clockwork orange Encodes a Transcriptional Repressor Important for Circadian-Clock Amplitude in Drosophila

Abstract

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer, CLOCK/CYCLE (CLK/CYC), transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function. Microarray studies have identified numerous rhythmically expressed transcripts, some of which are potential direct CLK targets. Here we demonstrate a circadian function for one such target, a bHLH-Orange repressor, CG17100/CLOCKWORK ORANGE (CWO). cwo is rhythmically expressed, and levels are reduced in Clk mutants, suggesting that cwo is CLK activated in vivo. cwo mutants display reduced-amplitude molecular and behavioral rhythms with lengthened periods. Molecular analysis suggests that CWO acts, in part, by repressing CLK target genes. We propose that CWO acts as a transcriptional and behavioral rhythm amplifier.

Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle

“…3). Moreover, previous studies showed that two copies of Cwo producing overexpression by tim-Gal4 (tim-Gal4/ þ ; UAS-cwo) generate a modest reduction of rhythmic power [12][13][14][15] , which can mimic the let-7 loss-of -function phenotype in this study, indicating that the let-7 mutant phenotype may be due to increased CWO expression. On the contrary, our results show that let-7 overexpression in Cwo cells lengthened circadian period, which is consistent with the effect of cwo-RNAi in both Cwo cells and Tim cells, and it was able to be partially rescued by Cwo overexpression, indicating regulation of Cwo by let-7.…”

“…CLK-CYC also activates two other feedback loops critical for the phase and amplitude of core oscillators and outputs. One feedback loop affecting Clk mRNA cycling is mediated by VRILLE (VRI) and PAR DOMAIN PROTEIN 1 expression 10,11 , and another is mediated by CLOCKWORK ORANGE (CWO), which is activated by CLK-CYC [12][13][14][15] . The per and tim genes receive two opposing signals form DNA-binding transcription factors-a direct activation from CLK-CYC and a direct repression from CWO.…”

“…In addition, CWO regulates the level of the CLK-CYC, presumably through its actions on each of three main loops 16 . So cwo contributes to sustaining a high-amplitude circadian oscillation in vivo [12][13][14] . The Drosophila rhythm strength is sensitive to the level and timing of cwo expression, although the amplitude of the cwo mRNA oscillation is much weaker than that of the other core clock genes 15 .…”

“…Drosophila rhythm strength is sensitive to cwo both in expression level and timing [12][13][14] . Here we report that the miRNA let-7 affects the locomotor rhythm through the circadian-relevant target gene cwo in the central clock.…”

Clk post-transcriptional control denoises circadian transcription both temporally and spatially

“…In Drosophila, the master genes Clock (Clk) and cycle (cyc) activate the circadian system by promoting rhythmic transcription of a number of key genes [2][3][4] . Three of these target gene products, PERIOD (PER) 5 , TIMELESS (TIM) 6 and CLOCKWORK ORANGE (CWO) [7][8][9] , repress CLK-CYC-mediated transcription on a daily basis. CLK activates transcription only while associated with CYC.…”