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. 2016 Aug 23;113(34):9623-8.
doi: 10.1073/pnas.1605045113. Epub 2016 Aug 9.

Histone Arginine Methylation in Cocaine Action in the Nucleus Accumbens

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Free PMC article

Histone Arginine Methylation in Cocaine Action in the Nucleus Accumbens

Diane M Damez-Werno et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms-such as histone acetylation and methylation on Lys residues-have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.

Keywords: ChIP-seq; Src; drug addiction; histone arginine (R) methylation; medium spiny neurons.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Repeated cocaine administration persistently represses PRMT6 expression in the NAc of mice, rats, and addicted humans. (A) Schematic for i.p. injection of saline or cocaine (20 mg/kg) in mice shown in the other panels. (B) NAc mRNA levels of the nine mammalian PRMTs after seven daily i.p. injections of saline (saline) or cocaine (repeated cocaine) or six daily saline treatments and one cocaine injection (acute cocaine). NAc mRNA levels are compared with the NAc of mice treated with seven daily i.p. saline injections. Shown are NAc mRNA levels of Prmt1 [F(2,69) = 4.36, P = 0.016; saline vs. acute: t(41) = 2.42, P < 0.05; saline vs. repeated: t(52) = 2.57, P < 0.01], Prmt2 [F(2,52) = 4.23, P = 0.02; saline vs. repeated: t (36) = 2.91, P < 0.01], Prmt5 [F(2,70) = 4.62, P = 0.013; saline vs. repeated: t(53) = 3.02, P < 0.01], Prmt6 [F(2,53) = 3.72, P = 0.03; saline vs. repeated: t (36) = 2.53, P < 0.01], Prmt8 [F(2,27) = 3.41, P = 0.049, saline vs. repeated: t (17) = 2.61, P < 0.01], and Prmt9 [F(2,27) = 6.31, P = 0.006; saline vs. repeated: t(27) = 3.49, P < 0.01] at 24 h after the last i.p. injection. n = 9–27. #P < 0.05; *P < 0.01. (C) NAc mRNA levels of PRMT6 [t(30) = 2.77, P = 0.014] at 28 d after the last i.p. injection. n = 14–16. #P < 0.05; Bonferroni correction, *P < 0.006. (D and E) Protein levels at 24 h after seven daily i.p. injections of either cocaine (20 mg/kg) (repeated cocaine) or saline (saline). (D) NAc protein levels of PRMT6 [t(26) = 2.44, P = 0.011). n = 9–18. *P < 0.05. (E) NAc protein levels of H3R2me2a [t(37) = 2.12, P = 0.02]. n = 7–30. *P < 0.05. (F) PRMT6 and H3R2me2a protein levels in rat NAc and Prmt6 mRNA levels in human postmortem NAc after cocaine exposure. Shwon are NAc rat protein levels of PRMT6 [t(14) = 2.51, P < 0.05] and H3R2me2a [t(14) = 2.38, P < 0.05] after 10 d of either saline or cocaine self-administration (SA) at 1 mg/kg/infusion for 2 h (FR1;TO30s), followed by 7 d of withdrawal with same animals given a single SA reexposure session with either saline or cocaine at 24 h after the reexposure test. n = 5–11. Also shown is the NAc human mRNA level of Prmt6 [t(25) = 2.16, P < 0.05]. n = 13–14. *P < 0.05. Data are presented as mean ± SEM.
Fig. 2.
Fig. 2.
Cell type-specific effects of PRMT6 in the NAc in cocaine action. (A and D) CPP testing with cocaine (7.5 mg/kg) using intra-NAc delivery of an HSV-PRMT6 construct (A, Left), an AAV-PRMT6-miR construct (A, Right), or an HSV-LS1L-PRMT6 construct (D). (A, Left) PRMT6 overexpression in the NAc increased time spent in the cocaine-paired CPP chamber [t(37) = 3.41, P = 0.0016]. n = 7–27. (A, Right) PRMT6 knockdown in the NAc decreased the time spent in the cocaine-paired CPP chamber [t(46) = 2.46, P = 0.017]. n = 13. (B) RiboTag methodology schematic. RiboTag mice (HA-tagged Rpl22) were crossed to D1-Cre or D2-Cre mice, resulting in expression of the HA-tagged Rpl22 only in cells expressing Cre (D1-Cre-RiboTag or D2-Cre-RiboTag mouse lines). Cell-specific ribosome-associated mRNA was isolated from the NAc of these mice using HA-tagged magnetic beads and then analyzed by qRT-PCR. (C) In D1-Cre-RiboTag and D2-Cre-RiboTag mice, NAc mRNA levels and fold change of Prmt6 at 24 h after seven daily i.p. injections of either cocaine (20 mg/kg) or saline. Shown are Prmt6 NAc mRNA levels in D1-Cre-RiboTag mice [t(21) = 2.09, P = 0.049] compared with saline-treated D1-Cre-RiboTag mice and in D2-Cre-RiboTag mice [t(20) = 2.46, P = 0.02] compared with saline-treated D2-Cre-RiboTag mice at 24 h after the last i.p. cocaine injection. n = 11. (D) Cell type-specific PRMT6 overexpression in D1-MSNs or D2-MSNs in the NAc oppositely regulated the time spent in the cocaine-paired CPP chamber. Shown is cocaine CPP in D1-Cre+ and D2-Cre+ mice and their respective WT littermate controls D1-Cre and D2-Cre, all of which were injected intra-NAc with HSV-LS1L-PRMT6. D1-Cre+ vs. D1-Cre: t(33) = 2.10, P = 0.04; n = 17–18. D2-Cre+ vs. D2-Cre: t(44) = 2.17, P = 0.03; n = 22–24. Data are presented as mean ± SEM. **P < 0.001, *P < 0.05.
