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ScienceDirect The neurobiological effects of stress as contributors to psychiatric disorders: focus on epigenetics Nadine Provencal1 and Elisabeth B Binder1,2 A large body of evidence describes the long term impact of stress on a number of biological systems and with it associated adverse health outcomes. This article will discuss the epigenetic mechanisms of the embedding of these long term changes, the differences in these mechanisms depending on the type and timing of stress exposure, including transgenerational effects as well as differences in the mechanisms for tissue specific versus more global epigenetic changes. A mechanistic understanding of the long term epigenetic consequences of stress may allow novel, targeted intervention and prevention strategies for psychiatric and other stress-associated disorders. Addresses 1 Department of Translational Research in Psychiatry, Max-Planck Institute of Psychiatry, Munich, Germany 2 Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Altanta, GA, USA Corresponding author: Binder, Elisabeth B ([email protected])
A number of studies have now shown that exposure to ELS is accompanied by changes within the epigenome [8,9]. The epigenome governs the accessibility of genes to transcriptional regulation using several mechanisms, which include DNA methylation, post-translational histone modifications and non-coding RNA signaling (see Glossary). Therefore, these processes are candidate mechanisms for long term, stress-exposure-induced effects on gene transcription and in consequence cell and circuit function [10–12]. This review will focus on the molecular mechanisms by which stress, especially ELS, may install long lasting epigenetic changes. Most of the studies on this topic to date have investigated DNA methylation as the epigenetic mark, but epigenetic changes such as DNA methylation and histone modifications usually go hand in hand to shape the chromatin landscape and transcriptional regulation [13,14].
Current Opinion in Neurobiology 2015, 30:31–37
Timing of the traumatic event
This review comes from a themed issue on Neuropsychiatry
Epigenetic regulation is a time-dependent and highly controlled process. The expression of genes involved in epigenetic remodeling is regulated over development. For example, the expression of DNA methyltransferases, the enzymes that add methyl residues to cytosine, are highest in developing and differentiating cells and reduced in cells that have reached their terminal differentiation but can still be detected even in post-mitotic cells such as neuronal cells [15]. The impact of environmental factors on the type and extent of epigenetic changes thus very likely depends on the developmental stage at which they occur. The timing of these environmental events therefore plays an important role in the long term epigenetic consequences of stressors. Early environmental factors such as maternal care in rats, early life stress paradigms in mice and childhood trauma in humans have repeatedly been shown to be associated with long term changes in global and regional DNA methylation and histone modification profiles [16,17,18]. The developing organism thus seems to be the most vulnerable to long lasting epigenetic effects of stress.
Edited by Steven Hyman and Raquel Gur
http://dx.doi.org/10.1016/j.conb.2014.08.007 0959-4388/# 2014 Published by Elsevier Ltd.
Exposure to stressful or traumatic life events, especially early in life (early life stress (ELS)), is one of the strongest risk factors for a number of psychiatric disorders, ranging from post-traumatic stress disorder (PTSD) over depression to bipolar disorder and schizophrenia [1–4]. Over the past decade, an ever growing body of evidence indicates that exposure to stressful life events can lead to long lasting changes in a number of systems including the endocrine system [5], the immune system [6] and brain structure and function [4,7]. These systemic changes maybe pathophysiologically linked to the increased risk and altered disease trajectory observed following ELS, although some might be independent of disease and represent adaptive changes of the organism that only become pathological in combination with additional environmental, developmental, physiological (e.g. puberty or pregnancy) or genetic factors. www.sciencedirect.com
Specific versus global changes Exposure to stress or trauma leads to both very specific as well as global responses ranging from the formation of fear responses to a particular stimulus, to organism-wide effects of catecholamines and glucocorticoids that prepare a number of organs to a physiological stress response. The epigenetic embedding of stress exposure will thus Current Opinion in Neurobiology 2015, 30:31–37
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Glossary Epigenomic marks involved in the acute and long lasting effects of stress. Histone tail modifications: The chromatin is responsible for DNA packaging around histone proteins and this is facilitated by the presence of post-translational modifications of histone tails, which include acetylation, methylation, ubiquitination, sumoylation and phosphorylation at specific residues. These histone modifications can either condense the chromatin into an inactive form or open the chromatin to allow gene transcription. These modifications are catalyzed by histone modifying enzymes that can also recruit other transcriptional regulatory elements to the DNA. Alterations in histone marks, such as histone 3 lysine 9 acetylation (H3K9), have been shown to be induced by ELS and these effects could be reversed using histone deacetylase (HDAC) inhibitors [16]. DNA methylation: A covalent modification of the cytosine residues that are located primarily but not exclusively at CpG dinucleotide sequences in mammals. DNA methylation can alter gene expression by recruiting or blocking the binding of transcriptional regulatory elements such as MeCP2. To accurately maintain the DNA methylation profiles and prevent a drift in the DNA methylation pattern during the life course, several biochemical elements come into play. DNMT1 maintains the methylation pattern during cell division by copying the parent strand whereas DNMT3a and 3b are responsible for de novo DNA methylation. DNA demethylation processes can occur passively through cell division as well as actively through the direct action of a demethylase or by DNA excision/repair-based mechanisms involving sequential catalytic steps as well as important modulators such as Gadd45. Altered DNA methylation profiles in response to ELS were observed not only in specific genes in neurons [16,25] and other tissues [18] but also on a genome-wide level [41]. DNA hydroxymethylation: A distinct chemical modification in cytosines, catalyzed by ten-eleven translocation (TET) proteins, are proposed to serve as an intermediate step in active demethylation but may also alter gene expression on their own and dynamically mediate behavioral adaptations [21]. Non-coding RNAs (ncRNAs): Regulate genes translation and transcription as well as chromatin stability. They include short microRNAs, PIWI-interacting RNAs and long non-coding RNAs. ncRNAs can regulate gene expression through post-transcriptional binding to the 30 UTR of mRNA, by directly binding to promoters at the DNA level and interfering with polymerases or by localizing transcriptionally repressive complexes onto the heterochromatin. Exposure to ELS was shown to alter miRNAs content in the sperm of the exposed fathers as well as in the brain of their offspring [54,55]. Together, all these marks act in concert to prime current but also future gene expression and translation modifications that can lead to diseases.
