Neuron
Review
Epigenetics of Stress-Related Psychiatric Disorders
and Gene 3 Environment Interactions
Torsten Klengel1,2 and Elisabeth B. Binder1,2,*
1Department
of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich 80804, Germany
of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA
*Correspondence: binder@psych.mpg.de
http://dx.doi.org/10.1016/j.neuron.2015.05.036
2Department
A deeper understanding of the pathomechanisms leading to stress-related psychiatric disorders is important
for the development of more efficient preventive and therapeutic strategies. Epidemiological studies indicate
a combined contribution of genetic and environmental factors in the risk for disease. The environment,
particularly early life severe stress or trauma, can lead to lifelong molecular changes in the form of epigenetic
modifications that can set the organism off on trajectories to health or disease. Epigenetic modifications are
capable of shaping and storing the molecular response of a cell to its environment as a function of genetic
predisposition. This provides a potential mechanism for gene-environment interactions. Here, we review
epigenetic mechanisms associated with the response to stress and trauma exposure and the development
of stress-related psychiatric disorders. We also look at how they may contribute to our understanding of the
combined effects of genetic and environmental factors in shaping disease risk.
Introduction
Psychiatric disorders and in particular stress-related psychiatric
disorders such as post-traumatic stress disorder (PTSD), major
depressive disorder (MDD), and anxiety disorders are multifactorial diseases influenced by both genetic predisposition and environmental factors (Stein et al., 2002; Sullivan et al., 2000).
Adverse life events, especially early in life, have consistently
been shown to strongly increase the risk for mood and anxiety
disorders in large epidemiological studies (Kessler et al., 1997).
Although severe forms of early adverse life events such as childhood abuse or neglect have been associated with the highest
rates of increased risk (Dube et al., 2001), other forms of early
adverse experiences, such as parental loss, bullying, or low socioeconomic status in childhood, were also shown to consistently increase risk for a number of psychiatric disorders (Kessler
et al., 2010). Finally, an increasing body of literature suggests
that prenatal adversity, in the form of stress or mood and anxiety
disorders of the mother, is also a risk factor for psychiatric disorders (Stein et al., 2014). A factor common to these early adversities is that they have all been associated with long-term
changes in the regulation of the stress hormone system (Lupien
et al., 2009), as illustrated in Figure 1, which may be causally
related to the development of disease. In addition to the strong
effects of the environment, there is a significant genetic contribution to the development of these disorders (Kendler et al., 2006;
Sullivan et al., 2000). However, strong main genetic effects have
not been observed for stress-related psychiatric disorders to
date, reflected by a lack of genome-wide significant associations
in studies with sample sizes that have led to robust genetic
association signals for schizophrenia and bipolar disorder
(Schizophrenia Working Group of the Psychiatric Genomics
Consortium, 2014; Ripke et al., 2013; Mühleisen et al., 2014).
The genetics of stress-related disorders are therefore confronted
with the so-called missing heritability that describes the lack of
strong effects in the kind of gene-association studies found in
twin and family studies (Lee et al., 2013). This is likely accounted
for by weak phenotype definitions potentially leading to a dilution
of genetic effects. Current diagnostic classification includes a
number of pathophysiological subtypes under the broad definitions of anxiety and depressive disorders. In addition, genetic
factors may have considerably smaller effect sizes compared
to schizophrenia where the explained variance by polygenic
factors has consistently increased with growing sample sizes
(Schizophrenia Working Group of the Psychiatric Genomics
Consortium, 2014). In MDD, anxiety disorders, and PTSD, the
reliable detection of such polygenic risk factors may need
much larger samples (Levinson et al., 2014).
Environmental factors as major triggers of stress-related disorders may lead to additional heterogeneity that is unaccounted
for in current genetic studies. Understanding the molecular
embedding of risk, conferred by adverse life events and how
these interact with genetic vulnerability, may be important for
the identification of the missing heritability observed in stress
disorders. One molecular mechanism that has come into focus
for mediating long-term environmental effects is epigenetics
(Slatkin, 2009).
Epigenetics subsumes mechanisms of functional control over
the genetic information without changing DNA sequence. These
mechanisms include the post-translational modification of
histone proteins as well as chemical modifications of single
nucleotides (most commonly in the form of DNA methylation
or hydroxymethylation at cytosine residues), which alter the
chromatin structure and thus the accessibility of the DNA to
transcriptional regulators. In the broader sense of epigenetic
regulation, these mechanisms also include the regulation of transcription and translation by non-coding RNAs, as schematically
represented in Figure 2. We here intentionally include regulation
by non-coding RNAs because of their ability to regulate transcriptional and translational output in post-mitotic neurons and
direct epigenetic modifiers to specific loci (Bird, 2007; Bonasio
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Figure 1. Stress and, in Particular, Early Life
Adversities Activate the Stress Hormone
System and May Epigenetically Program the
System toward a Lifelong Alteration of the
Hormonal Response to Even Minor
Stressors
Early life stress
Trauma exposure
CRH
AVP
GR GR
hsp90
POMC
Ultra-short
negative
feedback on
GR sensitivity
within the cell
GR GR
hsp90
FKBP5
FKBP4
FKBP5
ACTH
GR GR
hsp90
GRE
Adrenal gland
CORT
et al., 2010; Egger et al., 2004; Holliday, 2006; Jaenisch and Bird,
2003; Jenuwein and Allis, 2001; Peschansky and Wahlestedt,
2014). Some epigenetic modifications, especially DNA methylation, have been considered irreversible in the past, defining
cellular identity in a multicellular organism. By now it has been
shown that even stable chemical modifications, such as DNA
methylation, underlie highly dynamic regulation. This potential
reversibility makes these mechanisms suitable for encoding
the long-term impact of the environment also in post-mitotic tissue such as neurons (Sweatt, 2013). Although often depicted
separately for clarity, epigenetic mechanisms form a complex
interactive network with joint activities of different mechanisms
contributing to the same transcriptional regulation.
The field of epigenetics thus provides a possible molecular
framework of how genetic and environmental factors interact
and shape the risk for psychiatric disorders (Sweatt, 2013).
Epigenetics has been shown to play a decisive role in the
neuronal adaptations underlying learning and memory (Zovkic
and Sweatt, 2013), the response to environmental challenges
(Champagne, 2010; Jirtle and Skinner, 2007; McGowan and
Szyf, 2010; Zhang and Meaney, 2010) and the pathogenesis
of mental disorders (Bale et al., 2010; Jakovcevski and Akbarian, 2012; Mill and Petronis, 2007; Tsankova et al., 2007; Vialou
et al., 2013).
We here review the current knowledge on epigenetic modifications in response to environmental factors and their interaction
with genetic predisposition for stress-related diseases. In particular, we focus on the effects of childhood abuse and neglect, the
environmental factors conveying the most consistent increase in
risk, the epigenetic changes, and how they may relate to the
development of stress-related psychiatric disorders. We will be
discussing the current evidence of epigenetic mechanisms, in
particular DNA methylation, as a potential molecular link between environmental exposures and risk for psychiatric disorder,
as the vast majority of human studies have investigated this
modification with corresponding studies in laboratory animals,
and discuss how they can contribute to gene by environment
interactions (G3E).
