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Current Biology 17, 1–8, June 19, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.05.039 Report clockwork orange Encodes a Transcriptional Repressor Important for Circadian-Clock Amplitude in Drosophila Chunghun Lim, 1,2,3 Brian Y. Chung, 1,3 Jena L. Pitman, 1 Jermaine J. McGill, 1 Suraj Pradhan, 1 Jongbin Lee, 2 Kevin P. Keegan, 1 Joonho Choe, 2, * and Ravi Allada 1, * 1 Department of Neurobiology and Physiology Northwestern University Evanston, Illinois 60208 2 Department of Biological Sciences Korea Advanced Institute of Science and Technology Daejeon 305-701 Republic of Korea Summary 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 do- main protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function [1]. Microarray studies have identified numerous rhythmi- cally expressed transcripts [2–7], some of which are potential direct CLK targets [7]. 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. Results and Discussion Only two (out of five) microarray studies had initially identified CG17100 as a rhythmically expressed gene [4, 5]. To test whether CG17100 exhibits robust rhythms, we used real-time quantitative RT-PCR and found signif- icant rhythms in both 12 hr light/12 hr dark (LD) and con- stant dark (DD) conditions (Figures 1A and 1B). We also assayed CG17100 in Clk Jrk mutants and found that cwo levels are at trough levels, suggesting that CG17100 is a CLK-activated gene (Figure 1C). We identified a re- markable 20 CLK target CACGTG E box sequences in the 5 0 region and in the large first intron, suggesting direct CLK activation (Figure 1D). Given its potential clock function and the presence of an Orange domain, commonly found in basic helix-loop helix (bHLH) repres- sors [8], we dubbed it clockwork orange [9]. We then examined mutants containing transposon insertions—cwo e04207 (cwo e ) and cwo f05073 —in the first cwo intron (Figure 1D). To determine whether these in- sertions disrupt cwo, we performed qRT-PCR by using primers spanning this 7 kb intron. Amplification in homo- zygous mutants was reduced to w10% of wild-type levels (p < 0.001; Figure 1E). The only amplicon detected in the mutants was of wild-type size, although we probably failed to detect intron-containing tran- scripts because of the large size of the potential ampli- fied product (>7 kb). Assaying of levels of downstream exon 3 suggests that cwo e -containing transcript desta- bilizes the unspliced and/or misspliced transcript, resulting in reduced levels (Figure 1F). In cwo f , reduced apparent transcript amplification across intron 1 (Fig- ure 1E) probably reflects inefficient amplification of the large unspliced and/or misspliced product. Nonethe- less, because exon 1 contains the putative initiating ATG (Figure 1D), disruption of splicing between exon 1 and 2 in both cwo mutants would have a dramatic consequence on protein function. We then tested trans-heterozygous mutants (cwo e / cwo f ) as well as homozygous cwo mutants (cwo e / cwo e , cwo f /cwo f ) and observed dramatic reductions in the strength of behavioral circadian rhythms in DD (Fig- ures S1A–S1D and Table S1 in the Supplemental Data available with this article online; p < 0.001). Those flies that demonstrated detectable rhythms often had length- ened circadian periods with reduced strength (Table S1). Although rhythmicity is evident immediately upon transfer of flies to DD, rhythms dampen in DD (Figure 2C, Figure S1). This phenotype is recessive, and excision of either insertion can substantially improve mutant rhyth- micity and period (w24 hr; Table S1, Figure 2A, Fig- ure S1E; p < 0.05). We also performed complementation testing with flies heterozygous for a deletion that re- moves cwo, Df(3R)ED5495. Flies trans-heterozygous for cwo e and cwo f with Df(3R)ED5495 have very poor rhythms, indicating a failure to complement (Table S1, Figures 2A–2C, Figure S1F; p < 0.001). These data reveal a critical role for cwo in rhythm amplitude and addition- ally in setting period length. Under light-dark conditions, wild-type flies display a morning peak around the time of lights-on and an evening peak around the time of lights-off. Flies increase their activity in anticipation of these transitions, reflect- ing circadian-clock function. Quantitative analysis of morning anticipation indicates a reduction in the degree of anticipation—i.e., the magnitude of the activity increase preceding lights-on, in cwo e /Df(3R)ED5495 relative to heterozygous cwo e /+ or Df(3R)ED5495/+ con- trol flies (p < 0.05)—consistent with a long-period clock or other defect in LD clock function (Figures 2D–2F). To independently confirm the cwo phenotype, we also expressed a dsRNA targeting cwo in transgenic flies (Figure 1D). Expression using the circadian timGAL4 driver, which is expressed in all w100 pacemaker neu- rons controlling circadian behavior [10], also resulted *Correspondence: [email protected] (J.C.), r-allada@northwestern. edu (R.A.) 3 These authors contributed equally to this work. Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa- dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

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Current Biology 17, 1–8, June 19, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.05.039

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

Reportclockwork orange Encodesa Transcriptional Repressor Importantfor Circadian-Clock Amplitude in Drosophila

Chunghun Lim,1,2,3 Brian Y. Chung,1,3 Jena L. Pitman,1

Jermaine J. McGill,1 Suraj Pradhan,1 Jongbin Lee,2

Kevin P. Keegan,1 Joonho Choe,2,* and Ravi Allada1,*1Department of Neurobiology and PhysiologyNorthwestern UniversityEvanston, Illinois 602082Department of Biological SciencesKorea Advanced Institute of Science and TechnologyDaejeon 305-701Republic of Korea

Summary

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 theclock components period (per), timeless (tim), Par do-

main protein 1 (Pdp1), and vrille (vri), which feed backand regulate distinct features of CLK/CYC function [1].