likely have several components in each individual, both cell and brain circuit-specific as well as more global effects. The embedding of epigenetic changes in specific neuronal populations has been shown to be initiated by neuronal activation and underlies the mechanism of learning, often tested in the context of fear conditioning [19–22]. Such specific mechanisms may also be involved in mediating long term consequences of stress early in life and we illustrate this with two examples. Low levels of maternal care in rats lead to long lasting decreases in glucocorticoid receptor (GR) gene transcription in the hippocampus induced by an increase in DNA methylation in a specific promoter region of the gene [16]. Current Opinion in Neurobiology 2015, 30:31–37
A series of studies suggest that touch from the mother activates serotonin signaling and leads to the activation of the nerve growth factor-inducible protein A (NGFI-A) [23,24]. This transcription factor binds to a response element within the exon I7 sequence of the GR-promoter, influencing GR mRNA expression and leading to a local decrease in DNA methylation. This epigenetic effect of NGF1A depends on an interaction with the CREB binding protein, CBP. The NGF1A/CBP complex binds to the exon I7 sequence where CBP acts as a histone acetyltransferase and leads to increased levels of histone 3 lysine 9 acetylation (H3K9) at the exon I7 promoter. This process results in transcriptionally active chromatin and decreases DNA methylation levels. With low levels of maternal touch, such as in pups with low maternal care, this active demethylation does not take place, leading to a relative hypermethylation of the GR promoter and decreased transcription and altered stress hormone system reactivity. Similar mechanisms may be involved in the hypermethylation of the human orthologue site of this GR promoter (GR 1F) observed in postmortem hippocampus in suicide victims exposed to child abuse [25]. The methyl-binding protein MeCP2 plays an important role in activity-driven gene transcription in neuronal cells during the process of learning and memory storage [26] and this gene can also act more globally as a chromatinremodeling enzyme [27]. Neuronal activation has been shown to trigger the phosphorylation of MeCP2 and by this the release of the protein and other co-repressors from promoter sites, which can then be activated by transcription factor binding [28]. This mechanism may also mediate the sustained hypomethylation of the arginine vasopressin (AVP) gene promoter in the paraventricular nucleus (PVN) of mice exposed to early life stress by maternal separation [17]. AVP is a hypothalamic peptide essential in the regulation of the hypothalamus pituitary adrenal response. The CpG sites undergoing demethylation are located in the binding site of MeCP2. The authors showed that early life stress triggers the phosphorylation of MeCP2 leading to its dissociation from the promoter region and transcriptional activation of AVP expressing cells from the PVN. These differences in MeCP2 phosphorylation and protein occupancy at the promoter site are followed by differences in DNA methylation later in life, when these differences in MeCP2 phosphorylation are not apparent anymore, suggesting a sequential establishment of epigenetic marks. This early priming to demethylation by ELS exposure may be mediated by polycomb complexes and TET proteins [29]. Even though these data describe possible cascades of molecular events of how early environmental experiences can lead to transcriptional changes in a specific gene, it is unlikely that the broad range of behavioral, endocrine and molecular outcomes elicited by ELS are through changes www.sciencedirect.com
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in a single gene. In fact, the epigenetic response to maternal care [16] and also to exposure to child abuse [25] in the hippocampus appears to be coordinated across large stretches of DNA. These changes are not restricted to gene promoters but also occur in intragenic and intergenic regions, which are possible enhancer or repressor regions. These epigenetic changes are associated with non-random and gene-specific transcriptional adaptation [30,31]. Whether these epigenetic consequences are all mediated by the same specific mechanism as the changes in the GR gene is unlikely but a combination of transcription factors binding and their interaction with DNA binding proteins and specific epigenetic enzymes may be involved in this process. For examples, activity dependent changes in MeCP2 may lead to orchestrated changes in specific gene groups. Using MeCP2 knockout mice, Nuber et al. were able to show