Epigenetic mechanisms underlying pathobiology of alcohol use disorder

Abstract

Purpose of review

Chronic alcohol use is a worldwide problem with multifaceted consequences including multiplying medical costs and sequelae, societal effects like drunk driving and assault, and lost economic productivity. These large-scale outcomes are driven by the consumption of ethanol, a small permeable molecule that has myriad effects in the human body, particularly in the liver and brain. In this review, we have summarized effects of acute and chronic alcohol consumption on epigenetic mechanisms that may drive pathobiology of Alcohol Use Disorder (AUD) while identifying areas of need for future research.

Recent findings

Epigenetics has emerged as an interesting field of biology at the intersection of genetics and the environment, and ethanol in particular has been identified as a potent modulator of the epigenome with various effects on DNA methylation, histone modifications, and non-coding RNAs. These changes alter chromatin dynamics and regulate gene expression that contribute to behavioral and physiological changes leading to the development of AUD psychopathology and cancer pathology.

Summary

Evidence and discussion presented here from preclinical results and available translational studies have increased our knowledge of the epigenetic effects of alcohol consumption. These studies have identified targets that can be used to develop better therapies to reduce chronic alcohol abuse and mitigate its societal burden and pathophysiology.

I. Introduction

Alcohol has been used by humans for several millennia, renowned for its psychoactive effects that took on considerable medical and social consequences (1). Over time, production and acceptance of ethanol have become widespread to the point where alcohol is one of the most commonly used substances worldwide despite a recent meta-analysis that indicates that any amount of alcohol drinking contributes to disease burden and increases risk of mortality (2). In addition to medical consequences like increased risk of cancer and cardiovascular disease, alcohol drinking has negative societal effects such as decreased economic productivity, increased unintentional injuries including drunk driving accidents, and increased violence including physical and sexual assault (3). While these large-scale negative outcomes driven by alcohol drinking are readily appreciated, the pathobiological effects of ethanol on a molecular level have been relatively more complicated to elucidate (4). Unlike many other drugs of abuse that have discrete molecular targets, ethanol is a small permeable molecule that is rather promiscuous in its cellular and molecular interactions (5). We will briefly review known biological targets of ethanol before shifting focus to epigenetics and discussions of how both acute and chronic ethanol exposure modulate the epigenome in the brain and other tissues.

II. Molecular Targets of Ethanol

Ethanol interacts with various molecular targets including enzymes and ion channels, as well as epigenetic targets that produce histone and DNA chemical modifications and microRNA and non-coding RNA genetic regulatory elements (6). Adenylyl cyclase is an enzyme whose activity is enhanced acutely by ethanol (7). Adenylyl cyclase catalyzes the hydrolysis of ATP to cyclic adenosine 3′, 5′-monophosphate (cAMP) which then activates Protein Kinase A (PKA) (8). PKA is an important intracellular cAMP-dependent signaling molecule that is part of a cascade downstream of many G-protein coupled receptors in a variety of cell systems and acts via modulation of function of cAMP-responsive element binding (CREB) protein (9). Interestingly, pharmacological inhibition of PKA signaling in the central nucleus of amygdala (CeA) or nucleus accumbens (NAc) shell increases ethanol consumption, and decreased function of CREB in the amygdala is associated with alcohol-drinking behaviors in animal models (10–12). GABA_A receptors are chloride ion channels that mediate inhibitory neurotransmission, and ethanol enhances GABA_A receptor potentiation (13). By contrast, NMDA receptors are calcium ion channels that mediate excitatory glutamatergic neurotransmission, and ethanol inhibits NMDA receptor currents (14). Following chronic ethanol administration, neuroadaptations within GABAergic and glutamatergic neurons can lead to a state of hyperexcitability when ethanol is withdrawn leading to development of withdrawal symptoms (13). Detailed information about modulation of these molecular targets by ethanol exposure have been described in previous publications (4,13,15). While both acute and chronic ethanol consumption have myriad effects on signaling pathways and ion channel targets, the complex epigenetic mechanisms simultaneously regulating many genes in diverse tissues represent an emerging topic within the alcohol research field. Also, epigenetic processes have begun to explain how alcohol use can produce both sustained effects on the brain contributing to addiction and changes in the liver leading to steatohepatitis and hepatic carcinoma (16,17).

III. Epigenetic Mechanisms

Epigenetics is the study of environmental influence on transcriptomic changes (18), involving DNA and histone chemical modifications that alter expression of genes and related behaviors without changing underlying genetic sequences (19). Epigenetic alterations are heritable, stable, and their influence on gene expression has developmental and behavioral consequences (20). Various epigenetic mechanisms control the way DNA is packaged into chromatin and alter access of transcription factors (21). These mechanisms include methylation of DNA, covalent modifications of histone proteins such as acetylation, methylation, and phosphorylation, and changes in small and long non-coding RNAs expression that can influence gene transcription (22). Interestingly, several innovative techniques are emerging to examine epigenetic landscapes at the whole genome level using an unbiased approach to assess chromatin accessibility, three-dimensional chromatin interactions, or differentially modified histones or methylated DNA. These techniques include ATAC-Seq, ChIP-Seq, DNA bisulfite-sequencing, chromatin interaction analysis with paired-end tag, 3C/Hi-C, and others that can be synthesized together and combined with more traditional RNA-Seq and proteomics techniques to create rich epigenetic data sets for answering diverse questions of epigenetic regulation of gene expression (23,24).

