Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors

Abstract

Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

Figure 1

Engineered transcription factors targeting the FosB promoter bidirectionally regulate gene expression in NAc neurons via epigenetic manipulation. (a) Locations of FosB-ZFP binding relative to the FosB TSS. The location of functional SRF and CREB binding sites are shown. CpG: methylation sites analyzed in Fig. 1i. (b) HSV injection into the mouse NAc drives robust transgene expression in neurons. (c) The 6-finger ZFP35 recognizes the FosB promoter at approximately 250 bp upstream from the FosB TSS. (d) At 72 hours post-injection, FosB/ΔFosB mRNA expression in the NAc was significantly induced by HSV-FosB-ZFP35-p65 compared to control virus [FosB: t₇=2.37, *P=0.049; ΔFosB: t₇=3.83, *P=0.007; n=4, 5], , with a trend for ΔFosB induction by FosB-ZFP61-p65 [t₇=2.03, ^τP=0.076; n=4, 5]. Compared to controls, FosB/ΔFosB mRNA in the NAc were significantly repressed by HSV-FosB-ZFP35-G9a [FosB: t₆=4.84, *P=0.003; ΔFosB: t₆=3.40, *P=0.015; n=5, 3], -FosB-ZFP61-NFD [FosB t₇=2.39, *P=0.048; ΔFosB: t₇=2.18, ^τP=0.066; n=4, 5], and FosB-ZFP61-G9a [FosB: t₈=2.98, *P=0.017; ΔFosB: t₈=2.37, *P=0.047; n=5, 5]. HSV-G9a, -p65, -Klf4-ZFP-p65, VEGF-ZFP-G9a and -FosB-ZFP35-NFD had no effect on FosB/ΔFosB mRNA expression. Student's unpaired t-test P>0.05 for all other comparisons (see Supplementary Table 3). (e) Compared to HSV-FosB-ZFP35-NFD [n=9], NAc injection of HSV-FosB-ZFP35-p65 caused increased FosB/ΔFosB protein level [FosB: t₁₃=3.73, *P=0.003; ΔFosB: t₁₃=6.65, *P=0.000; n=6], while HSV-FosB-ZFP35-G9a had no effect at 72 hours post-injection [FosB: t₁₄=0.137, P=0.8929; ΔFosB: t₁₄=1.25, P=0.230; n=8. Representative western blot shown. Complete western blot shown in Supplementary Figure 5. (f) NAc injection of HSV-FosB-ZFP35-p65 causes enrichment of the activating mark H3K9/14ac at the FosB promoter at –1250 [t₈=2.65, *P=0.029; n=5, 5] and –250 bp [t₈=2.9, *P=0.021; n=5, 5] relative to the TSS compared to control HSV, with no change in tissue treated with HSV-FosB-ZFP35-NFD [t₁₀=0.00, P=1.00; n=6, 6] or at the cFos promoter [t₇=0.217, P=0.834; n=5, 4]. IgG control IP was undetectable in >80% of the sample wells by qRT-PCR. (g) NAc injection of HSV-FosB-ZFP35-G9a caused enrichment of the repressive mark H3K9me2 at the FosB promoter at –1250 [t₂₆=2.7, *P=0.011; n=14, 14], –500 [t₁₄=2.3, *P=0.041; n=7, 9], and –250 bp [t₁₃=2.9, *P=0.031; n=7, 8] relative to the TSS compared to control HSV, with no change in tissue treated with HSV-FosB-ZFP35-NFD [–1250: t₁₉=0.45, P=0.658, n=14, 7; –500: t₈=0.03, P=0.977, n=7, 3; –250: t₉=1.51, P=0.167, n=8, 3] or at the cFos promoter [t₁₆=0.42 P=0.680; n=7, 11]. IgG control IP was undetectable in >80% of the sample wells by qPCR. (h) NAc injection of HSV-FosB-ZFP35-G9a caused depletion of the repressive mark H3K9me3 [t₈=3.3, *P=0.011; n=5, 5] and enrichment of HP1α [t₇=2.5, *P=0.039; n=4, 4] at the FosB promoter –1250 bp upstream from the TSS compared to control HSV-FosB-ZFP35-NFD. (i) Bisulfite sequencing analysis performed on three CpG sites located –1141, –1101 and –1036 bp upstream of the FosB TSS, in a region that corresponds to the observed H3K9me2 and HP1α enrichment (See Fig. 1a). There was no difference in the percentage of methylated CpGs in NAc infected with HSV-FosB-ZFP35-G9a [n=19] compared to HSV-FosB-ZFP35-NFD [n=23]. Each row represents analysis performed on one clone. Data are presented as mean ± s.e.m.

