An organism’s behavioural and physiological and social milieu influence and are influenced by the epigenome, which is comprised predominantly of chromatin and the covalent modification of DNA by methylation. Epigenetic patterns are sculpted during development to shape the diversity of gene expression programmes in the organism. In contrast with the genetic sequence, which is determined by inheritance and is virtually identical in all tissues, the epigenetic pattern varies from cell type to cell type and is potentially dynamic throughout life. It is postulated that different environmental exposures could effect epigenetic patterns relevant for human behaviour. Because epigenetic programming defines the state of expression of genes, epigenetic differences could have the same consequences as genetic polymorphisms. Yet in contrast with genetic sequence differences, epigenetic alterations are potentially reversible. In the present chapter, we will discuss evidence that epigenetic processes early in life play a role in defining inter-individual trajectories of behaviour, with implications for mental health in adulthood.
Different cell types execute distinctive programmes of gene expression that are highly responsive to developmental, physiological, pathological and environmental cues. The combination of mechanisms that confer long-term programming on genes and can bring about a change in gene function without changing gene sequence are herein termed epigenetic changes. We propose a definition of epigenetics that includes any long-term change in gene function that persists even when the initial trigger is long gone that does not involve a change in gene sequence or structure. Thus a change in chromatin or DNA methylation in a post-mitotic neuron that lasts for a long period of time would be considered an epigenetic change even in the absence of cell division. This definition stands in contrast with some classical definitions of epigenetics that require heritability in dividing somatic cells or even through germ-line transmission across generations. However, the less strict definition of epigenetics proposed here is especially important in understanding long-term changes in gene function in the brain. As we will discuss, stable changes in chromatin, or DNA modification in post-mitotic neurons or dividing cells, could be environmentally driven, may occur in response to triggers at different points in life and are potentially reversible. In contrast, genetic differences are germ-line transmitted, virtually fixed and irreversible.
We have argued that much of the phenotypic variation seen in human populations might be caused by differences in long-term programming of gene function rather than the sequence per se, and any future study of the basis for inter-individual phenotypic diversity should consider epigenetic variations in addition to genetic sequence polymorphisms . In effect, epigenetic silencing and genetic silencing could have similar phenotypic consequences. Therefore mapping the epigenome is potentially as important as the mapping of the genome in our quest to understand phenotypic differences in humans. By extension, identifying epigenetic differences that are associated with behavioural pathologies have important implications for human health because they are potentially reversible .
The dynamic nature of epigenetic regulation, in contrast with the virtually static nature of the gene sequence, provides a mechanism for reprogramming gene function in response to changes in lifestyle trajectories. In this way, epigenetics may offer a mechanistic understanding of well-defined environmental effects on phenotypes. Parental care provides offspring with crucial information about the environment into which they are born . In the present chapter, we argue that differences in the quality of parental care have important consequences on offspring development via the programming of gene expression and lead to differences in health outcomes later in life. In what follows, we discuss evidence that stable epigenetic changes in gene expression programming occur as a result of differences in the quality of parental care early in life, and suggest that these effects may be nonetheless reversible. We will focus on two of the best-understood epigenetic mechanisms: chromatin remodelling and DNA methylation.