Epigenetic changes have been widely implicated in psychiatric disorders, and they provide a theoretical biological basis for the subjective environmental influences on a wide scope of brain diseases like post-traumatic stress disorder, depression, anxiety, and addiction (25,26). Epigenetic alterations have also been observed in many different types of cancers, including liver cancer associated with alcoholic liver disease (27,28). Recent studies have identified epigenetic pathways in the brain affected by alcohol exposure that modify gene expression and neuronal morphology in ways that are important for the development and maintenance of Alcohol Use Disorder (AUD), but existing AUD treatments do not currently target these emerging epigenetic pathways (6,22,29). By contrast, various agents that are developed to treat cancer are able to inhibit DNA methyltransferase (DNMT) or histone deacetylase (HDAC) enzymes, and current clinical trials are underway to treat various cancers including hepatocellular carcinoma (30). Various epigenetic mechanisms will now be discussed below in detail.

A. DNA Methylation

DNA is fundamentally composed of paired nitrogenous bases adenine and thymidine, or guanine and cytosine, stacked upon one another in a long chain double helix structure. CpG sites are regions of DNA where cytosine and guanine nucleotides are located next to each other and such sites can be methylated via DNMT enzymes to create 5-methylcytosine. Regions of the genome with elevated content of CpG dinucleotides are termed CpG islands and when these islands are highly methylated and located at gene promoters, gene expression is consequently reduced. DNMT1 is considered to be a maintenance methyltransferase, preserving DNA methylation patterns in dividing cells, whereas DNMT3a and DNMT3b participate in de novo DNA methylation (21,31–34). Once DNMT enzymes bind to DNA upon recognition of specific DNA sites, the methyl group donor S-adenosylmethionine (SAM) is recruited. Next, DNMTs incorporate the methyl group at the fifth carbon of the cytosine residue, and then DNMT enzyme and S-adenosylhomocysteine (SAH) substrate is released (32,33). Resultant methylation is a stable mark that, when present at gene promoters, mediates transcriptional repression by directly blocking access of DNA-binding proteins and recruiting co-repressors and other heterochromatin associated proteins, thereby blocking transcription factor access and preventing transcription (34,35). DNA methylation plays an important role in development where it is key for processes such as X-chromosome inactivation in females and genomic imprinting (36). While DNA methylation patterns are heritable and stable through multiple cellular divisions, they are no longer considered to be permanent modifications, as the endogenous enzymatic pathways for DNA demethylation via ten eleven translocation (TET) enzymes leading to subsequent hydroxymethylation, deamination, and base excision repair have since been elucidated (22,33,37). Nevertheless, DNA methylation remains the best known epigenetic mechanism playing a role in various disease-causing mutations, and it is one of several epigenetic features profoundly altered by ethanol exposure (22). We have described below how these processes are modulated by acute and chronic ethanol exposure and its role in pathophysiology of AUD.

B. Histone Modifications

DNA is packaged in the cell nucleus as chromatin, which consists of DNA wound around octamers of histone proteins responsible for organizing and packaging genetic material (38). DNA is wound around four pairs of the histone proteins H2A, H2B, H3, and H4 to create the nucleosome, which is the fundamental unit of DNA packaging consisting of 146 base pairs of supercoiled DNA (39). Nucleosomes are linked to one another via linker DNA bound to histone H1 proteins that stabilize nucleosome structure and overall chromatin architecture (40). While this elaborate structure for nuclear DNA packaging was initially conceptualized merely as a way to fit DNA within the space constraints of the microscopic cell nucleus, it has become clear that chromatin structure modulation via histone proteins plays a major role in regulation of gene expression (16,21,22,41,42). Histone proteins feature tails whose various amino acid residues can undergo post-translational modifications such as acetylation, phosphorylation, methylation and a number of other emerging modifications (43). These modifications fundamentally alter the chromatin architecture and, therefore, DNA accessibility, leading to increased or decreased gene expression depending on the type and location of histone modification (43). Large families of enzymes, so-called writers such as histone acetyltransferases (HATs) and lysine methyltransferases (KMTs), exist to add these histone modifications, while other enzymes, so-called erasers such as HDACs and lysine demethylases (KDMs), remove these histone modifications (41,44). Other groups of proteins called readers recognize the histone modifications and can serve to recruit additional epigenetic effectors to the chromatin to ultimately regulate gene expression (45). Overall, histones represent a large library of dynamically regulated proteins via reversible post-translational modifications that underlie control of gene expression (46). Furthermore, there is consistent evidence that alcohol exposure alters many histone modifications with resultant effects on behavior and disease development, and histone modifications therefore represent an intriguing pharmacological target for treatment of ethanol-induced pathology (6).

