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, 32 (48), 17454-64

Morphine Epigenomically Regulates Behavior Through Alterations in Histone H3 Lysine 9 Dimethylation in the Nucleus Accumbens

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Morphine Epigenomically Regulates Behavior Through Alterations in Histone H3 Lysine 9 Dimethylation in the Nucleus Accumbens

Haosheng Sun et al. J Neurosci.

Abstract

Dysregulation of histone modifying enzymes has been associated with numerous psychiatric disorders. Alterations in G9a (Ehmt2), a histone methyltransferase that catalyzes the euchromatic dimethylation of histone H3 at lysine 9 (H3K9me2), has been implicated recently in mediating neural and behavioral plasticity in response to chronic cocaine administration. Here, we show that chronic morphine, like cocaine, decreases G9a expression, and global levels of H3K9me2, in mouse nucleus accumbens (NAc), a key brain reward region. In contrast, levels of other histone methyltransferases or demethylases, or of other methylated histone marks, were not affected in NAc by chronic morphine. Through viral-mediated gene transfer and conditional mutagenesis, we found that overexpression of G9a in NAc opposes morphine reward and locomotor sensitization and concomitantly promotes analgesic tolerance and naloxone-precipitated withdrawal, whereas downregulation of G9a in NAc enhances locomotor sensitization and delays the development of analgesic tolerance. We identified downstream targets of G9a by providing a comprehensive chromatin immunoprecipitation followed by massively parallel sequencing analysis of H3K9me2 distribution in NAc in the absence and presence of chronic morphine. These data provide novel insight into the epigenomic regulation of H3K9me2 by chronic morphine and suggest novel chromatin-based mechanisms through which morphine-induced addictive-like behaviors arise.

