**4. DNA methylation changes associated with drugs of abuse**

Although DNA methylation is typically a stable epigenetic process, drugs of abuse have been shown to alter both DNA methylation and its associated enzymes. Much of this research has focused on DNA methylation in the nucleus accumbens (NAc), a brain region involved in reward and motivation learning [14]. A well-established marker of repeated exposure to drugs of abuse is an increase in the transcription factor ∆FosB protein in the NAc [15]. Acute or repeated cocaine administration decreases methylation at the *fosB* promoter in the NAc of rodents, which co-occurs with increases in *fosB* mRNA expression [16]. This may serve as a mechanism by which exposure to drugs of abuse produces stable increases in ∆FosB protein expression. Acute or chronic cocaine administration also increases *Dnmt3a* mRNA and MeCP2 protein expression in the NAc [16–18]. These increases are accompanied by decreases in psychostimulant reward as measured by conditioned place preference (CPP), a procedure in which an animal experiences a drug state while confined to one compartment of an apparatus and a neutral state while confined to an alternate compartment during conditioning, resulting in a shift in the animal's preference for the drug-paired compartment when given free access to both compartments. The decreased CPP effects are believed to be mediated by *Dnmt3a-* and MeCP2-induced silencing of genes that encode proteins that are needed for adaptation and functioning of NAc neurons [17, 18]. In addition, the TET enzyme that catalyzes DNA demethylation and subsequent transcriptional activation via conversion of 5mC to 5hmC [19] is decreased in the NAc in both rodents following cocaine administration and in postmortem tissue from human cocaine addicts [20]. Paradoxically, this decrease in TET is associated with increases in 5hmC expression at specific gene loci that have previously been linked to addiction [20]. Further investigation is needed to explain this complex pattern of epigenetic changes.

## **5. Histone modification**

In order for the long strands of DNA to fit within a cell's nucleus, DNA is tightly condensed into chromatin. Chromatin is made up of nucleosomes that contain a histone protein core comprised of two copies of each of four different histone proteins, H2A, H2B, H3, and H4, as well as 147 base pairs of DNA that is wrapped around the histone core (**Figure 1**). Chromatin can either be tightly (i.e., heterochromatin) or loosely packaged (i.e., euchromatin), where the former restricts and the latter permits gene expression. Chromatin is able to undergo dynamic remodeling by chemical modification of amino acid residues of the histone core proteins. Similar to DNA methylation, histone proteins can undergo post-translational addition or removal of one of several chemical groups via enzymatic reactions. There are more than 100 different posttranslational modifications that may occur and these changes correlate with either the activation or the suppression of gene expression.

Historically, DNA methylation was believed to be a permanent modification. However, demethylation of DNA can occur and also contributes to dynamic changes in gene expression. While passive demethylation in dividing cells may be due to malfunctioning of DNMT1, active demethylation occurs in both dividing and nondividing cells by enzymatic reactions. One reaction changes 5mC into a T, which is then recognized as a G/T mismatch. The mismatch activates a base excision repair (BER) pathway that utilizes thymine DNA glycosylase (TDG) and ultimately replaces T with a nonmethylated C [12]. Another reaction catalyzed by 10–11 translocation enzymes (TET) adds a hydroxyl (–OH) group to 5mC forming 5hmC. 5hmC itself has effects on gene expression and it can undergo further reactions that convert it back to a nonmethylated C [13]. Therefore, demethylation of DNA is generally correlated with an

