Delay in Feedback Repression
by Cryptochrome 1 Is Required
for Circadian Clock Function
Maki Ukai-Tadenuma,1,8 Rikuhiro G. Yamada,1,8 Haiyan Xu,4 Jürgen A. Ripperger,5 Andrew C. Liu,4and Hiroki R. Ueda1,2,3,6,7,*
1Laboratory for Systems Biology, RIKEN Center for Developmental Biology
2Functional Genomics Unit, RIKEN Center for Developmental Biology
3Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
4Department of Biological Sciences, The University of Memphis, Memphis, TN 38152-0001, USA
5Department of Medicine/Unit of Biochemistry, University of Fribourg, 5, rue du Musée, CH-1700 Fribourg, Switzerland
6Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
7Department of Mathematics, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto,
Kyoto 606-8502, Japan
8These authors contributed equally to this work
*Correspondence: uedah-tky@umin.ac.jp
DOI 10.1016/j.cell.2010.12.019

SUMMARY

Direct evidence for the requirement of delay infeedback repression in the mammalian circadianclock has been elusive. Cryptochrome 1 (Cry1), an
essential clock component, displays evening-time
expression and serves as a strong repressor atmorning-time elements (E box/E0 box). In this study,
we reveal that a combination of day-time elements(D box) within the Cry1-proximal promoter andnight-time elements (RREs) within its intronicenhancer gives rise to evening-time expression. A
synthetic composite promoter produced eveningtime expression, which was further recapitulated bya simple phase-vector model. Of note, coordinationof day-time with night-time elements can modulate
the extent of phase delay. A genetic complementation assay in Cry1/:Cry2/ cells revealed that
substantial delay of Cry1 expression is required torestore circadian rhythmicity, and its prolonged
delay slows circadian oscillation. Taken together,our data suggest that phase delay in Cry1 transcription is required for mammalian clock function.
INTRODUCTION
Circadian clocks are thought to consist of autoregulatory loops
with delayed transcriptional/translational feedback repressionin which delayed expression of clock components is critical for
maintaining circadian rhythmicity (Dunlap, 1999; Reppert andWeaver, 2002; Young and Kay, 2001). However, the underlying
molecular mechanism giving rise to such delay remains
268 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

unknown, hindering formal validation of its biological relevance.
In mammalian clocks, circadian transcriptional program ismediated through at least three clock-controlled DNA elements,
morning-time (E box/E0 box, or E/E0 box: CACGT[G/T]) (Gekakis,
1998; Hogenesch et al., 1997; Ueda et al., 2005; Yoo et al., 2005),
day-time (D box: TTA[T/C]GTAA) (Falvey et al., 1996; Ueda et al.,2005), and night-time elements (Rev-Erb/ROR-binding element,
or RRE: [A/T]A[A/T]NT[A/G]GGTCA) (Harding and Lazar, 1993;
Preitner et al., 2002; Ueda et al., 2002, 2005). The E/E0 box-mediated transcriptional program has a critical role in the core
autoregulatory loop of the mammalian circadian clock (Gekakis,
1998; Sato et al., 2006; Ueda et al., 2005). In this core loop,
bHLH-PAS transcription activators such as BMAL1 and CLOCK
form heterodimers that bind to E/E0 box cis-elements in the
promoter regions of their target genes, including the Per and
Cry genes; CRYs, in turn, form repressor complexes that physically associate with the BMAL1/CLOCK complex to inhibit E/E0
box-mediated transcription (Dunlap, 1999; Griffin et al., 1999;
Kume et al., 1999; Reppert and Weaver, 2002; Young and Kay,
2001). Thus, the CRYs play an integral role in the circadian clockby ‘‘closing’’ the core negative feedback loop.
Although Cry1/ mice and their SCN slices display circadianrhythms at the organismal and tissue levels, respectively, dissociated Cry1/ SCN neurons and fibroblasts are largely
arrhythmic. The issue of cell autonomy has been carefully examined in several recent studies (Brown et al., 2005; DeBruyne
et al., 2007; Liu et al., 2007). By contrast to Cry1-deficient cells,
dissociated Cry2/ SCN neurons and fibroblasts exhibit robust
rhythmicity, implying that CRY2 cannot substitute for Cry1
deficiency at the cellular level (Liu et al., 2007). Therefore, we
focused on transcriptional regulation of Cry1 gene.
CRY1 and its expression pattern play a pivotal role in thecore autoregulatory loop. Either overexpression of CRY1 orinterference of CRY1’s repressor activity on E/E0 box-mediatedtranscription can abolish circadian transcriptional oscillations(Sato et al., 2006; Ueda et al., 2005). Remarkably, Cry1 displays
delayed gene expression relative to other genes with E/E0 box
elements (Ueda et al., 2002, 2005). Circadian expression of
Cry1 peaks at evening phases in the SCN (CT12) (Ueda et al.,2002, 2005), which is much later than for typical morning-time
E/E0 box-regulated genes such as Rev-Erba, and is intermediate
between day-time D box- and night-time RRE-regulated genes
such as Per3 and Bmal1, respectively (Ueda et al., 2002,
2005). Dual roles of Cry1 as a strong repressor for E/E0 box
activity and a time delay mediator fit well with the current modelof the circadian clock, i.e., feedback repression with delay
may depend on the unique mode of transcriptional regulationof Cry1.
Previous studies identified an E0 box and an E box in Cry1’s
regulatory region (Ueda et al., 2005; Fustin et al., 2009) and two
RREs in its first intron (Ueda et al., 2005). In this study, we also
identified additional D boxes in the promoter region andconfirmed their functionality in conferring day-time expression(delayed phase relative to E box). We further discovered thata combination of the promoter containing E/E0 boxes and D boxes
with the first intron sequence of Cry1 containing RREs generated
delayed-phase expression of Cry1, in which the strength of nighttime elements (RREs) can modulate the extent of phase delay. Of
note, a simple phase vector model predicts that coordination
between day-time and night-time elements can determine theextent of phase delay. Based on this model, we generated anarray of Cry1 constructs that display different phases, and these
constructs were used in a genetic complementation assay torestore circadian oscillation in Cry1/:Cry2/ cells. Theseexperiments reveal that substantial delay of Cry1 expression isrequired to restore single-cell level rhythmicity and that
prolonged delay of Cry1 expression can slow circadian oscillation. These results suggest that phase delay in transcriptional
feedback repression is required for mammalian clock function.
RESULTS
Cry1 Promoter Confers Phase and Amplitude
Intermediate between Those Conferred by E/E0 Box
and D Box Circadian Elements
To examine Cry1 promoter activity, we generated a reporter
construct, P(Cry1)-Luc, in which a 1.5 kbp DNA fragment containing the Cry1 promoter was fused to the Luciferase (Luc)gene. Cry1 promoter-driven bioluminescence reached its peakat circadian time (CT) 9.60 ± 0.11 (n = 3, mean ± standard deviation), which was rather close to that of a D box-P(SV40)-Luc
reporter harboring three tandem repeats of D boxes fused toan SV40 promoter (CT10.42 ± 0.16), and delayed > 6 hr relative
to an E0 box-P(SV40)-Luc reporter harboring three tandem
repeats of E0 boxes fused to an SV40 promoter (CT3.53 ± 0.04)(Figure 1A and Figure S1A and Table S1 available online). On
the other hand, the Cry1 promoter produced a higher-amplitude
rhythm than that of the D box-P(SV40)-Luc reporter (Figure 1B).
The amplitude of E0 box-driven bioluminescence rhythms (Figure 1B, E0 box-P(SV40)-Luc) was even higher than those driven
by the Cry1 promoter. These data place the Cry1 promoter intermediate between D box and E0 box in both phase and amplitudeof driven rhythms and suggest that the Cry1 promoter might
contain both D box and E0 box elements.
D Box in Cry1 Promoter Confers Phase Delayand Day-Time Expression
We investigated the genomic sequences of the Cry1 promoter
and found five highly conserved regions, of which twosequences (50 -TTCAGAAA-30 and 50 -AAACGTGA-30 ) mostclosely resemble a D box according to position weight matrix
analysis. Interestingly, these sequences overlap with theconserved E0 box sequences in the promoter region (Figure 1C).
We designated this region of the Cry1 promoter as a Cry1proD
element and constructed a Cry1proD-P(SV40)-Luc reporter byfusing three tandem repeats of this element to an SV40
promoter. NIH 3T3 cells transiently transfected with thisconstruct showed circadian oscillation of bioluminescence witha peak at day-time (CT12.37 ± 0.05, n = 3; Figure 1D, Figure S1B,
and Table S1) and a relative amplitude between those of E0 box
and D box constructs (Figure 1E). Because the E0 box and two
putative D boxes in Cry1proD element overlap, it is not practical
to isolate each CCE for analysis individually. Instead, we tested
whether clock factors involved in E0 box- or D box-mediatedtranscription could activate or repress the Cry1proD element.
Cotransfection of E0 box activators BMAL1/CLOCK strongly
induced not only E0 box, but also Cry1proD activity in NIH 3T3cells (Figure 1F). Cotransfection of a D box repressor E4BP4
inhibited BMAL1/CLOCK induction of Cry1proD activity ina dose-dependent manner (Figure 1F). Interestingly, the E0 box
repressor CRY1 also inhibited this induction (Figure S1C), andthe D box activators DBP, HLF, and TEF (Mitsui et al., 2001)
also induced Cry1proD activity (Figure S1D). These results
suggest that Cry1proD is regulated by classical transcriptional
regulators of both D box and E/E0 box activities, consistent
with the observation that bioluminescence rhythms driven by
the Cry1 promoter display properties intermediate between
those driven by D box and E0 box constructs.
To confirm that Cry1 expression is delayed relative to E/E0 box
activity, we measured temporal mRNA profiles of endogenous
Per2, Bmal1, and Cry1 in NIH 3T3 cells expressing a P(Cry1)Luc or a P(Per2)-Luc reporter (Figure 1G, left). As shown, thephase of Luc mRNA driven by the Per2 promoter was almost
the same as that of endogenous Per2 mRNA (Figure 1G, right).
Furthermore, the phase of Luc mRNA driven by the Cry1
promoter was delayed relative to those of endogenous Per2mRNA or Luc mRNA driven by the Per2 promoter, with a phasedifference of 1–2 hr. These results further support the notionthat functional D boxes in the Cry1 promoter contribute to phasedelay of Cry1 expression.
Cry1 Intron Acts as an Enhancer to Confer Phase Delay
In addition to the phase delay caused by D boxes within theCry1 promoter, we also observed further phase delay of theendogenous Cry1 mRNA by at least 2 hr relative to Luc mRNAdriven by the Cry1 promoter (Figure 1G). The endogenous Cry1mRNA displayed 3–4 hr phase delay relative to endogenous
Per2 mRNA and 7–8 hr advance relative to Bmal1 mRNA.
This observation is consistent with previous reports (Baggs
et al., 2009; Etchegaray et al., 2003; Liu et al., 2008; Preitner
Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. 269

