Delta FosB mediates epigenetic desensitization of the c-fos gene after chronic amphetamine exposure

Abstract

The molecular mechanisms underlying the transition from recreational drug use to chronic addiction remain poorly understood. One molecule implicated in this process is DeltaFosB, a transcription factor that accumulates in striatum after repeated drug exposure and mediates sensitized behavioral responses to psychostimulants and other drugs of abuse. The downstream transcriptional mechanisms by which DeltaFosB regulates drug-induced behaviors are incompletely understood. We reported previously the chromatin remodeling mechanisms by which DeltaFosB activates the expression of certain genes; however, the mechanisms underlying DeltaFosB-mediated gene repression remain unknown. Here, we identify c-fos, an immediate early gene rapidly induced in striatum after acute psychostimulant exposure, as a novel downstream target that is repressed chronically by DeltaFosB. We show that accumulation of DeltaFosB in striatum after chronic amphetamine treatment desensitizes c-fos mRNA induction to a subsequent drug dose. DeltaFosB desensitizes c-fos expression by recruiting histone deacetylase 1 (HDAC1) to the c-fos gene promoter, which, in turn, deacetylates surrounding histones and attenuates gene activity. Accordingly, local knock-out of HDAC1 in striatum abolishes amphetamine-induced desensitization of the c-fos gene. In concert, chronic amphetamine increases histone H3 methylation on the c-fos promoter, a chromatin modification also known to repress gene activity, as well as expression levels of the H3 histone methyltransferase, KMT1A (lysine methyltransferase 1A, formerly SUV39H1). This study reveals a novel epigenetic pathway through which DeltaFosB mediates distinct transcriptional programs that may ultimately alter behavioral plasticity to chronic amphetamine exposure.

Figure 1.

ΔFosB desensitizes c-fos mRNA induction in striatum after chronic amphetamine exposure. A, Rats were treated with saline, acute amphetamine (4 mg/kg), or chronic amphetamine (7 d), and allowed to withdraw for 1–10 d. Rats that received chronic amphetamine received a challenge dose of either saline or amphetamine (4 mg/kg) and were analyzed 1 h later. The levels of c-fos mRNA observed after an acute dose of amphetamine were significantly reduced in rats that were exposed previously to chronic amphetamine and withdrawn from the drug for 1–5 d (ANOVA, significant effect of drug challenge, F_(1,35) = 132.57, p < 0.0001; significant effect of withdrawal, F_(5,35) = 9.24, p < 0.0001; Bonferroni post hoc, 1 d withdrawal vs acute, *p < 0.05, 3 d withdrawal vs acute, **p < 0.01; 5 d withdrawal vs acute, ***p < 0.001; n = 3–5). Moreover, at 1 d of withdrawal, the fold induction of c-fos versus acute was significantly blunted (^†p < 0.05), with a strong trend at 3 and 5 d of withdrawal. In rats treated with chronic amphetamine, after 5 d of withdrawal, there was a significant reduction in c-fos mRNA after a saline challenge compared with drug-naive controls (**p < 0.01). B, ChIP of striatal lysates found significantly more ΔFosB bound to the c-fos promoter after 1 and 5 d of withdrawal from chronic amphetamine (*p < 0.05, Student's t test; n = 4–5). C, Representative image of mouse striatum from the laser capture microscope before (top) or after (bottom) dissection of the infected region. D, Mice were given an acute dose of amphetamine and processed for laser capture microdissection. In striatum infected with AAV-ΔFosB compared with that infected with AAV-GFP, there was a significant decrease in c-fos mRNA (**p = 0.01, Student's t test; n = 3). No change was observed in β-tubulin mRNA (p > 0.05). Error bars indicate SEM.

Figure 2.

Recruitment of HDAC1 mediates ΔFosB action on c-fos. A, ChIP of striatal lysates revealed a significant increase in acetylated histone H4 on the promoter of c-fos 1 h after a challenge dose of amphetamine in drug-naive rats (ANOVA, significant effect of drug, F_(1,12) = 6.26, p < 0.05; Bonferroni post hoc, *p < 0.05; n = 5). This increase was not observed in rats exposed previously to chronic amphetamine (Amph) (p > 0.05). B, The HDAC inhibitor, sodium butyrate (400 mg/kg), reversed the amphetamine-induced reduction in c-fos mRNA observed after 5 d of withdrawal (ANOVA, significant effect of butyrate, F_(1,28) = 5.29, p < 0.05; Bonferroni post hoc, chronic amphetamine plus PBS vs chronic saline plus PBS, *p < 0.05, chronic amphetamine plus butyrate vs chronic saline plus PBS or butyrate, p > 0.05; n = 4–9). C, ChIP of striatal lysates revealed significantly more HDAC1 bound to the c-fos promoter (*p < 0.05, Student's t test; n = 5–6), but not the promoter of β-actin (p > 0.05), after 5 d of withdrawal from chronic amphetamine. D, HDAC1 was transfected into PC12 cells with either full-length FosB or ΔFosB. HDAC1 selectively immunoprecipitated with ΔFosB, not full-length FosB. E, In rats that received chronic electroconvulsive seizures (7 daily seizures), a condition known to increase ΔFosB several-fold, immunoprecipitation of HDAC1 pulled down significant levels of ΔFosB. This interaction was not observed in sham-treated animals. Blots are representative of two to three experiments. F, Serum stimulation increased c-fos mRNA significantly less in cells transfected with HDAC1 than with GFP (*p < 0.05; n = 3 independent experiments). G, Floxed HDAC1 mice whose striata were infected with either AAV-GFP or AAV-CreGFP on opposite sides of the brain were treated with chronic amphetamine (7 d, 4 mg/kg) and 5 d of withdrawal. We found significantly higher c-fos expression in cells infected with AAV-CreGFP, where HDAC1 had been floxed out, than in cells expressing AAV-GFP after a 2 mg/kg amphetamine challenge (*p < 0.05; n = 2–3). Significantly less Hdac1 mRNA was observed in AAV-Cre-infected neurons (***p < 0.001), whereas Hdac2 expression was unaffected (p > 0.05). Error bars indicate SEM.

Figure 3.

Histone methylation after chronic amphetamine exposure. A, ChIP of striatal lysates found significantly more H3K9me2 bound to the c-fos promoter after 5 d of withdrawal from chronic amphetamine (*p < 0.05, Student's t test; n = 3), whereas no change occurred on the β-actin promoter. B, Rats treated with chronic amphetamine and analyzed after 5 d of withdrawal have significantly higher Kmt1a/Suv39h1 mRNA levels compared with saline-treated rats (*p < 0.05; n = 3), whereas Hdac5 levels remain unchanged (p > 0.05). Error bars indicate SEM.