Cocaine epigenetically regulates gene expression via changes in histone post-translational modifications (HPTMs). We previously found that the immediate early gene Nr4a1 is epigenetically activated by cocaine in mouse brain reward regions. However, few studies have examined multiple HPTMs at a single gene. Bivalent gene promoters are simultaneously enriched in both activating (H3K4me3 (K4)) and repressive (H3K27me3 (K27)) HPTMs. As such, bivalent genes are lowly expressed but poised for activity-dependent gene regulation. In this study, we identified K4&K27 bivalency at Nr4a1 following investigator-administered cocaine in male and female mice. We applied sequential chromatin immunoprecipitation and qPCR to define Nr4a1 bivalency and expression in striatum (STR), prefrontal cortex (PFC), and hippocampus (HPC). We used Pearson’s correlation to quantify relationships within each brain region across treatment conditions for each sex. In female STR, cocaine increased Nr4a1 mRNA while maintaining Nr4a1 K4&K27 bivalency. In male STR, cocaine enriched repressive H3K27me3 and K4&K27 bivalency at Nr4a1 and maintained Nr4a1 mRNA. Furthermore, cocaine epigenetically regulated a putative NR4A1 target, Cartpt, in male PFC. This study defined the epigenetic regulation of Nr4a1 in reward brain regions in male and female mice following cocaine, and, thus, shed light on the biological relevance of sex to cocaine use disorder.

BACKGROUNDCocaine epigenetically regulates gene expression via changes in histone post-translational modifications (HPTMs). We previously found that the immediate early gene Nr4a1 is epigenetically activated by cocaine in mouse brain reward regions. HPTMs act combinatorically, yet few studies examine multiple HPTMs at a single gene. Bivalent gene promoters are simultaneously enriched in both activating (H3K4me3 (K4)) and repressive (H3K27me3 (K27)) HPTMs. As such, bivalent genes are lowly expressed but poised for activity-dependent gene regulation. In the current study, we defined regulation of K4&K27 bivalency at Nr4a1 following cocaine treatment in male and female mice. The inclusion of female mice can shed light on the epidemiological relevance of sex to cocaine use disorder.METHODSMale and female mice were injected with saline or cocaine (i.p. 20mg/kg). We applied sequential chromatin immunoprecipitation and qPCR to define Nr4a1 bivalency and expression in striatum (STR), prefrontal cortex (PFC), and hippocampus (HPC). Pearson’s correlation matrices quantified relationships within each brain region across treatment conditions for each sex.RESULTSWe defined K4&K27 bivalency at the Nr4a1 promoter in all three brain regions, in both sexes. In female STR, cocaine increased Nr4a1 mRNA, coupled to maintenance of Nr4a1 K4&K27 bivalency. In male STR, cocaine enriched repressive H3K27me3 and K4&K27 bivalency at Nr4a1 and failed to increase Nr4a1 mRNA. Furthermore, cocaine epigenetically regulated a putative NR4A1 target, Cartpt, in male PFC.CONCLUSIONThis study defined the epigenetic regulation of Nr4a1 in reward brain regions in male and female mice. Cocaine treatment in female mice increased Nr4a1 mRNA in STR, but there was no change in Nr4a1 H3K27me3 or K4&K27 promoter bivalency. Following cocaine treatment in male mice, Nr4a1 mRNA did not change in STR, HPC, or PFC, and Nr4a1 H3K27me3 and K4&K27 promoter bivalency increased in the STR.