A screening system that searches for clock-controlling factors

Abstract

Mammalian circadian rhythms are generated by a feedback loop in which positive (BMAL1:CLOCK) and negative (CRYs) components activate transcription for different periods. To investigate the molecular mechanism of the circadian system, we attempted to establish a screening method to identify clock-controlling factors. The reporter construct ( Bmal1-prom-Luc) was transfected into NIH3T3. In transient expression experiments using the reporter construct, the regulatory factors that affect Bmal1 transcription were screened. Using a real-time monitoring system for Bmal1 promoter activity, we examined whether the factors that regulate Bmal1 transcription affected the circadian changes in the reporter activity of the Bmal1 promoter. The real-time monitoring system showed that the transient co-expression of RORα with the Bmal1 reporter construct in NIH3T3 cells abolished the circadian changes of the reporter activity and RNAi against RORα attenuated the circadian expression of the reporter activity. These results indicate that RORα affects not only the level of Bmal1 transcription but also its circadian expression. [Jpn J Physiol 55 Suppl:S53 (2005)]