1344 Neuron 86, June 17, 2015 ª2015 Elsevier Inc.
GRE
GRE
The neuropeptides corticotrophin-releasing hormone (CRH) and vasopressin (AVP), released
from the hypothalamus in response to stress,
activate the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland,
finally leading to an increased systemic cortisol
secretion from the adrenal gland. Cortisol binds
to steroid receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR),
that act as transcriptional activators or repressors
in the nucleus through binding to glucocorticoid
response elements. This influences the expression of numerous genes involved in the stress
response, immune function, and metabolism.
Binding of the GR and transcriptional activation
of, for example, FKBP5 provide an ultrashort
feedback to the GR, terminating the stress
response and secretion of cortisol.
Modification of Epigenetic Profiles by Severe Stress and
Trauma in Early Life—Potential Mechanisms
In addition to a growing number of animal studies indicating
long-lasting epigenetic effects of early stressful environments,
a number of studies in humans now also suggest that such
mechanisms may play a role in stress-related psychiatric disorders. In contrast to animal studies that can focus on brain tissue,
most human studies have been performed in mixed tissues that
are accessible to molecular investigation, such as peripheral
blood and buccal cells, with only few studies investigating
post-mortem brain tissue. Initial studies followed hypothesisdriven, candidate-based approaches, but recent advances in
array- and sequencing-based techniques allowed the interrogation of epigenetic marks on a genome-wide level as recently reviewed in Klengel et al. (2014). Among the very first candidates
implicated in stress-related epigenetic regulation were genes
involved in the stress- or hypothalamus-pituitary-adrenal (HPA)
axis due to its prominent role in the pathophysiology of stressrelated disorders. Other candidate gene-driven studies were
led by initial findings from genetic and gene expression studies
investigating epigenetic modifications in genes involved in
monoaminergic or neurotrophic signaling. However, unbiased,
genome-wide studies have implicated epigenetic changes in
genes often unrelated to established candidates, implicating
alternative pathophysiological mechanisms. These studies suggest that epigenetic mechanisms are important in stress-related
disease, but they remain on a descriptive, associative level.
Largely due to the relative unavailability of human brain tissue,
very little is known about how these differences may be established and maintained into adulthood and how they could lead
to psychopathology. Furthermore, it remains unclear to what
extent changes in peripheral tissues reflect changes in the
CNS and which molecular processes may be shared across tissues. In the following paragraphs we highlight the potential
mechanisms known to date by which early life stress may lead
to a permanent imprint of the stressor onto the genome by
epigenetic modification, relying predominantly on non-human
literature, and these are summarized in Figure 3. We later focus
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A
Figure 2. Schematic Representation of Main
Features of Epigenetic Regulation by Posttranslational Histone Modification, DNA
Methylation, and Non-coding RNA
This overview explicitly reduces and simplifies
the complex and multifaceted mechanisms of
epigenetic regulation for clarity. More specialized
reviews for a deeper description of this matter are
given in the text.
(A) Histone modifications influence the condensation of the DNA around histone proteins and
regulate the accessibility of functional regions to
B
C
transcriptional regulators, through modification at
predominantly the N-terminal tails, altering the
spatial structure of the chromatin and the interaction with DNA-binding proteins. Contingent on
the location and the type of modification, this can
lead to a more condensed chromatin-repressing
active transcription (exemplified by histone H3,
lysine 27 dimethylation (H3K27me2) and histone
H3, lysine 9 trimethylation (H3K9me3)) or vice
versa to an open chromatin state facilitating
active transcription (exemplified by histone H3, lysine 4 trimethylation (H3K4me3) and histone H3, lysine 9 acetylation (H3K9ac)).
(B) DNA methylation predominantly at CG dinucleotides (CpG) can influence the spatial structure of the DNA and the binding of or repression of specific DNAbinding proteins to the DNA. The closed circles represent higher methylation at cytosine residues, and the open circles represent lower methylation. Methylation
around the transcription start site in the promoter and the first exon is usually accompanied by transcriptional silencing. DNA methylation at other regulatory
regions and in the gene body can also facilitate transcription. Not depicted here are other modifications such as hydroxymethylation.
(C) Non-coding RNAs that include, for example, miRNA can influence chromatin structure and protein binding to the DNA but also directly target transcription
and translation. Depicted here is the regulation of mRNA stability through binding of miRNAs at the 30 UTR of target mRNA that can lead to a decrease in mRNA
stability, a decrease in mRNA cleavage, and therefore a reduction in protein assembly.
on human epigenetic studies and how they shape our understanding of the development and treatment of psychiatric
disorders.
Neuronal Activation Leading to Post-translational
Modifications of Epigenetic Readers and Writers
The post-translational modification of epigenetic readers and
writers is an example of how stress can exert a long-term impact
on gene regulation. An example is methyl CpG binding protein 2
(MeCP2) that influences transcription in response to neuronal
activation. MeCP2 directly binds to methylated DNA, acting as
transcriptional repressor, but it has also been shown to interact
with other proteins such as cAMP response element-binding
protein (CREB) and in this conformation can trigger transcriptional activation as well (Chahrour et al., 2008). An activitydependent modification of MeCP2 appears to be involved in
the long-term de-repression of the arginine vasopressin (avp)
gene activity in response to maternal separation in mice. Murgatroyd et al. showed that maternal separation leads to the
phosphorylation of MeCP2 and thus to the dissociation from
the promoter region of the murine avp gene in the paraventricular
nucleus. Subsequently, the MeCP2 binding site is demethylated,
leading to a sustained transcriptional activation of the avp gene
by reduced binding of the MeCP2 repressor complex
(Figure 3A) (Murgatroyd et al., 2009). The early priming to demethylation by early life stress (ELS) exposure may be mediated by
polycomb complexes and ten-eleven translocation (TET)
proteins that attract DNA methyl transferases and histone deacetylases to ensure proper methylation status of the locus.
The binding of these proteins is reduced following stress-dependent phosphorylation and dissociation of MeCP2 (Murgatroyd
and Spengler, 2014). However, the exact signaling cascade
from the maternal separation to locus- and cell-type-specific
DNA demethylation remains unknown.
MeCP2 also targets other genes implicated in the regulation of
the HPA axis. Nuber et al. (2005) showed that MeCP2 knockout
mice have elevated mRNA expression levels of FK506 binding
protein 5 gene (fkbp5), serum and glucocorticoid-regulated
kinase 1 (sgk1), and other glucocorticoid-responsive genes
without substantially increased glucocorticoid plasma levels,
providing evidence that MeCP2 can function as a modulator of
glucocorticoid action in neuronal cells (Nuber et al., 2005).
More recent studies show that MeCP2 interacts with a plethora
of other chromatin-modifying enzymes depending on the phosphorylation status of the protein, which in turn leads to a directed
modulation of transcription of a given locus (Bellini et al., 2014;
Ebert et al., 2013). Stress-related post-translational changes of
this protein may thus have wide-ranging epigenetic effects.
Direct Transcriptional Regulation of Epigenetic Writers,
Readers, and Erasers by Stress
Stress can also directly influence the transcriptional regulation of
epigenetic writers, readers, and erasers. This has been shown
for the transcriptional repression of DNA (cytosine-5)-methyltransferase 1 (DNMT1), the main enzyme responsible for the
maintenance of DNA methylation, by glucocorticoid exposure
as a proxy for stress. Lee et al. demonstrated that in vitro exposure of a murine pituitary cell line but also in vivo exposure of
mice to the glucocorticoid analog dexamethasone correlated
with a dose-dependent decrease in dnmt1 expression and
reduced DNA methylation at the murine fkbp5 locus (Figure 3B)
(Lee et al., 2011). However, whether the demethylation of the
fkbp5 locus is directly connected to this reduction remains an
open question. In adult animals, stress has been shown to regulate dnmt expression in a brain-regions-specific manner (Elliott
et al., 2010; LaPlant et al., 2010).