Microarray studies have identified numerous rhythmi-cally expressed transcripts [2–7], some of which are

potential direct CLK targets [7]. Here we demonstratea circadian function for one such target, a bHLH-

Orange repressor, CG17100/CLOCKWORK ORANGE

(CWO). cwo is rhythmically expressed, and levelsare 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 thatCWO acts, in part, by repressing CLK target genes.

We propose that CWO acts as a transcriptional andbehavioral rhythm amplifier.

Results and Discussion

Only two (out of five) microarray studies had initiallyidentified CG17100 as a rhythmically expressed gene[4, 5]. To test whether CG17100 exhibits robust rhythms,we used real-time quantitative RT-PCR and found signif-icant rhythms in both 12 hr light/12 hr dark (LD) and con-stant dark (DD) conditions (Figures 1A and 1B). We alsoassayed CG17100 in ClkJrk mutants and found that cwolevels are at trough levels, suggesting that CG17100 isa CLK-activated gene (Figure 1C). We identified a re-markable 20 CLK target CACGTG E box sequences inthe 50 region and in the large first intron, suggestingdirect CLK activation (Figure 1D). Given its potentialclock function and the presence of an Orange domain,commonly found in basic helix-loop helix (bHLH) repres-sors [8], we dubbed it clockwork orange [9].

*Correspondence: [email protected] (J.C.), r-allada@northwestern.

edu (R.A.)3 These authors contributed equally to this work.

We then examined mutants containing transposoninsertions—cwoe04207 (cwoe) and cwof05073—in the firstcwo intron (Figure 1D). To determine whether these in-sertions disrupt cwo, we performed qRT-PCR by usingprimers spanning this 7 kb intron. Amplification in homo-zygous mutants was reduced to w10% of wild-typelevels (p < 0.001; Figure 1E). The only amplicondetected in the mutants was of wild-type size, althoughwe probably failed to detect intron-containing tran-scripts because of the large size of the potential ampli-fied product (>7 kb). Assaying of levels of downstreamexon 3 suggests that cwoe-containing transcript desta-bilizes the unspliced and/or misspliced transcript,resulting in reduced levels (Figure 1F). In cwof, reducedapparent transcript amplification across intron 1 (Fig-ure 1E) probably reflects inefficient amplification of thelarge unspliced and/or misspliced product. Nonethe-less, because exon 1 contains the putative initiatingATG (Figure 1D), disruption of splicing between exon1 and 2 in both cwo mutants would have a dramaticconsequence on protein function.

We then tested trans-heterozygous mutants (cwoe/cwof) as well as homozygous cwo mutants (cwoe/cwoe, cwof/cwof) and observed dramatic reductions inthe strength of behavioral circadian rhythms in DD (Fig-ures S1A–S1D and Table S1 in the Supplemental Dataavailable with this article online; p < 0.001). Those fliesthat demonstrated detectable rhythms often had length-ened circadian periods with reduced strength (TableS1). Although rhythmicity is evident immediately upontransfer of flies to DD, rhythms dampen in DD (Figure 2C,Figure S1). This phenotype is recessive, and excision ofeither insertion can substantially improve mutant rhyth-micity and period (w24 hr; Table S1, Figure 2A, Fig-ure S1E; p < 0.05). We also performed complementationtesting with flies heterozygous for a deletion that re-moves cwo, Df(3R)ED5495. Flies trans-heterozygousfor cwoe and cwof with Df(3R)ED5495 have very poorrhythms, indicating a failure to complement (Table S1,Figures 2A–2C, Figure S1F; p < 0.001). These data reveala critical role for cwo in rhythm amplitude and addition-ally in setting period length.

Under light-dark conditions, wild-type flies displaya morning peak around the time of lights-on and anevening peak around the time of lights-off. Flies increasetheir activity in anticipation of these transitions, reflect-ing circadian-clock function. Quantitative analysis ofmorning anticipation indicates a reduction in the degreeof anticipation—i.e., the magnitude of the activityincrease preceding lights-on, in cwoe/Df(3R)ED5495relative to heterozygous cwoe/+ or Df(3R)ED5495/+ con-trol flies (p < 0.05)—consistent with a long-period clockor other defect in LD clock function (Figures 2D–2F).

To independently confirm the cwo phenotype, we alsoexpressed a dsRNA targeting cwo in transgenic flies(Figure 1D). Expression using the circadian timGAL4driver, which is expressed in all w100 pacemaker neu-rons controlling circadian behavior [10], also resulted

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Figure 1. CG17100/clockwork orange Transcript Rhythms in Drosophila Heads with Real-Time PCR

(A and B) Quantitative real-time RT-PCR (A and B) experiments performed in light-dark (LD; [A]) and dark-dark (DD; [B]) conditions. The x axis

indicates either zeitgeber time (ZT; [A]), where ZT0 is lights-on, or circadian time (CT; [B]). For real-time experiments (A and B), relative transcript

levels have been normalized with the peak level (ZT or CT13) set to 100.

(C) Quantitative real-time RT-PCR analysis of cwo transcript levels in wild-type (+/+) and ClkJrk (Clk[Jrk]) mutants at ZT1 and ZT13. Relative tran-

script levels have been normalized with the wild-type peak (ZT13) set to 100.