Although DNA methylation is typically a stable epigenetic process, drugs of abuse have been shown to alter both DNA methylation and its associated enzymes. Much of this research has focused on DNA methylation in the nucleus accumbens (NAc), a brain region involved in reward and motivation learning [14]. A well-established marker of repeated exposure to drugs of abuse is an increase in the transcription factor ∆FosB protein in the NAc [15]. Acute or repeated cocaine administration decreases methylation at the *fosB* promoter in the NAc of rodents, which co-occurs with increases in *fosB* mRNA expression [16]. This may serve as a mechanism by which exposure to drugs of abuse produces stable increases in ∆FosB protein expression. Acute or chronic cocaine administration also increases *Dnmt3a* mRNA and MeCP2 protein expression in the NAc [16–18]. These increases are accompanied by decreases in psychostimulant reward as measured by conditioned place preference (CPP), a procedure in which an animal experiences a drug state while confined to one compartment of an apparatus and a neutral state while confined to an alternate compartment during conditioning, resulting in a shift in the animal's preference for the drug-paired compartment when given free access to both compartments. The decreased CPP effects are believed to be mediated by *Dnmt3a-* and MeCP2-induced silencing of genes that encode proteins that are needed for adaptation and functioning of NAc neurons [17, 18]. In addition, the TET enzyme that catalyzes DNA demethylation and subsequent transcriptional activation via conversion of 5mC to 5hmC [19] is decreased in the NAc in both rodents following cocaine administration and in postmortem tissue from human cocaine addicts [20]. Paradoxically, this decrease in TET is associated with increases in 5hmC expression at specific gene loci that have previously been linked to addiction [20]. Further investigation is needed to explain this complex pattern of epigenetic changes.

In order for the long strands of DNA to fit within a cell's nucleus, DNA is tightly condensed into chromatin. Chromatin is made up of nucleosomes that contain a histone protein core comprised of two copies of each of four different histone proteins, H2A, H2B, H3, and H4, as

**4. DNA methylation changes associated with drugs of abuse**

increase in DNA accessibility.

24 Recent Advances in Drug Addiction Research and Clinical Applications

**5. Histone modification**

**Figure 1.** DNA and histone chemical modifications. DNA (blue lines) wraps around pairs of histone proteins H2A, H2B, 3, and 4 (light pink and tan) that form an octomer histone protein core. (A) Closed chromatin, due to DNA meth‐ ylation (Me; red) and histone dimethylation on H3K9 (Me; orange), leads to transcriptional repression. (B) Open chro‐ matin, due to DNA hydroxymethylation (hMe; yellow) and histone acetylation (Ac; green), allows for transcription factors (TF; purple) to recruit RNA polymerase for transcription initiation.

A powerful technique for studying post-translational histone modifications is chromatin immunoprecipitation (ChIP). ChIP utilizes antibodies that bind specifically to chemically modified histone proteins, which can then be isolated along with the associated DNA (i.e., promoter regions, gene bodies, etc.) from the rest of the tissue. Next, histones and DNA segments are denatured and levels of specific DNA sequences are measured. This technique can be used to (1) determine which gene or gene promoter may be associated with a specific histone modification, (2) correlate changes in histone modification with expression of specific genes, and (3) suggest possible mechanisms for how a gene is turned on or off following an experimental manipulation (e.g., drug administration).

## **6. Histone modifications associated with drugs of abuse**

#### **6.1. Acetylation**

Acetyl groups are added to histone proteins, typically on a lysine residue, by histone acetyl transferases (HATs). Addition of acetyl groups leads to a more relaxed, less condensed chromatin state by negating the positive charge of the histone protein that is attracted to the negatively charged DNA (**Figure 1B**). Indeed, increases in drug-induced gene expression often positively correlate with the levels of histone acetylation.

Previous work has found widespread changes in acetylation of histone H3 and H4 subunits in the NAc following acute and repeated psychostimulant administration in rodents [21, 22], suggesting that many genes in the NAc may be primed for transcription, while others are suppressed. Acute cocaine administration induces expression of the IEGs *c-fos* and *fosB* in rodents [16, 23], and ChIP analysis revealed increases in H4 acetylation at the respective promoter regions of these genes [21]. With repeated cocaine administration, the increase in *fosB* expression is maintained and is associated with increases in H3 acetylation at the *fosB* promoter region [21]. This mechanism likely contributes to ∆FosB protein accumulation in the NAc following repeated drug exposure. Repeated cocaine administration also reduces the ability of cocaine to induce *c-fos* and this is accompanied by a reduction in H4 acetylation at the *c-fos* promoter region [21, 24]. Furthermore, chronic opiate administration decreases expression of another IEG, brain-derived neurotrophic factor (*Bdnf*), and decreases H3 acetylation at the *Bdnf* promoter in the ventral tegmental area (VTA) [25]. *Bdnf* is critical for the development and maintenance of synaptic structure and function [26, 27] and drugs of abuse exert their reinforcing effect primarily by activating mesocorticolimbic dopamine neurons that originate in the VTA and project to the NAc, prefrontal cortex (PFC), amygdala, and hippocampus [28, 29].

