Development and Psychopathology 25 (2013), 1279–1291 # Cambridge University Press 2013 doi:10.1017/S0954579413000618

Epigenetic mechanisms in the development of behavior: Advances, challenges, and future promises of a new field

TANIA L. ROTH University of Delaware

Abstract In the past decade, there have been exciting advances in the field of behavioral epigenetics that have provided new insights into a biological basis of neural and behavioral effects of gene–environment interactions. It is now understood that changes in the activity of genes established through epigenetic alterations occur as a consequence of exposure to environmental adversity, social stress, and traumatic experiences. DNA methylation in particular has thus emerged as a leading candidate biological pathway linking gene–environment interactions to long-term and even multigenerational trajectories in behavioral development, including the vulnerability and resilience to psychopathology. This paper discusses what we have learned from research using animal models and from studies in which the translation of these findings has been made to humans. Studies concerning the significance of DNA methylation alterations in outcomes associated with stress exposure later in life and dysfunction in the form of neuropsychiatric disorders are highlighted, and several avenues of future research are suggested that promise to advance our understanding of epigenetics both as a mechanism by which the environment can contribute to the development of psychiatric disorders and as an avenue for more effective intervention and treatment strategies.

Psychological and social–contextual factors are recognized for onstrated that DNA methylation alterations are biological re- their ability to produce profound effects on brain development sponses to psychological and social–contextual factors. This and plasticity and, in turn, for their influence on the develop- has afforded a new framework with which to understand ment of behavior. Mechanisms underlying these envi- how psychological and social–contextual factors can interact ronmentally driven phenomena are not fully understood, but with our biology and, in turn, a route through which behavior over the past decade the birth of epigenetics research has could arise. The dynamic nature of DNA methylation renders it caused a paradigm shift to occur with respect to our under- an ideal substrate that is responsive to environmental factors, standing of gene–environment interactions in this capacity. It whereas its stable nature renders it capable of maintaining sus- has become clear that epigenetic processes such as DNA tained changes in brain function and behavior that are a conse- methylation (Figure 1) actively regulate human genomes in re- quence of environmentally driven changes in gene regulation sponse to environmental input and are poised to do so not only and activity. The examination of risk and protective processes during early-life development but also as we develop through- and mechanisms in typical and atypical development is also out the life span. One alluring aspect of epigenetic regulation central to a developmental psychopathology perspective (Cic- of gene expression that has emerged is its capability to drive chetti, 1993, 2006; Sroufe & Rutter, 1984). To this end, evi- long-term and even multigenerational trajectories in behavioral dence is emerging that DNA methylation alterations could development. The birth of epigenetics research has thus pro- serve as mechanisms through which risk and protective factors vided an exciting new level of analysis for understanding tenets may operate to yield a phenotype. DNA methylation is also in- central to the discipline of developmental psychopathology. creasingly being recognized as a biological process that can in- Central to a developmental psychopathology perspective is fluence our psychological functioning from infancy to senes- the examination of the biological, psychological, and social– cence. Finally, because a focus on the boundary between contextual factors that facilitate adaptation and maladaptation normal and abnormal development is central to a develop- across the life span (Cicchetti, 1993; Cicchetti, 2006;Sroufe mental psychopathology perspective (Cicchetti, 1993, 2006; &Rutter,1984). As will be reviewed here, studies have dem- Sroufe & Rutter, 1984), DNA methylation is highlighted as a valuable measure informative of norms and aberrations pres- ent at the molecular level. Work on this article was supported by a grant from the University of Delaware Research Foundation and The National Institute of General Medical Sciences (1P20GM103653). The author thanks Dante Cicchetti, Jennifer Blaze, and Changes in DNA Methylation as a Function Kathryn Taylor for their comments in preparing this review. of Caregiving Environments Address correspondence and reprint requests to: Tania L. Roth, Depart- ment of Psychology, University of Delaware, 108 Wolf Hall, Newark, DE Although it has become clear over the past decade that epige- 19716; E-mail: [email protected] netic processes operate throughout the life span, the evidence 1279 1280 T. L. Roth

Evidence from rodent studies

In 2004, investigators brought forth evidence that challenged the previously held view that epigenetic mechanisms are static and unresponsive to the environment outside of embryonic de- velopment and periods of cellular differentiation. Data they presented indicated that methylation of DNA associated with the glucocorticoid receptor (GR) gene, a gene underlying stress responsivity through its regulation of hypothalamic–pi- tuitary–adrenal (HPA) activity, was directly associated with the type of caregiving experienced during the first postnatal week (Weaver et al., 2004). Specifically, they showed that adult male rats that had been reared by nurturing mothers that exhibited high levels of pup licking and grooming (LG) had low levels of methylation of DNA associated with the GR gene within their hippocampus, whereas adults who had been raised by low-LG mothers exhibited hypermethylation Figure 1. (Color online) DNA compaction and methylation. (a) DNA in the of GR DNA. These observations were consistent with GR nucleus is wrapped around histone proteins, forming what is referred to as gene expression patterns and anxiety-related behavior of the chromatin. This provides a highly ordered packaging state of DNA to serve animals. Animals with low methylation had higher expression as a building block of chromosomes. (b) DNA methylation occurs at cytosine residues of cytosine–guanine (CG) dinucleotides that are often clustered of the