Environmental influence on epigenetic inheritance atthe Avy allele

David IK Martin, Jennifer E Cropley, and Catherine M Suter

© 2008 International Life Sciences Institute

The material of inheritance can be more than DNA alone:epigenetic states are in some cases sufficiently stable in thegermline to produce epigenetic inheritance. The murineAvy allele is a prominent example of this phenomenon.Methyl donors in the maternal diet alter the epigeneticstate of the Avy allele in the somatic cells of offspring. Wehave found that methyl donors also influence the germ-line epigenetic state of Avy, inducing a heritable change.The finding raises the possibility that multigenerationalexposure to an environmental agent could produce acumulative and stable change in the phenotype of amammal.

In current usage, “epigenetics” refers to heritable(through mitosis or meiosis) alterations in gene expres-sion that are independent of DNA sequence.1 Essentially,epigenetics is a system of gene regulation based on theassociation of specific regions of DNA with a variety ofchromatin proteins and on covalent modification of DNA(cytosine methylation).2–6 These molecular accretions toDNA are a fundamental aspect of eukaryotic biology;they allow portions of complex genomes to be stored inan inactive state. They do not function independently.Rather, they form an interacting network that stabilizesthe active or the silent state. Histones associate with DNAto form the nucleosome, which is perhaps the fundamen-tal unit of epigenetic control. Modifications of histonetails may be critical in recruiting proteins that maintaineither the active or the silent transcription state, and evenfor determining cytosine methylation.6,7 Cytosine methy-lation is a covalent modification of DNA that recruits andbinds proteins that repress transcription; it functions inconcert with histone modifications to consolidate andmaintain repression of transcription initiation.3 A largenumber of proteins, exemplified by Polycomb and HP1 in

Drosophila and their homologues in other species, areinvolved in the formation or consolidation of heterochro-matin (stably condensed and transcriptionally silentchromatin).6,8

Eukaryotes use epigenetic modifications to reversiblyinactivate large regions of their genomes.1,8 Highereukaryotes use this mechanism in two important waysthat are fundamental to health and development. First,they suppress the activity of retrotransposons, genomicparasites that, when active, can disrupt genome function.9

Second, they silence genes that are not used in a specificdifferentiated cell type.10 Highly distinct cell types havethe same genotype, and their phenotypes are determinedlargely by epigenetic silencing of some genes and activityof others.

Epigenetic modifications are interposed between thegenome and its environment, and by their nature they aresusceptible to environmental influence. They may thus beviewed as a means for the environment (intracellular,organismal, or ecological) to impose stable changes onpatterns of gene expression.

The murine Avy allele is a model of epigenetic varia-tion and inheritance. In the Avy allele an inserted intrac-isternal A particle (IAP) acts as a controlling element,which expresses agouti by transcription from the longterminal repeat (LTR) of the IAP.11 A controlling element(the term was coined by McClintock12) may be defined asa transposable element that is able to influence theexpression of genes in the vicinity of its integration sitethrough any of a variety of mechanisms. In many casesthe epigenetic (transcriptional) state of the controllingelement determines its effects, and controlling elementsthus often act in mosaic patterns and exhibit complexpatterns of somatic and germline epigenetic inheritance.

Affiliations: DIK Martin, JE Cropley, and CM Suter, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, Australia. DIK Martin,Children’s Hospital Oakland Research Institute, Oakland, California, USA. CM Suter, Faculty of Medicine, University of New South Wales,Sydney, Australia.

Correspondence: DIK Martin, CHORI, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA. E-mail: dimartin@chori.org, Phone:+1-510-450-7974, Fax: +1-510-450-7910.

doi:10.1111/j.1753-4887.2008.00057.xNutrition Reviews® Vol. 66(Suppl. 1):S12–S14S12

The IAP inserted in the Avy allele acts by usurping tran-scriptional control of agouti. A promoter in the 5′ LTR ofthe IAP transcribes agouti: because the LTR is potentiallyactive in all somatic cells, the activity of the LTR promotersubstitutes a constitutive expression pattern for thenormal tight tissue-specific control exerted by the agoutipromoter. Agouti encodes agouti signaling protein (ASP),a paracrine signaling molecule that acts on the melano-cortin receptor and possibly other receptors as well.Ectopic expression of ASP produces a syndrome ofyellow fur, obesity, type II diabetes, and susceptibility to avariety of tumors. The epigenetic state of the IAP in theAvy allele is highly variable in somatic cells of isogenicmice and is reflected in CpG methylation of the LTRpromoter. As a result, the phenotypes of isogenic Avy micevary in a mosaic spectrum from fully affected to normal(lean and agouti, termed “pseudoagouti”).13

The epigenetic state of Avy is stable in somatic cells ofan individual mouse. Although the phenotypes of Avy

mice vary widely, each mouse retains the same phenotypethroughout life. But the epigenetic state of Avy is highlyunstable in the germline, so a mouse with a given Avy

phenotype will have offspring with a variety of differentphenotypes.13,14 Nevertheless, in the female germline theepigenetic state of Avy is sufficiently stable to produce ameasurable degree of transgenerational epigenetic inher-itance. Obese and fully yellow Avy females never havepseudoagouti offspring, but pseudoagouti females have15–20% pseudoagouti offspring; the difference is notattributable to any metabolic effects of the obese yellowphenotype itself. There is no apparent epigenetic stabili-zation with maintenance of the silent Avy in the femalegermline: we bred pseudoagouti daughters of pseudoag-outi females for a total of seven generations, and observedno shift in the proportion of pseudoagouti offspring.