E'-box-P(SV40)

3.53

4

5
2.5

5

P(Cry1)0
Normalized Bioluminescence
Low
High
-1
0
1

E'-box-P(SV40)

3.41

D-box-P(SV40)

10.38
0

1

2

Day

3

4

12
6
3

D-P(SV40)

Cry1proD-P(SV40)2

3

4Cry1proD-mut-P(SV40)

P(SV40)

Cry1 promoter-93 ~ -70+328

Cry1proD element
mouse : AAACACACTTCAGAAACGTGAGGT

Cry1proD-P(SV40)E'-P(SV40)
D-P(SV40)

0.4************************

human : AAACACACTTCAGAAACGTGAGGT
D-box consensus:

TTATGTAATTACGTAAAACGTG

E'-box consensus:
0

1

2

3

4

Day

P(Cry1)-Luc / NIH3T3

4

10000000

37500000

25000000

1

2500000

0

0

1

Day

2

3

0

18

12

6

0

Per2

Bmal1 Cry1

Luc

endogenous RNA

P(Per2)-Luc / NIH3T3

36000000

2

4000000

1

2000000

0

0

0

1

Day

2

3

Bioluminescence (counts/min)- + - +++ - + - +++ - + - +++ - + - +++ - + - +++
--+
--+
--+
--+
--+
E'-P(SV40)

1

mRNA/Gapdh (average=1)

Relative Luc Activity

-1208

G

15

0
BMAL1/CLOCK
E4BP4

mammal conservation

Bioluminescence (counts/min)

F

5

5

Normalized Bioluminescence
Low
High
-1
0
1

+328

Day

10

0

Cry1 exon1

(TSS)
-1

0.2

0.8
Phase (h)

12.37

0.4

chr10
-1208

E

15

Phase (h)Cry1proD-P(SV40)

Cry1proD-P(SV40)DP(Cry1)E'-P(SV40)
D-P(SV40)

0

Relative AmplitudeDay

3

D-P(SV40)

2

D-P(SV40)

1E'-P(SV40)

0

7.5

C

0.6

mRNA/Gapdh (average=1)

10.42

D-box-P(SV40)

B

Phase (h)10Relative Amplitude

12.5

9.60
Phase (h)Phase (h)P(Cry1)E'-P(SV40)

A

P(Cry1)-Luc
P(Per2)-Luc

Cry1 mRNA
Per2 mRNA
Bmal1 mRNA
Luc mRNA
LUC activity

Figure 1. The Cry1 Promoter Contains Both E/E0 Box and D Boxes(A) The phases of circadian transcriptional activities induced by P(Cry1), E0 box-P(SV40), and D box-P(SV40) promoters. Each promoter was fused witha Luciferase reporter gene (Luc) and transiently transfected into NIH 3T3 cells. Time series of bioluminescence expression were recorded in real time usinga photomultiplier tube (PMT). Heatmaps represent average promoter activities from three independent samples. Raw data were detrended for baseline andamplitude and then scaled into a range of 1 to 1 (left). Phases were estimated from the time series data by fitting a cosine wave (right).
(B) The relative amplitudes of circadian transcriptional activities induced by P(Cry1), E0 box-P(SV40), and D box-P(SV40) promoters.
(C) E0 box and its overlapping D boxes in Cry1 promoter. Genomic positions relative to the transcription start site (TSS, designated also as ‘‘1’’) of Cry1 gene areindicated along with evolutionary conservation scores among mammalian species. Colored letters indicate nucleotides matching the consensus sequence ofD box and E0 box.
(D) The phases of circadian transcriptional activities induced by Cry1proD-P(SV40), E0 box-P(SV40), and D box-P(SV40) promoters. The experiment was
performed as in (A).
(E) The relative amplitudes of circadian transcriptional activities induced by Cry1proD-P(SV40), E0 box-P(SV40), and D box-P(SV40) promoters.
(F) The Cry1proD element can be activated by BMAL1/CLOCK and repressed by E4BP4. Each of the indicated promoters was fused to a Luciferase reporter geneand transiently transfected into NIH 3T3 cells. Relative Luciferase activity for each promoter was scaled so that the activity without transcriptional regulation isnormalized to 1.
(A–F) Data are representative of two independent experiments. Error bars represent SD determined from three measurements for each sample (n = 3).
(G) mRNA expression patterns of endogenous Cry1, Per2, and Bmal1 and exogenous Luciferase (Luc). NIH 3T3 cells were transfected with P(Cry1)-Luc orP(Per2)-Luc reporter. Relative mRNA levels of each gene were measured. In parallel, transcriptional activities of P(Cry1) and P(Per2) promoters were monitored bybioluminescence recording. Phases of measured rhythm are indicated on the right. (Right) Error bars represent SEM (n = 3). (Left) Error bars represent SD (n = 3).
See also Figure S1 and Table S1.

et al., 2002; Sato et al., 2006; Ueda et al., 2005), which havespeculated that a phase delay would be generated by two functional RREs present in the intron regions of Cry1 (Ueda et al.,2005). To provide experimental evidence for the mechanism offurther phase delay of Cry1 expression, we focused on one of
the highly conserved regions of the Cry1 gene—the first intron,
270 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

which contains two RREs, designated here as R1 and R2(Figure 2A). These RREs are highly conserved and aligned ina head-to-head arrangement, perfectly matched to theconsensus RRE sequence ([A/T]A[A/T]NT[A/G]GGTCA). We
cloned a 1.03 kbp fragment containing the conserved intronic
RREs and inserted it into the P(Cry1)-Luc reporter plasmidto generate a P(Cry1)-Cry1 intron 1.03k-Luc reporter. Cells
expressing this reporter displayed a bioluminescence peak at
CT14.62 ± 0.20, whereas absence of the 1.03 kbp intron
sequence resulted in a peak at CT10.51 ± 0.30, a difference of4 hr (Figure 2B, Figure S2A, and Table S1). We next focusedon a highly conserved region of 336 bp within the 1.03 kbp intron
sequence for further analysis. Whereas cells expressing aP(Cry1)-Cry1 intron D336-Luc reporter exhibited a peak at
CT10.37 ± 0.24, those expressing P(Cry1)-Cry1 intron 336-Luc
peaked at CT14.32 ± 0.23 (Figure 2B, Figure S2A, and Table
S1), a 4 hr phase delay. Thus, Cry1 first intron sequences
containing RREs likely underlie the delayed phase of Cry1
expression. In addition, their effects appear to be independentof locations (Figure 2C, Figure S2B, and Table S1), suggestingthat this sequence functions as a transcriptional enhancer.
Next, we analyzed the regulatory regions for the presenceof their corresponding transcription factors in vivo using chromatin immunoprecipitation (ChIP) assays with time series
samples from mouse liver. Chromatin from wild-type, Dbp/
(Lopez-Molina et al., 1997), or Rev-Erba/ (Preitner et al.,2002) mice was immunoprecipitated by anti-BMAL1, anti-DBP,
or anti-REV-ERBa antibodies (Figure 2D). The levels of BMAL1
and DBP binding to Cry1proD displayed circadian oscillation inwild-type, Dbp/, or Rev-Erba/ mice, whereas DBP bindingin Dbp/ mice was significantly reduced (p < 0.01 by two-way
ANOVA) with residual signals potentially deriving from TEF
and/or HLF binding to the same element. On the other hand,no significant reduction was observed for the binding of DBP
to this region in Rev-Erba/ mice. The level of REV-ERBabinding to the Cry1 first intron region also displayed circadianoscillation in wild-type and Dbp/ mice, whereas it was significantly reduced in Rev-Erba/ mice (p < 0.01 by two-way
ANOVA). The levels of BMAL1 binding to the Dbp promoter
region displayed circadian oscillation, whereas there was only
background binding of DBP and REV-ERBa to this region.
This result confirmed that BMAL1 and DBP bind to the Cry1
promoter region, and REV-ERBa binds to the Cry1 first intron
region. The peak binding time of each transfactor is consistent
with previous reports of its in vivo binding or its nuclear accumulation (Lopez-Molina et al., 1997; Mitsui et al., 2001; Preitneret al., 2002; Ripperger and Schibler, 2006).
In addition to the biochemical interaction between the Cry1
promoter and D box trans-regulators described above, we also
examined the role of the D box using genetic approaches; wemeasured mRNA expression patterns from time course liver
samples of triple-knockout mice of PAR bZip genes (Tef, Hlf,and Dbp) (Gachon et al., 2004). Although these mice displayed
normal circadian behavior (possibly due to compensation
rendered by posttranslational mechanisms intracellularly and/orintercellular coupling of clock cells in vivo) (Gachon et al.,
2004; Lee et al., 2001; Liu et al., 2007), we found that Cry1’s circadian expression level was different from wild-type and its peak of
expression delayed (Figure 2E and Figure S2C), whereas those ofother measured clock genes (Bmal1, RevErba, and Per1) were
not. Importantly, the observed peak delay was reproducible
and significant in three independent experiments (p < 0.01 by
two-way ANOVA). These results further confirm that PAR bZip
genes are important for the proper phase of expression of Cry1.