Other studies have shown that early life stress can influence
the expression of histone deacetylases (HDACs)—enzymes
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Figure 3. Stress May Influence the
Epigenome on Different Levels and via
Distinct Mechanisms
(A) As shown for maternal separation, early life
stress impacts the post-translational modification
of the epigenetic modifier MeCP2 by phosphorylation, leading to a dissociation of the protein
complex from the DNA strand. Subsequently, DNA
is demethylated, influencing the ability of proteins
to bind to the DNA and repress transcription.
(B) Stress can also change the transcriptional
activity of epigenetic enzymes, thus leading to an
increased or decreased presence of the resulting
proteins and subsequent alterations of epigenetic
profiles.
(C) Binding of DNA-binding proteins such as transcription factors may change the underlying local
epigenetic pattern by itself.
(D) Stress also influences the expression of noncoding RNAs, such as miRNA, that in turn can
influence mRNA stability and translation of many
genes in a signaling pathway.
that lead to more condensed chromatin structure—in the murine
brain (Levine et al., 2012; Tesone-Coelho et al., 2015). Suri et al.
suggested an age-dependent expression of histone-modifying
enzymes in response to early life stress with a decreased expression in young animals and an increased expression in adult mice
when exposed to early life stress (Suri et al., 2014). This opposite
expression of HDACs over time was consistent with distinct
global mRNA expression profiles separating young from adult
animals exposed to early life stress. Likewise, Blaze and Roth
observed that adverse rearing conditions only led to minimal
differences in the expression of DNMTs, HDAC1, and MeCP2
in the medial prefrontal cortex of developing rats but found
that these changes increased and became significant in adulthood in a sex-specific manner (Blaze and Roth, 2013). Numerous
studies investigating the acute but also long-term consequences
of stress in rodents now show the importance of epigenetic
writers, readers, and erasers in establishing and maintaining
specific marks in response to stress, as reviewed by Peña
et al. (2014). These findings in animals are paralleled by less
extensive human studies. Sipahi et al., for example, investigated
the longitudinal DNA methylation profile of the genes encoding
DNMTs pre- and post-trauma exposure in peripheral blood cells.
They found that dnmt1 methylation increases after trauma
exposure only in individuals who went on to develop PTSD,
while dnmt3a and dnmt3b methylation, the enzyme subtypes mainly responsible for the establishment of genomic
DNA methylation pattern, increase in trauma-exposed individuals regardless of PTSD status, thus differentiating PTSDsusceptible and -resilient trauma-exposed individuals (Sipahi
et al., 2014). Overall, these data suggest that stress may induce
long-lasting epigenetic changes by altering the expression of
genes critically involved in epigenetic regulation. For now, it is
not clear, however, whether the expression changes that were
all observed in mixed tissues actually only apply to very distinct
cell types or are more global. How changes in the levels of
DNMTs or HDACs can actually lead to epigenetic modifications
at specific loci also requires further investigations, but interaction
with other proteins and transcription factors guiding these epige1346 Neuron 86, June 17, 2015 ª2015 Elsevier Inc.
netic proteins is a plausible explanation (Hervouet et al., 2009;
Joshi et al., 2013).
Activation of Transcription Factors that Lead to Local
Changes in the Epigenetic Profile
An additional molecular mechanism leading to long-term epigenetic changes in response to stress is the activation of specific
transcription factors that in turn lead to local changes in epigenetic profiles. Early reports on the transcription factor Sp-1
showed that binding of Sp-1 leads to a local inhibition of de
novo DNA methylation (Brandeis et al., 1994). Furthermore,
glucocorticoid receptor (GR) activation can lead to a local demethylation of GR response elements (GREs) (Thomassin et al.,
2001). The mechanism of GR-induced local demethylation has
not been fully understood, but the DNA repair machinery was
implicated in this process, allowing the replacement of methylated by unmethylated cytosines. This demethylation of GREs
subsequently facilitates the transcriptional effects of the GR on
the target gene (Figure 3C) (Kress et al., 2001, 2006). Another
example is the activation of the Nuclear Factor 1 A-type (NF1A)
transcription factor by maternal care in rodents. Weaver et al.
showed that high levels of maternal care in early life are linked
to serotonin signaling in the rat hippocampus with an increase
in expression of the transcription factor nerve growth factorinduced protein A (NGFI-A). This is the transcription factor that
binds to the I7 promoter of the rat GR gene, increasing its expression. Binding of NGFI-A leads to a decrease in methylation of the
promoter with subsequent higher transcription factor binding
and increased GR expression (Weaver et al., 2004; Zhang
et al., 2013). Recently, collaborative effects of increased expression and GR promoter binding of the methyl-CpG-binding
domain protein 2 (MBD2) and NGF1-A activation by maternal
care have been implicated in this demethylation (Weaver et al.,
2014).
Such processes of active demethylation maybe driven by the
TET methylcytosine dioxygenase proteins TET1, TET2, and
TET3, resulting in the oxidation of methylcytosine to hydroxymethylcytosine and further to formyl- and carboxylcytosine (Kohli
and Zhang, 2013). Hydroxymethylcytosine is an epigenetic
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mark that is most common in neuronal tissues and embryonic
stem cells. It is considered an intermediate step in DNA demethylation. It is suggested that the formation of intermediate cytosine modifications leads to a less stringent recognition of the
particular DNA sequence by the methylation maintenance proteins and methyl-binding proteins, possibly favoring a passive
demethylation of these sites.
How the specificity of a potential TET-mediated demethylation
is achieved remains unclear, but studies on interaction partners
have shown that ten-eleven proteins can bind to other regulatory
proteins, providing further evidence for sequence-specific regulation of DNA demethylation (Chen et al., 2013; Costa et al.,
2013; Guilhamon et al., 2013).
Clearly, transcription factor binding to the DNA does not
only facilitate the removal of methyl marks but also allows the
directed de novo methylation. This is achieved by interaction
with repressor proteins, chromatin remodeling enzymes, methyltransferases, and also small non-coding RNAs to open the chromatin and allow for remodeling, leading to either activation or
repression of transcriptional activity (Marchal and Miotto, 2015;
Meaney and Ferguson-Smith, 2010).
Signaling through Small Non-coding RNAs in Response
to Stress
Another pathway that has been implicated in generating longterm epigenetic signatures of environmental exposure is the
expression of small non-coding RNAs in the form of miRNAs
and their subsequent targeting of stress-relevant pathways. A
number of studies report stress-induced miRNA expression
changes (Jung et al., 2015; Meerson et al., 2010; Rinaldi et al.,
2010; Schouten et al., 2013; Smalheiser et al., 2011), and some
studies are able to link these changes to pathways such as the
HPA axis: Haramati et al. identified miR-34c to be upregulated
in a stress-dependent manner. One of the targets of miR-34c is
the 30 UTR of the corticotrophin-releasing hormone receptor 1
(crhr1) gene leading to a decreased crhr1 expression in response
to stress-dependent miR-34c activation (Figure 3D) (Haramati
et al., 2011). Micro RNAs also seem to be regulating the GR itself
through post-transcriptional effects in rodents that are also sensitive to stress exposure (Jung et al., 2015). By activating specific
miRNAs in the rodent brain, stress may thus influence the regulation of downstream genes that lead to an altered endocrine and
behavioral response to stress. Human post-mortem brain studies
implicate miRNA in depression with downregulation of specific
miRNAs in the prefrontal cortex of these patients (Smalheiser
et al., 2012). Interestingly, some of these changed miRNAs
were predicted to target DNMT3B, which in turn has been shown
to be upregulated in these samples. This suggests an interaction
between the short-term regulation of gene transcription via small
non-coding RNAs with the more long-lasting change conferred
by DNA methylation. As outlined in the next section, confirmation
of these models will require longitudinal studies that enable researchers to investigate the sequence of molecular changes in
response to stress or trauma.
