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A functional genomics strategy reveals clockwork orange as atranscriptional regulator in the Drosophila circadian clock
Akira Matsumoto, Maki Ukai-Tadenuma, Rikuhiro G. Yamada, Jerry Houl, Kenichiro D. Uno, Takeya
Kasukawa, Brigitte Dauwalder, Taichi Q. Itoh, Kuniaki Takahashi, Ryu Ueda, Paul E. Hardin, Teiichi
Tanimura and Hiroki R. Ueda
Genes & Dev. 2007 21: 1687-1700; originally published online Jun 19, 2007;
Access the most recent version at doi:10.1101/gad.1552207

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A functional genomics strategy reveals
clockwork orange as a transcriptional
regulator in the Drosophila circadian clock
Akira Matsumoto,3,9,11 Maki Ukai-Tadenuma,1,9 Rikuhiro G. Yamada,1,9 Jerry Houl,7,8
Kenichiro D. Uno,2 Takeya Kasukawa,2 Brigitte Dauwalder,8 Taichi Q. Itoh,4 Kuniaki Takahashi,6
Ryu Ueda,6 Paul E. Hardin,7 Teiichi Tanimura,4 and Hiroki R. Ueda1,2,5,10
1

Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe, Hyogo 650-0047, Japan;
Functional Genomics Unit, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe, Hyogo 650-0047, Japan; 3Center for
Research and Advancement in Higher Education, Kyushu University, Ropponmatu, Fukuoka 810-8560, Japan; 4Departmentof Biology, Faculty of Science, Kyushu University, Ropponmatu, Fukuoka 810-8560, Japan; 5Department of Bioscience,
Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; 6Genetic Strains Research Center,
National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan; 7Department of Biology and Center for Research on
Biological Clocks, Texas A&M University, College Station, Texas 77843, USA; 8Department of Biology and Biochemistry,
University of Houston, Houston, Texas 77204, USA
2

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 thesecandidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo.
Here we report the identification of novel clock gene candidates including clockwork orange (cwo), atranscriptional 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, we propose that the competition between cyclic CLK–CYC activity and theadjustable 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.
[Keywords: Functional genomics; circadian clock; Drosophila; in vivo RNAi; genome tiling array;
CHIP-on-chip]
Supplemental material is available at http://www.genesdev.org.
Received March 14, 2007; revised version accepted May 15, 2007.

The core molecular mechanism that mediates circadianoscillator function in Drosophila is thought to be composed of two interlocked transcriptional feedback loops:
a period (per)/timeless (tim) loop as a negative loop, anda Clock (Clk)/cycle (cyc) loop as a positive one (Hardin2005). While the framework of the molecular mechanism of circadian oscillation has been extensively elucidated, we still have not identified all the molecules required for feedback loop function. There have been sev-

9

These authors contributed equally to this work.
Corresponding authors.
10
E-MAIL uedah-tky@umin.ac.jp; FAX 81-78-306-3194.
11
E-MAIL akirarcb@mbox.nc.kyushu-u.ac.jp; FAX 81-92-726-4641.
Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.1552207.

eral approaches to study how gene expression is globally
regulated by the Drosophila clock mechanism using thesubtractive cDNA library screening (Van Gelder et al.
1995; Van Gelder and Krasnow 1996; Rouyer et al. 1997),
differential display of RT–PCR (Blau and Young 1999),
and genome-wide expression trap screening using luciferase reporter insertions (Stempfl et al. 2002). Recently,
high-density oligonucleotide probe arrays have been developed and applied as one of the most comprehensive
methods to evaluate how gene expression is regulated by
the circadian clock on a genome-wide basis (ClaridgeChang et al. 2001; McDonald and Rosbash 2001; Ceriani
et al. 2002; Lin et al. 2002; Ueda et al. 2002).
Although hundreds of clock-controlled genes wereidentified in Drosophila, the functional classification ofthese genes as components of the circadian clock mecha-

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Matsumoto et al.

nism remained for future studies. As a first step, coreclock genes, which encode critical components of feedback loops, must be distinguished from clock-regulated
output genes. An effective way of distinguishing core clock
genes from output genes is to observe the circadian phenotypes of mutants in these genes. Such a mutant screening
strategy has significantly facilitated analysis of the clock
mechanism in Drosophila (Konopka and Benzer 1971;
Sehgal et al. 1994; Allada et al. 1998; Price et al. 1998;
Rutila et al. 1998; Blau and Young 1999). However, there
are some disadvantages with this strategy when we apply it
at a genome-wide level. In particular, mutants are notavailable for many candidate genes, and even if they are,many of these mutants are lethal during development (Blauand Young 1999). In this study, we established a new functional genomic experimental strategy that can overcome
the disadvantages of more traditional mutant screening.
This strategy allowed us to identify additional core clock
gene candidates. Further analyses revealed that CG17100,
one of these candidates, is able to regulate its own expression to form a third feedback loop in the Drosophila
clock that interlocks with the two existing loops. Our
strategy of combining RNA interference (RNAi) screening and chromatin immunoprecipitation (ChIP)-on-chip
assays using genome tiling arrays represents a new functional genomics strategy for analyzing the function andtargets of transcription factors.
Results
Genome-wide tissue-specific knockdown analysis
in vivo reveals cwo as a clock gene candidate
We previously performed a comprehensive analysis ofcircadian gene expression in Drosophila heads by profiling whole-genome microarrays (Ueda et al. 2002). This
involved selecting a total of 200 genes with rhythmic
expression both in light–dark (LD) and constant darkness(DD) conditions (Supplementary Table 1). In order toscreen for core clock genes among these candidates, we
adopted an RNAi approach by using the double-stranded
RNA (dsRNA) of the target gene selectively induced by
the Gal4-UAS binary system (Pili-Floury et al. 2004). We
established a UAS-inversed repeat (UAS-IR) transgenic
strain carrying ∼500-base-pair (bp) fragments of a target
gene in a head-to-head manner into a pUAST-R57 plasmid (Fig. 1A) to express dsRNA for the target gene. The
target site of dsRNAi (500-bp fragment) was selected tominimize the possibility of off-target effects as calculated by dsCheck (http://dscheck.rnai.jp) (Naito et al.
2005). We generally established four to five transgeniclines, each of which carries UAS-IR at a different site of
the genome, then selected two lines that have no apparent insertional mutation. We mated UAS-IR transgenic
flies to the tim(UAS)-gal4 strain (Blau and Young 1999)
as a driver to induce a knockdown of the target gene’s
expression by RNAi in tim-positive cells, including
pacemaker neurons. Any abnormalities in locomotor
rhythms of these knockdown flies would reveal the target gene as an important element in maintaining circadian rhythmicity.

