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

Journal name:
Nature Neuroscience
Volume:
17,
Pages:
1720–1727
Year published:
DOI:
doi:10.1038/nn.3871
Received
Accepted
Published online

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.

At a glance

Figures

  1. Engineered transcription factors targeting the Fosb promoter bidirectionally regulate gene expression in NAc neurons via epigenetic manipulation.
    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 i. (b) HSV injection into the mouse NAc43 drove robust transgene expression in neurons. (c) The six-finger ZFP35 recognized the Fosb promoter ~250 bp upstream of the Fosb TSS. (d) At 72 h post-injection, Fosb/ΔFosb mRNA expression in the NAc was significantly induced by HSV-Fosb-ZFP35-p65 compared with control virus (Fosb, t7 = 2.37, *P = 0.049; ΔFosb, t7 = 3.83, *P = 0.007; n = 4, 5 mice per group), with a trend for ΔFosb induction by Fosb-ZFP61-p65 (t7 = 2.03, #P = 0.076; n = 4, 5). Compared with 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), HSV-Fosb-ZFP61-NFD (Fosb, t7 = 2.39, *P = 0.048; n = 4), and Fosb-ZFP61-G9a (Fosb, t8 = 2.98, *P = 0.017; ΔFosb, t8 = 2.37, *P = 0.047; n = 5, 5), with a trend for repression of ΔFosB by Fosb-ZFP61-NFD (ΔFosb, t7 = 2.18, #P = 0.066, n = 5). HSV-G9a, HSV-p65, HSV-Klf4-ZFP-p65, HSV-VEGF-ZFP-G9a and HSV-Fosb-ZFP35-NFD had no effect on Fosb/ΔFosb mRNA expression (Student's unpaired t test, P > 0.05 for all other comparisons; Supplementary Table 3). (e) Compared with HSV-Fosb-ZFP35-NFD (n = 9 ChIP samples), NAc injection of HSV-Fosb-ZFP35-p65 resulted in increased FosB/ΔFosB protein levels (FosB, t13 = 3.73, *P = 0.003; ΔFosB: t13 = 6.65, *P = 0.000; n = 6), whereas HSV-Fosb-ZFP35-G9a had no effect at 72 h post-injection (FosB, t14 = 0.137, P = 0.8929; ΔFosB, t14 = 1.25, P = 0.230; n = 8). Representative western blots are shown. Full-length western blots are shown in Supplementary Figure 5. (f) NAc injection of HSV-Fosb-ZFP35-p65 caused enrichment of the activating mark H3K9/14ac at the Fosb promoter at −1,250 (t8 = 2.65, *P = 0.029, n = 5, 5 ChIP samples) and −250 bp (t8 = 2.9, *P = 0.021, n = 5, 5) relative to the TSS compared with 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 Fos 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 −1,250 (t26 = 2.7, *P = 0.011, n = 14, 14 ChIP samples), −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 with control HSV, with no change in tissue treated with HSV-Fosb-ZFP35-NFD (−1,250, 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 Fos promoter (t16 = 0.42, P = 0.680 n = 7, 11). IgG control immunoprecipitation was undetectable in >80% of the sample wells by qPCR. (h) NAc injection of HSV-Fosb-ZFP35-G9a caused enrichment of H3K9me2 (t19 = 2.4, *P = 0.0238, n = 5, 5) depletion of the repressive mark H3K9me3 (t8 = 3.3, *P = 0.011, n = 5, 5 ChIP samples) and enrichment of HP1α (t7 = 2.5, *P = 0.039, n = 4, 4) at the Fosb promoter −1,250 bp upstream from the TSS compared with control HSV-Fosb-ZFP35-NFD. (i) Bisulfite sequencing analysis performed on three CpG sites located −1,141, −1,101 and −1,036 bp upstream of the Fosb TSS, in a region that corresponds to the observed H3K9me2 and HP1α enrichment (a). There was no difference in the percentage of methylated CpGs in NAc infected with HSV-Fosb-ZFP35-G9a (n = 19 clones) compared with HSV-Fosb-ZFP35-NFD (n = 23). Each row represents analysis performed on one clone. Data are presented as mean ±s.e.m.

