C H A P T E R

17
Alcohol and the Brain
An Epigenetic Viewpoint
Ketan Marballi, Igor Ponomarev, R. Dayne Mayfield,
and R. Adron Harris
Waggoner Center for Alcohol and Addiction Research, University of Texas–Austin, USA

O U T L I N E

General Introduction 349 Alcohol and DNA Methylation 352

Alcohol and Histone Modification 354
Epigenetics: A General Overview 349
Alcohol and microRNAs 355
Epigenetic Modifications:
Summary and Future Directions 355
An Overview 350
DNA Methylation 350 Acknowledgments 356
Histone Modifications 351
Noncoding RNAs 352 References 356

Alcohol, Epigenetics, and Gene Expression:

An Overview 352

GENERAL INTRODUCTION
Alcoholism is a serious health problem worldwide.
It is a significant cause of chronic disease and injury,
and according to the World Health Organization it
accounts for 4% of deaths worldwide every year (Yahn,
Watterson, & Olive, 2013). Data from the Centers of
Disease Control and Prevention have shown that alcohol
is the third lifestyle-related cause of death in the United
States, and unhealthy alcohol habits strongly correlate
with substantial increases in morbidity (Lotfipour et al.,
2013). Numerous research studies have shown that alco-
holism is a neurobiological disorder involving multiple
neurotransmitter circuitries, including the dopamine,
serotonin, glutamate, and GABA systems (Hillemacher,
2011). Epigenetic modifications such as DNA methyla-
tion influence expression of candidate genes in alco-
holism (Hillemacher, 2011). Chronic alcohol usage can
cause global changes in gene expression, particularly in
the brain, as supported by different in vitro and in vivo
studies. The brain is one of the few organs in which
changes in epigenetic status are not static, constantly
changing in response to different stimuli, including
changes in environment, exposure to drugs, and syn-
aptic activity (Crepaldi & Riccio, 2009). The aim of this
review is to summarize the different epigenetic modifi-
cations in the brain that have been shown to play a role
in alcohol dependence.
EPIGENETICS: A GENERAL OVERVIEW
The terms epigenetics/epigenome usually refer to
potentially heritable changes in the DNA that occur
without modifying the actual DNA sequence, in contrast
to mutations. These modifications take the form of dif-
ferent chemical groups that are added to the DNA or to
the histone proteins that the DNA surrounds. Together,
DNA and histones form chromatin that regulates gene
expression. Chromatin is made up of units called

Neurobiology of Alcohol Dependence

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349 © 2014 Elsevier Inc. All rights reserved.
350 17.  Alcohol and the Brain

FIGURE 17.1  An overview of chromatin structure and epigenetic modifications. The cartoon shows an overview of the hierarchy of chroma-
tin structure, starting with a whole chromosome that is made up of the histone octamer, surrounded by linker DNA. Amino acid residues on
histones such as lysine act as substrates for the addition of different epigenetic modifications exemplified by acetylation and methylation. DNA
can be modified by the addition of methyl groups on cytosine nucleotides. These modifications will interact to ultimately affect gene expression.

nucleosomes, consisting of a histone core made up of a
complex of eight histone proteins and approximately 147
base pairs of DNA that wrap around this complex. The
octamer consists of two units each of histones H2A, H2B,
H3, and H4, and histone H1 acts as a linker histone along
with the DNA. The histone core proteins have globular
carboxy or C-terminal domains, whereas the amino ter-
minal or N-terminal domains are more flexible and pro-
trude out from the nucleosome. The N-terminal region
consists of amino acids such as lysine and arginine. Most
of the chemical epigenetic modifications occur in this
region, including acetylation and methylation (Figure
17.1). These modifications act as regulators of chroma-
tin accessibility, essentially impacting how amenable the
chromatin is to the process of transcription. They also
act as docking sites for other protein complexes that
can recognize these chemical groups and recruit other
chromatin modifiers (Dawson & Kouzarides, 2012).
These modifications can cause epigenetic changes that
may impact the genome (the entirety of genetic material,
namely DNA) as well as the phenome (the sum total of
expressed traits and characteristics). Epigenetic changes
control functions ranging from basic developmental
phenomena, such as regulating cell determination and
differentiation in both dividing as well as nondividing
cells, to those that impact cellular processes on a global
level, including the interactions of environmental and
physiological conditions on the organism (Jablonka,
2012). Epigenetic modifications can occur as early as
fertilization (when there is extensive removal of methyl
groups in paternal DNA), followed by similar changes
in the zygote and embryo, and also during germ cell lin-
eage specification and organ-specific cell differentiation
(Cortessis et al., 2012).
