Epigenetic Regulation of Imprinting in Mouse Embryo

小鼠胚胎印记的表观遗传调控

• 批准号: 6734196
• 负责人: RICHARD M SCHULTZ
• 金额: $ 42.57万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6734196
• 关键词: DNA methylation; RNA interference; alleles; artificial fertilization; assistive reproductive technique; binding proteins; developmental genetics; double stranded RNA; egg /ovum; embryo /fetus culture; embryo /fetus transplantation; embryo implantation; ethology; gene expression; genetic enhancer element; genetic promoter element; genetic regulation; growth media; laboratory mouse; polymerase chain reaction; protein binding; protein structure function; single strand conformation polymorphism; transcription factor

The imprinted H19 gene is closely linked to the oppositely imprinted Igf2 gene in both mouse and humans. The imprinted expression of these genes is mediated by a 2 kb differentially methylated region (DMR) located 5' to the H19 gene. In vitro when the DMR is situated between an enhancer and promoter, it functions as a boundary that blocks the enhancer from engaging the promoter. The boundary function is likely mediated by the methylation-sensitive transcriptional regulatory protein CTCF. According to the boundary model, CTCF binds to the DMR on the maternal allele, thereby allowing the maternal H19 gene exclusive access to the enhancers. On the paternal chromosome, the hypermethylated DMR cannot bind CTCF. This hypermethylation results in both suppressing paternal H19 promoter activity and permitting the enhancers to engage exclusively the paternal Igf2 promoter. The model thus accounts for the observed maternal H19 expression and the reciprocal paternal Igf2 expression. Specific Aim 1 will use RNA interference (RNAi) in the mouse to test the hypotheses that (1) CTCF binding on the maternal allele is responsible for the exclusive maternal H19 expression, and (2) DNA hypermethylation and the interacting DNA methyl-binding proteins on the paternal allele mediate the repression of H19 and hence permits the exclusive paternal Igf2 expression. In addition, this aim will test the hypothesis that CTCF binding to the maternal DMR in the oocyte prevents DNA methylation and thereby ensures maternal H19 expression in the embryo. Understanding the expression of imprinted genes has implications for the practice of Assisted Reproductive Technology (ART). The practice of ART has increased dramatically during the past two decades and has resulted in the birth of tens of thousands of children. Subtle changes in the culture media for preimplantation mouse embryos can result in loss-of-imprinting of the normally imprinted and maternally expressed H19 gene. The resulting biallelic expression correlates with the loss of methylation in the DMR on the paternal allele. Although it is not known if such changes occur during the course of culture of human preimplantation embryos (and if there are long-term consequences), ART is occurring in the virtual absence of any basic research using an animal model. Specific Aim 2 will use the mouse as a model system to test the hypotheses that (1) embryo culture results in the differential loss-of-imprinting of imprinted genes, (2) this loss-of- imprinting is linked with the loss of DNA methylation of specific cytosines in the DMR, and (3) following implantation the ability of the embryo to restore the correct DNA methylation pattern and imprinted gene expression is coupled with successful development to term.

在小鼠和人类中，印记 H19 基因与相反印记 Igf2 基因密切相关。 这些基因的印记表达由位于 H19 基因 5' 的 2 kb 差异甲基化区域 (DMR) 介导。 在体外，当 DMR 位于增强子和启动子之间时，它充当边界，阻止增强子与启动子结合。 边界功能可能是由甲基化敏感转录调节蛋白 CTCF 介导的。 根据边界模型，CTCF 与母体等位基因上的 DMR 结合，从而允许母体 H19 基因独占增强子。 在父本染色体上，高度甲基化的 DMR 无法结合 CTCF。 这种高甲基化会抑制父本 H19 启动子活性，并允许增强子专门与父本 Igf2 启动子结合。 因此，该模型解释了观察到的母体 H19 表达和相反的父体 Igf2 表达。 具体目标 1 将在小鼠中使用 RNA 干扰 (RNAi) 来测试以下假设：(1) CTCF 与母体等位基因的结合负责母体 H19 的专有表达，以及 (2) DNA 高甲基化和相互作用的 DNA 甲基结合蛋白父系等位基因介导 H19 的抑制，因此允许排他性父系 Igf2 表达。 此外，该目标将检验以下假设：CTCF 与卵母细胞中母体 DMR 的结合可防止 DNA 甲基化，从而确保胚胎中母体 H19 的表达。 了解印记基因的表达对于辅助生殖技术（ART）的实践具有重要意义。 过去二十年里，辅助生殖技术的应用急剧增加，导致数以万计的儿童出生。 植入前小鼠胚胎培养基的细微变化可能导致正常印记和母体表达的 H19 基因印记丢失。 由此产生的双等位基因表达与父系等位基因上 DMR 甲基化的丧失相关。 尽管尚不清楚此类变化是否发生在人类植入前胚胎的培养过程中（以及是否会产生长期后果），但 ART 是在几乎没有使用动物模型进行任何基础研究的情况下发生的。具体目标 2 将使用小鼠作为模型系统来测试以下假设：(1) 胚胎培养导致印记基因的差异性印记丢失，(2) 这种印记丢失与 DNA 丢失有关DMR 中特定胞嘧啶的甲基化，以及 (3) 植入后胚胎恢复正确 DNA 甲基化模式和印记基因表达的能力与成功发育至足月相结合。