From a Short-Term Stress-Induced Imbalance to
Long-Lasting Dysregulation and Disease
An altered mRNA transcription following exposure to environmental impact can be seen as a short-term compensatory
reaction of the organism to maintain homeostasis and to overcome the environmental impact (McEwen and Gianaros, 2011).
These immediate responses at the transcriptional level do not
inevitably lead to long-lasting epigenetic changes. The longterm epigenetic changes in response to a qualifying environmental stressor require a sequence of short-term immediate
molecular responses leading to long-lasting epigenetic adjustments. Moderators of these long-lasting epigenetic changes
can be the quality, intensity, and timing of the stress exposure
and interaction with genetic factors. An example for such
concerted changes is the modification of the rodent arginine
vasopressin (avp) promoter in response to maternal separation
(Murgatroyd et al., 2009). Directly after a 10-day maternal separation period at postnatal day 10, the transcriptional activation of
AVP is detectable with changes in phosphorylation of MeCP2
and protein occupancy but without changes in the DNA methylation. At this time point, the epigenetic memory has not been
formed, and it is an intriguing question to ask if an early intervention e.g., by compensatory high maternal care, could prevent the
transition from short-term MeCP2 phosphorylation to DNA
methylation changes. The long-lasting epigenetic changes are
established in a subsequent step, engraving the short-term transcriptional change by creating a long-lasting epigenetic memory
by a reduced DNA methylation at the avp enhancer site for
MeCP2 in the paraventricular nucleus of the hypothalamus
(PVN) of early-life-stress-exposed mice. At this time point, the
differential phosphorylation of the MeCP2 protein that served
as an immediate molecular reaction to the environmental stimulus was not present anymore, and MeCP2 phosphorylation
was indistinguishable from control mice. These data suggest
that the immediate response via phosphorylation of MeCP2 is
subsequently replaced by DNA methylation changes that persist
over time. This example highlights that an understanding of factors leading to long-lasting modifications might help in improving
our abilities to prevent and treat stress-related disorders. It also
highlights the fact that longitudinal, prospective studies are
imperative to delineate the temporal sequence of molecular
events leading to disease, and that current studies, especially
in humans, often do not provide more than snapshots of the
respective disease conditions.
Differential Impact of Stress-Dependent Epigenetic
Modifications Depending on Developmental Stage
Besides type and intensity of early life stress, the timing of the
trauma is one of the most crucial factors determining epigenetic
changes and psychopathological outcome. In general, epigenetic mechanisms are dependent on developmental stage and
highly controlled as they play a major role in cell lineage determination but are also highly relevant in the adaptation of differentiated, post-mitotic neurons. Given the tremendous biological
differences between developmental stages in human life with regard to epigenetic regulation but also hormonal regulation and
neuronal connectivity, it is not plausible that there is a uniform
epigenetic response to stress across the lifespan. The early life
from pre-natal development until post-adolescence includes
the development and subsequent maturation of neuronal circuits
that support complex behavior, including language and cognition, but also pathways responsible for immune and hormone
regulation that impact stress vulnerability or resilience, and this
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period is more vulnerable to the detrimental effects of environmental stressors than any other periods in life (Fagiolini et al.,
2009; Fox et al., 2010; Kanherkar et al., 2014; McEwen, 2008).
Age dependence of stress vulnerability is illustrated by more
recent studies on the well-established example of changed GR
promoter methylation in response to maternal care or early life
stress. These suggest that a shift in the timing of the stressor
to adulthood does not lead to the same effects on the rodent I7
GR promoter, or the human analog GR 1F promoter, methylation
(Alt et al., 2010; Witzmann et al., 2012).
Developmental-stage-dependent vulnerability has also been
documented in human studies, and we will now focus on human
data in the following paragraphs. In extension of the rodent
studies, comparable age-dependent findings are seen for
stress-related changes on DNA methylation of FKBP5, where
GR activation early in neuronal cell development, but not after
differentiation, leads to lasting demethylation of GREs (Klengel
et al., 2013). Another study from our lab investigated the molecular signature of childhood abuse in individuals exposed to adult
trauma and suffering from PTSD (Mehta et al., 2013). We investigated peripheral gene expression and DNA methylation signatures in whole blood in these individuals and found evidence
for distinct underlying biological mechanisms of peripheral
blood gene expression changes between individuals suffering
from PTSD exposed or not exposed to childhood trauma. We
found not only differences in the pattern of gene expression
between childhood-trauma-exposed versus non-exposed individuals but also a higher overall contribution of DNA methylation
changes to the resulting expression patterns in early-life-traumatized individuals. The impact of early trauma on DNA methylation
was concentrated in regions that are important for regulation
of gene transcription, such as binding sites for enhancer or
repressor proteins, but mostly outside of classical promoter regions and that included the 30 UTR and the gene body. Similarly,
childhood, but not adult, socioeconomic status seems to impact
epigenetic profiles (Lam et al., 2012).
These data suggest that the developmental stage of the exposure to environmental risk factors is an important determinant of
their epigenetic effects. This dependence on the age variable is
possibly related to the developmental trajectory of expression of
epigenetic writers.
Direction, Genomic Localization, and Functional Effects
of Trauma-Related Epigenetic Changes
Localization and Direction—Stress-Related Epigenetic
Changes Affect the Whole Genome
A question that has not been addressed in this review up to now
is whether there are unifying concepts of stress-related epigenetic changes, such as a common direction of effects throughout
the genome (e.g., hyper- versus hypomethylation when focusing
on DNA methylation), a clustering of these changes to specific
genomic locations (e.g., promoter versus other regions), and,
finally, whether these changes lead to concerted changes in
gene expression in specific pathways.
