Appl Entomol Zool (2011) 46:391–397 DOI 10.1007/s13355-011-0052-z

ORIGINAL RESEARCH PAPER

bHLH-ORANGE family genes regulate the expression of E-box clock genes in Drosophila Taichi Q. Itoh • Teiichi Tanimura • Akira Matsumoto

Received: 1 November 2010 / Accepted: 2 May 2011 / Published online: 26 May 2011 Ó The Japanese Society of Applied Entomology and Zoology 2011

Abstract Drosophila has 13 basic helix-loop-helixORANGE (bHLH-O) family genes. One of the members, clockwork orange (cwo), which is the counterpart of mammalian clock genes Dec1 and Dec2, regulates the transcriptional feedback loops of circadian clock genes through binding to E-box sequences in target gene promoters. The goal of the current study was to determine the role of Drosophila bHLH-O proteins in circadian rhythms at the molecular and behavioral level. Promoter assays in cultured Drosophila S2 cells were carried out to investigate which of the known bHLH-O proteins directly regulates the transcription of clock genes. In addition to CWO, three other bHLH-O proteins, SIDE, Mb and Mc, suppressed E-box clock gene transcription in vitro. RNA interference (RNAi) was used to generate bHLH-O knockdown flies in pacemaker neurons, and then locomotor activity rhythm was measured. cwo knockdown flies exhibited a remarkable phenotype. To clarify the functional complementation in circadian regulation among CWO, SIDE, Mb and Mc, promoter activity in the presence of combinations of two bHLH-O genes and locomotor rhythm in double knockdown flies were examined. The results suggest that CWO

T. Q. Itoh Graduate School of Systems Life Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan T. Tanimura Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan A. Matsumoto (&) Department of Biology, Juntendo University School of Medicine, 1-1 Hiraga Gakuendai, Inzai, Chiba 270-1695, Japan e-mail: [email protected]

predominantly acts as a key factor of circadian regulation both in vitro and in vivo. Keywords Drosophila  Circadian rhythm  Clockwork orange  Basic helix-loop-helix-ORANGE

Introduction In Drosophila, the circadian oscillator is composed of three interlocked transcriptional feedback loops of clock genes. CLOCK/CYCLE (CLK/CYC) heterodimer activates the transcription of several clock genes, including period (per), timeless (tim), vrille (vri), PAR domain protein 1e (Pdp1e) and clockwork orange (cwo), through binding to E-box sequences in the promoter regions of these genes (Hardin 2006; Tomioka and Matsumoto 2009). PER/TIM inhibits its own transcription to constitute a negative feedback loop. VRI and PDP1e form another feedback loop to control the transcription of Clk, which in turn regulates its own transcription. CWO suppresses the transcription of all of the E-box clock genes listed above, including its own, comprising the third interlocked feedback loop (Kadener et al. 2007; Lim et al. 2007; Matsumoto et al. 2007; Richier et al. 2008). CWO is a member of the basic helix-loop-helixORANGE (bHLH-O) family, which are DNA-binding transcription factors that suppress numerous biological processes in invertebrates and vertebrates. bHLH-O proteins contain a tandem arrangement of the bHLH domain and an adjacent sequence known as the Orange domain (Davis and Turner 2001). bHLH-O proteins form homo- or heterodimers to regulate transcription through binding to a specific DNA sequence termed the E-box (Davis and Turner 2001). In Drosophila, there are 13 bHLH-O proteins,

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including CWO. It is not known whether bHLH-O proteins other than CWO are involved in the regulation of circadian rhythms. In the current study, we investigated the role of Drosophila bHLH-O proteins in circadian rhythms at the molecular and behavior levels.

