s u p p l e m e n ta r y i n f o r m at i o n

2

3

4

5

Repressor

6000000020000023451200000

2xUAS

150000RRE

11000004000000

50000
0CT17.71 ± 0.19
0

1

2

3

Day

4

5

Bioluminescence (counts/min)

00600000Relative Amplitude

Relative Amplitude

0.6

0200000100000

0.2
0

Day

4

5

1

2

3

4

5

0

2

3

2

3

4

5

3xRRE

800000

-1

40

24

30

CT2.6 ± 0.09

16

20

0

1

2

3

4

5

8

10

2xRRE

0

0

12

24

36

48

0

Time (h)1000000
5000004000000CT16.8 ± 0.08
0

1

2

3

4

0

5

2400002000001200001000000CT8.4 ± 0.14
01

CT16.7 ± 0.02
0300000

UAS

2

3

4

0

5

1

2

5

5xUAS

4

5

CT9.5 ± 0.21
0

1

2

3

Day

Relative Amplitude
2xCCE

0.4

3xCCE

0.3

4

32

40

24

30

16

20

8

10

0
0

12

24

36

48

Time (h)05Using dGAL4-VP16-Flag
Tbp-5’ ChIP product by anti-Flag
Tbp-5’ ChIP product by anti-V5
LUC activity
Using dGAL4-Flag
Tbp-5’ ChIP product by anti-Flag
Tbp-5’ ChIP product by anti-V5
LUC activity

0.2
0.1
0

1500000

3

UAS

Day

CT8.58 ± 0.171401500000

0.5

0

32

3

1

4xUAS

2000000
30360000CT9.09 ± 0.06
02500000
2

5

CT2.3 ± 0.09

300000

3xUAS
5xUAS

4

200000

2xUAS
4xUAS

0.4

3

400000400000

1xUAS

2

3xUAS

5000000.8

1Output2000000

3000006000000

Bioluminescence (10 counts min )1

1200000

Relative Amplitude

00

2xE’-box

600000-1

CT3.45 ± 0.08

Repressor

0

3xE’-box

1800000

3

40000

900000

Bioluminescence (10 counts min )2000000

1xUAS
Activator

80000

c
2400000

ChIP abundance (relative to Act-5’ )Bioluminescence (counts/min)E’-box

4000000b

Output

120000ChIP abundance (relative to Act-5’ )Activator

6000000

Bioluminescence (counts/min)

a

2

3

Day

4

5

1000000
5000000CT8.53 ± 0.23
0

1

2

3

4

5

Day

Supplementary Figure S1

Figure S1 Evaluating our in cellulo mammalian cell culture system. a,
Determination of number of UASs in the artificial promoter to regulate theoutput reporter gene. We first constructed reporter vectors, varying the number
of UAS (from 1 through 5) on the promoter driving a reporter gene (dLuc).
We transfected them with a morning activator and a night-time repressor into
NIH3T3 cells and then monitored the bioluminescence from the cells. The raw
bioluminescence data from two independent samples are shown. The relative
amplitudes of each output signal from two independent samples are also
shown (lower left panel). b, Determination of number of CCEs in the artificial
promoter to regulate the activator and repressor genes. We first constructed
two sets of vectors with two and three copies of the clock-controlled elements(CCE; E’-box from the Per2 gene, or RRE from the Bmal1 gene) on promotersdriving a reporter gene (dLuc), an artificial activator gene (dGal4-VP16) or an
artificial repressor gene (dGal4). We then monitored their promoter activities(either 2× or 3× E’-box, and either 2× or 3× RRE) as well as the output

Ukai-Tadenuma et al.

(UAS) from artificial transcriptional circuits with a morning activator and anight-time repressor. The raw bioluminescence data from two independent
samples are shown. The relative amplitudes of each output signal from two
independent samples are also shown (lower left panel). c, Negative controls
for quantification of GAL4-VP16-FLAG and GAL4-FLAG bindings to the UAS
in the artificial transcriptional circuits. After normalization for the amount of
input DNA, the amount of Tbp promoter (Tbp-5’) region immunoprecipitatedby anti-FLAG antibody (squares) and by anti-V5 antibody (triangles) were
quantified relative to a constitutively unbound region (Act-5’). These relative
amounts of Tbp-5’ ChIP products are indicated along with the LUC activity(line) for two different artificial transcriptional circuits using dGAL4-VP16FLAG (green) and dGAL4-FLAG (magenta). ChIP assays were performed withthe artificial transcriptional circuit (morning activator and night-time repressor)
in NIH3T3 cells at 4-h intervals for 24 h with anti-FLAG antibody and anti-V5
antibody as negative control.

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1© 2008 Macmillan Publishers Limited. All rights reserved.

s u p p l e m e n ta r y i n f o r m at i o n

b

2

3

4

5

Output

1

2

3

4

5

1800000

9000001200000

600000300000600000

RRE
0

1

2

3

4

5

0
900000200000600000100000
0

1

2

3

4

SV40
0

1

2

3

4

5

75000
50000
25000
0

5

TK
0

1

2

3

4

5

Day

300000100000

UAS
0

1

2

3

4

5

0

3000000

2400000

Output200000E’-box03000000D-box
02400000

1200000

00

morning (E’ -box)

1

day-time (D-box)

E’-box01000000

UAS
0

1

2

Day

3

4

5

SV40

180000500000120000250000

60000

0

0

1

30000

16000

20000

8000

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30000

1000000
0

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1800000

0

5

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3

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0

1

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5

16000
10000

0

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0

5

24000045000

160000

30000

80000

15000

8000
0

1

2

3

4

5

0

32000
24000
16000

1200000
600000
0

0

24000

20000

60000
0

0
32000

120000

0

1

2

3

4

0

5

0

1

2

Day

3

4

0

5

8000
0

1

2

3

4

5

0

Day

Day

Day

2000000

2400000

6000006000001500000

1800000

4000004000001000000

1200000

200000
0

0

1

2

3

4

500000200000

RRE
5

0

RRE
0

1

2

3

4

5

0

1800000

7500004500000

1200000

5000003000000

6000002500001500000

600000E’-box
0

1

2

3

4

5

0

Activator
Repressor

800000

D-box
0

1

2

3

4

5

320000240000160000

0

D-box
0

1

2

3

4

5

0

E’-box
0

1

2

3

4

5

300000

0

1

2

3

4

5

0

3000000

15000000

4500000

2000000

10000000

3000000

1000000

5000000

0E’-box
0

1

2

3

0

1

2

3

4

5

4

0

5

1500000

D-box
0

1

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3

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5

0

3000000

2400000

2400000

2000000

1600000

1600000

1000000

800000

RRE
0

UAS
0

1

2

3

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5

0

0

RRE
0

1

2

3

0

1

2

3

4

5

800000

UAS
1

2

3

4

5

0

UAS

Day

Day

24000012000020000045Day160000

80000

100000100000

50000
0

80000

D-box

1600002000001500000

Bioluminescence (counts/min)