Additionally, alcohol withdrawal in rodents reduces expression of the IEGs activity-regulated cytoskeleton protein (*Arc*) and *Bdnf* in the amygdala along with decreases in H3 acetylation at the respective gene promoters [30]. The amygdala is involved in processing emotional memories and it plays a critical role in alcohol-related behavior and anxiety [31]. Although these examples suggest functional links between degree of acetylation and associated gene expression, histone acetylation may occur even without changes in gene expression [32]. Therefore, further work into the causal role of acetylation in drug-induced gene expression is required.

#### **6.2. Methylation**

can be used to (1) determine which gene or gene promoter may be associated with a specific histone modification, (2) correlate changes in histone modification with expression of specific genes, and (3) suggest possible mechanisms for how a gene is turned on or off following an

Acetyl groups are added to histone proteins, typically on a lysine residue, by histone acetyl transferases (HATs). Addition of acetyl groups leads to a more relaxed, less condensed chromatin state by negating the positive charge of the histone protein that is attracted to the negatively charged DNA (**Figure 1B**). Indeed, increases in drug-induced gene expression often

Previous work has found widespread changes in acetylation of histone H3 and H4 subunits in the NAc following acute and repeated psychostimulant administration in rodents [21, 22], suggesting that many genes in the NAc may be primed for transcription, while others are suppressed. Acute cocaine administration induces expression of the IEGs *c-fos* and *fosB* in rodents [16, 23], and ChIP analysis revealed increases in H4 acetylation at the respective promoter regions of these genes [21]. With repeated cocaine administration, the increase in *fosB* expression is maintained and is associated with increases in H3 acetylation at the *fosB* promoter region [21]. This mechanism likely contributes to ∆FosB protein accumulation in the NAc following repeated drug exposure. Repeated cocaine administration also reduces the ability of cocaine to induce *c-fos* and this is accompanied by a reduction in H4 acetylation at the *c-fos* promoter region [21, 24]. Furthermore, chronic opiate administration decreases expression of another IEG, brain-derived neurotrophic factor (*Bdnf*), and decreases H3 acetylation at the *Bdnf* promoter in the ventral tegmental area (VTA) [25]. *Bdnf* is critical for the development and maintenance of synaptic structure and function [26, 27] and drugs of abuse exert their reinforcing effect primarily by activating mesocorticolimbic dopamine neurons that originate in the VTA and project to the NAc, prefrontal cortex (PFC), amygdala,

Additionally, alcohol withdrawal in rodents reduces expression of the IEGs activity-regulated cytoskeleton protein (*Arc*) and *Bdnf* in the amygdala along with decreases in H3 acetylation at the respective gene promoters [30]. The amygdala is involved in processing emotional memories and it plays a critical role in alcohol-related behavior and anxiety [31]. Although these examples suggest functional links between degree of acetylation and associated gene expression, histone acetylation may occur even without changes in gene expression [32]. Therefore, further work into the causal role of acetylation in drug-induced gene expression is

experimental manipulation (e.g., drug administration).

26 Recent Advances in Drug Addiction Research and Clinical Applications

positively correlate with the levels of histone acetylation.

**6.1. Acetylation**

and hippocampus [28, 29].

required.