Changes in environmental exposure, such as physical
activity, toxic agents, changes in diet, and social stressors
can all influence epigenetic regulation (Fleisch, Wright,
& Baccarelli, 2012). Epigenetics has been heralded as a
mechanism that may be able to elucidate the etiology of
complex diseases such as obesity, diabetes, schizophre-
nia, and disorders of addiction such as alcoholism, for
which changes in DNA sequence alone fail to explain
susceptibility and pathology (Fleisch et al., 2012). In
order to understand the role of epigenetics in alcohol-
ism, it is necessary to first review existing knowledge of
known epigenetic modifications.
EPIGENETIC MODIFICATIONS:
AN OVERVIEW
Epigenetic control of gene expression can be regulated
by two major pathways: through chemical modifications
such as DNA methylation or histone modifications, and
via novel epigenetic regulators such as small noncoding
RNAs, all of which can alter gene transcription.
DNA Methylation
The most well-characterized epigenetic DNA modi-
fication is methylation, which usually occurs at the 5’
position of cytosine residues (Nelson & Monteggia, 2011)

III.  GENE AND BEHAVIOR

 Epigenetic Modifications: An Overview 351
located at CpG sites in the vertebrate genome. These
methylated cytosines can occur in CpG-enriched por-
tions of the genome and are referred to as CpG islands.
It has been suggested that CpG islands occur at promoter
sites on 50% of the total genes present in the genome
(Hamilton, 2011). DNA methylation is believed to occur
at centromeres, telomeres, and repeat sequences and in
X-chromosome inactivation (Dawson & Kouzarides,
2012). Methylation is controlled by enzymes termed
DNA methyltransferases (DNMTs). There are three major
DNMTs in mammals. DNMT1 is a maintenance methyl-
ase that recognizes hemi-methylated DNA sequences and
methylates newly synthesized cytosines on the daughter
strand after replication or repair has occurred. On the other
hand, DNMT 3a, 3b are de novo methylases, which are
involved in establishing methylation signatures at desig-
nated spots in the genome and are particularly important
during embryogenesis (Dawson & Kouzarides, 2012).
DNA methylation acts as a signal for the recruitment of
methyl binding proteins such as methyl CpG–binding
protein 2 (MeCP2) and methyl binding domain–contain-
ing protein (MBD1). MBD1 contains an 80kDa methyl
CpG–binding domain (MBD) that is required for its chro-
mosomal binding (Nan, Meehan, & Bird, 1993; Nan, Tate,
Li, & Bird, 1996). In general, hypermethylation of genes
corresponds to transcription repression and is believed
to be mediated by different methyl-binding proteins
(such as MeCP2, MBD1, MBD2, and MBD3) interacting
with the hypermethylated DNA. One way these pro-
teins can regulate gene expression is by binding to RNA
polymerase, transcription factors to control initiation/
efficiency of transcription at hypermethylated promoters
(Ivanov, Kacevska, & Ingelman-Sundberg, 2012). Mice
that express lower levels of methyl binding proteins show
behavioral and social deficits, including deficits in learn-
ing and memory, signifying the importance of methyla-
tion in the brain (Nelson & Monteggia, 2011).
Histone Modifications
To date, approximately 16 different histone modifica-
tions have been identified. Key amongst these include
methylation, acetylation, sumoylation, phosphorylation,
and ubiquitination. These covalent modifications are car-
ried out by a number of enzymes such as methyltransfer-
ases (HMTs), histone acteyltransferases (HATs), kinases,
phosphatases, and multiple others (Ivanov et al., 2012).
