Review
DNA methylation: a permissive mark in memory
formation and maintenance
Ana M.M. Oliveira
Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg,
69120 Heidelberg, Germany
DNA methylation was traditionally viewed as a static mechanism required during cell fate determination. This view has been
challenged and it is now accepted that DNA methylation is involved in the regulation of genomic responses in mature
neurons, particularly in cognitive functions. The evidence for a role of DNA methylation in memory formation and main-
tenance comes from the increasing number of studies that have assessed the effects of manipulation of DNA methylation
modifiers in the ability to form and maintain memories. Moreover, insights from genome-wide analyses of the hippocampal
DNA methylation status after neuronal activity show that DNA methylation is dynamically regulated. Despite all the ex-
perimental evidence, we are still far from having a clear picture of how DNA methylation regulates long-term adaptations.
This review aims on one hand to describe the findings that led to the confirmation of DNA methylation as an important
player in memory formation. On the other hand, it tries to integrate these discoveries into the current views of how mem-
ories are formed and maintained.
It is now well established that gene transcription activation and
de novo protein synthesis are required for long-term forms of
neuronal plasticity and memory (Alberini and Kandel 2015;
Benito and Barco 2015). Changes in the expression of growth
factors, ion channels, ligand-gated receptors, and structural pro-
teins are necessary to support long-lasting functional and struc-
tural changes within a neuronal circuit. This functional and
structural remodeling is believed to underlie memory formation
and maintenance.
An increasing amount of evidence has convincingly demon-
strated that epigenetic mechanisms play an important role in
cognitive functions through the regulation of transcriptional re-
sponses (Barrett and Wood 2008; Graff and Tsai 2013; Zovkic
et al. 2013). Epigenetics include mechanisms of chromatin regu-
lation via covalent modifications of the DNA, such as DNA meth-
ylation, hydroxymethylation, and demethylation (Suzuki and
Bird 2008; Bhutani et al. 2011; Branco et al. 2012; Moore et al.
2013) as well as histone post-translational modifications (Strahl
and Allis 2000).
DNA methylation was traditionally viewed as a static mech-
anism with important roles in transcription repression in cell
fate determination, imprinting, female X-chromosome inactiva-
tion, and silencing of transposons during early development
(Suzuki and Bird 2008). This view has been challenged and it is
now accepted that DNA methylation plays also a critical role in
the regulation of genomic responses in mature neurons. This re-
view highlights the requirements for the enzymes responsible
for DNA methylation and demethylation in memory encoding.
In addition, it describes the data demonstrating dynamic changes
in the methylation status of postmitotic neuronal DNA in re-
sponse to experience. This review further aims to integrate these
findings into the current views of how memories are formed
and maintained.
Pharmacological and genetic evidence for
DNA methylation in memory formation
and maintenance
Early findings from the Sun and Greenberg laboratories showed
that DNA methylation and its reader methyl-CpG-binding pro-
tein 2 (MeCP2) are involved in the regulated expression of brain-
derived neurotrophic factor (Bdnf) upon neuronal activity in pri-
mary neuronal cultures (Chen et al. 2003; Martinowich et al.
2003). These studies provided the first hint that DNA methylation
plays a role in the regulation of neuronal activity-dependent gene
transcription and possibly in the mechanisms of synaptic plastic-
ity. They were corroborated and further expanded by subsequent
in vivo pharmacological and genetic studies targeted at the ma-
nipulation of DNA methylation processes that showed altered
plasticity mechanisms such as long-term potentiation (LTP) and
cognitive abilities.
Pharmacological studies
Pharmacological inhibition of DNA methyltransferases (Dnmts)
consistently resulted in impairments in plasticity and memory
formation. Hippocampal LTP induction and maintenance were
impaired in the presence of the Dnmt inhibitors, zeburaline,
and 5-aza-2-deoxycytidine (5-AZA) (Levenson et al. 2006). Also,
amygdalar LTP was shown to be impaired by 5-AZA treatment
(Monsey et al. 2011). Infusions into the amygdala of 5-AZA or
RG108 (a nonnucleoside inhibitor of Dnmts) (Brueckner et al.
