Repeated cocaine exposure is characterized by persistent changes in gene expression and altered neuronal morphology within the nucleus accumbens (NAc), a key component of the brain’s reward circuitry (1
). Chromatin remodeling is important in aberrant transcriptional changes in this brain region that may underlie aspects of cocaine addiction (3
). Cocaine regulation of chromatin structure in the NAc results, in part, from direct cocaine-induced modifications of the chromatin enzymatic machinery, leading to changes in histone acetylation and phosphorylation (4
); however, roles for enzymes controlling histone methylation have not yet been investigated.
A recent genome-wide promoter analysis using chromatin immunoprecipitation coupled to microarrays (ChIP-Chip) identified altered patterns of repressive histone H3 lysine 9 (H3K9) and 27 (H3K27) methylation at specific gene promoters in the NAc following repeated cocaine treatment (6
). We therefore profiled numerous lysine methyltransferases (KMTs) and demethylases (KDMs) that are known to control H3K9 or H3K27 methylation (). Only two enzymes, G9a and GLP, displayed persistent transcriptional regulation 24 hours after repeated cocaine administration, when the expression of both genes was significantly downregulated. Because G9a and GLP specifically catalyze the dimethylation of H3K9 (H3K9me2), their downregulation by cocaine is consistent with decreased global levels of euchromatic H3K9me2 observed at this time point (). In contrast, global levels of heterochromatic H3K27 methylation remained unaltered by repeated cocaine exposure (Fig. S1 in supporting online material
). Due to its high levels of catalytic activity both in vitro
and in vivo
), we set out to further investigate the functional significance of G9a repression following repeated cocaine exposure in the NAc. Levels of G9a protein, like levels of its mRNA, were significantly reduced 24 hours after repeated cocaine administration (Fig. S2
). Although G9a mRNA expression was reduced by 35% in the NAc, immunohistochemical analysis revealed a more modest 15% reduction in G9a protein levels, consistent with the observed 21% decrease in H3K9me2 after repeated cocaine administration (). G9a mRNA expression was also downregulated in this brain region by 20% following repeated self-administration of cocaine (Fig. S3
Fig. 1 Repeated cocaine represses G9a expression in NAc through a ΔFosB-dependent mechanism. (A) mRNA expression of H3K9/K27 KMTs and KDMs in NAc 24 hr after repeated cocaine. (B) H3K9me2 levels in NAc 24 hr after repeated cocaine. (C) Analysis of gene (more ...)
To identify whether changes in euchromatic H3K9me2 correlate with genome-wide alterations in gene expression in the NAc, we employed microarray analyses to examine gene expression profiles induced by a challenge dose of cocaine in animals with or without a history of prior cocaine exposure (see supplemental gene lists in Tables S1–S3
). Animals that had received repeated cocaine displayed dramatically increased gene expression 1 hour after a cocaine challenge in comparison to acutely treated animals (). This increased gene expression still occurred in response to a cocaine challenge given after 1 week of withdrawal from repeated cocaine. Consistent with previous reports, a small percentage of genes (~10%) displayed desensitized transcriptional responses following repeated cocaine administration (; see Table S1
). To directly investigate the role of G9a downregulation in the enhanced gene expression observed after repeated cocaine, mice received intra-NAc injections of Herpes simplex virus (HSV) vectors expressing either GFP or G9a and were treated with saline or repeated cocaine to determine whether G9a overexpression was sufficient to block the repeated cocaine-induced enhancement of gene expression. From a set of 12 randomly selected genes displaying heightened levels of expression following repeated cocaine, we observed that G9a significantly reduced the enhanced expression of 50% of these genes (Table S4
To identify upstream transcriptional events that mediate the repeated cocaine-induced repression of G9a expression, we investigated a possible role for ΔFosB, a highly stable splice product of the immediate early gene fosB
. ΔFosB accumulates in the NAc after repeated exposure to cocaine, where it has been linked to increased cocaine reward (11
). ΔFosB can act as either a transcriptional activator or repressor depending on the target gene involved (3
). Using bitransgenic NSE-tTA
mice, wherein ΔFosB expression can be induced selectively in the NAc and dorsal striatum of adult animals (13
), we examined the impact of ΔFosB expression on cocaine regulation of H3K9me2 and KMTs in the NAc. ΔFosB overexpression was sufficient to reduce levels of both H3K9me2 () and G9a expression (), thereby mimicking the effects of repeated cocaine. In contrast, ΔFosB did not reduce GLP expression in this brain region, and had no effect on SUV39H1 and EZH2, the principal trimethylating enzymes for H3K9 and H3K27, respectively (Fig. S4
). To confirm these data using an independent ΔFosB overexpression system, wildtype adult mice received bilateral intra-NAc injections of Adeno-associated virus (AAV) vectors expressing either GFP or ΔFosB. Viral-mediated overexpression of ΔFosB decreased levels of G9a expression in this brain region ().
