Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins

Abstract

Changes in gene expression contribute to the long-lasting regulation of the brain's reward circuitry seen in drug addiction; however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide chromatin changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings reveal several interesting principles of gene regulation by cocaine and of the role of DeltaFosB and CREB, two prominent cocaine-induced transcription factors, in this brain region. The findings also provide comprehensive insight into the molecular pathways regulated by cocaine-including a new role for sirtuins (Sirt1 and Sirt2)-which are induced in the nucleus accumbens by cocaine and, in turn, dramatically enhance the behavioral effects of the drug.

Fig. 1. Regulation of histone acetylation and methylation at gene promoters in the NAc by chronic cocaine

A. Venn diagrams of genes that show altered levels of H3 or H4 acetylation and H3 methylation (dimethyl-K9/K27) binding 24 hrs after chronic (7 days) cocaine administration. B. Patterns of cocaine-induced changes in H3 and H4 acetylation and H3 methylation at 6 representative gene promoters previously implicated in cocaine action.

Fig. 2. Regulation of ΔFosB and phospho-CREB binding at gene promoters in the NAc by chronic cocaine

A. Venn diagrams of genes that show significant levels of ΔFosB or phospho-CREB binding, or of H3/H4 acetylation or H3 methylation, after chronic (7 days) cocaine. B. Patterns of ΔFosB (green) and phospho-CREB (purple) binding at representative gene promoters after chronic cocaine (solid line) or saline (dotted line) treatment. Short bold lines on the x-axes indicate positions of consensus or near-consensus AP1 (red) or CRE (orange) sites. C. The top panel illustrates significant ΔFosB target genes from ChIP-chip (histogram) after chronic cocaine exposure and how expression of the encoded mRNAs are regulated upon inducible overexpression of either ΔFosB or its dominant negative antagonist ΔcJun in the NAc (heatmaps) (ρ = −0.09, p = 0.005). The bottom panel illustrates significant phospho-CREB target genes from ChIP-chip (histogram) after chronic cocaine exposure and how expression of the encoded mRNAs are regulated upon inducible overexpression of either CREB or its dominant negative antagonist mCREB (heatmaps) in the NAc (ρ = −0.3, p < 1E-16).

Fig. 3. Validation of sirtuins as a novel target for cocaine in the NAc

A. Changes in histone H3 and H4 acetylation, H3 K9/K27 methylation, and ΔFosB and phospho-CREB binding at the Sirt1 and Sirt2 gene promoters in the NAc after chronic (7 days) cocaine. A short red bold line along the x-axis indicates the position of an AP1 site. Significant changes are shown as solid lines. B. Quantitative ChIP confirmed cocaine-induced increases in H3 acetylation at the Sirt1 (left) and Sirt2 (right) gene promoters in an independent cohort of mice (p < 0.05, n = 3–6). Cocaine-induced ΔFosB binding was also confirmed for the Sirt2 promoter (p < 0.05, n = 3–6). This chromatin regulation is associated with significant increases in Sirt1 and Sirt2 (p < 0.05, n = 7–8) mRNA levels in the NAc. C. As well, SIRT1 and SIRT2 catalytic activity was significantly increased in NAc after chronic cocaine administration (p < 0.05, n = 7–8).

Fig. 4. Sirtuin regulates the electrical excitability of NAc neurons

A. Incubation (20 min) of acute NAc slices from adult mice with the sirtuin inhibitor, sirtinol (30 μM), caused a significantly higher rheobase compared to control (DMSO-treated) slices (ANOVA: F (2,13) = 24.64, p < 0.0001, Tukey’s post-hoc compared to DMSO, *p < 0.05). Conversely, slices incubated with 50 μM resveratrol, a sirtuin activator, exhibited a significant reduction in rheobase (*p < 0.05). B. A 100 pA injection into NAc neurons incubated with sirtinol (30 μM) elicits significantly fewer action potentials compared to control, while incubation with resveratrol (50 μM) results in significantly more firing than controls (ANOVA: F (2, 13) = 25.38, p< 0.0001, Tukey’s post-hoc compared to DMSO, *p < 0.05). C. Example traces from DMSO-, sirtinol-, and resveratrol-treated slices illustrate the robust physiological effects of manipulating sirtuins on NAc neurons.

