Epigenetics plays an important role in orchestrating key biologic processes. Epigenetic marks, including DNA methylation, histones, chromatin structure, and noncoding RNAs, are modified throughout life in response to environmental and behavioral influences. With each new generation, DNA methylation patterns are erased in gametes and reset after fertilization, probably to prevent these epigenetic marks from being transferred from parents to their offspring. However, some recent animal studies suggest an apparent resistance to complete erasure of epigenetic marks during early development, enabling transgenerational epigenetic inheritance. Whether there are similar mechanisms in humans remains unclear, with the exception of epigenetic imprinting. Nevertheless, a distinctly different mechanism—namely, intrauterine exposure to environmental stressors that may affect establishment of the newly composing epigenetic patterns after fertilization—is often confused with transgenerational epigenetic inheritance. In this review, we delineate the definition of and requirement for transgenerational epigenetic inheritance, differentiate it from the consequences of intrauterine exposure, and discuss the available evidence in both animal models and humans.—Van Otterdijk, S. D., Michels, K. B. Transgenerational epigenetic inheritance in mammals: how good is the evidence? FASEB J. 30, 2457‐2465 (2016). www.fasebj.org

ABBREVIATIONS

DNMT DNA methyltransferase DOHaD developmental origins of health and disease DPPA developmental pluripotency associated DZ dizygotic IAP intracisternal A particle lnRNA long noncoding RNA LTR long terminal repeat miRNA microRNA MZ monozygotic ncRNA noncoding RNA PGC primordial germ cell TET 10‐11 translocation

Epigenetics is the meiotically and mitotically heritable potential for gene expression that does not involve variation in the DNA sequence (1, 2). Epigenetic events are important in orchestrating key biologic processes, such as cell differentiation, genomic imprinting, and X‐chromosome inactivation (3–5). The primary mechanisms involved in epigenetic regulation include DNA methylation, posttranslational histone modification, chromatin remodeling, and RNA‐associated gene silencing by noncoding RNAs. Epigenetic mechanisms mediate diversified gene expression profiles to allow the generation of the variety of cells and tissues required in multicellular organisms. All cells in an organism contain essentially the same information, but different cell types vary in their phenotype, function, and expression profiles (6–9). Whereas an organism's genetic information does not change during its life span (with the exception of acquired mutations and DNA damage) epigenetic signatures are plastic (10–12) and can be modified in response to environmental and behavioral influences, such as nutrition, smoking, and air pollution (13–15). Alterations in the epigenome can result in subtle changes in cell differentiation or gene expression profiles or can result in cumulative detrimental effects in a cell and may compromise the normal function of the respective gene. Many diseases are associated with epigenetic changes. Epigenetic aberrations are most extensively studied in cancer, but variation in the epigenome has also been described in cardiovascular diseases, autoimmune diseases, metabolic disorders, and neurodegenerative diseases (16–18). Thus, although epigenetic variability enables relevant plasticity to adapt to environmental and lifestyle conditions, epigenetic aberrations may predispose to disease, and transferring the acquired epigenetic marks from parents to their offspring could affect the offspring's development, plasticity, and disease susceptibility. The epigenetic signature is erased in gametes and reset after fertilization, likely to prevent genome‐wide epigenetic inheritance. In mammals, genome‐wide DNA demethylation occurs in 2 steps during early development. The first complete demethylation occurs in the parental gametes when the DNA methylation marks are erased in 2 steps accompanied by the restoration of developmental potency (19). An active and rapid demethylation event mediated by 10‐11 translocation (TET) proteins is followed by a passive loss of DNA methylation marks during subsequent cell divisions. It has been thought that active demethylation mainly occurs in the paternal gametes and that the maternal gametes were mainly passively demethylated (20–22), because the maternal pronucleus may be protected from TET3‐mediated conversion by developmental pluripotency associated (DPPA)‐3 (also known as stella) (23). However, in a recent study, Guo et al. (24) suggested that both active demethylation by TET3 and passive demethylation are important in both parental gametes. Other mechanisms, such as the base excision repair systems, may also operate in conjunction with the TET proteins, to drive DNA demethylation (25). Following this first set of demethylation, reestablishment of DNA methylation marks commences during the establishment of the primordial germ cells (PGCs). The second general demethylation occurs after fertilization in the inner cell mass of the developing embryo (26). De novo methylation is established in the blastocyst associated with cellular differentiation. Notably, differentially methylated regions of imprinted genes and retrotransposable elements are exempted from this second de‐ and remethylation step. In both mice and humans, imprinted regions undergo rapid DNA demethylation during early germ‐line development (27–29), but they are protected from the second round of demethylation, which enables them to maintain their parent‐of‐origin methylation state (30–32). In mouse models some other repeats, such as intracisternal A particle (IAP), endogenous retroviral sequence‐1, and single copy sequences, have also been reported to escape epigenetic reprogramming (33–35). Whether this resistance to the second de‐ and remethylation pathway manifests an evolutionary path to ease transfer of epigenetic marks from parent to offspring remains unclear, but intergenerational inheritance may provide some functional advantages. For example, maintenance of methylation may be necessary to prevent transcriptional activity of the transposable elements and may reduce the risk of germ‐line mutations through dysregulation of adjacent genes (27). The evolutionary potential of the epigenetic code links back to theories of both Lamarck and Darwin. Lamarck proposed that the environment alters phenotype in a heritable manner, which is consistent with the concept that environmental exposures at critical developmental windows can promote epigenetic inheritance of epimutations in the germ line, which may increase phenotypic variation (36). Darwin argued that natural selection favors the survival or reproductive success of those with the greatest ability to adapt (36). This theory favors intergenerational plasticity and heritable adaptive phenotypic variation. Transgenerational epigenetic inheritance requires an incomplete erasure of the epigenetic signature during developmental reprogramming permitting the transfer of epigenetic marks from parents onto their offspring over subsequent generations. Most studies investigating this phenomenon are based on animal models, leaving it unclear whether transgenerational epigenetic inheritance exists in humans. Moreover, germ‐line inherited epigenetic traits must be differentiated from intrauterine exposures, as both may shape the neonates epigenetic profile and can result in similarities between parents and offspring in their epigenetic code and phenotypes. In this review, we will define the concept of transgenerational epigenetic inheritance, discuss the available data, and draw distinctions between transgenerational epigenetic inheritance and other phenomena, such as intrauterine exposures, and their implications for the establishment of the epigenome after fertilization.