Strength of Intronic RREs Correlates with Phase DelayTo determine whether the strength of RREs in 336 bp of Cry1 first
intron sequence correlates with the phase delay, we generated
an array of intron sequences harboring mutant RREs, including
deletion, mutation, and inversion of the two RREs (Figure 3A).
We inserted these mutant intron sequences into P(SV40)-Luc
vector to generate an array of P(SV40)-Cry1 intron 336-Luc
reporter constructs. As one measurement for the strength of
intronic RREs, we first examined transcriptional activation ofthese constructs by RORa, an activator of RRE, in a reporter
assay. We found that induced Luciferase activities varied significantly among constructs, ranging from strong induction by
wild-type RRE to almost no induction by double-mutant ordeleted RREs (R1 and R2) (Figure 3B). These results indicatethat the RREs within the intron sequence are functionally responsive to RORa and that intronic RREs of various strengths can beobtained from different RRE mutations.
As an independent measurement for the strength of intronic
RREs, we next examined the amplitude of circadian oscillations
expressed by these constructs in reporter rhythm assays (Figure S3A and Table S1). Rhythm amplitude was low when an
intron sequence of low RORa responsiveness was used to drive
reporter expression and high when an intron sequence of high
RORa responsiveness was used (Figures 3B and 3C). Overall,there was a significant positive correlation between the two
measurements for the strength of intronic RREs: RORa responsiveness and rhythm amplitude among the intronic RRE mutants(r2 = 0.95, p < 0.01; Figure 3D).
These mutant intron sequences allowed us to analyze quantitatively the role of intronic RREs in the phase delay mechanism.
Specifically, we examined how intronic RRE mutation affects
phase delay using a reporter rhythm assay (Figure 3E, Figure S3B,
and Table S1). We found that the observed phase delay significantly correlated with the first measurement for the strength of
the intronic RRE mutants, i.e., RORa responsiveness (r2 = 0.82,
p < 0.01; Figure 3F, top). Similarly, phase delay also correlated
well with the second measurement for the strength of the intronic
RRE mutants, i.e., the rhythm amplitude (r2 = 0.90, p < 0.01; Figure 3F, bottom). Taken together, these data suggest that thestrength of RREs correlates with the phase delay, further corroborating our finding that the RREs in the Cry1 intron act as anenhancer to further delay the phase conferred by Cry1 promoter.
Combination of Day- and Night-Time Elements
Produces Evening Phase Control
Given that the delayed expression of Cry1 is a combined effect of
its promoter and intron, we sought to understand whether thiscombinatorial effect is a general design principle in the circadiantranscriptional network or a mechanism unique to the transcriptional regulation of Cry1. We first asked whether the phase ofendogenous Cry1 expression could be synthesized using an
artificial promoter in clock cells. We constructed three setsof reporters, with each harboring one of the three CCEs (i.e.,
E/E0 box, D box, and RRE) in the presence or absence of the
RRE-containing intron sequence from the Cry1 gene (Figures
4A and 4B). Real-time bioluminescence recording of transfected
NIH 3T3 cells showed that the RRE-containing Cry1 intron
sequence, as expected, did not dramatically alter the phase of
Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. 271

-1 (TSS)A

23.8kbchr10

Cry1 gene region
mammal conservation

isolated 1.03kb region

isolated 336 base region

R1 R2

Flag Cry1
intronP(Cry1)

Luc

polyA

Phase (h)B
P(Cry1)

10.51

P(Cry1)-Cry1 intron 1.03k

14.62

336

P(Cry1)-Cry1 intron 336

14.32

336

P(Cry1)-Cry1 intron Δ336

10.37
0

1

2

3

4

Normalized Bioluminescence
Low
High

5

Day

-1

0

1

Phase (h)Cno Cry1 intron

10.04

P(Cry1)

Flag

Luc

polyA

upstream of P(Cry1)

12.22

Flag

Luc

polyA

downstream of P(Cry1)

14.94

P(Cry1)

336

Flag

Luc

polyA

inside ofthe coding sequence

13.91

P(Cry1)

Flag

336

Luc

downstream of
the coding sequence

12.61

P(Cry1)

Flag

Luc

0

1

2

3

4

336

P(Cry1)

polyA

336

polyA

5

Day

D

percent of input

2.5

anti-BMAL1

anti-DBP

Cry1 promoter

Cry1 promoter

0.15

0.3
0.2

0.1

0.1

0.05

1
0.5

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

Dbp-/-

Rev-Erbα-/-

wild typeDbp-/-

Rev-Erbα-/-

wild typeDbp-/-

Rev-Erbα-/-

Dbp promoter

2.5

0

0
0 4 8 12 16 20

wild type

percent of input

Cry1 1st intron

0.2

1.5

0
ZT(h) 0 4 8 12 16 20

Dbp promoter

0.4

2

Dbp promoter

0.2
0.15

0.3

1.5
0.2

0.1

0.1

0.05

1
0.5

0
ZT(h) 0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

Dbp-/-

Rev-Erbα-/-

wild type

E

0.4

2

anti-REV-ERBα

Cry1

0.015

0

0
0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

0 4 8 12 16 20

wild typeDbp-/-

Rev-Erbα-/-

wild typeDbp-/-

Rev-Erbα-/-

Bmal1

0.005

0.015

Rev-Er bα

Per 1

0.009

mRNA/Gapdh

0.004
0.01

0.003
0.002

0.005

0.01

0.006

0.005

0.003

0.001
0
ZT(h) 0 4 8 12 16 20

wild type0 4 8 12 16 20

Dbp-/-:Tef-/-:Hlf-/-

0

0 4 8 12 16 20

0 4 8 12 16 20

wild typeDbp-/-:Tef-/-:Hlf-/-

0

0 4 8 12 16 20

0 4 8 12 16 20

wild typeDbp-/-:Tef-/-:Hlf-/-

0

0 4 8 12 16 20

0 4 8 12 16 20

wild typeDbp-/-:Tef-/-:Hlf-/-

Figure 2. Cry1 Intron Acts as an Enhancer to Confer Phase Delay(A) The first intron of Cry1 contains RRE sequences. The marked 1.03 kbp and 336 bp of Cry1 first intron sequence, which are highly conserved in mammals, werecloned and examined in this study. Two RREs are indicated as R1 and R2, respectively.
(B) Cry1’s first intron confers phase delay. The Cry1 promoter was combined with the Cry1 1.03 kbp intron, 336 bp intron, or Cry1 intron D336 deletion mutant togenerate composite promoters. The experiment was performed as in Figure 1A.
(C) Cry1’s first intron sequence confers phase delay independently of its location. The 336 bp of Cry1 intron sequence was inserted upstream or downstream of
the Cry1 promoter and inside or downstream of the coding sequence. Data are representative of two independent experiments (B and C).
(D) Binding of BMAL1 (an E/E0 box regulator) and DBP (a D box regulator) to the Cry1 promoter region and REV-ERBa (a RRE regulator) to the Cry1 first intron
region in vivo. Chromatin from wild-type (gray), Dbp/ (orange), or Rev-Erba/ (purple) mice was prepared at 4 hr intervals from mice held in a 12 hr light/12 hr
dark cycle (LD 12:12). The binding of each regulator to its regulatory region was analyzed by ChIP with the indicated antibodies. Note that DBP binding in Dbp/
mice was significantly reduced (p < 0.01 by two-way ANOVA), with residual signals potentially deriving from TEF and/or HLF binding. Specific TaqMan probes

272 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

RRE-mediated reporter expression, albeit with an increase inamplitude (Figure S4). The intron sequence sometimes caused
double peaks for the E0 box-driven rhythms (Figure 4A and
Figure S4). When these rhythms were fitted to a circadian cosine
curve, we observed a reduction of the relative amplitude anda slight but reproducible phase advance (Figure 4A, Figure S4,
and Table S1). Importantly, the combination of D box in the
promoter and RRE-containing Cry1 intron sequence conferreda substantial phase delay of > 5 hr (CT14.48 ± 0.21) whencompared to the D box alone (CT9.31 ± 0.16) (Figure 4, Figure S4,
and Table S1). It is important to note that our result indicatesthat E/E0 boxes are dispensable for the generation of delayedphase expression of Cry1. This is because the synthetic
composite ‘‘D box + RRE’’ promoter (i.e., a combination ofa synthetic D box-driven promoter and RRE-containing Cry1
intron sequence) lacks functional E boxes, unlike the Cry1
promoter. Thus, the D box and the RRE can combine to generatea distinct intermediate phase.
We were able to recapitulate these experimental measurements in a simple model using ‘‘phase vectors.’’ A phase vector
represents phase and amplitude of the oscillation as direction
and length of the vector in polar coordinates. In this way, thecombination of two oscillations can be represented by the vector
sum of two corresponding phase vectors (Extended Experimental Procedures). We plotted measured oscillations (CCEwithout intron sequence and intron sequence without CCE) andobtained the summed phase vector of the CCE-intron sequence
combinations (Figure 4B, left three circles). Interestingly, thesummed phase vectors corresponded well with the measured
oscillations (Figure 4B, rightmost). These results support thenotion that combining two CCEs that otherwise function independently can be a general mechanism for generation of new phases
and, more specifically, the combined phase may be predicted, toa first-order approximation, by a vector sum.
Delayed Expression of Cry1 Restores Circadian
Rhythmicity in Cry1/:Cry2/ Cells
To address the functional importance of the RRE-mediatedphase delay, we employed cell-based genetic complementation,
testing for phenotypic rescue in arrhythmic Cry1/:Cry2/cells. We hypothesized that, if phase delay is an important property of Cry1, its delayed expression, peaking at evening-time,should restore circadian oscillations in these cells. To test thishypothesis, we established mouse embryonic fibroblasts from
Cry1/:Cry2/ double-knockout mice (van der Horst et al.,1999). Similar to negative control (Figure 5A, without Cry1),
Cry1 expression driven only by the Cry1 promoter, P(Cry1), did
not rescue circadian oscillations in these cells (Figure 5A,P(Cry1)). However, when Cry1 expression was regulated byP(Cry1)-Cry1 intron 336, which contains the Cry1 promoter andthe RRE-containing 336 bp of Cry1 intron sequence, its exogenous expression restored circadian rhythmicity in these cells,with a period length of 26.73 ± 0.19 hr (Figure 5A, Cry1 intron
336). The observed rescue capability was independent of the
CRY1 protein level, vector type, or method of DNA delivery(Figures S5A and S5B). Taken together, these results demonstrate that delay of Cry1 expression, conferred by the Cry1
intron, is required for rescue of circadian rhythmicity.
To further assess the contribution of delayed Cry1 expression
to the rescued circadian oscillation, we tested the rescue capability of the intronic RREs mutants that possess different RRE
strengths, as described above (Figure 3 and Figure 5A, nine
panels on the right, and Table S2). The ability of the intronic
RRE mutants to rescue rhythmicity, represented as amplitudeof circadian oscillations, significantly correlated with the strengthof intronic RREs, as measured by bioluminescence levels
derived from P(SV40)-Cry1 intron 336-Luc (r2 = 0.87, p < 0.01;
Figure 5B, left). Similarly, the rescue capability also correlatedwith another measurement of strength of intronic RREs, i.e.,
RORa responsiveness of the intronic RRE mutants (r2 = 0.97,
p < 0.01; Figure 5B right). More directly, the rescue capability
correlated with the phase delay conferred by the intronic
RRE mutants that was measured in bioluminescence rhythmsof P(Cry1)-Cry1 intron 336-Luc (r2 = 0.71, p < 0.01; Figure 5C).
It should be noted that the rescue capability in these experiments
does not correlate with either amplitude or basal bioluminescence levels of P(Cry1)-Cry1 intron 336-Luc (Figure S5C), suggesting that the rescue capability is most likely attributable tothe delayed phase of Cry1 expression conferred by RREs. To
directly confirm this, we demonstrated that the pure RREs,
when combined with Cry1 promoter, rescued circadian rhythmicity in Cry1/:Cry2/ cells, whereas Cry1 promoter alone
could not reliably rescue rhythms (Figure S5D and Figure 5).
The Cry1-rescued Cry1/:Cry2/ cells (a Cry2 knockout, inessence) showed a rather long period length of 27 hr (Figure 5A), which is consistent with previous reports showing that
Cry2/ single-knockout cells display long periods comparedto wild-type cells (24–25 hr) (Liu et al., 2007). We confirmedthat genetic complementation of Cry1 in Cry1/:Cry2/ cells
recapitulates the circadian phenotype in Cry2 single-knockout
cells, thus phenotypically validating the Cry1 rescue assay (Figure S5E and Table S3).
Cry1 Phase Delay Modulates Circadian Period Length
The genetic complementation assay expressing Cry1 of various
phases revealed that delay of Cry1 expression is required torestore circadian rhythmicity, consistent with the proposed designprinciple for circadian clocks, i.e., transcriptional/translational

