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

The nuclear receptor REV-ERBα is a key negative-feedback regulator of the biological clock. REV-ERBα 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-ERBα ligand, which enhances the recruitment of nuclear receptor co-repressor (NCoR) to REV-ERBα. In order to explore REV-ERBα action on resetting responses of the molecular clock, we first established the rhythmic transcription profile and expression level of REVERBα 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-ERBα 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-ERBα 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.