Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component

Abstract

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.[Keywords: Circadian; clk; Drosophila; transcriptional oscillations; chromatin] Supplemental material is available at http://www.genesdev.org.

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

“…We recently showed that Clk mRNA levels are regulated by microRNAs (miRNAs) 32 . To address the importance of Clk posttranscriptional regulation, we compared Clk mRNA levels with those of other circadian mRNAs using previously published microarray and RNA-seq data from fly heads and brains 7,41,42 . In these data sets Clk mRNA levels were the lowest of the core circadian components (Fig.…”

“…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.…”

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

“…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.…”