were used to detect the Cry1 promoter region, the Cry1 first intron region, or the Dbp promoter region (control region). ZT, Zeitgeber time. Mean and SD represent
three ChIP experiments.
(E) Circadian expression profiles of Cry1, Bmal1, Rev-Erba, and Per1 in vivo. RNA from liver of wild-type (gray) or Dbp/:Tef/:Hlf/ (orange) mice wasprepared at 4 hr intervals from mice held in LD 12:12 cycle. Relative mRNA levels of each gene were measured. Note that Cry1 expression in Dbp/:Tef/:Hlf/
mice was different from wild-type and its peak delayed. All RNA samples were normalized to Gapdh mRNA accumulation. Mean and SEM from two pools of three
mice each per time point. The representative data from three independent experiments was shown. The observed peak delay was reproducible and significant inthree independent experiments (p < 0.01 by two-way ANOVA).
See also Figure S2 and Table S1.

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. 273

P(SV40)

Flag Cry1
intron

Luc

B

polyA

20
Relative
Bioluminescence

A

R1 R2

336

15
10
5
0

0.4

Relative amplitude ofP(SV40)-Cry1 intron 336-Luc

inversion

mutationC

no Cry1 intron
wild type Cry1 intron 336
R1 deletion
R2 deletion
R1,2 deletion
R1 mutation
R2 mutation
R1,2 mutation
R1 inversion
R2 inversion
R1,2 inversion

0.3

0.2

0.1

0

1

2

3

mutation
Cry1 intron 336

D4

y = 0.018x + 0.052
r2= 0.95
0.3

0.2

0.1

0.0
0

5

Flag Cry1
intron

Luc

polyA

F
Phase (h)R1 R2

no Cry1 intron

10.63

wild type Cry1 intron 336

14.03

R1 deletion

11.70

R2 deletion

11.65

R1,2 deletion

10.36

R1 mutation

12.49

mutation

deletion

336

10

15

20

RORα-responsiveness5

Phase-delay
from P(Cry1)-Luc (h)P(Cry1)

inversion

0.4

Day

y=0.26x-0.21
r2=0.82
2.5

0

0

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20

-2.5

RORα-responsivenessinversion

5

R2 mutation

11.18

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9.61

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13.43

R2 inversion

10.48

R1,2 inversion

10.96

Normalized Bioluminescence
Low
-1

0

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Day

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0

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Phase-delay
from P(Cry1)-Luc (h)Edeletioncontrol element

Relative amplitude ofP(SV40)-Cry1 intron 336-Luc

deletion

RORα - + - + - + - + - + - + - + - + - + - + - + - + - + - +E'
D RRE (-) Wild R1 R2 R1,2 R1 R2 R1,2 R1 R2 R1 ,2

y = 14.86x - 1.01
r2= 0.90
2.5

0
0.0

0.1

0.2

0.3

0.4

5
-2.5

Relative amplitude ofP(SV40)-Cry1 intron 336-Luc

Figure 3. The Strength of Intronic RREs Correlates with Phase Delay(A) Mutant reporter constructs derived from P(SV40)-Cry1 intron 336-Luc. P(SV40)-Cry1 intron 336-Luc contains the 336 bp of Cry1 intron sequence (wild-type);
the RREs or R1/R2 within the Cry1 intron sequence were altered to generate three sets of Cry1 intron sequence mutants: deletions, mutations, and inversions.
Red arrow indicates the direction of R1 and R2. Purple, light blue, and white rectangles represent wild-type, mutated, and deleted RREs, respectively.
(B) RORa responsiveness of mutated intron sequences. Each reporter construct in (A) was transiently transfected into NIH 3T3 cells in the absence (–) or
presence (+) of RORa. Luciferase activities were scaled so that basal activity without RORa was 1.
(C) Relative amplitudes of circadian transcriptional activities induced by constructs presented in (A).
(D) Correlation between two measurements for the strength of intronic RREs, the RORa responsiveness (B), and the relative rhythm amplitude (C) of mutatedintron sequences.
(E) Mutant reporter constructs derived from P(Cry1)-Cry1 intron 336-Luc and their phases. The SV40 promoter in P(SV40)-Cry1 intron 336-Luc was replaced with
Cry1 promoter P(Cry1) to generate P(Cry1)-Cry1 intron 336-Luc. The RRE mutations in P(Cry1)-Cry1 intron 336-Luc are the same as in P(SV40)-Cry1 intron
336-Luc constructs in (A).
(F) Phase delay correlates with two measurements for the strength of intronic RREs, the RORa responsiveness and the relative rhythm amplitude. The RORa
responsiveness presented in (B) (top) and relative rhythm amplitudes presented in (C) (bottom) of mutated intron sequences are plotted against phase delay of P
(Cry1)-Cry1 intron 336-Luc activity relative to P(Cry1)-Luc activity presented in (E).
Data are representative of two independent experiments (B, C, and E). Error bars represent SD (n = 3) (B and C). Mean and SD (error bar) of two independent
experiments are shown (each experiment contains three samples; n = 3 unless otherwise indicated in Table S1) (D and F). See also Figure S3 and Table S1.

feedback repression with delay. This design principle further
predicts that Cry1 expression with a more prolonged delay canslow circadian oscillations. To test this prediction, we first attemp274 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

ted to generate constructs expressing Cry1 with prolonged
delays. According to the phase-vector model described above(Figure 4B), we should be able to generate evening-to-nightA
3× CCE

P(SV40)Flag Cry1
intronLuc

polyA

Phase (h)
R1 R2

336

336

3.27

E'-box-P(SV40)-Cry1 intron 336

2.13 *

D-box-P(SV40)

9.31

D-box-P(SV40)-Cry1 intron 336

14.48

RRE-P(SV40)

17.04

RRE-P(SV40)-Cry1 intron 336

16.32
0

1

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High
Normalized Bioluminescence336

E'-box-P(SV40)

1

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Predicted

Measured

0

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RRE + Cry1 intron 336

0

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E'-box + Cry1 intron 336

0
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6 18

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B

1.0

3
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0

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Phase (h)Figure 4. Combination of Intronic RREs with Known Circadian cis-Elements Gives Rise to Emergent Phases that Can Be Predicted by Phase
Vectors(A) Combination of the Cry1 intron sequence with known CCEs gives rise to emergent phases. A promoter was constructed by inserting 3 3 E0 box, 3 3 D box, or
3 3 RRE sequences in the upstream of P(SV40). In the reporter construct, Luciferase expression was under the control of the 3 3 CCE-P(SV40) promoter in theabsence or presence of the 336 bp of Cry1 intron sequence. The experiment was performed as in Figure 1A. Phases were estimated by fitting a cosine wave withcircadian period corresponding to maximum autocorrelation of the time series using detrended bioluminescence data. This method allowed phase estimation ofeven distorted wave form expressed by E0 box + Cry1 intron sequence (marked by asterisk).
(B) A phase vector model recapitulates the emergent phases. The phase vector of each CCE (E0 box, green arrow; D box, orange arrow; RRE, purple arrow) andCry1 intron sequence (black arrow) and the vector sum of the two phase vectors (center of colored ellipsoid) are plotted in the polar coordinate (left three circles).
The ellipsoidal disk represents 95% confidence region. The phase vectors (colored arrows of black border) represent measured circadian transcriptional activities
induced by the combined regulation of Cry1 intron sequence and each CCE (rightmost circle).
Data are representative of two independent experiments. See also Figure S4 and Table S1.

expression with a more prolonged delay by weakening the daytime promoter but keeping a constant strength of the night-timeenhancer of Cry1 intron sequence. Therefore, we generated anarray of day-time promoters with various strengths of D boxes,
containing 1, 2, or 3 tandem repeats of D boxes or Cry1proD
elements; we confirmed that these day-time promoters displayed
day-time phased bioluminescence rhythms of various relative
amplitudes, as expected (Figure 6A, Figure S6A, and Table S1).
We then generated another set of constructs by combining theseday-time promoters with the Cry1 intron sequence (Figure 6B, Figure S6B, and Table S1). These constructs displayed evening-tonight phases of bioluminescence rhythms (Figure 6B, Figure S6B,
and Table S1). Importantly, there was a significant correlation
between the observed phases and the predicted phases fromthe simple phase-vector model (r2 = 0.77, p < 0.01; Figure 6C,
rightmost panel).