Materials and methods Fly strains Flies were raised on standard glucose-cornmeal medium under a 12:12 h light:dark (LD) cycle at 25°C. The expression of bHLH-O genes were knocked down by RNA interferrence (RNAi) in a tissue-specific manner (Matsumoto et al. 2007). The Gal4 line pdf-gal4 (Renn et al. 1999), a kind gift from Jeffrey C. Hall, was used as a driver line with UAS-IR lines established at the National Institute of Genetics as a repsonder line. Briefly, knockdown flies were obtained by mating homozygous Gal4 females (pdfgal4/pdf-gal4) to each of the homozygous UAS-IR males (UAS-IR/UAS-IR). Because the knockdown effect can vary depending on the insertion site of the UAS-IR transgene, we typically analyzed two UAS-IR lines for each gene. Double knockdown flies were obtained by mating Gal4 females carrying UAS-IR as a target (for example, pdf-gal4/pdfgal4; UAS-A-IR1/UAS-A-IR1) to each other UAS-IR homozygous male (UAS-B-IR1/UAS-B-IR1). The knockdown effect was confirmed at two additional aspects other than circadian period in one of the bHLH-O genes, Side. Firstly, the genetic cross between Act5Cgal4/CyO (Ito et al., 1997) and UAS-Side-IR gave no Act5C-gal4/UAS-Side-IR progeny in which the expression of Side was knocked down in potentially all tissues in development, meaning the level of knockdown in the progeny at developmental stage is strong enough to induce lethality. Secondly, the expression level of Side mRNA was measured to decrease to 65% of normal in heads where dsRNAs of Side were induced by tim(UAS)gal4 (Blau and Young 1999) by the quantitative RT-PCR using ABI PRISM 7300 (Applied Biosystems, Foster City, CA). Recording of locomotor activity rhythms The locomotor activity of individual adult flies was measured using Drosophila activity monitors (Trikinetics Inc., Waltham, MA) for 3 days under LD, and then for approximately 2 weeks under constant darkness (DD). For each measurement, a single male fly was introduced into a measuring glass tube containing 1.5% agar with 10% glucose. The period of an individual fly was calculated by

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v2 periodogram analysis (Sokolove and Bushnell 1978) using Clocklab software (Actimetrics, Chicago, IL). Vector construction and promoter assays in cultured Drosophila cells The coding sequences of bHLH-O genes were subcloned individually into pAc5.1B-V5/His (Invitrogen, Carlsbad, CA) using the EFC-cloning method (de Jong et al. 2006). All primers used for cloning are listed in Table 1. Cultured Drosophila S2 cells were plated in 24-well tissue culture plates in Shields and Sang M3 insect medium (Sigma, St Louis, MO) supplemented with 12.5% fetal bovine serum (Biowest, Canada) and antibiotics (12.5 U/ ml penicillin, 12.5 mg/ml streptomycin; GIBCO, Grand Island, NY). S2 cells were transfected by Effectene Transfection Reagent (QIAGEN, Hilden, Germany) with 100 ng of pAct-Clk as an activator (Darlington et al. 1998), 100 ng of tim-luc (Darlington et al. 1998), which contains a typical E-box (McDonald et al. 2001) showing a relatively high expression (Ueda et al. 2002), and 10 ng of pAc5.1Rluc (Matsumoto et al. 2007) as a positive control for luciferase activity, along with 100 ng of pAc5.1-cwo and/or 100 ng of pAc5.1-bHLH-O (Matsumoto et al. 2007). As needed, the empty vector pAc5.1B-V5/His was used instead of pAc5.1-cwo or pAc5.1-bHLH-O to ensure that an equal amount of DNA was used for transfection in each well. Forty-eight hours after transfection, luciferase assays of transfected cells were carried out using a PiccaGene Dual SeaPansy Luciferase Assay kit (Toyo Ink, Tokyo, Japan) according to the manufacturer’s instructions. Firefly luciferase activity was measured with a BioOrbit 1254 luminometer (Thermo BioAnalysis, Tokyo, Japan) and then normalized to Rluc activity as a control for transfection efficiency. Assays were performed at least three times.

Results SIDE, Mb and Mc as well as CWO suppress the transcription of E-box clock genes Promoter assays were carried out to screen for potential regulators of E-box clock gene transcription among the Drosophila bHLH-O genes (Fig. 1). Mb, Mc and SIDE suppressed transcription, with Mb and Mc exhibiting similar effects as CWO, which suppressed transcription by 60% as compared to the positive control (Fig. 1). It is noteworthy that the suppressive effect of SIDE was approximately eight times as strong as that of CWO (Fig. 1). These results raised the possibility that these three proteins are regulators of E-box clock genes as well as CWO.

