The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Epigenetic gene regulation comprises the heritable changes in gene expression that occur in the absence of changes to the DNA sequence itself. Epigenetic mechanisms include chromatin folding and attachment to the nuclear matrix, packaging of DNA around nucleosomes, covalent modifications of histone tails (e.g. acetylation, methylation, phosphorylation), and DNA methylation. The influence of regulatory small RNAs and micro RNAs on gene transcription is also increasingly recognized as a key mechanism of epigenetic gene regulation. Conventional gene-environment interaction studies strive to understand how individuals with different genotypes respond to various environmental factors and how these responses change over time. Such research efforts have highlighted the important contribution of both genetic and environmental variability in human diseases. However, it has been argued that a full understanding of gene-environment interactions requires that epigenetic mechanisms be taken into account. Therefore, the interdisciplinary field of environmental epigenomics emphasizes the potential for nutritional and environmental factors to influence fetal, adult, and transgenerational epigenetic gene regulation, resulting in numerous phenotypic consequences.1 The viable yellow agouti (Avy) mouse model, in which coat color variation is correlated to epigenetic marks established early in development, has been used to investigate the impacts of nutritional and environmental influences on the fetal epigenome (Fig. 1A and B). The wild-type murine Agouti gene encodes a paracrine signaling molecule that produces either black eumelanin (a) or yellow phaeomelanin (A). Both A and a transcriptions are initiated from a developmentally regulated hair-cycle-specific promoter in exon 2 (Fig. 1A). Transient A expression in hair follicles during a specific stage of hair growth results in a sub-apical yellow band on each black hair shaft, causing the brown agouti coat color of wild-type mice.2 The Avy metastable epiallele resulted from the insertion of an intracisternal A particle (IAP) murine retrotransposon upstream of the transcription start site of the Agouti gene (Fig. 1A).2,3 A cryptic promoter in the proximal end of the Avy IAP promotes constitutive ectopic Agouti transcription not only in hair follicles, but throughout all cells, leading to yellow fur, as well as adult-onset obesity, diabetes, and tumorigenesis.4,5 Interestingly, CpG methylation in the Avy IAP correlates inversely with ectopic Agouti expression. The degree of methylation within the 5′ IAP long terminal repeat (LTR) varies dramatically among individual isogenic Avy/a mice, causing a wide variation in coat color ranging from yellow (unmethylated) to pseudoagouti (methylated) (Fig. 1B). The viable yellow agouti (Avy) mouse model. A) A contraoriented IAP insertion within pseudoexon 1A (PS1A) of the murine agouti gene contains a cryptic promoter (short arrowhead labeled Avy) that drives ectopic agouti expression. Transcription of A and a alleles initiates from a hair-cycle-specific promoter in exon 2 (short arrowhead labeled A, a). B) Genetically identical adult viable yellow agouti (Avy) mice representing the five coat color phenotypes. Yellow mice are hypomethylated at the transposable element upstream of the Agouti gene allowing maximal ectopic expression, whereas hypermethylation of this site silences ectopic agouti expression in the pseudoagouti animals. Mice that are predominately yellow are also clearly more obese than brown mice. Reprinted from Dolinoy et al. (2007)11 with permission. The Avy allele is the most extensively studied murine metastable epiallele. Metastable epialleles are identical alleles that are variably expressed due to epigenetic modifications that are established very early in development.6 They are most often associated with retroelements and transgenesis. Three of the identified murine metastable epialleles (Avy, AxinFu, CabpIAP) are associated with contraoriented IAP insertions.2,–8 The extent of DNA methylation at each allele is stochastic and dependent uponmaternal nutrition and environmental exposures during early development.3,–11 Approximately a thousand copies of IAP retrotransposons are present in the mouse genome,12 and about 40% of the human genome is comprised of transposable elements, of which approximately 9% are retrotransposons.13 The work summarized here utilizes the Avy mouse model as an epigenetic biosensor to characterize nutritional and environmental factors affecting epigenetic gene regulation and subsequent adult phenotype. First, the Avy model was recently employed to investigate the effects of a plant phytoestrogen on the fetal epigenome.9 Isoflavones represent a class of phytoestrogens present in soy and soy products that are active in multiple biological systems, including estrogen receptor- and non-estrogen receptor-mediated signaling pathways.14,15 Maternal dietary supplementation with genistein (250 mg/kg diet), the major isoflavone present in soy, shifted the coat color distribution of Avy/a offspring toward pseudoagouti (brown). This marked phenotypic change was mediated by increased DNA methylation of six CpG sites within the Avy IAP. The extent of DNA methylation in tissues from the three germ layers (brain, kidney, and liver) was correlated, indicating that genistein's influence on DNA methylation occurs during early embryonic development. Moreover, the genistein-induced hypermethylation persisted into adulthood, decreasing ectopic Agouti expression and protecting adult offspring from obesity. The observed effects of genistein on the epigenome serve as a plausible explanation for the lower incidence of certain cancers in Asians compared to Westerners16 as well as the increased cancer incidence in Asians who immigrate to the United States.17 In the f