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染色体灭活是两个x之一的过程 哺乳动物女性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，有两个X染色体和男性。 而 X灭活的基本特征已有30多年了， 这种孤独的精确分子，遗传和发育细节 范围染色体控制机制尚不清楚。 在过去的几个 多年来，定义已经取得了实质性进展和 了解X灭活中涉及的各种步骤： 通过X灭活的作用开始开发的初期 中心，颁布灭活信号和建立 以严格的顺式限制方式沿着染色体沿染色体的不活跃状态，并且 通过随后的细胞维持各种表观遗传状态 部门。 参与X失活的基因的强大候选者， xist，我们在1991年被我们描述。从那以后，遗传和 人类和小鼠都提出了发展证据 支持Xist在启动和建立中的作用 X灭活信号。 这些数据为进一步提供了一个框架 实验旨在检验Xist基因的假设及其及其 调节序列，两者都控制着最初的选择x 染色体以灭活并随后建立上下文 细胞核以区分活性和无活性X染色体。 本应用程序中描述的实验具有三个特定的目的： （1）确定X灭活中心的性质和角色 通过检查 在小鼠胚胎干细胞和/或中表达Xist基因 转基因小鼠，通过确定最近发现的效果 XIST突变是X灭活的启动，并通过 表征有多种情况的家庭中的其他此类突变 非随机X灭活； （2）确定发展环境 为了建立X灭活，通过研究的表达 早期受到小鼠的X失活的基因 当时的发展发生X失活。 这是为了 确定摆脱失活是否是启动失败， 建立或维持X灭活； （3）检查 X失活的染色体基础，通过研究 受到或逃脱的人和小鼠X染色体的基因 从X灭活和使用转基因小鼠专门测试 假设X在染色体水平上确定X灭活 域而不是单个基因。 提议 实验应提供确定的遗传和分子检测 假设Xist基因实际上是X失活的一部分 途径，并提供有关染色体，发育性的见解 和分子机制，从顺式控制X连锁基因 表达得以实现。