C. miRNA and ncRNA

Lastly, large swaths of the metazoan genome consist of non-coding sequences that are not eventually translated into functional proteins; this non-coding DNA was once considered “junk” but it has become increasingly clear that these non-coding sequences have roles in the regulation of gene expression (47). While these DNA sequences can themselves contain sequences that act as regulatory elements such as enhancers, silencers, and insulators, the functional regulation of gene expression is dependent on RNA polymerase II binding and transcription of various RNA species (48,49). MicroRNAs (miRNAs) are a family of noncoding RNAs defined by their short mature 21–23 base pair size that regulate protein synthesis by complementary base-pairing to target mRNA transcripts, although perfect complementarity isn’t always necessary (15,22,50). Since miRNAs interfere with gene expression without underlying alterations in the genetic sequence of the target, they are considered an epigenetic phenomenon (22). Long non-coding RNAs (lncRNAs), by contrast, are at least 200 base pairs in length, and growing evidence suggests mammalian cells produce thousands of these transcripts, that their presence is highly evolutionarily conserved, and they play key roles in various physiological processes such as development and pathophysiological ones like addiction and tumorigenesis (51–55). Overall, these non-translated RNA species represent a final class of epigenetic processes that shape gene expression and one that is increasingly associated with the pathobiological changes following alcohol exposure (56).

IV. Epigenetic Effects of Acute Ethanol

A. Introduction and Metabolism

Acute administration of ethanol has rapid and wide-ranging effects on epigenetic alterations and subsequent gene expression all over the body with notable behavioral and physiological effects in the brain and liver. Metabolism allows cells to produce energy necessary for cellular processes, and it is directly linked to epigenetic regulation via metabolites and metabolic enzymes that can modulate chromatin both directly and indirectly (57). The activities of the enzymes involved in dynamic epigenetic modifications are regulated in part by the availability of required substrates and cofactors (58). Ethanol is rapidly metabolized in the liver via both oxidative and non-oxidative pathways that generate many metabolites including acetate; these pools of ethanol-derived acetate can go on to influence local and global histone acetylation in various tissues (59–63). In the following sections, we will review the effects of acute ethanol exposure on epigenetic mechanisms in the brain and liver.

B. Epigenetic effects of Acute Ethanol in the Brain

Acute ethanol exposure activates numerous mechanisms that increase histone acetylation in the brain (64). For instance, in the amygdala of rats, ethanol (1g/kg) administration increases HAT CREB binding protein (CBP) expression while inhibiting HDAC activity, thereby contributing to increased histone H3K9 and H4K8 acetylation as well as increased synaptic plasticity-associated brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (Arc), neuropeptide Y (NPY), and prodynorphin (PDYN) and an anxiolytic phenotype one hour following ethanol treatment (65–69). Histone acetylation also plays a crucial role in the development of rapid tolerance to the anxiolytic effects of ethanol, and treatment with HDAC inhibitor Trichostatin A (TSA) reverses this tolerance phenomenon while driving upregulations of global histone acetylation and NPY expression (68,69). Recently, we demonstrated that acute ethanol also decreases histone methyltransferase G9a levels and H3K9me2 levels in the amygdala of rats where HDAC and G9a might interact dynamically to regulate the status of H3K9 modifications during acute ethanol exposure (70). Treatment with G9a inhibitor was able to reverse rapid tolerance to anxiolytic effects of acute ethanol via decreasing H3K9me2 levels and increasing NPY expression in the amygdala of rats (70). In the cortex and the hippocampus, acute ethanol also decreases HDAC expression and increases histone acetylation with less clear implications for the acute behavioral effects of ethanol (71). Furthermore, in the nucleus accumbens, several chromatin-modifying genes, including HAT Myst3, show greater induction with higher ethanol drinking (72). Acute ethanol produces ataxia that is associated with increased HAT expression and increased H3K27ac occupancy at the Fmr1 gene in the cerebellum, which is important in regulating glutamatergic neurotransmission (73). More recent studies have attempted to directly connect the metabolism of ethanol to the increase in brain histone acetylation. Using mass spectrometry and isotope-labelling, Mews et al. demonstrated that ethanol-derived acetate is directly incorporated into histones via chromatin-bound acetyl-CoA synthetase 2 (ACSS2) in the hippocampus, and this process is necessary for ethanol-related associative learning and is associated with transcriptomic changes (60). This mechanistic study links increases in brain histone acetylation following acute ethanol exposure directly to the ethanol-derived acetate, and, therefore, hippocampal ACSS2 may be an important target for altering ethanol-related memories that underlie relapse (60).

