN X CHROMOSOME INACTIVATION

人类 X 染色体失活的分子遗传学

• 批准号: 2022439
• 负责人: Huntington F Willard
• 金额: $ 28.12万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022439
• 关键词: DNA methylation; DNA replication; RNase protection assay; alleles; cytogenetics; developmental genetics; early embryonic stage; embryonic stem cell; fluorescent in situ hybridization; gene expression; gene induction /repression; gene mutation; genetic regulatory element; genetic transcription; genetically modified animals; human genetic material tag; laboratory mouse; molecular cloning; molecular genetics; northern blottings; nucleic acid sequence; polymerase chain reaction; regulatory gene; sex chromosomes; transfection

X chromosome inactivation is the process whereby one of the two X chromosomes in somatic cells of mammalian females is inactivated early in embryogenesis, presumably as a means of dosage compensation between females, with two X chromosomes, and males, with one. While the essential features of X inactivation have been known for over 30 years, the precise molecular, genetic, and developmental details of this lone- range chromosomal control mechanism remain unclear. Over the past few years, substantial progress has been made in the definition and understanding of the various steps involved in X inactivation: initiation early in development through the action of the X inactivation center, promulgation of the inactivation signal and establishment of the inactive state along the chromosome in a strictly cis-limited manner, and maintenance of the various epigenetic states through subsequent cell divisions. A strong candidate for a gene involved in X inactivation, XIST, was described by us in 1991. Since then, significant genetic and developmental evidence has been presented in both humans and mice that supports a role for XIST in the initiation and perhaps establishment of the X inactivation signal. These data provide a framework for further experiments designed to test the hypothesis that the XIST gene, with its regulatory sequences, both controls the initial choice of which X chromosome to inactivate and subsequently establishes a context within the nucleus to distinguish the active from the inactive X chromosome. The experiments described in this application have three specific aims: (1) to determine the nature of the X inactivation center and the role of XIST in initiating X inactivation, by examining the effect(s) of expressing the XIST gene in mouse embryonic stem cells and/or in transgenic mice, by determining the effect(s) of a recently discovered mutation in XIST on the initiation of X inactivation, and by characterizing additional such mutations in families with multiple cases of non-random X inactivation; (2) to determine a developmental context for the establishment of X inactivation, by studying the expression of genes subject to and escaping from X inactivation in mice early in development at the time X inactivation occurs. This is in order to determine whether escape from inactivation is a failure of initiation, establishment, or maintenance of X inactivation; and (3) to examine the chromosomal basis for X inactivation, by studying the distribution of genes on the human and mouse X-chromosomes that are subject to or escape from X inactivation and by using transgenic mice to specifically test the hypothesis that X inactivation is determined at the level of chromosomal domains rather than on an individual gene basis. The proposed experiments should provide a definitive genetic and molecular test of the hypothesis that the XIST gene is, in fact, part of the X inactivation pathway, as well as provide insights into the chromosomal, developmental, and molecular mechanisms by which cis-limited control of X-linked gene expression is achieved.

X染色体失活是两个X染色体之一的过程 雌性哺乳动物体细胞中的染色体早期失活 在胚胎发生中，大概是作为剂量补偿的一种手段 女性有两条X染色体，男性有一条。 虽然 X 失活的基本特征已为人所知 30 多年， 这个孤独的精确的分子、遗传和发育细节 范围染色体控制机制仍不清楚。 过去几年 多年来，在定义和 了解 X 失活涉及的各个步骤： 通过 X 失活作用在发育早期启动 中心，发布灭活信号并建立 沿着染色体以严格顺式限制的方式处于非活性状态，并且 通过后续细胞维持各种表观遗传状态 部门。 参与 X 失活的基因的有力候选者， XIST 是我们在 1991 年描述的。从那时起，显着的遗传和 人类和小鼠的发育证据均已提出 支持 XIST 在启动和可能建立中的作用 X失活信号。 这些数据为进一步 旨在检验 XIST 基因及其相关假设的实验 调控序列，都控制着 X 的初始选择 染色体失活并随后在其中建立上下文 细胞核以区分活性 X 染色体和非活性 X 染色体。 本申请中描述的实验具有三个具体目标： (1)确定X失活中心的性质及作用 XIST 在启动 X 失活中的作用，通过检查 在小鼠胚胎干细胞和/或 转基因小鼠，通过确定最近发现的一种或多种效应 X 失活开始时 XIST 发生突变，并且通过 描述具有多个病例的家庭中其他此类突变的特征 非随机 X 失活； (2)确定发展背景 通过研究 X 失活的表达 小鼠早期遭受和逃避 X 失活的基因 X失活发生时的发育。 这是为了 确定逃避失活是否是启动失败， X失活的建立或维持； (3) 检查 通过研究 X 失活的染色体基础 人类和小鼠 X 染色体上易受或逃避的基因 X 失活并使用转基因小鼠专门测试 假设 X 失活是在染色体水平上决定的 域而不是单个基因的基础上。 拟议的 实验应提供明确的遗传和分子测试 假设 XIST 基因实际上是 X 失活的一部分 途径，并提供对染色体、发育、 X连锁基因顺式限制控制的分子机制 表达就达到了。