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We first checked whether our knockdown strategy
could be applied to screen for clock genes with circadian
expression, namely, per, tim, vri, and Clk (Hardin 2005).
We established two independent transgenic lines of
UAS-per-IR and recorded locomotor activity rhythm intim(UAS)-gal4/UAS-per-IR trans-heterozygotes (Fig. 1B,left). All flies (n = 28) from both lines showed arrhythmicity under DD, suggesting that our knockdown strategy worked well in the circadian phenotype. As the pergene knockdown was reported to induce a long period
phenotype (Martinek and Young 2000), the effect wasmore severe in our case than in previous ones. We further checked the effect of RNAi-mediated knockdown
on other clock genes (Fig. 1B, left). While 15 out of 25 tim
knockdown flies became arrhythmic, the remaining 10
still showed normal rhythmicity. On the other hand,
RNAi-mediated knockdown of Clk and vri had little orno effect on locomotor activity rhythms since all flies
tested were rhythmic with ∼24-h periods (data notshown). RNAi works with different efficiencies with different genes. Perhaps our vri-IR or Clk-IR strains showed
little decrease in CLK and VRI levels, respectively. Alternatively, compared with perturbations of per and tim,
the fly clock system might be relatively resistant to perturbation of Clk and vri. In fact, moderate decreases in
CLK may have little impact on feedback loop function
since CLK activity is controlled at the post-transcriptional level (Yu et al. 2006), and heterozygous vri mutants show only modest increases of Clk mRNA (Glossop et al. 2003).
We screened an additional 133 genes and isolated fiveas novel core clock gene candidates (Fig. 1B, right; Table1). The deduced functions of the gene products ofthese candidates are very diverse; a transcription factor(CG17100), an ABC transporter (E23), a chromatin remodeling factor (Iswi), and a factor affecting cell proliferation (prod). The function of the one remaining geneproduct (CG5273) remains unknown. Among these candidates, we focused on the CG17100 gene, which has
been renamed clockwork orange (cwo) (Lim et al., 2007),since the locomotor phenotype was the strongest andmost stable among all of the candidates, with one cwo
RNAi transgenic line (cwo-IR2) showing a long period
phenotype (26.01 ± 0.07 h, n = 37) (Fig. 1B). In addition,
three out of 40 flies in this line were arrhythmic (Table
1). Although CG17100 was previously annotated as
sticky chordotonals 1 (stich1), Dubruille et al. (2002)
showed that the stich1 mutation was mapped to jumu, aneighboring gene to CG17100. Thus, we used cwo as a
new gene name for CG17100. To confirm that the CWO
protein level is reduced in the head of the knockdown
flies, an antibody was raised against full-length CWO
protein (see Materials and Methods). Western blot analyses with antisera against CWO protein confirmed thatthe expression of CWO protein reduced to less than onequarter level of wild type (Supplementary Fig. 2A,C). The
other line (cwo-IR3), which carries a UAS-IR insertion at
a different position of the genome from that of cwo-IR2,also showed a long period phenotype (25.61 ± 0.20 h,
n = 8). We also observed a similar circadian phenotype inDownloaded from www.genesdev.org on December 17, 2007 - Published by Cold Spring Harbor Laboratory Press

Genomics reveals the new clock gene cwo

Figure 1. A functional genomics strategy revealed cwo as a clock component. (A) Genome-wide tissue-specific knockdown analysisof clock-controlled genes in Drosophila. UAS-IR transgenic lines to express dsRNA for the target gene under the control of UAS were
established (Pili-Floury et al. 2004). Usually two independent insertion lines are established for one target gene. Each of the UAS-IR
lines was mated to tim(UAS)-gal4 to drive the expression of dsRNA specifically within clock cells. The locomotor activity of RNAitransgenic flies for 133 candidates among 200 clock-controlled genes was recorded in DD. UAS-TATA sequence or UAS sequence(yellow rectangle), SV40 late polyA site (white rectangle), ∼500-bp fragment of a target gene (red arrow), a tim promoter region (green
rectangle), and gal4 gene (pink arrow) are represented. (B) Typical locomotor activity in wild-type, knockdown flies of known clock
genes (left) and five new candidates (right). The names of the knocked-down genes are described at each actogram.

cwo RNAi transgenic flies with pdf-gal4 (Renn et al. 1999)
and gal1118 strains (Malpel et al. 2002) as drivers. As
GAL4 is commonly expressed in the pacemaker neuronsof a fly brain in three different driver strains (Blau andYoung 1999; Renn et al. 1999; Malpel et al. 2002), the target
cells of the circadian phenotype in cwo RNAi transgenic
flies are thought to be the central pacemaker neurons.
cwo is a new clock gene
To exclude the possibility of off-target effects of RNAi,
we also monitored the phenotype of a cwo mutant strain(f05073; Exelixis collection, Harvard University) that has
a transposon insertion in the first cwo intron. CWO ex-

pression was drastically reduced in homozygous f05073
flies (Fig. 2C; Supplementary Fig. 2B,D). Homozygous
f05073 flies had long period rhythms similar to cwo
RNAi transgenic flies, and a substantial portion of themwere arrhythmic (data not shown; see also accompanying paper by Kadener et al. 2007). In addition, two independent strains with overexpression of a dominant-negative type of CWO protein (⌬b-1 and ⌬b-4; see also Materials and Methods for details) also showed abnormal
rhythmicity similar to f05073; that is, a part of them
became arrhythmic (n = 15 in ⌬b-1 and n = 3 in ⌬b-4)
while the remaining showed a longer period (25.16 ± 0.12h, n = 9 in ⌬b-1; 24.86 ± 0.09 h, n = 18 in ⌬b-4) (Table 1;
Supplementary Fig. 1). In both strains, arrhythmicity oc-

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Matsumoto et al.

Table 1. Free-running periods of wild-type, knockdown, andcwo mutant flies

Gal4 driverIR lineWild-type

Period(mean ± SEM)
(h)

NR

NA

23.83 ± 0.07

26

0tim(UAS)-gal4

cwo-IR2
cwo-IR3
E23-IR1
E23-IR2
CG5273-IR1
CG5273-IR2
CG5273-IR3Iswi-IR1
Iswi-IR2
prod-IR1
prod-IR2
prod-IR3

26.01 ± 0.07a
25.61 ± 0.20a
24.73 ± 0.12a
24.81 ± 0.19a
24.30 ± 0.11a
24.52 ± 0.09a
24.93 ± 0.20a
24.48 ± 0.02a
24.86 ± 0.07a
24.19 ± 0.19a
25.48 ± 0.09a
24.25 ± 0.11a

37
8
25
11
13
13
6
30
15
8
6
11

3
0
0
0
0
0
0
2
0
1
0
1

pdf-gal4

cwo-IR2
cwo-IR3
E23-IR1⌬b-1⌬b-4

25.91 ± 0.30a
25.13 ± 0.08a
25.01 ± 0.09a
25.16 ± 0.12a
24.86 ± 0.09a

14
27
15
9
18

26
4
0
15
3

gal1118

cwo-IR3

25.99 ± 0.22a

130(NR) Number of rhythmic flies; (NA) number of arrhythmic
flies.
aSignificantly different from the period of wild-type (t-test,p < 0.05).

curred after 2–4 d in DD. These behavioral experiments
show that the quantitative and qualitative changes in
CWO have clear effects on circadian rhythms. Based onthese results, we conclude that decreased activity of the
CWO protein leads to the abnormalities in the circadianclock.
cwo encodes a basic helix–loop–helix(bHLH)-ORANGE family transcription factor
cwo encodes a candidate transcriptional repressor, with alength of 685 amino acids, belonging to the bHLHORANGE family (Fig. 2A; Davis and Turner 2001). The
CWO protein, however, lacks the tetrapeptide domainthat generally exists in the C terminus of Hey, hairy, orE(spl) subfamilies in the bHLH-ORANGE family (Davisand Turner 2001) and belongs to the Stra13 subfamily.
We found that the CWO protein is not only a structural
ortholog of DEC1 and DEC2 proteins in mammals but
also has a highly conserved sequence with the HES5 protein. DEC1 and DEC2 also lack the tetrapeptide domain(Davis and Turner 2001), are rhythmically expressed, andare capable of negatively regulating transcription ofclock-controlled genes through the E-boxes in mammals(Honma et al. 2002; Grechez-Cassiau et al. 2004). We
were also interested to find that Hes5 has a rhythmic
expression in the suprachiasmatic nucleus (SCN)(Supplementary Fig. 3). Hence, the three structural homologs of CWO protein in mammals can be regarded as