that glucocorticoid responsive genes, including Fkbp5 and Sgk1 are epigenetic targets of this protein leading to more global changes in glucocorticoid function in neuronal tissue [32]. In contrast to activation of specific neurons and cell type specific epigenetic changes, the effects of glucocorticoids, the main end effector of the stress response, are global and seen in many tissues and cell types. The global effects on gene transcription and cell function are likely associated with epigenetic changes in a number of tissues. The GR has been shown to induce stable demethylation in and around glucocorticoid receptor binding sites (GRE) leading to an increased transcriptional sensitivity of the target gene [33]. A similar mechanism may also mediate the lasting effects of childhood abuse on DNA demethylation at intronic GREs of the FKBP5 gene, a cochaperone regulating GR sensitivity, both in peripheral blood cells but also a hippocampal progenitor cell line [18]. These GR-induced local effects could explain some of the DNA methylation changes in response to stress exposure and similar mechanisms of local demethylation have been reported with other nuclear receptors (estrogen) and transcription factors [34–36]. Several mechanisms have been proposed for this transcription factor guided active demethylation and involve protein–protein interactions with methyl–DNA binding proteins and DNA repair mechanisms as well as an intermediate introduction of hydroxymethylation marks [37–40]. The different mechanisms of epigenetic consequences of stress exposure highlight the fact that some of these will be very specific to small numbers of cells in specific neural circuits while others may be seen across tissues and cell types (e.g. following GR activation). The latter type of mechanisms likely explain why ELS does not only have long term effects on brain function but also increases risk for metabolic disorders and has long lasting effects on the immune system [6]. Indeed, in rhesus macaques, the broad impact of maternal rearing in the first year of life on DNA methylation was seen in both the prefrontal cortex www.sciencedirect.com
and T cells, supporting the hypothesis that the response to early-life adversity is system-wide and genome-wide and persists to adulthood [41]. A handful of papers have now shown changes in DNA methylation in peripheral blood cells with early trauma in humans on a genome-wide level [42–44]. Suderman et al. specifically reported differential methylation in promoter regions of loci encoding microRNAs (miRNA) [43], in line with other studies showing the importance of miRNAs in modulating the stress response [45,46]. This may suggest that that a number of distinct epigenetic mechanisms could be involved in the biological embedding of postnatal stress.
Effects of stress across generations As introduced above, epigenetic consequences of stress exposure depend on the timing of the trauma, with earlier traumatic events likely having more profound impact. In fact, stress exposure of the mother during pregnancy has direct consequences on the set-point of a number of systems in the offspring, including the stress hormone system [47]. A series of studies suggest, that stress during pregnancy affects the expression of a number of key genes that may alter the permeability of the placenta to glucocorticoids possibly through increased DNA methylation of the 11b-hydroxysteroid dehydrogenase 2 (11b-HSD2) gene [48]. This enzyme converts excess cortisol/corticosterone to inactive cortisone. Decreases in this conversion rate due to decrease expression of this gene result in increased fetal exposure to glucocorticoids and lead to long term changes in neurodevelopment [48]. In addition, prenatal stress may also directly impact the function of enzymes involved in epigenetic regulation, such as the O-linked-N-acetylglucosamine transferase (OGT), which plays an important role in the regulation of chromatin remodeling proteins such as histones [49]. Changes in OGT may be linked to genome-wide epigenetic effects of prenatal stress [47]. Finally, exposure to stress may also have long term epigenetic consequences in the offspring independent of factors acting during gestation [50,51–53] and this maybe mediated by non-coding RNAs, especially miRNAs altered in the sperm of the exposed fathers. Such altered miRNAs profiles may install specific epigenetic marks in the brain of the offspring [54,55]. However, the exact mechanism how these changes in miRNAs lead to long term changes in DNA methylation, how exposure to stress changes the expression of miRNAs in sperm or whether similar mechanisms act in oocytes has not been elucidated yet.