These specific modifications can serve as signals for pro-
teins, such as the polycomb group of proteins (Niessen,
Demmers, & Voncken, 2009), which act as transcriptional
repressors. Each of these modifications occurs at specific
amino acids. For example, lysine residues can be acety-
lated, methylated, ubiquitinated, or sumoylated. The main
sites of histone acetylation in histone H3 include lysine 4, 9,
14, and 28 (Mandrekar, 2011). Histone acetylation usually
III.  GENE AND BEHAVIOR
corresponds with transcription activation mediated by
histone acetyl transferases (HATs), whereas deacetylation
corresponds with transcriptional repression carried out by
histone deacetylases (HDACs). Five families of HATs are
known, including HAT1, Gcn5/PCAF, MYST, p300 /CBP
(Creb-Binding Protein), and Rtt109 (Yuan & Marmorstein,
2013). Of these, the role of CBP has been well studied in
the brain, where CBP+/- mice or mice that lack its homo-
logue p300 show deficits in memory formation and object
recognition (Chan & La Thangue, 2001; Oliveira, Wood,
McDonough, & Abel, 2007). HATs usually function as
transcriptional coactivators and are recruited by transcrip-
tion factors serving in multi-subunit chromatin complex
regulation (Ellis, Atadja, & Johnstone, 2009). Antagonistic
to the action of HATs, histone deacetylation is usually
associated with a reduction in gene expression. HDACs
are divided into four families: class I (HDACs1–3 and 8),
class II (HDACs 4–10), class III (sirtuins 1–7), and class
IV (HDAC 11). Class I HDACs are primarily localized
in the nucleus, whereas Class II can localize to either the
nucleus or cytoplasm (Starkman, Sakharkar, & Pandey,
2012). Both families seem to have a tissue-specific pattern
of expression (Mandrekar, 2011). Class III HDACs require
NAD, whereas Classes I, II, and IV require Zn+2 (Kelly
& Marks 2005; Lee, Kim, Kummar, Giaccone, & Trepel,
2008). Altering HDAC expression has a significant impact
on brain function. For example, abrogating HDAC3 in the
dorsal hippocampus caused long term memory enhance-
ment, upregulation of histone H4K8 acetylation, and
increased expression of immediate early genes c-fos and
Nr4a2 (McQuown et al., 2011). HDAC2 knockout mice
show enhanced memory as well as higher levels of H4K5,
H4K12, and H2B acetylation in the hippocampus and
upregulation of genes, such as CaMKIIa and Creb, that
are important in memory formation (Guan et al., 2009).
HDAC inhibitors have therefore been proposed as thera-
peutic interventions for treatment of different psychiatric
and addiction disorders (Starkman et al., 2012).
Methylation is another well-studied type of histone
modification. Histone methylation (mono-, di-, or tri-
methylation) carried out by histone methyltransferases
(HMTs) occurs on lysine and arginine residues on his-
tones H3 and H4. It can function as a marker of activa-
tion/repression in the context of gene expression in a
residue-specific manner. Di- and tri-methylation of lysine
4 act as a signal for active transcription (H3K4me2 and
H3K4me3), whereas tri-methylation of lysine 9 and lysine
27 act as repressive signals (H3K9me3 and H3K27me3)
(Peterson & Laniel, 2004; Sims & Reinberg, 2006).
Increased histone H3 lysine 4 and 27 tri-methylation
(Huang et al., 2007) were reported in the prefrontal cortex
of schizophrenia patients. H3 methylation and acetyla-
tion have been associated with alterations in glutamater-
gic and GABAergic genes, thus pointing to the importance
of histone modifications in neuronal function.
352 17.  Alcohol and the Brain
Noncoding RNAs
Although enzymes and proteins involved in epi-
genetics such as HDACs or transcription factors can also
serve as epigenetic modulators, small or long noncoding
RNAs (ncRNAs), particularly miRNAs, have emerged as
particularly strong candidates of epigenetic regulators. It
has been estimated that ∼76% of the genome is actively
transcribed, and only 3% codes for proteins. This sug-
gests that noncoding RNAs likely constitute a significant
number of transcripts and play a crucial role in regulat-
ing gene expression. Long ncRNAs (>200 nucleotides in
length) usually control gene expression by DNA meth-
ylation. X chromosome inactivation is an example of epi-
genetic regulation mediated by long ncRNAs. Polycomb
proteins can bind to the ncRNA XIST (which is expressed
by the targeted X chromosome) and cause tri-meth-
ylation of histone H3 (H3K27), resulting in X chromo-
some silencing (Kaikkonen, Lam, & Glass, 2011). Small
ncRNAs such as miRNAs, on the other hand, are around
22 nucleotides in length. miRNAs can regulate multiple
developmental processes, including synaptic function
and neurogenesis (Papagiannakopoulos & Kosik, 2009;
Shen & Temple, 2009). Generation of miRNAs is a com-
plex process that requires multiple steps. The precursors
for miRNAs (known as pri-miRNAs) are usually several
thousand nucleotides in length, contain numerous stem
and loop structures, and are generated through transcrip-
tion by RNA polymerase II. The pri-miRNA is then con-
verted into a shorter pre-miRNA (about 70 nucleotides
in length) sequence by the microprocessor complex, con-
sisting of the protein Drosha and its cofactor DiGeorge
syndrome critical region gene 8 (DGCR8) in the nucleus.