Aside from histone acetylation, acute ethanol is known to alter expression of brain miRNAs including miR-494, which has been shown to be inhibited by acute ethanol in the amygdala and associated with ethanol-induced anxiolysis and increased expression of HATs CBP and p300 with resultant alterations in H3K9 acetylation (H3K9ac) levels (74). Interestingly, inhibition of miR-494 using antagomir in the CeA of rats produces anxiolysis and increases its target CBP and p300 expression thus mimicking the effects of acute ethanol in rats (74). Using MBD (Methyl-CpG-Binding-Domain) sequencing to probe the brain methylome following acute ethanol exposure, differential methylation at several genes implicated in inflammatory chemokine receptor CXCR4 and interleukin IL-7 signaling pathways was discovered, indicating DNA methylation in the brain is also altered by acute ethanol (75). It is clear that acute ethanol can produce myriad epigenetic effects in the brain with various resultant behavioral and physiological effects including anxiety, learning, ataxia and inflammation. Continuing to research these acute ethanol effects will help establish early epigenetic frameworks for understanding the development of alcohol-related pathologies in the brain and elsewhere.

C. Epigenetic effects of Acute Ethanol in the Liver

Binge alcohol consumption is known to alter many metabolic processes in the liver, where ethanol is metabolized to acetaldehyde via alcoholic dehydrogenase or microsomal ethanol oxidizing system (MEOS) and cytochrome P450 (CYP) and then oxidized to acetate by acetaldehyde dehydrogenase with NAD+ cofactor (76). Increases of alcohol metabolites in the liver lead to liver injury which can lead to alcoholic steatohepatitis, and, eventually cirrhosis (77). The effects of acute alcohol on the epigenome are immediate. In a study using mass-spectrometry to trace ethanol metabolism in the liver with ¹³C2 isotope labelled ethanol, acetylation sites on histones H3 and H4 were shown to be acetylated by the acetate derived from ethanol metabolism (78). Moreover, a different study has shown that alcohol and its metabolite, acetate, both increase H3K9ac but differentially regulate HDAC activity (79). In this study, alcohol increased HDAC activity and acetate decreased HDAC activity but only ethanol increased reactive oxygen species (ROS), suggesting that alterations in H3K9ac by ethanol are dependent on intermediate products of alcohol metabolism, namely ROS (79). This idea is further supported by a study where pretreatment of hepatocytes with ROS reducer N-acetylcysteine reduced ethanol-induced increases in H3K9ac whereas ROS inducer L-butathionine sulfoximine increased ethanol-induced H3K9ac (80). As such, the oxidative stress following acute ethanol exposure in the liver subsequently affects global histone H3 acetylation.

Furthermore, acute ethanol effects in the liver have been found to produce histone modifications in genome-specific locations that are strongly associated with regulation of lipid metabolism. PNPLA3 (Patatin-like phospholipase domain-containing protein 3) gene and protein expression, associated with increased levels of triacylglycerol, is epigenetically upregulated by increased H3K9ac at the PNPLA3 promoter in the mouse liver in an acute binge ethanol model of 3 intragastric doses of 3.5 g/kg ethanol at 12h intervals (81). This change in histone acetylation is potentially explained by downregulated HDAC isoform expression since another study using the same binge ethanol model at a slightly higher ethanol dose (4.5 g/kg) found decreased hepatic HDAC 1, 7, 9, 10, and 11 mRNA expression (82). Furthermore, this study showed acute ethanol leads to decreased association of HDAC9 with the fatty acid synthase (FAS) promoter, thereby increasing expression of FAS mRNA and protein (82). The same group found that HDAC inhibition attenuates liver steatosis, potentially by mediating HDAC3 inhibition of CPT1α expression, an enzyme in free fatty acid β oxidation (83). Overall, acute ethanol produces many observable epigenetic alterations with pathophysiological consequences for development of hepatic steatosis. However, in both the liver and the brain, most epigenetic studies have focused on chronic ethanol exposure, as this type of exposure leads to more pronounced pathobiological changes, such as addiction and cancer.

V. Epigenetic Effects of Chronic Ethanol

A. Introduction and Metabolic State in Ethanol Dependence

Chronic ethanol exposure produces widespread epigenetic disruptions with severe pathobiological consequences. Chronic ethanol consumption can lead to folate and vitamin B deficiencies with resulting decreases in SAM and increases in homocysteine (84,85). Reductions of SAM, a crucial methyl donor, decreases global DNA and histone methylation patterns and therefore alters gene expression (57,86–88). Furthermore, excessive chronic alcohol consumption promotes oxidative damage to DNA, and the resulting DNA repair mechanisms can demethylate 5-methylcytosine nucleotides (22,89). Overall, these pathways suggest the metabolic effects of chronic ethanol promote global hypomethylation of DNA and histones with direct consequences in the development of alcoholic liver disease and cancer (17,90,91). However, specific methylation patterns differ amongst different tissue and cell types, and global histone and DNA methylation status may not reflect gene-specific alterations with functional consequences (22). In the following sections, we will discuss chronic ethanol-mediated tissue-specific epigenetic alterations.

