Epigenetic Gene Regulation: Early Environmental Exposures

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Epigenetic gene regulation: early environmental exposures

Epigenetics is defined as the study of heritable “A growing body of evidence changes in gene expression that occur in the suggests that epigenetic gene absence of a change in the DNA sequence itself. regulation, including DNA These include DNA methylation, covalent modi- methylation and histone fications to histone tails, packaging of DNA around nucleosomes, chromatin folding and modifications, are also influenced chromatin attachment to the nuclear matrix. by the environment and may Additionally, the influence of regulatory play a role in the fetal basis of small/micro RNAs on gene transcription is an adult disease.” emerging mechanism of epigenetic gene regula- Dana C Dolinoy tion. The epigenome is particularly susceptible to Duke University Medical environmental influences during embryogenesis Center, Environmental epigenetics because the DNA synthetic rate is high and the Department of Radiation Oncology, University Program Traditional research on the combined effects of elaborate DNA methylation patterning required in Genetics and Genomics, genetics and the environment investigates how for normal tissue development is established dur- and Integrated Toxicology individuals differ in susceptibility to disease and ing this period. Furthermore, aberrant epigenetic Program, Box 3433, how susceptibility changes over time. The gene regulation has been proposed as a mecha- Durham, NC 27710, USA majority of these gene–environment interaction nism of action for nongenotoxic carcinogenesis [9], Tel.: +1 919 684 6203; studies focus on genetic sequence variants, imprinting disorders [10,11] and complex disorders Fax: +1 919 684 5584; including single nucleotide polymorphisms including schizophrenia [12] and asthma [13]. E-mail: [email protected] (SNPs), which influence toxicant uptake, Despite a growing consensus on the role of epi- metabolism and subsequent disease susceptibil- genetic gene regulation in susceptibility to ity. Others hold genetics constant to evaluate chronic diseases, we have yet to identify the the effects of differential nutritional, environ- majority of epigenetically labile regions of the mental and occupational exposures on health genome, fully characterize the important environ- and disease. However, it has become clear that mental exposures affecting the epigenome and while genetics and the environment play an determine the critical windows of vulnerability to important role in the manifestation of many dis- environmentally-induced epigenetic alterations. orders, they do not fully explain all variation in human disease susceptibility. Proof of concept: viable yellow However, a growing body of evidence suggests agouti mice that epigenetic gene regulation, including DNA Within human, mouse, and other animal spe- methylation and histone modifications, are also cies, individuals considered genetically identical, influenced by the environment and may play a such as monozygotic twins and isogenic mouse role in the fetal basis of adult disease [1]. Specifi- strains, often display phenotypic discordance in cally, the ‘early origins hypothesis’ postulates that various traits and disease susceptibility, even after nutrition and other environmental factors during controlling for environment. A handful of prenatal and early postnatal development pro- murine metastable epialleles have been identified gram the risks for adverse health outcomes in (Avy, AxinFu, CabpIAP) in which the activity of a adult life [2–4]. Developmental plasticity occurs retrotransposon controls expression of an adja- when environmental influences affect cellular cent gene (Figure 1A) [14–16]. Metastable epialleles pathways during gestation, enabling a single gen- are alleles that are variably expressed in geneti- otype to produce a broad range of adult pheno- cally identical individuals due to epigenetic types [1]. Recently, human epidemiologic and modifications that were established during early animal model data have suggested that develop- development [16]. Variable expressivity of murine mental plasticity is influenced by persistent epige- metastable epialleles results from stochastic part of netic adaptations that occur early in development DNA methylation of the retrotransposon, pro- in response to environmental factors [5–8]. ducing genetically identical individuals with