The epigenetic regulation of gene expression
Chromatin remodelling and targeting
The basic building block of chromatin is the nucleosome, which is made up of an octamer of histone proteins. The N-terminal tails of these histones are extensively modified by methylation, phosphorylation, acetylation and ubiquitination. The state of modification of these tails plays an important role in defining the accessibility of the DNA wrapped around the nucleosome core. It was proposed that the N-terminal tails of H3 and H4 histones that are positively charged form tight interactions with the negatively charged DNA backbone, thus blocking the interaction of transcription factors with the DNA. Modification of the tails neutralize their charge, thus relaxing the tight grip of the histone tails. Different histone variants, which replace the standard isoforms, also play a regulatory role and serve to mark active genes in some instances . The specific pattern of histone modifications was proposed to form a ‘histone code’, that delineates the parts of the genome to be expressed at a given point in time in a given cell type . The most investigated histone-modifying enzymes are HATs (histone acetyltransferases), which acetylate histone H3 and H4 at different residues, as well as other HDACs (histone deacetylases), which deacetylate histone tails. Histone acetylation is believed to be a predominant signal for an active chromatin configuration. Deacetylated histones are characteristic of chromatin associated with inactive genes. Histone tail acetylation is believed to enhance the accessibility of a gene to the transcription machinery, whereas deacetylated tails are highly charged and believed to be tightly associated with the DNA backbone, thus limiting accessibility of genes to transcription factors . A basic principle in epigenetic regulation is targeting. Histone-modifying enzymes are generally not gene-specific. Specific transcription factors and transcription repressors recruit histone-modifying enzymes to specific genes and thus define the gene-specific profile of histone modification . Transcription factors and repressors recognize specific cis-acing sequences in genes, bind to these sequences and attract the specific chromatin-modifying enzymes to these genes through protein–protein interactions. Signal transduction pathways, which are activated by cell-surface receptors, could serve as conduits for epigenetic change linking environmental triggers at cell-surface receptors with gene-specific chromatin alterations, leading to the reprogramming of gene activity.
DNA methylation and consequences for transcription
The DNA molecule itself can be chemically modified by methyl residues at the 5′ position of cytosine rings in the dinucleotide sequence CG in vertebrates (Figure 1). What distinguishes DNA methylation in vertebrate genomes is the fact that not all CGs are methylated in any given cell type, resulting in cell-type-specific patterns of methylation. Thus the DNA methylation pattern confers upon the genome its cell type identity. Since DNA methylation is part of the chemical structure of the DNA itself, it is more stable than other epigenetic marks and thus it has extremely important diagnostic potential in humans . A growing line of evidence supports the idea that, similar to chromatin modification, DNA methylation is also potentially reversible, even in predominantly non-dividing tissues such as the brain . The DNA methylation pattern is not copied by the DNA replication machinery, but by an independent enzymatic machinery termed DNMTs (DNA methyltransferases) (Figure 2).
DNA methylation in critical regulatory regions, including gene promoters and enhancers, serves as a signal to silence gene expression by two main mechanisms (Figure 3). The first mechanism involves direct interference of the methyl residue with the binding of a transcription factor to its recognition element in the gene. The interaction of transcription factors with genes is required for activation of the gene; lack of binding of a transcription factor would result in silencing of gene expression. The second mechanism is indirect. A certain density of DNA methylation in the region of the gene attracts the binding of methylated-DNA-binding proteins that leads to the formation of a ‘closed’ chromatin configuration and the silencing of gene expression. Thus aberrant methylation will silence a gene, resulting in loss-of-function, which will have a similar consequence to a loss-of-function by genetic mechanisms such as mutation, deletion or rearrangement. Although much of our current knowledge of the role of DNA methylation in gene regulation derives from studies of effects in promoter regions, it is worth noting that DNA methylation in other gene elements – including within exons, introns, and at 3′ ends of genes – plays important roles in regulating gene function . In addition, recent genome-wide analysis has revealved that most tissue-specific DNA methylation – that is, the DNA methylation pattern differentiating various tissue types such as the three embryonic cell-type lineages of spleen, liver and brain – occur in regions of moderate CpG density near, but not inside, CpG-rich ‘islands’. The possibility that these so-called ‘CpG shores’ are targets for epigenetic changes associated with environmental perturbations is an area of active interest [8a,8b].
The most controversial issue in the DNA methylation field is the question of whether the DNA methylation reaction is reversible. Several enzymatic activities were proposed to cause DNA demethylation. A G/T mismatch repair glycosylase functions as a 5-methylcytosine DNA glycosylase, recognizes methyl cytosines and cleaves the bond between the sugar and the base. The abasic site is then repaired and replaced with a non-methylated cytosine resulting in demethylation . An additional protein with similar activity was recently identified, the MBD4 (methylated DNA-binding protein 4) . MBD2b (a shorter isoform of MBD2) was shown to directly remove the methyl group from methylated cytosine in methylated CpGs , but this was contested by several groups . GADD45A, a damage response protein, was proposed to trigger active DNA demethylation through a repair-mediated process . However, this was also contested by a later study . More recently, it was proposed that DNMT3A acts as a demethylase, possibly through a mechanism which involves deamination .