This pre-miRNA is now exported into the cytoplasm by
the Exportin-5 (RAN GTP dependent transporter) where
it is processed by Dicer into short, double-stranded
miRNAs that are ∼22 nucleotides. The miRNA then
binds the RNA-induced silencing complex and guides
it to a specific mRNA target (based on sequence com-
plementarity) and cause translational/transcriptional
repression based on the extent of sequence complemen-
tarity. With about 197 (human) and 294 (mouse) different
miRNAs expressed in the brain (Ling et al., 2011; Shao
et al., 2010), it comes as no surprise that miRNAs have
been shown to mediate a variety of processes and path-
ways that can regulate behavior. For example, miRNAs
are involved in neural patterning (Ronshaugen, Biemar,
Piel, Levine, & Lai, 2005), and perturbed production of
miRNAs (from deficient Dicer and DGCR8 expression)
can alter dendrite growth and cause learning and mem-
ory deficits in vivo (Fenelon et al., 2011; Konopka et al.,
2010). Overall, the brain is subject to extensive epigen-
etic control via multiple mechanisms involving DNA,
histone modifications, and miRNA networks to regulate
gene expression. With a general understanding of these
III.  GENE AND BEHAVIOR
regulatory modifications, we can now delve deeper into
understanding their role in alcoholism.
ALCOHOL, EPIGENETICS, AND GENE
EXPRESSION: AN OVERVIEW
Although the brain is dynamically changing in term
of synaptic connections, it mainly consists of differenti-
ated neurons. Therefore it needs to employ approaches
that do not depend on cell division to maintain plastic-
ity. One way the brain accomplishes this is through plas-
ticity in the chromatin, which can then regulate neuronal
function (Crepaldi & Riccio, 2009). The brain reacts to
environmental changes, such as stress, toxins, and expo-
sure to alcohol and other drugs, by altering its chroma-
tin state, eventually causing synaptic alterations that in
turn regulate behavior. A complex web of epigenetic
modifications can modulate changes in gene expression
induced by alcohol and other drugs.
Alcohol and DNA Methylation
Much of the evidence linking alcohol exposure and
DNA methylation comes from in vitro and peripheral
tissue studies. Both human lymphoblast cell line and
peripheral blood cell studies have been valuable in
refining our understanding of genes implicated in alco-
holism. There are allele-specific effects on DNA meth-
ylation of genes involved in brain function, such as the
serotonin transporter (5-HTT or SLC6A4). Individuals
with a history of alcohol dependence had higher levels
of SLC6A4 expression, implying a synergism between
genetic and epigenetic factors (Philibert, Sandhu, et al.,
2008). Peripheral blood studies have shown that severity
of drinking has a negative correlation with methylation
of five defined CpG sites within the NMDA-receptor
2b (NR2B) promoter site, and NR2B expression was
increased in the first 24 hours of withdrawal (Biermann
et al., 2009). Chronic alcohol treatment caused a demeth-
ylation of CpG islands and was associated with increased
NR2B expression in mouse cortical neurons (Marutha
Ravindran & Ticku, 2004). Ethanol exposure caused a 5%
reduction in global CpG methylation in mouse embry-
onic fibroblasts and elicited proteosomal degradation of
DNA methyltransferases (DNMT1, 3a and 3b) as well
as methyl CpG–binding proteins (MeCp-2 and MBD-2
and -3; Mukhopadhyay, Rezzoug, Kaikaus, Greene,
& Pisano, 2013). A majority of these examples suggest
hypomethylation to correspond with an increase in gene
expression; however, this is not always true. Ethanol
downregulated expression of several cell cycle regula-
tion genes by causing DNA hypomethylation in neural
stem cell lines and in the mouse frontal cortex, an effect
that was enhanced in the presence of TGF-β (Hicks,
 Alcohol, Epigenetics, and Gene Expression: An Overview 353
Middleton, & Miller, 2010). Other factors such as gender
and ethnicity can also affect methylation status in alco-
hol dependence. For example, methylation status of the
monoamine oxidase A (MAOA) gene significantly cor-
related with both alcohol and nicotine dependence in
women but not men (Philibert, Gunter, Beach, Brody, &
Madan, 2008). Certain studies have shown that ethanol
can cause hypermethlyation. Most recently, a genome-
wide methylation study in alcoholism showed that there
were a higher number of genes that were hypermethyl-
ated between European American cases versus controls
(HTR3A, NCAM1, DRD4, MBD3, HTR2B, and GRIN1)
and between African American cases versus controls
(GABRB3 and POMC; Zhang et al., 2013). In addition,
three CpGs that were upstream of the μ-opioid recep-
tor gene (OPRM1, involved in mediating the rewarding
effects of alcohol) had significantly higher methylation
levels in alcohol-dependent versus control individuals
(Zhang et al., 2012). Overall, the majority of studies sup-
port the role of alcohol mediating changes in brain gene
expression via DNA methylation.