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being associated with the circadian clock, although circadian phenotypes of mutants in these genes have notyet been reported in vivo.
cwo mRNA is rhythmically expressed in the fly head,
and CWO protein is expressed in the oscillator
neurons
Our quantitative RT–PCR (Q-PCR) measurement revealed that temporal expression of cwo rhythmicallychanged in LD and DD in wild-type flies, peaking very
closely in phase with per and tim, which is consistent
with previous genome-wide expression analysis (Fig. 2B;
McDonald and Rosbash 2001; Ceriani et al. 2002; Lin etal. 2002; Ueda et al. 2002). The phase of cwo mRNA
cycling and lack of rhythmic expression in per01 and
ClkJrk mutants (Supplementary Fig. 4) suggest that cwo
is regulated by the same molecular mechanism as per
and tim. Like other CLK–CYC-dependent genes, per andtim are expressed in six groups of canonical brain oscillator neurons: dorsal lateral neurons (LNds), small andlarge ventral lateral neurons (sLNvs and lLNvs, respectively), and dorsal neurons 1, 2, and 3 (DN1, DN2, and
DN3) (Zerr et al. 1990; Hunter-Ensor et al. 1996). Brainsfrom wild-type flies collected at Zeitgeber time (ZT) 21
were coimmunostained with antisera against CWO andthe oscillator neuron marker PER. CWO immunoreactivity was detected in all three groups of DNs (Fig. 2C,
panels 1–3). Since PER is found in nuclei at this time
(Curtin et al. 1995), the overlap in CWO and PER immunostaining indicates that CWO is primarily or completely nuclear (Fig. 2C, panels 3,3m,6,6m). Likewise,CWO immunoreactivity is detected in nuclei fromsLNvs, lLNvs, and LNds (Fig. 2C, panels 4–6). This pattern of CWO immunostaining is consistent with reporter gene expression from an apparent cwo enhancer
trap line, R32 (Shafer et al. 2006). In homozygous f05073
mutants, CWO is not detected in any of the six canonicalgroups of brain oscillator neurons (Fig. 2C, panels
7–12m), consistent with Western results (Supplementary
Fig. 2). Given that sLNvs are necessary and sufficient for
behavioral rhythms (Renn et al. 1999; Grima et al. 2004),
our results suggest that cwo functions in brain oscillator
neurons to maintain behavioral rhythms.
cwo is controlled by CLK through canonical E-boxes
Oscillatory expression of cwo mRNA became arrhythmic in per01 and ClkJrk mutants (Supplementary Fig. 4),
and its expression profile was similar to those of per andtim mRNA in per01 and ClkJrk mutants (Fig. 3A). These
results strongly suggested that cwo is induced by CLK–CYC as in per and tim (Darlington et al. 1998). As CLK–CYC is known to bind E-boxes in the per and tim promoter regions (Hao et al. 1999; Darlington et al. 2000;
McDonald et al. 2001), we searched for E-boxes in the
cwo promoter region (Fig. 3B). The 2.8-kb promoter region of cwo contains five canonical E-boxes (CACGTG),
five E⬘-boxes (CACGTT), and 16 noncanonical E-box sequences (CANNTG other than CACGTG). The observed
frequencies of these elements within the 2.8-kb pro-

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Genomics reveals the new clock gene cwo

Figure 2. Temporal and spatial expression pattern of CWO protein. (A) cwo encodes a bHLH-ORANGE family protein. The CWO
protein, 685 amino acids in length, is multiply aligned with mouse DEC1, DEC2, and HES5 proteins. Conserved amino acids in bHLH
(green) and ORANGE (orange) domains are marked by blue and gaps are represented as “−”. (B) Temporal expression profiles of cwo(red square), per (black circle), and tim (blue triangle) mRNA in wild-type flies under LD and DD. Relative mRNA levels of theindicated genes were measured using a Q-PCR assay. GAPDH2 was used as an internal control. Data were normalized so that the
average copy number (n = 2) over 12 time points is 1.0. (C) Spatial expression pattern of CWO in adult brains. (Panel 1) CWO
immunostaining (green) in a 12-µm optical Z-stack through the right hemisphere of a wild-type brain. Three clusters of CWO-positive
cells are detected in the dorsal brain. A magnified view of the boxed region is shown in panel 1m. Arrows denote DN1s, DN2s, andDN3s. Arrowheads denote additional CWO immunostaining. (Panel 2) PER immunostaining (red) in the same region shown in panel
1. Three clusters of PER-positive cells are detected in the dorsal brain. A magnified view of the boxed region is shown in panel 2m.
Arrows denote the same cells described in panel 1m. (Panel 3) CWO and PER coimmunostaining in the same region shown in panels
1 and 2. Colocalization of CWO and PER immunofluorescence in this superimposed dual laser image is shown in yellow. A magnified
view of the boxed region is shown in panel 3m. Arrows denote the same cells described in panels 1m and 2m. Arrowheads denote CWO
staining in cells not expressing PER. (Panel 4) CWO immunostaining in a 22-µm optical Z-stack through the right hemisphere of awild-type brain. Three clusters of CWO-positive cells are detected in the lateral brain. A magnified view of the boxed region is shownin panel 4m. Arrows denote clusters of LNds, lLNvs, and sLNvs. Arrowheads denote additional CWO immunostaining. (Panel 5) PER
immunostaining in the same region shown in panel 4. Three clusters of PER-positive cells are detected in the lateral brain. A magnified
view of the boxed region is shown in panel 5m. Arrows denote the same cells described in panel 4m. (Panel 6) CWO and PER
coimmunostaining in the same region shown in panels 4 and 5. Colocalization of CWO and PER immunofluorescence is shown inyellow. A magnified view of the boxed region is shown in panel 6m. Arrows denote the same cells described in panels 4m and 5m.
Arrowheads denote CWO staining in cells not expressing PER. (Panel 7) CWO immunostaining in an 8-µm optical Z-stack through theright hemisphere of a f05073 mutant brain. No specific CWO immunostaining is detected. A magnified view of the boxed dorsal brain
region is shown in panel 7m. (Panel 8) PER immunostaining in the same region shown in panel 7. PER-positive DNs are detected in
the dorsal brain. A magnified view of the boxed region is shown in panel 8m. (Panel 9) CWO and PER coimmunostaining in the same
region shown in panels 7 and 8. Only PER immunofluorescence in DNs is seen in this superimposed dual laser image. A magnified
view of the boxed region is shown in panel 9m. (Panel 10) CWO immunostaining in a 24-µm optical Z-stack through the righthemisphere of a f05073 mutant brain. No specific CWO immunostaining is detected. A magnified view of the boxed region is shownin panel 10m. (Panel 11) PER immunostaining in the same region shown in panel 10. Three clusters of PER-positive cells are detectedin the lateral brain. A magnified view of the boxed region is shown in panel 11m. Arrows denote clusters of LNds, lLNvs, and sLNvs.
(Panel 12) CWO and PER coimmunostaining in the same region shown in panels 10 and 11. Only PER immunofluorescence is detected.
A magnified view of the boxed region is shown in panel 6m. Arrows denote the same cells described in panel 11m.
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Figure 3. cwo is directly controlled by circadian clock. (A) The averaged expression levels of cwo, per, and tim mRNA in wild-type
and arrhythmic mutant flies (per01 and ClkJrk). Temporal expression profiles in mutant flies are shown in Supplementary Figure 4. (B)Evolutionarily conserved E-box sequences in cwo promoter region. Relative positions of E-boxes to the transcription start site of cwo
are indicated. Multiple alignments of seven Drosophila species and its conservation score are represented (University of California at
Santa Cruz Genome Browser Database [Hinrichs et al. 2006] version dm2). (C) The cwo promoter is activated by CLK. (Top panel) The
three tandem repeats of an 18-bp fragment containing wild-type and mutant E-boxes were fused to a TATA sequence driving adestabilized luciferase (dLuc) reporter gene. Well-conserved base pairs (bold) and mutated base pairs (red) are also indicated. Relative
luciferase activities of cwo promoter, wild-type, and mutant E-box reporters in the presence of 0 and 100 ng of Clk are shown in thebottom panel. Signals were normalized with Rluc activity. Error bars represent the SEM (n = 3).

