Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS(PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.

The Drosophila circadian oscillator consists of interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLK and CYCLE (CYC) activate per and tim transcription at the same time as they repress Clk transcription, thus controlling the opposite cycling phases of these transcripts. CLK-CYC directly bind E box elements to activate transcription, but the mechanism of CLK-CYC-dependent repression is not known. Here we show that a CLK-CYC-activated gene, vrille (vri), encodes a repressor of Clk transcription, thereby identifying vri as a key negative component of the Clk feedback loop in Drosophila's circadian oscillator. The blue light photoreceptor encoding cryptochrome (cry) gene is also a target for VRI repression, suggesting a broader role for VRI in the rhythmic repression of output genes that cycle in phase with Clk.

The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.

Blue light activation of the photoreceptor CRYPTOCHROME (CRY) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. Here we show that acute arousal behavioral responses to blue light significantly differ in mutants lacking CRY, as well as mutants with disrupted opsin-based phototransduction. Lightactivated CRY couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K + ) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk −/− mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K + channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for CRY/Hk-coupled light response. Light activation of CRY is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor.phototransduction | potassium channel | redox | cryptochrome

In the fruit fly Drosophila melanogaster, CRYPTOCHROME (CRY) functions as a photoreceptor to entrain circadian oscillators to light-dark cycles and as a transcription factor to maintain circadian oscillator function in certain peripheral tissues. Given the importance of CRY to circadian clock function, we expected this protein to be expressed in all oscillator cells, yet CRY cellular distribution and subcellular localization has not been firmly established. Here we investigate CRY spatial expression in the brain using a newly developed CRY antibody and a novel set of cry deletion mutants. We find that CRY is expressed in s-LN v s, l-LN v s and a subset of LN d s and DN 1 s, but not DN 2 s and DN 3 s. CRY is present in both the nucleus and cytoplasm of these neurons, and its subcellular localization does not change over the circadian cycle. Although CRY is absent in DN 2 s and DN 3 s, cry promoter activity and/or cry mRNA accumulation can be detected in these neurons, suggesting that CRY levels are regulated post-transcriptionally. Oscillators in DN 2 s and DN 3 s entrain to environmental light-dark cycles, which implies that they are entrained indirectly by retinal photoreceptors, extra-retinal photoreceptors or other CRY expressing cells.