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393

Table 1 Primer sequences used in the cloning of bHLH-O genes

Table 1 continued

Primer name

Primer name

Sequence

To amplify hairy

To amplify Her

0

5 -GGAGACATCCGAAAATGGTTACCGGCGTAACA-3 0

5 -CGGGAAAGTCTACCAGGGCCGCCAGGGCTGC-3

0

0

To amplify dpn 50 -TACAAAATGGATTACAAAAACG-30

mc ATG-6

50 -CACAAAATGTCGTCGCTACAAATG-30

mc Stop

50 -CTACCAGGGACGCCAGACGTTC-30

mc ATG-6 EFC1

50 -GGAGACATCACAAAATGTCGTCGCTACAAATG-30

mc Stop EFC2

50 -CGGGAAAGTCTACCAGGGACGCCAGACGTTC-30 50 -TACACAATGGCCGTTCAGGGTC-30 50 -TCACCAGGGGCGCCAGACTTCTT-30

md ATG-6 EFC1

50 -GGAGACATTACACAATGGCCGTTCAGGGTC-30

md STOP EFC2

0

5 -CGGGAAAGTTCACCAGGGGCGCCAGACTTCTT-3

0

To amplify m3 m3 ATG-6

50 -TCGATCATGGTCATGGAGATGTC-30

m3 Stop

50 -TTACCAGGGCCTCCAGACGGGCT-30

m3 ATG-6 EFC1

50 -GGAGACATTCGATCATGGTCATGGAGATGTC-30

m3 Stop EFC2

50 -CGGGAAAGTTTACCAGGGCCTCCAGACGGGCT-30

To amplify m5 m5 ATG-6

50 -TACAAAATGGCACCACAGAGCA-30

m5 STOP

50 -TTACCAAGGGCGCCACATGGTTT-30

m5 ATG-6 EFC1

50 -GGAGACATTACAAAATGGCACCACAGAGCA-30

m5 STOP EFC2

50 -CGGGAAAGTTTACCAAGGGCGCCACATGGTTT-30

*

0.4 0.2

*

0

C

md ATG-6 md STOP

*

0.6

W O

To amplify md

0.8

ey

To amplify mc

1.0

er

50 -CGGGAAAGTTCACCAGGGACGCCACATGGGG-30

H

5 -GGAGACATAACAAAATGGTTCTGGAAATGG-3

mb Stop EFC2

8

mb ATG-6 EFC1

1.2

H

0

7

0

M

50 -TCACCAGGGACGCCACATGGGG-30

1.4

5

To amplify mb

M

50 -CGGGAAAGTTCACCAGGGTCGCCACATGCT-30

M

Side Stop EFC2

δ

50 -GGAGACATTGAGAAAATGACAGCAAAACGTG-30

50 -ACTTTCCCGGAAGGTAAGCCTATCCCTAA-30

3

Side ATG-6 EFC1

V5-S2 ECF2

M

50 -TCACCAGGGTCGCCACATGCT-30

50 -GAAGGTAAGCCTATCCCTAA-30

γ

Side Stop

V5-S2

M

50 -TGAGAAAATGACAGCAAAACGTG-30

β

Side ATG-6

50 -CTGATGGAGCGGCTTTGTGTC-30 50 -ATGTCTCCCTGATGGAGCGGCTTTGTGTC-30

M

To amplify Side

pAc5.1B-forA pAc5.1B-forA ECF1

E

5 -CGGGAAAGTCTACCACGGCCTCCAAGCGGAGC-3

0

M

0

mb Stop

50 -CGGGAAAGTCTACCAGGGCCTCCAGAGATTTG-30

D

50 -GGAGACATTACAAAATGGATTACAAAAACG-30

50 -AACAAAATGGTTCTGGAAATGG-30

Her Stop EFC2

ai ry

5 -CTACCACGGCCTCCAAGCGGAGC-3

dpn ATG-6 EFC1

mb ATG-6

50 -GGAGACATAGGCTTATGATGGCACGTCCCGAG-30

To amplify pAc5.1B

0

dpn Stop dpn Stop EFC2

Her ATG-6 EFC1

SI

0

50 -AGGCTTATGATGGCACGTCCCGAG-30 50 -CTACCAGGGCCTCCAGAGATTTG-30

pn

dpn ATG-6

Her ATG-6 Her Stop

H

50 -CTACCAGGGCCGCCAGGGCTGCT-30

D

hairy STOP

Relative luminescence unit (RLU)