In addition to in vitro models, studies using human
postmortem tissue have been invaluable in providing
insight into the role of alcohol on regulating gene expres-
sion in the brain. Ponomarev, Wang, Zhang, Harris, and
Mayfield (2012) identified global changes in epigenetic
modifications from postmortem brain samples of alco-
holics versus matched controls. Alcohol abuse correlated
with gene expression changes in different brain regions,
including the frontal cortex, basolateral, and central
nucleus of the amygdala (Ponomarev et al., 2012). Long
terminal repeat (LTR) regions containing retrotranspo-
sons (endogenous retroviruses, or ERV), which are con-
stitutively densely methylated, were found to be
hypomethylated and showed increased expression in
alcoholic brains compared to controls. Also, most of the
genes upregulated in the frontal cortex were GC rich in
content, and DNMT1 was shown to be downregulated
in all three brain regions, correlating with the hypometh-
ylation observed. Given the significant prevalence of
LTRs in the human genome, the study concluded that
global methylation changes are correlated with alcohol
dependence. A recent study utilized methylated DNA
immunoprecipitation hybridized on microarrays com-
posed of genomic promoter regions in frontal cortices of
alcoholics versus controls (Manzardo, Henkhaus, &
Butler, 2012). No differences in global methylation were
found between the two groups. Only about 20% of the
promoters showed differential methylation, with about
half being hypermethylated in alcoholics, for example,
histone gene HIST2H2AC. Another group studied the
role of alcohol abuse in gene expression changes in psy-
chiatric disorders, given the frequent occurrence of alco-
hol abuse as a comorbid factor in psychiatric patients
(Bolton, Robinson, & Sareen, 2009). Results suggested
III.  GENE AND BEHAVIOR
that alcohol usage affects levels of DNMT1 mRNA (con-
sistent with previous data by Ponomarev et al., 2012),
decreased levels of apolipoprotein B mRNA-editing
enzyme catalytic polypeptide-like (APOBEC-3C), and
increased levels of 10-11-translocation methylcytosine
dioxygenase (TET) mRNA (the latter two both involved
in the process of demethylation) in the frontal cortex of
psychotic patients (Guidotti et al., 2013). Thus studies
using human brain tissue provide evidence that meth-
ylation can be a key epigenetic mediator in alcoholism
and other psychiatric disorders.
In addition to studies in postmortem human brains, in
vivo studies of animal models of alcoholism supported a
role for methylation. For example, administration of the
DNMT1 inhibitor 5-azacitidine caused a 20% decrease in
alcohol intake in mice (Warnault, Darcq, Levine, Barak,
& Ron, 2013). Prenatal ethanol exposure in pregnant rat
dams produced significant changes in the epigenetic
profile of hypothalamic proopiomelanocortin (POMC)
neurons in their male offsprings, as shown by increased
levels of DNMT1 and methyl CpG–binding protein 2
(MeCP2), in addition to increased POMC gene methyla-
tion and decreased POMC mRNA levels (Bekdash,
Zhang, & Sarkar, 2013). Diet is a major contributor to the
methylation process. Alcohol-induced hypermethyl-
ation in rat hippocampus and prefrontal cortex was
decreased by treatment with choline, although choline
increased methylation in control rats (Otero, Thomas,
Saski, Xia, & Kelly, 2012), showing differential effects of
choline on methylation based on prior exposure to alco-
hol. These changes were reversed by addition of choline
in the mother’s diet during gestation, highlighting the
role of dietary supplements in regulating methylation.
Other dietary regulators include vitamins such as folic
acid (Williams, Berry, & Beerstecher, 1949), which can
contribute to the formation of S-adenosyl methionine
(SAM), the primary methyl group in DNA methylation
(Hamid, Wani, & Kaur, 2009). SAM has been shown to
attenuate effects of alcohol on gene expression. A recent
study found that rats fed an alcohol diet showed hyper-
methylation and concomitant upregulation of genes
(Cd14, Hspa1a, Irf1, Irak1, Irak2, Map3k7, Myd88, Pparα,
Ripk2, Tollip, and Traf6) compared to rats fed a com-
bined alcohol and SAM diet (Khachatoorian et al., 2013).
