Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation

Abstract

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine A vy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the A vy allele. We find that the somatic epigenetic state of A vy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts A vy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the A vy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mother's diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change.

Fig. 1.

The A^vy allele and the spectrum of A^vy phenotypes. (A) The A^vy allele carries an insertion of an IAP retrotransposon in an antisense direction in agouti pseudoexon 1A (3, 4). The A^vy transcript originates from a cryptic promoter in the 5′LTR of the IAP and is spliced to agouti coding exons 2, 3 and 4, which encode ASP (3). When the IAP is silent, agouti is transcribed from hair-cycle-specific promoters in exons 1B and 1C. (B) A^vy phenotypes are scored from 1 to 5 based on coat color. Fully yellow mice are scored as 1, and fully agouti mice are scored as 5. Phenotypes of mosaic mice range from mostly yellow (2) to mottled yellow/agouti (3) to mostly agouti (4).

Fig. 2.

Parent-of-origin effect of methyl donor supplementation. Parents were supplemented with methyl donors throughout pregnancy and offspring phenotypes scored as in Fig. 1B. (Left) Maternal transmission. Shown is the maternally derived A^vy allele (A^vy/a dam, a/a sire). (Right) Paternal transmission. Shown is the paternally derived A^vy allele (a/a dam, A^vy/a sire). Results are expressed as the percentage of supplemented offspring with the same phenotype as unsupplemented controls; the percentage of offspring of each coat color is shown beneath each graph.

Fig. 3.

Midgestation and germ-line effects of methyl donor supplementation. (A) F₁ phenotypes. Shown is the effect of methyl donor supplementation on mid-gestation supplemented offspring. Dams were supplemented with methyl donors from E8.5 to E15.5, and offspring phenotypes were scored as in Fig. 1B. Results are expressed as the percentage of supplemented offspring with the same phenotype as unsupplemented controls, and the percentage of offspring of each coat color is shown beneath each graph. (B) F₂ phenotypes. Shown is the heritable/grandparental effect of methyl donor supplementation. Phenotypes of F₂ offspring from pseudoagouti female mice shown in A are expressed as a percentage of control offspring (offspring from pseudoagouti dams that had never been supplemented) with the same phenotype. (C) A schematic diagram illustrating the effect of methyl donor supplementation in the germ line. Epigenetic changes to A^vy in primordial germ cells exposed to methyl donors during differentiation (Left) are maintained throughout gametogenesis and embryogenesis. Thus, pseudoagouti F₁ mice that are genetically and phenotypically identical but were subject to different diets in utero (Left vs. Right), can produce phenotypically different F₂ offspring.