B. Epigenetic Effects of Chronic Ethanol in the Brain

The development of psychiatric disorders like AUD is a multi-faceted process where genetic liabilities and environmental influences together contribute to phenotypes and behaviors (92). Here, we detail recent studies that have identified epigenetic pathways in the brain affected by chronic alcohol exposure that modify gene expression and neuronal morphology in ways that are important for the development and maintenance of AUDs (6,22,29). We have summarized the various epigenetic findings in the brain in relation to alcohol exposure in Table 1 and outlined them below.

Table 1:

Summarized findings of epigenetic effects of chronic ethanol exposure organized by species, chronic ethanol administration paradigm, and brain region.

Species/Model	Brain Region	Epigenetic Effect	Molecular/Behavioral Outcome
Mouse/ Ethanol Vapor	Prefrontal Cortex	↑Global H3K27me3 ↓Global H3K4me3	↓Calcium Signaling (93)
Mouse/ Ethanol Drinking	Hippocampus	↑H3K4me3	↑BDNF ↑Neurogenesis (94)
Rat/ Chronic Ethanol Treatment	Prefrontal Cortex	↑H3K4me3	↑GABA-Aα5 (95)
Mouse Primary Cortical Neurons/ Ethanol Vapor	Cortex	↑H3K9me2 ↓H3K9me3	↑NR2B (100)
Rat Ethanol Vapor	Hippocampus	↑H3K9ac	↑NR2B (101)
Rat/ Chronic Ethanol Liquid Diet	Amygdala	↑HDAC/↓HAT Activity ↓H3K9/H4K8 Acetylation	Anxiety during Withdrawal, reversed by TSA treatment (67)
Rat/ Chronic Ethanol Liquid Diet	Hippocampus	↑HDAC2 ↓H3K9ac	Depression-like behavior in withdrawal, reversed by SAHA treatment (102)
Rat/ Chronic Ethanol Liquid Diet	Ventral Tegmental Area	↑HDAC2 ↓H3K9ac	GABA hyposensitivity during withdrawal, reversed by SAHA treatment (103)
Rat/ Ethanol Vapor	Nucleus Accumbens	↑KDM6B ↓H3K27me3	↑IL-6 pathway (105)
Rat/ Ethanol Vapor	Prefrontal Cortex	↓PRDM2 ↓H3K9Me1	PRDM2 knockdown escalates drinking (106)
Rat/ Ethanol Vapor	Prefrontal Cortex	↑DNA Methylation	↓Syt2, escalated drinking (107)
Mouse/ Binge Ethanol Drinking	Nucleus Accumbens	↑DNMT1 ↑H4 Acetylation	DNMT and HDAC inhibitors reduce ethanol drinking (108)
Rat/ Adolescent Ethanol Treatment	Amygdala	↑DNMT3b ↑DNA Methylation	↓NPY and ↓BDNF, Anxiety; Restored by 5-Azacytidine (109)
Rat/ Adolescent Ethanol Treatment	Amygdala	↑HDAC2, ↑HDAC4, ↓H3K9ac	Higher alcohol intake, Anxiety, ↓BDNF and ↓Arc (113,114)
Rat/ Adolescent Ethanol Treatment	Amygdala	↓KDM6B, ↓CBP, ↑H3K27me3 ↓Arc eRNA, ↓Arc	Anxiety, ↓Arc (115)
Rat/ Adolescent Ethanol Treatment	Amygdala	↑miR-137 ↓LSD1, ↑H3K9me2	Higher alcohol intake, Anxiety, ↓BDNF, reversal by miR-137 antagomir infusion into CeA (116)
Rat/ Adolescent Ethanol Treatment	Hippocampus	↑HDAC, ↓CBP ↓H3K9ac	↓BDNF, ↓Neurogenesis, Reversal by TSA (117)
Rat/ Adolescent Ethanol Treatment	Prefrontal Cortex	↑H3/H4 acetylation ↑HAT activity ↑H3K4me2	↑cFos, ↑FosB ↑Cdk5 (118)
Post-Mortem AUD Subjects	Amygdala and Prefrontal Cortex	↑HMT expression ↑Global H3K4me3	↑Endogenous retroviruses (119)
Post-Mortem AUD Subjects	Prefrontal Cortex	↑DNA Methylation	↓NR3C1 (120)
Post-Mortem AUD Subjects	Cerebellum	↑DNA Methylation	↓GABA-Aδ (121)
Post-Mortem AUD Subjects with Adolescent onset	Amygdala	↑lncRNA BDNF-AS ↑H3K27me3, ↑EZH2	↓BDNF, ↓ARC (51)