moter region is significantly higher than expected for
random DNA sequences (p < 0.0001 for a canonicalE-box, p = 0.0101 for an E⬘-box, p = 0.0682 for a noncanonical E-box, and p = 0.0008 for all of these elements).
These elements were also observed within the 7.3-kb
first intron of cwo with significantly high frequencies(p = 0.0004 for six canonical E-boxes, p = 0.0024 for 10E⬘-boxes, p = 0.1338 for 35 noncanonical E-boxes, andp = 0.0020 for the total of them all). It is noteworthy that
only the cwo gene among 13,776 genes (20,406 transcripts) in the Drosophila genome has as many as fiveE-boxes within the 2.8-kb promoter region. Among these

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E-box related elements in the cwo gene’s promoter, wefound two canonical E-boxes (E-box1 and E-box2) to behighly conserved among several Drosophila species (Fig.
3B). We therefore examined whether CLK–CYC can induce gene expression through these E-boxes in Drosophila culture cell S2. Cotransfection with Clk strongly
induced luciferase expression with E-box2, while E-box1
and their mutants showed little induction, suggestingthat cwo can be directly regulated by CLK through Ebox2 (Fig. 3C). Recently, we found in a mammalian circadian clock system that neighboring sequences of canonical E-boxes (CACGTG) are important to function asDownloaded from www.genesdev.org on December 17, 2007 - Published by Cold Spring Harbor Laboratory Press

Genomics reveals the new clock gene cwo

the clock-controlled element (Y. Kumaki, M. Ukai-Tadenuma, K.D. Uno, J. Nishio, K. Masumoto, M. Nagano, T.
Komori, Y. Shigeyoshi, J.B. Hogenesch, and H.R. Ueda,
in prep.), which is consistent with previous results in
Drosophila showing that sequences flanking the canonical per CRS E-box are required for its function as a clock
regulatory element (Lyons et al. 2000). Based on thesefindings, we can hypothesize that some E-boxes withcertain neighboring sequences such as E-box2 in theabove are favored by CLK/CYC, and other E-boxes withdifferent neighboring sequences such as E-box1 are favorednot by CLK/CYC but by other bHLH transcription factors.
ChIP-on-chip assays reveal that CWO protein binds
to canonical E-boxes
The CWO protein has a bHLH known to act as a DNAbinding domain (Fig. 2A). In an attempt to identify potential targets of CWO protein at the genome-wide level,
we performed a ChIP assay using a Drosophila genome

tiling array (Manak et al. 2006). In this assay, Flag-tagged
CWO protein was induced in S2 cells and immunoprecipitated with its target DNA using anti-Flag antibody.
The precipitated DNA was then labeled and hybridized
to a tiling array. As a background control, we carried out
immunoprecipitation using anti-V5 antibody and performed subsequent tiling array experiments in the sameway. Potential candidates for binding sites were identified by TileMap (Ji and Wong 2005) as genomic sites
where signals from Flag-ChIP were significantly higher
than that from V5-ChIP. We were able to do this by comparing the signal intensity between the two sets of tilingarrays, one set for Flag-ChIP and the other set for V5ChIP (n = 2; two arrays for both sets). The subsequently
detected potential binding sites of CWO protein in the
Drosophila chromosome are illustrated in Figure 4A. Detailed information on all the detected sites is available
online at our Web site (see Materials and Methods).
Among the 1512 sites detected, we confirmed that the
CWO protein binds to the known clock genes vri and

Figure 4. CWO protein directly targets known clock genes. (A) Chromosomal view of potential CWO-binding sites (black vertical bar
on each chromosome) identified by ChIP assay on a Drosophila genome tiling array. The locations of vri, Pdp1, and cwo are indicatedas a green vertical bar. (B) Close view of potential cwo-binding sites on vri, Pdp1, and cwo genes. (Top panel) The fold changes between
signal (Flag) and background (V5) were plotted for each probe with the potential binding sites (blue box). (Bottom panel) Gene structures
are also indicated. We identified two independent binding sites in the Pdp1 promoter (Pdp1 #1 and #2). (C) Independent verificationof ChIP experiments. The relative abundance of immunoprecipitated chromosome regions was measured using a Q-PCR assay. Input
product (genomic DNA without ChIP) was used as an internal control. Act57B is used as a negative control. Error bars represent theSEM (n = 3). cwo, Pdp1, and vri promoters are significantly bound by CWO protein. (*) p < 0.05 in t-test. (D) Canonical E-box(CACGTG) recognized by CWO protein. The DNA sequence overrepresented in potential CWO-binding sites was identified by Weeder
and drawn by EnoLOGOS.

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Pdp1, both of which have the E-box sequence in theirpromoter region (Fig. 4B). We did not detect CWO binding in the promoter regions of per and tim, probably due
to the stringent threshold applied to the CWO ChIP
analysis, or alternatively reflecting the characteristics ofarrhythmic S2 cells that do not normally express per andtim mRNAs. We were also interested to find that CWO
protein strongly binds to its own promoter region (Fig.
4B). The significance of these results was verified by QPCR on each promoter region using the ChIP product asa template (Fig. 4C). Although we cannot exclude thepossibility that overexpression in this ChIP experimentmight reveal some false-positive interactions, our subsequent bioinformatics search for the consensus DNA sequence recognized by the CWO protein identified a sequence containing the canonical circadian E-box (CAC
GTG) (Fig. 4D), strongly implicating the CWO protein as
directly targeting the E-box. As the bHLH-ORANGE
family acts as a negative transcription factor, it is possible that the CWO protein not only negatively regulates
known clock genes that possess E-boxes in their promoter regions but also its own gene’s transcription. Thisidea led us to the hypothesis that cwo forms an autoregulatory negative feedback loop.
cwo negatively regulates the expression of clock genes
and forms an autoregulatory feedback loop
To test whether cwo forms an autoregulatory negative
feedback loop, we performed transcription assays using aluciferase reporter gene in S2 cells. The promoters of per,tim, vri, and Pdp1 were fully activated by CLK andstrongly suppressed by CWO (Fig. 5A). Although we cannot exclude the possibility that overexpression might reveal molecular interactions that are not significant at
physiological concentrations, this suppression effect isalso observed in vivo and clearly correlated with the activity of CWO as the early morning troughs of per, tim,
vri, and Pdp1 mRNA at ZT3 are significantly elevated in
cwo RNAi transgenic flies relative to those of wild-type
flies (Fig. 5B). To further investigate how cwo contributes to the clock system in vivo, we measured the temporal expression levels of per, tim, vri, and Pdp1 in cwo
RNAi transgenic flies. The amplitude of expression ofthese genes decreased to half the level found in wild-type
flies under LD conditions (Fig. 5C). The decreased amplitude of circadian oscillators in cwo RNAi transgenic
flies relative to wild-type flies has been also confirmed
under DD conditions (data not shown), suggesting thatcwo functions to produce a high-amplitude oscillation ofclock genes’ expression. Furthermore, we also found thatcwo strongly suppresses its own promoter (Fig. 5D). A
similar effect was observed when we used the E-box2-luc
construct as a suppression target. These results are consistent with the observation that trough levels of cwo
mRNA expression are elevated in cwo knockdown flies(Fig. 5E), which express reduced levels of CWO protein(Supplementary Fig. 2), and S2 cell experiments demonstrating that RNAi-mediated knockdown of cwo drastically reduces CWO levels without reducing cwo mRNA