We have proposed that the DNA methylation pattern is a balance of methylation and demethylation reactions that are responsive to physiological and environmental signals and thus forms a platform for gene–environment interactions  (Figure 1). There are now examples of active, replication-independent DNA demethylation during development, as well as in somatic tissues [17,18]. One example from our laboratory is that of the epigenetic regulation of the glucocorticoid receptor gene promoter in the brains of adult rats as a function of maternal care and pharmacological manipulations of the epigenetic machinery. As we will discuss in the following sections, these findings have implications for humans, because drugs and dietary constituents known to influence the epigenetic machinery are currently in widespread use.
The role of parental care in the epigenetic programming of gene expression
Maternal programming of the stress response in rodents
In the rat, the adult offspring of mothers that exhibit increased levels of pup LG (licking/grooming) (i.e. high LG mothers) over the first week of life show increased hippocampal GR (glucocorticoid receptor) expression, enhanced glucocorticoid feedback sensitivity, decreased hypothalamic corticotrophin releasing factor expression, and more modest HPA (hypothalamic–adrenal–pituitary) stress responses compared with animals reared by low LG mothers . Such differences occur naturally in populations of rodents living in research settings, and cross-fostering studies suggest direct effects of maternal care on both gene expression and stress responses . These studies showing sustained effects of early care that persist until adulthood, implicate the involvement of an epigenetic mechanism, since the fostering mother and not the biological genetic mother define the stress response of its adult offspring. We have demonstrated that, for example, the GR exon 17 promoter is programmed differently in the hippocampus of offspring of the high and low LG mothers and that differences which emerge between day 1 and 8 after birth remain stable thereafter. These differences include histone acetylation, DNA methylation and the occupancy of the promoter with the transcription factor NGFI-A (nerve growth factor-inducible protein A) (Figure 4) . Another intriguing recent study has provided evidence that other genes in the neural pathway mediating the stress response may be epigenetically regulated by early life stress, including arginine vasopressin expression in the hypothalamus .
Epigenetic contributions of parental care to mental health in humans
There has been a flurry of reports in the scientific literature pointing to a role for epigenetic changes in mental health in humans, from mental retardation, to schizophrenia to bipolar disorder and depression [1,21]. One example is that of the gene encoding REELIN, a pivotal protein in neuronal development and synaptogenesis and implicated in long-term memory. REELIN was found to be hypermethylated in the post-mortem brains of schizophrenia patients, and the methylation of the REELIN gene promoter was correlated with its reduced expression and increased DNMT1 expression in a subset of neurons in the prefrontal cortex [22,23]. These results suggest that gene-specific DNA methylation changes are associated with an increased risk of psychopathology.
At issue is whether the observed effects in these studies resulted from the genetic background of the individual, their environment, and/or pathological processes related to their mental disorder. We undertook a series of investigations of post-mortem brain tissue from individuals with well-characterized life histories to investigate the effects of early adversity in humans. Using established forensic psychiatric analyses, we focused on individuals with a history of severe physical or sexual abuse or neglect during childhood, which is common among suicide victims. Data in the literature had suggested that suicide might have a developmental origin, and it was known that adversity in early life is an important risk factor for suicide.
In a first published report of aberrant methylation associated with suicide, we showed that promoters of the genes encoding rRNA (ribosomal RNA) are heavily methylated in hippocampi from subjects who committed suicide relative to controls . Methylation of rRNA defines the fraction of rRNA molecules that are active in a cell, and the output of rRNA transcription defines, to a large extent, the protein synthesis capacity of a cell. We found that the genetic sequence of rRNA was identical in all subjects, and there was no difference in methylation between suicide victims and controls in the cerebellum, a brain region that is not normally associated with psychopathology. These data imply that epigenetic effects associated with psychopathology probably target particular neural pathways. Because all of the suicide victims and none of the controls had a history of severe abuse or neglect in childhood, the data suggest that severe adversity during early childhood may have been a contributing factor to the observed epigenetic differences. However, it was unclear in this study whether the observed abnormalities were a result of early adversity or whether they had emerged during adulthood as a result of the mental disorders associated with suicide. Therefore we undertook another study to address this question.
