InteractiveFly: GeneBrief

clockwork orange: Biological Overview | References

Gene name - clockwork orange

Synonyms - CG17100

Cytological map position-86B3-86B3

Function - transcription factor

Keywords - photoperiod response

Symbol - cwo

FlyBase ID: FBgn0259938

Genetic map position - 3R: 6,213,985..6,226,072 [+]

Classification - Helix-loop-helix domain, orange domain

Cellular location - nuclear

NCBI link: EntrezGene

cwo orthologs: Biolitmine
Recent literature
Zhou, J., Yu, W. and Hardin, P. E. (2016). CLOCKWORK ORANGE enhances PERIOD mediated rhythms in transcriptional repression by antagonizing E-box binding by CLOCK-CYCLE. PLoS Genet 12: e1006430. PubMed ID: 27814361
The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. This study shows that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.
Rivas, G. B. S., Zhou, J., Merlin, C. and Hardin, P. E. (2021). CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN. Curr Biol. PubMed ID: 34331859
The Drosophila circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, CWO target genes were identified that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.
Li, Q., Wang, L., Cao, Y., Wang, X., Tang, C. and Zheng, L. (2023). Stable Expression of dmiR-283 in the Brain Promises Positive Effects in Endurance Exercise on Sleep-Wake Behavior in Aging Drosophila. Int J Mol Sci 24(4). PubMed ID: 36835595
Sleep-wake stability is imbalanced with natural aging, and microRNAs (miRNAs) play important roles in cell proliferation, apoptosis, and aging; however, the biological functions of miRNAs in regulating aging-related sleep-wake behavior remain unexplored. This study varied the expression pattern of dmiR-283 in Drosophila and the result showed that the aging decline in sleep-wake behavior was caused by the accumulation of brain dmiR-283 expression, whereas the core clock genes cwo and Notch signaling pathway might be suppressed, which regulate the aging process. In addition, to identify exercise intervention programs of Drosophila that promote healthy aging, mir-283SP/+ (mir-23SP referes to mir-283sponge) and Pdf > mir-283SP flies were driven to perform endurance exercise for a duration of 3 weeks starting at 10 and 30 days, respectively. The results showed that exercise starting in youth leads to an enhanced amplitude of sleep-wake rhythms, stable periods, increased activity frequency upon awakening, and the suppression of aging brain dmiR-283 expression in mir-283SP/+ middle-aged flies. Conversely, exercise performed when the brain dmiR-283 reached a certain accumulation level showed ineffective or negative effects. In conclusion, the accumulation of dmiR-283 expression in the brain induced an age-dependent decline in sleep-wake behavior. Endurance exercise commencing in youth counteracts the increase in dmiR-283 in the aging brain, which ameliorates the deterioration of sleep-wake behavior during aging.
van den Berg, L., Kokki, K., Wowro, S. J., Petricek, K. M., Deniz, O., Stegmann, C. A., Robciuc, M., Teesalu, M., Melvin, R. G., Nieminen, A. I., Schupp, M. and Hietakangas, V. (2023). Sugar-responsive inhibition of Myc-dependent ribosome biogenesis by Clockwork orange. Cell Rep 42(7): 112739. PubMed ID: 37405919
The ability to feed on a sugar-containing diet depends on a gene regulatory network controlled by the intracellular sugar sensor Mondo/ChREBP-Mlx, which remains insufficiently characterized. This study presents a genome-wide temporal clustering of sugar-responsive gene expression in Drosophila larvae. Gene expression programs responding to sugar feeding were identified, including downregulation of ribosome biogenesis genes, known targets of Myc. Clockwork orange (CWO), a component of the circadian clock, is found to be a mediator of this repressive response and to be necessary for survival on a high-sugar diet. CWO expression is directly activated by Mondo-Mlx, and it counteracts Myc through repression of its gene expression and through binding to overlapping genomic regions. CWO mouse ortholog BHLHE41 has a conserved role in repressing ribosome biogenesis genes in primary hepatocytes. Collectively, these data uncover a cross-talk between conserved gene regulatory circuits balancing the activities of anabolic pathways to maintain homeostasis during sugar feeding.

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).

A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock

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, genome-wide functional screening using an RNA interference (RNAi) system was applied in vivo. This study reports 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, it is proposed 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 (Matsumoto, 2007).