2005) affected memory-related neurophysiological responses in
awake rats (Maddox et al. 2014). Similarly to its effects on synaptic
plasticity, pharmacological inhibition of Dnmts impacted memo-
ry formation. Miller and Sweatt (2007) showed that infusions of
zeburaline or 5-AZA into hippocampal area CA1, a region critical
Corresponding author: oliveira@nbio.uni-heidelberg.de
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for memory formation, immediately after contextual fear condi-
tioning training reduced rats’ ability to form long-term memory
(Miller and Sweatt 2007). These findings were confirmed in a
study by the same group in which the authors showed that zebura-
line or RG108 intra-CA1 infusions prior contextual fear condi-
tioning training impairs long-term memory formation but not
short-term memory (Lubin et al. 2008). Moreover, hippocampal
zeburaline treatment decreased long-term, but not short-term
place field stability (Roth et al. 2015). These studies have been ex-
tended to other forms of memory and brain regions. Intra-
amygdalar infusion of 5-AZA or RG108, for instance, induced
long-term memory impairments in auditory fear conditioning
without affecting short-term memory (Monsey et al. 2011;
Maddox et al. 2014). In addition, hippocampal or cortical admin-
istration of RG108 impaired long-term memory in a spatial object
recognition test (Mitchnick et al. 2015). Interestingly, DNA meth-
ylation has also been shown to be required for memory specificity
in a form of conditioning in the honeybee (Biergans et al. 2012,
2016). Thus, DNA methylation appears to be a conserved mecha-
nism required for long-term memory formation across brain re-
gions and species. The selective effect on long-term memory
further suggests that DNA methylation is involved in the regula-
tion of learning-dependent gene transcription required for long-
term memory formation.
The striking majority of studies have focused on the role of
DNA methylation in memory formation; however, Miller et al.
(2010) also investigated whether DNA methylation may also un-
derlie the maintenance of memory. In this study, the authors fo-
cused on the anterior cingulate cortex (ACC), a brain region
thought to hold the representation of remote memories upon
their formation in the hippocampus (Frankland et al. 2004). The
authors observed a persistent change in the methylation status
of the memory suppressor gene calcineurin (CaN) in the ACC after
contextual fear conditioning that was associated with reduced
CaN expression levels (Miller et al. 2010). Moreover, infusion of
Dnmt inhibitors into the mouse ACC 30 d after training disrupted
remote memory in a contextual fear conditioning task. These
findings suggest the intriguing possibility that—in contrast to
the processes underlying memory formation in the hippocampus,
where a short-term up-regulation of Dnmt activity is required—
the maintenance of memory via the ACC requires long-lasting
DNA methylation changes and continuous Dnmt activity
(Miller et al. 2010).
Genetic studies
The pharmacological evidence supporting a role for DNA methyl-
ation in memory formation and maintenance has been corrobo-
rated by genetic approaches targeted at disrupting the function
of DNA methyltransferases. For example, two studies used
Dnmt1 and Dnmt3a conditional knockout mice to address the
role of these enzymes in memory formation (Feng et al. 2010;
Morris et al. 2014). In the conditional knockout mouse used in
both studies, the gene deletion is restricted to the forebrain and
to the postnatal age. Feng and colleagues found that only in the
absence of both Dnmt1 and Dnmt3a did the mice exhibit memory
and LTP impairments. In contrast, Morris et al. found that knock-
out of Dnmt3a but not Dnmt1 elicited cognitive impairments.