Next, we asked whether evening-to-night Cry1 expression
with prolonged delay could slow circadian oscillations (Figure 6D
and Table S3). Interestingly, the periods of rescued circadianoscillations ranged from 27 to 31 hr. In particular, period length
correlated with the delay prolonged by weakening the day-timepromoter: the more the Cry1 phase was delayed, the longer
the rescued period (r2 = 0.81, p < 0.01; Figure 6E and see also
Figure S6C). We also confirmed that the period length did not
significantly correlate with either amplitude or basal activity of
Cry1 expression by using a different constitutive promoter (Figure S6D and Table S3). In addition, CRY1 protein level was not
responsible for the changes in rescued period (Figure S6E).
These results showed that Cry1 expression with a prolonged
delay slows circadian oscillations, further supporting the
proposed design principle of circadian clocks—transcriptional/translational feedback repression with delay.
Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. 275

B

0

6

deletion
1

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P(Cry1)12000000
8000000
4000000
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P(Cry1)-Cry1 intron 336

15000000
10000000
5000000
0

0

1

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Day

4

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6

R2

Relative amplitude of
rescued oscillationy = 1.87x + 0.14
r2= 0.87
0.6

0.3

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Day

no Cry1 intron
wild type Cry1 intron 336
R1 deletion
R2 deletion
R1,2 deletion
R1 mutation
R2 mutation
R1,2 mutation
R1 inversion
R2 inversion
R1,2 inversion

0.6

0.3

12

0

10000000

2

3

4

18

RORα-responsiveness

5

0

6

0

Day

C

6

6

15000000

y = 0.036x + 0.23
r2= 0.970515000000

0.0

Relative amplitude ofP(SV40)-Cry1 intron 336-Luc

R1,2

15000000

0.9

0.9

Relative amplitude of
rescued oscillation

0

R1

15000000Day
0.9

Relative amplitude of
rescued oscillation

Cry1-/-:Cry2-/- double KO MEF

6

Bioluminescence (counts/min)dLuc

Bioluminescence (counts/min)

P(Per2)

Bioluminescence (counts/min)

Plasmid DNA for reporter

10000000

mutation

Cry1

Bioluminescence (counts/min)

Flag Cry1
introninversion

Plasmid DNA for rescue
P(Cry1)

without Cry1
20000000

Bioluminescence (counts/min)

Bioluminescence (counts/min)

A

-2.5

0.6

0.3
y = 0.11x + 0.29
r2= 0.71
0.0
0.0

2.5

5.0

Phase-delay ofP(Cry1)-Cry1 intron 336-Luc (h)Figure 5. Delayed Expression of Cry1 Restores Circadian Rhythmicity in Cry1/:Cry2/ Cells(A) Genetic complementation of Cry1 rescues circadian oscillation in Cry1/:Cry2/ cells. A schematic diagram of Cry1 rescue constructs is shown on the left.
The composite promoter contains P(Cry1) and the 336 bp Cry1 intron sequence of wild-type or a mutant (deletion, mutation, or inversion of the R1 and R2
sequences as in Figure 3), which controls Cry1 expression. Cry1 rescue constructs were each cotransfected with a destabilized Luciferase reporter construct, P(Per2)-dLuc, into Cry1/:Cry2/ mouse embryonic fibroblast cells (left), followed by bioluminescence recording. Whereas mock-transfected Cry1/:Cry2/cells were completely arrhythmic and those expressing P(Cry1)-Cry1 were only transiently rhythmic during the first 2 days of recording, P(Cry1)-Cry1 intron 336Cry1 expression restored circadian oscillation with a period length of 26.73 ± 0.19 hr (bottom in the center column). Rescue effects varied among the intronic RRE
mutants (right nine panels). Data are representative of two independent experiments.
(B) Relative amplitude of rescued circadian oscillation correlates with the strength of intronic RREs. The relative amplitudes of rescued oscillation are plottedagainst two measurements for the strength of intronic RREs, the relative amplitudes of P(SV40)-Cry1 intron 336-Luc oscillation presented in Figure 3C, and the
RORa responsiveness presented in Figure 3B.
(C) Relative amplitude of rescued circadian oscillation correlates with phase delay. The relative amplitudes of rescued oscillations are plotted against the phasedelay of various P(Cry1)-Cry1 intron 336-Luc activities relative to P(Cry1)-Luc activity presented in Figure 3E.
Mean and SD (error bar) of two independent experiments are shown (each experiment contains three samples; n = 3 unless otherwise indicated in Table S1). See
also Figure S5 and Table S2.

Single-Cell Analysis Confirms the Importance of Cry1
Phase Delay in Feedback Repression
Arrhythmic phenotypes observed in population of cells might bedue to rapid damping of individual cells or lack of synchronization among individual cells. To discriminate between these
possibilities, we monitored bioluminescence levels in real timeat the level of single-cell resolution (Sato et al., 2006; Ukai
et al., 2007). As with whole-well assays, single-cell analysis
showed that most individual cells expressing Cry1 with a normal
delay, driven by the intron sequence containing wild-type RREs,
were robustly rhythmic, with a circadian period of 26.77 ± 0.12 hr
276 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

(Figures 7A and 7B and Table S3), whereas most cells expressing Cry1 without delay, driven by an intron sequence harboring
mutated RREs, were arrhythmic (Figure 7B and Movie S1). Moreover, individual cells expressing Cry1 with a prolonged delay
driven by the Cry1 intron sequence alone (i.e., in the absenceof Cry1 promoter) displayed long circadian periods of up to
32.00 ± 0.58 hr (Figures 7A and 7B and Table S3). The circadianoscillations in Figure 7A with delayed Cry1 expression
were statistically significant (p < 0.01 by autocorrelation) andreproducible in different series of experiments. Thus, singlecell analysis confirmed the circadian phenotypes observed in

A

3× CCE

P(SV40)

Flag

Luc

B

polyA

3× CCE

Flag Cry1
intronP(SV40)

Luc

polyA

Relative
Phase (h) amplitude

Phase (h)3×E'-box-P(SV40)

3.37

0.40

P(Cry1)

14.61

3×RRE-P(SV40)

18.55

0.26

3×D-box-P(SV40)

15.46

P(Cry1)

9.61

0.18

2×D-box-P(SV40)

16.60

3×D-box-P(SV40)

9.84

0.14

1×D-box-P(SV40)

17.35

2×D-box-P(SV40)

11.48

0.08

3×Cry1proD-P(SV40)

16.64

1×D-box-P(SV40)

---

0.06

2×Cry1proD-P(SV40)

16.56
16.61

3×Cry1proD-P(SV40)

13.43

0.15

1×Cry1proD-P(SV40)

2×Cry1proD-P(SV40)

12.70

0.18

P(SV40)1×Cry1proD-P(SV40)

12.00

0.11

---

0.06

P(SV40)
0

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phase

15

9
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12
Phase (h)D6Bioluminescence (counts/min)

P(SV40)

6

D-box-P(SV40)

20000000
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Cry1proD-P(SV40)

P(Cry1)20000000015

93× element

12
Phase (h)2× element
20000000

15000000

15000000

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16

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32

4

5

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30

28
y = 0.94x + 27.53
r2= 0.81-11500000015

Predicted phase (h)1× element

20000000016E200000000y = 1.22x - 3.54
r2= 0.77
17

0.4+ Cry1 intron 336

Bioluminescence (counts/min)

Bioluminescence (counts/min)

Bioluminescence (counts/min)+ Cry1 intron 336

00.2

P(Cry1)-Cry1 intron 336
3×D-box-P(SV40)-Cry1 intron 336
2×D-box-P(SV40)-Cry1 intron 336
1×D-box-P(SV40)-Cry1 intron 336
3×Cry1proD-P(SV40)-Cry1 intron 336
2×Cry1proD-P(SV40)-Cry1 intron 336
1×Cry1proD-P(SV40)-Cry1 intron 336

Amplitude

Amplitude

Predicted
phase
0.2

6

Period ofrescued oscillation (h)6Measured phase (h)Measured phase

Predicted phase

18

26

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Phase-delay ofcomposite promoters (h)

1

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P(SV40)P(Cry1)
3×D-box-P(SV40)
2×D-box-P(SV40)
1×D-box-P(SV40)
3×Cry1proD-P(SV40)
2×Cry1proD-P(SV40)
1×Cry1proD-P(SV40)

Figure 6. Prolonged Delay of Cry1 Expression Slows Circadian Oscillations in Cry1/:Cry2/ Cells(A) Promoters harboring various CCEs display different circadian phases. The promoters contain one, two, or three tandem copies of D box or Cry1proD element,
which were inserted into the P(SV40)-Luc vector to generate an array of reporter constructs.
(B) The 336 bp of Cry1 intron sequence confers phase delay to D box and Cry1proD element. Reporter constructs were generated similarly as in (A) except that the336 bp of Cry1 intron sequence was inserted. The experiment was performed as in Figure 1A (A and B).
(C) The measured phases conferred by the composite promoters are consistent with those predicted by phase vectors. (Left) The phase vectors of oscillations
driven by various promoters without the intron sequence (colored arrows) and those driven by the intron sequence (CT17.5) or P(Cry1) (CT10) (two blackarrows) are plotted with summed phase vectors (center of colored ellipsoidal disks). The ellipsoidal disk represents 95% confidence region. (Right) The summed
phase vectors in the left circle are plotted with phase vectors of measured oscillations driven by the composite promoters. (Rightmost) The predicted phases fromthe simple phase-vector model are plotted against the observed phases. Error bars represent SD (n = 3).
(D) Cry1 rescue of circadian oscillation in Cry1/:Cry2/ cells using synthetic composite promoters. The composite promoters presented in (B) were used todrive Cry1 expression. Cry1/:Cry2/ cells were cotransfected with a Cry1 expression construct and a P(Per2)-dLuc reporter.
(E) Prolonged phase delay of Cry1 expression correlates with period length of rescued oscillations. The period lengths of rescued oscillations are plotted against
the phase delay of various composite promoters’ activity relative to P(Cry1)-Cry1 intron 336-Luc activity presented in (B). Mean and SD (error bar) of two
independent experiments are shown (each experiment contains three samples; n = 3).
Data are representative of two independent experiments (A, B, and D). See also Figure S6, Table S2, and Table S3.