800000

OutputActivator

0

5

24000

d
Bioluminescence (counts/min)

Repressor

4

TK

32000

40000

180000Day

250000

Output

3

SV40

50000

240000

c

UAS
0

1

2

3

Day

4

5

0

80000

40000

UAS
0

1

2

Day

3

4

5

0

UAS
0

1

2

3

4

5

0

UAS
0

1

Day

Figure S2 Raw bioluminescence data from artificial transcriptional circuitswith various combinations of activators and repressors. a, Synthesis of daytime and night-time expressions in two different artificial transcriptional
circuits. Promoter activities of an activator, repressor, and output reporterin two different artificial transcriptional circuits: (left) morning activator
under E’-box control and night-time repressor under RRE control, and(right) day-time activator under D-box control and morning repressor
under E’-box control. b, High-amplitude transcriptional oscillation cannot
be generated by an activator or a repressor alone. (left) The activities of
the SV40 promoter and thymidine kinase (TK) promoter. The promoter
activities were monitored by bioluminescence from NIH3T3 cells, whichwere transfected SV40-dLuc and TK-dLuc. The transcriptional activityof TK promoter indicates lower expression than SV40 promoter (1:7.28± 0.70). (right) Promoter activities of the output reporter in six different
artificial transcriptional circuits. The promoters used to regulate theactivator and repressor genes were SV40 promoter (SV40, left panels) and
TK promoter (TK, right panels): morning activator or repressor alone (upper2

2

Repressor only
TK

2400007500002000000

Output02000000

300000

OutputBioluminescence (counts/min)

1000000

Bioluminescence (counts/min)

6000000

2000000

Bioluminescence (counts/min)

Activator

3000000

1500000

Repressor

40000010000000

4000000

Activator only

night-time (RRE)

a

2

Day

3

4

5

Supplementary Figure 2

Ukai-Tadenuma et al.

panels), day-time activator or repressor alone (middle panels) and nighttime activator or repressor alone (lower panels). c, Synthesis of various
phases from three basic circadian phases in artificial transcriptional
circuits. Promoter activities of an activator, a repressor, and an output
reporter in four different artificial transcriptional circuits: (left) nighttime activator under RRE control and day-time repressor under D-box
control, (middle left) night-time activator under RRE control and morning
repressor under E’-box control: (middle right) morning activator underE’-box control and day-time repressor under D-box control: and (right)
day-time activator under D-box control and night-time repressor under
RRE control. d, Promoter activity in artificial transcriptional circuits witha repressor in phase with an activator. Promoter activities of an activator,
repressor, and output reporter in three different artificial transcriptional
circuits: (left) morning activator and repressor both under E’-box control:(middle) day-time activator and repressor both under D-box control: and(right) night-time activator and repressor both under RRE control. The raw
bioluminescence data from two independent samples are shown.

www.nature.com/naturecellbiology© 2008 Macmillan Publishers Limited. All rights reserved.

s u p p l e m e n ta r y i n f o r m at i o nabRepressor : E’-box

Activator : RRE

20.6

22

22.2

24/0

20

RRE

20

4.7
18

10

16
14

4
6

RRE

x

12

x

'bo

E
E/

Activator : E’-box

8

2

bo

14

24/0

D-

10

16

x

12

4
6

bo

x

14

8

x

D-

bo

16

Activator : D-box

22

'bo

E
E/

18

Repressor : RRE

2

20

4
6

RRE

24/0

D-

16.6

x

'bo

E
E/

18

Activator : RRE 222c

Repressor : D-box

12

10

8

7.7

Activator : E’-box

11.0

Activator : D-box

Supplementary Figure 3

Figure S3 Transcriptional mechanisms to generate novel phases. Output
phases in artificial transcriptional circuits with a morning repressor(a), day-time repressor (b), or night-time repressor (c). The phase of an
output reporter (magenta lines and numbers) is indicated in circadianUkai-Tadenuma et al.

time (h) with the activator used (bold black). In artificial transcriptional
circuits in cellulo, the output phase could be changed according to
the input phase of the activator, even if that of the repressor was
unchanged.

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3© 2008 Macmillan Publishers Limited. All rights reserved.

s u p p l e m e n ta r y i n f o r m at i o n

Phase of repressor b
10

5

5

0

0

-5

-5

-10

Mean=1.01h-0.5 (h)-10Mean=0.55h

0

-5

-5

-5

-10

Mean=2.40h

Mean=2.86h-10Efficiency of transcription V1

Efficiency of transclation V2

Binding affinity of repressor 1/K R
10

10

10

10

5

5

5

5

5

5

0

0

0

0

0

0

-5

-5

-5

-5

-5

-5

Mean=2.80h-10

Mean=0.09h

10

5

5

0

0

Mean=2.24h-10

Mean=2.06h

Circadian expression of Repressor (R , R )

10

10

5

5

0

0

-5

-5

Phase of activator a-10

Mean=0h

Mean=0h

-10

-10
-5
0
5
10
Activator - Repressor (h)Phase of activator a
Phase of repressor b
Amplitude of activator α A
Amplitude of repressor α R
Baseline constant of activator γ A
Baseline constant of repressor γ R
Circadian expression of activator (α A,γ A )Circadian expression of repressor (α R, γ R)
Binding affinity of activator 1/ KA
Binding affinity of repressor 1/KR
Basal transcription β
Efficiency of transcription V1
Efficiency of transclation V2
Half-life of mRNA T1
Half-life of protein T2

0.5

1
1.5
Sensitivity (h)

