bHLH-ORANGE family genes regulate the expression of E-box clock genes in Drosophila

Drosophila has 13 basic helix-loop-helix-ORANGE (bHLH-O) family genes. One of the members, clockwork orange (cwo), which is the counterpart of mammalian clock genes Dec1 and Dec2, regulates the transcriptional feedback loops of circadian clock genes through binding to E-box sequences in target gene promoters. The goal of the current study was to determine the role of Drosophila bHLH-O proteins in circadian rhythms at the molecular and behavioral level. Promoter assays in cultured Drosophila S2 cells were carried out to investigate which of the known bHLH-O proteins directly regulates the transcription of clock genes. In addition to CWO, three other bHLH-O proteins, SIDE, M beta and M gamma, suppressed E-box clock gene transcription in vitro. RNA interference (RNAi) was used to generate bHLH-O knockdown flies in pacemaker neurons, and then locomotor activity rhythm was measured. cwo knockdown flies exhibited a remarkable phenotype. To clarify the functional complementation in circadian regulation among CWO, SIDE, M beta and M gamma, promoter activity in the presence of combinations of two bHLH-O genes and locomotor rhythm in double knockdown flies were examined. The results suggest that CWO predominantly acts as a key factor of circadian regulation both in vitro and in vivo.