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levels (Supplementary Fig. 5). Based on these results, we
propose that cwo negatively regulates the expression ofclock genes containing canonical circadian E-boxes, including cwo itself, to form an autonegative feedback loopthat functions to produce high-amplitude circadian transcription in Drosophila (Fig. 6).
Discussion
In this study, we adopted in vivo functional genomics
strategy to identify a new core component in Drosophila
clock. Our genome-wide screening using RNAi induced
by a Gal4-UAS binary system has several advantages
over previous clock mutant screenings and overcomessome of their limitations (Konopka and Benzer 1971;
Blau and Young 1999; Martinek et al. 2001). Our method
has proven to be one of the most concise and fastest ways
to directly investigate how target genes function in theclock system. Importantly, targeting of RNAi expression
using tissue-specific Gal4-driver lines allows us to avoid
inducing lethality when we knock down essential genes
for viability and development. Gene knockdowns of atleast three new candidates including cwo induced lethality when we constitutively and ubiquitously expressed
the dsRNA of the target gene (data not shown).
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 awider distribution of CWO protein within fly heads compared with PER protein since CWO is detected in additional cells within the fly brain (Fig. 2C). 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 thelevels of CLK–CYC activity, as shown in per01 and ClkJrk
mutants (Fig. 3A; Supplementary Fig. 4). 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 highamplitude oscillation.
Consistent with this hypothesis, impaired activity of
CWO leads to an elevated trough of per, tim, vri, andPdp1mRNA at ZT3 in cwo RNAi transgenic flies compared with those of wild-type flies (Fig. 5C). As the fly
circadian clock consists of complexly integrated negative and positive feedback loops (Fig. 6), 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 (Fig. 5B)
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

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Genomics reveals the new clock gene cwo

Figure 5. CWO protein regulates transcription of known clock genes. (A) CWO protein suppresses transcription of known clock
genes. Relative luciferase activities of per-luc, tim-luc, vri-luc, and Pdp1-luc in the presence of 0 and 100 ng of Clk and 0, 100, 400,
and 800 ng of cwo are represented. Signals were normalized with Rluc activity. Error bars represent the SEM (n = 3). (B) Expression ofper, tim, vri, and Pdp1 mRNA at the trough level (ZT3) in wild-type (black) and cwo RNAi (red) flies under LD. Relative mRNA levelsof each genes were measured with Q-PCR assay. GAPDH2 was used as an internal control. Data were normalized so that the average
copy number in cwo RNAi flies is 1.0. Error bars represent the SEM (n = 3). (C) Temporal expression profiles of per, tim, vri, and Pdp1
mRNA in wild-type (black circle) and cwo RNAi transgenic (red rectangle) flies under LD. The relative mRNA levels of each gene were
measured with a Q-PCR assay. GAPDH2 was used as an internal control. Error bars represent the SEM (n = 2). We note that samplingtime points (ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21) of temporal expression profile are different from ZT3 in B. The expected troughphase calculated from these temporal expression data is almost ZT3, suggesting that the two data are consistent with each other. Since
ZT1 and ZT5 are not exactly the trough phase, expression levels of clock genes at these timings are equal or slightly higher in wild-type
flies than those of cwo RNAi flies. (D) CWO protein suppresses its own transcription. Relative luciferase activities of cwo-luc, andE-box2-luc in the presence of 0 and 100 ng of Clk and 0, 100, 400, and 800 ng of cwo are represented. Signals were normalized with
Rluc activity. Error bars represent the SEM (n = 3). (E) Temporal expression profiles of cwo mRNA in wild-type (black circle) and cwo
RNAi transgenic (red rectangle) flies under DD. Relative mRNA levels of each gene were measured with a Q-PCR assay. GAPDH2 wasused as an internal control. Error bars represent the SEM (n = 2).

well as the decreased peak level of per expression (Matsumoto et al. 1999). Interestingly, the circadian phenotypes of this mutant are very similar to cwo knockdown
flies showing ∼26 h in period.
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 isone of the key factors in circadian oscillation, has been
extensively studied (Hardin 2005). This involves forma-

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corded and analyzed as described elsewhere (Matsumoto et al.
1999). The w1118 strain was used as wild type. Knockdown flies
were obtained by mating the Gal4 driver line to each of the
UAS-IR lines.
Transgenic flies carrying a dominant-negative type of CWO
proteinFigure 6. Transcriptional circuit underlying Drosophila circadian clock. Ellipsoids represent clock proteins, and rectangles
represent time-of-day-specific DNA elements. Activators andrepressors are represented in green and red, respectively. CWO
protein directly binds to E-boxes and functions as a repressor.

tion of a heterodimer between PER and TIM, both ofwhich lack a DNA-binding domain, that binds CLK–CYC to inhibit the DNA-binding activity of CLK–CYC(Lee et al. 1999; Yu et al. 2006). The direct suppression
mechanism through the E-box, however, had yet to beelucidated prior to this study. It has long been a mysteryas to how the constitutive expression of per and tim in
per01; tim01 double mutants can rescue rhythmicity atthe behavioral and molecular levels (Yang and Sehgal
2001). Our 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-boxmediated transcription, contributing to sustaining ahigh-amplitude circadian expression, and may compensate the molecular rhythmicity even when a functional
disorder occurs in other feedback loops.
Materials and methods
Genome-wide screening for rhythmically expressed genes
To extract the genes rhythmically expressed under both LD and
DD in fly heads, we used two statistical cosine filters—one forthe LD data set and the other for the DD data set obtained in aprevious report (Ueda et al. 2002). We first calculated the bestcross-correlation between 12-point time courses under LD ofeach probe set and cosine waves of defined periods and phases.
For this, we prepared cosine waves of nine test periodicities
from 20 to 28 h in increments of 1 h. Cosine waves for each test
period were considered over a total of 60 phases (i.e., peaks at 60
equally spaced times during the defined period), yielding a totalof 540 test cosine waves. We performed the same procedure forthe time courses under DD. Finally, we sorted all the probe setsby the sum of best correlations under LD and DD in a descending manner before selecting the top 200 probe sets for subsequent analysis.
Measuring locomotor activity of flies
Flies were kept under LD12:12 at 25°C and fed on standard
glucose-cornmeal medium. Locomotor activity rhythm was re-

