Dopamine D1 Receptor (D1R) Expression Is Controlled by a Transcriptional Repressor Complex Containing DISC1

Figures

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  Fig. 1. Upregulated D1r transcription in DISC1-deficient mice. a Relative transcript abundance of D1r in the striatum (n = 12), hippocampus (n = 6), and prefrontal cortex (n = 6) of adult (12-week-old) Disc1 (Δ2–3/Δ2–3) mice (right) and those of wild-type (WT) B6 mice (left) assayed by real-time qRT-PCR. b Relative transcript abundance of D1r in the striatum (n = 6), hippocampus (n = 10), and prefrontal cortex (n = 6) of Disc1-LI mice (right), compared with those of WT B6 mice (left). c Increased protein level of D1R in the striatum of adult Disc1-LI mice (n = 3). d Reduced transcript level of D1r in differentiated CAD cells upon overexpression of Flag-DISC1 (n = 8). e Decreased acetylation level of histone H3 in differentiated CAD cells upon Flag-DISC1 overexpression (n = 3). Anti-AcH3 (K9 and K14) antibody was used for IP. Columns represent mean ± SEM. *P < 0.05; ***P < 0.001; two-tailed t test

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  Fig. 2. Translocation of DISC1 into the nucleus via interaction with KLF16. a CoIP of KLF16-HA with Flag-DISC1 using anti-Flag antibody from differentiated CAD cell lysates. b Increased DISC1 protein level in the nucleus-enriched fractions of differentiated CAD cell lysates (n = 3). c Confocal images of Flag-DISC1 (red) and the Hoechst 33342 (blue) upon EGFP or KLF16-EGFP (green) overexpression. Mander’s overlap coefficients between Flag-DISC1 and Hoechst were calculated by the Cellsens software (Olympus) (n = 8 for EGFP, n = 10 for KLF16-EGFP, E15.5 mouse striatal cultured neurons at DIV10). The white line outlines the neuronal soma. The scale bar represents 5 μm. d Reduced transcript level of D1r upon DISC1, KLF16, and their combined overexpression in differentiated CAD cells (n = 8). e Reduced endogenous D1R protein level upon DISC1 and KLF16 co-expression (n = 3). f Enhanced ChIP-PCR signal of KLF16-EGFP at the D1r locus in DISC1-cotransfected CAD cells (n = 5). Anti-GFP antibody was used for IP. g Reduced acetylated H4 level at the D1r locus upon DISC1, KLF16, and their combined overexpression in differentiated CAD cells (n = 4). Anti-AcH4 (K5, K8, K12, and K16) antibody was used…

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  Fig. 3. Recruitment of SIN3A corepressor by the KLF16-DISC1 complex at the D1r locus. a Enhanced ChIP-PCR signal of SIN3A-Myc at the D1r locus upon co-expression of KLF16 in differentiated CAD cells (n = 3). Antibody against GFP and Myc were used for IP. b Reduced acetylated H4 level at the D1r locus upon KLF16 and SIN3A overexpression in differentiated CAD cells (n = 3). Anti-AcH4 (K5, K8, K12, and K16) antibody was used for IP. c Reduced transcript abundance of D1r upon KLF16 and SIN3A overexpression in differentiated CAD cells (n = 6). d Reduced endogenous D1R protein level upon KLF16 and SIN3A overexpression in differentiated CAD cells (n = 4). e CoIP of KLF16-HA with endogenous DISC1 and SIN3A from differentiated CAD cell lysate. f Enhanced coIP of KLF16-HA with endogenous SIN3A upon Flag-DISC1 overexpression in differentiated CAD cells (n = 3). Anti-SIN3A antibody was used for IP. g Confocal images of Flag-DISC1 (red, left bottom), SIN3A (blue), Hoechst (cyan), KLF16-EGFP (green), and their merged images (bottom) of the nucleus of cultured mouse striatal neuron at DIV10. Scale bars represent 5 μm. h Pearson’s correlation coefficients between DISC1 and SIN3A, between DISC1 and…

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  Fig. 4. Effects of DISC1-deficiency on D1R signaling and related behaviors. a Enhanced phosphorylation of ERK1/2. Primary cultured mouse striatal neurons (DIV10) from Disc1-LI or wild-type mouse embryos (E15.5) were treated with DA (10 μM) for 30 min. SCH23390 (10 μM) was pretreated for 15 min. b, c Altered locomotor sensitization to cocaine in wild-type mice (b) and Disc1-LI mice (c). Mice were treated with saline or cocaine (20 mg/kg) in combination with SCH23390 (0.3 mg/kg) as indicated. Data represent total travel distances relative to that on day 1 (n = 6 for WT + cocaine and WT + cocaine + SCH23390; n = 7 for Disc1-LI + cocaine + SCH23390; n = 8 for the others). d, e Comparison of locomotion between wild-type mice and Disc1-LI mice on day 2 (d) and day 5 (e). f Cocaine CPP scores of wild-type (n = 11) and Disc1-LI mice (n = 8) and the effect of pretreatment of SCH23390 (n = 7 for each). Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; two-way ANOVA with post-hoc Bonferroni test (b, c); one-way ANOVA with post hoc Tukey test (a, d, e, f)