Bidirectional role of orphan nuclear receptor RORalpha in clock gene transcriptions demonstrated by a novel reporter assay system.

Circadian rhythms are generated by an extremely complicated transcription-translation feedback loop. To precisely analyze the molecular mechanisms of the circadian clock, it is critical to monitor multiple gene expressions and/or interactions with their transcription factors simultaneously. We have developed a novel reporter assay system, the tricolor reporter in vitro assay system, which consists of green- and red-emitting Phrixothrix luciferases as dual reporters and blue-emitting Renilla luciferase as internal control. We have successfully employed this system in analyzing the effects of clock gene products on the enhancer elements of Per1 and Bmal1 promoters. The results indicate that the orphan nuclear receptor RORalpha regulates bidirectionally Bmal1 (positively) and Per1 (negatively) transcriptions simultaneously.