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.
Built upon 20 newly identified DMRs that potentially play a role in the pathogenesis of about 20 diseases, our project aims to determine imprinting mechanisms controlling five novel DMR- controlled loci, which will reveal the changed methylation patterns of DMRs in altering imprinted expression in response to environmental exposure. Studies directed at the determination of the underlying mechanisms to regulate the developmental plasticity (via genomic imprinting) and the critical windows of susceptibility to exogenous environmental factors will allow for prevention strategies to protect the unborn fetus.