The identified clock gene cwo is a transcriptional repressor and exhibits oscillatory expression under LD and DD, although the amplitude of this cwo mRNA oscillation in whole fly head is relatively modest (approximately twofold) compared with other transcriptional repressors such as per, tim, and vri. This may reflect a wider distribution of Cwo protein within fly heads compared with Per protein since Cwo is detected in additional cells within the fly brain. Alternatively, Cwo function within the circadian oscillator may not require rhythms in the levels of Cwo protein, precluding the need for high-amplitude cwo mRNA cycling. Importantly, cwo expression levels consistently reflect the levels of CLK-Cyc activity, as shown in per01 and ClkJrk mutants. Hence, the antagonistic competition between the cyclic CLK-Cyc activity and the adjustable threshold imposed by Cwo protein can be expected to keep E-box-mediated transcription within the controllable range of its activity, therefore rendering it more robust in generating high-amplitude oscillation (Matsumoto, 2007).

Consistent with this hypothesis, impaired activity of Cwo leads to an elevated trough of per, tim, vri, and Pdp1mRNA at ZT3 in cwo RNAi transgenic flies compared with those of wild-type flies. Since the fly circadian clock consists of complexly integrated negative and positive feedback loops, this possible direct effect of impaired Cwo activity may propagate and induce the indirect effects in the fly clock system. The observed lower peak level of per, tim, vri, and Pdp1 RNA expression in cwo RNAi transgenic flies might reflect the indirect effects of impaired Cwo activity. It is noteworthy that similar indirect effects on the oscillatory expression of per were observed in the hypomorphic timrit mutant, which exhibited a decreased activity of transcriptional repression by Per-TIM as well as the decreased peak level of per expression. Interestingly, the circadian phenotypes of this mutant are very similar to cwo knockdown flies showing ~26 h in period (Matsumoto, 2007).

Isolation of cwo as a new clock component and subsequent identification of a new negative feedback loop in this study revealed that transcriptional regulation through E-boxes is more complex than previously thought. The indirect autoregulatory negative feedback mechanism by Per and Tim through E-boxes, which is one of the key factors in circadian oscillation, has been extensively studied. This involves formation of a heterodimer between Per and Tim, both of which lack a DNA-binding domain, that binds Clk-Cyc to inhibit the DNA-binding activity of Clk-Cyc. The direct suppression mechanism through the E-box, however, had yet to be elucidated prior to this study. It has long been a mystery as to how the constitutive expression of per and tim in per01; tim01 double mutants can rescue rhythmicity at the behavioral and molecular levels. The finding that cwo, one of the Clk-Cyc target genes, can suppress the expression of a group of clock genes through binding to E-boxes suggests a new pathway for the negative feedback regulation in Drosophila clock. This negative regulation directly affects E-box-mediated transcription, contributing to sustaining a high-amplitude circadian expression, and may compensate the molecular rhythmicity even when a functional disorder occurs in other feedback loops (Matsumoto, 2007).

clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer Clk/Cyc transcriptionally activates the clock components per, 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. This study demonstrates a circadian function for one such target, a bHLH-Orange repressor, Clockwork Orange. 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. It is proposed that Cwo acts as a transcriptional and behavioral rhythm amplifier (Lim, 2007).

This study demonstrates an in vivo role for Cwo in the Drosophila circadian clock. The data demonstrate reduced morning anticipation, lengthened periods, and damping rhythms in DD. Given that these alleles may not be nulls, it cannot be determined definitively whether Cwo is essential for clock function. Nonetheless, the data argue strongly for a Cwo role in driving high-amplitude transcriptional oscillations. Indeed, the strength of the observed phenotypes is comparable to or greater than those of loss-of-function alleles in the PDP1/VRI feedback loop. Mechanistic analysis suggests this may be accomplished, in part, by binding to CLK target E boxes and repressing E box-driven transcription (Lim, 2007).

It is interesting to compare the Cwo repressor with the well-studied transcriptional repressor Per. Both are rhythmically expressed, are Clk activated in vivo, and in turn repress Clk activation in S2 cells, and genetic disruption leads to circadian molecular and behavioral phenotypes in both. Interestingly, both display differential effects on Clk target genes. In per01, vri, and Pdp1ɛ, transcripts are at wild-type peak levels consistent with Per's proposed repressor function, whereas the per transcript or transcription is intermediate between peak and trough. Reduced per transcription has been explained by low Clk levels in per01, but then why do vri and Pdp1ɛ levels remain at peak levels? In cwo mutants, vri and Pdp1ɛ transcripts are elevated at trough times, whereas per transcript is reduced only at peak times. One possible explanation for the complexity of per regulation is that full repression by Per and/or Cwo may be required to get subsequent full per activation. Alternatively, Cwo and/or Per may activate per transcription under some conditions (Lim, 2007).