INTRAUTERINE EXPOSURES Any environmental stressor that acts during early development may affect the establishment of the epigenome and in turn the individual's phenotype in the short and long term. For this reason there has been considerable interest among the Developmental Origins of Health and Disease (DOHaD) community in exploring the epigenetic underpinnings of exposures during pregnancy that affect later‐life susceptibility to chronic disease (37, 38). The fetus is exposed in utero to some of the same environmental stressors as the mother. As a result, intrauterine experiences may induce fetal reprogramming via alterations in the epigenetic code before birth, with no inheritance from the parents. The epigenome is thought to be particularly vulnerable to environmental factors during embryogenesis, which becomes evident from numerous studies that have reported serious consequences in later life caused by intrauterine stressors. In animal studies, altered levels of the offspring's stress response, glucose metabolism, blood pressure, cholesterol metabolism, and cardiac energy metabolism were related to maternal diet, stress, and even traumatic exposures during pregnancy, and these associations were complemented by differential epigenetic patterns and differentially expressed genes (35, 39–43). Maternal behavior may also affect offspring epigenetics. The levels of maternal licking, grooming, and arched‐back nursing in mouse pups have been reported to influence their level of fearfulness. The impact of maternal care on the development of stress reactivity has been suggested to be mediated by changes in the levels of expression of specific genes in brain regions that regulate behavioral and endocrine responses to stress (44), and maternal behavior has been suggested to alter DNA methylation and chromatin structure in these pups (44, 45). However, these observations still have to be confirmed by other research groups. In humans, parental nutritional and smoking behavior during pregnancy affect the offspring's risk of cardiovascular and metabolic diseases, schizophrenia, and antisocial personality disorders (46–49); however, any epigenetic involvement remains less clear. Whereas maternal smoking has consistently been linked to demethylation of the AHRR gene, the methylation status of the human NR3C1 gene in newborns is sensitive to prenatal maternal mood, and periconceptional exposure to famine has been associated with lower DNA methylation of the IGF2 gene in adulthood (50–52), examples of epigenetic differences linking prenatal exposures and adult disease outcomes are still missing, primarily because of the lack of availability of biospecimens at birth in longitudinal studies.