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. 277

Bioluminescence (counts/min)A

P(Cry1)-Cry1 intron 336
R1,2 mutation-Cry1

P(Cry1)-Cry1 intron 336-Cry1

P(SV40)-Cry1 intron 336
R1,2 mutation-Cry1

P(SV40)-Cry1 intron 336-Cry1

15000000

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Activity (% of maximum)

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Figure 7. Single-Cell Analysis Confirms the Requirement of Cry1 Phase Delay

RRE(A) P(Per2)-dLuc bioluminescence levels in transfected Cry1/:Cry2/ cells as recorded. The P(Per2)-dLuc reporter and a Cry1 expression construct as indicatedwere cotransfected into Cry1/:Cry2/ cells, and bioluminescence expression was recorded with a PMT. Data from three independent samples are shown.
(B) P(Per2)-dLuc bioluminescence levels in transfected individual Cry1/:Cry2/ cells as recorded by a luminescence microscope (n = 100). Reporter activities
from each cell were normalized so that the maximum and minimum bioluminescence values are 100% and 0%, respectively. The mean reporter activity for all of theanalyzed single cells at each time point is indicated by a thick black line (top row). Time series of bioluminescence expression shown in the top row were redrawn asheatmaps (bottom row). Each row in the heatmap represents a time series of P(Per2)-dLuc reporter activities from a single cell. The corresponding p value ofrhythmicity at the period of maximum autocorrelation was evaluated for each time series and is depicted on the right. One-hundred cells were randomly selected
and individually analyzed. Data are representative of two independent experiments (A and B).
(C) The roles of phase delay in Cry1 expression. Through regulation of Cry1 expression, the promoter and intron primarily affect the amplitude and period of theclock system, respectively.

278 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

Cry1-rescued Cry1/:Cry2/ cells at the cell population level,
which lends strong support for our finding that delay of Cry1
expression is required for circadian clock function.
DISCUSSION
Cry1 Phase Control Mechanism
In this report, we provided experimental data, as well as model
predictions, for a ‘‘combinatorial regulatory mechanism’’ toexplain the delayed expression of Cry1. We newly identified D
boxes, which overlap with the E/E0 box and confer phase delay
over E/E0 box activity. In addition, we also demonstrated thatthe previously identified RREs in the first intron (Ueda et al.,2005) can confer the additional phase delay in Cry1 expression.
Furthermore, we observed that the synthetic pure RREs, in
combination with Cry1 promoter, rescued circadian rhythmicity
in Cry1/:Cry2/ cells with statistical significance (p < 0.01;
Figure S5D). Together with the observation that the combinationof pure D box and Cry1 intron sequence also rescued rhythms(Figure 6D), we conclude that the RRE and D box elements
can recapitulate the basic function of Cry1 intron and Cry1
promoter, respectively. It should be noted that the possible
contribution from unknown elements in Cry1 intron 336
sequence could not be completely excluded because the relative amplitude of the pure RRE elements (combined with Cry1
promoter) is slightly lower than that of the wild-type Cry1 intron
336 (combined with Cry1 promoter, Figure S5D).
General Design Principles for New Phases
As revealed in this study, these multiple distinct regulatory sites(i.e., two RREs in first intron and the E/E0 box and D boxes in the
promoter region) function in a coordinated fashion to generate
substantial phase delay, leading to evening-time expression.
Interestingly, in an effort to study design principles of thecircadian clockwork, we employed a simple phase-vector modelin which the new evening-time could be predicted by the
combination of two component phase vectors. Although thephase-vector model was not used for phase prediction in ourprevious study (Ukai-Tadenuma et al., 2008), such a model isalso applicable to this previous study when we take into accountthe time delay associated with transcription/translation of regulator proteins and the Luciferase reporter (r2 = 0.99, p < 0.01;
Figures S7A and S7B and Table S4). These results show thata new phase can be generated through combinatorial synthesisof either two transcriptional regulators or two clock-controlled
DNA elements and also can be described, at least to a first-orderapproximation, by a phase-vector model. Taken together, thiscombinatorial regulatory mechanism for the generation of newcircadian phases of transcription represents a general designprinciple underpinning the complex system behavior.
Although the phase-vector model predicts the phase ofa synthesized oscillation, it is only a first-order approximation.

For example, there are some discrepancies between predicted
and measured amplitudes. Also, the wave form generated by
the combination of E0 box-driven promoter and the Cry1 intron
sequence appeared like a ‘‘two-peak’’ wave form (Figure S4,
upper-left), indicative of nonlinear effects or involvement of yet
unknown factors. In detailed analysis, we found that the 11 hr
period oscillation was actually a significant component in the
two-peak wave form (Figure S4, upper-right; p < 0.01). This is
the first demonstration of synthesized ultradian rhythms, which
may lead to insights into mechanisms of ultradian gene expression with harmonic periods, as recently reported (Hughes et al.,2009).
Delayed Cry1 Expression Contributes to Clock
Robustness
In this study, we focused on the level of transcriptional regulationand demonstrated the importance of delay in feedback repression at the intracellular level. Constitutive expression of Cry1
abolished circadian rhythmicity in wild-type cells (Ueda et al.,2005) and failed to restore circadian oscillation in arrhythmic
Cry1/:Cry2/ cells (Figure S6D and Figure 7A), suggestingthat rhythmic expression of Cry1 is important for clock function.
We revealed that the transcriptional oscillation of Cry1 with acorrect phase with substantial delay was sufficient and required
to rescue circadian oscillation in arrhythmic Cry1/:Cry2/cells (Figure 5) and that transcriptional oscillation of Cry1 witha prolonged delay slows circadian oscillation (Figure 6). Importantly, we also confirmed that the amount (baseline) of CRY1
protein was not responsible for the changes in amplitude (Figure S5A) and period (Figure S6E) of rescued oscillations. These
results suggest that the phase of Cry1 expression is responsible
for the changes in rescued amplitude and period rather than theamount (baseline) of CRY1 protein. Because we confirmed thesignificant linear correlation between transcriptional activities
and protein levels (p < 0.01), when monitored by firefly
Luciferase, and CRY1 protein amounts, when monitored byfusion Renilla Luciferase, irrespective of cell types (r2 = 0.93 in
NIH3T3 and r2 = 0.90 in Cry1/:Cry2/ cells), we speculated
that phase of CRY1 protein level would be responsible for theamplitude and period of rescued oscillations.
It should be noted that the CRY1 protein expression levels in
our experiments are within a certain range (Figure S5A and Figure S6E), and we do not exclude (and our current results are notin conflict with) the notion that CRY1 protein amounts may affectthe parameters of clock function when CRY1 protein levels drastically differ from those in our experimental system, as previously
reported (Baggs et al., 2009; Ueda et al., 2005). In addition, we
do not exclude the possibility that other regulatory mechanisms
such as posttranscriptional modifications (Lee et al., 2001; Liuet al., 2008) play important roles in attaining the robustness ofthe clock. For example, rhythmic expression of PER2 is recently
reported to play a prominent role in CRY1 function (Chen et al.,(D) A schematic diagram of a minimal circuit for the mammalian circadian transcriptional network. The network can be represented by a simple circuit, consisting of
two transcriptional activations (green arrows) and four transcriptional repressions (red arrows) on three regulatory elements (three rectangles).
(E) The minimal circuit envisaged as a composite of two distinct oscillatory network motifs: (1) A repressilator that is composed of three repressions (left) and (2)a delayed negative feedback loop, which is composed of two activations and one repression (right). See also Figure S7, Movie S1, and Table S3.

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. 279

2009). And PER2 is phosphorylated by CKId/3, which is also an
essential mammalian clock component (Lee et al., 2009) andhas been recently implicated in temperature compensation (Isojima et al., 2009). In addition, it has been reported that constant
supply of membrane-permeable CRY1 and CRY2 proteins
rescued circadian oscillation in Cry1/:Cry2/ cells (Fanet al., 2007). In line with this observation, our single-cell analysis
indicated that a fraction of individual cells transfected with Cry1driven by a constitutive promoter exhibited weak circadian oscillation even though the rhythms are rather transient (Figure 7B),
implying that constant Cry1 expression might partially rescue
circadian clock function. This qualitatively less-robust clockfunction is probably attributable to posttranscriptional and posttranslational mechanisms (Lee et al., 2001; Liu et al., 2008).
Even in this context, it appears that phase delay in rhythmic
Cry1 expression may contribute to the robustness of clock function by ensuring properly timed nuclear translocation of CRY
proteins. This idea is strongly supported by our results presented
in this study: delayed Cry1 expression via D box-mediatedtranscription (i.e., from Cry1 promoter) allowed partial rescue,and further delay via the RREs from the Cry1 intron restoredcircadian rhythmicity with amplitude and persistence comparable to wild-type cells.
Design Principle for a Circadian Transcriptional
Network
Delayed feedback repression is one of the most prevailing but as
yet unverified design principles for a circadian transcriptional
network. This design principle predicts that decreased delay
dampens circadian oscillations and that prolonged delay slows
down circadian oscillations (Figure 7C, Figures S7C–S7F, andExtended Experimental Procedures) (Bernard et al., 2006; Lewis,
2003; Novak and Tyson, 2008). The results presented in thisstudy are consistent with the two predictions from the delayed
feedback repression, suggesting that it is an applicable designprinciple in the mammalian circadian transcriptional network.
A Minimal Circuit for a Circadian Transcriptional
Network
In a previous effort to identify a minimal circuit of the complex
autoregulatory transcriptional networks in the mammalian circadian clock, we showed that day-time promoter activity can bereconstructed by combining a morning-time activator anda night-time repressor and night-time promoter activity by
combining a day-time activator and a morning-time repressor(Ukai-Tadenuma et al., 2008). In this study, we succeeded insynthesizing the evening-time phase control of transcription.
Our previous and current results suggest that the complex
mammalian transcription network can be reduced to a relatively
simple diagram (Figure 7D) that would consist of three regulatory
elements and six transcriptional regulations (two activations andfour repressions). It is noteworthy that this diagram can be envisaged as a composite of two distinct oscillatory network motifs(Figure 7E). The first oscillatory network motif is composed ofthree repressions (i.e., E/E0 box to RRE, RRE to D box, andD box to E/E0 box), comprising a cyclic negative feedback
loop—a repressilator (Elowitz and Leibler, 2000). The second
oscillatory network motif is composed of two activations (i.e.,
280 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