The reason for the discrepancy between the two studies is not
clear. However, differences in the gender and age of the mice
used and the genetic background might account for the distinct
findings. In other studies, temporally and spatially controlled
genetic manipulations have confirmed a role for Dnmt3a in hip-
pocampus-dependent long-term memory formation (Oliveira
et al. 2012, 2015; Mitchnick et al. 2015). The Dnmt3a genomic lo-
cus contains two genes, Dnmt3a1 and Dnmt3a2: the promoter of
Dnmt3a2 is located in the 6th intron of Dnmt3a and gives rise to
a transcript encoding the Dnmt3a2 protein, which is identical
to Dnmt3a1 except that it lacks 219 amino acids at its N-terminus
(Chen et al. 2002). Dnmt3a2 is a neuronal activity-regulated im-
mediate early gene whose expression is strictly dependent on
NMDA receptor activation. In contrast, Dnmt3a1 appears to be
expressed at higher levels, and its expression is not regulated by
neuronal activity (Oliveira et al. 2012). Other studies have ob-
served the learning-dependent activation of Dnmt3a expression.
However, the isoform being analyzed was not distinguished
(Levenson et al. 2006; Miller and Sweatt 2007; Monsey et al.
2011; Morris et al. 2014; Mitchnick et al. 2015).
Dnmt3a2 knockdown in the adult mouse hippocampus im-
pairs hippocampal long-term memory formation and extinction
(Oliveira et al. 2012, 2015). Moreover, basal and learning-induced
Dnmt3a2 expressions are reduced in the aging hippocampus and
reestablishing Dnmt3a2 levels rescues aging-dependent cognitive
decline (Oliveira et al. 2012). Furthermore, a facilitating effect of
Dnmt3a2 in cognitive function was not only observed in aged
mice (Oliveira et al. 2012) but also in young adult cognitively nor-
mal mice (Oliveira et al. 2015). Whether Dnmt3a1 also plays a role
in memory formation remains to be investigated.
Interestingly, Mitchnick and co-authors found a dissociation
in the function of the different Dnmts between the hippocampus
and the perirhinal cortex. More specifically, hippocampal knock-
down of Dnmt3a caused long-term memory deficits in an object-
place recognition test. In contrast, knockdown of Dnmt1 in the
perirhinal cortex, but not of Dnmt3a or Dnmt3b, caused long-
term memory impairments (Mitchnick et al. 2015). These results
suggest that different epigenetic mechanisms may operate dif-
ferently within distinct brain regions during long-term memory
formation, and highlights the need to analyze each region
individually.
Taken together, pharmacological and genetic evidence
emerging from different laboratories consistently showed that in-
hibition of Dnmt function, and particularly that of Dnmt3a, im-
pairs hippocampal long-term plasticity and memory formation.
Thus, the activity of Dnmts appears to be permissive for long-term
memory formation. These findings appear counterintuitive at first
given the view that DNA methylation of promoter regions is
associated with transcriptional repression, according to which
blocking DNA methylation should enhance, not impair, memory
formation. However, the newly emerging picture is that the func-
tion of DNA methylation is dependent on the genomic location.
How DNA methylation regulates genomic responses in the con-
text of plasticity is not totally understood.
DNA methylation and expression
of plasticity-related genes
Given the function of DNA methylation in transcriptional regula-
tion, several studies have focused on the role of DNA methylation
in regulating the expression of memory-related genes during
long-term memory formation. DNA methylation changes in these
genes upon learning have indeed been identified (Miller and
Sweatt 2007; Lubin et al. 2008; Miller et al. 2010; Mizuno et al.
2012). These included both de novo methylation and demethyla-
tion, demonstrating that DNA methylation is dynamic, and pro-
viding evidence for the existence of active demethylation in
postmitotic cells. Memory formation requires both the activation
of memory promoting genes and the inhibition of memory
suppressor genes (Abel and Kandel 1998; Abel et al. 1998), and
DNA methylation/demethylation has been proposed as a poten-
tial molecular mechanism for achieving this dual regulation. In
particular, pioneer work from the Sweatt laboratory reported pro-
moter demethylation in genes required for memory formation,
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such as reelin, but also the methylation of promoters of memory
suppressor genes such as the phosphatase PP1 (Miller and Sweatt
2007).