  2. HSV-Fosb-ZFP35-p65 and -G9a in the NAc specifically regulate FosB/[Delta]FosB expression.
    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 −1,250 bp (t5 = 2.75, *P = 0.040, n = 3, 4 ChiP samples) and −250 bp (t5 = 2.69, *P = 0.043, n = 3, 4) without changes in the activating marks H3K9/14ac (−1,250, 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 (−1,250, 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 (−1,250, 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 with the control (HSV-Fosb-ZFP-NFD). (b) cDNA was generated from NAc injected with HSV-Fosb-ZFP35-p65, HSV-Fosb-ZFP35-G9a or HSV-Fosb-ZFP35-NFD. qRT-PCR was used to measure expression of potential off-target genes (Supplementary Table 2) in samples that showed 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 mice) 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 were normalized to HSV-GFP (Student's unpaired t test, P > 0.05 for all other comparisons; Supplementary Table 3). (c) HSV-Fosb-ZFP35-G9a in the NAc did 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 ChiP samples). Data are presented as mean ±s.e.m.

  3. Cocaine induction of FosB/[Delta]FosB protein expression and endogenous transcription factor binding is blocked by HSV-Fosb-ZFP35-G9a in the NAc.
    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 HSV-Fosb-ZFP35-G9a and treated with repeated cocaine (20 mg per 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 (sal), cocaine (coc)) (F1,28 = 6.6, *P = 0.016, n = 8 mice per group); no main effects of virus (F1,28 = 0.98, P = 0.330, n = 8) or drug (F1,28 = 1.18, P = 0.286, n = 8) alone). Cocaine enhances FosB+ cell levels in 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) 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). (e) Compared with 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). (f) Compared with saline, repeated cocaine treatment resulted in enrichment of phospho-SRF(S103) at the Fosb promoter in NAc injected with HSV-Fosb-ZFP35-NFD (t1,6 = 2.7, *P = 0.034, n = 4, 4 ChiP samples) compared with repeated saline with only a modest effect of HSV-Fosb-ZFP-G9a in blocking this enrichment (t(1,8) = 1.9, #P = 0.097; n = 5, 5). (g) NAc injection of HSV-Fosb-ZFP-G9a caused enrichment of HP1α at the Fosb promoter compared with HSV-Fosb-ZFP-NFD under cocaine conditions (t1,7 = 1.24, *P = 0.0002, n = 4, 5) and a trend under saline treatment conditions (t1,8 = 1.97, #P = 0.085, n = 4, 5). Data are presented as mean ±s.e.m.

  4. Engineered transcription factors bidirectionally modulate cocaine- and stress-evoked behaviors.
    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), HSV-Fosb-ZFP35-p65 (n = 9), HSV-Fosb-ZFP35-NFD (n = 9) or control virus (n = 5 mice per group (10 mg per kg) or n = 10 (5 mg per kg)). (b) Social behavior was measured 24 h 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 per kg), locomotor behavior sensitized 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 (F3,18 = 4.00, *P = 0.024) on locomotor behavior. Among cocaine-treated mice, there was a main effect of virus (F1,10 = 8.81, P = 0.026) and a trend for an interaction between virus and day (F2,8 = 3.33, P = 0.077) such that GFP locomotor behavior was enhanced above Fosb-ZFP35-G9a levels by treatment day 6 (t1,10 = 2.61, P = 0.026). (d) At low doses (5 mg per kg), cocaine-induced locomotor behavior sensitized over time with Fosb-ZFP35-p65 in NAc (repeated measures: interaction between day, treatment and virus, F3,32 = 4.71, *P = 0.008). Among cocaine-treated mice, there was an interaction between virus and day (F2,15 = 7.08, P = 0.003) and a trend for a main effect of day among Fosb-ZFP35-p65 (F1,32 = 2.85, P = 0.053), but not Fosb-ZFP35-GFP cocaine-treated animals. Among cocaine-treated animals, Fosb-ZFP35-p65 enhanced locomotor behavior above Fosb-ZFP35-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, similar to 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 (F1,28 = 0.13, P = 0.944). There was no effect of day among cocaine-treated mice (F1,28 = 0.86, P = 0.471). HSV-GFP data from d are shown. (f) Heat maps show representative locomotor data in the chamber for mice over the course of repeated cocaine exposure. (g) H3K9me2 was significantly enriched at −1,250 bp from the Fosb TSS in depressed humans (t22 = 2.19, P = 0.040, n = 8, 17 subjects per group) compared with levels in control subjects. (h) Fosb-ZFP35-G9a in the NAc reduced exploration of the open arm in the elevated plus maze compared with control virus (t12 = 2.36, *P = 0.036, n = 7 mice per group). (i) HSV-Fosb-ZFP35-G9a in the NAc blocked increased exploration of a novel aggressor mouse after exposure to subthreshold social defeat, compared with control virus (t12 = 3.1, *P = 0.009, n = 7 mice per group) with no effect of HSV-Fosb-ZFP35-NFD (t12 = 3.2, *P = 0.008, n = 7). (j) Representative heat maps of social interaction after a subthreshold defeat stress showed a preference for the interaction zone when a target mouse was present for mice injected with control virus and HSV-Fosb-ZFP35-NFD, but not HSV-Fosb-ZFP35-G9a. Data are presented as mean ±s.e.m.