Figure 2

HSV-FosB-ZFP35-p65 and -G9a in the NAc specifically regulate FosB/ΔFosB expression. (a) Induction of H3K9me2 at the FosB promoter by HSV-FosB-ZFP-G9a in NAc occurs relative to the TSS at –1250 bp [t₅=2.75, *P=0.040; n=3, 4] and –250 bp [t₅=2.69, *P=0.043; n=3, 4] without changes in the activating marks H3K9/14ac [–1250: t₅=1.49, P=0.197; –500: t₅=0.50, P=0.636; –250: t₅=0.39, P=0.715; n=3, 4] or H3K4me3 [–1250: t₅=0.37, P=0.728; –500: t₅=0.24, P=0.820; –250: t₅=0.22, P=0.835; n=3, 4] or the repressive mark H3K27me3 [–1250: t₅=0.46, P=0.666; –500: t₅=0.59, P=0.583; –250: t₅=0.23, P=0.831; n=3, 4] as compared to the control (HSV-FosB-ZFP-NFD). (b) cDNA was generated from NAc injected with HSV-FosB-ZFP35-p65, -G9a or NFD. qRT-PCR was used to measure expression of potential off-target genes (See Supplementary Table 2) in samples that show regulation of FosB/ΔFosB by HSV-FosB-ZFP35-p65 [FosB: t₇=4.73, *P=0.002; ΔFosB: t₇=4.83, *P=0.002; n=4, 5] and HSV-FosB-ZFP35-G9a [FosB: t₈=2.40, *P=0.043; ΔFosB: t₈=2.88, *P=0.021; n=5, 5] but not HSV-FosB-ZFP35-G9a [FosB: t₈=0.38, P=0.715; ΔFosB: t₈=0.52, P=0.619; n=5, 5]. Data are normalized to HSV-GFP. Student's unpaired t-test P>0.05 for all other comparisons (see Supplementary Table 3). (c) HSV-FosB-ZFP35-G9a in the NAc does not cause H3K9me2 enrichment at off-target loci as measured by qChIP using primers that flank the off-target binding site [Ptprn2: t₄=0.03, P=0.97; Pygo1: t₅=0.10, P=0.922; Sardh: t₄=0.93, P=0.407; Syncrip: t₄=0.34, P=0.775; Tlr12: t₄=1.80, P=0.152; n=3, 3].

Figure 3

Cocaine induction of FosB/ΔFosB protein expression and endogenous transcription factor binding is blocked by HSV-FosB-ZFP35-G9a in the NAc. (a) Mice were injected intra-NAc with HSV-FosB-ZFP35-NFD or -G9a and treated with repeated cocaine (20 mg/kg) or saline. (b) Cocaine induction of FosB/ΔFosB+ cells in the NAc is suppressed by FosB-ZFP35-G9a [two-way ANOVA: interaction between virus (NFD, G9a) and drug (saline, cocaine) (F(1,28)=6.6, *P=0.016, n=8); no main effects of virus (F(1,28)=0.98, P=0.330, n=8) or drug (F(1,28)=1.18, P=0.286, n=8) alone]. Cocaine enhances FosB+ cell levels within NFD-infected tissue [t₁₄=2.48, *P=0.027; n=8] but not G9a-infected tissue [t₁₄=1.20, P=0.291; n=8]. Among animals receiving cocaine, G9a repressed FosB/ΔFosB+ cells [t₁₄=2.25, *P=0.041; n=8]. (c) Representative images from mice infected with HSV-FosB-ZFP35-NFD in the left hemisphere and HSV-FosB-ZFP35-G9a into the right hemisphere. Ac, anterior commissure. (d) Compared to saline, repeated cocaine treatment caused enrichment of phospho-CREB(S133) at the FosB promoter in NAc injected with HSV-FosB-ZFP35-NFD [t₁₇=2.38, *P=0.029; n=9, 11], but not with HSV-FosB-ZFP-G9a [t₁₅=0.09, P=0.932; n=8, 9]. (e) No change was observed in total CREB at the FosB promoter after cocaine treatment in NAc injected with either virus [NFD: t₁₈=1.55, P=0.138; n=9, 11; G9a: t₁₅=1.13, P=0.274; n=8, 9]. (f) Compared to saline, repeated cocaine treatment causes enrichment of phospho-SRF(S103) at the FosB promoter in NAc injected with HSV-FosB-ZFP35-NFD [t(1, 6)= 2.7, p=0.034; n=4, 4] compared to repeated saline with only a modest effect of HSV-FosB-ZFP-G9a in blocking this enrichment [t(1, 8)= 1.9, ^τP=0.097; n=5, 5]. (g) NAc injection of HSV-FosB-ZFP-G9a causes enrichment of HP1α at the FosB promoter compared to HSV-FosB-ZFP-NFD under cocaine conditions [t(1, 7)= 1.24, *P=0.0002, n=4, 5] and a trend under saline treatment conditions [t(1, 8)= 1.97, ^τP =0.085; n=4, 5].

