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, a genome-wide approach was designed and clockwork orange (cwo) was identified 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 (Kadener, 2007). To other studies similarly identified Clockwork orange an a transcriptional repressor that inhibits Clk-mediated activation (Matsumoto, 2007; Lim, 2007).

In Drosophila, the genes Clock (Clk) and cycle (cyc) sit at the top of a genetic hierarchy governing circadian rhythms and promote the rhythmic transcription of several key clock genes. The protein products of two of these target genes, Per (period) and Tim (timeless), repress Clk-Cyc-mediated transcription toward the end of every cycle and thereby repress their own synthesis. The comparable event in mammals involves cryptochromes (CRYs) as well as Pers as the major transcriptional repressors. A second transcriptional feedback loop in flies affects Clk mRNA cycling and involves vri (vrille) and Pdp1 (Par domain protein 1), two other direct targets of the Clk-Cyc heterodimer (Kadener, 2007 and references therein).

The mechanism by which Per represses Clk-driven transcription is still uncertain. Recent reports indicate that cyclical Clk target gene expression may be the result of cyclical DNA binding, both in Drosophila and mammals (Ripperger, 2006; Yu, 2006). Moreover, recent evidence suggests that Drosophila Per may deliver the kinase DBT (doubletime) to the Clk-Cyc dimer. Clk phosphorylation likely ensues, with the subsequent disassociation of Clk-Cyc from target E-boxes. Although comparable biochemical detail is lacking for mammalian clocks, activation-repression cycles generate high-amplitude mRNA oscillations in both systems and are proposed to be important for behavioral oscillations. At least in the fly system, there is good genetic evidence that this is the case (Kadener, 2007 and references therein).

A recent report suggests an additional 'active' repression mechanism, as important changes in chromatin structure escort circadian transcriptional oscillations in mammals and Neurospora (Ripperger, 2006; Belden, 2007). In mammals, these modifications appear to follow the CRY-Per repression events and may enhance the oscillation amplitude of various cycling mRNAs. It is likely that similar phenomena take place in the Drosophila system (Kadener, 2007).

It is curious that all known bona fide direct targets of the master gene Clk are involved in transcriptional regulation (per, vri, tim, Pdp1). It was therefore reasoned that finding additional Clk direct targets might identify new biochemical pathways relevant to central clock function or perhaps reinforce the centrality of transcriptional regulation. This study reports the isolation and characterization of a new core clock component: clockwork orange (cwo). cwo transcription is activated by Clk-Cyc and repressed by Per-Tim. As a consequence, cwo mRNA oscillates with an amplitude and phase comparable with other Clk direct targets; for example, vri, Pdp1, per, and tim. cwo is prominently expressed in circadian brain neurons and cooperates with Per to repress Clk-Cyc-mediated transcription. Characterization of flies deficient in cwo activity demonstrates that the protein is essential for robust oscillations of core clock mRNAs as well as persistent behavioral rhythms with wild-type periods. Because the oscillation amplitude of direct clock transcripts is specifically affected in cwo-deficient strains, the core transcriptional feedback loop appears essential for circadian rhythms in Drosophila. As cwo orthologs (Dec1 and Dec2) are possible pacemaker components in mammals (Honma, 2002), this view may also extend to other animal systems (Kadener, 2007).

To find additional Clk direct targets, a transgenic fly line was generated expressing a Clk-glucocorticoid receptor fusion protein (ClkGR) and expressed it in clock neurons (tim-gal4; UAS-ClkGR fly strain). To identify direct Clk targets, fly heads were cultured and stimulated with dexamethasone in the presence of the protein synthesis inhibitor cycloheximide. A parallel experiment was carried out in S2 cells. This approach identified candidate direct Clk targets, which were ranked according to targetness (TGT). Among the 28 genes that passed a stringent cutoff criterion were the four known direct targets as well as other genes described as cyclically expressed or affected in the Clk mutant Jrk. However, 60% were not previously connected to Drosophila rhythms, and many of these were also activated by Clk in S2 cells. Since overexpression studies can reveal nonphysiological targets, some of these candidates were tested in an independent assay. To this end, luciferase reporter constructs were constructed from the promoters of three new targets and they were tested in S2 cells with vri-luciferase as a positive control. All were strongly activated when cotransfected with a Clk-expressing plasmid (pAc-Clk) and repressed by Per cotransfection (Kadener, 2007).

