Mapping of binding regions for the circadian regulators BMAL1 and CLOCK within the mouse Rev-erbalpha gene.

Two major interconnected feedback loops govern circadian rhythmicity in mammals. The transcriptional activators BMAL1/MOP3 and CLOCK drive one loop by binding to E-box motifs in the regulatory regions of circadian target genes. Time-of-day-dependent inhibition of the activity of BMAL1 and CLOCK by PERIOD (PER) and CRYPTOCHROME (CRY) proteins provokes consequent rhythmic gene expression. In this fashion, the Per and Cry genes can feedback onto their own synthesis. The orphan nuclear receptor Rev-erbalpha manifests the second feedback loop. Genetically linked to the molecular oscillator, it was identified as a repressor of the Bmal1 and Clock genes. In an attempt to understand the circadian regulation of the mouse Rev-erbalpha gene, we developed a screening technique for BMAL1 and CLOCK binding regions consisting of chromatin-immunoprecipitation. Here we show that the mouse Rev-erbalpha gene contains at least two binding regions for the transcriptional activators close to its previously identified promoter region and in its first intron. However, our data also indicate that not all potential E-box motifs in vitro are occupied in vivo.