Fig. 2: Promoter activity of DISC1 is enhanced by CLOCK and BMAL1 through E-box sequences.

a DISC1 promoter was cloned into pGL3 vector to monitor promoter activity by luciferase expression. Luciferase assay was performed in HEK293 cells overexpressing CLOCK or together with BMAL1 (n = 3, biological replicates). b Luciferase assay with DISC1 promoter sub-regions: distal part (−982 to −624 bp relative to TSS), middle part (−623 to −324 bp relative to TSS), and proximal part (−323 to +47 bp relative to TSS). Luciferase assay was performed in HEK293 cells overexpressing CLOCK and BMAL1 (n = 3, biological replicates). Each group was normalized to the respective control (empty vectors were transfected). c Luciferase assay with E-box-mutated DISC1 promoter. Luciferase assay was performed in HEK293 cells overexpressing CLOCK and BMAL1 (n = 3, biological replicates). Each group was normalized to the respective control (empty vectors were transfected). d Luciferase assay conducted using the distal part of DISC1 promoter with E-box mutations in HEK293 cells overexpressing CLOCK and BMAL1 (n = 3, biological replicates). Each group was normalized to the respective control (empty vectors were transfected). e Chromatin immunoprecipitation (ChIP) was performed in HEK293 cells transfected with CLOCK or BMAL1 or both to assess direct binding on E-box sequences of DISC1 promoter (n = 3, biological replicates). GFP antibody was utilized to pull down GFP-BMAL1. qPCR was used to quantify the E-box sequences precipitated by GFP-BMAL1. Red arrows indicate estimated primer binding sites for qPCR. For CLOCK and BMAL1 transfection, RFP-CLOCK-myc and GFP-BMAL1 constructs were used for (a–d), and (e). RLA represents relative luciferase activity. Data are means with SEM. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, one-way ANOVA and Tukey’s post-hoc test for (a–c), (d), and (e).