GR gene and exhibited stress resilience, whereas animals within gene regulatory regions. DNA methylation is an epigenetic mecha- with higher methylation had lower gene expression and in- nism typically associated with gene suppression, as methyl groups can inter- creased anxiety-like behavior. Through a series of cross-foster- fere with the binding of transcription factors necessary to promote gene tran- ing studies, they were able to demonstrate that the levels of GR scription and/or recruit repressor proteins that promote a compact chromatin promoter methylation were determined by the mother’s behav- state not permissive to gene transcription. However, there are reports of DNA methylation being associated with active gene transcription (Chahrour et al., ior during the postnatal period and were not a product of the 2008; Uchida et al., 2011). biological mother’s behavioral predilection. These data were key in providing an association between the levels of caregiv- ing behavior and DNA methylation of the GR gene promoter. Finally, in an effort to help establish a causal link between the available at present indicates that environmentally driven observed epigenetic modifications, gene expression patterns, DNA methylation changes are especially robust and long and adult behavior, they demonstrated that pharmacologically lived when they occur during sensitive periods (i.e., prenatal manipulating methylation patterns removed group differences or early postnatal development). Cross-disciplinary studies in DNA methylation, histone acetylation (another epigenetic have long indicated that sensitive periods during develop- mark), gene expression, and behavior. ment are heightened epochs of brain plasticity where environ- Since this landmark study, laboratories have continued to mental factors are able to shape (i.e., program) neural circuits link caregiver experiences with DNA methylation patterns. to direct structural and functional aspects of brain and behav- We have also learned that the effects of the caregiving envi- ior for the life span. Although it has been appreciated for ronment on DNA methylation are not exclusive to the GR some time that the long-term consequences of early-life ex- gene, as other genes within the hippocampus (and other brain periences represent so-called epigenetic influences, it has regions, as discussed below) show similar sensitivity to the only been within the past decade that empirical evidence quality of the caregiving environment. For example, maternal that directly supports this notion has been presented. Such LG behavior affects g-aminobutyric acid (GABA) inhibitory paradigm-shifting studies have forced us to recognize the sen- circuits, as males reared by low-LG mothers show reduced sitivity of DNA methylation at specific gene loci within the hippocampal levels of the rate-limiting enzyme in GABA syn- brain to the quality of early-life experiences. Furthermore, thesis (glutamic acid decarboxylase [GAD1]), an effect these studies have illustrated the ability of parental care (in- shown to be associated with increased methylation of GAD1 cluding the lack thereof) to canalize DNA methylation pat- promoter DNA (Zhang et al., 2010). Other studies have shown terns with multigenerational consequences for the genome that infant male rats experiencing repeated separation from and behavior. The following discussion examines develop- their mother and nest environment show altered methylation mental studies with rodent models that have made it clear and expression of estrogen receptor (ERb) DNA (a gene that DNA methylation is a molecular process sensitive to that encodes a receptor responsive to estrogen; Wang, Meyer, early-life environmental input. These studies have also illus- & Korz, 2012) and increased methylation (and therefore re- trated that experience-induced changes in DNA methylation duced expression) of the synaptic plasticity gene reelin within occur in brain circuitry with functional relevance to behav- the hippocampus (Qin et al., 2011). Furthermore, it has been ioral trajectories and psychopathology. demonstrated that epigenetic changes can occur on a much Epigenetics and behavioral development 1281 broader genomewide scale within the hippocampus in re- tory cytokine interleukin 10 gene mRNA within microglia of sponse to maternal LG behaviors (McGowan et al., 2011). the nucleus accumbens, an effect established during the first Experience-induced changes in DNA methylation are a postnatal week and maintained via decreased methylation of mechanism by which early-life caregiving experiences can DNA associated with the interleukin 10 gene (Schwarz, also produce long-lasting alterations in function of the HPA Hutchinson, & Bilbo, 2011). Increased maternal care in this axis, particularly at the level of the hypothalamus. Increased study also produced animals that were more resilient to rein- LG behavior of male infant rats, which improves learning and statement of morphine-conditioned place preference. memory capacity in adulthood, has been shown to reduce ex- pression of and methylation of the corticotropin releasing Evidence from human studies hormone (Crh) gene in the paraventricular nucleus (PVN) of the hypothalamus (Korosi et al., 2010; McClelland, Kor- In human epigenetic studies (which have primarily focused on osi, Cope, Ivy, & Baram, 2011). The impact of infant (male adults), it is difficult to distinguish methylation patterns that and female) separation on depressive-like behaviors and may have developed as a response to the caregiving environ- Crh and GR gene expression in the hypothalamus have ment from those that occurred as a consequence of additional been linked to DNA methylation profiles (Chen, Evans, experiences throughout life. Functional polymorphisms are et al., 2012; Franklin et al., 2010). Furthermore, male mice predicted to add an extra layer of complexity to understanding show hypomethylation of arginine vasopressin (AVP) DNA how behavioral outcome is moderated by early-life experi- and increased AVP gene expression in the PVN, effects that ences. Furthermore, there is the question of the validity of pe- coincide with increased corticosterone secretion, both at basal ripheral measures of DNA methylation (obtained from blood conditions and in response to stress, as well as an attenuated samples or cheek swabs) in accurately reflecting the complex- memory capacity a year after experiencing repeated separa- ity of DNA methylation patterns within the brain (i.e., gene- tions from their mother (Murgatroyd et al., 2009). specific and region-specific responses). Nevertheless, studies Maternal care