Mouse X Inactivation

小鼠 X 灭活

• 批准号: 6822453
• 负责人: Christine M. Disteche
• 金额: $ 35.63万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6822453
• 关键词: DNA methylation; Turner' s syndrome; binding sites; clinical research; developmental genetics; early embryonic stage; electroporation; fluorescent in situ hybridization; gene dosage; gene expression; gene induction /repression; gene rearrangement; genetic mapping; genetic regulation; genetically modified animals; human tissue; laboratory mouse; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein binding; sex chromosomes; tissue /cell culture

DESCRIPTION (provided by applicant): X inactivation results in the silencing of most genes on the mammalian X chromosome. However, some genes escape silencing and are thus expressed from both alleles in females. Turner syndrome is thought to result in part from haploinsufficiency of these escape genes. We have previously shown that there are few escape genes in mouse and that silencing precedes escape in early embryogenesis. Each mouse escape gene is embedded within silenced chromatin, while human escape genes are often grouped in larger domains. Our search for chromatin boundary elements between an escape gene (Smcx) and an adjacent activated gene has identified CTCF, a zinc finger protein known to have insulator function, as binding to this region in mouse. CTCF does not bind to the corresponding region in human where the two adjacent genes escape. We have also found that, even in early embryogenesis, DNA methylation is very low at Smcx CpG island including at the CTCF binding sites, suggesting that this protein may prevent the establishment of DNA methylation. Our goal is to characterize escape domains on the X chromosome and to investigate the role of CTCF and epigenetic modifications in the establishment of domains. In the present proposal we outline experiments to (1) search for insulator elements at other regions of the genome that separate escape genes from silenced genes; (2) to examine the chromatin structure of a well defined region of escape and surrounding regions in terms of histone modifications and intergenic transcripts and to follow epigenetic modifications during embryogenesis (3) to construct mice containing mutations at the endogenous CTCF binding sites and mice with inserts of large X chromosome segments containing escape genes, with and without mutations at the associated CTCF binding sites. These functional approaches are aimed at determining the chromatin configuration of escape domains and the role of CTCF in escape and DNA methylation.

描述（由申请人提供）：X 失活导致哺乳动物 X 染色体上的大多数基因沉默。然而，一些基因逃脱了沉默，因此在雌性中从两个等位基因中表达。特纳综合征被认为部分是由于这些逃逸基因的单倍体不足造成的。我们之前已经表明，小鼠体内几乎没有逃逸基因，并且在早期胚胎发生中沉默先于逃逸。每个小鼠逃逸基因都嵌入在沉默的染色质中，而人类逃逸基因通常分组在更大的域中。我们对逃逸基因 (Smcx) 和相邻激活基因之间的染色质边界元件的搜索发现，CTCF（一种已知具有绝缘子功能的锌指蛋白）与小鼠的该区域结合。 CTCF 不与人类中两个相邻基因逃逸的相应区域结合。我们还发现，即使在早期胚胎发生中，Smcx CpG 岛（包括 CTCF 结合位点）的 DNA 甲基化也非常低，表明该蛋白可能阻止 DNA 甲基化的建立。 我们的目标是表征 X 染色体上的逃逸域，并研究 CTCF 和表观遗传修饰在域建立中的作用。在本提案中，我们概述了以下实验：（1）在基因组的其他区域寻找将逃逸基因与沉默基因分开的绝缘体元件； (2) 根据组蛋白修饰和基因间转录物检查明确的逃逸区域和周围区域的染色质结构，并跟踪胚胎发生过程中的表观遗传修饰 (3) 构建内源 CTCF 结合位点含有突变的小鼠和具有含有逃逸基因的大 X 染色体片段的插入，在相关的 CTCF 结合位点有或没有突变。这些功能方法旨在确定逃逸域的染色质构型以及 CTCF 在逃逸和 DNA 甲基化中的作用。