vy Figure 1. The viable yellow agouti (Avy) mouse model. methylation in the A IAP correlates inversely with ectopic agouti expression. The degree of methylation varies dramatically among individual A. CpG sites 1–9 isogenic Avy/a mice, causing a wide variation in IAP Avy coat color ranging from yellow (unmethylated) to 3´ 5´ pseudoagouti (methylated) [21]. A,a Recently, the Avy model has been used as an 5´ 3´ epigenetic biosensor for determining whether ~15 kb maternal nutritional supplementation affects the 1A PS1A 2 fetal epigenome. Cooney and colleagues [8] and ~100 kb Waterland and colleagues [19] demonstrated that maternal dietary methyl supplementation with B. extra folic acid, vitamin B12, choline and betaine shifts the coat color distribution of the offspring towards the pseudoagouti phenotype. Waterland and colleagues further demonstrated that the shift in coat color distribution was caused by increased methylation near the Avy retrotransposon [19]. Methylation profiles were highly correlated in tissues from ectodermal (brain and tail), endodermal (liver) and meso- dermal (kidney) lineages, indicating that methylation profiles at the Avy locus are established prior to embryonic stem cell differentiation. In (A) A contra-oriented IAP insertion within pseudoexon 1A of the murine agouti gene contains a cryptic promoter (short arrowhead labeled Avy) that drives addition, methylation in day 21 of life (d21) tis- ectopic Agouti expression. Transcription of A and a alleles initiates from a hair- sues is correlated to methylation in d100 tissues, cycle specific promoter in exon 2 (short arrowhead labeled A, a). (B) Genetically demonstrating that methylation of this meta- identical 3-month old viable yellow agouti mice representing the five coat color stable epiallele is efficiently maintained over phenotypes. Yellow mice are hypomethylated at the transposable element time. Subsequently, Cropley and colleagues upstream of the agouti gene,allowing maximal ectopic expression, whereas reported that nutritional influences on coat color hypermethylation of this site silences ectopic agouti expression in the distribution in Avy mice are inherited in the F2 pseudoagouti animals. Mice that are predominately yellow are also clearly more obese than brown mice. Maternal supplementation with methyl donating generation via germline epigenetic modifications compounds, such as folic acid, or the phytoestrogen, genistein, is associated [23]. Pseudoagouti female offspring of mothers with a coat color distribution shift toward the brown pseudoagouti phenotype, supplemented with methyl donors (folic acid, resulting in population level protection from adult onset diseases. Reprinted betaine, choline, methonine and vitamin B12) with permission from [17]. produced more pseudoagouti offspring than Avy: Viable yellow agouti; IAP: Intracisternal A particle; PS1A: Pseudoexon 1A. pseduoagouti females who were fed a normal control diet. This striking shift in coat color varying phenotypes. Interestingly, the normal indicates that increased methylation at the Avy distribution of variable expressivity can be locus persists into the F2 generation, even shifted at these metastable epialleles following though only the grandmother received nutrient early exposure to various environmental supplementation. Thus, it is clear that environ- factors [8,17–19]. mental effects on the epigenome can be inherited One model for such epigenetically-based in the mammalian germline. phenotypic variability is the viable yellow agouti More recently, Dolinoy and colleagues demon- (Avy) mouse (Figures 1A and B), in which coat color strated that maternal dietary supplementation of variation is influenced by epigenetic marks estab- mice with the phytoestrogen genistein, at levels lished early in development. The Avy allele comparable with humans consuming high soy resulted from the insertion of an intracisternal A diets, shifted the coat color of offspring toward particle (IAP) murine retrotransposon upstream pseudoagouti by increasing methylation of the of the transcription start site of the agouti gene Avy retrotransposon. Methylation profiles were (Figure 1A) [19,20]. A cryptic promoter in the prox- consistent across tissues from the three germ layer imal end of the Avy IAP promotes constitutive lineages, supporting the postulate that the meth- ectopic agouti transcription, leading to yellow ylation profiles genistein is influencing are also fur, obesity and tumorigenesis [21,22]. CpG established very early in development. Moreover,