also promotes epigenetic changes of addi- have reported associations between DNA methylation changes tional genes and epicenters of stress regulation, cognitive con- and early-life caregiving experiences. trol, addiction, and maternal behavior. For example, some of Akin to the rodent literature, the human GR gene (Nr3c1)ap- our work has shown that infant rats repeatedly exposed to pears equally susceptible to experience-induced and long-last- an adverse caregiving environment exhibit significant ing changes in DNA methylation. For example, increased methylation of brain-derived neurotrophic factor (Bdnf) DNA methylation of Nr3c1 and ribosomal RNA genes (and a DNA in their prefrontal cortex that either persists throughout corresponding decrease in transcript levels) were found in hip- (DNA associated with exon IX) or evolves (exon IV) during pocampal samples derived from adult males with a history of development (Roth, Lubin, Funk, & Sweatt, 2009). Aberrant childhood abuse (Labonte´,Yerko,etal.,2012; McGowan caregiving behaviors were elicited by the combination of envi- et al., 2008, 2009). In another study linking the effects of ronmental novelty and resource deprivation (lack of nesting early-life adversity with epigenetic modification of the human material), factors in our hands and those of others capable GR gene, parental loss, childhood maltreatment, and disruptions of producing abnormal caregiving behaviors that include a in parental care were found to be associated with increased high proportion of rough handling, pup stepping on and drag- Nr3c1 promoter DNA methylation (as determined from leuko- ging, active avoidance (neglect), and decreased LG of pups cyte DNA) and attenuated cortisol responses to a stress chal- (Ivy, Brunson, Sandman, & Baram, 2008; Raineki, Corte´s, lenge (Tyrka, Price, Marsit, Walters, & Carpenter, 2012). Epi- Belnoue, & Sullivan, 2012; Roth et al., 2009; Roth & Sulli- genetic changes in response to early-life experiences also van, 2005). Adult DNA methylation profiles in our study appear to occur on a much broader, genomewide scale within were also found to coincide with reduced Bdnf gene expres- the hippocampus. Male individuals with histories of severe sion and aberrant maternal behavior. Helping to establish a abuse during childhood have been shown to exhibit genome- link between the methylation and gene changes, we also dem- wide changes in hippocampal DNA methylation, including onstrated that the gene deficits could be reversed through phar- hypermethylation of over 200 gene promoters and hypometh- macological manipulation of the methylation patterns. ylation of around 100 gene promoters, many of which are in- In the hippocampus and nucleus accumbens of adolescent volved in neural plasticity (Labonte´, Suderman, et al., 2012). rats (age 35 days), methylation of mu-opioid receptor Epigenetic regulation of other gene loci has been linked to (Oprm1) DNA has been linked to the quantity of pup LG be- childhood experiences. For example, antisocial behavior in havior (Hao, Huang, Nielsen, & Kosten, 2011). In addition, females with a history of childhood sexual abuse coincides low-LG mothers are known to yield female offspring that ex- with both genotype and hypermethylation of DNA associated hibit decreased ER-a expression and a corresponding increase with the promoter region of the gene encoding the serotonin in methylation associated with the ER-a promoter in the me- transporter (Beach, Brody, Todorov, Gunter, & Philibert, 2011; dial preoptic area (MPOA) of the hypothalamus, whereas the Vijayendran, Beach, Plume, Brody, & Philibert, 2012). In- opposite is true of high-LG mothers (Champagne et al., creased stress responsivity in maternally and socially isolated 2006). Finally, work has shown that increased maternal care infant macaques has likewise been linked to genotype and hy- of male rats increases adulthood expression of anti-inflamma- permethylation of the gene encoding the serotonin transporter 1282 T. L. Roth

(Kinnally et al., 2010, 2011). Children (male and female) 2011). Consumption of a high-fat diet during pregnancy aged 7 to 10 years and raised in institutional care show greater has been shown to produce offspring that prefer sucrose methylation of DNA associated with a number of genes in- and fat in adulthood, a phenotype that is accompanied by volved in controlling serotonin and glucocorticoid biosynth- DNA hypomethylation and increased expression of several esis, immune responses, and cell-signaling pathways in- reward-related genes (including the Oprm1 and dopamine re- volved in memory formation than do children of the same uptake transporter) within the nucleus accumbens or prefron- age who were raised by their biological parents (Naumova tal cortex (Vucetic, Kimmel, Totoki, Hollenbeck, & Reyes, et al., 2012). Finally, adolescent and adult DNA methylation 2010). Studies are beginning to indicate that the epigenetic levels, including those of genes known to play an important consequences of prenatal stress likely occur on a broad ge- role in cell-signaling pathways within the brain, have been nomewide scale (Mychasiuk, Ilnytskyy, Kovalchuk, Kolb, & linked to maternal and paternal stress levels during early Gibb, 2011) and that stress does not have to be experienced childhood years (Borghol et al., 2012; Essex et al., 2011). directly by the mother in order for it to affect her offspring. One report has shown global DNA methylation (frontal cor- tex and hippocampus), gene expression, and behavioral al- Changes in DNA Methylation as a Function of the terations in offspring of pregnant rats that were simply housed Prenatal Environment with another female who was repeatedly subjected to stress The intrauterine environment is also recognized as a major outside of the home cage (Mychasiuk, Schmold, et al., 2011). contributor to normal growth and development of an individ- ual, and disturbances at this critical time are recognized for Evidence from human studies their ability to adversely affect behavioral development and the long-term mental health of offspring. Across-species Methylation status of the human genome appears equally sen- studies have shown that one way prenatal stress and environ- sitive to a variant of prenatal factors. For example, infants mental factors can alter developmental trajectories is through born to mothers who reported high levels of depression and changes in DNA methylation. anxiety during their third trimester of pregnancy exhibit in- creased methylation of the Nr3c1 promoter in cord blood cells (Oberlander et al., 2008). Methylation status of the Evidence from rodent