The Direction of Stress-Induced Epigenetic
Modifications
Most studies on stress-related epigenetic changes show a highly
diverse response of the epigenome to stress rather than global
1348 Neuron 86, June 17, 2015 ª2015 Elsevier Inc.
up- or downregulation of DNA methylation or histone modifications. Follow-up studies in both rat and human post-mortem
hippocampus tissue of GR promoter methylation with early environmental stressors initially investigated larger genomic regions
surrounding the GR locus (McGowan et al., 2011; Suderman
et al., 2012). These studies revealed that the early environmental
exposure was associated with both hyper- and hypomethylation
across larger stretches of DNA of about 100 kb, and this was
seen both with maternal care in rats as well as with child abuse
in humans. The overall distribution of hyper- and hypomethylation was also shown to be roughly equal. Investigating rat hippocampal tissue, Suderman et al. could show that DNA methylation
changes were mirrored by changes in histone modification, with
transcription-enhancing marks such as histone acetylation and
methylation. DNA methylation increased in exonic regions, and
DNA methylation decreased at promoter regions in rats exposed
to higher maternal care. These studies have been taken to a
genome-wide level by Labonté et al. (2012), who investigated
the promoter methylation profiles in post-mortem hippocampal
tissue of men having experienced childhood abuse or not and
who compared these profiles with RNA expression profiles. Using cell-sorted neuronal and non-neuronal fractions for validation, the authors were able to show that differential methylation
in promoter regions occurs mainly in the neuronal fraction, suggesting a significant impact of childhood abuse on neuronal
epigenetic regulation, specifically in genes related to neuronal
plasticity (Labonté et al., 2012). Moreover, Labonté reported
twice as many hypermethylated promoters than hypomethylated
regions. Other studies have investigated DNA from peripheral
blood. Here, higher methylation levels were reported by Naumova et al. investigating methylation levels using the promotercentric Infinium HumanMethylation27 BeadChip array in a small
study of institutionalized children compared to controls (Naumova et al., 2012). These results are supported by results from
our lab on patients with PTSD that report an increased number
of hypermethylated regions with early trauma at gene loci, which
show transcriptional differences with early trauma (Mehta et al.,
2013). Another study, investigating the effects of early trauma in
peripheral blood DNA using promoter-targeted methylated DNA
immunoprecipitation (MeDIP), reported opposite effects, with
more hypomethylated than hypermethylated regions (Suderman
et al., 2014). These results have to be interpreted with care
because all of the methods used are biased to certain genomic
regions, especially CG dinucleotide (CpG)-rich promoter areas,
and sample sizes were relatively small.
The genome-wide investigations of the epigenetic effects of
early trauma seem to support concerted epigenetic changes
with exposure to early life stress. Sudermann et al. report that
with child abuse, hyper- versus hypomethylation clusters are
of at least 1 Mb in peripheral blood (Suderman et al., 2014). In
another genome-wide study on the effects of childhood
maltreatment on peripheral blood using Illumina 450k methylation arrays, Yang et al. found that 74% of the differential
methylated CpGs are located at low-methylation sites (i.e., sites
with methylation levels below 20%). Those CpGs and mediumlevel methylated CpGs (between 20% and 80%) exhibit higher
methylation levels in response to childhood maltreatment. In
contrast highly methylated CpGs (above 80%) showed the
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opposite effect, with lower methylation levels in maltreated children (Yang et al., 2013).
However, there is currently a lack of understanding how these
diverse effects on epigenetic regulation may arise following an
environmental impact. We can only speculate that inherent
differences in the mechanisms driving the changes exist. As
detailed above, different environments may lead to the activation
of a specific or even multiple signal transduction cascades with
epigenetic changes at regulatory regions in the genome. Subsequently, global changes in gene expression then may induce
epigenetic changes across larger genomic regions.
The Genomic Location of Stress-Induced Epigenetic
Changes
As with the direction of the effects, changes related to early
stress are observed across all genomic regions and are not
concentrated around CpG islands (regions > 200 bp with a
high frequency of CpG sites, often located at promoter regions),
for example. However, as noted above, all studies to date have
to be interpreted with care because no comprehensive methylome-wide sequencing data have been generated from subjects
with early environmental exposure, and all used techniques,
even if assessing on a genome-wide level, are biased to specific
areas in the genome by design. Nonetheless, these studies
suggest that stress-related epigenetic changes may be preferentially located outside of classical CpG islands and promoter
regions. We could show in peripheral blood DNA that childhood-abuse-related differentially methylated regions were
enriched in regions outside the classical gene promoter, such
as gene bodies, and were also less likely to be located in CpG
islands, shores, and shelves (regions up to 2 kb and 4 kb, respectively, around CpG islands) and more likely in regions termed
open sea (isolated regions in the genome that contain fewer
CpGs) (Mehta et al., 2013). A similar distribution of environment-sensitive CpGs was found in a study investigating the
effect of an in utero exposure to a natural disaster on the methylome of offspring, with < 10% of the detected changes in the
CpG island and > 50% in open sea regions (Cao-Lei et al.,
2014). The study by Yang et al. (2013) found low- to mediummethylated CpGs most sensitive to the impact of childhood
maltreatment. These are located in regulatory regions such as
downstream enhancers or CpG island shores, distinct and not
classical promoters, or CpG islands that are often not methylated (Yang et al., 2013). In addition, Teh et al. (2014) showed
that the effect of in utero exposure to variable environmental
conditions results in differential methylation of variable methylated regions that tend to be located just outside of CpG islands,
gene bodies, and intergenic regions (Teh et al., 2014). This suggests a role of more long-range regulatory elements in the epigenetic response to early environmental stressors such as child
abuse, maternal care, and other early environmental factors.
Here additional annotation with functional genomic elements,
such as transcription factor binding sites, as well as better mapping of the 3D structure of chromatin will likely yield important
mechanistic insights.
The Functional Effects on Gene Transcription
Epigenetic DNA regulation by methylation involves the following
basic concept: increased methylation leads to a reduced mRNA
expression, and decreased methylation leads to enhanced tran-
scription. This only reliably occurs at sites close to the transcription start, surrounding the first exon. This is not the case in other
genomic locations, with examples showing that an increased
methylation can facilitate the expression of a certain gene
depending of where methylation occurs (Wu et al., 2010), what
type of methylation is installed (Sérandour et al., 2012), and
which DNA-binding proteins are influenced by this mark (Niesen
et al., 2005). This already implies that the effects of stress-related
epigenetic changes, often located outside the promoter area, will
present variable correlations of DNA methylation and gene
expression changes. For example, our group could show that
although DNA methylation at gene promoters negatively correlated with gene expression, the differentially methylated CpG
sites in the gene body correlated with gene expression both
positively as well as negatively, suggesting a bidirectional functional output of gene body methylation on gene enhancer and
repressor regions. Similar patterns have been observed in other
studies (Mehta et al., 2013; Teh et al., 2014).
An important point to note is that changes in DNA methylation
due to early stress may not directly correlate with baseline gene
expression but could lead to poised states that will determine
future transcriptional responses following a specific environmental stimulus. Such a possibility has been suggested in a
study by Lam et al. where variation in DNA methylation at
specific CpG sites was not associated with baseline gene transcription but with the inflammatory response to ex vivo toll-like
receptor stimulation in peripheral blood monocytes (Lam et al.,
2012).
Another question is whether different types of DNA modifications may lead to different transcriptional effects, such as for
methylcytosine versus hydroxymethylcytosine. Most studies
currently do not distinguish between these two, and assays
based on bisulfite conversion will measure both DNA methylation and hydroxymethylation. Here, future studies investigating
both of these changes in parallel will be most informative. In
addition, DNA methylation outside of CpG dinucleotides may
add an additional layer of epigenetic code not investigated so
far in the context of stress-related changes (Guo et al., 2014).