TRANSGENERATIONAL EPIGENETIC INHERITANCE Intergenerational epigenetic inheritance is the transfer of epigenetic marks from the gametes to the embryo for 1 generation. It requires incomplete erasure of the parental epigenetic marks, thus avoiding epigenetic reprogramming in the gametes and during early embryo development. For transgenerational epigenetic inheritance to occur, epigenetic marks and phenotypes must be transferred across subsequent generations. In a gestating female, the phenotypic changes would have to be maintained for at least 4 generations. When the gestating mother (F0) is exposed to an environmental challenge, her embryo (F1) and the already developing germ line (F2) of the embryo, are also directly exposed (Fig. 1). Thus, a third generation's phenotype may result from the grandmother's experiences via intrauterine exposure and does not represent inheritance. In investigations of exposures operating before gestation via the maternal germ line or of inheritance via the paternal germ line, the third‐generation offspring phenotype is sufficient to establish transgenerational epigenetic inheritance (53, 54). Figure 1 Open in figure viewer Epigenetic inheritance via the female and male germ line. If a gestating mother (F0, blue) is exposed to environmental stressors, her fetus (green) and its already developing germ line (red) are also directly exposed. As a result, phenotypes observed up to the F2 generation (red) may result from the grandmother's experience. The F3 generation (yellow) is the first generation that has not been exposed to these environmental stressors, thus phenotypes observed in this generation could represent transgenerational epigenetic inheritance. If inheritance via the male or the maternal germ line before gestation is investigated, environmental stressors may affect the F0 generation (blue) and the developing germ line (red). As a result, the first generation that could represent transgenerational epigenetic inheritance is F2 (yellow). Epigenetic marks may be inherited through several pathways. We will discuss these in the sections below.

IMPRINTING The strongest evidence for transgenerational epigenetic inheritance in mammals is genomic imprinting. Imprinted genes are expressed by only one of the 2 parentally inherited alleles, whereas the other parental allele is silenced by epigenetic mechanisms in a parent‐of‐origin‐specific manner. The parental specificity of the active allele and silenced allele has been faithfully maintained throughout generations. To establish these parental imprints, the germ cells must first lose their inherited maternal and paternal imprints, and the parental imprints must then be reestablished in an allele‐specific manner during gamete formation (55, 56). Maternal DNA methylation imprints are established during oogenesis at different time points, depending on the imprinted gene loci, whereas paternal imprints are established during spermatogonial differentiation in the adult testis (57). Once established these epigenetic marks are able to resist postfertilization global epigenetic reprogramming in the preimplantation embryo through the interaction of the chromatin and DNA‐modifying factors zinc finger protein (ZFP)‐57 with tripartite motif containing (TRIM)‐28, which are attracted to methylated imprinting control regions (58), or by specific factors, such as DPP A3, that prevent DNA demethylation by binding H3K9me2 and blocking TET3 activity (23). Because of the selective nature of epigenetic reprogramming in the preimplantation embryo, inheritance of parental imprints by the new embryo is permissive. Since the imprinted regions are able to resist the second wave of reprogramming, errors that may occur during the first wave of erasure of parental imprints are maintained in the embryo. Several disorders, such as Beckwith‐Wiedemann, Prader‐Willi, and Angelman syndromes, are caused by loss of imprinting that results in biallelic expression of the respective imprinted gene. Although these imprinting defects may also be caused by spontaneous epimutations, Buiting et al. (59) reported that loss of imprinting in humans may be the result of a failure to erase the parental imprint. In their study, imprinting defects in a subset of patients with Angelman syndrome occurred on the chromosome inherited from the maternal grandparents, whereas, in a subset of patients with Prader‐Willi syndrome, the imprinting defect occurred at the chromosome inherited from the paternal grandmother. While incomplete erasure of the parental imprint may explain some of these phenomena, the observed imprinting defects may also have occurred after the erasure of parental imprints or could result from the patient's genetic background (59).