studies gene encoding the serotonin transporter (SLC6A4) is likewise In one of the earliest studies of gestational stress (using a regi- affected by maternal depression, as increased maternal de- men that included restraint stress and exposure to predator pressed mood scores have been associated with decreased odor or loud noises), prenatally stressed male (but not female) maternal (peripheral leukocytes) and infant (umbilical cord) offspring when adult were found to exhibit marked changes SLC6A4 promoter methylation (Devlin, Brain, Austin, & in expression of the Crh and GR genes, reduced methylation Oberlander, 2010). An unbalanced diet consumed during of Crh DNA in both the hypothalamus and amygdala, and in- pregnancy (one that is linked to elevated blood pressure and creased HPA-axis responsivity and a depressive-like pheno- cortisol in adult offspring) produces methylation of the GR type (Mueller & Bale, 2008). Data since have been consistent gene (Drake et al., 2012). In addition, maternal alcohol and in showing the ability of stress experienced directly by the tobacco use are factors known to alter methylation of DNA mother to render epigenetic consequences in offspring. Re- associated with genes important in placental growth and de- straint stress has been shown to produce high levels of velopment (Suter et al., 2011; Wilhelm-Benartzi et al., 2011). Dnmt1 and 3a (genes encoding enzymes that are responsible for adding methyl groups to DNA) mRNA in the frontal cor- Changes in DNA Methylation as a Function tex and hippocampus of male offspring (Matrisciano et al., of Later-Life Events 2012). This same study showed that prenatally stressed mice also displayed increased methylation of DNA associated The studies highlighted in the previous sections are consistent with synaptic plasticity genes (including reelin) and several with the notion that epigenetic alterations caused by adversity schizophrenic-like behaviors, including hyperactivity, defi- within the intrauterine or caregiving environments may pro- cits in social interaction, altered prepulse inhibition, and vide a basis for the concept of developmental origins of memory capacity. Restraint stress of pregnant rats has also re- health and disease. Adversity experienced later in life is like- cently been associated with changes in placental and fetal wise recognized for its ability to render lasting psychological HSD11B2 mRNA and methylation, a gene whose product and health costs, and emerging evidence suggests that helps protect the fetus against the damaging effects of stress changes in gene expression mediated by DNA methylation hormones (Jensen Pen˜a, Monk, & Champagne, 2012). have significance in these outcomes as well. In utero experiences also affect epigenetic regulation of genes and circuitry associated with addiction and motivation. Evidence from rodent studies Altered DNA methylation of the dopamine receptor 2 gene has been found in the nucleus accumbens of adult rats that In parallel to developmental studies, research efforts aimed at were exposed to in utero glucocorticoids (Rodrigues et al., understanding molecular mechanisms underlying memory Epigenetics and behavioral development 1283 processes and stress outcomes have illustrated the sensitivity changes permissive for Gdnf transcription. These studies of the adult rodent brain to environmentally driven epigenetic are beginning to provide a glimpse of the epigenetic attributes alterations. It is interesting that these experience-driven of stress resilience versus stress susceptibility. changes appear to occur with similar selectivity for gene net- works and circuitry as that affected by early-life adversity. Evidence from human studies Much of the pioneering work in regard to DNA methylation alterations in memory formation has utilized contextual fear To date, there has only been one study that investigated conditioning, a behavioral paradigm that produces robust changes in DNA methylation immediately following a spe- and long-lasting fear memories in rodents. Data illustrate cific stressful event in humans (Unternaehrer et al., 2012). that hours following fear conditioning when the fear memory Study participants were adults who had been exposed to is being formed (consolidated) there are changes in hippo- war adversities in early childhood and subjected to the Trier campal DNA methylation that include demethylation and Social Stress Test (TSST). Each participant had blood sam- transcriptional activation of the memory enhancing gene ples drawn at a time point prior to the TSST, 10 min following reelin (Miller & Sweatt, 2007) and both DNA methylation completion of the TSST, and 90 min after the TSST. Exam- and demethylation of DNA associated with the Bdnf gene ination of DNA methylation patterns of two stress-related within the hippocampus (Lubin, Roth & Sweatt, 2008). There genes, oxytocin receptor and Bdnf, revealed stress-evoked are also changes in methylation of DNA associated with DNA methylation changes across targeted sequences of the calcineurin within the prefrontal cortex, an effect that is sus- oxytocin receptor gene, with very little change detected in tained for at least several weeks (Miller et al., 2010). Bdnf DNA (though change was detected at one Bdnf CG Alterations in hippocampal DNA methylation appear to site). Although their study population was based on indi- serve as markers not only of memory but also of exposure viduals who might have increased susceptibility to DNA to chronic stress (Chertkow-Deutsher, Cohen, Klein, & methylation changes (due to early-life stress and/or aging), Ben-Shachar, 2010; Roth, Zoladz, Sweatt, & Diamond, their within-subject design provides seminal evidence regard- 2011). For example, repeated exposure to predatory and so- ing the ability of acute psychosocial stress in humans to have cial instability stress produces rodents that show learning immediate epigenetic consequences. Work highlighted in the and memory deficits, increased anxiety-like behavior, and next section demonstrates DNA methylation alterations with glucocorticoid abnormalities weeks to months following the reference to experiencing a number of lifetime stressful initiation of the stress regimen (Zoladz, Conrad, Fleshner, events or trauma exposure in posttraumatic stress disorder & Diamond, 2008; Zoladz, Fleshner, & Diamond, 2012). (PTSD). We have recently shown that these behavioral abnormalities coincide with robust alterations in hippocampal Bdnf DNA Psychiatric Disorders With Aberrant DNA methylation (that vary by subregion) and gene expression Methylation (Roth et al., 2011). Other types of stress exposure