(D) Full-length transcript profile and domain organization of wild-type cwo. White and gray boxes indicate untranslated and protein-coding

regions, respectively. Positions of the various cwo transposon insertions used are shown as black triangles above the diagram. The genomic

region used for the UAS-cwoRNAi construct is shown above the transcript profile (labeled as RNAi). Arrows over the diagram denote the location

of the primer sets used in real-time PCR experiments. Asterisks indicate the physical location of canonical E box elements (CACGTG) within the

promoter and first intron, as identified by Fly Enhancer (http://genomeenhancer.org/fly). The figure has been drawn to scale, and all units are

provided in kilobases.

(E and F) cwo transcript levels in wild-type (+/+; =100) and homozygous cwo mutants cwoe (e/e) and cwof (f/f). Primer sets spanning either exons

1 and 2 (E) or exon 3 (F), as shown in (D), were used to measure relative transcript levels. n experiments R 3. Error bars represent standard error of

the mean (SEM).

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Figure 2. Circadian and Diurnal Behavior of

cwo Mutants under Constant-Darkness and

Light-Dark Conditions

(A–C) Behavior under light-dark and con-

stant-darkness conditions of cwoe/+ (A)

(n = 80), Df(3R)5495/+ (B) (n = 89), and cwoe/

Df(3R)5495 (C) (n = 43). White and black

boxes indicate light and dark periods. Gray

boxes indicate subjective day in constant

darkness.

(D–F) Normalized activity profiles during

diurnal conditions of cwoe/+ (D) (n = 77),

Df(3R)5495/+ (E) (n = 79), and cwoe/

Df(3R)5495 (F) (n = 35). Light and dark bars

indicate activity during the light and dark

phase, respectively. n experiments = 2–4. Nu-

merical values indicate measures of morning

anticipation. Error bars represent SEM.

in period-lengthening phenotypes (Table S2; p < 0.001).In contrast, cwo overexpression by timGAL4 resultedin only a modest reduction of rhythmic power whencompared to the timGAL4/+ control (Table S2). TheRNAi phenotypes are due to specific knockdown ofcwo because RNAi directed against GFP (GFPRNAi)does not result in detectable phenotypes (data notshown), the cwoRNAi period phenotype can be partiallyrescued by wild-type cwo (p < 0.001), and cwoRNAi spe-cifically reduces cwo, but not cyc—a bHLH family mem-ber—transcript levels (Figure S2). Period effects appearto be mediated by a core set of PIGMENT DISPERSINGFACTOR (PDF)-expressing ventral lateral pacemakerneurons (LNv) because the pdfGAL4 driver leads tolengthened periods in combination with UAScwoRNAi

#44 (p < 0.001) [11]. pdf-promoter-driven GAL80 [12],a GAL4 inhibitor, can block the period-lengtheningeffects seen with timGAL4 (p < 0.001). These data areconsistent with reports that cwo may be expressedspecifically in pacemaker neurons [13]. We also ana-lyzed the expression of a GAL4 enhancer trap, c632a[14], inserted just upstream of the cwo transcriptionstart site (Figure 1D) and find expression in the PDF+LNv (Figure S3).

We next assayed whole-head transcriptional oscilla-tions of three CLK target genes, vrille (vri), Pdp13, andperiod (per; see Figure S4), over the first day of constantdarkness in cwo mutants (Figure 3A). These whole-headmolecular rhythms largely reflect clock function in theeye as opposed to the w100 brain neurons that drivebehavior. In different cwo mutants, we observed ele-vated vri transcript levels at trough times CT1 (cwoe/cwof, cwof/cwof only) and CT5 (all cwo mutants tested,

p < 0.05). In all cwo mutants, we also found reducedtranscript levels at the peak CT13 (p < 0.05). Weobserved similar results when examining another CLKtarget, Pdp13. Pdp13 levels exhibited increased troughlevels at CT1 and CT5 in cwo mutants (Figure 3B),consistent with a CWO role as a repressor of CLK-activated transcripts.

per mRNA (Figure 3C) and pre-mRNA levels(Figure S5A) were also altered but with reduced peaklevels at CT13 in cwo mutants (p < 0.05). Clk expressionin cwo mutants is comparable to wild-type levels, indi-cating that reduced peak per levels cannot be explainedby reduced Clk expression (Figure S5B). cwo mutanteffects were not evident by DD day 4, although oscilla-tions were also not detectable because of damping ofeye clocks (data not shown). The finding of transcriptphenotypes on DD day 1 when behavioral phenotypesare subtle suggest that eye clocks may be more sensi-tive to cwo loss than behaviorally relevant pacemakerneurons. Importantly, two CLK target genes, vri andPdp13, show increased transcript levels at trough timesin cwo mutants.

cwo encodes for bHLH-Orange (bHLH-O) proteins thatare often DNA-binding transcriptional repressors [8].Interestingly, two proteins implicated in control of mam-malian circadian rhythms, Dec1 and Dec2, are alsobHLH-O proteins that can repress mouse Clock action[15]. However, genetic inactivation of Dec1 (also knownas Stra13) does not have a core clock phenotype [16].Definitive tests of the in vivo function of Dec1 and Dec2will require the analysis of double-knockout animals.

To determine whether CWO can repress CLK function,we cotransfected cwo and Clk into Drosophila S2 cells.