Such pronounced and specific regulation of G9a prompted us to investigate whether altering G9a expression specifically in NAc neurons regulates behavioral responses to cocaine. Wildtype mice received intra-NAc injections of HSV vectors expressing GFP or G9a and were then analyzed using an unbiased cocaine conditioned place preference paradigm, which provides an indirect measure of drug reward. Viral overexpression of G9a in NAc neurons was confirmed following behavioral testing (). G9a overexpression significantly decreased preference for cocaine in comparison to animals overexpressing GFP (), and increased H3K9me2 levels in the NAc (). Overexpression of a catalytically dead mutant of G9a (G9aH1093K) (14
) did not affect cocaine preference () and had no affect on H3K9me2 levels in this brain region ().
Fig. 2 G9a in NAc regulates cocaine-induced behavioral plasticity. (A) Representative image of HSV-mediated transgene expression in NAc. Cartoon of the coronal brain slice was taken from the mouse brain atlas. (B) Conditioned place preference for cocaine and (more ...)
To further study the role of G9a in the behavioral effects of cocaine, and more specifically to mimic the repeated cocaine-induced repression of G9a expression in the NAc, adult G9afl/fl
) received intra-NAc injections of AAV vectors expressing Cre recombinase or GFP as a control. AAV-Cre knockdown of G9a in the NAc, which was confirmed immunohistochemically (Fig. S5
), significantly increased the effects of cocaine in place conditioning experiments and decreased H3K9me2 levels in the NAc (). A commercially available pharmacological inhibitor of G9a and GLP, BIX01294 (15
), was used to ascertain whether enzyme inhibition similarly affects behavioral responses to cocaine. Indeed, pharmacological inhibition of G9a and GLP significantly increased preference for cocaine and decreased H3K9me2 in the NAc ().
Repeated administration of cocaine increases the density of dendritic spines on NAc medium spiny neurons (17
), a process associated with functional changes at excitatory glutamatergic synapses onto these neurons (18
) and sensitized behavioral responses to the drug (17
). We thus hypothesized that downregulation of G9a activity in the NAc by repeated cocaine might mediate cocaine’s ability to regulate the dendritic spine density of NAc neurons. Using chromatin immunoprecipitation (ChIP) with an anti-G9a antibody, we identified several putative G9a gene targets in the NAc, each of which has previously been implicated in cocaine-induced dendritic plasticity () (20
). We found that repeated cocaine administration significantly decreased G9a binding, as well as levels of H3K9me2, at these gene promoters (). In contrast, acute cocaine administration rapidly recruited G9a to some of these same gene promoters, consistent with increased G9a expression observed in the NAc 1 hour after an acute dose of cocaine (Fig. S6
). Although G9a binding at specific gene promoters correlates with changes in its expression, it remains unclear as to whether such events are mediated by altered global levels of G9a in the NAc and/or by differences in G9a recruitment following acute vs.
repeated cocaine administration.