To summarize, postmortem data suggests that alco-
hol causes DNA hypomethylation, whereas some of the
in vivo data point to DNA hypermethylation. These
opposing effects seem to occur in a cell/tissue/organ-
specific manner and may also be temporal in nature. The
dogma of increased DNA methylation being synony-
mous with gene repression is changing and giving rise
to a far more complex pattern of gene regulation via
methylation. One important point to consider is the
potential difference in methylation patterns between
peripheral and brain tissues. Barker, Zhang, Wang,
354 17.  Alcohol and the Brain
Taylor, and Zhang (2013) compared effects of alcohol-
induced DNA methylation of the serotonin receptor 3a
(Htr3a) gene in mice using nine different brain regions
and trunk blood. Alcohol consumption did not alter
methylation of the Htr3a promoter CpGs in six of the
nine brain regions (Barker et al., 2013). However, changes
in promoter methylation (both increases and decreases)
were observed that were brain region, blood, and CpG
specific. In some brain regions, such as the dorsomedial
striatum, methylation inversely correlated with Htr3a
expression. This points to not only variations in gene
expression between brain and peripheral tissue, but dif-
ferential brain regional variation as well. Given that
DNA methylation and methyl-binding proteins have
been implicated in complex neuronal processes such as
long term potentiation (Nelson & Monteggia, 2011),
methylation may serve as a means of fine-tuning gene
expression and may effectively enable different regions
of the brain to specifically modulate behavior.
Alcohol and Histone Modification
In Vitro Models
Alcohol-induced histone modifications have been
reported in the brain, liver, and gastrointestinal system
(Shukla et al., 2008). Alcohol treatment induced acetylation
at lysine 9 in histone H3 (H3AcK9) in primary rat hepato-
cyte cultures (Park, Miller, & Shukla, 2003). Knockdown of
the GCN5 HAT caused global changes in gene expression
(891 transcripts differentially expressed) in hepatoma cells
transfected with alcohol dehydrogenase 1 (Choudhury
et al., 2011). SLC44A2 (a putative choline transporter
gene) was upregulated in response to alcohol treatment
(Choudhury et al., 2011). In human SK-N-MC neuroblas-
toma cells and fetal-derived primary neurons exposed to
ethanol, an upregulation of HDAC activity, induction of
HDAC1,3 gene expression, and concomitant increase in
expression of the serotonin gene were observed (Agudelo,
Yoo, & Nair, 2012), and these changes were reversed by
treatment with the HDAC inhibitor trichostatin A (TSA).
Studies using human SH-SY5Y neuroblastoma cells
showed that prolonged ethanol exposure significantly
increased H3K27me3 and H3K9Ac levels in the promoter
region of the prodynorphin (PDYN) gene (D’Addario
et al., 2011). A chronic intermittent ethanol paradigm
increased levels of the NMDA-receptor 2B (NR2B) gene
and increased H3K9Ac levels near the NR2B promoter
in mouse cortical neurons. Levels of G9a, Suv39 h1, and
HDAC1–3 in the chromatin of the NR2B gene promoter
decreased and were believed to contribute to the H3K9Ac
increase reported earlier (Qiang, Denny, Lieu, Carreon, &
Li, 2011). Neural stem cells exposed to a binge-like etha-
nol dose showed decreased migration, differentiation, and
gene expression analysis, revealing a number of genes
involved in glutamate transmission, axon extension, and
III.  GENE AND BEHAVIOR
cell cycle regulation that were downregulated in the pres-
ence of ethanol (Zhou et al., 2011). This downregulation
was similar to the actions of the methylation inhibitor
5-azacitidine (5-AzaC), and sites altered by methylation
were also shown to be binding sites for transcription fac-
tors (Zhou et al., 2011). Overall, in vitro data suggests inter-
play of histone methylation and acetylation modifications.
This is indicative of a more integrated process of control-
ling gene expression whereby a single histone modifica-
tion does not predicate the direction of gene expression,
but instead a combination of different modifications
directs gene expression and cellular function.
In Vivo/Animal Models
Alcohol exposure in rats increased H3K9 and H4K8
acetylation in the brain (Pandey, Ugale, Zhang, Tang, &
Prakash, 2008). Alcohol induced anxiety-like behavior,
with a concomitant decrease in HDAC activity. It also
increased levels of CREB-binding protein (CBP) and
neuropeptide Y (NPY), coinciding with the increased
histone acetylation. However, anxiety-like behavior dur-
ing alcohol withdrawal showed the opposite effects,
that is, decreased acetylation and decreased expression
of these proteins in the amygdala. The HDAC inhibitor
Trichostatin A (TSA) reversed these effects during with-
drawal, restoring mRNA and protein levels of NPY and
preventing the anxiety-like behavior (Pandey et al., 2008).