REPETITIVE RETROTRANSPOSONS Maintaining genomic stability is vital for mammalian survival, and several diseases are a consequence of the inability to maintain genomic stability. For example, cancer is associated with site‐specific hypermethylation of CpG islands at promoters and global DNA hypomethylation at repetitive and satellite regions, such as retrotransposons (60–62). One of the best‐studied long terminal repeat (LTR) retrotransposons in mice is the IAP element. IAPs are retroviruslike repetitive DNA elements that possess an LTR region that functions as a promoter. These IAP elements have been suggested to provide a potential pathway of epigenetic inheritance. They are under the control of DNA methyltransferase 1 (DNMT1), as transcript levels are elevated in mouse embryos that are DNMT1 deficient (63). CpG islands located close to an IAP showed consistently high methylation levels across all developmental stages (64), and methylation of the LTR sequences of most of the IAP element copies in the mouse genome persisted through the wave of demethylation that occurs in the preimplementation embryo. A significant fraction of these IAP genomes remained essentially unreprogrammed, even in PGCs (27, 65, 66). Because of the ability of IAPs to resist the second wave of reprogramming, IAPs are a potential way of epigenetic inheritance in mammals, and several studies have investigated this in mice. IAPs were reported to be essential in the variable methylation affecting the inheritance of coat color and tail length in Agouti viable yellow and Axinfused alleles in mice (66, 67), and methylation of the Agouti viable yellow gene in offspring can be modulated by the availability of methyl donors in the maternal diet (68). In utero exposure to methyl donors by the F1 generation in pseudoagouti mice resulted in F2 generation mice with pseudoagouti phenotypes (69). Supplemented mice were more variable in their methylation levels, with the highest variation in mice that were supplemented over several subsequent generations. Supplementation also affected the offspring's disease susceptibility: supplemented offspring were protected from developing obesity and diabetes mellitus (70). It is important to note, however, that inheritance of these traits by subsequent and unexposed generations has not been shown. Also, these mouse experiments were performed with inbred mouse strains, which may differ in their ability to reprogram epigenetic marks at IAPs after fertilization (67), and studies performed in one inbred mouse strain may not be reproducible in other strains. Thus, genetic variation may be an alternative explanation for these phenomena rather than epigenetic inheritance. Moreover, the resistance of IAPs to demethylation during development may be a protective trait that prevents IAP retrotransposition, which may induce mutations (65). Thus, the maintenance of DNA methylation patterns may be a bystander rather than the driver, and it is an exceptional situation, as it does not occur on a genome‐wide level. Even though a large portion of the human genome is composed of transposable elements, a similar mechanism has not been identified in humans. However, transposable elements in humans are also highly methylated (71) and in a recent study, Tang et al. (72) reported that some retrotransposon‐associated and single‐copy regions in the human genome resist DNA demethylation. As a result, it is possible that factors affecting the degree of methylation cause variable gene expression in humans similar to that in mice.