E/E0 box to D box and D box to RRE) and one repression (i.e.,
RRE to E/E0 box), comprising a delayed negative feedback
loop. It is interesting to note that oscillatory properties of both
network motifs were experimentally suggested by synthetic
approaches (Elowitz and Leibler, 2000; Stricker et al., 2008).
Therefore, further experimental and theoretical analyses of thecomposite of these oscillatory network motifs lie ahead.
EXPERIMENTAL PROCEDURES
Preparation of Embryonic Fibroblasts from Cry1/:Cry2/Double-Knockout Mice
Cry1/:Cry2/ double-knockout mice (van der Horst et al., 1999) were
carefully kept and handled according to the RIKEN Regulations for Animal
Experiments. The dissociated cells (mouse embryonic fibroblasts [MEF]from Cry1/:Cry2/ double-knockout mice; Cry1/:Cry2/ cells) weresuspended and cultured in DMEM (Invitrogen) supplemented with 10% FBS(JRH Biosciences) and antibiotics (see Extended Experimental Proceduresfor details).
Real-Time Circadian Reporter Assay Using NIH 3T3
Cells and Cry1/:Cry2/ Cells
Real-time circadian assays were performed as previously described (Satoet al., 2006; Ueda et al., 2005) with the following modifications. NIH 3T3 cells
were transfected with the Luciferase reporter plasmids. Cry1/:Cry2/ cells
were transfected with pGL3-P(Per2)-dLuc reporter plasmid (Sato et al., 2006)
and each Cry1 gene expression vector. The cells were stimulated by 10 mM(NIH 3T3) or 30 mM (Cry1/:Cry2/ cells) forskolin (Fermentek), and the bioluminescence was measured at 30 C (see Extended Experimental Proceduresfor details).

SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, seven
figures, four tables, and one movie and can be found with this article online at
doi:10.1016/j.cell.2010.12.019.
ACKNOWLEDGMENTS
This research was supported by an intramural Grant-in-Aid from the RIKEN
Center for Developmental Biology (CDB) (H.R.U.), Uehara Memorial Foundation (H.R.U.), The Mitsubishi Foundation (H.R.U.), the President’s Fund fromRIKEN (H.R.U.), KAKENHI (Grant-in-Aid for Scientific Research) on Priority
Areas ‘‘Systems Genomics’’ from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (H.R.U.), the National Science Foundation(IOS-0920417) (A.C.L.), and the SNF/FNS (J.A.R.). We thank Ueli Schibler,
Urs Albrecht, and Frédéric Gachon for their valuable reagents; Akira Yasui
and Gijsbertus T.J. van der Horst for Cry1/:Cry2/ double-knockout
mice; Hideki Ukai and Koh-hei Masumoto for technical support in establishment of Cry1/:Cry2/ cells; Yohei Koyama for critical comments on thetheoretical analysis of delayed negative feedback loop; Hajime Tei for Cry1
and Cry2 expression vectors; and David K. Welsh for critical reading of themanuscript.
H.R.U. and A.C.L. designed the research scheme. M.U.-T. constructedmost of materials and performed real-time luminescence assays. R.G.Y. conducted bioinformatic, statistic, and theoretical analyses. H.X. constructed thevirus vector and performed the corresponding real-time luminescence assays.
J.A.R. performed ChIP analysis and mRNA accumulation analysis. All authors
discussed the results and commented on the manuscript text.
Received: May 21, 2010
Revised: August 21, 2010
Accepted: December 10, 2010
Published online: January 13, 2011