Later findings from Miller et al. (2010) suggest that this
mechanism may not be restricted to memory formation but
may also be involved in regulating memory persistence. Specifi-
cally, the authors observed a significant persistent increase in
the methylation of the memory suppressor gene CaN in the
ACC after contextual fear conditioning.
An increasing body of evidence convincingly demonstrates
the existence of active DNA demethylation in postmitotic neu-
rons. Recently, it has been shown that the ten-eleven-trans-
location (Tet) family of dioxygenases is involved in DNA
demethylation via the formation of an intermediate, 5-hydroxy-
methylcytosine (5 hmC), in the adult mouse brain (Guo et al.
2011b). Interestingly, 5 hmC has also been proposed to be an epi-
genetic mark in its own right (Branco et al. 2012; Li et al. 2014).
Consistent with an important role for Tet enzymes in cognitive
function, a few studies have already demonstrated memory and
extinction impairments upon interference with Tet family mem-
bers (Kaas et al. 2013; Rudenko et al. 2013; Li et al. 2014). Other
molecules proposed to be involved in active DNA demethylation
include members of the growth arrest and DNA-inducible 45
(Gadd45) family and Methyl-CpG-binding domain protein 4
(Mbd4) ( for recent reviews on mechanisms of active DNA de-
methylation see Gavin et al. 2013; Li et al. 2013; Lister and
Mukamel 2015). Gadd45b and -g are known to be regulated by
neuronal activity and learning (Ma et al. 2009; Leach et al.
2012; Sultan et al. 2012). There is also indication from two studies
that used Gadd45b global knockout mice (Gupta et al. 2005) that
this putative demethylase plays a role in learning and memory
(Leach et al. 2012; Sultan et al. 2012). Because the two studies
obtained contrasting results, however, it is not clear whether
Gadd45b facilitates or constrains fear memory formation.
Genome-wide analysis of DNA methylation
changes triggered by neuronal activity
Most studies examining DNA methylation changes after neuronal
activity or learning have predominantly focused on the expres-
sion analysis of preselected genes and in the methylation analysis
of their promoters. Genome-wide studies show that this type of
analysis is rather limited, because most activity-regulated changes
occur outside gene promoters (Guo et al. 2011a; Halder et al.
2016). Activity-dependent changes in DNA methylation in the
adult dentate gyrus have been shown to be enriched at both inter-
genic and intronic regions (Guo et al. 2011a). This finding was
recently confirmed by another study that examined DNA methyl-
ation changes in the hippocampal CA1 region and the ACC after
contextual fear conditioning (Halder et al. 2016). Thus, genome-
wide analysis appears necessary to obtain a clearer picture of how
DNA methylation plays a role in memory formation. In agree-
ment with the genetic evidence that the enzymes responsible
for de novo DNA methylation or demethylation are required for
memory formation, and consistent with the idea that the two pro-
cesses are necessary for long-term neuronal adaptations, these
studies also demonstrated rapid de novo DNA methylation and
demethylation in the hippocampus in response to increased neu-
ronal activity (Guo et al. 2011a) or fear conditioning (Halder et al.
2016). Halder and colleagues found that learning-induced DNA
methylation changes significantly colocalized with H3K27ac re-
gions, indicating that at least some of these changes are targeted
to cis-regulatory regions. In agreement with a possible role in reg-
ulating gene expression, a fraction of the genes that are differen-
tially expressed after fear conditioning were also differentially
methylated. In this study, DNA methylation analysis was per-
formed in the CA1 region of the hippocampus, an area critical
for memory formation, and in the ACC, a region important for
long-term memory storage. Consistent with these functions, sig-
nificant DNA methylation changes were identified in CA1 only
at early time points after learning (1 h) and not at 4 wk. In con-
trast, in the ACC, DNA methylation changes were detected at
both time points. These findings are in agreement with the study
of Miller et al. (2010) that found persistent changes in the DNA
methylation status of the CaN gene promoter in ACC after contex-
tual fear conditioning. Interestingly, in the study by Halder et al.