RNA‐DEPENDENT PROCESSES Epigenetic inheritance may also be mediated by noncoding RNAs (ncRNAs), such as microRNA (miRNA) or long noncoding RNA (lnRNA), and RNA‐dependent processes may contribute to the transmission of acquired traits in mammals. ncRNAs have key functions during early development; they control embryonic gene expression, promote developmental transitions, and maintain developmental states (73). Animal studies suggest that initial inductions resulting in a modification of the early embryonic genome to miRNAs may be sufficient to induce a heritable phenotype mediated through miRNAs. Mouse experiments have shown that stress before breeding affects the expression profiles in sperm miRNA and the responsiveness of stress by the offspring (74, 75). However, these alterations are present only in sperm of the F1 generation and not in later generations. Two other studies suggested that an initial induction involving a modification of the expression of critical miRNAs could be inherited both paternally and maternally by the next‐generation offspring and could affect the offspring's phenotype in mice (76, 77). Both studies found specific miRNA sequences in the sperm of the affected animals, which was supported by an increase in transcription in the study by Rassoulzadegan et al. (76). Wagner et al. (77) reported that this phenotype could even be transmitted for at least 3 generations. Another study by Padmanabhan et al. (78), reported that a Mtrr deficiency in grandparents and parents, leading to a defect in folate metabolism, resulted in epigenetic effects on the daughter's maternal environment and their gametes, leading to congenital abnormalities. An effect of this Mtrr mutation was found up to 5 generations in the wild‐type offspring. It remains unclear which mechanism underlies this observation, but it may be epigenetic inheritance, even though other possibilities cannot be excluded. For example, the initial mutation in the gene may lead to a perturbed regulation of the nucleotide biosynthesis pathway that may result in deficiencies in DNA repair mechanisms and potentially genetic mutations (79). Also, the epigenetic mark underlying the stability of the modified phenotypes observed in these studies and the exact role of the miRNAs has yet to be elucidated. To our knowledge, no data are currently available on the potential role of lnRNAs in transgenerational epigenetic inheritance. In human sperm, miRNA expression profiles differed between smoking and nonsmoking volunteers (80), but it remains unknown whether these miRNA profiles are maintained during early embryogenesis. In a study on human parent‐offspring triads, regulatory scores of most miRNAs correlated between parents and offspring, suggesting a possible heritability of miRNAs (81). However, in humans, consistency in miRNA profiles across subsequent generations has not been reported, nor is there any other proof of miRNA‐induced epigenetic inheritance to date.

HISTONE RETENTION Histones are small proteins that constitute major building blocks of the chromatin structure; they have a positively charged central fold domain and terminal tails. The tails are crucial for normal function of cellular processes, as they are targets for posttranslational modifications, such as acetylation, methylation, phosphorylation, and ubiquitination. A negative charge of the histone tail will result in an open chromatin structure, allowing access to the transcription machinery, whereas positively charged histone tails provide the ability to fold the chromatin, protecting it from transcription. Histones have been reported to be an important factor in early life development, as embryos deficient of certain histone methyltransferases display severe growth retardation and early lethality (82, 83). To date, little support is available for a role of histones in the process of transgenerational epigenetic inheritance. Studies in both mice and humans include only early stage embryos, and thus it remains unclear what the impact of these modifications is for the offspring in later stages of development. In mice, specific histone modifications were found to undergo reprogramming during early embryo development. Hyperacetylated histone H4, Me(Arg17)[3H], and Me(Arg3)H4 marks were removed during the metaphase in eggs and early embryos, whereas the histone marks Me(Lys9)[3H], Me(Lys4)[3H], and Ph(Ser1)H4/H2A were reported to be stable until at least the blastocyst stage of development (84). A study with human sperm suggested that epigenetic inheritance may transit through an incomplete replacement of histones by protamines during gametogenesis (85). In this study, correlations were observed between H3K4me in sperm and early expression in the embryo. A human embryo study suggested that constitutive heterochromatin marks in the embryo are transmitted and retained from human spermatozoa (86). However, it remains to be clarified whether these histone marks are maintained during later embryonic development and whether they can be propagated into subsequent generations.