have been shown to engage Studies in the previous sections were chosen to demonstrate DNA methylation as determined from central (Elliott, Ezra- the remarkable ability of environmental input, both during Nevo, Regev, Neufeld-Cohen, & Chen, 2010; LaPlant and outside of periods of early-life development, to alter et al., 2010; Sterrenburg et al., 2011; Uchida et al., 2011) DNA methylation. These studies make it clear that DNA or peripheral (Tung et al., 2012) measures. Social defeat stress methylation alterations (both short- and long-lived) are bio- is associated with a lasting decrease in methylation of Crh logical responses to psychological and social–contextual fac- DNA in the hypothalamus of animals that develop social tors. Furthermore, these observations are consistent with the avoidance behavior (Elliott et al., 2010). It is interesting hypothesis that epigenetic marking of genes could underlie that mice in this study that did not show these epigenetic mod- aspects of psychopathology, especially in regard to neuropsy- ifications also did not develop social avoidance behavior fol- chiatric disorders that can be associated with early-life stress lowing defeat stress. Stress-susceptible (inbred BALB/c) and and abnormal brain development and function. To this end, in stress-resilient (C57BL/6) male mice are known to show be- this section I summarize evidence linking the presence havioral differences following chronic exposure to mild of DNA methylation alterations with schizophrenia (SZ), stressors, and a study recently attributed these phenotypes PTSD, and mood disorders. to differential epigenetic marking of glial cell derived neuro- trophic factor (Gdnf) DNA within the nucleus accumbens SZ (Uchida et al., 2011). Although both strains of mice were shown to display increased methylation of the Gdnf promoter Increased Dnmt1 expression and decreased expression of following chronic stress, stress-susceptible mice (BALB) reelin and GAD1 are common findings in the cortex of indi- showed additional epigenetic changes that lead to repression viduals with SZ (Guidotti et al., 2007; Ruzicka et al., 2007; of Gdnf transcription, including gene promoter interactions Veldic et al., 2004, 2007; Veldic, Guidotti, Maloku, Davis, with histone deactylases (HDACs). In contrast, mice that & Costa, 2005). Deficits in reelin and GAD1 levels have adapted to chronic stress (B6) showed additional epigenetic been linked to altered methylation levels (Abdolmaleky 1284 T. L. Roth et al., 2005; Connor & Akbarian, 2008; Grayson et al., 2005; genes that play a role in neuronal growth, development, and Huang & Akbarian, 2007), and genomewide epigenetic ap- cholinergic transmission (Sabunciyan et al., 2012)have proaches on cortical tissue suggest there are many additional been found in the brains (within the frontal cortex, amygdala, gene loci with altered DNA methylation in SZ, including and PVN) of individuals who committed suicide and/or had other gene families related to GABAergic and neurotrophic been diagnosed with major depression. Altered levels of (Bdnf) function (Connor & Akbarian, 2008; Mill et al., Dnmt mRNA (Higuchi et al., 2011) and Bdnf DNA 2008). Peripheral measures of DNA methylation implicate methylation (Fuchikami et al., 2011) have likewise been epigenetic marking of the monoamine oxidase A gene promo- found in peripheral measures in patients with major depres- ter (Chen, Zhang, Zhang, Shen, & Xu, 2012), DNA associ- sion. Other findings in depressed patients include increased ated with several serotonin receptors (Abdolmaleky et al., methylation of genes involved in brain development and me- 2011; Carrard, Salzmann, Malafosse, & Karege, 2011; Gha- tabolic processes (Uddin, Koenen, et al., 2011), decreased dirivasfi et al., 2011), and the catechol-O-methyltransferase methylation of genes, such as the inflammatory gene interleu- gene (Abdolmaleky et al., 2006; Lott et al., 2012; Melas kin 6 (Uddin, Koenen, et al., 2011), and high methylation of et al., 2012; Nohesara et al., 2011). Finally, a genomewide the angiotensin converting enzyme gene (Zill et al., 2012). analysis of DNA methylation on peripheral blood DNA sam- ples obtained from monozygotic twin pairs discordant for Evidence for the Heritable Nature of Acquired DNA major psychosis showed numerous loci with DNA Methylation Changes methylation differences between twins discordant for SZ and bipolar disorder (Dempster et al., 2011). Data from the previous sections are also consistent with the no- tion that if DNA methylation is an operational mechanism sub- serving an organism the ability to adapt to environmental fac- PTSD tors, then the heritable nature of epigenetic changes could Peripheral measures of methylation show strong associations allow information acquired during one’s lifetime to be passed between child abuse, total life stress, methylation of DNA as- on to offspring. Intriguing datafrom animal studies provide sup- sociated with genes related to immune function and inflam- port that this does occur. As pointed out in an earlier section, in- mation, and the diagnosis of PTSD (Smith et al., 2011; Uddin fant experiences with nurturingmothers(high-LG behavior)de- et al., 2010). An interaction between methylation status of the creases methylation of DNA associated with ER-a gene in the SLC6A4 (Koenen et al., 2011)orMAN2C1 (Uddin, Galea, MPOA, producing increased gene expression and responsivity et al., 2011) genes and the number of traumatic events experi- to hormonally primed maternal behavior (Champagne et al., enced has been reported in PTSD. Assessment of serum DNA 2006). As a consequence of these epigenetic modifications, off- methylation patterns in genomic repetitive elements (LINE-1 spring and grand-offspring from high-LG mothers become and Alu) of US military service members pre- and postde- high-LG mothers themselves (Champagne, 2008;Champagne ployment and with or without PTSD diagnosis suggest &Meaney,2007). In this model, transmission of behavior DNA methylation operative as a resilience or vulnerability and DNA methylation patterns represent experience-dependent factor (Rusiecki et al., 2012). inheritance mediated by maternal care itself. There is also emerging evidence of a “double hit” model, in As evidence of the ability of early-life adversity to produce which having a risk allele and promoter DNA methylation may multigenerational effects on DNA methylation, we have more accurately capture molecular risk of PTSD. For example, shown that offspring born to females that were maltreated 9-repeat allele carriers of the dopamine transporter (SLC6A3) (in their infancy) likewise