Histone Tail Modification
Most DNA in eukaryotic cells is densely packed in chromatin, where 147 base pairs (bp) are wrapped around a nucleosome core in ~1.7 superhelical turns67
. Nucleosomes are composed of octamers that contain four histone dimers, one each of histones H2A, H2B, H3, and H4, with H1 binding to spans of non-nucleosomal DNA. Numerous types of posttranslational modifications of the N-terminal tails of histones alter chromatin compaction to create more “open” (euchromatin, which is transcriptionally permissive) vs.
“closed” (heterochromatin, which is transcriptionally repressive) states68
Many residues in the tails of histones are covalently modified in numerous ways, resulting in a complex “code” that is thought to control the accessibility of individual genes to the transcriptional machinery69
. Histone acetylation, which negates the positive charge of lysine residues in the histone tail, is associated with transcriptional activation. This process is controlled by histone acetyltransferases (HATs) and HDACs, each of which comprises multiple enzyme classes whose expression and activity are exquisitely regulated67
. Histone methylation has been associated with both transcriptional activation and repression depending on the particular residue and the extent of methylation70,71
: both lysine and arginine residues can be methylated by several families of histone methyltransferases (HMTs), and this reaction can be reversed by equally diverse histone demethylases. Histone tail modifications also include phosphorylation, ubiquitination, sumoylation, ADP ribosylation, among many others67
. The prospect of deciphering the histone code is daunting, given the seemingly infinite number of possible patterns of histone modifications, and the possibility that a particular pattern may have varying meaning depending on the individual gene involved. Nevertheless, new tools are accelerating progress in mapping the epigenetic state of individual gene promoters and the genome as a whole, and future research will determine the feasibility of identifying functionally meaningful chromatin codes72
Multiple drugs of abuse induce changes in histone acetylation in brain, and evidence has begun to accumulate that these modifications underlie some of the functional abnormalities found in addiction models66,70
. First, global (i.e., total cellular) levels of H3 and H4 acetylation are increased in the NAc after acute or chronic exposure to cocaine65,73
, and gene promoters that show increased H3 vs.
H4 acetylation have been mapped genome-wide32
. Despite these global increases, many genes show decreased histone acetylation after chronic cocaine, raising a key question as to what governs gene-specific acetylation changes in the face of global modifications. Another key question concerns the precise intracellular signaling cascades through which cocaine induces changes in histone acetylation — there is some information that such changes may be specific to D1-type MSNs and involve regulation of growth factor-associated kinases74,75
. Second, alcohol withdrawal has been demonstrated to increase HDAC activity and reduce histone acetylation in the mouse amygdala76
, and the commonly abused inhalant benzyl alcohol regulates potassium channels that are tied to alcohol tolerance via H4 acetylation in Drosophila77
. Third, exposure to Δ9
-THC, the active ingredient in marijuana, increases HDAC3 in trophoblast cells78
. However, this alteration was absent in a genome-wide screen of brain tissue from Δ9
, demonstrating that experiments on cell lines can yield effects that are very different from those found in a complex heterogeneous tissue like the brain. These data highlight the need for further research to define the effects of drugs of abuse on histone acetylation in brain in a region- and cell type-specific manner and to identify the specific HAT and HDAC subtypes and intracellular signaling pathways that mediate this regulation in vivo
Experimental alterations in histone acetylation potently affect addiction-related behaviors. Short-term administration of non-specific HDAC inhibitors, either systemic or intra-NAc, potentiates place conditioning and locomotor responses to psychostimulants and to opiates65,73,80
. More prolonged HDAC inhibition has been reported to induce changes in the opposite direction81,82
, perhaps through adaptations that oppose initial enzyme inhibition. Studies of specific HDAC isoforms have yielded interesting information: overexpression of HDAC4 or HDAC5 decreases behavioral responses to cocaine73,80
, whereas genetic deletion of HDAC5 hypersensitizes mice to the chronic (but not acute) effects of the drug80
. Likewise, mutant mice with reduced expression of CBP, a major HAT in brain, exhibit decreased sensitivity to chronic cocaine83
. Much additional work is needed to define the influence of specific HAT and HDAC subtypes on addiction-related phenomena.
The potential complexity involved is indicated by recent findings on sirtuins, which are considered Class III HDACs but in reality influence many non-histone proteins. Genome-wide studies of chromatin alterations in the NAc after chronic cocaine revealed upregulation of two sirtuins, SIRT1 and SIRT2. Pharmacological inhibition of sirtuins decreases cocaine place preference and self-administration, whereas activation increases rewarding responses to cocaine32
. SIRT1 and SIRT2 induction is associated with increased H3 acetylation and increased ΔFosB binding at their gene promoters32
, suggesting that sirtuins are downstream targets of ΔFosB. Work is now needed to identify the proteins that are affected by cocaine-induced regulation of these sirtuins. For example, sirtuins deacetylate several transcription factors such as forkhead box (FoxO) proteins, and serve scaffolding functions by contributing to transcriptional repressive complexes84,85
, processes which now warrant study in cocaine models. These findings illustrate the ability of genome-wide efforts to identify fundamentally new mechanisms involved in drug action.