We next examined the GR gene promoter in the hippocampus of human suicide victims and controls . Again, all of the suicide victims and none of the controls had a history of childhood abuse or severe neglect. A third group was comprised of suicide victims with a history that was negative for childhood abuse or neglect. We found evidence that, as in the animal model described above, the GR was epigenetically regulated in the human brain, and associated with altered GR gene expression. Hypermethylation of the GR gene was found among suicide victims with a history of abuse in childhood, but not among controls or suicide victims with a negative history of childhood abuse. The data suggest that epigenetic processes might mediate the effects of the social environment during childhood on hippocampal gene expression, and that stable epigenetic marks, such as DNA methylation, might then persist into adulthood and influence vulnerability for psychopathology through effects on intermediate levels of function such as activity of the HPA axis that regulates the stress response. However, we should remain cautious about this interpretation, because it is still unclear whether the epigenetic aberrations were present in the germ-line, whether they were introduced during embryogenesis or whether they were truly changes occurring during early childhood. Future studies, particularly of monozygotic twins who share virtually the same genotype will provide a way to examine epigenetic discordance in humans as a function of differences in their environment [26,27].
The potential reversibility of the epigenetic programming of gene expression
Epigenetic programming by maternal care in rats is reversible in adulthood
The idea that epigenetic programming could be reversible in adulthood depends upon the assumption that the enzymatic machineries required to generate a new methylation pattern are present in adult tissue. We injected the HDACi (HDAC inhibitor) TSA (trichostatin A) into the brain of adult rats to test the hypothesis that the machineries required to modify chromatin and DNA methylation were found in neurons and associated with the GR exon 17 promoter. TSA injected into brains of adult offspring of low LG maternal care mothers increased acetylation, reduced methylation and activated GR exon 17 promoter at levels indistinguishable from those of adult offspring of high LG maternal care mothers and reduced stress responsivity to the levels of high LG offspring (Figure 4) . We similarly reasoned that if the DNA methylation and chromatin state is in a dynamic equilibrium even in adult neurons, it should be possible to revert the epigenetic programming in the other direction towards increased methylation, leading to a reversal of the maternal programming of GR expression and HPA responses to stress. We therefore injected methionine, a methyl donor and the precursor of SAM (S-adenosyl-methionine), into the brain of the adult offspring of different maternal care mothers. Methionine treatment of the offspring of high LG mothers changed the DNA methylation state of the GR exon 17 promoter and expression of GR in the hippocampus, as well as increasing their stress responsiveness and reducing the time that these animals spent in the centre of an open field, a measure of anxiety .
Because methionine alone does not methylate DNA but is converted into the methyl donor SAM in the DNA methylation reaction, the DNMTs must be poised to methylate GR exon 17 promoter (Figure 4). Taken together, the TSA and methionine experiments support the basic hypothesis that epigenetic programmes in the brain are maintained by a dynamic equilibrium of methylating and demethylating enzymes, a balance that could be shifted by agents which either inhibit demethylation reactions or stimulate DNMTs. Thus despite the remarkable stability of epigenetic programmes (Figure 2) they are nevertheless reversible.
Are changes in epigenetic signalling reversible in humans?