50 -CCGAAAATGGTTACCGGCGTAACA-30

hairy STOP EFC2

50 -CGGGAAAGTTCAATAGGCCATCTCGGCGCCCC AA-30

Hey STOP EFC2

hairy ATG-6 hairyATG-6 EFC1

Sequence

Fig. 1 The suppressive effect of bHLH-O proteins on tim transcription as measured by luciferase reporter gene assay. Relative luciferase activity of tim-luc in the presence of 100 ng of pAc-Clk and the indicated pAc5.1B-bHLH-O expression plasmids. Signals were normalized to Rluc activity and then to the control signal of cells transfected with 100 ng of tim-luc and pAc-Clk. Error bars represent SEM (n = 3). SIDE, Mb and Mc suppressed the transcription of E-box genes. Asterisks represents p \ 0.01 versus control (Dunnett’s multiple range test)

Flies with single knockdown of Side, mb and mc showed a normal rhythm

To amplify m7 m7 ATG-6

50 -AACACAATGGCCACCAAATACG-30

m7 Stop

50 -TTACCAGGGACGCCACACCTGTC-30

m7 ATG-6 EFC1

50 -GGAGACATAACACAATGGCCACCAAATACG-30

m7 Stop EFC2

50 -CGGGAAAGTTTACCAGGGACGCCACACCTGTC-30

To amplify m8 m8 ATG-6

50 -ACAAAAATGGAATACACCACCAAG-30

m8 Stop

50 -TTACCAGGGGCGCCACAAGGGCT-30

m8 ATG-6 EFC1

50 -GGAGACATACAAAAATGGAATACACCACCA-30

m8 Stop EFC2

50 -CGGGAAAGTTTACCAGGGGCGCCACAAGGG-30

To amplify Hey Hey ATG-6

50 -CCACAAATGGATCACAACATGC-30

Hey STOP

50 -TCAATAGGCCATCTCGGCGCCCCAA-30

Hey ATG-6 EFC1

50 -GGAGACATCCACAAATGGATCACAACATGC-30

We next examined the locomotor activity rhythms of the bHLH-O gene knockdown flies (Table 2). The pdf-gal4 flies as the negative control exhibited a normal period of about 24 h, while the cwo knockdown flies as the positive control exhibited a longer period of over 25 h (Table 2; Fig. 2). The difference in mean period compared to that of pdf-gal4 was within 1 h for almost single knockdown lines, although one each of the m7 knockdown line had a slightly longer period than pdf-gal4 (Dunnett’s multiple range test, p \ 0.01). These results imply that among the bHLH-O proteins, CWO predominantly affects locomotor activity rhythm in vivo. However, we could not exclude the

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Table 2 The circadian periods of single knockdown flies by pdf-gal4 Subfamily

Strain

Period (mean ± SEM) h

NR

NA

Control

pdf-gal4

24.17 ± 0.07

24

1

Stra13

cwo-IR3

25.70 ± 0.12*

62

0

hairy

dpn-IR4

24.41 ± 0.14

15

0

E(spl)

Hey

h-IR1

24.01 ± 0.07

22

0

h-IR2

24.02 ± 0.14

19

0

Side-IR1

24.10 ± 0.09

13

0

Side-IR2 mb-IR2

24.48 ± 0.20 24.56 ± 0.12

13 12

0 2

mb-IR4

24.07 ± 0.13

25

0

mc-IR1

23.99 ± 0.06

17

0

mc-IR4

24.33 ± 0.11

19

0

md-IR1

24.09 ± 0.09

14

2

md-IR2

24.00 ± 0.08

16

0

m3-IR1

24.08 ± 0.11

14

1

m3-IR3

24.43 ± 0.15

20

2

m5-IR1

24.20 ± 0.13

15

3

m7-IR1

24.95 ± 0.11*

17

3

m7-IR2

24.51 ± 0.15

16

0

m8-IR1

24.17 ± 0.10

20

0

m8-IR3

24.46 ± 0.07

21

0

Her-IR2

23.95 ± 0.09

16

0

Her-IR3 Hey-IR1

24.35 ± 0.13 24.33 ± 0.11

29 23

0 0

Hey-IR3

24.35 ± 0.20

13

0

NR number of rhythmic flies recorded, NA number of arrhythmic flies recorded * Significantly different from the period of pdf-gal4 (Dunnett’s multiple range test, p \ 0.01)

pdf-gal4 (24.4h)