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cwo cDNA lacking the “basic” region (cwo⌬b) of the bHLH
domain was obtained by a PCR-based method using ⌬b-forwardand ⌬b-reverse 5⬘-phospholylated primer sets with the cwo
cDNAs cloned into a pIB/V5-His-TOPO plasmid (Invitrogen) as
a template. Then a cwo⌬b BamHI fragment was cloned into the
BglII site of the pUAS-T vector. The UAS-cwo⌬b dominantnegative flies were obtained by P-element-mediated transformation. We established four UAS-⌬b lines, each of which carries
the transposon insertion at different sites of genome. Using ⌬b-1
and ⌬b-4 lines, both of which have the insertion on the third
chromosome, pdf-gal4; UAS-cwo⌬b flies were established andrecorded for their locomotor activities.
Controls of genome-wide knockdown functional screening
in vivo
The gal4 driver lines used in this study have been reported toshow no rhythm abnormalities (Blau and Young 1999; Renn etal. 1999; Malpel et al. 2002). The behavior of driverless IR lines
for 20 different target genes (∼15% of target genes) were recorded
as a small-scale pilot study and displayed no rhythm abnormalities, which indicates that the IR construct alone does not cause
rhythm abnormalities. Independent lines for each transgenewith clock cell drivers were recorded in the full screening andshow similar results. The possibility that each insert in independent lines for the same transgene would give similar results
makes it unlikely that the phenotype is due to the insert alone.
The genetic background of the knockdown lines (w1118) displayed normal rhythms as shown in Table 1.
Primer sequences for cwo⌬b⌬b-forward, 5⬘-GATCGCATGAACTCCTGCCTGGCGGACC
TG-3⬘; ⌬b-reverse, 5⬘-TCCGGTGGCATATTCAGCATCGTCCTCGCT-3⬘.
CWO antibody production and immunolocalization
Sequences coding for the CWO ORF were inserted into the
pET28a vector as an NcoI/HindIII fragment, creating a fusion
protein with a HIS-tag at the C terminus. The NcoI site contains
the CWO translation start codon. The HindIII site was inserted
8 bp upstream of the natural translation stop codon by PCRmediated mutagenesis (primer sequence, CCGAAGCTTCCAGCTCTGGTTCATGGCC). CWO protein expression was
induced in BL21 cells using isopropyl ␤-D-1-thiogalactopyranoside (IPTG). The cells were lysed and sonicated in 6 M guanidium hydrochloride (pH 8.0). The cell lysate was filteredthrough a 0.45-µm filter and passed over a HiTrap Chelating HP
column (Amersham Biosciences) charged with copper sulfate.
Purified protein was eluted with increasing concentrations ofimidazole. Pooled protein fractions were dialyzed against 0.05
M ammonium carbonate, frozen, and lyophilized. Purified
CWO was resuspended in 6 M urea and used to immunize
guinea pigs for antibody production (Cocalico Biological).
Immunofluorescent detection of CWO and PER in brainsfrom wild-type and homozygous f05073 mutant flies collected
at ZT21 was carried out as described (Houl et al. 2006). CWO
antibody (GP27) was diluted 1:1000, and rabbit PER antibody
preabsorbed against per01 embryos was diluted 1:15,000. Alexa
Fluor 488-conjugated anti-guinea pig and Alexa Fluor 568-con-Downloaded from www.genesdev.org on December 17, 2007 - Published by Cold Spring Harbor Laboratory Press

Genomics reveals the new clock gene cwo

jugated anti-rabbit secondary antibodies (Molecular Probes/Invitrogen) were diluted to a final concentration of 1:200. Brains
were optically scanned at a thickness of 2 µm using an Olympus
FV1000 confocal microscope, and images were processed using
Adobe Photoshop. Images are representative of 12 brain hemispheres from wild-type and 12 brain hemispheres from f05073
mutants.
Q-PCR of temporal expression level
Q-PCR was performed using ABI Prism 7700 and SYBR Green
Reagents (Applied Biosystems). Wild-type flies were sampled
every 4 h in LD and DD for 2 d (n = 2). tim(UAS)-gal4; cwo-IR2RNAi transgenic flies were sampled every 4 h in LD (n = 2).
per01 and ClkJrk mutants were sampled every 6 h in LD (n = 2).
Total RNA was purified from ∼100 heads at each time point as
described elsewhere (Ueda et al. 2002). cDNA was synthesizedfrom 0.5 µg of total RNA using random 6-mer (Promega) andSuperScript II Reverse Transcriptase (Invitrogen) according to
the standard protocol. The amplified DNA sample was measured for verification of the ChIP experiment. Samples contained 1× SYBR Green Master Mix (Applied Biosystems), 0.5 µM
primers, and 1/40 synthesized cDNA in a 25-µL volume. The
PCR conditions were as follows: 10 min at 95°C, then 40 cyclesof 15 sec at 94°C, 30 sec at 59°C, and 1 min at 72°C. Absolute
cDNA abundance was calculated using the standard curve obtained from Drosophila genomic DNA. GAPDH2 expression
levels were quantified and used as the internal control.
Primer sequences for Q-PCR of Drosophila cDNA
cwo-forward, 5⬘-ATCTGCGCCCAAGTGTACCT-3⬘; cwo-reverse, 5⬘-TGCTTCTCCTCCATTTCCATTAAC-3⬘; per-forward, 5⬘-ATAAGCACAACGACGAGATGGA-3⬘; per-reverse,
5⬘-GAGCCTCCTCTTTTTATCCCGT-3⬘; tim-forward, 5⬘-CA
CACCATCTTCGAGCTGAATAA-3⬘; tim-reverse, 5⬘-AGTT
GTTGATTTGATCGCATTTG-3⬘; Pdp1␧-forward, 5⬘-GCAACTGGTAATGGAAATGGTG-3⬘; Pdp1␧-reverse, 5⬘-CTGTTCAAATGGTTGTGATGCTC-3⬘; vri-forward, 5⬘-CATCACTACAGCCAGCAGAAGC-3⬘; vri-reverse, 5⬘-ATATTGGATAGCCGGACGTTGT-3⬘; GAPDH2-forward, 5⬘-CTACCTGTTCAAGTTCGATTCGAC-3⬘; GAPDH2-reverse, 5⬘-AGTGGACTCCACGATGTATTCG-3⬘.
Primer sequences for Q-PCR of mouse cDNA
Hes5-forward, 5⬘-TCTCCACGATGATCCTTAAAGGA-3⬘; Hes5reverse, 5⬘-CAAAATCGTGCCCACATGC-3⬘; Tbp-forward, 5⬘GTTGTGCAGAAGTTGGGCTTC-3⬘; Tbp-reverse, 5⬘-TCACAGCTCCCCACCATGTT-3⬘.
Primer sequences for Q-PCR of ChIP products
cwo-5⬘-forward, 5⬘-GAGTAACCCGAGCCCGTTTT-3⬘; cwo5⬘-reverse, 5⬘-ATACGACCGGTTGGTTGCTT-3⬘; Pdp1-5⬘#1forward, 5⬘-GGATTGGTTCCCTATTCTCGG-3⬘; Pdp1-5⬘#1reverse, 5⬘-GCAGAGCTTTGCTATTGGCC-3⬘; Pdp1-5⬘#2-forward, 5⬘-TCTTGCGTCGGCACTTGAG-3⬘; Pdp1-5⬘#2-reverse,
5⬘-TTATCGAGCACCCCAACATTT-3⬘; vrille-5⬘-forward, 5⬘TCGCAATCGAAATCGGACTC-3⬘; vrille-5⬘-reverse, 5⬘-ACG
CACACACACGTAAGGTGAC-3⬘; Act75B-5⬘-forward, 5⬘-GATACGATCTCCACGCTCGATC-3⬘; Act75B-5⬘-reverse, 5⬘-GG
AATACCATATAGCATATAGCGCAG-3⬘.

