FIGURE 3.

Dual roles of zebrafish Per2 are mediated by E-boxes and RORE boxes. A, schematic of the DNA construct aanat2-luc wherein the aanat2 promoter contains one E-box. B, schematic of the DNA construct bmal1b-luc wherein the bmal1b promoter harbors the two RORE motifs (in red), which are identical in human, mouse, and zebrafish. C, Per2 inhibits the Clock1a-Bmal1b heterodimer-mediated aanat2 transcription through E-box. The activities of the E-box containing aanat2 promoter are activated by co-transfection of Clock1a and Bmal1b but repressed by Per2. The effects of both activation and repression are lost without the E-box. D, Per2 enhances Rorαa- or Rorαb-mediated bmal1b expression. Both Rorαa and Rorαb can activate bmal1b transcription. The activation efficacy of Rorαa is ∼2-fold that of Rorαb. Rev-erbα outcompetes Rorαa or Rorαb to repress bmal1b expression. Per2 reverses the inhibitory effects of Rev-erbα to enhance bmal1b expression. All effects are lost without the two RORE motifs. E, zebrafish Rev-erbα outcompetes Rorα to repress bmal1b in a dose-dependent manner. F, zebrafish enhances bmal1b expression in a dose-dependent manner. G, the inhibitory effect of Rev-erbα was not significant when RORE1 was mutated. H, the inhibitory effect of Rev-erbα still persisted when RORE2 was mutated. The Dual-Luciferase reporter system was used in the experiments. ***, p < 0.001, unpaired two-tailed Student's t test. Error bars represent S.E.