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Research ArticleNew Research, Disorders of the Nervous System

Fosb Induction in Nucleus Accumbens by Cocaine Is Regulated by E2F3a

Hannah M. Cates, Casey K. Lardner, Rosemary C. Bagot, Rachael L. Neve and Eric J. Nestler
eNeuro 20 March 2019, 6 (2) ENEURO.0325-18.2019; DOI: https://doi.org/10.1523/ENEURO.0325-18.2019
Hannah M. Cates
1Nash Family Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029
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  • ORCID record for Hannah M. Cates
Casey K. Lardner
1Nash Family Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029
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Rosemary C. Bagot
1Nash Family Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029
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Rachael L. Neve
2Gene Delivery Technology Core, Massachusetts General Hospital, Cambridge, Massachusetts 02139
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Eric J. Nestler
1Nash Family Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029
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  • Figure 1.
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    Figure 1.

    E2F3 binding at the Fosb gene promoter is regulated in NAc by cocaine. A, E2F consensus sequence and a schematic diagram of Fosb. Primer sets designed against putative E2F binding sites noted with golden double arrowheads. B, E2F3 binding, not E2F1 or E2F4, was increased following cocaine administration 500 bp upstream of the Fosb TSS (interaction of drug and gene position was observed by two-way ANOVA: F(9,60) = 13.35, p < 0.0001; Cocaine vs saline at −500 bp via post hoc analysis: t(8) = 9.904, ****p < 0.0001). All data points have error bars representing ± SEM. C–F, Significant binding of E2F3 over an IgG control was observed and significantly increased by cocaine in (C) NAc (F(9,60) = 13.35, ****p < 0.0001, t(8) = 9.904, ****p < 0.0001). Significant binding of E2F3 was observed but was not affected by cocaine administration in (D) PFC, binding over an IgG control (F(1,16) = 48.44, ****p < 0.0001), no main effect of drug observed (F(1,16) = 1.789, p = 0.1998); (E) VTA, binding over an IgG control (F(1,10) = 7.024, *p = 0.0243), no main effect of drug observed (F(1,10) = 0.029, p = 0.869); or (F) CPu, binding over an IgG control (F(1,16) = 25.113, ****p < 0.0001), no main effect of drug observed (F(1,16) = 1.12, p = 0.305). N = 4–5, with three mice pooled per sample for all qChIP assays.

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    Figure 2.

    E2F3a is expressed in NAc D1 MSNs and overexpression of E2F3a in NAc regulates expression of Fosb isoforms in NAc. A, Expression of E2f3a and E2f3b in FACS-isolated D1 MSNs. Main effects of both drug treatment (F(1,9) = 9.624, p = 0.0127) and transcript variant (F(1,9) = 6.845,*p = 0.028) were observed. A significant increase in E2f3a expression was confirmed via post hoc analysis (E2f3a: t(5) = 4.186, **p = 0.0047; E2f3b: t(4) = 0.3328, p = 0.9999). N = 3–4. B, Expression of E2f3a and E2f3b in FACS-isolated D2 MSNs. A main effect of transcript was observed (F(1,9) = 15.489, **p = 0.0034) but no effect of drug treatment (F(1,9) = 0.0171, p = 0.8987). N = 3–4. C, NAc cross section indicating site of injection for viral-mediated gene transfer. D, qPCR from mouse NAc injected with HSV-GFP, HSV-E2F3a, or HSV-E2F3b. Normalized to GFP (Gapdh used as housekeeping gene in ΔΔCt method). A main effect of virus was observed (F(2,29) = 9.332, p = 0.0007). E2F3a, but not E2F3b, increased both Fosb and ΔFosb mRNA expression by post hoc analysis (E2F3a vs GFP–Fosb: t(10) = 3.138, **p = 0.0117; E2F3a vs GFP–ΔFosb: t(9) = 2.868, *p = 0.0229; E2F3b vs GFP–Fosb: t(10) = 1.336, p = 0.5757; E2F3b vs GFP–ΔFosb: t(10) = 0.575, p = 0.9999; N = 8). E, qPCR from mouse PFC injected with HSV-GFP or HSV-E2F3a. Normalized to GFP. No main effect of virus was observed (F(1,19) = 1.637, p = 0.2162; N = 6). F, qPCR from mouse NAc injected with HSV-miR-LacZ or HSV-miR-E2F3a and treated with either saline or acute cocaine (1 injection of 20 mg/kg). Normalized to miR-LacZ + saline. A main effect of drug treatment was observed (F(3,20) = 3.297, p = 0.0415) and post hoc analysis confirmed that acute cocaine increased ΔFosb expression and viral knockdown of E2F3a did not significantly blunt this effect (miR-LacZ + saline vs miR-LacZ + cocaine: t(9) = 2.492, *p = 0.0508, miR-LacZ + saline vs miR-E2f3a + cocaine: t(9) = 1.223, p = 0.9999; miR-LacZ + cocaine vs miR-E2f3a + cocaine: t(10) = 1.331, p = 0.9999; N = 5–7).