AUD, Alcohol use disorder; Arc, Activity regulated cytoskeleton-associated protein; BDNF, Brain-derived neurotrophic factor; BDNF-AS, BDNF antisense; CBP, CREB binding protein;Cdk5, Cyclin dependent kinase 5; DNMT, DNA methyltransferase; EZH2, Enhancer zeste homolog 2; eRNA, Enhancer RNA; GABA, γ Aminobutyric acid;H3K27me, H3K27 trimethylation; HDAC, Histone deacetylase; H3K4me2, H3K4 dimethylation; H3K4me3, H3K4 trimethylation; H3K9ac, H3K9 acetylation; HAT, Histone acetyltransferase; HMT, Histone methyltransferase; NR3C1, Glucocorticoid receptor gene; miR-137, microRNA-137; NR2B, N-methyl-D-aspartate receptor 2B subunit; KDM6B, Lysine demethylase 6B; lncRNA, Long non-coding RNA; LSD1, Lysine-specific histone demethylase 1A;TSA, Trichostatin A

Despite chronic alcohol consumption and metabolic disturbances leading to global methylation changes, many studies in the brain have found divergent methylation modifications. For instance, a recent genome-wide study found a 3-week history of ethanol vapor exposure increased global H3K27 trimethylation (H3K27me3) and reduced global H3K4 trimethylation (H3K4me3) in the prefrontal cortex (93). While DNA methylation typically represses gene expression, histone modifications can be activating or repressing depending on the type and location of the modification (42,43). H3K27me3 is repressive whereas H3K4me3 is activating, indicating that this study found evidence for chronic ethanol causing genome-wide gene expression changes via two histone methylation mechanisms (93). Furthermore, chronic ethanol consumption increased H3K4me3 andH3K27me2 at different promoter regions of BDNF correlating with increased expression and increased neurogenesis in the hippocampus (94). Another study found that chronic drinking increased the expression of GABA-Aα5 in the prefrontal cortex by inducing its H3K4me3, an activating mark with transgenerational stability that may underlie offspring’s vulnerability to excessive ethanol drinking (95). Indeed, another study found male but not female offspring of chronic ethanol-exposed sires had reduced ethanol consumption and increased Bdnf in the VTA that was associated with DNA hypomethylation at the Bdnf promoter region compared to control sires; furthermore, Bdnf DNA hypomethylation in the VTA could be traced back to the chronic ethanol-exposed sires (96). In general, since a recent meta-analysis found the heritability of AUD to be approximately 50% (97), changes in parental chromatin structure and epigenetic marks due to chronic ethanol may be transmissible and account for altered responses to ethanol and susceptibility to development of AUD (98). In this review, we focus on transcriptional changes and associated epigenetic alterations with behavioral and pathophysiological consequences in subjects with direct ethanol exposure, as the majority of these studies do not examine transgenerational heritability. A recent review by Rompala and Homanics offers a detailed examination of the intergenerational epigenetic effects of ethanol (99).

Other studies find chronic ethanol leads to hypomethylation and altered acetylation of histones with corresponding changes in brain region-specific gene expression. For example, five days of chronic ethanol vapor treatment in mouse neurons decreased expression of several histone methyltransferases with concurrent reductions in global H3K9 di- and trimethylation, typical gene silencing marks, both globally and at the specific NR2B glutamatergic receptor subunit gene (100). By contrast, examining NR2B in the rat hippocampus during withdrawal from 16 weeks of chronic ethanol drinking found increased expression levels of NR2B mRNA and protein with increasing symptoms of ethanol withdrawal, driven by changes in H3K9ac (101). During withdrawal after chronic ethanol exposure, anxiety-like behavior is associated with decreases in acetylation of H3 and H4 and corresponding decreases in HAT CBP and increases in HDAC activity in the amygdala (67). HDAC inhibitor TSA reverses these epigenetic changes and blocks the development of ethanol withdrawal-induced anxiety-like behavior (67). Similarly, the same chronic ethanol exposure reduced H3K9ac and increased HDAC2 in the hippocampus; these changes were associated with depression-like behavior and reversed with HDAC inhibitor SAHA treatment (102). Furthermore, SAHA normalized GABA hyposensitivity and H3K9ac protein levels in the VTA during withdrawal from the same chronic ethanol diet (103). The role of HDAC2 in the amygdala has also been investigated in genetic alcohol preference and innate anxiety-like behaviors using alcohol preferring (P) and non-preferring (NP) rats (104). Innate HDAC2 levels but not other HDAC isoforms (HDAC1, HDAC3, HDAC4, HDAC5, and HDAC6) are higher in the CeA of P rats as compared with NP rats. The CeA of P rats also display global and Bdnf- and Arc-specific deficits in H3K9ac levels and reduced protein expression of BDNF and ARC (104). HDAC2 siRNA infusion into the CeA was able to attenuate alcohol intake and anxiety-like behaviors in P rats, increase CeA occupancy of H3K9/14ac at Bdnf and Arc genes, and correct the deficits in BDNF and Arc protein expression in the CeA of P rats (104). These data mechanistically implicate HDAC2 in the CeA in regulating alcohol-drinking and anxiety-like behaviors.