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cwo represses CLK activation of several clock-genepromoters (Figure 4A; p < 0.005). CWO does not directlyinteract with CLK, nor does it significantly affect CLKlevels (data not shown). Interestingly, cwo transfectionalone also reduced baseline activity of CLK target pro-moters (Figure 4B; p < 0.05). Importantly, CWO doesnot repress (or only weakly represses) promoters thatare not CLK activated: Clk itself and the heterologousthymidine kinase (TK) promoter (Figure 4B). CWO alsoselectively represses a CACGTG E box-containingpromoter but not other artificial promoters (Figure S6;p < 0.005). CWO repression depends on intact CLKtarget CACGTG sequences (Figure 4C; p < 0.005). Toindependently test whether CWO acts through E boxes,we expressed a form of CWO fused to the VP16 tran-scriptional-activation domain. This CWO-VP16 fusion

Figure 3. Altered Rhythmic Expression of vri, Pdp13, and per in cwo

Mutants

Quantitative real-time RT-PCR analysis of vri (A), Pdp13 (B), and per

(C) expression during the first day of DD. The x axis indicates circa-

dian time. Wild-type (+/+) levels at CT 13 (peak) are set to 100 and

indicated as a closed line. cwo mutants cwoe/cwof (e/f), cwof/

Df(3R)ED5495 (f/Df), and cwof/cwof (f/f) are indicated as dashed

lines. Data for f/f are not shown for vri but are similar to e/f. Statistical

significance (p < 0.05) is indicated with a 1 for comparing +/+ with

e/f, 2 for comparing +/+ with f/Df, and 3 for comparing +/+ with f/f.

n experiments R 3 except f/Df CT5,9,17,21, where n = 2. Error

bars represent SEM.

activates transcription in an E box-dependent manner(p < 0.001). Gel-shift analyses with recombinant CWOindicated specific binding to a CACGTG E box but notmutant E box probes. This binding is partially competedby an unlabeled E box, but not a mutated E box frag-ment, and is super shifted by GST antibodies (Figure 4D).We found similar results with extracts from FLAG-tagged CWO bHLH-domain-transfected 293T cells(Figure S7). These results suggest that CWO specificallybinds to E boxes and represses CLK-activated pro-moters. Importantly, these in vitro results are consistentwith our in vivo data indicating elevated vri and Pdp13

transcript levels in cwo mutants at times of maximumrepression (Figures 3A and 3B).

Here we have demonstrated an in vivo role for CWO inthe Drosophila circadian clock. Our data demonstratereduced morning anticipation, lengthened periods, anddamping rhythms in DD. Given that these alleles maynot be nulls (Figures 1E and 1F), we cannot determinedefinitively whether CWO is essential for clock function.Nonetheless, our data argue strongly for a CWO rolein driving high-amplitude transcriptional oscillations.Indeed, the strength of the observed phenotypes iscomparable to or greater than those of loss-of-functionalleles in the PDP1/VRI feedback loop [17–19]. Mecha-nistic analysis suggests this may be accomplished, inpart, by binding to CLK target E boxes and repressingE box-driven transcription. Two other groups havealso found similar in vivo results for CWO (M. Rosbashand H. Ueda, personal communications).

It is interesting to compare the CWO repressor withthe well-studied transcriptional repressor PER. Bothare rhythmically expressed [20], are CLK activated invivo [21], and in turn repress CLK activation in S2 cells[22], and genetic disruption leads to circadian molecularand behavioral phenotypes in both. Interestingly, bothdisplay differential effects on CLK target genes. Inper01, vri, and Pdp13, transcripts are at wild-type peaklevels consistent with PER’s proposed repressor func-tion [18], whereas the per transcript or transcription is in-termediate between peak and trough [20, 23]. Reducedper transcription has been explained by low Clk levels inper01, but then why do vri and Pdp13 levels remain atpeak levels? In cwo mutants, vri and Pdp13 transcriptsare elevated at trough times, whereas per transcript isreduced only at peak times (Figure 3). One possibleexplanation for the complexity of per regulation is thatfull repression by PER and/or CWO may be required toget subsequent full per activation. Alternatively, CWOand/or PER may activate per transcription under someconditions.

The identification of clockwork orange further empha-sizes the pivotal role of the Clk gene in the circadianclock. CLK appears to directly activate five clock com-ponents, all of which feed back and control Clk gene ac-tivity at distinct steps (Figure S3). PER/TIM regulateCLK/CYC DNA binding [24, 25], PDP1/VRI control Clktranscription [18, 26], whereas CWO is activated byCLK, and feeds back by binding and repressing throughCLK/CYC target sites. Taken together, the multiplicity offeedback controls highlights the central role of Clk, oneconsistent with a master-regulator function [27].

We propose that CWO and CLK are principally in-volved in regulating pacemaker amplitude [28], whereas

clockwork orange, a Novel Drosophila Clock Gene5

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Figure 4. CWO Specifically Represses and Binds Clock Gene Promoters

(A and B) S2 cells were cotransfected with reporter plasmids (1 mg except 0.1 mg for vri-luc) and the increasing amounts (0.25 mg and 1 mg) of

expression vector for FLAG-tagged CWO (AcnF/CWO) in the presence (A) or absence (B) of expression vector for V5-tagged CLK (AcV5/CLK;

10 ng for per-luc, 0.25 ng for tim-luc and Pdp1-luc, 1 ng for vri-luc).

(C) S2 cells were cotransfected with reporter plasmids (1 mg), and the expression vector for FLAG-tagged CWO or VP16-activation-domain-fused

CWO (1 mg). Activation fold was calculated by normalizing values to luciferase activity in the presence of reporter plasmid, which was set to 1,

whereas repression fold was calculated by inversely normalizing them.

For (A–C), n experiments = 3, and standard deviations are depicted by error bars.