Epigenetic mechanisms in gene environment interactions A number of studies have shown that the long term consequences of ELS can be moderated by genetic variation [56]. Such gene environment interactions may be mediated by genotype-specific epigenetic changes. We have shown that a functional genetic variant in the FKBP5 gene, which alters the 3D structure of the Current Opinion in Neurobiology 2015, 30:31–37
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Figure 1
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Epigenetic alterations Stress exposure Current Opinion in Neurobiology
Induction, transmission and possible reversibility of early life stress consequences during development. (1) Maternal stress exposure during gestation is likely to affect the fetus by altering epigenetic trajectories during development. This could be mediated, for example through the direct action of maternal GC on the placenta with epigenetic changes in the OGT and 11b-HSD2 genes which may lead, among other consequences to an increase fetal GC exposure which in turn has long term effects on several systems. (2) Stress and trauma in childhood have been shown to affect the epigenetic status of various genes in many tissues, including genes relevant for stress hormone system regulation such as NR3C1 and FKBP5. These epigenetic effects are likely to persist to adulthood and further alter the stress hormone response, neuronal networks and the immune system and influence risk for psychiatric disorders. (3) The exposure to stress at various developmental stages is likely to be registered also in the germ cells, possibly leading to altered miRNAs content in the sperm allowing transmission of such epigenetic signals to the next generation. (4) Since epigenetic alterations are potentially reversible by either drug or environmental interventions, these negatives outcomes may be avoided. Interventions inducing global epigenetic changes using drugs such as HDACs or DNMTs inhibitors but also behavioral interventions could possibly reverse the phenotype by inducing global as well as specific epigenetic changes allowing a resetting of dysregulated systems and potentially facilitating relearning of maladaptive behaviors. In addition, targeting the gene products and pathways dysregulated by these changes may allow specific interventions that could also have long term epigenetic effects by altering GC activity for example. GC: glucocorticoids, OGT: O-linked-N-acetylglucosamine transferase, 11b-HSD2: 11b-hydroxysteroid dehydrogenase 2, GR: glucocorticoid receptor, FKBP5: FK506 binding protein 5, HPA: hypothalamic–pituitary–adrenal, HDACs: histone deacetylases, DNMTs: DNA methyltransferases, miRNAs: microRNAs. Current Opinion in Neurobiology 2015, 30:31–37
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gene, leads to an increased induction of FKBP5 mRNA and protein following GR activation. This resulted in prolonged cortisol release following stress exposure. If the stress is abuse or maltreatment during childhood, this increased GR activation following stress likely leads to a long lasting demethylation of a GRE in this gene and further transcriptional depression of FKBP5 [18]. This has been associated with an increased risk for a number of psychiatric disorders [57]. Child abuse related DNA demethylation is only observed in carriers of the FKBP5 allele associated with increased transcriptional activation following stress. It is very likely, that genetic polymorphisms that directly alter the stress-induced binding of specific transcription factors will also impact the long term consequences of stress, and that this mechanism is not restricted to specific candidate genes. As described above, binding of transcription factors can be associated with changes in DNA methylation and if this binding is altered by genetic factors, this will also impact its epigenetic consequences. It is now clear that DNA methylation is not only a stable pattern induced during development to specify cell function but also responsive to environmental stimuli. This makes it likely that early life stress associated changes could be reversible, at least to some degree. Effects of drugs interfering with epigenetic regulation, such as histone deacetylase inhibitors have been shown to reverse early experience associated changes in DNA methylation and behavioral changes [16]. Recently, epigenetic changes following effective psychotherapy in patients with PTSD have been reported [58], suggesting that trauma-associated epigenetic changes maybe reversible, or that positive environment can install epigenetic changes on their own that may counteract the effects of stress-induced changes. The described studies, while offering exciting insight in how stress conveys risk for long term negative health outcomes, also highlight current challenges in our research approaches. Human studies are mostly cross sectional, but only longitudinal studies will be able to establish the sequence of events. The effects of positive environmental exposure are under-investigated as is the possible reversibility of stress-induced epigenetic changes. To establish true causality of these epigenetic changes we need animal models that allow specific epigenetic modification or the prevention of the establishment of such changes following stress. Overall, these studies indicate that the genome responds to stress exposure in a genome-wide but orchestrated way that leads to the transcriptional regulation of functionally related genes. A deeper understanding of the factors that are involved in the establishment, maintenance and reversibility of such genome-wide and still specific epigenetic changes will be an important next step in the field, and may open novel therapeutic and preventive strategies (see Figure 1). www.sciencedirect.com
Conflict of interest statement Nothing declared.
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49. Chen Q, Chen Y, Bian C, Fujiki R, Yu X: TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 2013, 493:561-564. 50. Franklin TB, Russig H, Weiss IC, Graff J, Linder N, Michalon A, Vizi S, Mansuy IM: Epigenetic transmission of the impact of early stress across generations. Biol Psychiatry 2010, 68: 408-415. One of the first papers to show that ELS is transmitted to the following generations through the male germ cells and that DNA methylation alterations observed in Crhr2 and Mecp2 genes in sperm of the exposed generation as well as in the brain of the subsequent generation could be part of the mechanism for this transgenerational effect. 51. Morgan CP, Bale TL: Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. J Neurosci 2011, 31: 11748-11755. 52. Mueller BR, Bale TL: Sex-specific programming of offspring emotionality after stress early in pregnancy. J Neurosci 2008, 28:9055-9065. 53. Dias BG, Ressler KJ: Parental olfactory experience influences behavior and neural structure in subsequent generations. Nat Neurosci 2014, 17:89-96.