Figure 4

Engineered transcription factors bidirectionally modulate cocaine- and stress-evoked behaviors. (a) Locomotor activity was assessed during repeated cocaine exposure in mice injected intra-NAc with HSV-FosB-ZFP35-G9a [n=7], -p65 [n=9], -NFD [n=9] or control virus [n=5 (10mg/kg) or n=10 (5mg/kg)]. (b) Social behavior was measured 24 hours after subthreshold social defeat in mice injected with HSV-FosB-ZFP35-G9a or control virus into the NAc. (c) At high doses of cocaine (10 mg/kg), locomotor behavior sensitizes over time and this effect was blocked by HSV-FosB-ZFP35-G9a in NAc. Repeated measures ANOVA revealed an interaction between day, cocaine, and virus [F(3,18)=4.00, *P=0.024] on locomotor behavior. Among cocaine treated mice, there is a main effect of virus [F(1,10)=8.81, *P=0.026] and a trend for an interaction between virus and day [F(2,8)=3.33, ^τP=0.077] such that GFP locomotor behavior is enhanced above FosB-ZFP35-G9a levels by treatment day 6 [t(1,10)=2.61, *P =0.026]. (d) At low doses (5 mg/kg), cocaine-induced locomotor behavior sensitizes over time with FosB-ZFP35-p65 in NAc [repeated measures: interaction between day, treatment, and virus [F(3,32)=4.71, *P=0.008]. Among cocaine treated mice, there is an interaction between virus and day [F(2,15)=7.08, *P=0.003] and a trend for a main effect of day among FosB-ZFP35-p65 [F(1,32)=2.85, ^τP=0.053] but not GFP cocaine treated animals. Among cocaine-treated animals, FosB-ZFP35-p65 enhances locomotor behavior above GFP levels by treatment day 4 [t₁₇=2.58, *P=0.020] through day 16 [t₁₇=2.92, *P=0.009]. (e) NAc injection of HSV-FosB-ZFP35-NFD, like controls, did not display cocaine locomotor sensitization to a low dose of cocaine. Repeated measures failed to find an interaction among day, treatment, and virus [F(1,28)=0.13, P=0.944]. There is no effect of day among cocaine-treated mice [F(1,28)=0.86, P=0.471]. HSV-GFP data are the same as in (Fig. 4d). (f) Heat maps show representative locomotor data within the chamber for mice over the course of repeated cocaine exposure. (g) H3K9me2 is significantly enriched at –1250 bp from the FosB TSS in depressed humans [t₂₂=2.19, p=0.040, n=8, 17] compared to levels in control subjects. (h) FosB-ZFP35-G9a in the NAc reduced exploration of the open arm in the elevated plus maze, compared to control virus [t₁₂=2.36, *P=0.036; n=7]. (i) HSV-FosB-ZFP35-G9a in the NAc blocked increased exploration of a novel aggressor mouse after exposure to subthreshold social defeat, compared to control virus [t₁₂3.1, *P=0.009; n=7] with no effect of HSV-FosB-ZFP35-NFD [t₁₂=3.2, *P=0.008; n=7]. (j) Representative heatmaps of social interaction after a subthreshold defeat stress show a preference for the interaction zone when a target mouse is present for mice injected with control virus and HSV-FosB-ZFP35-NFD, but not HSV-FosB-ZFP35-G9a.