(D) Electrophoretic-mobility-shift assay for CWO binding to E boxes (CACGTG). An increasing amount (10- and 50-fold molar excess) of unla-

beled competitors containing wild-type (E box) or a mutant E box (mE box) or 2 mg of anti-GST or anti-FLAG was preincubated with GST-fusion

proteins prior to the addition of labeled probe. C1 indicates shift by GST-CWO bHLH:DNA complex; C2 indicates supershift by

GST-bHLH:DNA:anti-GST antibody complex.

the PER/TIM loop plays a pre-eminent role in dictatingperiod or phase of the rhythms. Interestingly, our CWOresults are similar to those of Clk mutants in both fliesand mice in which reduced-amplitude circadian rhythmsare observed [21, 29, 30]. Mutants in per and tim (time-less) and their phosphorylation regulators [31] can leadto large (>2 hr) period changes while largely sparingrhythmicity. Given their evolutionary conservation, wepredict that genetic inactivation of both Dec1 andDec2 will reveal similar roles in mammals.

Experimental Procedures

Plasmids

Total RNA from adult fly heads was isolated with the TRIzol reagent

(Invitrogen) and reverse-transcribed with the M-MuLV reverse tran-

scriptase according to the manufacturer’s instructions (Roche). The

cwo cDNA was amplified by PCR with the appropriate primer set,

inserted into pAcnF for N-terminally FLAG-tagged expression in

S2 cells, and confirmed by sequencing. It was also inserted into

pAcVP16 for VP16-activation-domain fusion-protein expression in

S2 cells. The cDNA corresponding to the CWO bHLH domain (aa

1–126) was subcloned into pFLAG-CMV2 (Sigma) for N-terminally

FLAG-tagged expression in mammalian cells and pGEX4T-1 (Amer-

sham Biosciences) for glutathione-S-transferase (GST)-fusion-

protei expression in bacteria. The dClk cDNA [24] were similarly

inserted into pAc5/V5-His (Invitrogen) for V5- and His-tagged

expression in S2 cells. The per-luc, tim-luc, dClk-luc, and tk-luc

constructs were described previously [22]. Promoter regions of vri

gene (from 22.8 kb to 48 bp relative to the transcriptional start

site) [17] and Pdp1 gene (from 23.5 kb to 196 bp relative to the tran-

scriptional start site) were amplified from fly genomic DNA, inserted

into pGL3-basic (Promega)—which was modified to remove a puta-

tive binding site of E4BP-4/VRI/PDP1 [18, 32]—and designated

as vri-luc and Pdp1-luc, respectively. The promoter region from

21.0 kb to 196 bp of Pdp1 gene was similarly subcloned and desig-

nated as Pdp1-Ebox-luc. This region contains one canonical E box

element (CACGTG) at 2758 bp upstream from the transcriptional

start site; this E box element was mutated (CCCGGG) in the Pdp1-

mutEbox-luc construct.

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Drosophila Stocks

All flies were reared with standard cornmeal-yeast-agar medium

at 25�C under 12 hr light/12 hr dark (LD) cycles. pdf-GAL4, tim-

GAL4-62, and pdf-GAL80 were described previously [10, 12, 33].

UAS-GFPRNAi [34] was obtained from Bloomington Drosophila Stock

Center. For CWO overexpression in a transgenic fly via the GAL4/

UAS system [35], cwo cDNA was inserted into pUAST that was mod-

ified to express N-terminally FLAG-tagged CWO. The RNAi con-

struct for cwo gene was designed according to the genomic cDNA

hybrid method [36]. cDNA corresponding to the second and third

exons of cwo gene (nt 60–460) and genomic DNA including the

cDNA with the internal and adjacent 30 introns were ligated

together into the pUAST. Transgenic constructs were injected with

pUCHsppD2-3 into w1118 embryos, from which several germline

transformants were established.

Behavioral Analysis

Locomotor activity of individual male flies was measured with

Drosophila Activity Monitors (Trikinetics). Monitoring conditions in-

cluded LD cycles for 2–5 days, followed by constant dark (DD) cycles

for a week. Data were analyzed with ClockLab analysis software

(Actimetrics) with the significance level of the c2 periodogram set

to a = 0.05. Flies with a c2 statistic R10 over the significance line

were scored as rhythmic. Results from at least two independent

experiments were averaged. Normalized activity plots for LD and

DD were generated by normalizing the average activity of each

individual fly to 1. Flies with little or no activity over the final day of

the analysis, or throughout the entire analysis, were considered

potentially sick and removed. To calculate values of the morning-

anticipation, we determined the largest 2 hr increase in normalized

average activity of each genotype over the last 6 hr of the dark

phase. For all genotypes, the largest 2 hr increase in activity oc-

curred between zeitgeber time 22 (ZT22) and ZT24. Anticipation-

index values were compared between genotypes and their appropri-

ate controls with one-way ANOVA and Tukey post-hoc tests at a

significance level of p = 0.05.

Cell Culture and Transient Transfection

Drosophila Schneider 2 (S2) cells were maintained in Shields and

Sang M3 insect medium (Sigma) supplemented with 10% fetal

bovine serum and 1% penicillin-streptomycin (Invitrogen). 293T

cells were maintained in Dulbecco’s modified Eagle’s medium

(Invitrogen) supplemented with 10% fetal bovine serum. Cells were

transiently transfected with the standard calcium-precipitation

method. The quantity of total DNA used in transfection was kept

constant by including an appropriate blank vector. For reporter as-

says, cells were harvested at 36 hr after transfection and luciferase

assays were performed according to manufacturer’s instructions

(Promega). In parallel, cell extracts used in the luciferase assays

were immunoblotted with V5 antibody to routinely monitor the con-

stant expression of V5-tagged dCLK protein.