  5. A suite of Fosb-ZFPs bidirectionally regulate Fosb expression in vitro.
    Supplementary Fig. 1: A suite of Fosb-ZFPs bidirectionally regulate Fosb expression in vitro.

    (a) FosB/ΔFosB mRNA was significantly induced by several FosB-ZFP-p65 and -G9a constructs expressed in N2a cells and harvested after 48 hours. FosB-ZFP-NFD constructs activated gene expression to a lesser extent or not at all. Data are normalized to mock transfected cells. Complete statistics are available in Supplementary Table 3. Student's unpaired t-test: *P<0.05, *P<0.10. Data are presented as mean ± s.e.m.

  6. HSV-ZFPs specifically infect neurons in NAc and inhibit basal Fosb expression.
    Supplementary Fig. 2: HSV-ZFPs specifically infect neurons in NAc and inhibit basal Fosb expression.

    (a) HSV-GFP specifically infects DARPP-32 positive neurons in the NAc. White arrows indicate double labeled cells. (b) NAc injection of HSV-FosB-ZFP35-G9a repressed expression of FosB/ΔFosB in HSV infected (GFP+) cells [t13=3.55, *P=0.033; n=7, 8] compared to HSV-FosB-ZFP35-G9a (72 hours post HSV injection). Data are presented as mean ± s.e.m.

  7. Regulation of Fosb expression and reward behavior by Fosb-TALEs and a suite of additional catalytic domains fused to Fosb-ZFP35.
    Supplementary Fig. 3: Regulation of Fosb expression and reward behavior by Fosb-TALEs and a suite of additional catalytic domains fused to Fosb-ZFP35.

    (a) Locations of FosB-ZFP and -TALE binding relative to the FosB TSS. The location of functional SRF and CREB sites are shown. (b) FosB/ΔFosB mRNA expression in the NAc was significantly induced by HSV-FosB-TALE2-VP64 [FosB: t8=3.03, *P=0.016; ΔFosB: t8=6.40, *P=0.000; n=5], and -FosB-TALE3-VP64 [FosB: t8=2.79, *P=0.023; ΔFosB: t8=4.01, *P=0.004; n=5] compared to control virus. (c) The binding sites of the 6-finger ZFP35 and 17-RVD (repeat variable diresidue) TALE1 recognize the FosB promoter at overlapping sites approximately 250 bp upstream from the FosB TSS. (d) FosB-ZFP35-p65 [FosB: t4=5.91, *P=0.004; ΔFosB: t4=26.11, *P=0.000; n=3], -p65x2 [FosB: t4=2.30, *P=0.000; ΔFosB: t4=3.46, *P=0.026; n=3], -VP16 [FosB: t4=9.04, *P=0.001; ΔFosB: t4=7.45, *P=0.002; n=3], and -VP64 [FosB: t4=19.40, *P=0.001; ΔFosB: t4=7.45, *P=0.001; n=3] and FosB-TALE1-VP64 [FosB: t4=6.80, *P=0.002; ΔFosB: t4=15.17, *P=0.000; n=3] activate FosB/ΔFosB mRNA levels when expressed in Neuro2a cells and harvested after 48 hours. Data are normalized to mock transfected cells. (e) HSV-FosB-TALE1-VP64 in NAc sensitizes cocaine-induced hyperactivity over time. There is a significant interaction between day, cocaine treatment, and virus [F(3,32)=3.42, *P=0.029]. TALE1-VP64 sensitizes the effect of cocaine on locomotor activity [main effect of day among TALE1-VP64 [F(3,35)=9.92, *P=0.000, n=10], but not GFP control [F(3,36)=2.05, P=0.126, n=9]. HSV-GFP data are the same as in Fig. 4d-e. Heat maps show representative locomotor data within the chamber for mice over the course of repeated cocaine exposure. Data are presented as mean ± s.e.m.