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the genistein-induced hypermethylation persisted offspring with tail kinks. Recently, DNA into adulthood and protected Avy animals from methylation in Avy gametes, zygotes and blasto- adult-onset obesity [17]. cysts was assessed to determine if methylation was Until now, the Avy model has been employed the transgenerationally inherited epigenetic chiefly as a biosensor for epigenetic alteration mark [37]. Interestingly, DNA methylation was following maternal nutritional exposure; how- absent from the blastocyst, indicating that an epi- ever, the opportunity exists to utilize this model genetic mechanism other than DNA methylation as an epigenetic biosensor for xenobiotic, behav- is the inherited mark. ioral, pharmaceutical and physical environmental factors. Moreover, for environmental agents “These findings indicate that you that shift the coat color distribution toward the are not only what you eat, but yellow (unmethylated) phenotype, it will be what your mother and important to determine whether coexposure with methyl donors and/or genistein negates the grandparents ate as well.” hypomethylation effect. The determination that nutritional supplementation counteracts epi- Incomplete germline reprogramming of epigenetic hypomethylating effects would hold tre- genetic marks implies that environmental- mendous potential for public health prevention induced changes in the epigenome may be inher- and intervention. ited transgenerationally, even in the absence of continued exposure. These findings indicate that Early environmental exposures, you are not only what you eat, but what your the fetal epigenome & mother and grandparents ate as well. A flurry of transgenerational inheritance recent animal and human epidemiological studies Prenatal and early postnatal environmental fac- supports this tenet. Drake and colleagues tors, including nutritional supplements [5,8,17–19], reported the transgenerational inheritance of low xenobiotic chemicals [6,24,25], behavioral birth weight and adult diabetes in rats following cues [26,27], reproductive factors [28,29], and even maternal or paternal exposure to abnormal levels low-dose radiation [30], have been linked to of glucocorticoids [38]. The authors hypothesized altered epigenetic programming and subsequent that germline epigenetic reprogramming rather changes in gene-expression patterns. Further- than genetic mutations is responsible for the more, epigenetic alterations have been observed observed phenotypic changes. Subsequently, in response to adolescent and adult exposure to Newbold and colleagues reported trans- environmental factors [31,32]. These important generational inheritance of tumor susceptibility observations have recently been summarized [33]. in mice following perinatal maternal exposure to Therefore, in this editorial I have focused on cur- diethylstilbestrol, a synthetic estrogenic rent literature demonstrating transgenerational chemical [39]. Increased risk is thought to result inheritance of epigenetic marks influenced by from both genetic and epigenetic mechanisms, environmental exposures. including decreased methylation in uterine genes Epigenetic marks, including CpG methylation, such as lactoferrin and c-fos. Koturbash and col- are generally stable in somatic cells; however, dur- leagues also recently observed global decreases in ing at least two developmental time periods, the methylation, reduced DNA methyltransferase epigenome undergoes extensive reprogramming. concentration, and reduced methyl binding pro- These critical windows of development include tein levels in mouse offspring of parents who were gametogenesis as well as early preimplantation both exposed to radiation [40]. These epigenetic embryos [34]. Importantly, some genomic loci fully mechanisms are postulated to underlie observed or partially escape epigenetic reprogramming dur- transgenerational genome instability and carcino- ing gametogenesis, leading to transgenerational genesis risk following parental radiation exposure. inheritance of phenotype via epigenetic, not Finally, using epidemiological, crop yield and genetic mechanisms. In the late 1970s breeding economic data from Sweden, Pembrey and col- studies involving both Avy and axin-fused (AxinFu) leagues reported sex-specific male germline trans- mice revealed inheritance of coat color [35] or tail generational inheritance of health endpoints, kink phenotype [36], respectively. For example, including increased mortality [41]. Researchers pseudoagouti Avy mothers but not fathers produce hypothesize underlying epigenetic and sex-linked more pseudoagouti offspring. In contrast, pene- genetic mechanisms are associated with these trant AxinFu mothers and fathers produce more inherited phenotypes. futurefuture sciencescience groupgroup www.futuremedicine.com 7 EDITORIAL – Dolinoy

