The mammalian circadian clock has evolved as an adaptation to the 24-h light/darkness cycle on earth. Maintaining cellular activities in synchrony with the activities of the organism (such as eating and sleeping) helps different tissue and organ systems coordinate and optimize their performance. The full extent of the mechanisms by which cells maintain the clock are still under investigation, but involve a core set of clock genes that regulate large networks of gene transcription both by direct transcriptional activation/repression as well as the recruitment of proteins that modify chromatin states more broadly.
Kimberly H Cox and Joseph S Takahashi
Gérard Triqueneaux, Sandrine Thenot, Tomoko Kakizawa, Marina P Antoch, Rachid Safi, Joseph S Takahashi, Franck Delaunay, and Vincent Laudet
Rev-erbα is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erbα mRNA isoforms, namely Rev-erbα1 and Rev-erbα 2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erbα1) and P2 (Rev-erbα2) contain several E-box DNA sequences which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK–BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3–6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK–BMAL1 heterodimer also regulates the zebrafish Rev-erbα gene. In line with these data Rev-erbα circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erbα circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erbα in the circadian clock output.