(2016), gene ontology analysis indicated that the differentially
methylated and expressed genes in CA1 were enriched for the
functional categories “ion channels” and “transcription regula-
tion.” At 4 wk after fear conditioning, differentially methylated
and expressed genes in the ACC were enriched for functional cat-
egories associated with morphology regulation. These findings
may indicate that DNA methylation mechanisms regulate synap-
tic transmission and gene transcription during hippocampal
memory formation and cortical morphological arrangements re-
quired for memory maintenance.
Both the Guo et al. (2011a) and Halder et al. (2016) studies
found that activity-dependent changes in DNA methylation
were also associated with the generation of alternative splice var-
iants. This is in agreement with a previously established role for
DNA methylation in RNA splicing regulation (Shukla et al.
2011) and suggests that this mechanism may also play a role dur-
ing memory formation.
Although DNA methylation changes were enriched in inter-
genic and intragenic regions (Guo et al. 2011a; Halder et al. 2016)
and although they colocalized with putative cis-regulatory re-
gions (Halder et al. 2016), these methylation changes did not al-
ways correlate with gene transcription changes (Guo et al.
2011a; Li et al. 2014), indicating that the role played by neuronal
activity triggered de novo DNA methylation and demethylation
may not only reside in direct regulation of expression of associat-
ed genes (Guo et al. 2011a) or even always be associated with tran-
scriptional changes. Moreover, DNA methylation changes may
precede or persist beyond the time when transcriptional changes
are observed (Li et al. 2014). As previously proposed (Guo et al.
2011a; Baker-Andresen et al. 2013), an intriguing possibility is
that DNA methylation changes, in addition to directly regulating
gene expression at some loci, may also be responsible for creating
a primed, more permissive environment for subsequent genomic
regulation. In such a scenario, a “primed genome” could be more
readily activated, inducing stronger or faster transcriptional re-
sponses. Priming mechanisms could include, for instance, setting
the environment for transcription factor binding (Domcke et al.
2015; Schubeler 2015). Also, de novo DNA methylation could pro-
vide a substrate for subsequent epigenetic marks. Consistent with
this idea, it was recently observed that Dnmt3a knockout leads to
reductions in both 5 mC and 5 hmC, suggesting that de novo DNA
methylation generates the 5 mC substrate for the activity of en-
zymes responsible for DNA hydroxymethylation, such as Tets
(Colquitt et al. 2014). Moreover, an interplay between DNA meth-
ylation and histone post-translational modifications (Vaissiere
et al. 2008) may also occur. Evidence for such interaction during
memory consolidation already exists (Miller et al. 2008; Monsey
et al. 2011). Miller et al. (2008) and Monsey et al. (2011) showed
that pharmacological inhibition of Dnmts impaired training-
dependent histone H3 acetylation, and that DNA methylation
and histone acetylation may work in concert during fear memory
consolidation in the hippocampus and amygdala, respectively.
In order to thoroughly analyze the interdependence and
timecourse of the various epigenetic marks, it would be interest-
ing to perform a detailed temporal analysis after a learning event
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of the various epigenetic marks in conjunction with transcription
factor occupancy and gene expression studies. As technology
stands, the costs and labor associated with such a study are unfor-
tunately prohibitive.
Possible roles for DNA methylation in memory
formation and maintenance
Cellular-level memory consolidation
The formation of long-term memory is strictly dependent on the
activation of gene transcription and de novo protein synthesis
(Alberini and Kandel 2015). A learning event, such as exposure
to a new environment or training in a fear conditioning test, trig-
gers rapid activation of transcription in the hippocampus. This
early transcriptional response, which occurs within the first
hour after the learning stimulus, is dominated by the expression
of immediate early genes (IEGs). The expression of IEGs is depen-
dent on constitutively expressed transcription factors (TFs) such
as cAMP responsive element binding protein (Creb), myocyte en-
hancer factor-2 (Mef2), and serum response factor (SRF) that are
activated by post-translational modifications downstream from
signaling cascades regulated by neuronal stimulation. Among
the rapidly regulated IEGs are a number of TFs, including FBJ oste-
osarcoma oncogene (c-Fos), neuronal PAS domain protein 4
(Npas4), and early growth response 1 (Egr1), that are responsible
for driving a secondary transcriptional
response and regulating the expression
of effector genes. The effectors genes, in
turn, are thought to be required for the
plastic changes associated with learning
and memory (Benito and Barco 2015).