  8. Differential regulation by Fosb-ZFPs in vitro and in vivo.
    Supplementary Fig. 4: Differential regulation by Fosb-ZFPs in vitro and in vivo.

    (a) Expression of FosB-ZFP35-G9a differs between N2a cells and NAc [t6=4.50, *P=0.004; n=3,5]. (b) Titration of the amount of FosB-ZFP35-G9a expression in N2a cells leads to a reduction in the amount of induced mRNA expression of ΔFosB but not FosB. Using a 2-tailed Pearson correlation, we found a significant correlation between μg of transfected DNA and mRNA fold change of ΔFosB [R12=0.83, *P=0.001] but not FosB [R12=.66, P=0.665]. Data are presented as mean ± s.e.m.

  9. Full blots of experiment shown in Figure 1e.
    Supplementary Fig. 5: Full blots of experiment shown in Figure 1e.

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Author information

  1. Present address: Department of Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, Arizona, USA

    • Deveroux Ferguson
  2. Present address: Department of Physiology, Michigan State University, East Lansing, Michigan, USA.

    • Feng Zhang

Affiliations

  1. Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Elizabeth A Heller,
    • Hannah M Cates,
    • Catherine J Peña,
    • Haosheng Sun,
    • Ningyi Shao,
    • Jian Feng,
    • Sam A Golden,
    • Jessica J Walsh,
    • Michelle Mazei-Robison,
    • Deveroux Ferguson,
    • Scott J Russo,
    • Li Shen &
    • Eric J Nestler
  2. National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.

    • James P Herman
  3. Department of Pharmacology and System Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

    • Jessica J Walsh &
    • Ming-Hu Han
  4. Sigma Aldrich, Saint Louis, Missouri, USA.

    • Scott Knight,
    • Mark A Gerber &
    • Christian Nievera
  5. Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    • Carol S Tamminga
  6. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Rachael L Neve
  7. Sangamo Biosciences Inc., Richmond, California.

    • H Steve Zhang
  8. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Deveroux Ferguson &
    • Feng Zhang

Contributions

E.A.H. designed and executed the biochemical, molecular and behavioral experiments (viral packaging constructs, qRT-PCR expression analysis, chromatin immunoprecipitation (mouse and human), immunohistochemical preparation and behavioral assays). H.M.C. and H.S. generated viral packaging constructs, conducted expression analysis and prepared chromatin. C.J.P. and S.A.G. analyzed immunohistochemical data. N.S. and L.S. conducted genome-wide sequence analysis. J.F. and E.A.H. performed DNA methylation sequencing. S.A.G. and S.J.R. generated human chromatin. E.A.H., H.M.C., H.S., J.J.W., M.M.-R., D.F. and M.-H.H. performed stereotaxic mouse surgery. J.P.H. generated mouse locomotor heat maps. C.S.T. prepared human postmortem brain tissue. R.L.N. generated HSV viral vectors. H.S.Z. generated the G9a catalytic domain construct. S.K., M.A.G. and C.N. generated the ZFP constructs. F.Z. generated the TALE constructs. C.J.P., E.A.H. and H.M.C. analyzed the data conducted statistical analyses. E.A.H. and E.J.N. discussed the data and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: A suite of Fosb-ZFPs bidirectionally regulate Fosb expression in vitro. (261 KB)

    (a) FosB/ΔFosB mRNA was significantly induced by several FosB-ZFP-p65 and -G9a constructs expressed in N2a cells and harvested after 48 hours. FosB-ZFP-NFD constructs activated gene expression to a lesser extent or not at all. Data are normalized to mock transfected cells. Complete statistics are available in Supplementary Table 3. Student's unpaired t-test: *P<0.05, *P<0.10. Data are presented as mean ± s.e.m.