DISCUSSION Transgenerational epigenetic inheritance is a topic of great interest as the potential relevance of epigenetics for DOHaD is being scrutinized. However, many questions remain. If transgenerational inheritance is present in the form of incomplete erasure, it remains uncertain whether it is an error or there is an evolutionary advantage to maintaining epigenetic marks reflecting the parents' experience. It has been observed in animal experiments and in epidemiologic studies in humans that the impact of nutrition, smoking, irradiation, and even traumatic exposures in the parents and grandparents may affect the (grand)children's phenotype or risk of diseases, and similarities in the epigenetic profile between parent and offspring have been found (39–43, 46–52, 87–91). These observations may be caused by intrauterine exposures, germ‐line‐mediated transmission of phenotypic traits through genetic mutations, mutations in the DNA repair mechanisms, or genetic mutations in epigenetic modifiers, rather than to the continuity of epigenetic marks between generations. Moreover, a shared environment may explain parental and sometimes even grandparental, similarities in the epigenetic profile, rather than transgenerational epigenetic inheritance. A condition for transgenerational epigenetic inheritance is that the epigenetic mark and the associated phenotype are maintained for at least 4 generations in a gestating female and for at least 3 generations if pregestational exposures or inheritance via the male germ line are investigated (53, 54). Few studies have considered the maintenance of epigenetic marks for these durations. Wagner et al. (77) observed in a mouse experiment that miRNAs were maintained over 3 generations of offspring, and a study by Padmanabhan et al. (78) in mice suggested that an Mtrr mutation may impact epigenetic inheritance for up to 5 generations. Nonetheless, these studies were performed in animals that were genetically inbred, and it remains unclear whether these results are reproducible in other mouse strains. The most convincing examples of potential pathways of transgenerational epigenetic inheritance come from mouse studies suggesting resistance to epigenetic reprogramming by repetitive retrotransposons or inheritance of miRNA profiles. Differences in the epigenome between humans and mice do not permit direct inference to humans (92, 93), and to date there is no evidence of transgenerational epigenetic inheritance in humans, except for the parent‐of‐origin specificity of genomic imprinting. Some histones have been reported to be stably inherited from the parents to the offspring during the first phases of embryonic development; correlations in the regulatory effect scores of miRNAs were observed between parents and offspring; and in imprinting disorders, some of the imprinting defect in humans may be the result of a failure to erase the parental imprint. However, in all these examples, it remains unclear whether those marks are preserved during later stages of development and in subsequent generations and other possible explanations for the observed effects cannot be excluded, such as the influence of genetic background. The genetic contribution to DNA methylation was investigated by Gertz et al. (94) in 3 human generations. They reported that most variation in DNA methylation in the genome can be explained by genotype and that the genetic influence exceeds the influence of imprinting on genome‐wide methylation levels. Their data are further supported by twin studies, where evidence was found for within‐pair epigenetic variability, including both methylation and gene expression analyses, in multiple tissues at birth (95–97). Methylation and gene expression differences within monozygotic (MZ) twin pairs were observed to be smaller than those observed for dizygotic (DZ) twins (95, 96), suggesting a genetic contribution to methylation and gene expression profiles. MZ dichorionic twins, each with their own placenta, displayed greater within‐pair expression discordance than did MZ monochorionic twins who shared a placenta (97), likely influenced by the intrauterine environment. These observations are in line with the concept that perceived inheritance of DNA methylation is driven by genetics and intrauterine exposure rather than transgenerational epigenetic inheritance.

FUTURE PERSPECTIVE The study of transgenerational epigenetic inheritance in humans is more complex than in animals for several reasons. First, because transgenerational epigenetic inheritance has to be maintained for several generations, longitudinal multigenerational studies are required with biospecimens available for 3 to 4 generations. To date, no such studies have been conducted. Second, studies in humans are characterized by individual variation, stochastic differences, and ethical restrictions and thus differ profoundly from the controlled environment of animal studies. Last, the heterozygosity of the human population makes it difficult to distinguish between genetics and epigenetics. Only when individuals are truly genetically identical and exhibit a range of phenotypes that are heritable may these be attributed to epigenetic variation. These factors make it hard to study transgenerational epigenetic inheritance and its potential players in humans. To fully understand transgenerational epigenetic inheritance, the influence of intrauterine exposures and a shared postnatal environment on the epigenetic signature has to be studied in greater detail to enable a clear distinction between these exposures and transgenerational epigenetic inheritance. Future studies may focus on understanding how epigenetic marks during early life development are established, in addition to identify epigenetic marks that are maintained over subsequent generations. Shedding light on these processes may help to understand whether, and how, parental epigenetic marks influence offspring's health and disease susceptibility through transgenerational epigenetic inheritance and intrauterine exposures.

CONCLUSIONS Several processes that may be part of intergenerational epigenetic inheritance have been identified in mice. However, much uncertainty remains as to whether these processes are truly epigenetically inherited or are influenced by other factors, such as intrauterine exposures and genetics, and whether these epigenetic marks can be maintained over several subsequent generations. As a result, proof of principle of a widespread transgenerational epigenetic inheritance is lacking to date. Because of differences in the epigenome between mice and humans and the limited number of studies performed in humans, the concept of transgenerational epigenetic inheritance in humans remains equivocal.