In a series of recent reports, Anway and col- imprinted genes display monoallelic, parent-of- leagues report transgenerational reproductive toxic- origin-dependent expression, which is regulated by ity, decreased epididymal sperm count, and a combination of DNA methylation, histone reduced sperm motility in male rats following modification, and antisense transcript epigenetic maternal exposure to the endocrine active fungicide mechanisms. The development of genome-wide vinclozolin and the pesticide methoxyclor [42]. The approaches, including expression arrays and novel reduced reproductive outcomes were observed not bioinformatic tools [47], to determine those epi- only in the first generation, but also in generations genetically labile targets in the human genome that two to four. The high frequency of these patholo- are involved in the etiology of chronic diseases is gies, coupled with a sustained high risk of develop- critical to the development of prevention, screening them in subsequent generations, led to the ing and therapeutic strategies. While the Human exclusion of genetic mutation in their etiology. In Epigenome Project [101] has achieved advances in addition, as the animals aged, a number of adult the categorization of genome-wide DNA methyla- onset disorders were observed transgenerationally, tion patterns of human genes in major tissues [48], including breast cancer and prostate disease [43]. this approach will be incomplete without further These transgenerational effects were associated with identification of environmentally responsive inherited epigenetic marks, including DNA meth- epigenetically labile loci in the human genome. ylation, in several genes in the male germ line [42,44]. “Unlike genetic mutations, Incorporation of epigenomics into epigenetic profiles are potentially personalized healthcare reversible. Therefore, epigenetic The mounting evidence summarized in this editorial emphasizes the potential for environmental approaches for prevention and factors to influence fetal, adult and transgenera- treatment, such as nutritional tional epigenetic gene regulation, resulting in supplementation and/or numerous phenotypic consequences. Nevertheless, pharmaceutical therapies, may be despite the weight of this evidence, little is know about the genes in the human genome that are developed to counteract negative most susceptible to environmentally induced epi- epigenomic profiles.” genetic changes. Analysis of epigenetic profiles of monozygotic twins offers confirmation that epige- Once the identification of key genomic loci is netic dysregulation results in differential pheno- accomplished, epigenetic approaches for screening type in genetically identical humans [45,46]. For and diagnosis will become highly useful in ena- example, epigenetic profiles of monozygotic twin bling clinicians to identify at-risk individuals prior sets, as measured by global CpG methylation and to disease onset. For example, screening individu- histone H3 and H4 acetylation, exhibited greater als at an early age for epigenetically susceptible divergence in older twins as well as twins who had disease profiles will allow for closer monitoring spent more than 50% of their lives apart [45]. These and more frequent follow-up. Additionally, unlike findings indicate that the adult environment influ- genetic mutations, epigenetic profiles are poten- ences epigenetic gene regulation as individuals age. tially reversible. Therefore, epigenetic approaches Additionally, a twin discordant for a set of birth for prevention and treatment, such as nutritional defects known as caudal duplication syndrome supplementation and/or pharmaceutical thera- exhibited markedly decreased methylation of the pies, may be developed to counteract negative epi- AXIN 1 gene when compared with her affected genomic profiles. The future of epigenomics twin sister [46]. Whether this phenotypic differ- therapy holds tremendous potential for not only ence, as well as others displayed in monozygotic individualized healthcare, but also population- twin sets, stems from intrauterine environment wide diagnostic screening and prevention remains to be determined. strategies, an exciting possibility indeed. However, environmental epigenetic research has revealed that two distinct sets of epigenetically Acknowledgements labile genes link early environmental exposures to This work was supported by NIH grants ES13053, adult disease. Genes with metastable epialleles ES08823, ES015165, and T32-ES07031. The author have highly variable expression due to stochastic declares no competing financial interests and thanks Randy allelic changes in the epigenome. In contrast, L Jirtle for critical reading of the manuscript.

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