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54. Gapp K, Jawaid A, Sarkies P, Bohacek J, Pelczar P, Prados J, Farinelli L, Miska E, Mansuy IM: Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nat Neurosci 2014, 17:667-669. 55. Rodgers AB, Morgan CP, Bronson SL, Revello S, Bale TL: Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation. J Neurosci 2013, 33:9003-9012. One of the first papers to describe transgenerational transmission of ELS possibly through altered miRNAs content in sperm. 56. Karg K, Burmeister M, Shedden K, Sen S: The serotonin transporter promoter variant (5-HTTLPR), stress, and depression meta-analysis revisited: evidence of genetic moderation. Arch Gen Psychiatry 2011, 68:444-454. 57. Zannas AS, Binder EB: Gene–environment interactions at the FKBP5 locus: sensitive periods, mechanisms and pleiotropism. Genes Brain Behav 2014, 13:25-37. 58. Yehuda R, Daskalakis NP, Desarnaud F, Makotkine I, Lehrner AL, Koch E, Flory JD, Buxbaum JD, Meaney MJ, Bierer LM: Epigenetic biomarkers as predictors and correlates of symptom improvement following psychotherapy in combat veterans with PTSD. Front Psychiatry 2013, 4:118.
Current Opinion in Neurobiology 2015, 30:31–37
ScienceDirect The neurobiological effects of stress as contributors to psychiatric disorders: focus on epigenetics Nadine Provencal1 and Elisabeth B Binder1,2 A large body of evidence describes the long term impact of stress on a number of biological systems and with it associated adverse health outcomes. This article will discuss the epigenetic mechanisms of the embedding of these long term changes, the differences in these mechanisms depending on the type and timing of stress exposure, including transgenerational effects as well as differences in the mechanisms for tissue specific versus more global epigenetic changes. A mechanistic understanding of the long term epigenetic consequences of stress may allow novel, targeted intervention and prevention strategies for psychiatric and other stress-associated disorders. Addresses 1 Department of Translational Research in Psychiatry, Max-Planck Institute of Psychiatry, Munich, Germany 2 Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Altanta, GA, USA Corresponding author: Binder, Elisabeth B ([email protected])
A number of studies have now shown that exposure to ELS is accompanied by changes within the epigenome [8,9]. The epigenome governs the accessibility of genes to transcriptional regulation using several mechanisms, which include DNA methylation, post-translational histone modifications and non-coding RNA signaling (see Glossary). Therefore, these processes are candidate mechanisms for long term, stress-exposure-induced effects on gene transcription and in consequence cell and circuit function [10–12]. This review will focus on the molecular mechanisms by which stress, especially ELS, may install long lasting epigenetic changes. Most of the studies on this topic to date have investigated DNA methylation as the epigenetic mark, but epigenetic changes such as DNA methylation and histone modifications usually go hand in hand to shape the chromatin landscape and transcriptional regulation [13,14].
Current Opinion in Neurobiology 2015, 30:31–37
Timing of the traumatic event
This review comes from a themed issue on Neuropsychiatry
Epigenetic regulation is a time-dependent and highly controlled process. The expression of genes involved in epigenetic remodeling is regulated over development. For example, the expression of DNA methyltransferases, the enzymes that add methyl residues to cytosine, are highest in developing and differentiating cells and reduced in cells that have reached their terminal differentiation but can still be detected even in post-mitotic cells such as neuronal cells [15]. The impact of environmental factors on the type and extent of epigenetic changes thus very likely depends on the developmental stage at which they occur. The timing of these environmental events therefore plays an important role in the long term epigenetic consequences of stressors. Early environmental factors such as maternal care in rats, early life stress paradigms in mice and childhood trauma in humans have repeatedly been shown to be associated with long term changes in global and regional DNA methylation and histone modification profiles [16,17,18]. The developing organism thus seems to be the most vulnerable to long lasting epigenetic effects of stress.
Edited by Steven Hyman and Raquel Gur
http://dx.doi.org/10.1016/j.conb.2014.08.007 0959-4388/# 2014 Published by Elsevier Ltd.