Both pharmacological inhibition of the
first transcriptional wave (for review, see
Korzus 2003) and interference with the
function of activity-regulated TFs induce
robust impairments in memory forma-
tion (Jones et al. 2001; Fleischmann
et al. 2003; Katche et al. 2010; Rama-
moorthi et al. 2011), demonstrating the
importance of this step. The high rele-
vance of this first event for the formation
of memories has resulted in an almost
exclusive research focus on this early
transcriptional response. However, a
few studies have also described a second
wave of expression (8–24 h after learn-
ing) for some activity-regulated genes
in the hippocampus (Arc, cFos, Egr1,
Bdnf) (Igaz et al. 2002; Bekinschtein
et al. 2007; Katche et al. 2010; Nakayama
et al. 2015). Interestingly, only those
memories that are persistent exhibit a
second wave of IEG expression (Katche
et al. 2010), and inhibition of this wave
impairs memory maintenance, but not
memory formation (Bekinschtein et al.
2007; Katche et al. 2010; Nakayama
et al. 2015). Together, these findings sug-
gest that additional rounds of trans-
cription and protein synthesis may be
necessary for the persistence of memory.
Moreover, although merely speculative
due to the lack of experimental evidence,
the multiple rounds of transcription acti-
vation may reflect the communication between the hippocampus
and neocortical regions that is believed to occur during the pro-
cess of systems-level memory consolidation (see below).
As described in the above sections, pharmacological and ge-
netic manipulation of DNA methylation modifiers demonstrates
their requirement in memory formation and maintenance.
Moreover, neuronal activity induces genome-wide de novo DNA
methylation and demethylation in the adult mouse hippocam-
pus. Therefore, it is now well accepted that DNA methylation
mechanisms play a role in memory formation and maintenance.
However, a clear picture of how DNA methylation regulates mem-
ory processes is still elusive. Bringing together the current knowl-
edge of the mechanisms underlying memory formation and
maintenance and the findings concerning DNA methylation,
I propose that DNA methylation players that are activated by
neuronal activity (including Dnmt3a2, Gadd45b/g, and possibly
others) could have at least a twofold role in memory formation
and maintenance in the hippocampus. They could, on the one
hand, generate a primed and more permissive epigenome state
that could facilitate future transcriptional responses and on the
other hand, directly regulate the expression of genes that set the
strength of the neuronal network connectivity, this way altering
the probability of reactivation of the same network (Fig. 1).
In the first wave of learning-dependent transcription, the ex-
pression of the genes coding for Dnmt3a2 (Oliveira et al. 2012;
DVC Brito and AMM Oliveira, unpubl.), Gadd45b/g (Leach et al.
2012; Sultan et al. 2012; DVC Brito and AMM Oliveira, unpubl.),
Figure 1. Hypothetical model for the regulation of hippocampal transcriptional responses during
memory formation and maintenance by DNA methylation-dependent mechanisms. Gene transcription
activation and de novo protein synthesis are necessary for the formation and maintenance of memory.