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

Mean=0.10h

Phase of repressor b

Amplitude of activator A-0.5 (h)×0.5

Amplitude of repressor R

0.8

0.7
0.6

0.8

0.6

0.5
0.4

0.6

0.4

0.4

0.3
0.2

Baseline constant of activator A

0.2

0.2

0.1

0

+0.5 (h)

-10
-5
0
5
10
Activator - Repressor (h)

×2

-10
-5
0
5
10
Activator - Repressor (h)Binding affinity of repressor 1/K R

0

0

0
-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)Basal transcription 

Efficiency of transcription V1

Efficiency of transclation V2

Baseline constant of repressor R

Binding affinity of activator 1/K A

0.6

0.7

0.7

0.6

0.6

0.6

0.6

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0.5

0.5

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0

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0

0

0

0

-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)Half-life of protein T2

Half-life of mRNA T1
0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0

0
-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)Supplementary Figure 4

-10
-5
0
5
10
Activator - Repressor (h)

d

0.1

0.1

2

-10
-5
0
5
10
Activator - Repressor (h)

0.6

0.5

Mean=0h

-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)0-10Mean=0.09h

-10
-5
0
5
10
Activator - Repressor (h)

0

b

-10
-5
0
5
10
Activator - Repressor (h)Circadian expression of Activator (A , A)-10

Mean=0.10h-5-5Basal transcription 

-10
-5
0
5
10
Activator - Repressor (h)Half-life of protein T2

10

-10

-10

-10
-5
0
5
10
Activator - Repressor (h)Mean=2.38h

-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)Relative amplitude

Output - Activator (h)0Binding affinity of activator 1/K A

-10
-5
0
5
10
Activator - Repressor (h)Output - Activator (h)

5

0

10

-10

Relative amplitude

5

10

Half-life of mRNA T1

0.1
0
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-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)

10
5
0
-5
-10
Activator - Repressor (h)Phase of activator a
Phase of repressor b
Amplitude of activator α A
Amplitude of repressor α R
Baseline constant of activator γ A
Baseline constant of repressor γ R
Circadian expression of activator (α A,γ A )Circadian expression of repressor (α R, γ R)
Binding affinity of activator 1/ KA
Binding affinity of repressor 1/KR
Basal transcription β
Efficiency of transcription V1
Efficiency of transclation V2
Half-life of mRNA T1
Half-life of protein T2

Ukai-Tadenuma et al.

Figure S4 Sensitivity of the output phase and the relative amplitude to eachparameter. a, The sensitivity of the output phase over -12 < a-b < 12 isshown when each parameter value was changed by 1 hour (a and b) or 4-fold(the others). X-axis represents the advance of a repressor compared to an
activator (h) (phase difference between an activator and repressor), while
Y-axis represents the delay of an output reporter compared to an activator(h) (phase difference between an output reporter and activator). The effectsof simultaneous increases in both absolute amplitude and base-line levelsof an input activator (αA and γA) or repressor (αR and γR) are also shown inthe boxed-panel (inbox). The black curve shows a simulation model withrepresentative parameter values of one hundred fitted parameter sets; theother curves show changed values for sensitive analysis. b, The simulatedsensitivity of the output phase to the parameter describing each process.
Each bar shows the average phase difference obtained by changing its
original parameter value by 1 hour (parameter a and b) or 4-fold (other

4

10

5

-10

×2

Baseline constant of activator A

10

-10
-5
0
5
10
Activator - Repressor (h)

-10
-5
0
5
10
Activator - Repressor (h)Relative amplitude+0.5 (h)Amplitude of repressor R

10

Baseline constant of repressor R-10

Relative amplitude

×0.5

Parameters

Output - Activator (h)

-10
-5
0
5
10
Activator - Repressor (h)cAmplitude of activator A

Output - Activator (h)Phase of activator a
10

Parameters

Output - Activator (h)

a

0

0.05

0.1
0.15
0.2
0.25
Sensitivity (Relative Amplitude)

0.3

parameters). Each error bar represents standard error. These values were
calculated from 100 simulations with trimming of the 5% outliers. c, Thesensitivity of the relative amplitude over -12 < a-b < 12 is shown when eachparameter value was changed by 1 hour (a and b) or 4-fold (the others).
X-axis represents the advance of a repressor compared to an activator(h) (phase difference between an activator and repressor), while Y-axis
represents the relative amplitude of oscillation of output protein (dLUC)
activity. The black curve shows a simulation model with representative
parameter values of one hundred fitted parameter sets; the other curves
show changed values for sensitive analysis. d, The simulated sensitivityof the relative amplitude to the parameter describing each process. Eachbar shows the average relative amplitude difference obtained by changing
its original parameter value by 1 hour (parameter a and b) or 4-fold (otherparameters). Each error bar represents standard error. These values were
calculated from 100 simulations with trimming of the 5% outliers.

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Supplementary Text

Determination of number of the galactose upstream activating sequences.
The number of the galactose upstream activating sequences (UASs) was determined by
the following experiments.

We constructed reporter vectors with varying numbers of

UAS (from 1 through 5) on the promoter driving dLuc output, reporter, transfected thevector into NIH3T3 cells and then monitored bioluminescence from the transfected cells(Supplementary Fig. 1a).

We observed that reporter vectors with 1 or 2×UAS did notexhibit high-amplitude oscillations, while those with 3, 4, and 5×UAS produced
high-amplitude oscillations in comparable phases.

In this manuscript, we adopt

4×UAS because outputs with 4×UAS results in the relative amplitudes with the smallest
variation (Supplementary Fig. 1a lower left).

Determination of number of the clock-controlled elements.
To determine the number of clock-controlled elements (CCE) that we should use todrive activator/repressor expression, we constructed two sets of vectors containing
either two or three copies of the CCEs on promoters driving dLuc reporter or artificial
activators/repressors, and monitored their promoter activities and transcriptional output(Supplementary Fig. 1b).

Via bioluminescence, we observed that promoter activities

and outputs for both 2 and 3×CCE exhibited comparable phases and relative amplitudes(Supplementary Fig. 1b lower left).

Although we could have used 2xCCE, we opted

to use 3×CCE in our system, because it has been the construct used since our preceding
works1.

© 2008 Macmillan Publishers Limited. All rights reserved.

Comparison of the previous predictions and the numerical experimental data
The predictions of repressor-antiphasic-to-activator and repressor-precedes-activatormechanisms in the previous report1 is well matched with our numerical experimental
data although we should give attention to the difference of the transcription activity thatwas evaluated in the previous model and the output protein activities that were
measured in our experiment.