Several other studies demonstrated the role of histone methylation mechanisms in alcohol dependence and escalation in drinking. For example, in the NAc of alcohol dependent rats, decreased mRNA expression and increased protein expression of lysine demethylase KDM6B is associated with decreased H3K27me3 levels and upregulation in the IL-6 inflammatory signaling pathway (105). Also, a history of ethanol vapor exposure reduced dorsomedial prefrontal cortical histone methyltransferase Prdm2 expression and corresponding H3K9me1, an activating histone mark; a knockdown of this gene in ethanol-naïve rats recapitulated dependent-like phenotypes of increased ethanol consumption and stress-induced reinstatement of ethanol seeking (106). Similarly, this group showed post-dependent rats with increased chronic ethanol intake had increased DNA methylation resulting in decreased expression of several important synaptic genes in the prefrontal cortex, with a specific lentiviral knockdown supporting a mechanistic role for Syt2 in aversion-resistant drinking (107). Interestingly, recent evidence has shown that these DNA methylation changes can be pharmacologically targeted with DNMT inhibitors such as 5-azacytidine to reduce chronic ethanol drinking in mice (108). Furthermore, 5-azacytidine treatment can reverse excessive drinking and anxiety behaviors seen in rats exposed to adolescent ethanol, presumably through normalization of amygdalar Bdnf and Npy expression via upstream DNA methylation (109).

Chronic exposure to ethanol during adolescence is an intriguing area of research, as ethanol can interfere with normal epigenetic reprogramming events associated with brain maturation, and alcohol use during this key developmental window has persistent effects that significantly raise the risk of AUD development later in life (110–112). Adolescent intermittent ethanol (AIE) exposure to ethanol in rats, designed to mimic teenage binge drinking patterns, produces persistent epigenetic changes in histone acetylation and histone and DNA methylation in the amygdala with functional relevance for the anxiety and high-drinking phenotypes observed in these rats in adulthood (109, 112–114). These epigenetic changes lead to decreased amygdaloid BDNF and Arc expression that along with AIE-induced anxiety-like and alcohol drinking behaviors in adulthood can be reversed with HDAC inhibitor TSA treatment (113). Furthermore, epigenetic mechanisms also regulate expression of enhancer RNA (eRNA) in the amygdala and have been implicated in the AIE-induced susceptibility to adult anxiety. The deficits in Arc expression depend on decreased Arc eRNA and reduced Kdm6b expression, as RNA knockdown of these targets in ethanol-naïve rats provoked anxiety and recapitulated epigenetic phenotypes of AIE in adulthood (115). Additionally, for BDNF regulation, miR-137 is increased in adult amygdala after AIE and this miRNA regulates a histone demethylase Lsd1 which alters H3K9me2 to control BDNF expression at the Bdnf IV promoter (116). Furthermore, inhibition of miRNA-137 in the CeA was able to reverse epigenetic changes and attenuated AIE-induced anxiety-like and alcohol drinking behaviors (116). Outside of the amygdala, the same adolescent ethanol exposure resulted in increased hippocampal HDAC and decreased CBP activity, thereby reducing H3K9ac and resulting in decreased BDNF expression and neurogenesis (117). In prefrontal cortex after repeated binge-like exposure to ethanol in adolescent rats, activating H3K4me2 is increased at the promoters of transcription factors linked to synaptic plasticity and learning (118). Overall, these preclinical studies illustrate the complexity of epigenetic modifications in the brain following chronic alcohol exposure that contribute to complex AUD pathology, including aversion-resistant drinking, comorbid-anxiety, withdrawal symptoms, and altered neurogenesis and synaptic plasticity. It is important to bear in mind the methodological differences in route, timing, and length of ethanol administration and withdrawal as well as brain region and species differences, as these important details help contextualize various discrepancies in the above results.

In human post-mortem brain tissue, where we know the subjects were diagnosed with AUD, several studies have characterized epigenetic alterations in the brain. Ponomarev et al. found increased global H3K4me3 in the brains of alcohol use disorder subjects compared to healthy controls paired with increased histone methyltransferase expression in the amygdala and frontal cortex (119). On the other hand, when looking at specific genomic loci, chronic alcohol drinking results in significantly increased cortical DNA methylation of the glucocorticoid receptor NR3C1 exon variant 1_H associated with reduced NR3C1 mRNA and protein expression levels in the prefrontal cortex of AUD human subjects compared with controls (120). Similarly, in the cerebellum, increased DNA methylation at the δ subunit of the GABAA receptor was associated with reduced mRNA and protein levels of alcohol use disorder subjects (121). Since DNA methyltransferase expression was unchanged while DNA demethylation enzyme TET1 was decreased, the authors suggest chronic ethanol reduces DNA demethylation (121). In subjects who started drinking in adolescence, increased lncRNA BDNF-As expression in the amygdala is associated with decreased BDNF mRNA expression and gene-specific increased H3K27me3 and EZH2, suggesting early drinking leads to epigenetic reprogramming that increases risk for AUD development (51) similar to preclinical models (112). Furthermore, while the above studies in AUD patients use post-mortem tissue to evaluate neuroepigenetic consequences of chronic ethanol drinking, emerging studies are evaluating epigenetic signatures in the blood of living AUD patients at different withdrawal time points which could serve as corollaries for epigenetic disruptions in the brain and liver (122,123). Once again, these studies highlight differences in global versus gene specific epigenetic dysregulation following chronic ethanol consumption and further emphasize the important role of epigenetic modifications in neuropathology of AUD.