(1) Following a learning stimulus, an initial transcription wave is induced. The basal epigenetic state of
the neuron likely dictates the permissiveness for the activation of the first wave of transcription. For in-
stance, DNA demethylases (such as Tet1 [Rudenko et al. 2013] and possibly others) may be required to
hold the promoter and/or enhancer of activity-regulated genes in a hypomethylated state in order to
permit rapid activation of transcription. (2) The first wave of transcription includes the up-regulated ex-
pression of activity-dependent transcription factors (TFs) (cFos, Npas4, Egr1) and DNA methylation
modifiers (Dnmt3a2 (Oliveira et al. 2012), Gadd45b/g (Ma et al. 2009; Leach et al. 2012; Sultan
et al. 2012), and possibly others). These TFs and DNA methylation modifiers may work in concert to
regulate the expression of downstream effector molecules. (3) In addition to regulating early learning-
driven transcriptional responses, DNA methylation players may also modify the epigenome, priming it
(4) for later waves of gene expression (for instance, second waves of IEG expression, proposed to be
important for memory maintenance), or (5) for responses triggered by subsequent stimulations
(such as memory recall or the presentation of a new stimulus). (6) Finally, DNA methylation players,
the expression of which are not regulated by neuronal activity, could instead be s ubject to regulation
at the level of activity, stability, or subcellular localization (possibly via post-translational modifications),
thus providing for another contribution to the learning-triggered transcription responses and/or epige-
nome priming. TFs, transcription factors; IEG, immediate early gene.
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and activity-regulated TFs (e.g., c-Fos, Npas4, Egr1) is activated
simultaneously. Activity-regulated TFs and epigenetic modifiers
may then act in concert during the regulation of the secondary
transcriptional responses driven by these TFs (Fig. 1, step 2).
Plausible mechanisms include establishing the environment
for the binding of the activity-regulated TFs, transcription facilita-
tion by de novo DNA methylation within target gene bodies, ac-
tive DNA demethylation, or the establishment of 5 hmC marks.
Moreover, the activity-regulated epigenetic molecules could
prime the genome in a way that would render permissive for the
second wave of IEG transcription and further hypothetical rounds
of transcription possibly through similar mechanisms (Fig. 1,
steps 3, 4, 5). It would be interesting to investigate if the activi-
ty-regulated epigenetic molecules are induced in the later hippo-
campal transcriptional waves that underlie memory persistence.
This could function as a propagation mechanism to ensure effi-
cient transcriptional responses necessary for the maintenance of
memory.
Regarding the regulation of the early transcriptional re-
sponse triggered by learning, it is highly unlikely that this re-
sponse is dependent on the pool of activity-regulated epigenetic
modifiers. The activation of expression of IEGs and epigenetic
modifiers occurs simultaneously; therefore, interdependency is
not possible. Thus, it is likely that the first transcriptional wave de-
pends on the resting epigenomic state of the neuron or on the ac-
tivity of the basal pool of epigenetic modifiers (Fig. 1, step 1).
Active DNA demethylation during basal conditions could be re-
quired for the maintenance of TF-binding sites (such as promoters
and enhancers) in a hypomethylated state (Rudenko et al. 2013).
This would allow for a rapid transcriptional response upon neuro-
nal stimulation. Another intriguing possibility is that the activity
or even the sub-cellular localization of the DNA methylation
writers and erasures—rather than their expression levels—are reg-
ulated by neuronal activity. In such a scenario, the rapid regula-
tion of DNA methylation players could play a role in the
regulation of the transcriptional responses triggered by neuronal
stimulation (Fig. 1, steps 1, 6). One could envisage that post-
translational modifications could serve as a molecular mechanism
for such fast regulation, and regulation by post-translational mod-
ifications of Dnmts has been shown in other cell types (Denis et al.
2011). Whether this also occurs in neurons has never been
investigated.
On a network level, it is now believed that a memory trace
is allocated to a subpopulation of neurons (neuronal ensembles)
and that its consolidation is associated with the strengthening
of the synaptic connections between this set of neurons (Josse-
lyn et al. 2015; Ryan et al. 2015). These changes in connectivity
are thought to increase the likelihood that the same neuronal
population will be reactivated during the recall of the memory.
