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gene is a target of the circadian clock pacemaker
Laboratoire de Biologie Moléculaire et Cellulaire, CNRS UMR 5161, Ecole Normale Supérieur de Lyon, 46 allée d’Italie, 69364 Lyon cedex, France
1 Howard Hughes Medical Institute, Northwestern University, Department of Neurobiology and Physiology, 2205 Tech Drive, Evanston, IL 60208, USA
2 Laboratoire de Physiologie des Membranes Cellulaires, CNRS UMR 6078, Université de Nice-Sophia Antipolis, Chemin du Lazaret, 06 238 Villefranche-sur-mer, France
(Requests for offprints should be addressed to V Laudet; Email: Vincent.Laudet{at}ens-lyon.fr)
* (G Triqueneaux and S Thenot contributed equally to this work)
Rev-erb
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-erb mRNA isoforms, namely Rev-erb1 and Rev-erb 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-erb1) and P2 (Rev-erb2) 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-erb gene. In line with these data Rev-erb 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-erb circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erb in the circadian clock output.
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