Due to transcription and translation of an output reporter,

the simulation model with representative parameter values (black line in Fig. 5a) in ourcurrent manuscript has an approximately 4-hour delay from the previous model. Thisdelay is comparable to the delay estimated from our experiment in Fig. 2a and b, where
the peak-time of dGAL4-VP16 binding to UAS and peak-time of Luciferase mRNA are
about 3.0-hour and 2.0-hour before that of output Luciferase activity, respectively, andthus the delay between transcription activity and output protein was estimated as about
2.0–3.0 hours.

Sensitivity analysis of the phase and relative amplitude of the output oscillations
We calculated the sensitivity of the phase and the relative amplitude of the output
oscillation to each parameter by changing the parameter's value by 4-fold (from 1/2- to
2-times the original parameter value) (Supplementary Fig. 4a, c), except for the input
phase of the transcriptional factors, which we changed by 1 h (from a 0.5-h delay to
0.5-h in advance of the original phase).

By repeating this procedure for 100 fitted

parameter sets, we also evaluated the average contribution of each process(Supplementary Fig. 4b, d).

2© 2008 Macmillan Publishers Limited. All rights reserved.

As expected from our in cellulo mammalian cell culture system results (Figs.
4a-c), the phase difference of input transcription factors can contribute to the output
phase in silico (Supplementary Fig. 4b).

For example, a 1-h alteration in the phase ofan activator or a repressor led to ~1 h changes in the output phase (0.98 h for a and 0.62
h for b).

In addition, the absolute amplitude and base-line level of the input

transcription factor phases contributed to the output phase: a 4-fold parameter change in
the absolute amplitude (αA and αR) or the absolute base-line level (γA and γR) of anactivator or repressor could alter the output phase by 1 ~ 2 h (0.95 h for αA, 1.77 h for αR,
0.70 h for γA and 1.03 h for γR).

However, the effects of absolute amplitudes andbase-line levels on output phase seemed to cancel out when they were both increased
simultaneously. This was because the effects of simultaneous increases of both absolute
amplitude and base-line levels of the inputs are smaller than independent increases inabsolute amplitude or base-line levels (0.33 h for αA and γA, and 0.80 h for αR and γR and
see also Supplementary Fig. 4a inbox).

We noted that binding affinities of the input

activator or repressor onto the promoters (1/KA, or 1/KR) are theoretically equivalent tothe simultaneous increase of αA and γA, or αR and γR, respectively (0.33 h for 1/KA and
0.80 h for 1/KR).

We also noted that the half-life of the mRNA or protein of a reporter(1.07 h for T1 and 2.14 h for T2) contributed more to the output phase than the reporter'stranscription or translation processes (<0.01 h for β, V1 and V2), which implies that thedegradation process can cause output phases to be fairly continuous, as is observed invivo2-5.
As for the contribution of input transcription factors to the relative amplitude of
the output oscillations (Supplementary Fig. 4d), we find that the absolute amplitudes
and base-line levels of the inputs have larger effects than the phases of the inputs (0.15
for αA, 0.12 for αR, 0.12 for γA and 0.08 for γR, 0.03 for a, 0.03 h for b). Interestingly,
these effects of absolute amplitudes and base-line levels of the inputs do not seem to3© 2008 Macmillan Publishers Limited. All rights reserved.

cancel out much when both are increased simultaneously since the effects ofsimultaneous increase of both absolute amplitude and base-line levels of the inputs are
similar to, or greater than, those of either absolute amplitude or base-line levels of the
inputs (0.16 for αA and γA, and 0.14 for αR and γR and see also Fig. 5d).

Hence, weobserve a marked contrast to the output phase, implying that absolute circadian
expression levels of the inputs seem to play a key role in controlling the relative
amplitudes of outputs.
To recap, we noted: 1) that binding affinity of the input activator or repressor
onto the promoters (1/KA, or 1/KR) are theoretically equivalent to the simultaneous
increase of αA and γA, or αR and γR, respectively (0.16 for 1/KA and 0.14 for 1/KR), and;2) that the half-life of the mRNA or protein of a reporter (0.08 for T1 and 0.28 h for T2)
also contributed to the relative amplitude of the output

Interestingly, however, basaltranscription is more important for the relative amplitude of the output than theefficiencies of transcription and translation (0.15 for β, <0.001 for V1 and V2).

4

© 2008 Macmillan Publishers Limited. All rights reserved.

Supplementary Methods

Construction of pCMV-dGal4-VP16-Flag and pCMV-dGal4-Flag.

To construct

Flag-tagged activator and repressor plasmids regulated by CMV promoter, theFlag-PEST coding DNA fragment, amplified from the pCMV-dGal4 plasmid (SeeMethods in main text) by PCR with forward(5'-GGCCGCAGGTACCGACTACAAGGATGACGATGACAAGAGCCATGGCTTCCC
GCCGGCGGTG-3') and reverse(5'-TTATTCAGGTACCCACATTGATCCTAGCAGAAGC-3') primers containing the
KpnI recognition sequence (under line) and Flag coding sequence (italics), was inserted
into the VP16-fused and original pBIND vectors (Promega, See Methods in main text),
previously digested with KpnI. These products were termed pCMV-dGal4-VP16-Flagand pCMV-dGal4-Flag, respectively, and used for Western Blot analysis.

Measurement of protein half-lives of dGAL4-VP16-FLAG and dGAL4-FLAG.
NIH3T3 cells (American Type Culture Collection) were grown in DMEM (Invitrogen)supplemented with 10% FBS (JRH Biosciences) and antibiotics (25 U/ml penicillin, 25
mg/ml streptomycin; Invitrogen).

Cells were plated at 1×106 cells per dish in 90-mm

dishes 24 h before transfection.

These cells were transfected with FuGene6 (Roche)

according to the manufacturer's instructions. The cells in each well were transfectedwith 12 µg of pCMV-dGal4-VP16-Flag or pCMV-dGal4-Flag.
in each well were harvested and plated in 6 35-mm dishes.

After 24 h, the cells

After 48 h, the cells weretreated with cycloheximide (CHX, SIGMA) at a final concentration of 100 µg/ml until
their harvest. The treated cells were harvested at 0, 2, 4 and 6 h and used for Western
Blot Analysis.

