Jennifer R. Weidman

Maternal genistein alters coat color and protects Avy mouse offspring from obesity by modifying the fetal epigenome.

Genistein, the major phytoestrogen in soy, is linked to diminished female reproductive performance and to cancer chemoprevention and decreased adipose deposition. Dietary genistein may also play a role in the decreased incidence of cancer in Asians compared with Westerners, as well as increased cancer incidence in Asians immigrating to the United States. Here, we report that maternal dietary genistein supplementation of mice during gestation, at levels comparable with humans consuming high-soy diets, shifted the coat color of heterozygous viable yellow agouti (Avy/a) offspring toward pseudoagouti. This marked phenotypic change was significantly associated with increased methylation of six cytosine–guanine sites in a retrotransposon upstream of the transcription start site of the Agouti gene. The extent of this DNA methylation was similar in endodermal, mesodermal, and ectodermal tissues, indicating that genistein acts during early embryonic development. Moreover, this genistein-induced hypermethylation persisted into adulthood, decreasing ectopic Agouti expression and protecting offspring from obesity. Thus, we provide the first evidence that in utero dietary genistein affects gene expression and alters susceptibility to obesity in adulthood by permanently altering the epigenome.

Metastable Epialleles, Imprinting, and the Fetal Origins of Adult Diseases

Epigenetics is the study of the heritable changes in gene expression that occur without a change in the DNA sequence itself. These heritable epigenetic changes include chromatin folding and attachment to the nuclear matrix, packaging of DNA around nucleosomes, histone modifications, and DNA methylation. The epigenome is particularly susceptible to dysregulation during gestation, neonatal development, puberty, and old age. Nevertheless, it is most vulnerable to environmental factors during embryogenesis because the DNA synthetic rate is high, and the elaborate DNA methylation patterning and chromatin structure required for normal tissue development is established during early development. Metastable epialleles are alleles that are variably expressed in genetically identical individuals due to epigenetic modifications established during early development and are thought to be particularly vulnerable to environmental influences. The viable yellow agouti (Avy) allele, whose expression is correlated to DNA methylation, is a murine metastable epiallele, which has been used as an epigenetic biosensor for environmental factors affecting the fetal epigenome. In this review, we introduce epigenetic gene regulation, describe important epigenetic phenomenon in mammals, summarize literature linking the early environment to developmental plasticity of the fetal epigenome, and promote the necessity to identify epigenetically labile genes in the mouse and human genomes.

Cancer Susceptibility: Epigenetic Manifestation of Environmental Exposures

Cancer is a disease that results from both genetic and epigenetic changes. Discordant phenotypes and varying incidences of complex diseases such as cancer in monozygotic twins as well as genetically identical laboratory animals have long been attributed to differences in environmental exposures. Accumulating evidence indicates, however, that disparities in gene expression resulting from variable modifications in DNA methylation and chromatin structure in response to the environment also play a role in differential susceptibility to disease. Despite a growing consensus on the importance of epigenetics in the etiology of chronic human diseases, the genes most prone to epigenetic dysregulation are incompletely defined. Moreover, neither the environmental agents most strongly affecting the epigenome nor the critical windows of vulnerability to environmentally induced epigenetic alterations are adequately characterized. These major deficits in knowledge markedly impair our ability to understand fully the etiology of cancer and the importance of the epigenome in diagnosing and preventing this devastating disease.

Genes flanking Xist in mouse and human are separated on the X chromosome in American marsupials.

X inactivation, the transcriptional silencing of one of the two X chromosomes in female mammals, achieves dosage compensation of X-linked genes relative to XY males. In eutherian mammals X inactivation is regulated by the X-inactive specific transcript (Xist), a cis-acting non-coding RNA that triggers silencing of the chromosome from which it is transcribed. Marsupial mammals also undergo X inactivation but the mechanism is relatively poorly understood. We set out to analyse the X chromosome in Monodelphis domestica and Didelphis virginiana, focusing on characterizing the interval defined by the Chic1 and Slc16a2 genes that in eutherians flank the Xist locus. The synteny of this region is retained on chicken chromosome 4 where other loci belonging to the evolutionarily ancient stratum of the human X chromosome, the so-called X conserved region (XCR), are also located. We show that in both M. domestica and D. virginiana an evolutionary breakpoint has separated the Chic1 and Slc16a2 loci. Detailed analysis of opossum genomic sequences revealed linkage of Chic1 with the Lnx3 gene, recently proposed to be the evolutionary precursor of Xist, and Fip1, the evolutionary precursor of Tsx, a gene located immediately downstream of Xist in eutherians. We discuss these findings in relation to the evolution of Xist and X inactivation in mammals.