C. Epigenetic Effects of Chronic Ethanol in the Liver

The epigenetic mechanisms of chronic alcohol use and its outcome, alcoholic liver disease, are well studied, and chronic ethanol is known to modify the epigenome of cells in the liver via DNA and histone methylation, histone acetylation modification, and miRNAs (124–126). Throughout many organ systems, chronic alcohol alters autophagy, a process of maintaining homeostasis in response to stress, via epigenetic mechanisms such as alterations in Sirtuin deacetylase or G9a methyltransferase activity (127). In the liver, chronic alcohol impairs methionine synthase, altering SAM/SAH levels leading to DNA hypomethylation (87). Alcohol affects availability of SAM, and reductions in SAM levels lead to altered DNMT activity and DNA hypomethylation (128). Furthermore, chronic alcohol may increase HDAC11 activity in Kupffer cells in mice exposed to chronic alcohol, leading to decreases in IL-10, an anti-inflammatory cytokine (129). Also, miR-155 is implicated in increases of inflammatory responses in Kupffer cells and increased responsiveness to gut-derived endotoxin by inhibiting negative regulators of the TRL4 pathway (129). Chronic ethanol alters lipid metabolism in the liver, and PCSK9, a gene heavily implicated in the regulation of FAS, PPARα and CPT1, and inflammatory signaling, may be regulated by alterations in methylation of the promoter region of the gene (122,130). The many epigenetic alterations during chronic alcohol use contribute to the long-term pathobiological outcomes in the liver and digestive system associated with alcohol use disorder.

VI. Conclusion

With increasing knowledge of the complex gene regulatory mechanisms that encompass the wide range of epigenetic processes, the multifarious effects of ethanol consumption have come to be appreciated from a new perspective (Figure 1) that wasn’t possible mere decades ago when non-protein coding regions of the genome were considered junk. The growth of the alcohol and epigenetics field and the studies described in this review clearly demonstrate the pivotal role epigenetic alterations play in mediating alcohol’s biological actions in the brain and the liver (Figure 1). These changes can jump start or promote pathobiological processes such as cancer, liver disease, and addiction, and identifying these changes can help in rational drug design. Currently used pharmacologic agents with primarily epigenetic targets include DNMT inhibitors like citabine and 5-azacytidine and HDAC inhibitors like vorinostat and romidepsin, but the pathologies these drugs are FDA-approved to treat are myelodysplastic syndrome and cutaneous t-cell lymphoma respectively, malignancies that are not associated with ethanol abuse (131,132). Preclinical studies clearly advocate use of HDAC and DNMT inhibitors in attenuating alcohol intake and related behavioral phenotypes (64). Targeting these pharmacological treatments to specific tissues, brain regions, and cell types represent broad challenges in successfully translating this research to humans, but advanced drug delivery and gene therapy systems using nanoparticles, viruses, or CRISPR-Cas9 are intriguing options for targeted epigenetic therapies (133,134). Continuing to further our understanding of the epigenetic mechanisms altered by acute and chronic ethanol will help in the design of better medications for the pathologies that specifically result from ethanol consumption.

Figure 1:

Model depicting the alcohol induced changes in epigenetic processes and related changes in gene expression and pathobiology in brain and liver. Both acute and chronic ethanol exposure modulate epigenetic targets in the liver and the brain. These modifications lead to epigenetic reprogramming via histone and DNA chemical modifications that may play a role in changes in biological pathways and pathophysiology related to AUD. DNA methyltransferases (DNMTs) and DNA demethylases (TETs) regulate DNA methylation; hypomethylated DNA is more readily transcribed. Enzymes such as histone acetyltransferases (CREB binding protein [CBP] and p300, etc.) and lysine methyltransferases (G9a, etc.) add histone modifications. Enzymes such as HDACs (Histone deacetylases) and sirtuins remove acetyl group whereas lysine demethylases remove methyl group from histone. microRNAs (miR-494, miR-155, etc.) bind to mRNAs regulate expression of gene targets. **For expanded information regarding effects of chronic ethanol exposure on epigenetic mechanisms in the brain, refer to Table 1.