Fig. 5. Sirtuins regulate behavioral responses to cocaine

A. Systemic administration of the sirtuin agonist, resveratrol (20 mg/kg ip, dissolved in 5% hydroxypropyl β-cyclodextrin vehicle), increases the rewarding effects of cocaine (5 mg/kg) in the conditioned place preference paradigm (p < 0.05, n = 9–12). B. Intra-NAc delivery of the sirtuin antagonist, sirtinol (50 μM in 5% hydroxypropyl β-cyclodextrin), decreases the rewarding effects of 10 mg/kg cocaine (right). Data are expressed as mean ± s.e.m. (n = 9–12 in each group), *p < 0.05 by t-test. C. Intra-NAc delivery of sirtinol (100 μM) in rats that were trained to self-administer cocaine significantly reduced the number of cocaine infusions at the threshold dose of 62 μg/infusion (*p < 0.05, n = 5–7). D. The sirtinol-induced decrease in cocaine self-administration was specific to the active (cocaine-associated) nose poke apertures, as they behaved normally at the inactive apertures. E. Sirtinol significantly reduces ERK1/2 phosphorylation under depolarizing conditions in acute NAc slices ex vivo (*p < 0.05, n = 4).

Fig. 6. Molecular pathway analysis of the genomic effects of cocaine in the NAc

Chronic cocaine-induced molecular changes in the NAc were identified by ChIP-chip for changes in acetylated H3 and H4, H3 dimethyl-K9/K27, ΔFosB, and phospho-CREB binding followed by rigorous statistical analysis (>3.1SD) and Ingenuity molecular pathway examination. The Key defines the different types of regulation shown in the figure. Alterations in second messenger (A) and growth factor (B) regulated pathways are shown. Red indicates modifications associated with gene activation (increased histone acetylation or decreased methylation); bright green, gene repression (decreased histone acetylation or increased methylation). Dark green arrows indicate genes that show significant alterations in ΔFosB binding, purple arrows phospho-CREB binding. See Supplemental Information for definitions of the gene abbreviations used in the figure.

Fig. 6. Molecular pathway analysis of the genomic effects of cocaine in the NAc

Chronic cocaine-induced molecular changes in the NAc were identified by ChIP-chip for changes in acetylated H3 and H4, H3 dimethyl-K9/K27, ΔFosB, and phospho-CREB binding followed by rigorous statistical analysis (>3.1SD) and Ingenuity molecular pathway examination. The Key defines the different types of regulation shown in the figure. Alterations in second messenger (A) and growth factor (B) regulated pathways are shown. Red indicates modifications associated with gene activation (increased histone acetylation or decreased methylation); bright green, gene repression (decreased histone acetylation or increased methylation). Dark green arrows indicate genes that show significant alterations in ΔFosB binding, purple arrows phospho-CREB binding. See Supplemental Information for definitions of the gene abbreviations used in the figure.

Fig. 6. Molecular pathway analysis of the genomic effects of cocaine in the NAc

Chronic cocaine-induced molecular changes in the NAc were identified by ChIP-chip for changes in acetylated H3 and H4, H3 dimethyl-K9/K27, ΔFosB, and phospho-CREB binding followed by rigorous statistical analysis (>3.1SD) and Ingenuity molecular pathway examination. The Key defines the different types of regulation shown in the figure. Alterations in second messenger (A) and growth factor (B) regulated pathways are shown. Red indicates modifications associated with gene activation (increased histone acetylation or decreased methylation); bright green, gene repression (decreased histone acetylation or increased methylation). Dark green arrows indicate genes that show significant alterations in ΔFosB binding, purple arrows phospho-CREB binding. See Supplemental Information for definitions of the gene abbreviations used in the figure.