Histone methylation is directly regulated by drugs of abuse as well: global levels of histone 3 lysine 9 dimethylation (H3K9me2) are reduced in the NAc after chronic cocaine37
and a genome-wide screen revealed alterations in H3K9me2 binding on the promoters of numerous genes in this brain region32
; both increases and decreases were observed, indicating again that epigenetic modifications at individual genes often defy global changes. The global decrease in H3K9me2 in the NAc is likely mediated by cocaine-induced downregulation of two HMTs, G9a and G9a-like protein (GLP), which catalyze H3K9me237
. These adaptations mediate enhanced responsiveness to cocaine, as selective knockout or pharmacological inhibition of G9a in the NAc promotes cocaine-induced behaviors, whereas G9a overexpression has the opposite effect. G9a likewise mediates the ability of cocaine to increase the spine density of NAc MSNs37
). Interestingly, there is a functional feedback loop between G9a and ΔFosB: ΔFosB seems to be responsible for cocaine-induced suppression of G9a, and G9a binds to and represses the fosb
promoter, such that G9a downregulation may promote the accumulation of ΔFosB observed after chronic cocaine37
. In addition, G9a and ΔFosB share many of the same target genes.
Chronic cocaine also downregulates H3K9me3, a mark of heterochromatin, specifically in the NAc and this change is associated with a decrease in the total amount of heterochromatin in NAc MSN nuclei and an increase in the volume of these nuclei86
. Genome-wide mapping of H3K9me3 after chronic cocaine indicates that most of the cocaine regulation of this mark occurs at non-genic regions, including at repetitive line elements, which are consequently induced by cocaine86
. Although the functional implications of this regulation are not yet known, these findings highlight the profound effects that cocaine exerts on the genome within NAc neurons.
Studies are now needed to examine the actions of other drugs of abuse on these histone endpoints, as well as the effect of drugs on many other types of histone modifications known to regulate eukaryotic gene expression in other systems, in addiction models. Examples include recent, preliminary observations of chronic cocaine regulation of histone arginine methylation and poly-ADP ribosylation, of several families of chromatin remodeling proteins, and of histone variant subunits in the NAc, all of which illustrate the complexity of epigenetic changes associated with drug exposure87–90
Moreover, it will be important to relate drug-induced modifications of histones, occurring at specific drug-regulated genes, with the recruitment of numerous additional proteins that ultimately constitute the transcriptional activation or repression complexes that mediate such regulation. For example, early studies have demonstrated that cocaine induction of CDK5 in the NAc involves a cascade of events which include binding of ΔFosB to the Cdk5
gene promoter, followed by the recruitment of CBP, increased H3 acetylation, and the recruitment of specific chromatin remodeling factors, such as transcription activator BRG173
(). Such activation also involves reduced repressive histone methylation at this promoter, which is mediated via cocaine suppression of G9a. In contrast, a very different cascade mediates chronic amphetamine repression of the c-fos
gene. Here, ΔFosB binds to the c-fos
promoter and recruits HDAC1 and SIRT1, and presumably numerous other proteins91
. Also, chronic amphetamine induces increased repressive histone methylation at the c-Fos promoter, perhaps mediated via increased G9a binding37
. It is interesting that such increased G9a binding occurs despite the global decrease in G9a expression, once again highlighting gene-specific changes that occur on top of global modifications. Understanding the molecular basis of such gene-specific modifications — e.g., why ΔFosB triggers a cascade of transcriptional activation when it binds to one promoter, but a cascade of transcriptional repression when it binds to another — is a crucial goal of current research. To date, these efforts have been pursued on a protein-by-protein basis, which is experimentally painstaking. A major need in the field is to develop tools to analyze the complete protein complexes recruited to individual genes in concert with drug exposure.
Epigenetic basis of drug regulation of gene expression
Methylation of DNA occurs at the 5′ position of cytosine nucleotides, with the resulting methyl group projecting into the major groove of the DNA double helix92
. In mammals, this occurs almost exclusively in 5′-CpG-3′ sequences and methylation is common throughout the genome — ~3% of all cytosines in human DNA are methylated93
— with proper cytosine methylation required for normal development, genetic imprinting, and X-chromosomal inactivation94
. CpG sequences are not evenly dispersed throughout the genome, but rather concentrated in regions termed CpG islands. These are CG-rich regions that overlap with the promoters of 50–60% of human genes and are typically methylated to a much lower extent than CpG dinucleotides found outside of islands95
. CpG methylation is catalyzed by a family of enzymes termed DNA methyltransferases (DNMTs), some of which are responsible for maintenance of DNA methyl states whereas others perform de novo
. The process of demethylation is less well understood, and may utilize DNA repair mechanisms, such as growth arrest and DNA damage-inducible protein GADD45 (Gadd45)93
and methylcytosine dioxygenase Tet196–98
. A variant of DNA methylation, 5-hydroxycytosine methylation, also seems to be important in gene regulation99,100
but has not yet been investigated in addiction models.