Quantitative RT-PCR

Fly heads were isolated at the indicated time points and total RNA

was isolated with TRIzol reagent (Invitrogen). After the removal of

contaminating genomic DNA by DNase I digestion, it was reverse-

transcribed by the M-MuLV reverse transcriptase and oligo(dT)

primer according to the manufacturer’s instructions (Promega).

Alternatively, total RNA from heads was directly amplified with the

QuantiTect SYBR green RT-PCR kit (QIAGEN). Semiquantitative

PCR was performed under nonsaturating conditions, and PCR prod-

ucts were resolved by 1% agarose gel electrophoresis. The relative

amount of clock gene transcripts was quantified as described previ-

ously [37]. For quantitative RT-PCR experiments, the following

primer sets were used: for rp49, RP49-F 50-CGACGCTTCAAGGGA

CAGTATC-30 and RP49-R 50-TTACGACACCAAACGATCGA-30; for

vri (exon 3), Vri-F 50-TGTTTTTTGCCGCTTCGGTCA-30 and Vri-R 50-

TTACGACACCAAACGATCGA-30; for Pdp1 (exon 1–2), Pdp1RD-F

50-GAACCCAAGTGTAAAGACAATGCG-30 and Pdp1RD-R 50-CTGG

AAATACTGCGACAATGTGG-3; for per (exon 1–2), Per-F 50-CAGC

AGCAGCCTAATCG-30 and Per-R 50-GAGTCGGACACCTTGG-30;

for per pre-mRNA (intron 1), Per[915–936]-F 50- AACCCCTACGATT

TGGATAGCC-30 and Per[1046–1067]-R 50- TGGATAACAGTCGCAT

AACCCG-30; for Clk (exon 8), Clk-F 50-TACTGCGTGAGGATATCG-30

and Clk-R 50-GTTGTTGTTCTGGTTGC-30; for cwo (exon 1–2), Cwo-F

50-CCCTATTGGAACGAGACGAA-30 and Cwo-R 50-GGCATATTCAG

CATCGTCCT-30; and for cwo (exon 3), Cwo(3)-F 50-CCGTATCGAGA

AGACGGAGA-30 and Cwo(3)-R 50-GCATGTGAACGTCGTAGAGG-30.

Circadian-Cell Expression of cwo

The c632a-GAL4 enhancer trap line (Fly-Trap, J. Douglas Armstrong)

was identified as having circadian expression by eGFP as part of an

unrelated behavioral screen. The insertion site of the GAL4 was then

mapped to the promoter region of cwo with inverse PCR (Dr. Eric

Spana, Model Systems Genomics Group, Duke University). Male

files 5–7 days old expressing c632a-GAL4-driven UAS-eGFP were

dissected, fixed by 3.7% paraformaldehyde, and then immuno-

stained with rabbit-anti-pigment dispersing-factor primary antibody

[38] at a 1:10,000 concentration and donkey-anti-rabbit Alexa Fluor

594 secondary antibody (Invitrogen, Molecular Probes) at a 1:500

concentration. Whole-mount brains were mounted in 80% ultra-

pure glycerol (Invitrogen) and were imaged with laser-scanning

confocal microscopy (Nikon).

Electrophoretic-Mobility-Shift Assay

293T cells in 6-well plates were transfected with blank vector or

expression vector for FLAG-tagged CWO bHLH domain (aa 1–

126). Cells were harvested 24 hr after transfection and lysed in hypo-

tonic lysis buffer (10 mM HEPES [pH 7.9] and 1.5 mM MgCl2) at 4�C

for 15 min. After centrifugation, nuclear proteins were prepared by

extracting the pellet with high-salt lysis buffer (20 mM HEPES [pH

7.9], 500 mM KCl, 10% glycerol, 1.5 mM MgCl2, 0.3 mM EDTA,

0.2 mM dithiothreitol, 0.1% Nonidet P-40, and 1 mM phenylmethyl-

sulfonyl fluoride) at 4�C for 30 min and stored at 270�C before

use. Synthetic oligonucleotides containing an E box (50-AAAGCCGC

CGCTCACGTGGCGAACTGCGTG-30) or a mutated E box (mE)

(50-AAAGCCGCCGCTCAGCTGGCGAACTGCGTG-30) were labeled

with g-32P ATP by use of T4 polynucleotide kinase and annealed

to complementary strands. Labeled probe (approximately 200 fmol

per reaction) was incubated with 5 mg of nuclear extract or 50 ng

of bacterially purified GST-fusion proteins in binding buffer (4 mM

HEPES [pH 7.9], 100 mM KCl, 2% glycerol, 0.3 mM MgCl2,

0.06 mM EDTA, 0.04 mM dithiothreitol, 0.02% Nonidet P-40,

0.2 mM phenylmethylsulfonyl fluoride, and 1 mg poly[dI-dC]) at

room temperature for 30 min. For a competition assay, a 10- or

50-fold molar excess of unlabeled probes containing a wild-type

or mutated E box was preincubated with the proteins prior to the

addition of labeled probe. For a supershift assay, 2 mg of FLAG

antibody (Sigma) or GST antibody (Upstate) was preincubated

with the proteins prior to the addition of labeled probe. The reactions

were terminated by electrophoresis on a 6% native polyacrylamide

gel in Tris-borate-EDTA running buffer. The gel was dried and

subjected to autoradiography.

Statistical Analysis

Time-point and genotype experiments were performed with one-

way ANOVA with Tukey post-hoc tests at significance level p = 0.05.

Supplemental Data

Seven figures and two tables are available at http://www.

current-biology.com/cgi/content/full/17/12/---/DC1/.