Exposure to stressful or traumatic life events, especially early in life (early life stress (ELS)), is one of the strongest risk factors for a number of psychiatric disorders, ranging from post-traumatic stress disorder (PTSD) over depression to bipolar disorder and schizophrenia [1–4]. Over the past decade, an ever growing body of evidence indicates that exposure to stressful life events can lead to long lasting changes in a number of systems including the endocrine system [5], the immune system [6] and brain structure and function [4,7]. These systemic changes maybe pathophysiologically linked to the increased risk and altered disease trajectory observed following ELS, although some might be independent of disease and represent adaptive changes of the organism that only become pathological in combination with additional environmental, developmental, physiological (e.g. puberty or pregnancy) or genetic factors. www.sciencedirect.com
Specific versus global changes Exposure to stress or trauma leads to both very specific as well as global responses ranging from the formation of fear responses to a particular stimulus, to organism-wide effects of catecholamines and glucocorticoids that prepare a number of organs to a physiological stress response. The epigenetic embedding of stress exposure will thus Current Opinion in Neurobiology 2015, 30:31–37
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Glossary Epigenomic marks involved in the acute and long lasting effects of stress. Histone tail modifications: The chromatin is responsible for DNA packaging around histone proteins and this is facilitated by the presence of post-translational modifications of histone tails, which include acetylation, methylation, ubiquitination, sumoylation and phosphorylation at specific residues. These histone modifications can either condense the chromatin into an inactive form or open the chromatin to allow gene transcription. These modifications are catalyzed by histone modifying enzymes that can also recruit other transcriptional regulatory elements to the DNA. Alterations in histone marks, such as histone 3 lysine 9 acetylation (H3K9), have been shown to be induced by ELS and these effects could be reversed using histone deacetylase (HDAC) inhibitors [16]. DNA methylation: A covalent modification of the cytosine residues that are located primarily but not exclusively at CpG dinucleotide sequences in mammals. DNA methylation can alter gene expression by recruiting or blocking the binding of transcriptional regulatory elements such as MeCP2. To accurately maintain the DNA methylation profiles and prevent a drift in the DNA methylation pattern during the life course, several biochemical elements come into play. DNMT1 maintains the methylation pattern during cell division by copying the parent strand whereas DNMT3a and 3b are responsible for de novo DNA methylation. DNA demethylation processes can occur passively through cell division as well as actively through the direct action of a demethylase or by DNA excision/repair-based mechanisms involving sequential catalytic steps as well as important modulators such as Gadd45. Altered DNA methylation profiles in response to ELS were observed not only in specific genes in neurons [16,25] and other tissues [18] but also on a genome-wide level [41]. DNA hydroxymethylation: A distinct chemical modification in cytosines, catalyzed by ten-eleven translocation (TET) proteins, are proposed to serve as an intermediate step in active demethylation but may also alter gene expression on their own and dynamically mediate behavioral adaptations [21]. Non-coding RNAs (ncRNAs): Regulate genes translation and transcription as well as chromatin stability. They include short microRNAs, PIWI-interacting RNAs and long non-coding RNAs. ncRNAs can regulate gene expression through post-transcriptional binding to the 30 UTR of mRNA, by directly binding to promoters at the DNA level and interfering with polymerases or by localizing transcriptionally repressive complexes onto the heterochromatin. Exposure to ELS was shown to alter miRNAs content in the sperm of the exposed fathers as well as in the brain of their offspring [54,55]. Together, all these marks act in concert to prime current but also future gene expression and translation modifications that can lead to diseases.
likely have several components in each individual, both cell and brain circuit-specific as well as more global effects. The embedding of epigenetic changes in specific neuronal populations has been shown to be initiated by neuronal activation and underlies the mechanism of learning, often tested in the context of fear conditioning [19–22]. Such specific mechanisms may also be involved in mediating long term consequences of stress early in life and we illustrate this with two examples. Low levels of maternal care in rats lead to long lasting decreases in glucocorticoid receptor (GR) gene transcription in the hippocampus induced by an increase in DNA methylation in a specific promoter region of the gene [16]. Current Opinion in Neurobiology 2015, 30:31–37
A series of studies suggest that touch from the mother activates serotonin signaling and leads to the activation of the nerve growth factor-inducible protein A (NGFI-A) [23,24]. This transcription factor binds to a response element within the exon I7 sequence of the GR-promoter, influencing GR mRNA expression and leading to a local decrease in DNA methylation. This epigenetic effect of NGF1A depends on an interaction with the CREB binding protein, CBP. The NGF1A/CBP complex binds to the exon I7 sequence where CBP acts as a histone acetyltransferase and leads to increased levels of histone 3 lysine 9 acetylation (H3K9) at the exon I7 promoter. This process results in transcriptionally active chromatin and decreases DNA methylation levels. With low levels of maternal touch, such as in pups with low maternal care, this active demethylation does not take place, leading to a relative hypermethylation of the GR promoter and decreased transcription and altered stress hormone system reactivity. Similar mechanisms may be involved in the hypermethylation of the human orthologue site of this GR promoter (GR 1F) observed in postmortem hippocampus in suicide victims exposed to child abuse [25]. The methyl-binding protein MeCP2 plays an important role in activity-driven gene transcription in neuronal cells during the process of learning and memory storage [26] and this gene can also act more globally as a chromatinremodeling enzyme [27]. Neuronal activation has been shown to trigger the phosphorylation of MeCP2 and by this the release of the protein and other co-repressors from promoter sites, which can then be activated by transcription factor binding [28]. This mechanism may also mediate the sustained hypomethylation of the arginine vasopressin (AVP) gene promoter in the paraventricular nucleus (PVN) of mice exposed to early life stress by maternal separation [17]. AVP is a hypothalamic peptide essential in the regulation of the hypothalamus pituitary adrenal response. The CpG sites undergoing demethylation are located in the binding site of MeCP2. The authors showed that early life stress triggers the phosphorylation of MeCP2 leading to its dissociation from the promoter region and transcriptional activation of AVP expressing cells from the PVN. These differences in MeCP2 phosphorylation and protein occupancy at the promoter site are followed by differences in DNA methylation later in life, when these differences in MeCP2 phosphorylation are not apparent anymore, suggesting a sequential establishment of epigenetic marks. This early priming to demethylation by ELS exposure may be mediated by polycomb complexes and TET proteins [29]. Even though these data describe possible cascades of molecular events of how early environmental experiences can lead to transcriptional changes in a specific gene, it is unlikely that the broad range of behavioral, endocrine and molecular outcomes elicited by ELS are through changes www.sciencedirect.com
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in a single gene. In fact, the epigenetic response to maternal care [16] and also to exposure to child abuse [25] in the hippocampus appears to be coordinated across large stretches of DNA. These changes are not restricted to gene promoters but also occur in intragenic and intergenic regions, which are possible enhancer or repressor regions. These epigenetic changes are associated with non-random and gene-specific transcriptional adaptation [30,31]. Whether these epigenetic consequences are all mediated by the same specific mechanism as the changes in the GR gene is unlikely but a combination of transcription factors binding and their interaction with DNA binding proteins and specific epigenetic enzymes may be involved in this process. For examples, activity dependent changes in MeCP2 may lead to orchestrated changes in specific gene groups. Using MeCP2 knockout mice, Nuber et al. were able to show that glucocorticoid responsive genes, including Fkbp5 and Sgk1 are epigenetic targets of this protein leading to more global changes in glucocorticoid function in neuronal tissue [32]. In contrast to activation of specific neurons and cell type specific epigenetic changes, the effects of glucocorticoids, the main end effector of the stress response, are global and seen in many tissues and cell types. The global effects on gene transcription and cell function are likely associated with epigenetic changes in a number of tissues. The GR has been shown to induce stable demethylation in and around glucocorticoid receptor binding sites (GRE) leading to an increased transcriptional sensitivity of the target gene [33]. A similar mechanism may also mediate the lasting effects of childhood abuse on DNA demethylation at intronic GREs of the FKBP5 gene, a cochaperone regulating GR sensitivity, both in peripheral blood cells but also a hippocampal progenitor cell line [18]. These GR-induced local effects could explain some of the DNA methylation changes in response to stress exposure and similar mechanisms of local demethylation have been reported with other nuclear receptors (estrogen) and transcription factors [34–36]. Several mechanisms have been proposed for this transcription factor guided active demethylation and involve protein–protein interactions with methyl–DNA binding proteins and DNA repair mechanisms as well as an intermediate introduction of hydroxymethylation marks [37–40]. The different mechanisms of epigenetic consequences of stress exposure highlight the fact that some of these will be very specific to small numbers of cells in specific neural circuits while others may be seen across tissues and cell types (e.g. following GR activation). The latter type of mechanisms likely explain why ELS does not only have long term effects on brain function but also increases risk for metabolic disorders and has long lasting effects on the immune system [6]. Indeed, in rhesus macaques, the broad impact of maternal rearing in the first year of life on DNA methylation was seen in both the prefrontal cortex www.sciencedirect.com
and T cells, supporting the hypothesis that the response to early-life adversity is system-wide and genome-wide and persists to adulthood [41]. A handful of papers have now shown changes in DNA methylation in peripheral blood cells with early trauma in humans on a genome-wide level [42–44]. Suderman et al. specifically reported differential methylation in promoter regions of loci encoding microRNAs (miRNA) [43], in line with other studies showing the importance of miRNAs in modulating the stress response [45,46]. This may suggest that that a number of distinct epigenetic mechanisms could be involved in the biological embedding of postnatal stress.
Effects of stress across generations As introduced above, epigenetic consequences of stress exposure depend on the timing of the trauma, with earlier traumatic events likely having more profound impact. In fact, stress exposure of the mother during pregnancy has direct consequences on the set-point of a number of systems in the offspring, including the stress hormone system [47]. A series of studies suggest, that stress during pregnancy affects the expression of a number of key genes that may alter the permeability of the placenta to glucocorticoids possibly through increased DNA methylation of the 11b-hydroxysteroid dehydrogenase 2 (11b-HSD2) gene [48]. This enzyme converts excess cortisol/corticosterone to inactive cortisone. Decreases in this conversion rate due to decrease expression of this gene result in increased fetal exposure to glucocorticoids and lead to long term changes in neurodevelopment [48]. In addition, prenatal stress may also directly impact the function of enzymes involved in epigenetic regulation, such as the O-linked-N-acetylglucosamine transferase (OGT), which plays an important role in the regulation of chromatin remodeling proteins such as histones [49]. Changes in OGT may be linked to genome-wide epigenetic effects of prenatal stress [47]. Finally, exposure to stress may also have long term epigenetic consequences in the offspring independent of factors acting during gestation [50,51–53] and this maybe mediated by non-coding RNAs, especially miRNAs altered in the sperm of the exposed fathers. Such altered miRNAs profiles may install specific epigenetic marks in the brain of the offspring [54,55]. However, the exact mechanism how these changes in miRNAs lead to long term changes in DNA methylation, how exposure to stress changes the expression of miRNAs in sperm or whether similar mechanisms act in oocytes has not been elucidated yet.