24

24

12

12

Time of day

cwo-IR3 (26.0h)

24

LD

LD

DD

DD

24

12

24

12

24

Time of day

Fig. 2 cwo knockdown flies show a longer period phenotype. Actograms of pdf-gal4 (left panel) as a control and cwo knockdown flies (right). The number in parentheses represents the free-running period of the corresponding fly. Adult flies were entrained in LD cycle for 3 days, and then kept in constant darkness (DD). The horizontal bars in white and black represent the light and dark regime of the LD cycle, respectively. White vertical bar indicates LD; black vertical bar indicates DD

possibility that bHLH-O proteins could interact together to functionally complement each other in the regulation of circadian rhythms.

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The suppressive effects in combination among SIDE, Mb, Mc and CWO in vitro To determine whether any of the bHLH-O proteins interacted with each other to regulate the transcription of E-box clock genes, we co-transformed S2 cells with different pairs of bHLH-O expression vectors and then measured luciferase activity using the tim-luc reporter gene. The suppressive effects of CWO plus Mb, CWO plus Mc, and Mb plus Mc were significantly different from those of only, CWO, Mb and Mc (Table 3; Dunnett’s multiple range test, p \ 0.05). However, there was no evidence of a synergistic effect of co-transfection with combinations of CWO, Mb and/or Mc, and the suppressive effects of CWO plus Mb, CWO plus Mc, and Mb plus Mc were consistent with an additive effect of these combinations. While the effects of CWO plus SIDE, SIDE plus Mb and SIDE plus Mc were significantly different from those of only CWO, Mb and Mc (Dunnett’s multiple range test, p \ 0.01), the effects were not significantly different from those of only SIDE (Table 3). The suppressive effect of SIDE was too strong to prohibit an evaluation of any interactions between SIDE and the other bHLH-O proteins (Table 3). These results suggest that CWO, Mb and Mc function independently to regulate the transcription of E-box clock genes in vitro, and the suppressive effect of SIDE is completely epistatic over the other three. bHLH-O genes altered circadian periods in combinational knockdown with cwo To determine whether the bHLH-O proteins interacted in vivo to functionally complement each other, we generated double knockdown flies of all combinations of the four genes and then measured their locomotor activity rhythms. Double knockdown flies of cwo with any of the other three bHLH-O genes (Side, mb and mc) exhibited longer periods than their parental strains, including cwo single knockdown flies (Tables 2, 4; Fig. 3; Dunnett’s multiple range test, p \ 0.01). In the case of double knockdown of mb and mc, a slightly longer period was observed as compared to the periods of parental strains (Tables 2, 4; Fig. 3; Dunnett’s multiple range test, p \ 0.05). Double knockdown flies of Side with either mb or mc exhibited normal rhythmicity (Tables 2, 4; Fig. 3). Thus, three bHLH-O genes, Side, mb and mc, of which the single knockdown fly exhibited normal rhythmicity, could be classified into two groups by the phenotypes in combinational knockdowns among these. One is mb and mc, which affects the circadian period in double knockdown, and the other is Side, showing no effect in any case.

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Table 3 Comparisons of transcriptional suppressions by combinations of CWO, SIDE, Mb and Mc to those by single expressions as measured by luciferase assay Combinational expression

Single expression CWO (0.80 ± 0.07)

SIDE (0.01 ± 0.00)

Mb (0.79 ± 0.04)

Mc (0.48 ± 0.06)

CWO plus SIDE (0.08 ± 0.01)

p \ 0.01

n.s.

–

–

CWO plus Mb (0.42 ± 0.06)

p \ 0.01

–

p \ 0.01

–

CWO plus Mc (0.21 ± 0.02) SIDE plus Mb (0.01 ± 0.00)

p \ 0.01 –

– n.s.

– p \ 0.01

p \ 0.05 –

SIDE plus Mc (0.01 ± 0.00)

–

n.s.

