Molecular mechanisms of circadian timekeeping in Drosophila

Molecular and genetic studies in the fruit fly Drosophila melanogaster have revealed that the circadian timekeeping mechanism is based on feedback loops in gene expression. These feedback loops, which are highly conserved in mammals, can be divided into temporally distinct transcriptional activation, repression, and reactivation phases. Determining how different phases of the transcriptional feedback cycle are controlled and integrated is necessary to understand the molecular basis of circadian timekeeping. In Drosophila, feedback loop function is initiated when hypophosphorylated CLOCK (CLK) forms a heterodimer with CYCLE (CYC) and binds E-boxes to activate transcription of clock genes including period (per), timeless (tim), vrille (vri), PAR domain protein 1 epsilon (Pdp1 epsilon), and clockwork orange (cwo). Such activation is reduced in cwo mutants, suggesting that CWO supports CLK-CYC dependent transcription. After PER accumulates to high levels during the night, complexes containing PER, DOUBLETIME (DBT) kinase, and possibly TIM, bind to and remove CLK-CYC from E-boxes to repress transcription. PER-CLK binding isn't sufficient to remove CLK-CYC from E-boxes, but PER-DBT-mediated hyperphosphorylation of CLK by one or more unidentified kinase(s) coincides with CLK-CYC release from E-boxes, which implies that CLK phosphorylation promotes CLK-CYC release from E-boxes. After lights-on, PER is degraded due to DBT-dependent phosphorylation, thereby releasing CLK-CYC. Over the course of 4-6 h, phosphorylated CLK is then replaced by transcriptionally competent hypophosphorylated CLK to initiate the next cycle of transcription.