5© 2008 Macmillan Publishers Limited. All rights reserved.

For Western Blot Analysis, each nuclear and cytoplasmic fraction was
extracted from the harvested cells with NE-PER Nuclear and Cytoplasmic Extraction
Reagents (PIERCE). Each cell lysate was applied to a lane of polyacrylamide gels.
For immunoblot analysis, proteins were transferred to PVDF membranes.

Anti-FLAG

M2 monoclonal Antibody-Peroxidase Conjugate (SIGMA) was diluted 2000-fold andused for detection of dGAL4-VP16-FLAG and dGAL4-FLAG protein.

Anti-Tubulinα

monoclonal antibody (clone DM1A, LAB VISION) was diluted 330-fold, and used for
detection of Tubulinα protein as internal control. For visualization, ECL Plus Western
Blotting Detection System (GE Healthcare) and LAS-1000 (FUJIFILM) were usedaccording to the manufacturer's instructions.

Construction of plasmid pG1-dLuc, pG2-dLuc, pG3-dLuc and pG5-dLuc.

To

construct the output reporter plasmids, the pG5Luc (containing five UAS) plasmid in
the CheckMate Mammalian Two-Hybrid system (Promega) was modified to containone, two or three tandem repeats of the galactose upstream activating sequences (UAS)
and a gene encoding destabilized luciferase (dLuc) as follows.

pG5Luc was digested

with XbaI (partial digestion) and EcoRI (for remaining one UAS), XhoI and NheI (forremaining two UAS), or XhoI and EcoRI (for remaining three UAS). These digested
vectors were blunted by T4 DNA Polymerase, and ligated with itself.

The products

and pG5Luc (Promega) were then digested with SphI and SalI, ligated to the SphI-SalI
fragment from the SV40-dLuc plasmid, which contains the PEST sequence of the dLuc1
and termed pG1-dLuc (containing one UAS), pG2-dLuc (containing two UAS),
pG3-dLuc (containing three UAS), and pG5- dLuc (containing five UAS). These
constructs were used as the reporter plasmids for the artificial transcription circuits.

6© 2008 Macmillan Publishers Limited. All rights reserved.

Real-time circadian reporter assays using reporter vectors with UAS (1-5×).
Real-time circadian assays were performed as previously described1,6 with the following
modifications.

NIH3T3 cells (American Type Culture Collection) were grown in

DMEM (Invitrogen) supplemented with 10% FBS (JRH Biosciences) and antibiotics(25 U/ml penicillin, 25 mg/ml streptomycin; Invitrogen).

Cells were plated at 1×105

cells per well in 35-mm dishes 24 h before transfection.

These cells were transfectedwith FuGene6 (Roche) according to the manufacturer's instructions.

The cells in eachwell were transfected with three plasmids (0.4 µg of dLuc reporter plasmid, 0.025 µg of
p3×E'-box-SV40-dGal4-VP16, and 0.1 µg of p3×RRE-SV40-dGal4).

As the dLuc

reporter plasmid, we used p3×E'-box-SV40-dLuc and p3×RRE-SV40-dLuc1 formonitoring the promoter activity of the artificial transcription factors, or pG1-dLuc,
pG2-dLuc, pG3-dLuc, pG4-dLuc , and pG5-dLuc for monitoring the transcriptional
output in the artificial transcriptional circuits. The amount of transfected plasmid was
adjusted to 2.0 µg with empty vector.

After 72 h, the media in each well was replacedwith 2 ml of culture medium (DMEM/10% FBS) supplemented with 10 mM HEPES(pH 7.2, Invitrogen), 0.1 mM luciferin (Promega), antibiotics, and 0.01 µM forskolin(Nacalai Tesque).

Bioluminescence was measured with photomultiplier tube (PMT)

detector assemblies (LM2400, Hamamatsu Photonics).

The modules and cultures

were maintained in a darkroom at 30°C and interfaced with computers for continuous
data acquisition.

Photons were counted for 1 min at 12-min intervals.

Construction of plasmid p2×CCE-SV40-dLuc, p2×CCE-SV40-dGal4-VP16 andp2×CCE-SV40-dGal4.

To construct activator or repressor plasmids regulated by twotandem repeated clock-controlled element (CCE), we modified the pCMV-dGal4-VP16
or pCMV-dGal4 vectors as follows. The oligonucleotides containing two tandem
repeat sequences of CCEs were annealed, and inserted into MluI/BglII site of the7© 2008 Macmillan Publishers Limited. All rights reserved.

SV40-dLuc vectors1. These products (p2×CCE-SV40-dLuc plasmids) were digested
with KpnI and HindIII, blunted by T4 DNA polymerase, and inserted into the BglII and
NheI site of pCMV-dGal4-VP16 or pCMV-dGal4. The end products,
p2×CCE-SV40-dGal4-VP16 or p2×CCE-SV40-dGal4, and the intermediate productp2×CCE-SV40-dLuc were used as the activator or repressor plasmids for the artificialtranscription circuits, and as the reporter plasmid for promoter activity, respectively.

The oligonucleotide sequence for two tandem repeated clock-controlled element.
2× Per2 E'-box-forward:
5'-CGCGGCGCGCGCGGTCACGTTTTCCACTATGTGACAGCGGAGGGCGCGCG
CGGTCACGTTTTCCACTATGTGACAGCGGAGG-3'
2× Per2 E'-box-reverse:
5'-GATCCCTCCGCTGTCACATAGTGGAAAACGTGACCGCGCGCGCCCTCCGCTGTCACATAGTGGAAAACGTGACCGCGCGCGC-3'
2× Bmal1 RRE-forward:
5'-CGCGAGGCAGAAAGTAGGTCAGGGACGAGGCAGAAAGTAGGTCAGGGA
CG-3'
2× Bmal1 RRE-reverse:
5'-GATCCGTCCCTGACCTACTTTCTGCCTCGTCCCTGACCTACTTTCTGCCT-3'

Real-time circadian reporter assays using artificial activator and repressor derived
by 2×CCE-SV40 promoters.

Real-time circadian assays were performed aspreviously described1,6 and as mentioned above with the following modifications.

8© 2008 Macmillan Publishers Limited. All rights reserved.