Acknowledgments

We thank Michael Rosbash and Hiroki Ueda for communication of

results prior to publication; Monica Villar for expert technical sup-

port; Eric Spana (Duke University) for inverse PCR analysis; Terrance

Lee for behavioral-analysis-software improvements; the Blooming-

ton Drosophila Stock Center, Harvard Exelixis Drosophila Stock

Collection, J. Douglas Armstrong (c632a), and Michael Rosbash

for fly strains; and Justin Blau (dClk-luc, tk-luc), Choogon Lee

(dClk cDNA), Charles Weitz (54bp-luc), and Steve Kay (per-luc,

tim-luc) for clones. This work is supported by the following grants:

the Brain Research Center (grant # M103KV010003-06K2201-

00310) of the 21st Century Frontier Research Program funded by

the Ministry of Science and Technology, the Republic of Korea

(J.C.); the Basic Research Promotion Fund (KRF-2005-201-

C00035) of the Korea Research Foundation funded by the Ministry

clockwork orange, a Novel Drosophila Clock Gene7

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

of Education and Human Resources Development, the Republic of

Korea (J.C.); the Protein Network Research Center at Yonsei Univer-

sity funded by the Ministry of Science and Technology, the Republic

of Korea (J.C.); the National Institutes of Health (R.A.); and the March

of Dimes (R.A.).

Received: March 20, 2007

Revised: May 8, 2007

Accepted: May 18, 2007

Published online: June 7, 2007

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Supplemental Data S1

clockwork orange Encodesa Transcriptional Repressor Importantfor Circadian-Clock Amplitude in Drosophila

Chunghun Lim, Brian Y. Chung, Jena L. Pitman,Jermaine J. McGill, Suraj Pradhan, Jongbin Lee,

Kevin P. Keegan, Joonho Choe, and Ravi Allada

Figure S1. Circadian and Diurnal Behavior of cwo Mutants

Normalized activity profiles during diurnal (left) and constant-darkness (right) conditions of +/+ (A), cwoe/cwoe (B), cwof/cwof (C), cwoe/cwof (D),

cwof/+ (E), and cwof/Df(3R)5495 (F). Light and dark bars indicate activity during the light and dark phase. White and black boxes indicate light and

dark periods. Gray boxes indicate subjective day in constant darkness. Values on diurnal activity profiles indicate largest 2 hr increase in nor-

malized average activity of each genotype over the last 6 hr of the dark phase (morning-anticipation index—see Experimental Procedures for

details). n = 24–93 (diurnal), and n = 13–86 (constant darkness). n experiments = 2–5 (diurnal, constant darkness). Error bars (diurnal) represent

SEM.

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

Figure S2. Expression of UAS-cwoRNAi Transgenes by timGAL4

Driver Reduce Expression of Endogenous cwo Gene Transcript

RT-PCR shown from total RNA of adult heads at ZT 3 with primer

sets specific for cwo, cyc, and pdf gene.

Figure S3. The cwo Enhancer-Trap GAL4

Line, c632a, Labels Circadian Pacemaker

Cells

(A) One hemisphere showing c632aGAL4-

driven UASeGFP expression throughout the

brain. Large and small ventral-lateral neurons

(in box) are expanded in (B), (C), and (D). Note

additional expression in pars intercerebralis

(left arrow) and circadian dorsal-lateral neu-

rons (right arrow).

(B) eGFP expression in ventral-lateral neu-

rons.

(C) PDF expression in ventral-lateral neurons.

(D) Merged image showing eGFP and pig-

ment-dispersing factor (PDF) expression in

large and small ventral-lateral neurons.

S2

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

Figure S4. Multiple Modes of Transcriptional

Feedback in the Drosophila Circadian Clock

The CLK/CYC heterodimer binds the E box

(CACGTG) and activates transcription of

per, tim, Pdp1, and vri. PER and TIM repress

CLK/CYC E box binding; PDP1 and VRI acti-

vate and repress, respectively, Clk transcrip-

tion. CLK also activates clockwork orange

(cwo), which competes for the E box to

repress transcription of CLK target genes.

Figure S5. Rhythmic Expression of per pre-mRNA and Clk in cwo Mutants

Quantitative real-time RT-PCR analysis of per pre-mRNA (A) and Clk (B) expression during the first day of DD. The x axis indicates circadian time.

Wild-type (+/+) levels at CT 13 (peak) are set to 100 and indicated with a blue line. cwo mutants cwoe/cwof (e/f) and cwof/cwof (f/f) are indicated by

red and green lines, respectively. Statistical significance (p < 0.05) is indicated with a 1 for comparing +/+ with e/f and 2 for comparing +/+ with f/f.

n experiments R 3. Error bars represent SEM.

S3

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

Figure S7. CLOCKWORK ORANGE Specifi-

cally Binds E Boxes

Nuclear extracts from 293T cells transfected

with blank vector (FLAG-CMV2) or expres-

sion vector for FLAG-tagged CWO bHLH

domain (aa 1–126) were incubated with radio-

labeled oligonucleotides containing a wild-

type or mutant E box at room temperature

for 30 min. Where indicated, a 50-fold molar

excess of unlabeled probes containing wild-

type or mutant E box or 2 mg of FLAG anti-

body was preincubated with the nuclear

extracts prior to the addition of labeled

probe. The reactions were terminated by

electrophoresis on a 6% native polyacryl-

amide gel in Tris-borate-EDTA running buffer.

The gel was dried and subjected to autoradi-

ography.

C1 indicates shift by FLAG-CWO bHLH:DNA

complex; C2 indicates supershift by FLAG-

CWO bHLH:DNA:anti-FLAG antibody com-

plex; one asterisk indicates E box-dependent

shift by a nuclear protein in 293T cells; and

two asterisks indicate E box-independent

shift by a nuclear protein in 293T cells.