Epigenetic mechanisms in gene environment interactions A number of studies have shown that the long term consequences of ELS can be moderated by genetic variation [56]. Such gene environment interactions may be mediated by genotype-specific epigenetic changes. We have shown that a functional genetic variant in the FKBP5 gene, which alters the 3D structure of the Current Opinion in Neurobiology 2015, 30:31–37
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Figure 1
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Epigenetic alterations Stress exposure Current Opinion in Neurobiology
Induction, transmission and possible reversibility of early life stress consequences during development. (1) Maternal stress exposure during gestation is likely to affect the fetus by altering epigenetic trajectories during development. This could be mediated, for example through the direct action of maternal GC on the placenta with epigenetic changes in the OGT and 11b-HSD2 genes which may lead, among other consequences to an increase fetal GC exposure which in turn has long term effects on several systems. (2) Stress and trauma in childhood have been shown to affect the epigenetic status of various genes in many tissues, including genes relevant for stress hormone system regulation such as NR3C1 and FKBP5. These epigenetic effects are likely to persist to adulthood and further alter the stress hormone response, neuronal networks and the immune system and influence risk for psychiatric disorders. (3) The exposure to stress at various developmental stages is likely to be registered also in the germ cells, possibly leading to altered miRNAs content in the sperm allowing transmission of such epigenetic signals to the next generation. (4) Since epigenetic alterations are potentially reversible by either drug or environmental interventions, these negatives outcomes may be avoided. Interventions inducing global epigenetic changes using drugs such as HDACs or DNMTs inhibitors but also behavioral interventions could possibly reverse the phenotype by inducing global as well as specific epigenetic changes allowing a resetting of dysregulated systems and potentially facilitating relearning of maladaptive behaviors. In addition, targeting the gene products and pathways dysregulated by these changes may allow specific interventions that could also have long term epigenetic effects by altering GC activity for example. GC: glucocorticoids, OGT: O-linked-N-acetylglucosamine transferase, 11b-HSD2: 11b-hydroxysteroid dehydrogenase 2, GR: glucocorticoid receptor, FKBP5: FK506 binding protein 5, HPA: hypothalamic–pituitary–adrenal, HDACs: histone deacetylases, DNMTs: DNA methyltransferases, miRNAs: microRNAs. Current Opinion in Neurobiology 2015, 30:31–37
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Epigenetic and stress-related disorders Provencal and Binder 35
gene, leads to an increased induction of FKBP5 mRNA and protein following GR activation. This resulted in prolonged cortisol release following stress exposure. If the stress is abuse or maltreatment during childhood, this increased GR activation following stress likely leads to a long lasting demethylation of a GRE in this gene and further transcriptional depression of FKBP5 [18]. This has been associated with an increased risk for a number of psychiatric disorders [57]. Child abuse related DNA demethylation is only observed in carriers of the FKBP5 allele associated with increased transcriptional activation following stress. It is very likely, that genetic polymorphisms that directly alter the stress-induced binding of specific transcription factors will also impact the long term consequences of stress, and that this mechanism is not restricted to specific candidate genes. As described above, binding of transcription factors can be associated with changes in DNA methylation and if this binding is altered by genetic factors, this will also impact its epigenetic consequences. It is now clear that DNA methylation is not only a stable pattern induced during development to specify cell function but also responsive to environmental stimuli. This makes it likely that early life stress associated changes could be reversible, at least to some degree. Effects of drugs interfering with epigenetic regulation, such as histone deacetylase inhibitors have been shown to reverse early experience associated changes in DNA methylation and behavioral changes [16]. Recently, epigenetic changes following effective psychotherapy in patients with PTSD have been reported [58], suggesting that trauma-associated epigenetic changes maybe reversible, or that positive environment can install epigenetic changes on their own that may counteract the effects of stress-induced changes. The described studies, while offering exciting insight in how stress conveys risk for long term negative health outcomes, also highlight current challenges in our research approaches. Human studies are mostly cross sectional, but only longitudinal studies will be able to establish the sequence of events. The effects of positive environmental exposure are under-investigated as is the possible reversibility of stress-induced epigenetic changes. To establish true causality of these epigenetic changes we need animal models that allow specific epigenetic modification or the prevention of the establishment of such changes following stress. Overall, these studies indicate that the genome responds to stress exposure in a genome-wide but orchestrated way that leads to the transcriptional regulation of functionally related genes. A deeper understanding of the factors that are involved in the establishment, maintenance and reversibility of such genome-wide and still specific epigenetic changes will be an important next step in the field, and may open novel therapeutic and preventive strategies (see Figure 1). www.sciencedirect.com
Conflict of interest statement Nothing declared.
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