Real-Time Analysis of the Circadian Oscillation of the Rev-Erb β Promoter

Aim: Rev-Erb β gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erb β that generate and determine the phase of the circadian oscillation remain unclear. Methods: We analyzed the Rev-Erb β promoter by luciferase reporter assays, real-time bioluminescence monitoring assays and electrophoretic mobility shift assays. Results: Luciferase reporter assays indicated that only the 5’ region and exon 1 have obvious promoter activity. Real-time bioluminescence monitoring assays revealed that E1, E2, E3, D boxes are important for maintenance of the amplitude of Rev-Erb β oscillation. Based on EMSA results, REV-ERBβ binds ROREs in the Bmal1 promoter region and inhibits Bmal1 promoter activity. Conclusion: We provide direct evidence that three E-boxes and one D-box located in the first intron are crucial for the phase of circadian oscillation in Rev-Erb β expression and that the sequences upstream from its transcription start site function as a promoter with no circadian regulation. We also found that the E1 box affects the Rev-Erb β oscillation phase. Our results offer new insight into the role of Rev-Erb β in the circadian rhythm system.