DNA methylation is generally considered to repress gene transcription through recruitment of corepressor complexes (e.g., HDACs, HMTs) that can sterically hinder the transcriptional machinery or modify nucleosome structure. Such complexes involve several DNA methyl-binding domain proteins (MBDs)93
, which are required for normal cell growth and development. Indeed, mutations in methyl CpG binding protein 2 (MeCP2), a prominent MBD, cause the majority of Rett Syndrome cases and are found in a small number of patients with other autism spectrum disorders94
There are multiple known links between DNA methylation and addiction. Cocaine self-administration increases MeCP2 expression in the NAc101
and dorsal striatum102
, and lentiviral knockdown of MeCP2 in the dorsal striatum (but not the NAc) decreases drug intake under extended but not limited access conditions66
. Hypomorphic Mecp2
mutant mice show reduced locomotor sensitization and place conditioning with chronic amphetamine103
, however, the same study reported that viral knockdown of MeCP2 in the NAc increases amphetamine place conditioning whereas local overexpression decreases this behavioral response104
. The reasons for this discrepancy are unclear, but it seems likely that developmental abnormalities in the mutant mice, or the effects of reduced Mecp2
expression in other brain regions, explain these differences. These findings therefore emphasize the importance of utilizing inducible and brain region-specific tools.
Two possible mechanisms for the actions of MeCP2 in drug reward have been proposed. First, a reduction in MeCP2 prevents amphetamine-mediated increases in NAc dendritic spine density while increasing the number of GABAergic synapses103
. This is complemented by a GABAergic interneuron-specific increase in MeCP2 phosphorylation in the NAc, which regulates its transcriptional activity and correlates strongly with behavioral sensitization to amphetamine103
. An alternative model suggests that MeCP2 represses the transcription of specific microRNAs (see below), resulting in reduced repression of brain-derived neurotrophic factor (BDNF)105
, which is also a target for CREB. BDNF has previously been described to promote cocaine self-administration106
, consistent with the MeCP2 data. Though these models are not mutually exclusive, further work is necessary to integrate them with our growing understanding of the multiple brain regions and cell types involved in reward behaviors.
A direct link between CpG methylation and addiction involves DNMT3a. Repeated cocaine administration dynamically regulates DNMT3a expression in the mouse NAc, with decreases seen during early phases of withdrawal and sustained increases seen at later time points82,107
. Experimental reduction of DNMT3a activity in the adult NAc, achieved either via viral-mediated local knockout in floxed Dnmt3a mice or via local infusion of a DNMT inhibitor, increases behavioral responses to cocaine, whereas DNMT3a overexpression in this region decreases these responses, but also has the paradoxical effect of increasing NAc MSN spine density107
, similar to the effects of MEF2 manipulation in this brain region60
. A major goal of current research is to identify the specific genes whose methylation status changes in response to chronic cocaine and consequently regulates cellular and behavioral adaptations to the drug.
These observations that chronic cocaine alters DNMT3a and MBDs in the NAc and dorsal striatum raise the possibility that drug-induced changes in DNA methylation might also occur in germ cells and be passed onto to subsequent generations to regulate the propensity of the offspring for addictive behaviors. Such trans-generational transmission of DNA methylation changes and resulting behavioral plasticity remains highly speculative, although recent research has demonstrated robust effects of adult cocaine exposure in rats on cocaine responses in their progeny108
Gene Priming and Desensitization
Ongoing studies of chromatin regulation in addiction models support the view that epigenetic modifications at individual genes, in addition to underlying stable changes in the steady-state levels of mRNA expression of certain genes, alter the inducibility of many additional genes in response to some subsequent stimulus in the absence of changes in baseline expression levels. Although such studies are still in relatively early stages of development, these types of latent epigenetic changes can be viewed as “molecular scars” that dramatically alter an individual’s adaptability and contribute importantly to the addicted state.
