Epigenetic Regulation of Imprinting in Mouse Embryo

印迹H19基因与小鼠和人类中相反印迹的Igf2基因紧密相连。 这些基因的印记表达由位于H19基因5'端的2kb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，它起到阻止增强子与启动子接合的边界的作用。 边界功能可能是由甲基化敏感的转录调控蛋白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基因排他性地接近增强子。 在父本染色体上，高甲基化的DMR不能结合CTCF。 这种超甲基化导致抑制父本H19启动子活性和允许增强子专门接合父本Igf2启动子。 因此，该模型解释了观察到的母体H19表达和相互的父本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结合在母体等位基因上负责排他性母体H19表达，以及（2）DNA超甲基化和父本等位基因上的相互作用DNA甲基结合蛋白介导H19的抑制，从而允许排他性父本Igf2表达。 此外，这一目的将检验CTCF与卵母细胞中的母体DMR结合防止DNA甲基化从而确保胚胎中的母体H19表达的假设。 了解印记基因的表达对辅助生殖技术（ART）的实践有影响。 在过去二十年中，抗逆转录病毒疗法的做法急剧增加，导致成千上万的儿童出生。 植入前小鼠胚胎的培养基中的细微变化可导致正常印迹和母系表达的H19基因的印迹丢失。 所得的双等位基因表达与父本等位基因上DMR中甲基化的丧失相关。 虽然目前还不知道这种变化是否发生在人类植入前胚胎的培养过程中（以及是否有长期的后果），但ART是在几乎没有任何使用动物模型的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检验以下假设：（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DMR中特定胞嘧啶的DNA甲基化丢失有关，以及（3）植入后，胚胎恢复正确的DNA甲基化模式和印记基因表达的能力与成功的DNA甲基化结合在一起。发展到长期。

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.