The period E-box is sufficient to drive circadian oscillation of transcription in vivo

The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In c ell culture, an 18 bp E-box element from the period promoter is regulated b y five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS p roteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERI OD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the s ame 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clk(jrk) mutation maintained rhythmic expression from the E-box alt hough at a lower level than wild type. Homozygous mutant Clk(jrk) animals h ad drastically lowered and arrhythmic expression. In a per(01) background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. Th e per E-box DNA element and cognate binding proteins can confer per-like te mporal and spatial expression. This demonstrates in vivo that the known cir cadian genes that form the core of the circadian oscillator in Drosophila i ntegrate their activities at a single DNA element.