**6. Histone modifications associated with drugs of abuse**

In contrast to histone acetylation, methylation can be associated with both transcriptional activation and repression, depending on which histone residue is modified. For example, dimethylation of histone H3 Lysine 9 (i.e., H3K9me2) is commonly associated with transcrip‐ tional repression. One histone methyltransferase that catalyzes H3K9me2, G9a, is decreased in the NAc following both chronic cocaine and opiate administration [33, 34]. It should be noted that this also occurs following chronic social stress in mice that produces depressivelike behavioral phenotypes, including decreased social interaction and increased anhedonia [35]. Similarly, G9a is decreased in postmortem NAc tissue of clinically depressed patients [35]. Decreases in G9a are associated with increases in cocaine and morphine CPP [33, 34]. Inter‐ estingly, G9a opposes expression of ∆FosB [33, 34]. In turn, ∆FosB represses G9a expression, creating a feedback loop that perpetuates its own expression through disinhibition. Similarly, postmortem NAc brain tissue of human cocaine addicts exhibits decreases in G9a expression [36] and increases in ∆FosB expression [37], providing further support for a functional link between these two molecules. In addition to specific genes, compelling work using ChIP and high-throughput sequencing of the associated genome has shown that cocaine-induced downregulation of H3K9me2 expression preferentially occurs in nongenic regions of chromo‐ somes [38], suggesting additional roles of histone methylation that may be independent of traditional effects on specific protein-coding genes.

## **7. Noncoding RNAs**

Before the 1990s, noncoding RNA was often referred to as "junk DNA" that was thought to have little relevance to biological function. A growing body of research over the past 25 years has shown that noncoding RNAs have pivotal roles in almost every cellular process investi‐ gated. One class of noncoding RNAs that has received much attention is microRNAs (miR‐ NAs). miRNAs are small transcripts (~22 nucleotides) that regulate gene expression posttranscriptionally. They are transcribed from DNA in a manner similar to protein-coding transcripts, where transcription factors recognize promoter sequences upstream of miRNA genes and initiate transcription (**Figure 2**). Once transcribed, the several hundred nucleotide long transcript folds and binds to itself, producing a stem-loop hairpin structure referred to as a pri-miRNA. The enzyme, Drosha, trims the pri-miRNA into a smaller form known as premiRNA (~70 nucleotides long). Pre-miRNA is then transported out of the nucleus into the cytoplasm, where the loop portion of the pre-miRNA is cleaved by the enzyme Dicer [39]. Now as a double-stranded RNA molecule, it is unwound and one strand joins with several proteins to form the miRNA-induced silencing complex (miRISC). The mature miRNA binds to complementary sequences in the 3′ untranslated region (UTR) of a target mRNA where miRISC causes either translational repression, deadenylation, or endonucleolytic cleavage of the target mRNA, preventing its expression [40]. Importantly, the mature miRNA needs only ~6–8 complementary nucleotides for which to base pair with the 3′ UTR of the target mRNA, and therefore, one miRNA can target several hundreds of different mRNAs in a given cell. For this reason, miRNAs have been regarded as "master regulators" of gene expression. Changes

**Figure 2.** MicroRNA processing and function. MicroRNAs are transcribed similarly to protein-coding RNAs, except they form a stem-loop structure following transcription (i.e., pri-miRNA). The enzyme, Drosha, trims the ends of the stem (i.e., pre-miRNA) to prepare for exportation from the nucleus via Exportin 5. Once in the cytoplasm, Dicer cleaves the loop of the pre-miRNA that produces a double-stranded RNA. One strand (i.e., mature miRNA) is chosen to be incorporated into the miRNA-induced silencing complex (miRISC). Upon binding to a complementary sequence in the 3′ untranslated region of a target mRNA, either translational repression, deadenylation, or endonucleolytic cleavage may occur. All three mechanisms lead to decreases in protein product.

in miRNA expression can therefore lead to widespread changes in gene expression and alteration in several cellular signaling cascades. Other types of noncoding RNAs include: (1) PIWI-interacting RNA (piRNA), which regulates sperm development, (2) small nuclear RNA (snRNA), which regulates mRNA splicing, and (3) long noncoding RNA (lncRNA), which has widespread effects on chromatin modification and transcription [41].
