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

Abstract

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 (A(vy/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.

Figure 1

Methylation status of CpG sites within the A^vy IAP in genetically identical A^vy/a littermates. (A) A contraoriented IAP insertion within pseudoexon 1A (PS1A) of the murine Agouti gene. A cryptic promoter (short arrow labeled A^vy) drives ectopic Agouti expression. CpG sites 1–9 are oriented in the 3′ to 5′ direction with respect to the IAP insertion, as shown. Transcription of A and a alleles initiates from a hair-cycle–specific promoter in exon 2 (short arrow labeled A,a). (B) Pseudoagouti animals exhibit the highest degree of CpG methylation at sites 4–9. Bisulfite sequencing reveals increasing intensity of the cytosine lane at CpG sites 4–9 within the A^vy IAP in genetically identical A^vy/a animals representing the five coat classes. (C) Genetically identical week-15 A^vy/a mouse littermates representing the five coat-color phenotypes.

Figure 2

Coat-color distribution and methylation of CpG sites 4–9 of the A^vy IAP in offspring whose mothers were fed unsupplemented and genistein-supplemented (250 mg genistein/kg diet) diets. (A) Coat-color distribution of A^vy/a offspring born to 15 unsupplemented and 12 genistein-supplemented litters. (B) Genomic sequence containing nine CpG sites located between the cryptic Agouti promoter and the IAP promoter (blue arrow in Figure 1A) at the 5′ end of the contraoriented A^vy IAP. CpG sites 1–9 are numbered and marked by gray boxes. (C) Box plots representing the percentage of cells methylated at sites 4–9 in unsupplemented (n = 52) and genistein-supplemented (n = 44) A^vy/a offspring. Ends of the boxes indicate the interquartile range representing the 25th to 75th percentiles of the data; horizontal lines within each box indicate median; and dashed horizontal lines represent average percent methylation of CpG sites 4–9 according to coat-color phenotype.

Figure 3

Mediational regression analysis (Baron and Kenny 1986) of genistein diet, A^vy IAP methylation, and coat color. (A) CpG methylation variable specified as regional methylation of CpG sites 4–9. Genistein diet significantly influences coat color (top); however, the relationship is reduced when regional A^vy methylation is included in the regression model (bottom). (B) CpG methylation variable specified as individual CpG site 4. When CpG site 4 methylation is included in the model (bottom), the direct effect of diet on coat color is abrogated, indicating that methylation at site 4 plays a strong role in mediating the genistein effect on coat color.

Figure 4

Average A^vy IAP methylation as a function of coat color, tissue type, and age. Abbreviations: B, brain; K, kidney; L, liver; T, tail. Average methylation across CpG sites 1–9 in day 150 tissues derived from ectodermal (B and T), mesodermal (K), and endodermal (L) tissues from five genistein-supplemented A^vy/a animals representing the five coat-color phenotypes is correlated with that in day 21 tail tissue (Pearson’s r > 0.9 and p < 0.05 for each correlation).

Figure 5

Variation of average body weight among animal coat-color class over time. Significant weight differences among coat-color classes start at week 25 and continue through adulthood. Pseudoagouti animals exhibit normal body weight compared with overweight yellow, slightly mottled, mottled, and heavily mottled animals due to hyper-methylation in the A^vy IAP region, which shuts off ectopic Agouti transcription. By shifting the offspring population coat-color distribution toward brown pseudoagouti animals, genistein supplementation significantly increases the incidence of normal-body-weight animals.

Figure 6

One carbon metabolism pathway. Abbreviations: 5MTHF, 5-methyltetrahydrofolate; THF, tetrahydrofolate. The availability of methyl groups for DNA methylation is increased by provision of excess methyl donors and cofactors, including folate, choline, and SAM, which is the major methyl donor for DNA, RNA, protein, and lipid methylation.