Ligand modulation of REV-ERBalpha function resets the peripheral circadian clock in a phasic manner

The nuclear receptor REV-ERBalpha is a key negative-feedback regulator of the biological clock. REV-ERBalpha binds to ROR elements of the Bmal1 (Arntl) promoter and represses Bmal1 transcription. This stabilizing negative loop is important for precise control of the circadian pacemaker. In the present study, we identified a novel synthetic REV-ERBalpha ligand, which enhances the recruitment of nuclear receptor co-repressor (NCoR) to REV-ERBalpha. In order to explore REV-ERBalpha action on resetting responses of the molecular clock, we first established the rhythmic transcription profile and expression level of REV-ERBalpha in Rat-1 fibroblasts. When applied at different phases of the circadian oscillation to cell models containing stably transfected Bmal1::Luc or Per2::Luc, the REV-ERBalpha ligand induced phase-dependent bi-directional phase shifts. When the phase changes were plotted against time, a clear phase response curve was revealed, with a significant peak-to-trough amplitude of ca. 5 hours. The phase-resetting effect was also observed when the compound was applied to primary lung fibroblasts and ectopic lung slices from transgenic PER2::Luc mice. Therefore, similar regulation of REV-ERBalpha function by endogenous ligands, such as heme, is likely to be an important mechanism for clock resetting. In addition, we identify a new means to generate phasic shifts in the clock.