Comparative phylogenetic analysis reveals multiple non-imprinted isoforms of opossum Dlk1

Imprinted genes are monoallelically expressed in a parent-of-origin manner and were previously identified in both marsupials and eutherians, but not in monotremes. Phylogenetic comparison of imprinted domains is a powerful tool for investigating the molecular and adaptive evolution of this unique gene regulatory mechanism. Herein, we report that multiple transcripts of Dlk1 (Delta, Drosophila, Homolog-like 1) are expressed in the opossum, but none are imprinted. Thus, we provide the first example of a reciprocally imprinted gene domain in which imprinting evolved in a common ancestor to eutherian rather than therian mammals. Moreover, the reciprocally imprinted Meg3 (Maternally Expressed Gene 3), found downstream of Dlk1 in eutherian mammals, is absent in the opossum. We propose that the Meg3 sequence integrated into the eutherian Dlk1 domain via a LINE-1 element and that Dlk1 became imprinted in eutherian mammals only after this downstream integration. These findings clearly demonstrate that imprinted genes did not all evolve before the divergence of marsupials and eutherians.

Imprinting of Opossum Igf2r in the Absence of Differential Methylation and Air

Phylogenetic comparison of extant mammals with divergent imprint status is a powerful method for identifying critical components of imprint regulation at individual loci. The entire genomic region of Igf2r in the imprinted marsupials, Didelphis virginiana and Monodelphis domestica, and the nonimprinted monotreme, Ornithorhynchus anatinus, was isolated and sequenced. Genetic and epigenetic comparisons of over 160 kb of sequence were then performed in five distinct mammalian species. Surprisingly, opossum Igf2r is imprinted and maternally expressed despite the absence of the intron 2 CpG island (CpG2), antisense Igf2r RNA (Air) and differential methylation of the promoter (CpG1) required for imprinting of this gene in mice. These findings demonstrate that the genomic elements necessary for imprinted Igf2r expression in eutherians are not required for imprinting of this locus in metatherians. Thus, the regulatory mechanisms of Igf2r imprinting did not evolve convergently within the Therian subclass of mammals.

Convergent and divergent evolution of genomic imprinting in the marsupial Monodelphis domestica

BackgroundGenomic imprinting is an epigenetic phenomenon resulting in parent-of-origin specific monoallelic gene expression. It is postulated to have evolved in placental mammals to modulate intrauterine resource allocation to the offspring. In this study, we determined the imprint status of metatherian orthologues of eutherian imprinted genes.ResultsL3MBTL and HTR2A were shown to be imprinted in Monodelphis domestica (the gray short-tailed opossum). MEST expressed a monoallelic and a biallelic transcript, as in eutherians. In contrast, IMPACT, COPG2, and PLAGL1 were not imprinted in the opossum. Differentially methylated regions (DMRs) involved in regulating imprinting in eutherians were not found at any of the new imprinted loci in the opossum. Interestingly, a novel DMR was identified in intron 11 of the imprinted IGF2R gene, but this was not conserved in eutherians. The promoter regions of the imprinted genes in the opossum were enriched for the activating histone modification H3 Lysine 4 dimethylation.ConclusionsThe phenomenon of genomic imprinting is conserved in Therians, but the marked difference in the number and location of imprinted genes and DMRs between metatherians and eutherians indicates that imprinting is not fully conserved between the two Therian infra-classes. The identification of a novel DMR at a non-conserved location as well as the first demonstration of histone modifications at imprinted loci in the opossum suggest that genomic imprinting may have evolved in a common ancestor of these two Therian infra-classes with subsequent divergence of regulatory mechanisms in the two lineages.