region of vri (2.5 kb), Pdp1 (4.1 kb), and cwo (2.75 kb) was amplified by PCR from a w1118 fly genome and subcloned into apCRII vector (Invitrogen). The XbaI–SpeI fragment of vri or the
Pdp1 promoter region was then cloned into the NheI site of thepGL3-basic vector, while the HindIII fragment of the cwo promoter region was cloned into the HindIII site of the pGL3-basic
vector. For Figure 3C, oligonucleotides (Hokkaido System Science) containing three tandem repeats of wild-type or mutant
E-boxes from the cwo promoter region were annealed and inserted into the HindIII site upstream of TATA-Luc, which contains a minimal promoter region of the hsp70 gene at the
HindIII–NcoI site of pGL3-basic. A pAc5.1-Rluc reporter was
also constructed by inserting the Rluc (Renilla luciferase) geneof the pRL-CMV vector (Promega) into pAc5.1B (Invitrogen).
Drosophila S2 cells were maintained in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% FBS(Bioserum) and antibiotics (12.5 U/mL penicillin, 12.5 mg/mL
streptomycin; GIBCO). S2 cells were plated onto six-well plates
at 4 × 106 cells per well density the day before transfection. The
following day, S2 cells were transfected using Effectene Transfection Reagent (Qiagen) with 100 ng of the promoter reporter
construct in the presence of 0 and 100 ng of pAct-Clk (Darlington et al. 1998), and 0, 100, 400, and 800 ng of pAc5.1-Flag-cwo
by a standard transfection method. The total amount of transfected plasmid was equaled to 1.1 µg by pAc5.1B empty vector(Invitrogen). Additionally, 5 ng of an Rluc reporter vector,
pAc5.1-RLuc, were added into each transfection as an internal
control for transfection efficiency. Forty-eight hours after transfection, transfected cells were harvested and assayed with theDual-Luciferase Reporter Assay System (Promega). Luciferase
activity was normalized by Rluc activity.
Primer sequences for amplifications of vri, Pdp1, and cwo
promoter region
vriG3564-forward, 5⬘-GCTAGGGAACAAACGCAATAAGTG3⬘; vriG6085-reverse, 5⬘-TCGAAAGAAGGCTGAATCGCAA3⬘; Pdp1G30257-forward, 5⬘-CATACACAGGCACAAGACC
GTT-3⬘; Pdp1G35327-reverse, 5⬘-TTCGTGTGGTCAGGGAATGCAC-3⬘; cwoG0973-forward, 5⬘-TAAGCTTAGCCATG
CAATCGGGGTAAACAG-3⬘; cwoG3681-reverse, 5⬘-TAAGC
TATGTACGGGCGAGCGAGCAACGTG-3⬘.
Oligonucleotide sequences
E-box1-forward, 5⬘-CGCGACGTTTCACGTGTTTTATACGTTTCACGTGTTTTATACGTTTCACGTGTTTTAT-3⬘; E-box1reverse, 5⬘-GATCATAAAACACGTGAAACGTATAAAACACGTGAAACGTATAAAACACGTGAAACGT-3⬘; E-box2-forward,
5⬘-CGCGCAACAACACGTGACCAACCAACAACACGTGACCAACCAACAACACGTGACCAAC-3⬘; E-box2-reverse, 5⬘-GAT
CGTTGGTCACGTGTTGTTGGTTGGTCACGTGTTGTTGG
TTGGTCACGTGTTGTTG-3⬘; E-box1-mutant-forward, 5⬘-CGC
GACGTTTACCGGTTTTTATACGTTTACCGGTTTTTATACGTTTACCGGTTTTTAT-3⬘; E-box1-mutant-reverse, 5⬘-GATCATAAAAACCGGTAAACGTATAAAAACCGGTAAACGTATAAAAACCGGTAAACGT-3⬘; E-box2-mutant-forward, 5⬘-CGCGCAACAAACCGGTACCAACCAACAAACCGGTACCAAC
CAACAAACCGGTACCAAC-3⬘; E-box2-mutant-reverse, 5⬘-GA
TCGTTGGTACCGGTTTGTTGGTTGGTACCGGTTTGTTG
GTTGGTACCGGTTTGTTG-3⬘.
Construction of cwo expression vector

Transfection and luciferase assay
A luciferase reporter plasmid was constructed based on a pGL3basic vector (Promega). For Figure 5, A and B, each promoter

To construct the Flag-tagged cwo expression vector, a pMT/V5His-B plasmid (Invitrogen) was digested with EcoRV and XhoIand blunt-ended with T4 DNA polymerase. An HU2 cassette

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containing a Kozak sequence, start codon, Flag tag sequence,
I-SceI recognition sequence, PI-PspI recognition sequence, andstop codon (all of them in-frame) was amplified from a pMU2
plasmid (H. Ukai, T.J. Kobayashi, M. Nagano, K. Masumoto, M.
Sujino, T. Kondo, K. Yagita, Y. Shigeyoshi, and H.R. Ueda, inprep.) by PCR and inserted into the digested sites of pMT/V5His-B and termed pMT-Flag. The full coding sequence of cwo
was amplified by PCR from the head cDNA of a w1118 fly witha forward primer containing an I-SceI recognition sequence anda reverse primer containing a PI-PspI recognition sequence. PCR
products were digested with I-SceI and PI-PspI and inserted into
a pMT-Flag vector and termed pMT-Flag-cwo.
Primer sequences for HU2 cassette amplification
HU2-forward, 5⬘-GTGACACTATAGAATACAAGC-3⬘; HU2reverse, 5⬘-CCGCACCCGACATAGATTC-3⬘.
Primer sequences for ORF amplification
cwo-forward, 5⬘-ATTACCCTGTTATCCCTAATCCGCCCAT
CCGGTCAAG-3⬘; cwo-reverse, 5⬘-ACCCATAATACCCATAATAGCTGTTTGCCACCAGCTCTGGTTCATGGC-3⬘.
ChIP assay with Drosophila genome tiling chipS2 cells were transfected with pMT-Flag-cwo (1.9 µg) or pMTFlag (1.9 µg) and pCoHygro (0.1 µg; Invitrogen, for selection)using Effectene (Qiagen) according to the manufacturer’s instructions. Hygromycin-B (300 µg/mL final concentration; Invitrogen) was added 5 d after transfection. After 3 wk, the established stable cell lines were used for the ChIP assay.
Cells (4 × 107) were collected and stimulated by copper sulfate
to induce fusion genes. Cells were harvested 24 h after stimulation. ChIP was then performed according to the previously
described method (Yamashita et al. 2002) with some modifications. In this report, we used Chromatin Immunoprecipitation
Assay Kits (Upstate Biotechnology), anti-Flag M2 antibody(Sigma), and anti-V5 antibody (Sigma). Protein G-Sepharose(Amersham) was used for precipitation of antibody/protein immune complexes. The resulting precipitated DNA was amplified by adaptor-mediated PCR (Cawley et al. 2004) and thenfragmented to 50–150 bp by DNase I (Epicentre) in One-Phor-All
buffer (Pharmacia). The distribution of fragmented DNA size
was verified on a 2% agarose gel. The fragmented DNA wasthen end-labeled with GeneChip Labeling Reagent (Affymetrix)
by using terminal deoxynucleotidyltransferase (Roche) in TdT
buffer (Roche) and CoCl2 (Roche). To prepare the hybridization
cocktail, 5 µg of labeled DNA fragment were mixed with final
1× MES, 3 M TMAC, 0.02% Triton (Sigma), 50 pM Oligo B2, 1×
Hybridization Control (Affymetrix), and 100 µg/µL Herring
Sperm DNA (Promega). Two-hundred microliters of Hybridization cocktail were applied to a Drosophila genome tiling array(Affymetrix) and hybridized for 18 h at 45°C with 45 rpm rotation. Stain and wash was executed by a Fluidics station 450 with
the EukGE-WS2 version 4 program. Arrays were scanned by a
GeneChip Scanner 7G (Affymetrix).
Western blot analysis of CWO in cwo RNAi fly
Fly heads were homogenized with a pestle, and protein extracts
were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). The extracts of 40 heads were applied to
one lane of a polyacrylamide gel. For immunoblot analysis, proteins were transferred to PVDF membranes (Invitrogen) and incubated with diluted antibodies in blocking solution. Anti-