Figures

Figure 1.
Figure 1.
Regulation of repressive H3K9 and H3K27 methylation by morphine administration in the NAc. a, C57BL/6J mice were injected intraperitoneally once daily with either saline or 20 mg/kg morphine for 7 d. Acute morphine-treated animals received six injections of saline, followed by a single intraperitoneal injection of 20 mg/kg morphine. Animals were analyzed 24 h after the last injection, and NAc tissue was collected for qPCR analysis for KMTs and KDMs for H3 lysine 9 and lysine 27. b, c, Animals were injected intraperitoneally once daily (20 mg/kg) for 1, 3, 5, or 7 d or for 7 d with 10 mg/kg morphine. Animals were killed 24 h after the last injection, and NAc tissue was collected for Western blot analysis for G9a and H3K9me2. d, Animals were injected with an escalating morphine (Esc Mor) paradigm (20, 40, 60, 80, 100, 100 mg/kg morphine every 8 h), and killed 24 h after the last injection. NAc tissue was collected for Western blot analysis for G9a and H3K9me2. e, NAc tissue was collected 24 h after 7 d of 20 mg/kg intraperitoneal injections, and Western blot analysis was performed to examine levels of H3K9me1, H3K9me3, and H3K27me3. Dorsal striatum tissue was also collected to examine levels of H3K9me2. Data were expressed as mean ± SEM. Student's t test or one-way ANOVA was performed, and, assuming significance in the main effect, post hoc test were also performed. *p < 0.05, **p < 0.01 compared with saline-treated animals.
Figure 2.
Figure 2.
G9a regulates morphine-induced behaviors. a, TUNEL/immunohistochemical analysis of NAc sections after HSV–GFP or HSV–G9a surgery, along with negative and positive controls. b, Western blot analysis for various chromatin modifications after HSV–G9a infusion into the NAc. c, CPP. Animals were trained to pair morphine or saline with two contextually distinct chambers for 3 d after viral-mediated gene transfer with HSV–G9a or HSV–GFP, and CPP scores were calculated as the difference in time spent between the morphine- and saline-paired chambers. Two-tailed Student's t test, *p < 0.05. d, Locomotor sensitization after G9a overexpression. Animals were habituated in a locomotor chamber after saline injections. Morphine (10 mg/kg, s.c.) was then administered daily, alternating between the locomotor box and their home cage for 7 d. Animals (HSV–GFP, white squares; HSV–G9a, black triangles) were monitored for locomotor activity for 30 min on days 1, 3, 5, and 7. A two-way ANOVA was used, followed by Bonferroni's post hoc analysis. Day: F(3,60) = 13.59, p < 0.001; virus: F(1,60) = 28.98, p < 0.001; day × virus: F(3,60) = 2.893, p < 0.05. **p < 0.01, post hoc test. e, Locomotor sensitization after Cre-mediated G9a knockdown in the NAc of G9afl/fl mice. Mice (HSV–GFP, white squares; HSV–Cre, black triangles) were monitored for locomotor activity for 30 min for 5 consecutive days of morphine treatment (5 mg/kg). Two-way ANOVA; day: F(4,76) = 1.004, NS; virus: F(1,76) = 15.54, p < 0.001; day × virus:F(4,76) = 0.7863, NS. *p < 0.05, post hoc test. f, Physical withdrawal. Mice were injected intraperitoneally with escalating doses of morphine (20, 40, 60, 80, 100, and 100 mg/kg) every 8 h for 2.5 d. Two hours after the last morphine injection, naloxone (1 mg/kg) was administered subcutaneously. Withdrawal behaviors (jumps, wet dog shakes, tremors, ptosis, diarrhea, and weight loss) were then monitored for 30 min. Bonferroni's post hoc test, *p < 0.05, **p < 0.01. g, h, Analgesic tolerance. A hotplate test was used, in which the latency for paw lick was recorded. The antinociceptive response was calculated as a percentage of MPE, where MPE = (test − control latency)/(cutoff − control) × 100, and responses were normalized to the first day. Repeated morphine injections (15 and 20 mg/kg, s.c., for g and h, respectively) were given daily for 4 d, and analgesia was measured 30 min after each drug dose. For g, HSV–GFP, white squares; HSV–G9a, black triangles. A two-way ANOVA followed by Bonferroni's post hoc analysis were performed. day: F(3,57) = 12.32, p < 0.001; virus: F(1,57) = 4.17. p < 0.05 *p < 0.05, post hoc test. For h, HSV–GFP, white squares; HSV–Cre, black triangles. A two-way ANOVA followed by Bonferroni's post hoc analysis were performed. virus: F(1,36) = 7.20, p < 0.05. *p < 0.05, post hoc test.
Figure 3.
Figure 3.
Chronic morphine regulates the genome-wide distribution of H3K9me2 in NAc. a, The global distribution of H3K9me2 peaks was not found to be different between saline- and morphine-treated animals. Combined, ∼80 and 20% of the H3K9me2 peaks were shown to be distributed throughout intergenic and genic regions of the genome, respectively. b, H3K9me2 is inversely correlated with basal gene expression in NAc. c, Morphine treatment results in differential enrichment of H3K9me2 at localized regions throughout the genome, as identified by diffReps analysis (see Materials and Methods). There is a modest enrichment of morphine-regulated peaks in genic regions compared with the overall global distribution, as well as significantly more morphine downregulated H3K9me2 peaks compared with upregulated peaks. d, Distribution within genic regions shows that the morphine-regulated peaks occur distally from TSSs (preferentially in downstream gene bodies) and are centered within exons. An example of this is illustrated for the FosB gene using IGV (Integrative Genomics Viewer) genome browser software, demonstrating that downregulation of H3K9me2 across the gene occurs mostly within exons.
Figure 4.
Figure 4.
Chronic morphine regulates H3K9me2 and G9a binding at, and expression of, glutamate receptor genes. a, Summary of glutamate signaling genes that were regulated in H3K9me2 binding after chronic morphine treatment, as analyzed by ChIP-seq results. b–f, All glutamatergic-associated genes displaying differential H3K9me2 enrichment after chronic morphine were studied for expression changes after morphine treatment by qPCR in independent tissue samples. b, Several genes in a, which did not show concerted changes in H3K9me2, did not show altered mRNA expression levels. The exception was grip1 (c), which showed an increase in expression after morphine treatment. In contrast, the three genes in a that did display concerted changes in H3K9me2 also show commensurate changes in mRNA expression levels (d–f). Most of these latter genes also exhibited equivalent changes in G9a binding. Representative areas along the gene in which H3K9me2 and corresponding G9a binding changes after morphine treatment are shown in c–f. E, Exons; I, introns. Student's t tests were performed for qPCR validation and G9a ChIP between saline- and morphine-treated animals. #0.05 < p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001.

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