TSA also increased ethanol consumption in C57BL/6NCrl
mice, and ethanol intake affected gene expression levels
in the nucleus accumbens (NAc), including several genes
involved in chromatin remodeling (Wolstenholme et al.,
2011). A recent study by Warnault, Darcq, Levine, Barak
and Ron (2013) showed that administering the DNMT
inhibitor (5-AzaC) decreased alcohol drinking, whereas
the HDAC inhibitor SAHA reduced binge-like drink-
ing in a dose-specific manner (Warnault et al., 2013). In
line with these findings, excessive alcohol consumption
increased DNMT1 levels and decreased histone H4 acety-
lation in the NAc of mice (Warnault et al., 2013). The NAc
seems to be a critical region in controlling alcohol depen-
dence, with higher levels of glutamate being observed
in the NAc of alcohol-dependent patients compared to
controls and glutamate and glutamine levels in the NAc
and cingular cortex, respectively, showing a positive cor-
relation with drinking behavior (Bauer et al., 2013).
Two major studies have modeled epigenetics in fetal
alcohol spectrum disorders (FASD). Both these studies
use ethanol exposure in the time period of postnatal days
two to 12, which models the third trimester of human
pregnancy, in which severe cerebellar damage (one of
the hallmarks of FASD) occurs (Green, 2004). Postnatal
day seven rats that had previously been exposed to etha-
nol showed a decrease in histone acetyltransferase
CBP levels and a concomitant decrease in acetylation
of H3 and H4 in the cerebella (Guo et al., 2011).
 Summary and Future Directions 355
Inhibition of the lysine dimethyltransferase (G9a) prior
to ethanol treatment in postnatal day seven mouse pups
rescued alcohol-mediated neurodegeneration and nor-
malized the increased levels of H3K9me2 and H3K27me2
in the developing mouse brain (Subbanna et al., 2013).
A majority of the in vivo studies discussed previously
point to histone acetylation and transcriptional comodu-
lators, such as CBP, in regulation of alcohol-mediated
epigenetic effects.
Human Postmortem Brain Studies
Human postmortem studies have also shown changes
in histone gene expression in alcoholics. For example,
CBP and CREB mRNA levels decreased in alcoholic
brains (Ponomarev et al., 2012). On the other hand,
expression of genes involved in transcription corepres-
sor complexes (TCC) increased in alcoholics (Liu et al.,
2006; Ponomarev et al., 2012). Thus several of the changes
observed in human postmortem brains agree with the
findings from animal models of alcoholism. The study
of the interrelationship between epigenetics and alcohol
in human brain tissue is still in its infancy, and further
studies are warranted to analyze the complex epigenetic
modifications involved in the alcoholic brain.
Alcohol and microRNAs
microRNAs (miRNAs) are short, noncoding RNAs
that are capable of controlling chromatin modification,
DNA methylation, mRNA degradation, and repres-
sion of mRNA translation (Bartel, 2009). One of the first
reports of ethanol action on miRNA showed that at doses
representative of chronic alcohol users ethanol caused a
downregulation in levels of miR-21, -335, -9, and -153
in neurosphere cultures (Sathyan, Golden, & Miranda,
2007). These miRNAs have specific functions in control-
ling proliferation and apoptosis, suggesting that ethanol
may alter these functions indirectly by altering miRNA
expression. Furthermore, miR-9 was upregulated in
rat adult brains following ethanol exposure, whereas
expression of specific BK-channel (calcium- and voltage-
gated channels) splice variants was downregulated
(Pietrzykowski et al., 2008). Increased expression of miR-
375 was reported in pharyngeal/laryngeal tumors that
correlated with alcohol use (Avissar, McClean, Kelsey,
& Marsit, 2009). Studies of alcoholic liver disease have
shown that ethanol can produce overexpression of
miR-212 and cause gut leakiness via regulation of tight
junction proteins, such as ZO-1, which is a miR-212 tar-
get (Tanget et al., 2008). In addition, ethanol-induced
miR-199 can target ET-1 and HIF-1 in endothelial cells
(Yeligar, Tsukamoto, & Kalra, 2009). Microarray studies
have shown that ethanol exposure causes global changes
in miRNA expression in primary cortical neurons (Guo,
Chen, Carreon, & Qiang, 2012) in agreement with similar
III.  GENE AND BEHAVIOR
changes seen in postmortem brain samples of alcoholics
(Lewohl et al., 2011). Recently, Gorini, Nunez, Roberts,
and Mayfield (2013) identified region-specific changes in
miRNA levels in the cerebral cortex and midbrain using
a chronic intermittent ethanol exposure paradigm in
vivo and revealed miRNA 146b as a top alcohol-respon-
sive miRNA (Gorini et al., 2013). This miRNA has been
shown to negatively regulate Toll-like receptor-4 signal-
ing (Curtale et al., 2013). miRNAs have emerged as key
mediators of gene and protein expression, and future
research will determine the long-term effects of alcohol
usage on brain miRNA expression and epigenetic regu-
lation in the brain.