Fig. 3 G9a in NAc regulates cocaine-induced dendritic spine plasticity. (A) Quantitative G9a ChIP in NAc from animals treated acutely or repeatedly with cocaine, at 1 or 24 hr, respectively. APRT was used as a negative control. Data are presented as the relative (more ...)
Based on G9a’s regulation of numerous plasticity-related genes in the NAc, we examined directly whether maintenance of G9a expression in this brain region following repeated cocaine treatment was sufficient to block cocaine-induced dendritic spine formation. Using a cocaine treatment protocol previously demonstrated to promote dendritic spine induction in the NAc (20
), we examined spine density in animals injected with either HSV-GFP or HSV-G9a. In agreement with previous findings, we observed a significant increase in dendritic spine density in the NAc following cocaine treatment, an effect that was blocked completely by G9a overexpression (). G9a overexpression alone was not sufficient to decrease NAc dendritic spine density in the absence of cocaine. To complement these data, G9afl/fl
mice received intra-NAc injections of HSV-Cre and spine density was quantified and compared to animals receiving HSV-GFP in the absence of cocaine. Knockdown of G9a expression significantly increased spine density on NAc medium spiny neurons ().
Given the evidence that G9a downregulation in the NAc after repeated cocaine is mediated by ΔFosB, we next examined whether this transcription factor is likewise involved in the regulation of NAc dendritic spines. Although ΔFosB has not previously been linked causally to such dendritic plasticity, several of its targets, including Cdk5 and NFκB subunits, have been so implicated (20
), and ΔFosB’s persistent expression in NAc neurons correlates with increased dendritic spine density after repeated cocaine treatment (27
). First, we found that induction of ΔFosB in bitransgenic mice in the absence of cocaine, which downregulated G9a and H3K9me2 expression (), decreased G9a binding to numerous plasticity-related genes, many of which have also been shown to be direct targets of ΔFosB itself () (3
). We next showed that viral overexpression of ΔFosB in the NAc significantly increased dendritic spine density under basal conditions, similar to that observed following repeated cocaine administration (). Conversely, overexpression in the NAc of ΔJunD, a dominant negative mutant protein that antagonizes ΔFosB transcriptional activity, blocked the ability of repeated cocaine to increase dendritic spine formation in the NAc ().
Our observation that ΔFosB regulates G9a expression in the NAc, and that ΔFosB and G9a regulate some of the same target genes, led us to examine other interactions between ΔFosB and G9a. After acute cocaine, when G9a levels were increased, binding of G9a to the fosB
gene was increased, while after repeated cocaine, when G9a expression was suppressed, G9a binding to the fosB
gene was decreased (). Such decreased G9a binding after repeated cocaine was not observed for c-fos,
where G9a binding is increased by repeated cocaine (Fig. S7
). This is consistent with the fact that, unlike fosB
is repressed, not induced, by chronic psychostimulant exposure (5
). ΔFosB overexpression in bitransgenic mice was sufficient to significantly decrease G9a binding to the fosb
gene (). Furthermore, G9a overexpression was sufficient to reduce increased ΔFosB expression following repeated cocaine administration (Table S4
). These data suggest an autoregulatory loop whereby G9a initially limits induction of ΔFosB by acute cocaine administration. However, as ΔFosB accumulates with repeated drug exposure, it represses G9a and thereby potentiates its own further induction.
In conclusion, we have demonstrated that histone lysine methylation in the NAc is critically involved in regulating neuronal gene expression in response to cocaine. Repression of G9a and H3K9me2 following repeated cocaine administration promotes cocaine preference, in part, through the transcriptional activation of numerous genes known to regulate aberrant forms of dendritic plasticity. Gaining a better understanding of the genes being regulated through such mechanisms will improve our knowledge of the complex biological basis of drug addiction and could aid in the development of more effective treatments of addictive disorders.