Cry1 circadian phase in vitro: wrapped up with an E-box.

The circadian timing of gene expression is determined by transcriptional regulation through upstream response elements present throughout the genome. Central to this regulation are the actions of a core group of transcriptional activators and repressors, which act through, and are themselves regulated by, a small set of canonical circadian response elements. Among these, the E-box (CACGTG) is crucial for daytime transcriptional activity. The mammalian Period (Per1-3) and Cryptochrome (Cry1-2) genes are E-box-regulated genes, but in peripheral tissues peak Cry1 mRNA expression is delayed by several hours relative to that of Per. It has been proposed that this delay originates from interactions between the proximal E-box and retinoic acid-related orphan receptor response elements (RORE) present in the Cry1 promoter. By using real-time luciferase reporter assays in NIH3T3 cells the authors show here that a proximal 47-bp E-box containing region of the Cry1 promoter is both necessary and sufficient to drive circadian Cry1 transcription with an appropriate phase delay (around 4 h) relative to Per2. The results therefore suggest that, at least in this in vitro model of the clock, RORE are not necessary for the appropriate circadian regulation of Cry1 expression and rather suggest that sequences surrounding the proximal E-boxes confer gene-specific circadian phasing.