showed altered Bdnf DNA gene show an increased risk of lifetime PTSD when in con- methylation patterns (Roth et al., 2009). We used cross-fos- junction with high methylation present in the SLC6A3 promo- tering studies to address whether the appearance of DNA ter (Chang et al., 2012). Methylation of genes within the pitui- methylation alterations in this generation of infants was due tary adenylate cyclase-activating polypeptide (PACAP) to the mother’s caregiving behaviors. Our results revealed system, a system responsive to cellular stress and implicated that the changes in DNA methylation were not simply a in neurotrophic function, also appears to predict PTSD diagno- product of the postnatal experience, as they were not easily re- sis. Females with high plasma PACAP levels and greater versed by altering the caregiving environment. One intrigu- methylation of the PACAP receptor gene (ADCYAP1R1) con- ing explanation for our results is the phenomenon of germ- taining a polymorphism show strongest PTSD symptoms and line inheritance, and though we have yet to address this notion physiological fear responses (Ressler et al., 2011). in our model, other studies have directly demonstrated herita- ble epigenetic and behavior changes via the germ-line that are independent of maternal care. Suicide and mood disorders In an earlier section, the ability of infant separation to Epigenetic phenomena have similarly been associated with produce lasting changes in depressive-like behaviors suicide and depression. Dnmt mRNA alterations (Poulter and methylation of the Crh gene (Franklin et al., 2010)was et al., 2008), increased Bdnf DNA methylation (Keller discussed. From this same study, anxiety and depression-re- et al., 2010), and altered methylation patterns of numerous lated behaviors, as well as methylation changes, were also Epigenetics and behavioral development 1285 found to be present in offspring and grand-offspring derived how are these changes then translated into altered gene net- from males that had experienced the initial separation (Frank- works, circuitry, and behavior? lin et al., 2010; Weiss et al., 2011). This is an important obser- vation because in rodents the males do not provide infant care Although it is clear that environmental factors have direct ef- and are typically removed from a cage once successful mating fects on gene expression and behavior via alterations in DNA has occurred. Subsequent studies from this line of work have methylation that appear to have some brain region specificity, revealed deficits in cognition and social interaction transmit- we do not at present understand how this may be the case. We ted through the F3 generation but also stress resiliency, sug- do know that postmitotic neurons express the enzymatic ma- gesting both positive and negative long-term and transgenera- chinery necessary for modifying chromatin and DNA tional effects of early-life stress (Franklin et al., 2011). Paternal methylation states (Brown, Weaver, Meaney, & Szyf, 2008; transmission of stress-related behaviors induced by social de- Feng, Chang, Li, & Fan, 2005;Maetal.,2009), providing feat have also recently been demonstrated (Dietz et al., 2011). a susceptible basis for environmentally driven epigenetic Another line of evidence supporting epigenetic transmis- modifications. Brain region, and cell-type specificity for sion of acquired information comes from that manipulating that matter, would require qualitative, quantitative, and spatial the prenatal environment. Fetal-alcohol-exposed rats exhibit arrangement of mechanisms capable of orchestrating re- deficits in expression of the proopiomelanocortin (POMC), sponses to similar stimuli, which appears to be in place. In an effect that coincides with increased methylation of the the hippocampus for example, isoforms of Dnmt are known POMC promoter and persists in F2 and F3 generation males to vary by subregion (Brown et al., 2008) and cell type (Govorko, Bekdash, Zhang, & Sarkar, 2012). Offspring of (Feng et al., 2005; Siegmund & Connor, 2007). Although pregnant guinea pigs treated with synthetic glucocorticoids most studies have utilized a candidate gene approach to iden- show sustained alterations in central nervous system DNA tify specific sites of DNA methylation changes (and rightly methylation and gene expression (within the cerebellum), so, because this is a more sensitive approach for detecting an effect likewise present in the next generation (Crudo methylation changes), an increasing number of studies that et al., 2012). Providing some of the strongest evidence of have utilized genomewide approaches indicate changes are the phenomenon of germ-line epigenetic inheritance, includ- occurring at numerous gene loci. This would suggest changes ing altered stress responses for three to four generations re- in gene networks, with functional implications for disrup- moved from the initial environmental insult, are studies ex- tions at the circuit and behavioral levels. How DNA meth- posing pregnant dams to endocrine disruptors (Crews et al., ylation changes orchestrate these downstream effects is not 2012; Skinner, Anway, Savenkova, Gore, & Crews, 2008) known, and it is certainly an opportunity for future focus. or high-fat diets during gestation (Dunn & Bale, 2011). Do experiences within early-life developmental windows pro- duce the greatest effects on DNA methylation? Questions and Challenges in the Field Animal studies over the past decade have provided fascinat- Studies exposing either developing or adult animals to a vari- ing insight into the relationship between environmental fac- ety of stressors demonstrate the remarkable ability of the cen- tors, experience-driven DNA methylation patterns, and be- tral nervous system to alter its epigenetic status in response to havioral outcomes. Discoveries as summarized in Figure 2 environmental factors. At the same time, this shows that ex- are consistent with the notion that epigenetic mechanisms un- periences do not have to be confined to early-life develop- derlie cause–effect relations in regard to neurobehavioral de- mental periods in order to evoke DNA methylation changes. velopment and later stress-driven abnormalities. As applied Whether sensitive periods early in life truly represent height- to human development and psychopathology, epigenetic ened periods of vulnerability to these types of changes is an is- studies in humans also provide compelling evidence for sue that has not been systematically addressed. Expression pro- DNA methylation alterations as a biological substrate through files of Dnmt1, 3a, and 3b genes are