Real-time analysis of the circadian oscillation of the Rev-Erbβ promoter

Abstract

Aim: The Rev-Erbβ gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erbβ that generate and determine the phase of the circadian oscillation remain unclear. Methods: We analyzed the Rev-Erbβ 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-Erbβ 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-Erbβ 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-Erbβ oscillation phase. Our results offer new insight into the role of Rev-Erbβ in the circadian rhythm system.