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For visualization, the ECL Plus Western Blotting Detection System (Amersham) and LAS-3000mini (FujiFilm) were used according to the manufacturers’ instructions.
Western blot analysis of CWO protein and Q-PCR of cwo
mRNA
Drosophila S2 cells were plated onto six-well plates at 4 × 106
cells per well density the day before transfection. The following
day, S2 cells were transfected using Effectene Transfection
Reagent (Qiagen) with 500 ng of pMT-Flag-cwo, 200 ng ofpWAGAL4 (a kind gift of Y. Hiromi), 300 ng of pUAS-T empty
plasmid, and pUAS-cwo-IR by a standard transfection method.
Additionally, 20 ng of pMT-Flag-GFP were added into eachtransfection as an internal control for transfection efficiency. A
total amount of transfected plasmid was equaled to 1.1 µg by
pAc5.1B empty vector (Invitrogen). Forty-eight hours after
transfection, the recombinant proteins’ expression was inducedwith 500 µM copper sulfate. Twenty-four hours after the induction, transfected cells were harvested and used for Western blot
analysis and Q-PCR.
For Western blot analysis, each nuclear and cytoplasmic fraction was extracted from the harvested cells with NE-PER
Nuclear and Cytoplasmic Extraction Reagents (Pierce), andeach cell lysate of 4 × 106 cells was applied to a lane of polyacrylamide gel. For immunoblot analysis, proteins were transferred to PVDF membranes. Anti-Flag M2 monoclonal Antibody-Peroxidase Conjugate (Sigma) was diluted 2000-fold andused for detection. For visualization, the ECL Plus Western Blotting Detection System (Amersham) and LAS-3000mini (FujiFilm)
were used according to the manufacturers’ instructions.
Q-PCR was performed using ABI Prism 7700 and SYBR Green
Reagents (Applied Biosystems). Total RNA was purified fromthe harvested cells using TRIzol reagent (Invitrogen). cDNA wassynthesized from 0.25 µg of total RNA with random 6-mer (Promega) and SuperScript II Reverse Transcriptase (Invitrogen) according to the standard protocol. Samples contained 1× SYBRGreen Master Mix (Applied Biosystems), 0.5 µM primers, and1/20 synthesized cDNA in a 25-µL volume. The PCR conditions
were as follows: 10 min at 95°C, then 40 cycles of 15 sec at
94°C, 30 sec at 59°C, and 1 min at 72°C. Absolute cDNA abundance was calculated using the standard curve obtained from
Drosophila genomic DNA, pMT-Flag-cwo, and pMT-Flag-GFP
plasmid. GAPDH2 or Flag-GFP expression levels were quantified and used as the internal control.
Primer sequences for Q-PCR
cwo-forward, 5⬘-ATCTGCGCCCAAGTGTACCT-3⬘; cwo-reverse, 5⬘-TGCTTCTCCTCCATTTCCATTAAC-3⬘; GAPDH2forward, 5⬘-CTACCTGTTCAAGTTCGATTCGAC-3⬘; GAP
DH2-reverse, 5⬘-AGTGGACTCCACGATGTATTCG-3⬘; Flagforward, 5⬘-GACTACAAGGATGACGATGAC-3⬘; cwo-reverse2, 5⬘-ACTGGAGACAGCTCTCATAC-3⬘; GFP-forward,
5⬘- CCATGGCCAACACTTGTCAC-3⬘; GFP-reverse, 5⬘- TCC
GGATAACGGGAAAAGC-3⬘.
Tiling array data analysis
For the identification of transcription factor-binding sites(TFBSs) of cwo, we used data obtained from four arrays: two forDownloaded from www.genesdev.org on December 17, 2007 - Published by Cold Spring Harbor Laboratory Press

Genomics reveals the new clock gene cwo

Flag-antibody and two for V5-antibody. The four sets of arraydata were analyzed by TileMap (Ji and Wong 2005). In the resulting BED file, we selected TFBSs with a score of 999 (thehighest score) for further analysis to achieve high stringency.
Detailed information for these detected TFBSs is available online at https://alcyone.cdb.riken.jp/papers/cwo. Array data including raw data are available at Gene Expression Omnibus(http://www.ncbi.nlm.nih.gov/geo) via series accession number
GSE7569. To find the binding motif of cwo, we first selected
TFBSs within 2 kb of the transcription start sites of known
genes. We then inputted the corresponding genomic sequences
to Weeder (Pavesi et al. 2004) running on a local computer. The
sequence logo in Figure 4D was drawn by EnoLOGOS (Workman et al. 2005).
Statistical significance of E-box frequencies in cwo promoter
To assess the statistical significance of observing the canonicalE-box, E⬘-box, and noncanonical E-box frequencies in the ∼2.8kb promoter region of cwo (∼2550 bp upstream of and ∼250 bp
downstream from the TSS), we generated 10,000 random sequences with the length of 2.8 kb, then counted occurrences ofcanonical E-boxes, E⬘-boxes, and noncanonical E-boxes in thosesequences, respectively. The random sequences were generated
so that the dinucleotide frequency was same as that of promoter
regions in the Drosophila genome. Nonoverlapping 7021 promoter regions were sampled and used for calculating dinucleotide frequency. For the analysis of the first intron, 11,637
nonoverlapping first intron regions were sampled and used for
calculating dinucleotide frequency. Then we generated 10,000
random sequences with a length of 7.3 kb.

Acknowledgments
We thank Kenji Tomioka, Taishi Yoshii, Miki Shimohigashi,
Ayami Matsushima, and Yasuyuki Shimohigashi for useful discussions; Yasushi Hiromi for providing pWAGAL4; and Steve
A. Kay for providing pAct-Clk, per-luc, and tim-luc plasmids.
The tim(UAS)-gal4 strain was a kind gift of Justin Blau; pdf-gal4
was donated by Jeffrey C. Hall; gal1118 was donated by Francois
Rouyer; and a cwo-deficient strain, f05073, was donated by the
Exelixis collection, Harvard University. We thank Ueli Schibler
for critical reading of this manuscript and also thank Michael
Rosbash and Ravi Allada for sharing information before publication. We thank Kiyo Kimura, Makiko Haruta, Kazuko Fujitani, and Yukoko Sado for technical assistance and Douglas Sippand Michael Royle for proofreading of the manuscript. We
thank Philipp Kapranov, Srinka Ghosh, Michael Paumen, andTom R. Gingeras for technical support on both the ChIP assay
and data analysis with a Drosophila genome tiling chip. This
work is supported in part by an intramural Grant-in-Aid from
CDB (to H.R.U.); the President’s Fund from RIKEN (to H.R.U.);a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no.
17570134 and no. 18016025 to A.M., no. 17017032 and National
BioResource Project to R.U., and Systems Genomics Project to
H.R.U.); a Grant-in-Aid from the Uehara Memorial Foundation(to H.R.U.); and an NIH grant NS052854 (to P.E.H.).

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