NIH3T3 cells were plated at 1×105 cells per well in 35-mm dishes 24 h before
transfection, and were transfected with FuGene6. The cells in each well weretransfected with three plasmids (0.4 µg of dLuc reporter plasmid, 0.025 µg of
p2×E'-box-SV40-dGal4-VP16, and 0.1 µg of p2×RRE-SV40-dGal4).

As the dLuc

reporter plasmid, we used p2×E'-box-SV40-dLuc and p3×RRE-SV40-dLuc1 formonitoring the promoter activity of the artificial transcription factors, and pG4-dLuc formonitoring the transcriptional output in artificial transcriptional circuits.
of transfected plasmid was adjusted to 2.0 µg with empty vector.

The amount

After 72 h, the media

in each well was replaced with 2 ml of culture medium including 0.01 µM forskolin.
Bioluminescence was measured with photomultiplier tube (PMT) detector assemblies(LM2400) in a darkroom at 30°C.

Construction of p3×E'-box-SV40-dGal4-VP16-Flag andp3×RRE-SV40-dGal4-Flag.

To construct Flag-tagged activator and repressor

plasmids regulated by E'-box and RRE, the Flag-PEST coding DNA fragment,
amplified from the pCMV-dGal4 plasmid (See Methods in main text) by PCR withforward(5'-GGCCGCAGGTACCGACTACAAGGATGACGATGACAAGAGCCATGGCTTCCC
GCCGGCGGTG-3') and reverse(5'-TTATTCAGGTACCCACATTGATCCTAGCAGAAGC-3') primers containing the
KpnI recognition sequence (under line) and Flag sequence (italics), was inserted into
the KpnI sites of p3×E'-box-SV40-dGal4-VP16 and p3×RRE-SV40-dGal4 vectors (SeeMethods in main text), that were previously digested with KpnI. These products weretermed p3×E'-box-SV40-dGal4-VP16-Flag and p3×RRE-SV40-dGal4-Flag, and used
for Chromatin Immunoprecipitation assay.

9© 2008 Macmillan Publishers Limited. All rights reserved.

Chromatin Immunoprecipitation (ChIP) assay and Quantitative PCR of ChIP
pruduct.

NIH3T3 cells (American Type Culture Collection) were grown in DMEM

(Invitrogen) supplemented with 10% FBS (JRH Biosciences) and antibiotics (25 U/ml
penicillin, 25 mg/ml streptomycin; Invitrogen). Cells were plated at 1×106 cells perdish in 14 90-mm dishes 24 h before transfection. These cells were transfected with
FuGene6 (Roche) according to the manufacturer's instructions.

The cells in each dishwere transfected with three plasmids ({8 µg of pG4-dLuc, 4.8 µg of
p3×E'-box-SV40-dGal4-VP16-Flag and 19.2 µg of p3×RRE-SV40-dGal4} and {8 µgof pG4-dLuc, 4.8 µg of p3×E'-box-SV40-dGal4-VP16 and 19.2 µg of
p3×RRE-SV40-dGal4-Flag}).

After 24 h, the cells in each well were harvested andplated in 14 90-mm dishes and two 35-mm dishes. After 48 h, the media in each dishwas replaced with 10 ml of culture medium (DMEM/10% FBS) supplemented with 10
mM HEPES (pH 7.2, Invitrogen), antibiotics, and 0.01 µM forskolin (Nacalai Tesque),and cultured at 30°C.

After 12, 16, 20, 24, 28, 32, 36 h from the stimulation, thetreated cells were fixed with 1% paraformaldehyde by a set of two dishes and used for
Chromatin Immunoprecipitation.

In the same time, bioluminescence was measured

with photomultiplier tube (PMT) detector assemblies (LM2400, Hamamatsu Photonics)
with the cells in 35-mm dishes, which was replaced with 2 ml of culture medium(DMEM/10% FBS) supplemented with 10 mM HEPES (pH 7.2, Invitrogen), 0.1 mM
luciferin (Promega), antibiotics, and 0.01 µM forskolin (Nacalai Tesque). The
modules and cultures were maintained in a darkroom at 30°C and interfaced withcomputers for continuous data acquisition.

Photons were counted for 1 min at 12-minintervals.
Chromatin Immunoprecipitation assays with these NIH3T3 cells were
performed as previously described7 with the following modifications. In this report,

10© 2008 Macmillan Publishers Limited. All rights reserved.

we used Chromatin Immunoprecipitation Assay Kits (Upstate Biotechnology),
anti-FLAG M2 antibody (Sigma) and anti-V5 antibody (Sigma).

Protein G-Sepharose(GE Healthcare) was used for precipitation of antibody/protein immune complexes.
The resulting precipitated DNA was quantified by quantitative PCR.

Quantitative

PCR was performed using ABI Prism 7700 and Power SYBR Green Reagents (Applied
Biosystems).Samples contained 1×Power SYBR Green Master Mix (Applied
Biosystems), 0.5 µM primers and 1/25 ChIP product DNA in a 10 µl volume. The
PCR conditions were as follows: 10 min at 95ºC, then 45 cycles of 15 s at 94ºC, 30 s at
59ºC and 1 min at 72ºC.

Absolute DNA abundance was calculated using the standard

curve obtained from NIH3T3 cell's genomic DNA and pG4-dLuc plasmid. The Act
promoter (Act-5') and Tbp promoter (Tbp-5') regions were used as the internal control.

Primer sequence for Quantitative PCR of ChIP product.
UAS-forward: 5'-TAGGCTGTCCCCAGTGCAAG-3'
UAS-reverse: 5'-CGATAGAGAAATGTTCTGGCAC-3'
Act-5'-forward: 5'-CCAAGAGGCTCCCTCCACA-3'
Act-5'-reverse: 5'-GTGCAAGGGAGAAAGATGCC-3'
Tbp-5'-forward: 5'-GAGAGCATTGGACTCCCCAG-3'
Tbp-5'-reverse: 5'-AGCACCCACATGGCTACTCAC-3'

Quantitative PCR of dLuc mRNA.

NIH3T3 cells were transfected on the same

condition as ChIP assay (as described above), and harvested at same timing of ChIP
assay.

Total RNAs were purified from the harvested cells using TRIzol reagent(Invitrogen), and treated with DNaseI with RNeasy kit (QIAGEN) and RNase-free

11© 2008 Macmillan Publishers Limited. All rights reserved.

DNase set (QIAGEN) according to the standard protocol.