detectable in neurons which a variety of factors can alter individuals for the long throughout the life span, though Dnmt3a is known to peak dur- haul (Figure 2). Although studies are being published at a ing the early postnatal period (Brooks, Marietta, & Goldman, rapid pace, behavioral epigenetics is still considered an 1996;Fengetal.,2005;Gotoetal.,1994). To directly answer emerging scientific field, and there are many unresolved is- this question, studies would need to incorporate a life span ap- sues and challenges that are certain to attract additional re- proach to provide information regarding when epigenetic mod- search. This final section serves as a forum to propose a hand- ifications are more likely to occur, or perhaps be attenuated, in ful of questions (arranged in no order of importance) that response to a specific stimulus or experience. Worth consider- might drive future research (both with animal models and hu- ing in this context is that it is possible that a much broader gene mans) to more clearly elucidate the role of epigenetics in de- pool could be at play during sensitive periods (such as in early velopment and psychopathology. postnatal development or adolescence), whereas a more re- stricted gene pool is responsive to later-life factors. What are the natural pathways by which environmental stim- With relevance to the importance of utilizing a life span uli preferentially alter DNA methylation in brain regions, and approach, evidence has emerged that experiences do not al- 1286 T. L. Roth

Figure 2. (Color online) Summary of psychological and social–contextual factors known to produce DNA methylation alterations. (Top) Experiences known to produce behavioral change in both developing and adult rodents that have been linked to altered DNA methylation and gene expression profiles. These epigenetic changes occur in many brain regions with known relevance to psychopathology, several of which are depicted here. (Bottom) Many of these animal findings have translated to similar observations in humans. A growing number of psychiatric disorders, several of which are listed here, have also been associated with DNA methylation alterations. ways translate into immediate changes in DNA methylation Our knowledge regarding the role of epigenetic modifications but that changes can evolve after an appreciable delay. For ex- in mediating behavioral effects has been mostly derived from ample, separation of infant mice produces changes in AVP rodent models that exclude any confounds of genetic variability DNA methylation that are not detectable during infancy, and where the behavior outcome is fairly uniform. It is impor- but rather, seem to evolve over the course of development tant to consider that in humans, however, genetic polymor- only to be present within adult tissue (Murgatroyd et al., phisms exist, and it is often the case that experiences that pro- 2009). Adding further complexity, we also know that DNA duce a particular outcome in some people do not in others. methylation patterns naturally change with maturation and Thus, functional polymorphisms are predicted to add an extra aging processes (Numata et al., 2012; Penner et al., 2010; layer of complexity to understanding how behavioral outcome Siegmund & Connor, 2007). Both of these phenomena likely is moderated by life experiences, and it may be the case that have significance in terms of “stress incubation” and direct polymorphisms serve as predispositions that make individuals versus indirect trajectories leading to later behavior change more susceptible to subsequent epigenetic alterations and dis- or the onset of psychiatric disorder. ease. The flip side of this is that certain polymorphisms could also serve as protective factors against epigenetic alterations. Are better animal models needed to fully realize the transla- Although keeping animals in constant conditions to under- tional implications of DNA methylation? stand the association between an early-life experience and later Epigenetics and behavioral development 1287 outcome has obvious value, this experimental design excludes plasticity, learning and memory, and stress-related behaviors the reality of complex social environments and the summing in rodents (for a review, see Abel & Zukin, 2008). effects of total life experiences that we know profoundly influ- In several of the aforementioned developmental studies, ence human behavior. One way that animal research could ad- investigators have successfully modified patterns of DNA dress this issue is to incorporate both early-life and later-life methylation in the adult brain that were associated with stress exposures in various models to determine outcomes early-life experiences (Roth et al., 2009; Weaver et al., associated with cumulative stress and epigenetic loads. Fur- 2004). The ability of epigenetic therapy to reverse gene deficits thermore, it should be evident from the developmental animal (and behavior) that were either set up in infancy or in cogni- studies reviewed here that rodent models have focused on tively impaired animals is a remarkable finding in its own right. mother–pup interactions and how the dynamics within this Since these studies have only examined the effects of epige- context can influence DNA methylation and subsequent devel- netic therapy on gene patterns or behavior on a rather short- opment. In humans it is not the case that only mothers interact time scale (hours to days later), whether this strategy would with their children, and a broader social context (fathers, be successful as a standalone treatment to stably (i.e., life- grandparents, childcare facilities, etc.) has an ability to leave long) reverse aberrant DNA methylation and behavior patterns its imprint on DNA methylation and gene expression patterns. is not known. Animals would need to be followed for long pe- Animal models with communal nesting and environmental riods of time (another point arguing for a life-span design in complexity may be a way to capture this. studies) and, perhaps, through multiple generations. This ap- proach is not only necessary to help us understand trajectories Does DNA methylation play an important role in why males of psychiatric disorders, but to understand when, where, and and females often have different outcomes from the same how to successfully intervene. Whether behavioral therapies, experience? such as environmental or social enrichment, would be better suited for such a long-term application, or in combination Some of the evidence reviewed in earlier sections suggests that with drugs targeted at epigenetically modifying programs of epigenetic