It also has to be acknowledged that epigenetic changes in
stress-related psychiatric disorders clearly do not have the
magnitude of DNA methylation changes in other fields, such as
cancer research, and rather subtle changes in regulatory regions
and specific neural cell types likely influence the behavioral
phenotype. DNA methylation differences often below 10% are
commonly seen, and these are close to the detection limits of
the assays used. Moreover, the techniques available for detection of DNA methylation on a genome-wide level might introduce
biases with regard to the magnitude of methylation changes and
location by the inherent design of the assays. By using the 450k
Illumina array or methods such as reduced representation
bisulfite sequencing (RRBS) or tiling arrays, researchers might
actually miss relevant signals that are located in areas that are
not covered by the respective assay, as most of these assays
are biased toward CpG-rich regions and promoter areas. In addition, most studies use mixed tissues with, most likely, only a
fraction of cells actually being modified by stress and accounting
for behavioral changes, thus diluting the effect of epigenetic
modification. Here, a novel method that has successfully been
Neuron 86, June 17, 2015 ª2015 Elsevier Inc. 1349
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used to profile neurons is single-cell RNA sequencing, which
could help in identifying the susceptible subset of cells (Zeisel
et al., 2015).
Tissue Specificity of Stress-Induced Changes
Stress is an organism-wide response, with overlapping as well
as distinct systems acting on different organs. Although, for
example, effects of the stress hormone on the glucocorticoid receptor can affect a large number of different tissues, changes
involving specific neural circuit activation will likely not have
cross-tissue correlation. Here, we will discuss the question of
whether peripheral tissue can be used to interrogate effects of
stress in relationship to psychiatric disease.
Tissue Specificity Is Likely Dependent on Mechanisms
by which Stress Induces Changes
Human neuropsychiatric studies bear the disadvantage that the
primary tissue of interest, the brain, is usually not available. It is
still controversial whether findings from peripheral tissues can
be meaningful with regard to pathomechanisms. Studies now
suggest that although a number of signaling pathways are very
specific to the brain, others are common across tissues, and
here extrapolations from one tissue to the other may be possible.
One example is the activation of the GR, which is expressed in
different isoforms across tissues and which is functionally active
through transcriptional activation and repression of up to 10%–
20% of all genes in the human genome (Oakley and Cidlowski,
2013). The activation of the HPA axis by stress leads to a global
increase of cortisol. Although tissue-specific GR binding sites
and poised states have been described, some sites show common activation across tissues (John et al., 2011). GR activation
may thus lead to epigenetic adaptations across tissues. Ewald
et al. (2014) could show that GR agonist exposure leads to correlated DNA methylation changes within the rodent fkbp5 locus in
both blood and brain and that DNA methylation levels in blood
predicted DNA methylation and gene expression of fkbp5 in
the hippocampus. Interestingly, different intronic GREs were
affected in blood and brain.
We have recently demonstrated that the allele-specific demethylation in FKBP5 in peripheral blood cells following childhood
abuse is paralleled by similar findings from in vitro studies of
GR activation in a human hippocampal progenitor cell line. We
observe the demethylation of a functional GRE in intron 7 of
the gene, with the same three CpGs showing demethylation in
peripheral blood cells with child abuse as well as with pharmacological GR activation in neuronal cells. Interestingly, this GRE, as
identified by chromatin immunoprecipitation sequencing (ChIPseq) (Wang et al., 2012), contains six CpG sites, of which three
lie outside the three predicted consensus GRE binding sites
and three lie either directly within or between consensus binding
sites. Only the latter three show this demethylation, further suggesting the importance of GR activation in this process (Klengel
et al., 2013).
Similar cross-tissue effects seem to be observed with the
GR 1F promoter, and changes in DNA methylation within the
NGF1-A binding sites are reported both in post-mortem hippocampus and peripheral blood (Turecki and Meaney, 2014; Zhang
et al., 2013). Whereas the mechanism of this hypermethylation
has been delineated in the hippocampus, it is not clear whether
1350 Neuron 86, June 17, 2015 ª2015 Elsevier Inc.
similar mechanisms, such as activation of NGF1-A and subsequent changes in DNA-binding proteins, also occur in blood
cells. Genome-wide studies in rhesus macaques indicate that
a number of tissues are also likely to be affected by differences
in early-life-rearing experiences, with significant DNA methylation changes observed in both the prefrontal cortex and
T cells. However, the number of specific sites actually overlapping between the two tissues was very limited (Provençal
et al., 2012). Nevertheless, although these examples provide
some evidence for a cross-tissue signature of, in this case,
activation of GR and related stress pathways, specific signatures
in peripheral tissues may not be observable for all psychiatric
disorders.
In conclusion, although early experience most likely affects a
number of tissues, and epigenetic effects could thus be
observed not only in the brain, more studies are necessary to
delineate which changes are tissue-specific and which are
seen across several different tissues. Recent studies suggest
that, in addition to peripheral blood cells, DNA derived from
cheek swabs as well as saliva may also be used in epigenetic
studies for psychiatric disease (Smith et al., 2015). Here, the
collection is easy and often the only possible way to access
DNA in children, but more studies about the potential usefulness
of this tissue are warranted. In fact it has been shown that epigenetic profiles from both saliva and buccal epithelial cell DNA are
distinct from those observed in peripheral blood in the same individual (Jiang et al., 2015; Smith et al., 2015). While the use of
saliva or buccal cell DNA is promising and may allow large-scale
epigenetic studies in epidemiological samples, it is important to
acknowledge that in these tissues, there often is a mixture of
epithelial and blood cells that needs to be accounted for. Bioinformatics methods have been developed with this aim in mind
(Guintivano et al., 2013; Houseman et al., 2012; Jaffe and Irizarry,
2014; Lam et al., 2012).
Finally, peripheral blood cells may indeed give direct mechanistic insights into the brain as a number of studies have now
implicated immune changes as one possible contributor to the
pathophysiology of psychiatric disorders. For example, the
most recent genome-wide association studies (GWAS) metaanalysis in schizophrenia showed an enrichment of variants in
enhancer elements active in immune cells, supporting the hypothesis of immune-related pathologies as risk factors for the
disorder (Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014). In fact, some studies suggest that immune changes can directly influence brain activity (Kronfol and
Remick, 2000), and immune system activation can trigger
stress-related disorders in a subset of patients (Felger and Lotrich, 2013). Stress-induced activation of the immune system
leads to the release of cytokines and other signaling molecules
that in turn can enter the brain and activate or modulate a broad
range of neurotransmitter systems, neuroendocrine function,
synaptic plasticity, and circuits that regulate mood and anxiety
(Capuron and Miller, 2011). Such changes in immune cell
composition but also activation status may directly reflect on
the epigenetic profile. So, whereas correcting for changes in immune cell composition (Houseman et al., 2012) may be important
for some questions, it could also hide effects where immune cell
composition changes are causally related to disease.
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Gene by Environment Interaction as a Unifying Concept
The past years have seen an increasing convergence of the
exploration of genetic and environmental risk factors, acknowledging the well-known impact of the environment and, in
particular, traumatic experiences in childhood on stress-related
disorders (Heim et al., 2010) but also the inter-individual variability of outcomes following exposure to such events. This is
highlighted in an ever-growing interest in gene by environment
interaction (G3E) studies in psychiatry, a field that has gained
momentum following a number of landmark studies by the
team of Caspi and Moffitt (Caspi and Moffitt, 2006) and in which
the interplay of usually common genetic variants with a broad
spectrum of environmental factors on psychiatric outcomes
was investigated. Although most of the current literature focuses
on the combined effects of genetic variants and the detrimental
effects of trauma, negative life events, and their long-term
sequelae on mental health following the diathesis-stress model,
G3E also includes by definition the genetic moderation of the
effects of positive and protective environmental factors (Belsky
and Pluess, 2013). A better understanding of the interplay of
both harmful and advantageous as well as individual and shared
environmental factors with individual genetic variation will
contribute to explain individual differences of risk or resilience
trajectories (Klengel and Binder, 2013; Manuck and McCaffery,
2014).