Very little is known about the ability of the behavioural, pharmacological or social environment to reverse epigenetic changes influencing mental health in humans. What little we do know comes primarily from studies of the effects of drugs that tap in to the epigenetic machinery and are used in the treatment of mental disorders. For example, valproic acid, a long-established anti-epileptic and mood stabilizer, is also an HDACi , suggesting a possible role for HDACi in treating mental disorders such as schizophrenia and bipolar disorder. Valproic acid has been shown to have some effect in alleviating psychotic agitation as an adjunct to antipsychotics in schizophrenia . Another example is that of the effects of SAM in mood disorders . As mentioned above, central infusion of l-methionine, a precursor of SAM, increases DNA methylation of the promoter of the GR gene in rodents. The fact that SAM, which similarly enhances DNA methylation, is effective in the treatment of depression is apparently contradictory to this effect of methionine. However, SAM is a methyl residue donor not only for the DNA methylation reaction, but also for other enzymatic reactions. For example, creatine is produced from SAM and guanidinoacetate, and SAM treatment increases phosphocreatine levels in the brain. This effect may also contribute to the antidepressive effect of SAM because decreased phosphocreatine levels have been reported in bipolar depression . It is becoming clear that we need to consider these issues in the future when assessing the safety of drugs, nutraceuticals and dietary habits, as DNA methylation in the brain has both pharmacological and toxicological implications.
Summary and future prospects
We, along with others, have hypothesized that the social environment early in life has a long-lasting impact on mental and physical health trajectories via epigenetic marking of specific genes [1,33,34]. However, one important aspect of the basic epigenetic mechanisms reviewed in the present chapter is that although the epigenetic markings are long-lasting they are nevertheless potentially reversible. Studies on the reversal of maternal effects on DNA methylation in rats using either TSA or methionine suggest that neurons express the machinery necessary for methylation and demethylation in adulthood (Figure 4). DNA methylation can be altered through sustained alterations of chromatin structure such as histone acetylation. Genome-wide scales of analysis using microarray and high-throughput sequencing have begun to identify the epigenomic signatures of basic processes of cellular phenotype and alterations that accompany pathological conditions that include mental disorders and cancer. Such analyses will provide a more comprehensive view of how cellular signalling may be modulated by epigenetic processes. In turn, the findings will allow us to pose the fascinating question: what is the degree to which such processes remain sensitive to environmental regulation throughout life? In the present chapter, we have focussed on the epigenetic consequences of social adversity early in life and its association with clear deleterious behavioural outcomes, such as suicide in humans. However, although much work remains to be done, there is also some indication that positive early social experience can have a mitigating effect on stress responses later in life via epigenetic mechanisms, suggesting a protective role for positive early parental care . Other data suggest that dietary manipulations that affect the availability of the methyl donors during development had a protective effect against endocrine disruption . Taken together, these data suggest that social and dietary interventions might activate signalling pathways in the brain that would result in a change in either the targeting or activity of the epigenetic machinery and thus a change in epigenetic markings. In this way, epigenetics could serve as a bridge between the biological sciences and the social sciences, allowing a truly integrated understanding of human health and behaviour.
• We propose that the DNA methylation and chromatin structure are found in a dynamic balance throughout life, which is maintained and defined by sequence-specific factors that deliver histone modification and DNA-modification enzymes to genes.
• We propose that the direction of the DNA methylation reaction is defined by the state of chromatin and, as such, factors that target specific chromatin-modification events to genes define the direction of the DNA-methylation equilibrium by either recruiting DNA methylation enzymes or by facilitating demethylation.
• Epigenetic programming in the brain of rodents by maternal care during the first week of life is a highly stable yet reversible process that results in long-term changes in gene expression.
• In our studies, we found that aberrant DNA methylation of the rRNA promoter as well as the GR promoter lead to decreased transcription of each gene, and that this effect was associated with a history of early childhood abuse or neglect in humans.
• Many of the phenotypic variations seen in human populations might be caused by differences in the long-term programming of gene function rather than the sequence per se, and any future study of the basis for inter-individual phenotypic diversity should consider epigenetic variations in addition to genetic sequence polymorphisms.
• The fact that changes in chromatin and DNA methylation are potentially reversible processes provides a wide platform for research into pharmacological and therapeutic manipulations with known epigenetic effects from drugs used to treat mental illness, such as valproate, to dietary supplements such as SAM.
Work from the M.S. laboratory is supported by the Canadian Institute of Health Research and the Sackler Foundation.
- © The Authors Journal compilation © 2010 Biochemical Society