The cDNA was synthesizedfrom 0.25 µg of total RNA with random 6mer (Promega) and Superscript II Reverse
Transcriptase (Invitrogen) according to the standard protocol.

Quantitative PCR was

performed using ABI Prism 7700 and Power SYBR Green Reagents (Applied
Biosystems).

Samples contained 1×Power SYBR Green Master Mix (Applied

Biosystems), 0.5 µM primers and 1/20 synthesized cDNA in a 10 µl volume. The
PCR conditions were as follows: 10 min at 95ºC, then 45 cycles of 15 s at 94ºC, 30 s at
59ºC and 1 min at 72ºC.

Absolute cDNA abundance was calculated using the standard

curve obtained from NIH3T3 cell's genomic DNA and pG4-dLuc plasmid.

GapdhmRNA expression levels were quantified and used as the internal control.

Primer sequence for Quantitative PCR of mRNA.
Luciferase-forward: 5'-CTTACTGGGACGAAGACGAACAC-3'
Luciferase-reverse: 5'-GAGACTTCAGGCGGTCAACG -3'
Gapdh-forward: 5'-CAAGGAGTAAGAAACCCTGGACC-3'
Gapdh-reverse: 5'-CGAGTTGGGATAGGGCCTCT-3'

Peak-time estimation of transcription factors' binding and Luciferase mRNA.
First, we chose the largest points in the latter 4 time points (24 h - 36 h) for thedGAL4-VP16-FLAG ChIP and the Luciferase mRNA experiments, and the largest
point in the former 4 time points (12 h - 24 h) for the dGAL4-FLAG ChIP experiment.
Next, we fitted a quadratic function to 3 time points around the largest point for eachexperiment, and identified the top of the function as a peak-time of the experiment.
The peak-times were converted into circadian time by using periods of simultaneously

12© 2008 Macmillan Publishers Limited. All rights reserved.

measured Luciferase activities (21.66 for the Luciferase mRNA experiments, and 20.92
for the ChIP experiments), by the function: (the raw peak-time / the period of theLuciferase activity) * 24.

Construction of plasmid p3×CCE-TK-dGal4-VP16 and p3×CCE-TK-dGal4. To
construct activator or repressor plasmids regulated by thymidine kinase (TK) promoter,
we modified the pCMV-dGal4-VP16 or pCMV-dGal4 vectors as follows.

We

amplified the TK promoter sequences from pMU2-P(TK) (unpublished, the origin of
TK promoter was phRL-TK (Promega)) by PCR with a forward primer containing an
BglII recognition sequence (5'-ATATATAGATCTCTAGCCCGGGCTCGAGATC-3')
and a reverse primer containing a NheI recognition sequence(5'-GTAGTAGCTAGCCCGTTATAGTTACTGCAGAAGC-3').

The PCR productwas then digested with BglII and NheI, and inserted in the pCMV-dGal4-VP16 or
pCMV-dGal4 vectors, that were previously digested with BglII and NheI. To drive theartificial activator or repressor by CCE, the oligonucleotides containing three tandem
repeat sequences of CCEs were annealed, and inserted into BglII site of thepTK-dGal4-VP16 or pTK-dGal4 vectors.

The end products,

p3×CCE-TK-dGal4-VP16 and p3×CCE-TK-dGal4 plasmids, were used as the activatoror repressor plasmids for the artificial transcription circuits.

The oligonucleotide sequence for three tandem repeated clock-controlled element.
3× Per2 E'-box-forward:
5'-GATCGCGCGCGCGGTCACGTTTTCCACTATGTGACAGCGGAGGGCGCGCG

13© 2008 Macmillan Publishers Limited. All rights reserved.

CGGTCACGTTTTCCACTATGTGACAGCGGAGGGCGCGCGCGGTCACGTTTTCCACTATGTGACAGCGGAG-3'
3× Per2 E'-box-reverse:
5'-GATCCTCCGCTGTCACATAGTGGAAAACGTGACCGCGCGCGCCCTCCGCTGTCACATAGTGGAAAACGTGACCGCGCGCGCCCTCCGCTGTCACATAGTGG
AAAACGTGACCGCGCGCGC-3'
3× Per3 D-box-forward:
5'-GATCCCCGCGCGTTATGTAAGGTACTCGCCCGCGCGTTATGTAAGGTACTCGCCCGCGCGTTATGTAAGGTACTCG-3'
3× Per3 D-box-reverse:
5'-GATCCGAGTACCTTACATAACGCGCGGGCGAGTACCTTACATAACGCGCG
GGCGAGTACCTTACATAACGCGCGGG-3'
3× Bmal1 RRE-forward:
5'-GATCAGGCAGAAAGTAGGTCAGGGACGAGGCAGAAAGTAGGTCAGGGACGAGGCAGAAAGTAGGTCAGGGACG-3'
3× Bmal1 RRE-reverse:
5'-GATCCGTCCCTGACCTACTTTCTGCCTCGTCCCTGACCTACTTTCTGCCTCGTCCCTGACCTACTTTCTGCCT-3'

Re-annotation of clock-controlled genes.

The previously published genome-wide

expression data for the liver 2 were re-analyzed and re-annotated to find transcription
factors that showed circadian expression.

Each probe set that was identified as aclock-controlled gene in the previous report 2, was annotated by using link information

14© 2008 Macmillan Publishers Limited. All rights reserved.

in the Entrez Gene database (gene name, gene symbol, and Entrez Gene ID), link
information in the TRANSFAC database (FACTOR, MATRIX, consensus binding sites,
CLASS ID, and upstream transcription factors), the Affymetrix annotation file (InterProID), and information in the mammalian promoter/enhancer database (clock-controlledelements, E-box, D-box, and RRE) (Kumaki et al., submitted). The annotation table isshown in Supplementary Table 1 online.

Parameter search.

First, the initial parameter set in the formulas of our theoreticalmodel was randomly chosen except for KA, KR (=1/2), a (=0), b and n (=1).

