Exposure-altered gene expression in five candidate imprinted loci for adult disease

 描述（由申请人提供）：来自1944-45年荷兰饥饿冬季动物和个体研究的越来越多的证据支持早期胚胎的环境暴露导致成年期疾病发展风险增加的概念（巴克假说）。例如，小鼠胚胎暴露于内分泌干扰化学物质双酚A（BPA）已知会导致许多疾病，包括神经/行为障碍。基因组印记（一个表观遗传过程，以控制父母的起源特异性表达的基因）已被理论上作为这些影响的主要决定因素。Igf 2差异甲基化区域（DMR;印记的标志）的甲基化在荷兰饥荒中幸存的个体中持续变化。我们对“巴克假说”的机制的理解受到了阻碍，因为我们只关注少数几个印记基因座（即Igf 2）。然而，这是不可能的，所有20个“巴克病”是由于仅仅是 Igf 2表达的变化;特别是许多“巴克病”与已知的印记基因无关。使用两种新的方法（所谓的NORED和标签镶嵌性），它允许一个分析DNA甲基转移酶（DNMT）敲除（KO）胚胎干细胞（ESC）的碱基分辨率甲基化组，我们已经成功地确定了20个新的DMR代表17个“候选”印记位点。有趣的是，这17个假定的印记基因座中有5个与人类的神经系统疾病有关。需要进一步的研究来证实这些假定的基因座含有印记基因，并确定环境胁迫改变的DMR是否（以及如何）改变这些基因座内这些印记基因的表达，从而有助于“巴克病”的发病机制。“为此，我们确认了至少一个位点（即，D2 hgdh）通过揭示其等位基因甲基化模式，发现5个DMR的等位基因甲基化决定了绝缘子CTCF的结合，从而潜在地影响印迹基因的组蛋白修饰。此外，BPA的胎儿暴露改变了多个DMR，包括选定的DMR。基于我们令人兴奋的初步数据，我们假设DMR决定了CTCF的等位基因特异性结合，CTCF绝缘随后控制了5个位点中不同印记基因启动子处的不同组蛋白修饰以进行调节。最后，BPA在这些基因座中改变的DMR改变了基因表达，从而导致神经系统疾病的发病机制。我们将通过三个互补的目标来测试我们的假设：SA 1，确定推定的DMR是否以组织特异性方式动态改变基因表达。SA 2，确定五个新位点的印迹机制。SA 3，确定BPA诱导的新型DMR改变是否会导致发育期间组织特异性基因表达的改变。这些研究的结果应阐明来自五个“候选”基因座的puericum印记基因如何在正常生长和发育过程中或在环境胁迫改变的条件下促进发育可塑性，并为制定保护未出生胎儿的预防战略提供信息。

 DESCRIPTION (provided by applicant): Increasing evidence from studies of animals and of individuals who suffered from the Dutch Hunger Winter of 1944-45 supports the concept that environmental exposure of early embryos causes increased risk of development of disease in adulthood (Barker hypothesis). For example, exposure of mouse embryos to the endocrine disrupting chemical Bisphenol A (BPA) is known to cause many disorders, including neurological/behavioral disorders. Genomic imprinting (an epigenetic process to control parent-origin-specific expression of genes) has been theorized as the major determinant of these effects. Methylation of Igf2 differentially-methylated-region (DMR; a hallmark of imprinting) is persistently changed in individuals surviving the Dutch famine. Our understanding of the mechanism(s) underlying the "Barker hypothesis" has been hampered by focusing only on a few imprinted loci (i.e. Igf2). However, it is unlikely that all 20 "Barker diseases" are due solely to changes in Igf2 expression; particularly as many "Barker diseases" have no association with known imprinted genes. Using two novel approaches (so-called NORED and Tag-mosaicity), which allow one to analyze base-resolution methylomes of DNA methyltransferase (DNMT) knockout (KO) embryonic stem cells (ESCs), we have successfully identified 20 novel DMRs representing 17 `candidate' imprinted loci. Intriguingly, five of these 17 putative imprinted loci are linked to neurological disorders in humans. Further studies are needed to confirm that these putative loci contain imprinted genes and to determine whether (and then how) environmental exposure-altered DMRs change expression of these putatively imprinted genes within these loci, thereby contributing to the pathogenesis of "Barker diseases." To this end, we confirmed at least one locus (i.e., D2hgdh) via revealing its allelic methylation patterns and found that alleli methylation of five DMRs dictates the binding of insulator CTCF for potentially effecting histone modifications of imprinted genes. Furthermore, fetal exposure of BPA altered multiple DMRs including selected DMRs. Based on our exciting preliminary data, we hypothesized that DMRs dictate the allele-specific binding of CTCF and CTCF insulation subsequently controls the distinct histone modifications at promoters of different imprinted genes in five loci for regulatio. Lastly, BPA exposure-altered DMRs in these loci change gene expression, thereby contributing to the pathogeneses of neurological disorders. We will test our hypothesis through three complementary aims: SA1, Determine whether the putative DMRs dynamically alter gene expression in a tissue-specific manner. SA2, Determine the mechanisms of imprinting of five novel loci. SA3, Determine whether BPA-induced alterations in novel DMRs result in altered tissue-specific gene expression during development. The results of these studies should elucidate how putatively imprinted genes from five `candidate' loci contribute to developmental plasticity during normal growth and development or under environmental exposure-altered conditions and inform the development of prevention strategies to protect the unborn fetus.