SUMMARY AND FUTURE DIRECTIONS
The field of epigenetics has rapidly advanced over the
past few years. The ENCyclopedia Of DNA Elements
(ENCODE) project has been key in catapulting our
understanding of the genome and epigenome. It has
been calculated that about 56% of the genome is highly
enriched in modified histones, supporting a significant
role for epigenetics in constitutive gene expression and
therefore normal cellular function. Given the complex-
ity of epigenetic regulators and their modifications, an
intricate array of processes for regulation of gene expres-
sion has emerged. Improved techniques for analyzing
epigenetic changes have further fueled growth of this
field, including bisulphate sequencing, ChIP-seq and
Ribo-seq, profile proteins associated with histone marks,
and transcriptome assays (Telese, Gamliel, Skowronska-
Krawczyk, Garcia-Bassets, & Rosenfeld, 2013).
Alcoholism is a complex disease that is subject to
multiple genetic and environmental factors. It also
appears to be comorbid with other disorders such as
psychiatric illness and cancer. Multiple epigenetic
mechanisms control gene expression, usually in a cell/
tissue-specific manner (Figure 17.2). For example, based
FIGURE 17.2  Factors contributing to epigenetic changes. The
figure depicts different factors contributing to epigenetic modifications
that can alter gene expression in a cell/tissue-specific manner, leading
to changes in behavior.
356 17.  Alcohol and the Brain
on human postmortem tissue studies DNA and histone
methylation seem to be the predominant epigenetic
modifications in the brain, whereas acetylation, meth-
ylation, and phosphorylation have been observed in the
liver. This does not necessarily imply that epigenetic
changes in the brain are limited to methylation, but it
may instead reflect a limitation of current methodology
to detect all modifications in tissue sensitive to degra-
dation. Support for this comes from studies in neurons
in vitro in which acetylation was detected (Qiang
et al., 2011).
It is important to keep in mind that alcoholism, as a
complex disease, involves multiple interconnected path-
ways. Metabolites of alcohol, such as acetaldehyde, can
cause DNA damage (Langevin, Crossan, Rosado,
Arends, & Patel, 2011; Rulten, Hodder, Ripley, Stephens,
& Mayne, 2008), and neuroimmune regulation is another
important pathway implicated in alcoholism (Blednov
et al. 2012; Crews, Zou, & Qin, 2011; Liu et al., 2004). The
combined effects of alcohol on epigenetic and inflamma-
tory processes are examples of pathways that may be
interconnected to modulate genetic regulation on a
global scale without affecting DNA sequence. The conse-
quences of these interactions may be far reaching,
affecting not only the brain but also other organs in
which inflammatory mechanisms could be potentially
damaging.
The ultimate goal of epigenetic research is to discover
successful therapies for addictions such as alcoholism
and other complex diseases. As noted by Ponomarev
(2013), current literature shows increased levels of his-
tone acetylation due to acute alcohol exposure, and
withdrawal is characterized by increased deacetylation,
therefore providing a strong rationale for the use of
HDAC modulators as therapeutic targets. However, cur-
rent studies also suggest brain region–specific changes
in epigenetic modifications, reinforcing the need for tar-
geted therapy for specific regions or even specific neuro-
nal subpopulations. Dietary and other environmental
factors are additional considerations in designing poten-
tial therapeutic agents to target epigenetic regulation of
alcoholism.
The human genome is highly enriched in modified
DNA and histones, providing a prime template for epi-
genetics in regulating constitutive gene expression and
therefore normal cellular function. Alcohol’s role in
altering these mechanisms will no doubt continue to fuel
significant advancements in the genetics of alcohol
addiction, highlighting pathways for gene modulation
without any change in DNA sequence.
Acknowledgments
The authors would like to express their sincere gratitude towards Dr.
Jody Mayfield for her valuable editorial comments and suggestions.
We would also like to acknowledge funding support provided by a
III.  GENE AND BEHAVIOR
NIH, NIAAA grant from the Integrative Neuroscience Initiative on
Alcoholism to RAH (AA013518), a K award to IP (AA017234), and a
DOD grant to RAH (W81XWH-11-1-0245).
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