Fig. 3.
Fig. 3.
H3R2me2a is regulated genome-wide in the NAc after repeated cocaine. (A) Averaged genome-wide heatmap profile of three independent biological replicates of H3R2me2a binding under saline (Lower) and cocaine (Upper) conditions. Each line of the x-axis represents a single gene, arranged in order of decreasing H3R2me2a-binding enrichment in saline conditions. The y-axis represents the genomic region from 5′ to 3′ with 2,000 bp upstream of TSSs and downstream of transcription end sites (TESs). (B) Pie chart showing ChIP-seq genome-wide distribution patterns of significant differential H3R2me2a-binding sites in the NAc between saline and cocaine conditions. Arrows represent sites showing a significant cocaine-induced increase (red) or decrease (blue) in H3R2me2a binding. Cocaine induced 337 differential H3R2me2a-binding sites, among which 129 were decreased and 208 were increased with cocaine. (C) Heatmap of correlation between cocaine-induced H3R2me2a differential binding sites (diffReps) and seven other chromatin modification differential binding sites (H3K4me1, H3K4me3, H3K9me2, H3K9me3, H3K27ac, H3K27me3, and H3K36me3). Binding up/down (red/blue arrow) indicates differential binding sites increased/ decreased after cocaine. The shade of green indicates the odds ratio (i.e., observation/expectation) of enrichments. The P values of enrichments were determined using Fisher’s exact test and are labeled in red. (D) NAc mRNA levels of nine genes characterized by a cocaine-induced decrease in H3R2me2a binding and an increase in H3K4me3 binding. NAc mRNA levels are compared with those of saline-treated animals for Cobll1 [t(27) = 1.759, P = 0.0433], Fryl [t(27) = 1.784, P = 0.0662], Mxd1 [t(27) = 2.108, P = 0.022], Srcin1 [t(27) = 2.535, P = 0.008], and Usp46 [t(27) = 2.151, P = 0.02]. n = 14–15. (E) Repression of gene expression in the NAc with local PRMT6 overexpression achieved with intra-NAc infusion of HSV-PRMT6 vs. HSV-GFP controls. Mxd1, t(6) = 2.108, P = 0.0126; Srcin1, t(8) = 2.921, P = 0.0096 compared with respective HSV-GFP. n = 4–5. Data are presented as mean ± SEM. #P < 0.1, *P < 0.05, **P < 0.01.
Fig. 4.
Fig. 4.
Srcin1 induction in the NAc opposes cocaine-induced behaviors. (A, Left) p140Cas-associated protein, p140Cap (also known as Srcin1) intracellular signaling cascade (40). (A, Right) NAc protein levels of Srcin1 [t(41) = 2.32, P = 0.013], the inactive (phosphorylated at tyrosine Y residue 527) form of Src (pY527Src) [t(35) = 2.14, P = 0.02], the active (phosphorylated at tyrosine Y residue 416) form of Src (pY416Src), total Src, the phosphorylated at tyrosine 397 focal adhesion kinase (pY397FAK) [t(37) = 2.18, P = 0.02], the Src-dependent phosphorylated at tyrosine 925 FAK (pY925FAK) [t(44) = 2.03, P = 0.02], and total FAK. n = 16–24. (B) Srcin1 overexpression in the NAc decreased the time spent in the cocaine-paired CPP chamber [t(18) = 2.40; P = 0.03]. n = 9–13. (C and D) Overexpression of Srcin1 in the NAc decreases the reinforcing efficacy to self-administer cocaine in rats. Rats were injected intra-NAc with HSV-GFP or HSV-Srcin1 and tested 2 d later for 3 consecutive days. n = 5–6. (C) The threshold procedure paradigm was used to generate dose–response curves for cocaine and determine cocaine reinforcing efficacy. Dose–response curves occurred within a single session, and data were averaged from three sessions and presented as lever press responses vs. dose. Two-way ANOVA; virus effect: F(1, 80) = 19.74, P < 0.0001; dose effect: F(9, 80) = 6.71, P < 0.0001; interaction effect: F(9, 80) = 1.35, P = 0.22; dose GFP vs. Srcin1: t(8) = 3.75, P < 0.01. (D) Overexpression of Srcin1 in the NAc of rats decreases the motivation to self-administer cocaine. Pmax (price at which maximal responding occurs): t(8) = 3.31, *P < 0.05. (E) Cell type-specific Srcin1 overexpression in D2-MSNs in the NAc decreased the time spent in the cocaine-paired CPP chamber. Shown is cocaine CPP in D1-Cre+ and D2-Cre+ mice and their respective WT littermate controls (D1-Cre and D2-Cre), all of which were injected intra-NAc with HSV-LS1L-Srcin1. D1-Cre+ vs. D1-Cre: t(9) = 0.88, P = 0.43; n = 5–6. D2-Cre+ vs. D2-Cre: t(20) = 2.38, P = 0.04; n = 9–13. Data are presented as mean ± SEM. *P < 0.05.

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