Figure S6. Selective CLOCKWORK ORANGE

Repression of a Clock Target Promoter

293T cells in 6-well plates were cotransfected

with reporter plasmids (1 mg) and with in-

creasing amounts of CWO expression vector

(0.25 mg; + and 1 mg; ++). Repression fold was

calculated by inversely normalizing values to

luciferase activity in the presence of reporter

plasmid, which was set to 1. Results repre-

sent the averages of three independent

experiments, and standard deviations are

depicted by error bars. Artificial promoters

contain synthetic tandem repeats of tran-

scription-factor binding sites. These include

ATF/CREB (ATFx3-luc), Sp1 (5xSP1-luc),

E2F (2xE2F-luc), p53 (G13-luc), TCF (OT),

STAT (2xSIE-luc), and mouse CLOCK/

BMAL1 (54bp-luc).

S4

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039

Table S1. Circadian Behavior in clockwork orange Mutants

Genotype Period 6 SEM Power 6 SEMa nb %Rc

+/+ 23.9 6 0.0 101.5 6 5.0 86 97

cwoe/+ 23.9 6 0.0 121.6 6 5.7 77 100

cwof/+ 24.0 6 0.1 98.3 6 5.4 75 97

cwoe/cwoe 26.1 6 0.4 11.5 6 3.6 13 38

cwof/cwof 25.3 6 0.4 24.7 6 11.2 18 33

cwoe/cwof 26.4 6 0.1 24.3 6 3.3 50 62

Df(3R)5495/+ 24.0 6 0.1 77.6 6 5.7 79 89

cwoe/Df(3R)5495 26.5 6 0.1 8.8 6 2.0 35 26

cwof/Df(3R)5495 26.2 6 0.2 23.0 6 3.6 35 69

cwoe excision #1 24.2 6 0.1 82.1 6 10.7 29 83

cwoe excision #2 24.3 6 0.1 65.9 6 9.8 15 80

cwoe excision #3 24.5 6 0.1 94.5 6 14.4 17 94

cwoe excision #4 24.4 6 0.1 92.0 6 8.6 34 91

cwoe excision #5 24.2 6 0.1 72.8 6 9.5 26 92

cwof excision #1 24.3 6 0.1 109.3 6 11.3 24 100

cwof excision #2 24.0 6 0.1 61.1 6 6.3 55 78

cwof excision #3 24.1 6 0.1 74.7 6 9.9 25 84

a Power is a measure of rhythmic strength.b n indicates number of flies analyzed.c %R indicates percent flies with detectable rhythmicity.

Table S2. Circadian Behavior in clockwork orange RNAi Flies

Genotype Period 6 SEM Power 6 SEMa nb %Rc

pdf-GAL4/+ 23.7 6 0.0 116.1 6 5.0 30 100

tim-GAL4-62/+ 24.2 6 0.1 47.2 6 5.6 32 84

UAS-cwo #2-4 /+ 23.6 6 0.1 101.1 6 8.4 28 100

tim-GAL4-62/UAS-cwo #2-4 23.6 6 0.1 42.8 6 7.6 14 93

UAS-cwoRNAi #44 23.8 6 0.1 36.4 6 4.8 31 81

pdf-GAL4/+; UAS-cwoRNAi #44 25.2 6 0.1 71.9 6 7.2 26 96

tim-GAL4-62/+; UAS-cwoRNAi #44 26.9 6 0.1 30.7 6 3.1 100 70

tim-GAL4-62/pdf-GAL80; UAS-cwoRNAi #44 24.6 6 0.1 26.8 6 4.0 41 66

tim-GAL4-62/UAS-cwo #2-4; UAS-cwoRNAi #44 25.8 6 0.1 39.4 6 3.9 61 82

UAS-cwo #3-3/+ 23.5 6 0.1 72.7 6 10.8 12 92

UAS-cwo #3-3 23.1 6 0.1 41.0 6 7.2 21 81

pdf-GAL4; UAS-cwo #3-3 24.2 6 0.1 80.2 6 12.0 22 86

tim-GAL4-62/+; UAS-cwo #3-3/+ 23.6 6 0.1 49.2 6 7.5 29 86

tim-GAL4-62/+; UAS-cwo #3-3 23.9 6 0.1 16.5 6 4.4 21 43

tim-GAL4-62/pdf-GAL80; UAS-cwo #3-3 23.9 6 0.1 37.7 6 7.1 19 74

UAS-cwo #3-7/+ 23.5 6 0.1 103.4 6 10.3 11 100

UAS-cwo #3-7 23.5 6 0.1 81.8 6 7.2 24 100

pdf-GAL4; UAS-cwo #3-7 24.1 6 0.0 64.6 6 5.8 56 91

tim-GAL4-62/+; UAS-cwo #3-7/+ 23.7 6 0.1 55.0 6 6.2 29 93

tim-GAL4-62/+; UAS-cwo #3-7 24.3 6 0.1 18.4 6 3.8 20 50

tim-GAL4-62/pdf-GAL80; UAS-cwo #3-7 23.9 6 0.1 30.1 6 7.3 16 75

a Power is a measure of rhythmic strength.b n indicates number of flies analyzed.c %R indicates percent flies with detectable rhythmicity.

S5

Please cite this article in press as: Lim et al., clockwork orange Encodes a Transcriptional Repressor Important for Circa-dian-Clock Amplitude in Drosophila, Current Biology (2007), doi:10.1016/j.cub.2007.05.039