Among putative direct Clk targets was a gene encoding a transcription factor with an Orange domain, CG17100, called clockwork orange (cwo). cwo was previously misannotated as stich1. cwo belongs to a family of transcriptional repressors (basic helix-loop-helix-O [bHLH-O]) involved in various aspects of cell physiology and metabolism (Davis, 2001). Importantly, two close mouse relatives, dec1 and dec2, are circadianly expressed in the suprachiasmatic nucleus and regulate circadian gene expression (Honma, 2002; Hamaguchi, 2004; Li, 2004; Sato, 2004). cwo mRNA oscillates in a circadian manner. cwo mRNA cycles with a phase (peak around 14 h Zeitgeber time [ZT14]) (two hours into the night) that resembles those of per, tim, Pdp1, and vri mRNAs. These Clk direct targets contain numerous Clk-Cyc-binding elements (E-boxes) in their promoters. E-boxes are necessary for transcriptional oscillations and have been shown in some cases to mediate Clk activation followed by Per repression. There are six E-boxes within the promoter of cwo, 2 kb upstream of the transcriptional start site, and 15 E-boxes within the first intron. Moreover, cwo mRNA was regulated in clock mutant strains like characterized direct target genes, namely, low and high mRNA levels in the Clk mutant Jrk and the per01 mutant, respectively (Kadener, 2007).

To examine cwo spatial expression, a UAS-GFP line was crossed with an enhancer trap fly line containing a GAL4-coding sequence in the promoter region of cwo. Costaining with anti-PDF antisera (PDF is a neuropeptide specific for pacemaker cells) showed strong GFP expression in brain pacemaker neurons. Recent independent reports confirm this observation: A LacZ enhancer trap in this same gene is prominently expressed in clock cells  that is, in all Per-expressing cells (Shafer, 2006)  and comparable data with an anti-Cwo antibody are presented in an accompanying paper by Matsumoto (2007) (Kadener, 2007).

cwo encodes a transcriptional repressor, which synergizes with Per and inhibits Clk-mediated activation. Consistent with this function, the mRNA profiles of Clk direct target genes manifest high trough values and low amplitude oscillations in mutant flies. Because rhythmicity fails to persist in DD and there is little or no effect on average mRNA levels in the 5073 strain, one of the insertion lines, transcriptional oscillation amplitude appears 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 cwo homologs (Dec1 and Dec2) in mammals suggests that a similar feedback mechanism exists in mammals (Kadener, 2007).

This study used a genome-wide approach to identify candidate Clk targets from fly heads. Intriguingly, a significant fraction of these genes are nonoscillating. Because the S2 cell assays predict that most of these genes are probably bona fide Clk targets, they may reflect a noncircadian role of Clk. Accordingly, a recent study reported that Clk expression is not restricted to circadian neurons in the fly brain (Houl, 2006). In contrast, cwo mRNA cycles and is expressed in circadian neurons (see also Matsumoto, 2007). Moreover, the cwo mRNA profile is similar to that of the other core clock components, since the gene is activated by Clk-Cyc and repressed by Per. It is suggested that cyclical transcription of cwo probably contributes to circadian changes in the level of Cwo similar to other direct Clk-Cyc targets (Kadener, 2007).

The two cwo insertion strains have no detectable cwo mRNA. Both also have long periods, which fail to persist after 4-5 d in DD. The penetrance of these two phenotypes, however, is not identical: 100% of flies have long periods, whereas 50%-75% are arrhythmic after 4 d in DD. This suggests that these two phenomena are separable and that the long periods are not due to the weak rhythms. The circadian phenotype is slightly more severe in the 4027 strain but is accompanied by a high mortality of flies. In contrast, 5073 homozygous flies and 5073/4027 trans-heterozygous flies show no life-span effect and have comparable phenotypes; that is, long rhythms and 75% arrhythmic flies after 4 d in DD. This indicates that both rhythm features are determined by the absence of cwo expression (Kadener, 2007).