activity plays an important role in sex specificity and genes, is also a question open for investigation. susceptibility to stress. This may be due to developmental win- dows in which some genes have the potential to be differentially Can peripheral measures really tell us anything about the marked between male and females. For example, DNA brain? methylation of the ER-a gene is higher in female rodents than in males at postnatal day 1 (Schwarz, Nugent, & McCarthy, DNA methylation alterations continue to be linked to psychi- 2010). mRNA and protein levels of Dnmt3a (Kolodkin & atric disorders, and this has led to the idea that peripheral Auger, 2011)andMeCP2 (Kurian, Forbes-Lorman, & Auger, measures of DNA methylation (including from blood sam- 2007) within the infant amygdala are higher in females than ples or cheek swabs) can serve as a proxy for brain changes in males, but by postnatal day 10 these sex differences have dis- that have already occurred or even as a predictive biomarker appeared. Dnmt3a is highest in the female and male cortex at for the probability of some outcome or individual responsiv- postnatal day 10, whereas Dnmt1 is highest after weaning ity to clinical treatment. As highlighted in an earlier section, a (Westberry, Trout, & Wilson, 2010). Within the MPOA, gene growing body of work has noticeably demonstrated a link be- promoters involved in sexual differentiation of the brain show tween blood-DNA methylation profiles and the risk for and differential epigenetic patterns between males and females at diagnosis of psychiatric disorders. Since there is accumulat- both an embryonic and early postnatal stage (Matsuda et al., ing evidence that epigenetic modifications occur with speci- 2011). It stands to reason that developmental epigenetic studies ficity across tissue and cell types (Davies et al., 2012;Iwa- that incorporate both sexes will reveal substantial etiological in- moto et al., 2011; Ladd-Acosta et al., 2007), however, formation regarding sex-specific development of behavior. questions arise regarding the ability of peripheral measures of DNA methylation to serve as an accurate and reliable proxy Can we target the epigenome for behavioral benefits? for changes occurring the brain and, thus, the viability of using DNA methylation as a biomarker. A new type of therapy aimed at correcting epigenetic “de- Although the link between peripheral and central mea- fects” has received considerable attention. In so-called epige- sures has not been unequivocally established, evidence we netic therapy, investigators aim to use drugs like 5-azacyti- do have at present suggests blood-DNA methylation profiles dine, zebularine, sodium butyrate, and trichostatin A to do show promise of serving as valuable diagnostic biomark- relax chromatin structure in order to provide gene transcrip- ers. For example, it was shown in one study that methylation tion accessibility. The ability of these drugs to correct epige- patterns of the rodent catechol-O-methyltransferase gene netic patterns was first recognized in cancer treatment, but the were directly correlated in peripheral blood mononuclear scope of their application has continued to be broadened to cells and the prefrontal cortex (Ursini et al., 2011). However, include enhancing cognition and treating specific symptoms methylation in the peripheral blood mononuclear cells did not of various psychiatric disorders. HDAC inhibitors, for exam- correlate with patterns in the hippocampus (although they ple, have been successfully used to reverse deficits in synaptic were in the same direction) or striatum. In the largest cross- 1288 T. L. Roth tissue and interindividual comparison of blood and brain cioeconomic disparities and child advocacy programs aimed methylation patterns in humans, investigators found that at helping infants and young children cope with early-life ad- though DNA methylation at specific gene loci were found versity. What we have learned regarding epigenetics and to differ between tissue types (blood, cortex, cerebellum, health outcomes has previously under-appreciated legal and etc.), strong correlations between DNA methylation for blood ethical implications, which could include litigations regard- and brain tissue were present (Davies et al., 2012). ing multigenerational, environmentally driven health effects. Finally, in the realm of medical policy this should include policy decisions regarding diets (including infant formula) Conclusions and chemical exposure, medical experiences such as in vitro Paradigm-shifting research in the past decade has provided fertilization, and funding of research aimed at early detection evidence that epigenetics serve as candidate pathways by and interventions to restore normal brain function and health. which experiences can leave their mark on genes to drive sus- In conclusion, epigenetic studies over the past decade tained changes in behavior. Although we still lack a complete have contributed fascinating insight into the role of DNA understanding of the cause-and-effect role of epigenetic methylation (and chromatin for that matter) as a rich source mechanisms in health outcomes and disease, evidence is clear for interactions between our environment and static genome. that epigenetic alterations are biological consequences of The birth of epigenetics research has provided an exciting early-life and later-life environmental input. Furthermore, new level of analysis for understanding tenets central to the dis- the evidence at hand suggests these alterations likely play a cipline of developmental psychopathology. Further research role in the development and enduring nature of psychopathol- using human study populations and animal models, especially ogy. Although a complete picture has yet to emerge, our those with clinical relevance, promises a greater understanding charge now is to incorporate the insights we have gained of the regulatory role of epigenetic processes in aspects of brain from these studies into programs and policies, an action point development relevant to the etiology of and resilience to psy- increasingly making its way into the literature (Birnbaum & chiatric disorders. The current momentum in the field also Jung, 2011; Hackman, Farah, & Meaney, 2010; Rothstein, points toward the applicability of using epigenetics, along Cai, & Marchant, 2009). These include those of the public with genetic polymorphisms, in providing individualized med- health sector, such as programs designed to help alleviate so- icine and predicting more effective responses to treatments.

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