–

p \ 0.01

Mb plus Mc (0.29 ± 0.00)

–

–

p \ 0.01

p \ 0.05

Number in parentheses under the sample name shows the mean relative luciferase activity of tim-luc ± SEM (N = 3) The luciferase activity was normalized to Rluc activity and then to the control signal of cells transfected with 100 ng of tim-luc and 100 ng of pAc-Clk without pAc5.1-bHLH-O Statistical analysis was done by Dunnett’s multiple range test n.s. not significant, – not determined

Table 4 Comparisons of the circadian period of double knockdown strain to those of its parent strains in pdf-gal4 background Double knockdown stain

Parental strain cwo-IR3 (25.70 ± 0.12 h, NR = 62, NA = 0)

Side-IR2 (24.48 ± 0.20 h, NR = 13, NA = 0)

mb-IR2 (24.56 ± 0.12 h, NR = 12, NA = 2)

mc-IR1 (23.99 ± 0.06 h, NR = 17, NA = 0)

cwo-IR3 9 Side-IR2 (26.88 ± 0.21 h, NR = 23, NA = 0)

p \ 0.01

p \ 0.01

–

–

cwo-IR3 9 mb-IR2 (26.50 ± 0.21 h, NR = 23, NA = 0)

p \ 0.01

–

p \ 0.01

–

cwo-IR3 9 mc-IR1 (26.13 ± 0.11 h, NR = 32, NA = 0)

p \ 0.05

–

–

p \ 0.01

Side-IR2 9 mb-IR2 (24.21 ± 0.05 h, NR = 36, NA = 0)

–

n.s.

p \ 0.05

–

Side-IR2 9 mc-IR1 (24.42 ± 0.07 h, NR = 23, NA = 4)

–

n.s.

–

p \ 0.01

mb-IR2 9 mc-IR1 (24.92 ± 0.09 h, NR = 26, NA = 6)

–

–

p \ 0.05

p \ 0.01

Numbers in parentheses under the strain name shows the mean period ± SEM, numbers of rhythmic and arrhythmic flies as NR and NA, respectively Statistical analysis was done by Dunnett’s multiple range test n.s. not significant, – not determined

Discussion Here, we showed that the bHLH-O genes are involved in the regulation of circadian rhythms in Drosophila. SIDE, CWO, Mb and Mc suppress the promoter activity of the E-box clock gene in vitro. The single knockdown of cwo, that of m7, and the double knockdown in combinations among cwo, mb, mc and Side altered the circadian period in vivo. Among these genes, considering the effectiveness of cwo in vitro and in vivo, it is possible that CWO is

predominantly involved in the generation of circadian rhythms. Co-transfection of Mb and Mc had a suppressive effect in vitro (Table 3), and the corresponding double knockdown flies showed a longer period (Table 4). These results suggest that Mb and Mc are involved in a generation of circadian rhythms through suppression of E-box clock gene expression. However, their single knockdown flies showed a normal rhythmicity. This can be explained if we assume that Mb and Mc functionally complement to each other.

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cwo-IR3 x Side-IR2 (26.3h) LD

Side-IR2 x mγ -IR1 (24.3h)

LD DD

DD

24

12

24

12

24

24

Time of day cwo-IR3 x mγ -IR1 (25.7h)

LD

12

24

12

24

Time of day Side-IR2 x mβ -IR2 (24.3h)

LD

DD

24

12

24

12

24

DD

24

12

Time of day

24

12

24

Time of day

cwo-IR3 x mβ -IR2 (26.1h)

LD

mγ -IR1 x mβ -IR2 (25.4h)

LD DD

DD

24

12

24

12

Time of day

24

24

12

24

12

24

Time of day

Fig. 3 Actograms of bHLH-O gene double knockdown flies. The knockdown genes are designated by the mating scheme above each actogram. The number in parentheses represents the period of the corresponding fly. Adult flies were entrained to the LD cycle for 3 days and then kept in constant darkness (DD). The horizontal bars in white and black represent the light and dark regime of the LD cycle, respectively. White vertical bar indicates LD; black vertical bar indicates DD. The effect of double knockdown of cwo with any other gene was stronger than that of the corresponding single knockdown parental strains (see Tables 2 and 4). mb and mc double knockdown flies exhibited a slightly longer period than the corresponding single knockdown parental lines