It has to be noted, though, that G3E studies that rely only on
statistical interactions are fraught with possible methodological
problems that need to be carefully considered when interpreting
these studies (Almli et al., 2014; Dudbridge and Fletcher, 2014;
Duncan and Keller, 2011; Keller, 2014; Manuck and McCaffery,
2014; Munafò et al., 2014). A molecular and systemic understanding of G3E will thus be important to develop improved
methods and assessments to avoid many of the concerns that
may lead to confounding in G3E studies. The integration of
epigenetic mechanisms as outlined in the previous sections
could support molecular models for G3E.
Given the replicated G3E of genetic variants in FKBP5 with
childhood abuse, we asked whether this interaction also influences the epigenetic response to childhood abuse. In fact, we
were able to show that childhood abuse is associated with demethylation of FKBP5 itself in a genotype-dependent response.
Carriers of the allele that confers risk for later psychopathology
also facilitate the demethylation of functional GREs in FKBP5
when individuals were exposed to childhood trauma. In contrast,
carriers of the protective genotype exhibit a more stable epigenetic configuration even when exposed to severe trauma. In
this case, the SNP leads to differential 3D structure of the
gene, with a GRE in intron 2 only coming into direct contact
with the transcription start site in carriers of the risk allele. This
is accompanied by higher FKBP5 induction with stress and
changes in the GR feedback that are associated with higher
cortisol levels following stress. This increased activation of the
GR following early trauma is then followed by a demethylation
of a second GRE in intron 7 and a further increase in stimulated
FKBP5 transcription. In this case, the long-term epigenetic
response is linked to the individual’s genetic predisposition via
subsequent systemic changes in the stress hormone system
(Klengel et al., 2013). The importance of allele-specific epige-
netic changes with environmental exposure is supported on a
broader scale by the study of Teh et al. These authors investigated the genome and epigenome of neonates and found over
1,400 regions that were highly variable across individuals. A
quarter of these were best explained by genetic variation only,
while three-quarters were explained by an interaction between
genotype and in utero environment, suggesting that allele-specific environmental effects occur throughout the genome and
will only accumulate with age (Teh et al., 2014).
Severe stress and trauma may induce allele-specific epigenetic changes by different mechanisms. This can include very
specific effects of sequence changes in a transcription factor
binding site of a specific gene facilitating or impeding epigenetic
effects following the activation of the transcription factors or
direct changes of CpGs into other dinucleotides, with the
possibility of propagating sequence-specific DNA methylation
changes (Mill et al., 2008). Furthermore, DNA variants can alter
the stress-induced activation of epigenetic writers. On the other
hand, there may also be indirect effects, such as for FKBP5,
where the DNA sequence changes lead to changes in the stress
hormone system and to subsequent differences in the epigenetic
effects of GR activation.
Epigenetic Modifications as a Potential Target of
Psychiatric Therapy
The reversibility of environmentally induced epigenetic marks
establishes the possibility to directly or indirectly interfere with
these imprints to reverse or ameliorate disease status. It should
be noted, however, that the use of drugs targeting epigenetic
modifications in psychiatry is highly speculative at the moment.
The bidirectional regulation of DNA methylation and other epigenetic marks leads to the question whether unidirectional drugs
such as DNMT modulators or HDAC inhibitors can actually be
used for the treatment of stress-elicited psychiatric disorders
(Narayan and Dragunow, 2010; Szyf, 2009). At the same time,
currently used antidepressants and related drugs, such as valproic acid, possess epigenetic effects (Göttlicher et al., 2001).
Although the growing literature—in particular, from rodent
models—suggests the involvement of epigenetic mechanisms
in disease development, the location and direction of the epigenetic alterations are highly variable, challenging the idea of a
single or even multiple epigenetic drugs that could restore the
complex pattern of epigenetic states to a pre-disorder or resilient
state. Among the major challenges is the fact that the specific
targeting of epigenetic marks within the genome and even within
the gene loci themselves is crucial to the functional outcome on
gene regulation, as marks have opposing effects on transcription
depending on their location. In addition, a safe manipulation of
epigenetic states needs to be specific to the cell type and brain
region that is responsible for disease development, which is
often unknown. Although we learned much from using DNMT
pan-inhibitors or HDAC inhibitors with respect to basic concepts
of memory, learning, and environmental epigenetics (BahariJavan et al., 2014; Zovkic and Sweatt, 2013), these global
modulators are not likely to deliver the temporal and spatial
needs for a targeted epigenetic influence of psychiatric disorders. It is also possible that the administration of such drug inhibitors will be associated with severe side effects, possibly also
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with long-term effects. It remains an open question whether
different subtypes of HDACs and DNMTs will have distinct
cell-specific roles in stress-related disorders, which may be addressed by subtype-specific inhibitors delivered to the cell type
of interest. However, such specific drugs are lacking (Fischer,
2014).
Epigenetic modifiers might be used not only to change signatures in general or at specific genes but also to guide the
epigenome toward a higher plasticity or to re-open windows of
enhanced plasticity to facilitate the reversion of pathological
epigenetic adaptations by conventional medications or psychotherapy (Sweatt, 2009). Evidence for a successful application of
this strategy in humans was generated by using the HDAC inhibitor valproate for relearning absolute pitch, the ability to identify
the correct pitch of a sound without reference. The authors
propose that interfering with HDAC leads to a re-opening of a
window of increased plasticity in the auditory system (Gervain
et al., 2013). Finally, epigenetic studies may give insight into
basic disease mechanisms and pathways disturbed in psychiatric disorders. These may then be directly targeted. If such an
approach is tried with FKBP5, for which small molecule antagonists have been developed, that could reduce the genetically and
epigenetically driven overactivity of this gene in trauma-exposed
risk allele carriers. Early in vivo studies of these antagonists show
promising behavioral effects in laboratory animals (Gaali et al.,
2015).
In addition to being a potential therapeutic target, epigenetic
marks could be useful in predicting and also monitoring therapeutic approaches (Guintivano et al., 2014a, 2014b; Powell
et al., 2013; Roberts et al., 2014; Yehuda et al., 2013). For
example, the peripheral blood methylation status at the GR locus
that is altered in response to trauma exposure may predict the
response to prolonged exposure psychotherapy, as proposed
in a small pilot treatment study. This suggests that epigenetic
changes in response to the traumatic event could predict further
environmental modification, in this case by psychotherapeutic
intervention (Yehuda et al., 2013). Roberts et al. observed an
increasing DNA methylation at the serotonin transporter locus
in individuals with anxiety disorders responding to cognitive
behavioral therapy as compared to non-responders, who actually showed a decrease in DNA methylation (Roberts et al.,
2014). Moreover, in a recent study by Powell et al., DNA methylation at the IL6 locus predicted response to classical antidepressant treatment in the Genome-Based Therapeutic Drugs for
Depression (GENDEP) cohort, suggesting that epigenetic
profiling before treatment could be used to reduce the likelihood
of treatment failure by selecting the appropriate drug (Powell
et al., 2013). Although these studies provide interesting evidence
for potential biomarkers, careful interpretation of their relevance
for central mechanisms is necessary.