A role for DNA methylation in the formation and stabilization
of neuronal ensembles associated with a particular memory
has never been investigated. It is tempting to speculate that
DNA methylation is involved in the modulation of the strength
of synaptic connections within the neuronal ensemble and con-
sequently sets the likelihood for reactivation of the same neuro-
nal population to occur during memory recall. As mentioned
above, DNA methylation changes in the hippocampus triggered
by neuronal activity or learning preferentially occur at genes
that regulate signal transduction pathways and synaptic func-
tion and were also associated with the generation of alternative
splice variants (Guo et al. 2011a; Halder et al. 2016). It is there-
fore plausible that the gene pool controlled by de novo DNA
methylation and demethylation confers unique functional
properties to neurons that allow them to alter their connectivity
and excitability, hence determining the likelihood of their
reactivation.
Systems-level memory consolidation
Although much is known about the mechanisms underlying
memory formation, the mechanisms underlying maintenance
and long-term storage of memories are much less explored. It
has been postulated that neocortical regions are responsible for
the long-term storage of information (Frankland et al. 2004).
Through a mechanism known as systems consolidation, it is
thought that recent memories are initially encoded in the hippo-
campus and neocortex and, with the passage of time, become in-
dependent of the hippocampus. The hippocampus is thought to
guide the gradual stabilization of the information in the neocor-
tex through hippocampal–cortical communication (Squire et al.
2015). Recently, the neural replay of the learning activity pattern
during sleep phases was proposed as a neural mechanism un-
derlying this dialogue (O’Neill et al. 2008). However, molecular
correlates of systems-level memory consolidation are largely un-
known. Lesburgueres et al. (2011) suggested that a cortical “tag-
ging process,” which may be epigenetic in nature, occurs at the
time of learning and facilitates the hippocampal– cortical com-
munication necessary for the stabilization and restructuring of
the neocortex during remote memory storage (Lesburgueres
et al. 2011). The authors further suggested that cortical synaptic
tags may function as early and persistent marks of activity that
are necessary to ensure the progressive rewiring of cortical net-
works that underlies remote memory storage. In support of the
idea that epigenetic mechanisms may be involved in the setting
of synaptic tags, the authors observed increased acetylation of his-
tone H3 in the cortex and demonstrated that increasing histone
acetylation (through inhibition of histone deacetylases) in the rel-
evant cortical region at an early phase of memory consolidation
improved the “tagging process” and provided for enhanced re-
mote memory. These findings suggest that histone acetylation fa-
cilitated the expression of putative molecular tags that could
potentially support the stabilization and restructuring of memo-
ry-associated cortical networks. It is tempting to speculate that
the maintenance of expression of these cortical tags depends
also on Dnmt activity. This way, in agreement with the study
from Miller et al. (2010), inhibiting Dnmt activity in the anterior
cingulate cortex would impair remote memory maintenance.
Intriguingly, Halder et al. (2016) recently showed that 1 h after
contextual fear conditioning training DNA methylation changes
occur in both the CA1 region of the hippocampus and the ACC.
At 4 wk after training, a time point that corresponds to remote
memory, DNA methylation changes are still present in the ACC,
but not in the hippocampus. Moreover, in the ACC, genes that
were differentially methylated and expressed were functionally
associated with structural changes. These findings provide sup-
port for a role of DNA methylation in the cortical rewiring neces-
sary for stabilization of remote memory. Although the role of DNA
methylation in systems-level consolidation has only been as-
sessed in two studies (Miller et al. 2010; Halder et al. 2016) the cor-
relation between the spatiotemporal pattern of DNA methylation
changes and the systems-level consolidation theory is striking. As
already proposed by the authors, DNA methylation could repre-
sent the molecular mnemonic substrate in memory formation
and maintenance.
Conclusions and open questions
It appears now consensual that DNA methylation plays a central
role in cognitive function. The manipulation of DNA methyla-
tion-related mechanisms consistently impacts memory processes.
In light of the current literature, DNA methylation seems to regu-
late the expression of genes with relevant functions in neuronal
transmission and structure in brain regions critical for memory.
DNA methylation: a permissive mark in cognition
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