The slow clock is not only manifest by a period phenotype in DD but also by a late activity phase in LD. More specifically, 5073/5073 flies have delayed anticipation of the lights-on transition. This is consistent with cwo acting in the pdf-expressing neurons, since these cells are both responsible for the morning anticipation in LD and period determination in DD. The parsimonious interpretation is preferred that the delayed phase is caused by a slow central molecular oscillator rather than an output defect. More support for this hypothesis comes from the delayed mRNA profiles as well as the delayed phase-response curve (PRC). Although aberrant PRCs often reflect defects in light perception, it has been suggested that they can also reflect a fast or slow central oscillator. In this view, the wider PRC delay zone reflects a slower clock and in particular the broader transcriptional peak. Taken together with the 5073 mRNA curves, Cwo may preferentially function to repress transcription at the end of each cycle. In contrast, the more potent advance zone of the 5073 PRC may reflect an underlying weaker circadian oscillator (Kadener, 2007).

Expression of a cwo transgene in tim-expressing cells restored a 24-h period to the mutant genotype. In contrast, cwo overexpression using the pdf-gal4 as well as the tim-gal4 driver had no effect on the period of an otherwise wild-type strain. This adds to the evidence that the long period phenotype is due to the absence of functional cwo. Although the rescue also improves the rhythm strength of the cwo-deficient host strain, it is not as strong as that of wild-type flies. Moreover, cwo overexpression combined with heterozygosity for the 5073 or 4027 chromosomes also gives rise to weak rhythms. It is suspected that rhythm strength is sensitive to the levels and timing of cwo expression. The UAS transgene lacks the cwo 5' and 3' untranslated (UTR) regions, which are unusually long (2.6 and 1.5 kb, respectively) and probably contribute to post-transcriptional regulation of Cwo expression (Kadener, 2007).

In the current model, high-amplitude oscillations of tim, per, vri, and Pdp1 mRNAs levels are due to cyclical activation and repression of the Clk-Cyc heterodimer. Recent reports from mammals suggest that there is a daily change in chromatin structure, which parallels the Clk-BMAL (Clk-Cyc equivalent) activation cycle. Moreover, circadian chromatin remodeling has recently been reported in the Neurospora system. How these changes are generated and/or linked to the activation-repression cycle is not known (Kadener, 2007).

However, based on the link between bHLH-O proteins and histone deacetylase recruitment, it is suggested that Cwo helps build a repressive chromatin structure during the end of a cycle not unlike the one observed at mammalian circadian promoters. This explains the higher trough values as well as the long period and delayed mRNA decline in the 5073 strain. Per probably recruits the kinase Doubletime (Dbd) to the Clk-Cyc dimer, resulting in diminished Clk-Cyc affinity for DNA; this should favor Cwo binding to E-boxes and corepressor recruitment. Similarly, Cwo activity may aid Clk-Cyc inactivation by Per-DBT, as observed in the S2 cells experiments. Since closed chromatin structures are necessary for full activation by several transcription factors, this may also help explain the lower mRNA peak of most Clk direct targets in the 5073 strain. This lower mRNA peak could also be an indirect or 'system' effect, since peak direct Clk-target mRNA levels in this strain are comparable (~60%) with the levels observed in other repressor mutant strains, namely, per01 and tim01 (Kadener, 2007).

Although mRNA and transcriptional oscillations were proposed long ago to be essential for circadian clock function, recent evidence strongly indicates that they are dispensable in cyanobacteria. Consistent with this notion, there is evidence in the fly system that some rhythmicity persists without per and tim transcriptional cycling. Importantly, behavioral rhythms and probably other Clk direct target genes still undergo oscillations in these strains. It is suggested that the Cwo feedback system contributes to this residual rhythmicity (Kadener, 2007).

As shown in this study, the absence of Cwo has two effects on the mRNA profiles of tim, per, and vri: late repression and low-amplitude oscillations. It is proposed that the former is responsible for the phase change in LD and period change in DD, whereas the latter causes the weak rhythmicity phenotype. Because mRNA oscillation amplitude is affected with little or no effect on average mRNA levels in the 5073 strain, transcriptional regulation appears essential for persistent DD rhythms, which fail after several days in the cwo mutant genotypes. In this view, the weak mRNA amplitude is interpreted to be the cause of the weak rhythms. This adds to the evidence supporting the direct involvement of transcriptional oscillations in the timekeeping process. It is noted that the possibility cannot be ruled out that the weak rhythmicity is a consequence of an additional role of cwo in the output pathway. Although there are no comparable genetic results in mammalian systems, the similar expression profiles as well as the conservation of Dec1 and Dec2 with cwo suggest that a comparable feedback mechanism with behavioral effects exists in mammals (Kadener, 2007).