The identification of clockwork orange further emphasizes the pivotal role of the Clk gene in the circadian clock. Clk appears to directly activate five clock components, all of which feed back and control Clk gene activity at distinct steps. Per/Tim regulate Clk/Cyc DNA binding, PDP1/VRI control Clk transcription, whereas Cwo is activated by Clk, and feeds back by binding and repressing through Clk/Cyc target sites. Taken together, the multiplicity of feedback controls highlights the central role of Clk, one consistent with a master-regulator function (Lim, 2007).

It is proposed that Cwo and Clk are principally involved in regulating pacemaker amplitude, whereas the Per/Tim loop plays a pre-eminent role in dictating period or phase of the rhythms. Interestingly, these Cwo results are similar to those of Clk mutants in both flies and mice in which reduced-amplitude circadian rhythms are observed. Mutants in per and tim (timeless) and their phosphorylation regulators can lead to large (>2 hr) period changes while largely sparing rhythmicity. Given their evolutionary conservation, it is predicted that genetic inactivation of both Dec1 and Dec2 will reveal similar roles in mammals (Lim, 2007).

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

MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. This study identified microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of Clockwork Orange (Cwo). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian Prothoracicotropic hormone (PTTH) and Clock-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH Ecdysteroid receptor. Thus, this study has found a regulatory cycle involving PTTH, a direct target of Clock, and PTTH-driven miRNA let-7 (Chen, 2014).


Search PubMed for articles about Drosophila Clockwork orange

Belden, W. J., Loros, J. J., and Dunlap, J. C. (2007). Execution of the circadian negative feedback loop in Neurospora requires the ATP-dependent chromatin-remodeling enzyme CLOCKSWITCH. Mol. Cell 25: 587-600. PubMed ID: 17317630

Chen, W., Liu, Z., Li, T., Zhang, R., Xue, Y., Zhong, Y., Bai, W., Zhou, D. and Zhao, Z. (2014). Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle. Nat Commun 5: 5549. PubMed ID: 25417916

Davis, R. L. and Turner, D. L. (2001). Vertebrate hairy and Enhancer of split related proteins: Transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 20: 8342-8357. PubMed ID: 11840327

Hamaguchi, H., et al. (2004). Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system. Biochem. J. 382(Pt 1): 43-50. PubMed ID: 15147242

Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y., and Honma, K. 2002. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature 419: 841-844. PubMed ID: 12397359

Houl, J. H., Yu, W., Dudek, S. M., and Hardin, P .E. (2006). Drosophila CLOCK is constitutively expressed in circadian oscillator and non-oscillator cells. J. Biol. Rhythms 21: 93-103. PubMed ID: 16603674

Kadener, S., Stoleru, D., McDonald, M., Nawathean, P. and Rosbash. M. (2007). Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component. Genes Dev. 21(13): 1675-86. PubMed ID: 17578907

Li, Y., Song, X., Ma, Y., Liu, J., Yang, D. and Yan, B. (2004). DNA binding, but not interaction with Bmal1, is responsible for DEC1-mediated transcription regulation of the circadian gene mPer1. Biochem. J. 382(Pt 3): 895-904. PubMed ID: 15193144

Lim, C., Chung, B. Y., Pitman, J. L., McGill, J. J., Pradhan, S., Lee, J., Keegan, K. P., Choe, J. and Allada, R. (2007). Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila. Curr. Biol. 17(12): 1082-9. PubMed ID: 17555964

Matsumoto, A., et al. (2007). A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock. Genes Dev. 21(13): 1687-700. PubMed ID: 17578908

Ripperger, J.A. and Schibler, U. (2006). Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions. Nat. Genet. 38: 369-374. PubMed ID: 16474407

Sato, F., et al. (2004). Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation. Interaction with BMAL1. Eur. J. Biochem. 271(22): 4409-19. PubMed ID: 15560782

Yu, W., Zheng, H., Houl, J. H., Dauwalder, B., and Hardin, P. E. (2006). Per-dependent rhythms in Clk phosphorylation and E-box binding regulate circadian transcription. Genes Dev. 20: 723-733. PubMed ID: 16543224

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date revised: 18 February 2024

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