In the present study, we observed a statistically significant shift in coat-color phenotype and adult body weight distribution among genetically identical offspring whose mothers received a diet supplemented with 250 mg/kg diet of genistein. The shifts in coat color and body weight were mediated by increased methylation at CpG sites 4–6 located immediately upstream of the cryptic promoter region of the Avy IAP upstream of the transcription start site of the Agouti gene. Hypermethylation in the genistein-supplemented population results in decreased ectopic Agouti expression, which reduces yellow phaeomelanin production and protects against adult-onset obesity.
In addition to assessing average methylation over the Avy
retrotransposon region, we also determined individual methylation levels at each of the nine CpG sites. The enhanced significance of site 4 coupled with the general increase in methylation closer to the cryptic Avy
promoter suggests that site 4 represents a boundary to methylation spreading and may be particularly important in determining the epigenetically regulated mosaicism in Avy
mouse coat color. Our data indicate that site 4 methylation principally mediates the effect of genistein supplementation on Avy
coat color. This finding is consistent with methylation status of a single CpG in the glucocorticoid receptor gene promoter principally mediating the effect of maternal caregiving behavior on long-term stress responsiveness in rats (Weaver et al. 2004
The low variability in CpG methylation among the three germ layer tissues relative to high variability between individual animals indicates that the establishment of epigenotype at the Avy
IAP, which genistein is influencing, occurs early in embryonic development. Furthermore, the concordance between Avy
methylation in day 21 tail and that in the various tissues of the same animal at day 150 demonstrates that genistein-induced epigenetic changes persist to adulthood. The phenomenon of high interindividual coupled with low inter-tissue variability in methylation may represent a common characteristic of epigenetically labile genes in the mouse and human genomes whose expression is controlled by DNA methylation established early in development (Waterland and Jirtle 2004
). Consequently, future studies using the Avy
mouse model should more thoroughly investigate the role of stem cells not only in determining cell differentiation early in life but also in promoting cell differentiation during pubertal development.
Body weight data indicate that enhanced IAP methylation in the genistein-supplemented offspring increased the probability that ectopic Agouti expression is silenced, leading to a decreased incidence of adult-onset obesity. Using the Avy mouse model, we have demonstrated for the first time that maternal dietary supplementation is associated with not only altered fetal methylation patterns but also methylation-dependent susceptibility to disease. This finding supports the hypothesis that environmental and nutritional influences on the establishment of epigenetic gene regulatory mechanisms in early life influence adult metabolism and chronic disease susceptibility.
The lack of an association between genis-tein supplementation and SAM or SAH levels indicates that genistein affects DNA methylation through a mechanism that is independent of the one-carbon metabolism pathway. Genistein and other isoflavones interact with the estrogen receptor to enhance histone acetylation (Hong et al. 2004
). Therefore, his-tone acetylation may open up the IAP region for methylation, leading to transcriptional deactivation. Whether genistein’s enhancement of DNA methylation is beneficial or deleterious may depend on other environmental factors, such as whether the local food supply is supplemented with folic acid. Because folate is an important cofactor in one-carbon metabolism, individuals who are exposed to folic acid fortification and consume a diet high in soy may experience an additive or even synergistic effect on DNA methylation. Given the recent demonstration of the ability of environmental influences to induce epigenetic changes in the early postnatal period (Weaver et al. 2004
), such an interaction could be particularly worrisome for infants fed soy formula diets in which genistein intake relative to body weight reaches levels higher than those used in the present study (Setchell et al. 1997
The results of our study have a number of other important implications. First, the biologic importance of establishing genomic methylation patterns during early development suggests that it is essential to determine the effects of environmental factors on the epigenome during prenatal and early postnatal development, rather than just in adults. For example, insulin-like growth factor 2 (IGF2
) loss of imprinting, which places individuals at increased risk of developing colon cancer, is not caused by exposure to adult environmental factors (Cruz-Correa et al. 2004
). Rather, it is a trait that is either inherited and/or induced by environmental influences early in embryonic development (Jirtle 2004
). Nutritional effects on the fetal epigenome may therefore underlie the long-term cardioprotection of rats born to mothers supplemented with soy during pregnancy (Souzeau et al. 2005
). Second, phytoestrogen content in laboratory animal feed is highly variable (Degen et al. 2002
). Therefore, genistein’s effect on fetal DNA methylation patterns could significantly influence the interpretation of hormone and other rodent assay studies (Brown and Setchell 2001
; Naciff et al. 2004
; Wang et al. 2005
) as well as confound the interpretation of gene expression arrays and DNA methylation studies. Finally, it needs to be determined whether the relatively high genistein intake of infants consuming soy formulas is beneficial or has unintended deleterious effects on the human epigenome, especially in the United States and other countries where the food supply is fortified with folic acid.
This is the first study to demonstrate that exposure to dietary genistein in utero
, at levels present in human adult populations consuming high-soy diets, affects coat color and reduces population incidence of obesity by altering the epigenome in mice. Thus, an active ingredient in soy enhances the early establishment of DNA methylation. In addition to single-nucleotide polymorphisms affecting environmentally responsive genes, our findings show that early nutritional and environmentally induced epigenetic modifica-tions can provide an alternative mechanism for varying individual susceptibilities to environmental agents. Our results also suggest a plausible explanation for the lower incidence of certain cancers in Asians compared with Westerners (Chang et al. 2001
; Lee et al. 1991
) as well as the increased cancer incidence in Asians who immigrate to the United States (Ziegler et al. 1993