The somatic epigenetic state of the Avy allele can beshifted by in utero exposure to high doses of methyldonors.15,16 When Avy dams are fed a diet supplementedwith a number of methyl donors, the spectrum of pheno-types in their offspring is shifted so that more of the pupshave higher proportions of agouti fur. Because thepseudoagouti phenotype is associated with CpG methy-lation in the LTR promoter responsible for ectopicexpression, it has been supposed that methyl donors actby increasing methylation of the LTR.15,16

We asked if maternal methyl donor supplementationaffects the germline epigenetic state of Avy.17 Our strategywas to expose mice to methyl donor supplementation inutero, withdraw the supplement at weaning, and thenobserve the spectrum of phenotypes in offspring of themice who were exposed to methyl donor supplementa-tion in utero (the F2 generation). In our initial experi-ments we found that methyl donor supplementationaffected the offspring only when the Avy allele was con-

tributed by the sire.We designed our experiment to maxi-mize the chances of observing an effect on the germlinestate of Avy, by breeding only F1 females who had thepseudoagouti phenotype associated with silence andsoma-wide methylation of the IAP inserted in the Avy

allele. It is the silent epigenetic state that is inherited in thefemale germline, and pseudoagouti females are thosemost likely to carry germ cells with a silent Avy allele(while yellow females are likely to have none, and thusnothing to inherit). We mated Avy/a males with a/afemales and provided pregnant dams with a NIH-31 dietwith the following elements per kg: 15 g choline, 15 gbetaine, 7.5 g L-methionine, 150 mg zinc, 15 mg folicacid, and 1.5 mg vitamin B12 to pregnant dams. The dietwas administered during mid-gestation, when the pri-mordial germ cells that will give rise to the F2 generationare differentiating. Methyl donor supplementation waswithdrawn after this period. Controls were mice bred inthe same way on an unsupplemented NIH-31 diet. As inprevious reports, exposure to methyl donor supplemen-tation during midgestation shifted Avy phenotypes in themice exposed as fetuses (the F1 generation). We selectedpseudoagouti F1 females and bred them without furthermethyl donor supplementation. The shift in the spectrumof phenotypes was retained in their (F2) offspring. Thisresult indicates that in utero exposure to methyl donorschanged the epigenetic state of Avy in germline cells, andthat the altered state was retained through subsequentgametogenesis and embryogenesis. The result suggeststhat a pregnant mother’s diet may have an influence onthe phenotypes of her grandchildren, independent of thediet consumed by her children or grandchildren.17

Although our experiment demonstrated an effect ofthe germline epigenetic state of Avy, it did not demon-strate that the induced change was stable for more thanone generation: the Avy allele in the somatic cells of the F2generation had not passed through the stage of primor-dial germ cell differentiation when many epigeneticmarks are erased and reset.We are continuing this experi-ment to ask if continuous methyl donor supplementationresults in a cumulative effect, i.e., if multigenerationalexposure amplifies the rather modest effect of methyldonor supplementation. Cumulative effects would beconsistent with maintenance of the induced changethrough the life cycle; this can be confirmed by withdraw-ing supplementation and asking if the effect persists formore than one generation.

Discussions of the mechanism by which methyldonors affect the Avy allele have generally assumed thatmethyl donors directly increase methylation of the IAP,thereby silencing it and changing the phenotypes ofmice.15,16 We have carried out extensive bisulphite allelicsequencing of the IAP in the region of the LTR fromwhich transcription of agouti is initiated. We find that

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CpG methylation of the LTR is incomplete even when it istranscriptionally silent (i.e., in pseudoagouti mice), incomparison to highly similar IAPs selected at randomfrom the mouse genome. We speculate that this incom-plete methylation is related to the unstable germline stateof the Avy allele. In utero exposure to supplementarymethyl donors does not increase the density of CpGmethylation in the silent LTR, either in the F1 or the F2generation. This result is entirely consistent with earlierreports of higher average Avy methylation in mice exposedto methyl donors.We interpret it as indicating that methyldonor supplementation does not directly increase methy-lation of Avy, but increases the probability of the IAPshifting to a silent state in early embryogenesis, thus shift-ing the proportion of phenotypes in a population. Methyldonors increase the pool of S-adenosylmethionine, whichmakes methyl groups available to a variety of proteins.The induced silencing of Avy may stem from methylationof some protein, for example a histone, which is involvedin the stochastic choice of active and silent epigeneticstates.

DNA has long been regarded as the sole material ofinheritance, and it is a stable molecule that changes veryinfrequently. Epigenetic inheritance presents the prospectof a system of inheritance based on a complex assortmentof proteins associated with DNA; its rules will be com-pletely different from those of mendelian inheritance. Ifdiet can change the epigenetic state of a gene in the ger-mline, this raises the possibility that stable alterations inphenotype can be induced by the environment without achange in genotype. The implications for the health ofpopulations, and for species evolution, make it importantto understand the mechanisms of the phenomena.

Acknowledgments

Declaration of interest. The authors have no relevantinterests to declare.

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