  • Figure 3.
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    Figure 3.

    Viral-mediated E2F3a overexpression leads to increased E2F3 binding at Fosb promoter in NAc. A, E2F3 binding was increased at 500 bp upstream of the Fosb TSS after E2F3a overexpression (interaction of virus and gene position by two-way ANOVA: F(3,35) = 3.619, p = 0.0224; E2F3a vs GFP at −500 bp via post hoc analysis: t(7) = 3.285, **p = 0.0093). B, H3K4me3 enrichment was not affected by E2F3a overexpression (no main effect of virus observed: F(1,40) = 0.057, p = 0.8126). N = 5–6, with three mice pooled per sample for all qChIP assays.

Tables

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    Table 1.

    ChIP-qPCR Primers along the Fosb gene

    Location on FosbForwardReverse
    −1250 ATGGGACTCAGGTTGTCAGG AGCCAGGGCTACACAGAGAA
    −500 GAGTTGCACCTTCTCCAACC GGCCCAGTGTTTGTTTGGTA
    −250 ATGGCTAATTGCGTCACAGG ACCTCCCAAACTCTCCCTTC
    Exon IV CAACCTGACGGCTTCTCTCT CGGGTTTGTTTGTTTTGTTTG
    • These qChIP primers were used to amplify three putative E2F consensus sequences in Fosb: 500 and 250 bp upstream of the TSS, and a site within Exon IV. A sequence 1250 bp upstream of the TSS was used as a negative control.

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    Table 2.

    RT-qPCR Primers for Fosb transcripts and Gapdh for probing mRNA expression

    TranscriptForwardReverse
    Fosb GTGAGAGATTTGCCAGGGTC AGAGAGAAGCCGTCAGGTTG
    ΔFosb AGGCAGAGCTGGAGTCGGAGAT GCCGAGGACTTGAACTTCACTCG
    Gapdh AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA
    • These qPCR primers were used to amplify and assess expression of Fosb and ΔFosb mRNA. Gapdh was used as a housekeeping gene in the ΔΔCt method.

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    Table 3.

    Statistical table

    FigureData structureType of testPower
    a Fig. 1B,CNormal distribution4 × 4 ANOVA0.0621
    b Fig. 1DNormal distribution2 ×2 ANOVA0.0595
    c Fig. 1ENormal distribution2 ×2 ANOVA0.0595
    d Fig. 1FNormal distribution2 ×2 ANOVA0.0595
    e Fig. 2ANormal distribution2 ×2 ANOVA0.0558
    f Fig. 2BNormal distribution2 ×2 ANOVA0.0558
    g Fig. 2DNormal distribution2 ×3 ANOVA0.0692
    hΔFosb/Fosb mRNA ratioNormal distributionStudent’s t test0.0661
    i Fig. 2ENormal distribution2 ×2 ANOVA0.0607
    j Fig. 2FNormal distribution2 ×4 ANOVA0.0651
    k Fig. 3ANormal distribution2 ×2 ANOVA0.0607
    l Fig. 3BNormal distribution2 ×2 ANOVA0.0607
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Fosb Induction in Nucleus Accumbens by Cocaine Is Regulated by E2F3a
Hannah M. Cates, Casey K. Lardner, Rosemary C. Bagot, Rachael L. Neve, Eric J. Nestler
eNeuro 20 March 2019, 6 (2) ENEURO.0325-18.2019; DOI: 10.1523/ENEURO.0325-18.2019

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Fosb Induction in Nucleus Accumbens by Cocaine Is Regulated by E2F3a
Hannah M. Cates, Casey K. Lardner, Rosemary C. Bagot, Rachael L. Neve, Eric J. Nestler
eNeuro 20 March 2019, 6 (2) ENEURO.0325-18.2019; DOI: 10.1523/ENEURO.0325-18.2019
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Keywords

  • addiction
  • cocaine
  • E2F3a
  • ∆FosB
  • nucleus accumbens
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