Rhythmic transcription of Bmal1 stabilizes the circadian timekeeping system in mammals

In mammals, the circadian clock consists of transcriptional and translational feedback loops through DNA cis-elements such as E-box and RRE. The E-box-mediated core feedback loop is interlocked with the RRE-mediated feedback loop, but biological significance of the RRE-mediated loop has been elusive. In this study, we established mutant cells and mice deficient for rhythmic transcription of Bmal1 gene by deleting its upstream RRE elements and hence disrupted the RRE-mediated feedback loop. We observed apparently normal circadian rhythms in the mutant cells and mice, but a combination of mathematical modeling and experiments revealed that the circadian period and amplitude of the mutants were more susceptible to disturbance of CRY1 protein rhythm. Our findings demonstrate that the RRE-mediated feedback regulation of Bmal1 underpins the E-box-mediated rhythm in cooperation with CRY1-dependent posttranslational regulation of BMAL1 protein, thereby conferring the perturbation-resistant oscillation and chronologically-organized output of the circadian clock.