Open Questions and Future Directions
Epigenetic modifications are emerging as an integral part of the
molecular events leading to the development of stress-related
psychiatric disorders, in particular in interaction with environmental adversities such as childhood trauma and genetic predisposition. We are only at the very beginning of understanding the
temporal and spatial complexity of different layers of epigenetic
1352 Neuron 86, June 17, 2015 ª2015 Elsevier Inc.
regulation in psychiatric disorders, with animal models of stressrelated disorders providing invaluable insights into the possible
underlying mechanisms. Even though we understand basic
mechanisms in reprogramming epigenetic patterns in response
to environmental factors, the inherent complexity of these events
in combination with genetic variations prevent, to date, a specific
therapeutic intervention based on these principles. Most critical
will be longitudinal studies delineating the chain of molecular
events following stress and trauma leading to either risk or resilience and identifying the relevant cell types and tissues. Here,
birth cohort studies with longitudinal samples of several tissues
provide important insights but will need to be complemented by
animal experiments for access to brain tissue.
Which Regions in the Genome Are Epigenetically StressSensitive?
While we have focused on exemplary mechanisms and target
genes, stress-induced activation of epigenetic mechanisms
will remodel chromatin on a global scale but, nevertheless,
in very specific stress-responsive regions. Here, the current
progress in understanding the basic mechanisms of, e.g., DNA
methylation need to guide efforts to apply these concepts to
stress-related psychiatric disorders. For example, overall DNA
methylation is reduced in regulatory regions compared to the
majority of fully methylated cytosines across the genome, and
Stadler et al. (2011) identified genomic regions that are characterized by a comparatively low density of CG dinucleotides
(CpGs), open chromatin marks, and enhancer activity, which
are prone to epigenetic modification by transcription factor
binding. Here, the developmentally dependent activity of DNAbinding proteins may shape the DNA methylation profile in these
regulatory regions of the genome (Blattler and Farnham, 2013;
Feldmann et al., 2013; Stadler et al., 2011). However, the mechanisms underlying methylation patterns around specific transcription factor binding sites in regions with a low CpG density
remain unclear (Baubec and Schübeler, 2014). Importantly, unbiased, genome-wide methods will be necessary to map these
mechanisms, and integrative approaches combining the mapping of several epigenetic mechanisms with RNA expression in
different cell types will be critical for a better understanding of
these mechanisms (Kundaje et al., 2015). In the context of
G3E, it will also be important to delineate how epigenetic mechanisms contribute to the regulation of long-range enhancers and
the impact of genetic variants in these regions on altered chromosomal looping and transcription regulation. Long-range enhancers have gained increasing importance in schizophrenia
(Bharadwaj et al., 2014; Roussos et al., 2014) and will likely be
highly relevant in stress-related disorders.
Cell Specificity of Epigenetic Changes and Relationship
to Systemic Changes
As mentioned above, all current epigenetic studies are using
mixtures of cells. In the future, single-cell profiling, as possible
for RNA expression (Zeisel et al., 2015), may lead to important insights, which changes are specific, and which are shared across
different cell types and in extension tissues. In human studies,
lack of online monitoring of changes in neurons will always
hamper a deeper understanding of the dynamic epigenetic
changes associated with the development of risk versus resilience to stress- or trauma-related disease. Here, patient-derived
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pluripotent cell models and brain organoids, as briefly discussed
below, may offer some insights. In addition, a number of studies
have tried to correlate peripheral epigenetic changes with
brain function using neuroimaging approaches. For example,
increased promoter methylation of the serotonin transporter
gene in peripheral blood predicted increased threat-related
amygdala reactivity, and increased methylation of the same
sites predicted decreased mRNA expression in post-mortem
amygdala tissue (Nikolova et al., 2014). To date, however, the
mechanistic insights from studies correlating peripheral epigenetic changes with brain imaging are limited. The development
of positron emission tomography ligands based on compounds
targeting epigenetic mechanisms, such as, for example, HDAC1
inhibitors, may also further our understanding of the contribution
of these mechanisms in stress-related psychiatric disorders
(Wang et al., 2013).
Manipulating Epigenetic Pattern through Genomic
Engineering
The extension of using the protein-guided transcription activator-like effectors (TALEs) and the RNA-guided clustered
regularly interspaced short palindromic repeats (CRISPR)/
Cas9 systems to manipulate epigenetic marks may allow
temporally and spatially controlled alterations of not only genetic sequences but also epigenetic signatures at DNA loci
known to play a role in stress-related disorders (Maeder et al.,
2013). By fusing epigenome-modifying proteins to the Cas9
protein, the RNA-guided system could allow the targeted
manipulation of epigenetic states and thus the design of studies
to understand the consequences of, for example, specific DNA
methylation changes in response to early life stress. Given a
successful control over off-target effects that would strongly
limit the precise usage of this system, this may allow a multiplexed targeting of specific loci that are related to the pathophysiology of stress-evoked mental disorders, but it is unclear
at the moment if the manipulation of even multiple loci across
the genome is sufficient to revert the concerted and multilayered epigenetic changes across the genome in response to
childhood abuse.
Induced Pluripotent Stem Cells—A Possible Model for
Studying Neuronal Epigenetics in Stress-Related
Disorders?
The inaccessibility of neuronal tissue is one major drawback in
human epigenetic studies, although similarities between central
nervous and peripheral tissue can be found, as discussed above,
and immune cells may also be of primary interest. The generation
of neuronal cells using induced pluripotent stem cells (iPSCs)
also represents a promising future avenue for this kind of
research. Brennand et al. were able to provide evidence that
neurons created from fibroblast cells from schizophrenia patients recapitulate some cellular and molecular phenotypes
related to schizophrenia (Brennand et al., 2011). While the genetic identity of the derived cells with the patient enables personalized investigations, it is unclear at the moment the degree to
which the epigenetic profile will resemble that of the patient
(Hjelm et al., 2013). Genetically driven epigenetic changes are
likely to be recapitulated, possibly also as a response to different
developmental programs. It is not known, however, whether acquired environmental changes will also be observed in the iPSCs
(Roessler et al., 2014). In addition, especially for further differentiations to neurons, only a very short span of cell and organism
development can be recapitulated in the dish, so epigenetic
differences only triggered during later development or after birth
may be very difficult to model in these cells. Overall, more
research in this area will be necessary for a full assessment of
the power of these model systems.
Epigenetic Effects In Utero and across Generations
Although this review focuses on postnatal epigenetic effects of
stress, developments in recent years suggest that maternal
stress during pregnancy can lead to early epigenetic programming of the fetus (Oberlander et al., 2008) and that parental
stress exposure prior to conception may be transmitted to the
next generation in the germline via epigenetic mechanisms
(Dias and Ressler, 2014; Rodgers et al., 2013), with the findings
of transgenerational inheritance in mammals being controversially discussed (Grossniklaus et al., 2013). So far, the relevance
of ancestral environmental exposure for psychiatric disorders in
decedents remains elusive. Such effects of in utero exposure to
stress and transgenerational effects, while only convincingly
documented in animals so far, need to be kept in mind when
interpreting human studies where data are often collected
cross-sectionally or do not include information on pregnancy
or parental stress and trauma.
Overall, epigenetic mechanisms and their role in stress-related
psychiatric disorders are a rapidly developing field, may yield
important insights in the pathophysiology of these disorders,
and may provide a mechanistic understanding of G3E. As
further studies fill the gap in cell- and tissue-specific investigations over time and methylome-wide, the associated epigenetic
changes may offer the possibility for development of biomarkers
and novel treatment strategies for stress-related psychiatric
disorders.
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