  2. Supplementary Figure 2: HSV-ZFPs specifically infect neurons in NAc and inhibit basal Fosb expression. (621 KB)

    (a) HSV-GFP specifically infects DARPP-32 positive neurons in the NAc. White arrows indicate double labeled cells. (b) NAc injection of HSV-FosB-ZFP35-G9a repressed expression of FosB/ΔFosB in HSV infected (GFP+) cells [t13=3.55, *P=0.033; n=7, 8] compared to HSV-FosB-ZFP35-G9a (72 hours post HSV injection). Data are presented as mean ± s.e.m.

  3. Supplementary Figure 3: Regulation of Fosb expression and reward behavior by Fosb-TALEs and a suite of additional catalytic domains fused to Fosb-ZFP35. (282 KB)

    (a) Locations of FosB-ZFP and -TALE binding relative to the FosB TSS. The location of functional SRF and CREB sites are shown. (b) FosB/ΔFosB mRNA expression in the NAc was significantly induced by HSV-FosB-TALE2-VP64 [FosB: t8=3.03, *P=0.016; ΔFosB: t8=6.40, *P=0.000; n=5], and -FosB-TALE3-VP64 [FosB: t8=2.79, *P=0.023; ΔFosB: t8=4.01, *P=0.004; n=5] compared to control virus. (c) The binding sites of the 6-finger ZFP35 and 17-RVD (repeat variable diresidue) TALE1 recognize the FosB promoter at overlapping sites approximately 250 bp upstream from the FosB TSS. (d) FosB-ZFP35-p65 [FosB: t4=5.91, *P=0.004; ΔFosB: t4=26.11, *P=0.000; n=3], -p65x2 [FosB: t4=2.30, *P=0.000; ΔFosB: t4=3.46, *P=0.026; n=3], -VP16 [FosB: t4=9.04, *P=0.001; ΔFosB: t4=7.45, *P=0.002; n=3], and -VP64 [FosB: t4=19.40, *P=0.001; ΔFosB: t4=7.45, *P=0.001; n=3] and FosB-TALE1-VP64 [FosB: t4=6.80, *P=0.002; ΔFosB: t4=15.17, *P=0.000; n=3] activate FosB/ΔFosB mRNA levels when expressed in Neuro2a cells and harvested after 48 hours. Data are normalized to mock transfected cells. (e) HSV-FosB-TALE1-VP64 in NAc sensitizes cocaine-induced hyperactivity over time. There is a significant interaction between day, cocaine treatment, and virus [F(3,32)=3.42, *P=0.029]. TALE1-VP64 sensitizes the effect of cocaine on locomotor activity [main effect of day among TALE1-VP64 [F(3,35)=9.92, *P=0.000, n=10], but not GFP control [F(3,36)=2.05, P=0.126, n=9]. HSV-GFP data are the same as in Fig. 4d-e. Heat maps show representative locomotor data within the chamber for mice over the course of repeated cocaine exposure. Data are presented as mean ± s.e.m.

  4. Supplementary Figure 4: Differential regulation by Fosb-ZFPs in vitro and in vivo. (254 KB)

    (a) Expression of FosB-ZFP35-G9a differs between N2a cells and NAc [t6=4.50, *P=0.004; n=3,5]. (b) Titration of the amount of FosB-ZFP35-G9a expression in N2a cells leads to a reduction in the amount of induced mRNA expression of ΔFosB but not FosB. Using a 2-tailed Pearson correlation, we found a significant correlation between μg of transfected DNA and mRNA fold change of ΔFosB [R12=0.83, *P=0.001] but not FosB [R12=.66, P=0.665]. Data are presented as mean ± s.e.m.

  5. Supplementary Figure 5: Full blots of experiment shown in Figure 1e. (355 KB)

PDF files

  1. Supplementary Figures and Tables (18.9 MB)

    Supplementary Figures 1–5 and Supplementary Tables 1–3

  2. Supplementary Methods Checklist (125 KB)

    Reporting Checklist for Nature Neuroscience

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