The other possibility is that Mb and Mc are involved in the development of pacemaker neurons through regulation of E-box genes. m7 knockdown flies had a slightly longer period phenotype of close to 25 h (Table 2). Although we cannot exclude the possibility that m7 is involved in the regulation of circadian rhythms, the effect is likely to be indirect, because M7 had no effect on clock gene promoter activity in vitro (Fig. 1). SIDE showed the most remarkable effect among the bHLH-O proteins in vitro (Fig. 1; Table 3). However, the period of locomotor rhythms was normal in Side knockdown flies (Table 2), in agreement with the result of the previous study (Nagoshi et al. 2010). Double knockdown flies of Side and any other bHLH-O gene, with the exception of cwo, also exhibited normal rhythmicity (Table 4). Interestingly, Side mRNA is reported to enrich 44.9-fold in clock cells as compared to other neurons in the adult brain (Nagoshi et al. 2010). Although we cannot estimate the exact knockdown level of Side in pacemaker neurons, our results lead us to assume two working hypotheses about the function of Side to E-box genes. One is that the functional expression level of Side in pacemaker neurons may be so low that circadian rhythms could not be

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affected in our knockdown experiment. Another hypothesis is that SIDE may be related to E-box clock genes only in development. In this case, there may be a compensatory molecular mechanism that counteracts the suppressive effects of SIDE on the expression of clock genes in pacemaker neurons of adults. There are several reports that bHLH-O proteins are pivotal during Drosophila development (Davis and Turner 2001). Thus, one of the next important issues is to distinguish a direct effect on circadian transcription by bHLH-O genes from an indirect one in development. vri and Pdp1, both of which are E-box clock genes having an effect on development as well as on the generation of circadian rhythms, are expressed at a certain embryonic stage (George and Terracol 1997; Lin et al. 1997). per has also been reported to be expressed in the embryonic CNS, with no detectable expression until the first instar larvae stage (Siwicki et al. 1988). CWO may heterodimerize to function in circadian regulation because proteins of the bHLH-O family in Drosophila have been reported to heterodimerize with each other to suppress transcription in various biological processes through binding to E-box sequences (Davis and Turner 2001). Based on our results in vivo and in vitro, SIDE, Mb and Mc are the potential primarily candidates for the partner of CWO. The other possibility is that CWO may homodimerize to function in circadian regulation. In this case, because CWO does not have a WRPW motif, which mediates interactions of bHLH-O proteins with various co-factors, including GRO/TLE (Fisher et al. 1996; Grbavec and Stifani 1996; Fisher and Caudy 1998), CWO could suppress the transcription of clock genes without co-factors, possibly by acting as a competitor of CLK/CYC for binding to the E-box. Alternatively, there may be an as-yet unidentified domain within CWO that interacts with co-factors that mediate transcriptional suppression. In mammals, the cwo homologs Dec1 and Dec2 have been shown to be components of the circadian clock (Honma et al. 2002). However, the molecular mechanism by which DEC1 and DEC2 suppress the transcription of clock genes through E-box sequences is unknown. For example, it is unclear whether DEC1 and DEC2 recruit co-factors, or whether DEC1 and DEC2 heterodimerize to bind to the E-box. Further analyses are needed to clarify the molecular mechanisms of transcriptional suppression by CWO orthologs in circadian clock regulation, not only in Drosophila, but in mammals as well. Our results have a significant contribution to issues in applied entomology as well, especially the sterile insect technique (SIT), because the mating activity of the target insects of SIT sometimes reveals circadian rhythms (Miyatake 2011). In the melon fly, Bactrocera cucurbitae, the molecular analyses of clock genes to control the mating

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rhythm have been elucidated based on the molecular information in Drosophila (Matsumoto et al., 2008; Fuchikawa et al. 2010). Since bHLH-O family genes are essential in animals (Davis and Turner 2001), our results can be applied to the molecular analysis of circadian rhythms in the melon fly and other insects as an SIT target. Acknowledgments We thank Jeffrey C. Hall for the pdf-gal4 lines and Steve A. Kay for the pAct-Clk and tim-luc plasmids. UAS-IR strains were obtained from Genetic Strains Research Center, National Institute of Genetics. We thank Kiyo Kimura, Makiko Haruta and Kyoko Sakamoto for technical assistance, Yoshitaka Fukada and Masami Shimoda for helpful discussion, and Kenji Tomioka for critical reading of an earlier version of this manuscript. This work is supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to A. M. and T. T.

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