As

multiplying by KA, KR is equivalent to dividing by both αA, αR and γA, γR , in the formula
T(t), KA and KR were fixed to a constant (1/2) to eliminate ambiguity. If the minimum
amplitude of an output with a specific initial parameter set (obtained by changing thephase of the repressor) was too low or too high (<1/100 or >100-fold of a mean
amplitude of an activator and a repressor), the initial parameter set was discarded and a
new parameter set was randomly searched again.
Second, one parameter other than KA, KR, a, b, n was chosen, and then ten
different values of the parameter were generated within 30-fold of its original value.
For each value of the parameter, graphs like that in Fig. 5a and b were drawn bychanging the value of b. The first graph (Fig. 5a) shows relationships between phasedifferences of the activator and the repressor (activator phase – repressor phase) andphase differences of the activator and the output protein (output protein phase –activator phase).

The second graph (Fig. 5b) shows relationships between phasedifference of the activator and the repressor and the relative amplitude of the output
protein.

The points in these graphs represent observed outputs of various

combinations of activators and repressors.

The parameter value that had the lowest

15© 2008 Macmillan Publishers Limited. All rights reserved.

least squares distance to the measured experimental points was chosen as the new valueof the parameter.

We did not use *-marked points because these outlier points havelarger (>4-fold amounts of the activator than the repressor) differences between
amounts of the activator and the repressor (Fig. 5c), and also seem to have lower
relative amplitudes.

This second fitting procedure was repeated until no more

improvements were observed, and the final parameter set was recorded as a fitted
parameter set.

By repeating these procedures, we found 100 fitted parameter sets.

Sensitivity analysis.

All parameters except for n were analyzed for the degree towhich the output phase and the relative amplitude was sensitive to them.

To calculate

the sensitivity of the output phase to a certain parameter, the value of the parameter waschanged within 1 hour (0.5-hour advance and 0.5-h delay) for a or b, or changed within
a 4-fold (one-half and double the original parameter value) if it was one of the otherparameters.

For each parameter change, the maximum changes in phase difference

between the activator and the output were calculated for each b within -12 < b < 12, andthe average value of the maximum phase change (or the change of the relative
amplitude) was calculated over -12 < b < 12 and defined as the "phase change" (or"relative amplitude change") due to the parameter change.

This procedure was

performed for each parameter set in 100 fitted parameter sets.

The average and SEMof the phase change due to the parameter change were then calculated over the 100
fitted parameter sets with trimming of the 5% outliers, and defined as the measures ofsensitivity of the corresponding parameter change.

Construction of p3×E'-box-SV40-Dbp and p3×RRE-SV40-E4bp4.

To construct

Dbp and E4bp4 gene expression vectors regulated by E'-box and RRE, the Dbp and

16© 2008 Macmillan Publishers Limited. All rights reserved.

E4bp4 coding sequence were amplified from the pMU2-Dbp and pMU2-E4bp4
plasmids (Kumaki et al., submitted) by PCR with forward(5'-CTCGAAGGAGAGGCCACCATGGAC-3') and reverse(5'-GCACCCGACATAGATTCATTAACCC-3') primers, and phosphorylated byMighty Cloning kit <Blunt End> (TaKaRa). The vectors for insertion of these PCR
fragment were amplified from the p3×E'-box-SV40-dGal4 and p3×RRE-SV40-dGal4
plasmids (See Methods in main text) by invert PCR with forward(5'-GGTACCTGAATAACTAAGGCCGCTTCC-3') and reverse(5'-CAGGAGGCTTGCTTCAAGCTGGC-3') primers, and blunted by T4 DNA
Polymerase, and then dephosphorylated by Bacterial Alkaline Phosphatase.

The

dGal4 coding region was deleted by this treatment. The Dbp and p3×E'-box-SV40
PCR fragments, and E4bp4 and p3×RRE-SV40 PCR fragments were ligated,
respectively.

These products were termed p3×E'-box-SV40-Dbp and p3×RRE-

SV40-E4bp4, and used for perturbation experiment.

Perturbation experiments of natural transcriptional circuits to verify thepredictions.

Real-time circadian assays were performed as previously described1,6

with the following modifications.

NIH3T3 cells (American Type Culture Collection)

were grown in DMEM (Invitrogen) supplemented with 10% FBS (JRH Biosciences)
and antibiotics (25 U/ml penicillin, 25 mg/ml streptomycin; Invitrogen).

Cells wereplated at 1×105 cells per well in 35-mm dishes 24 h before transfection.

These cells

were transfected with FuGene6 (Roche) according to the manufacturer's instructions.
The cells in each well were transfected with two plasmids (0.5 µg of
p3×D-box-SV40-dLuc1 reporter plasmid, 0, 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 µg of
p3×E'-box-SV40-Dbp or p3×RRE-SV40-E4bp4). The amount of transfected plasmid
was adjusted to 2.0 µg with empty vector.

After 72 h, the media in each well was

17© 2008 Macmillan Publishers Limited. All rights reserved.

replaced with 2 ml of culture medium (DMEM/10% FBS) supplemented with 10 mM
HEPES (pH 7.2, Invitrogen), 0.1 mM luciferin (Promega), antibiotics, and 0.01 µM
forskolin (Nacalai Tesque).

Bioluminescence was measured with photomultiplier tube

(PMT) detector assemblies (LM2400, Hamamatsu Photonics).

The modules andcultures were maintained in a darkroom at 30°C and interfaced with computers forcontinuous data acquisition.

Photons were counted for 1 min at 12-min intervals.

18© 2008 Macmillan Publishers Limited. All rights reserved.

References1.

Ueda, H.R. et al. System-level identification of transcriptional circuits

underlying mammalian circadian clocks. Nat Genet 37, 187-192 (2005).
2.

Ueda, H.R. et al. A transcription factor response element for gene expression

during circadian night. Nature 418, 534-539 (2002).
3.

Akhtar, R.A. et al. Circadian cycling of the mouse liver transcriptome, as

revealed by cDNA microarray, is driven by the suprachiasmatic nucleus. Curr Biol 12,
540-50 (2002).
4.

Panda, S. et al. Coordinated transcription of key pathways in the mouse by thecircadian clock. Cell 109, 307-20 (2002).
5.

Storch, K.F. et al. Extensive and divergent circadian gene expression in liverand heart. Nature 417, 78-83 (2002).
6.

Sato, T.K. et al. Feedback repression is required for mammalian circadianclock function. Nat Genet 38, 312-319 (2006).
7.

Yamashita, M. et al. Identification of a Conserved GATA3 Response Element

Upstream Proximal from the Interleukin-13 Gene Locus. J. Biol. Chem. 277,
42399-42408 (2002).

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