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.

印迹的H19基因与小鼠和人类中的IGF2基因相反的IGF2基因密切相关。 这些基因的印记表达是由位于H19基因5'的2 kb差异甲基化区（DMR）介导的。 在体外，当DMR位于增强子和启动子之间时，它的作用是阻止增强子吸引启动子的边界。 边界功能可能是由甲基化敏感的转录调节蛋白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基因独家访问增强子。 在父亲染色体上，高甲基化的DMR不能结合CTCF。 这种高甲基化既导致抑制父亲H19的启动子活性，又允许增强子完全参与父亲IGF2启动子。 因此，该模型解释了观察到的母体H19表达和相互父亲IGF2的表达。 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. 此外，该目标将检验以下假设：CTCF与卵母细胞中的母体DMR结合可防止DNA甲基化，从而确保母体H19在胚胎中的表达。 了解印迹基因的表达对辅助生殖技术（ART）的实践有影响。 在过去的二十年中，艺术实践急剧增加，并导致成千上万的儿童出生。 植入前小鼠胚胎的培养基的细微变化会导致正常印记和母体表达的H19基因的损失。 所得的双重表达与父亲等位基因DMR中甲基化的丧失相关。 尽管尚不清楚这种变化是否在人类植入前胚胎的培养过程中发生（并且是否存在长期后果），但在使用动物模型的任何基础研究中，实际上正在发生艺术。 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成功发展。