Genomic sensitization in response to G9a repression following repeated cocaine exposure

Cocaine-induced alterations in gene expression cause changes in neuronal morphology and behavior that may underlie cocaine addiction. We identified an essential role for histone 3 lysine 9 (H3K9) dimethylation and the lysine dimethyltransferase G9a in cocaine-induced structural and behavioral plasticity. Repeated cocaine administration reduced global levels of H3K9 dimethylation in the nucleus accumbens. This reduction in histone methylation was mediated through the repression of G9a in this brain region. To identify whether changes in H3K9me2 correlated 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. 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. These data suggest that repeated, but not acute, cocaine exposure results in persistent sensitized genomic responses to a cocaine challenge, indicating that sensitized behavioral responses to repeated cocaine are likely the result of G9a-dependent alterations in global transcriptional responses to cocaine. Four groups (3 independent biological replicates per group) were utilized for this microarray study, totaling 12 microarrays (see 'Treatment protocol' for cocaine treatment information). 1 hour following the last cocaine injection, animals were rapidly decapitated and brains were removed and placed on ice. Dissections of nucleus accumbens were taken using a 15-gauge needle punch and were quickly frozen on dry ice until RNA was extracted. Bilateral punches were pooled from four animals per replicate, totaling 12 mice per group. RNA isolation, microarray processing, and data analysis were performed as previously described. Briefly, RNA was isolated and purified and was checked for quality using Agilent’s Bioanalyzer. Reverse transcription, amplification, labeling and hybridization to Illumina MouseWG-6 v2.0 arrays were performed using standard procedures. Raw data were background subtracted and quantile normalized using Beadstudio software. Normalized data were analyzed using GeneSpring software and genelists were generated using significance criteria of a 1.3 fold change cutoff coupled with a non-stringent p-value cutoff of p < 0.05.