Fig. 1: Circadian nuclear receptor REV-ERBα modulates 5-HT rhythmicity in an antagonistic manner with PET-1.

a Schematic representation of the mouse Tph2 (mTph2), rat Tph2, and human Tph2 (hTph2) promoter regions with PET-1 binding sites (blue bars)¹² and putative REV-ERBα binding sites (red bars), which are predicted using the PROMO, Genomatix, and TFsitescan databases. b Effects of REV-ERBα and/or PET-1 on the 2.7-kb mTph2 promoter or 1.9-kb hTph2 promoter activity. Dose-dependent repression of the each Tph2 promoter by REV-ERBα in functional competition with a PET-1 nuclear activator at the indicated ratios was assessed. c Assessment of target amplicon(s) on the Tph2 promoter for relative binding of REV-ERBα or PET-1 on the mTph2p and hTph2p via a ChIP-qPCR analysis. Differentiated PC12 cells were transfected with EGFP-REV-ERBα- and/or HA-PET-1-expressing constructs at the indicated ratios (n = 3). d Schematic representation of WT and designed site-directed deletions of REV-ERBα or PET-1 binding sites on mTph2 promoter regions (target deletion sites are indicated with underlines). Transcriptional activities of WT (e), RRE mut 1, RRE mut 2 (f), PRE mut 3, and PRE mut 4 (g) of mTph2 promoters upon REV-ERBα and/or PET-1 expression were measured (n = 3). Functional studies of RORα transcription factors on mTph2- (h) or mBmal1- (i) promoter-driven luciferase reporter in competition with REV-ERBα. For the promoter assays, the Tph2 promoter-driven luciferase reporter indicated that the transcription factor(s) and pRL-TK were co-transfected into differentiated PC12 cell lines (REV: REV-ERBα, PET: PET-1). Luciferase activity (A.U.) was normalized using the renilla activity (pRL-TK), which was used as the internal control. For promoter assays, data are presented as mean ± SEM, and the significance was assessed by one-way ANOVA followed by Dunnett’s post-hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns = not significant.