Such priming and desensitization of genes is evident in a recently published microarray study37
. Numerous desensitized genes were identified: ~10% of genes whose transcription is induced acutely in the NAc by cocaine are no longer induced by a cocaine challenge after prior chronic exposure to the drug (). Conversely, numerous genes are primed: genes that are not affected by acute cocaine become inducible after a chronic course of cocaine, with ~3-fold more genes being induced in cocaine-experienced animals. The mechanisms underlying such gene desensitization and priming remain incompletely understood; our hypothesis is that epigenetic mechanisms are crucial (). A subset of primed genes show reduced binding of G9a and H3K9me2 at their promoters in the NAc, suggesting the involvement of this epigenetic mark37
. Desensitization of the c-fos
gene in the NAc, discussed above and depicted in , involves stable increases in the binding of ΔFosB, G9a, and related co-repressors, which—although not affecting steady-state levels of c-Fos
mRNA—dramatically repress its inducibility to subsequent drug exposure91
A major need for the field is to now investigate many additional chromatin mechanisms that are recruited by drug exposure to mediate gene priming and desensitization and to understand the detailed mechanisms that target those particular genes. The goal of such studies would be to identify “chromatin signatures” that underlie such long-lasting regulation. The prominence of gene priming and desensitization indicates that studies of steady-state mRNA levels per se
would miss important aspects of drug regulation that are not captured at the particular time point examined. For example, the aforementioned microarray study37
measured mRNA levels 1 hr after a cocaine challenge, and preliminary evidence suggests that a partly distinct set of genes show evidence of priming and desensitization at 4 hr. These observations highlight the unique utility of genome-wide assays of chromatin regulation, as such assays would reveal priming and desensitization more globally32
Increasing attention has focused on a variety of non-coding RNAs that are important in biological regulation109
. These include microRNAs, which are generally around 22 bp long, are found in all mammalian cells, and are post-translational regulators that bind to complementary sequences on target mRNAs to repress translation and thus silence gene expression. Like histone modifications and DNA methylation, expression of microRNAs can alter the transcriptional potential of a gene in the absence of any change to the DNA sequence, and thus can be considered an epigenetic phenomenon. Several recent studies have implicated microRNAs in addiction behaviors, and miRNAs altered by drugs of abuse have been shown to regulate the expression of many proteins strongly linked to addiction110
Cocaine self-administration in rats reportedly increases expression of the microRNA miR-212 in striatum, and experimentally increasing miR-212 levels in this region decreases cocaine reward111
. The actions of miR-212 depend on upregulation of CREB, which is known to decrease the rewarding effects of cocaine (see above), and more recent work demonstrates that MeCP2 may interact homeostatically with miR-212 to control BDNF expression and cocaine intake105
. It has been proposed that this CREB–miR-212–MeCP2–BDNF mechanism is at least partially responsible for cocaine tolerance and escalating intake. miR-124 and miR-181a are also regulated in brain by chronic cocaine, where they are decreased and increased, respectively112
. miR-124 overexpression in the NAc reduces cocaine place conditioning, whereas overexpression of miR-181a has the opposite effect113
, suggesting that drug regulation of these microRNAs may also act as mechanisms of tolerance and escalating intake. Like miR-212, miR-124 and miR-181a may operate through the CREB–BDNF pathway, as miR-124 overexpression downregulates both of these genes. However, these microRNAs have also been shown to affect the expression of the dopamine transporter, so their mechanisms of action are likely to be complex114
. Finally, arginine exporter protein ARGO2 — which is important in microRNA-mediated gene silencing — along with several specific microRNAs have recently been implicated in cocaine regulation of gene expression selectively in the D2 subclass of striatal MSNs115
Other drugs of abuse have been linked to microRNAs as well. Opioid receptor activation downregulates miR-190 in cultured rat hippocampal neurons in a beta-arrestin2-dependent manner116
, and the let-7
family of microRNA precursors is upregulated by chronic morphine exposure in mice117
. Interestingly, the μ opioid receptor is itself a direct target for let-7, and the resulting repression of the receptor has been suggested as a novel mechanism for opiate tolerance117
. In zebrafish and in cultured immature rat neurons, morphine decreases miR-133b expression, and this might influence dopamine neuron differentiation114
. Additionally, both acute and chronic alcohol exposure upregulates miR-9 in cultured striatal neurons, and this may contribute to alcohol tolerance through regulation of large-conductance Ca2+
. miR-9 seems to preferentially downregulate BK channel isoforms that are sensitive to alcohol potentiation, perhaps shifting BK channel expression toward more tolerant subytpes119
. miR-9 also targets the D2 dopamine receptor119
, and so probably influences alcohol reward.
In the future, next-generation sequencing of microRNAs in several brain regions after exposure to drugs of abuse will be essential to uncover regulation of specific microRNAs and eventually the genes they regulate. Indeed, this process has already begun, as such screens are revealing numerous mcicroRNAs regulated in the NAc after chronic cocaine115,120
. For example, cocaine regulation of the miR-8 family suggests novel mechanisms for drug-induced alterations in the neuronal cytoskeletal and synaptic structure120
. Exploring this mechanism in drug-induced regulation of NAc dendritic morphology is an important line of future investigation.