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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Real-Time Circadian Reporter Assay Using NIH 3T3 Cells
Real-time circadian assays were performed as previously described (Sato et al., 2006; Ueda et al., 2005) with the following modifications. NIH 3T3 cells (American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% FBS (JRH
Biosciences) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin, Invitrogen). Cells were plated at 1 3 105 cells perwell in 35-mm dishes 24 hr before transfection. These cells were transfected with FuGene6 (Roche) according to the manufacturer’s
instructions. The cells in each well were transfected with 0.4 mg of the each Luciferase reporter plasmids. The amount of transfected
plasmid was adjusted to 2.0 mg with empty vector. After 72 hr, the media in each well 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 10 mM forskolin (Fermentek). Bioluminescence was measured with photomultiplier tube detector assemblies (LM2400R, 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.
Transfection and Luciferase Assay
NIH 3T3 cells (American Type Culture Collection) were maintained in DMEM (Invitrogen) supplemented with 10% FBS (JRH Biosciences) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Invitrogen). One day prior to transfection, cells were plated
onto six-well plates at a density of 2 3 105 cells per well. The following day, cells were co-transfected using FuGene6 (Roche) with 0.4
mg of a Luciferase reporter plasmid in the presence of the following constructs as indicated in respective figures: 0 or 0.4 mg each ofpMU2-Bmal1 (Kumaki et al., 2008) and pMU2-Clock (Kumaki et al., 2008), and 0, 0.8, 1.6 or 3.2 mg of pMU2-E4bp4 (Kumaki et al.,2008) (for Figure 1F); 0 or 0.4 mg each of pMU2-Bmal1 and pMU2-Clock, and 0 or 1.6 mg of pMU2-Cry1 (For Figure S1C); 0 or 0.8 mg ofpMU2-Dbp (Kumaki et al., 2008), pMU2-Tef, pMU2-Hlf (for Figure S1D); and 0 or 0.4 mg of pMU2-Rora (Kumaki et al., 2008) (for Figure 3B). Empty vector was used to make up the total amount of DNA to 4.0 mg per well. Additionally, 50 ng of a phRL-SV40 plasmid(Renilla luciferase (RLuc) reporter vector, Promega) was added into each transfection as an internal control for transfection efficiency.
Forty-eight hours after transfection, cells were harvested and assayed with Dual-Luciferase Reporter Assay System (Promega).
Luciferase activity was normalized by Rluc activity.
mRNA Expression Measurement of Endogenous Cry1, Per2, and Bmal1 and Exogenous Luciferase by Quantitative
PCR
Temporal mRNA profiles of endogenous Per2, Bmal1, and Cry1 in transfected NIH 3T3 reporter cells expressing a P(Cry1)-Luc or a P(Per2)-Luc reporter were measured by quantitative PCR. We sampled the transfected NIH 3T3 cells every 4 hr over 3 days after
synchronizing cells by forskolin stimulation.
NIH 3T3 cells were grown in DMEM (Invitrogen) supplemented with 10% FBS (JRH Biosciences) and antibiotics (100 U/ml penicillinand 100 mg/ml streptomycin; Invitrogen). NIH 3T3 cells were plated at a density of 9 3 105 cells per well in 12 of 90-mm dishes. The
following day, NIH 3T3 cells were transfected using FuGene6 (Roche) with 5.4 mg of pMU2-P(Per2)-Luc or pMU2-P(Cry1)-Luc
plasmid according to the manufacturer’s instructions. Empty vector was used to make up the total amount of DNA to 18.0 mg perdish. After 24 hr, cells in each dish were harvested and plated in 76 of 35-mm dishes. After 48 hr, medium in each dish 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 10 mM forskolin (Fermentek), and subsequently cultured at 30 C. Cells were sampled every 4 hr for 3 days (n = 3
dishes). Total RNA was prepared from 35-mm dishes at each time point using TRIzol reagents (Invitrogen), and purified with RNeasykit (QIAGEN) and RNase-free DNase set (QIAGEN) according to the manufacturer’s instructions. The cDNA was synthesized from0.25 mg of total RNA with random 6-mer (Promega) and Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using ABI Prism 7700 and Power SYBR Green Reagents (Applied Biosystems). Samples contained 1 3 Power SYBR Green Master Mix (Applied Biosystems), 0.5 mM primers and 1/20 synthesized cDNAin a 10 ml volume. PCR conditions were as follows: 10 min at 95 C, then 45 cycles of 15 s at 94 C, 30 s at 59 C and 1 min at 72 C.
Absolute cDNA abundance was calculated using a standard curve obtained from genomic DNA and pMU2-Luc plasmid DNA. Gapdh
expression levels were quantified and used as an internal control.
The expression phases were obtained from analyses of temporal mRNA profiles by fitting a cosine wave of 21.5 hr period, as estimated from circadian bioluminescence rhythms monitored in parallel.
Primer Sequences Used in Quantitative PCR
Cry1 30 -UTR
Forward primer, 50 -TGAGGCAAGCAGACTGAATATTG-30 ; reverse, 50 -CCTCTGTACCGGGAAAGCTG-30 .
Per2 30 -UTR
Forward primer, 50 -TGTGCGATGATGATTCGTGA-30 ; reverse, 50 -GGTGAAGGTACGTTTGGTTTGC-30 .
Bmal1 30 -UTR
Forward primer, 50 -CCACCTCAGAGCCATTGATACA-30 ; reverse, 50 -GAGCAGGTTTAGTTCCACTTTGTCT-30 .
Gapdh
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Forward primer, 50 -CAAGGAGTAAGAAACCCTGGACC-30 ; reverse, 50 -CGAGTTGGGATAGGGCCTCT-30 .
Luciferase
Forward primer, 50 -CTTACTGGGACGAAGACGAACAC-30 ; reverse, 50 -GAGACTTCAGGCGGTCAACG-30 .
Chromatin Immunoprecipitation from Mouse Liver Chromatin
Chromatin samples from mouse liver were obtained from Dbp/ mice (Lopez-Molina et al., 1997) (kind gift from U. Schibler, Geneva)
or Rev-Erba/ mice (Preitner et al., 2002) (kind gift from U. Albrecht, Fribourg). In brief, mouse liver was homogenized in 1 3 PBS
supplemented with 1% formaldehyde (Ripperger and Schibler, 2006). Following 5 min incubation at RT, the reaction was quenchedby addition of three volume of ice-cold 2.4 M sucrose, 10 mM HEPES pH 7.6, 15 mM KCl, 0.5 mM spermidine, 0.15 mM spermine,
150 mM glycine, 1 mM DTT. The sample was layered on top of a 2.05 M sucrose/ 10% glycerol cushion in the same buffer, and centrifuged for 1 hr at 4 C and 100,000 3 g. The pellet containing purified nuclei was resuspended in 1% SDS, 20 mM Tris-HCl pH 7.4,
150 mM NaCl, 2 mM EDTA, and sonicated five times using a micro-tip device for the ultrasonic homogenizer 300V/T (BioLogics).
The dilution conditions, the pre-clearing step, the co-immunoprecipitation conditions, the washing conditions, the reversal of thecross-links and the subsequent purification of the DNA fragments, and the antibodies (anti-BMAL1, anti-DBP and anti-REVERBa) used have been described (Schmutz et al., 2010; Wuarin and Schibler, 1990). The co-immunoprecipitated DNA-fragments
were quantified using TaqMan RT-PCR using a RotorGene 6200 RT-PCR machine (Corbett Life Science) and EX Taq PCR mastermix (TaKaRa Bio, Inc.). Specific TaqMan probes were used to detect the Cry1 promoter region, the Cry1 1st intron region, or the
Dbp promoter region (control region).
Probe Sequences Used in Quantitative PCR
FAM, 6-carboxyfluorescin; TAMRA, 6-carboxytetramethylrhodamine; BHQ1, black hole quencher1.
Dbp Promoter (Control Region)
Forward, 50 -ACACCCGCATCCGGTAGC-30 ; reverse, 50 -CCACTTCGGGCCAATGAG-30 ; probe, 50 -FAM-CGCGCAAAGCCATGTG
CTTCC-TAMRA-30 .
Cry1 Promoter
Forward, 50 -CGCAACCGGTCCCGAAGCA-30 ; reverse, 50 -ACCACCGGCACCTCACGTTT-30 ; probe, 50 -FAM-TCAGACCCCCGCG
TGCGCCG-BHQ1-30 .
Cry1 1st intron
Forward, 50 -TCATTGTGATGGGAGTATGC-30 ; reverse, 50 -TCCAAAAGATGATTTCAACA-30 ; probe, 50 -FAM-CACCCACTGGTTG
CTATAGCGA-BHQ1-30 .
Quantitative PCR from Mouse Liver RNA
RNA samples from mouse liver were obtained from PAR bZip triple knockout mice (Dbp/:Tef/:Hlf/) (Gachon et al., 2004) andwild-type mice (kind gift from F. Gachon, Lausanne). In brief, total RNA was extracted from liver tissue using RNA-Bee (AMS Biotechnology) according to the manufacturer’s instructions. ssDNA complementary to the RNA starting from hybridized random hexamer
primers was synthesized with SuperScript II (Life Technology Corporation) according to the manufacturer’s instructions. The products were quantified using TaqMan RT-PCR using a RotorGene 6200 RT-PCR machine (Corbett Life Science) and EX Taq PCR
master mix (TaKaRa Bio, Inc.). All RNA samples were normalized to Gapdh mRNA accumulation.
Probe Sequences Used in Quantitative PCR
FAM, 6-carboxyfluorescin; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binder.
Cry1
Forward, 50 -CTGGCGTGGAAGTCATCGT-30 ; reverse, 50 -CTGTCCGCCATTGAGTTCTATG-30 ; probe, 50 -FAM-CGCATTTCACATA
CACTGTATGACCTGGACA-TAMRA-30 .
Bmal1
Forward, 50 -CCAAGAAAGTATGGACACAGACAAA-30 ; reverse, 50 -GCATTCTTGATCCTTCCTTGGT-30 ; probe, 50 -FAM-TGACCCTCATGGAAGGTTAGAATATGCAGAA-TAMRA-30 .
Rev-Erba
Forward, 50 -CATGGTGCTACTGTGTAAGGTGTGT-30 ; reverse, 50 -CACAGGCGTGCACTCCATAG-30 ; probe, 50 -FAM-ACGTGGCC
TCAGGC-MGB-30 .
Per1
Forward, 50 -ACCAGCGTGTCATGATGACATAC-30 ; reverse, 50 -CTCTCCCGGTCTTGCTTCAG-30 ; probe, 50 -FAM-CCGTCCAGGG
ATGCAGCCTCTG-TAMRA-30 .
Dbp
Forward, 50 -CGTGGAGGTGCTTAATGACCTTT-30 ; reverse, 50 -CATGGCCTGGAATGCTTGA-30 ; probe, 50 -FAM-AACCTGATCCCG
CTGATCTCGCC-TAMRA-30 .
Tef
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Forward, 50 -GCCGAGCTTCGCAAGGA-30 ; reverse, 50 -ACAGGTTACAAGGGCCCGTACT-30 ; probe, 50 -FAM-ACACGATGGTCTTGCACTTGCCCA-TAMRA-30 .
Hlf
Forward, 50 -AGGCGCAGAAAGAACAACATG-30 ; reverse, 50 -GAATGAGGCCCGGATTGC-30 ; probe, 50 -FAM-CGGCCAAGCGCT
CCCGTG-TAMRA-30 .
Gapdh
Forward, 50 -CATGGCCTTCCGTGTTCCTA-30 ; reverse: 50 -CCTGCTTCACCACCTTCTTGA-30 ; probe, 50 -FAM-CCGCCTGGAGAAA
CCTGCCAAGTATG-TAMRA-30 .
Preparation of Embryonic Fibroblasts from Cry1/:Cry2/ Double-Knockout Mice
Cry1/:Cry2/ double knockout mice (van der Horst et al., 1999) were carefully kept and handled according to the RIKEN Regulations for Animal Experiments. Embryos of Cry1/:Cry2/ double knockout mice at E13-14 were excised and washed several
times with Hanks’ balanced salt solution. After the placenta and internal organs were completely removed, the embryos were cut
into small pieces, and the cells were dissociated by incubation in 0.05% trypsin solution with 0.53 mM EDTA for 30 min at 37 C.
The dissociated cells (mouse embryonic fibroblasts (MEF) from Cry1/:Cry2/ double knockout mice; Cry1/:Cry2/ cells)
were suspended and cultured in DMEM (Invitrogen) supplemented with 10% FBS (JRH Biosciences) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin, Invitrogen).
Real-Time Circadian Reporter Assay Using Cry1/:Cry2/ Cells
Real-time circadian assays were performed as described above with the following modifications. Cry1/:Cry2/ cells were plated
at 4 3 105 cells per well in 35-mm dishes 24 hr before transfection. The cells in each well were transfected with FuGene6 with 3.95 mg
of pGL3-P(Per2)-dLuc reporter plasmid (Sato et al., 2006) and 0.15 mg of each Cry1 gene expression vector. The amount of transfected plasmid was adjusted to 4.1 mg with empty vector. After 72 hr, the medium in each well was replaced with 2 ml of culture
medium (DMEM/10% FBS) supplemented with 10 mM HEPES (pH 7.2), 0.1 mM luciferin, antibiotics, and 30 mM forskolin (Fermentek). Bioluminescence was measured with photomultiplier tube detector assemblies (LM2400R, Hamamatsu Photonics) at 30 C.
Real-Time Circadian Reporter Assay and Measurement of Rluc Activity Using Rluc Fused Cry1
Real-time circadian assays were performed as described above with the following modifications. Cry1/:Cry2/ cells were plated
at 4 3 105 cells per well in two 35-mm dishes 24 hr before transfection. The cells in each two well were transfected with FuGene6(Roche) with 3.95 mg of pGL3-P(Per2)-dLuc reporter plasmid (Sato et al., 2006) in the presence of the following constructs as indicated in respective figures: 1.5, 0.45 or 0.15 mg (for Figure S5A) and 0.3, 0.2 or 0.15 mg (for Figure S6E) of each Cry1-Rluc fusion
gene expression vector. The amount of transfected plasmid was adjusted to 5.5 mg with empty vector. After 24 hr, the cells ineach two well were harvested and plated in two 35-mm dishes. After 48 hr, bioluminescence was measured with photomultipliertube detector assemblies (LM2400R, Hamamatsu Photonics) at 30 C with the cells in each one 35-mm dishes, which was replacedwith 2 ml of culture medium (DMEM/10% FBS) supplemented with 10 mM HEPES (pH 7.2), 0.1 mM luciferin, antibiotics, and 30 mM
forskolin (Fermentek). In the same time, cells in each another well were harvested and measured Rluc activity with Dual-Luciferase
Reporter Assay System (Promega). To measure the average of CRY1 protein levels over a circadian period, the timing of measurement was just before the stimulation where the population of cells had a mixture of different circadian phases.
Real-Time Single-Cell Bioluminescence Imaging
Cry1/:Cry2/ cell samples were prepared as described above with the following modifications. Six-well culture plate was sealedwith coverslips, placed on the stage of a luminescence microscope (Luminoview LV100, Olympus), and kept at 30 C in a heatedchamber (Olympus). Bioluminescence was imaged using a 10 3 objective (10 3 /0.40 U Plan SApo, Olympus) and transmitted toa cooled CCD camera (ORCA-II ER, Hamamatsu Photonics) mounted on the bottom port of the microscope. Readout noise was
reduced by using 2 3 2 binning of the 672 3 512 pixel array. Four samples were measured at the same experiment. Time-lapse
images were collected at 120 min intervals with 28 min exposures per sample by a computer using image analysis software (Metamorph, Molecular Devices).
Image Processing of Real-Time Single-Cell Bioluminescence Images
Hot pixels and cosmic rays were removed by applying spatial and temporal local median filters, respectively using Matlab (MathWorks). The position of a hot pixel was detected by comparing the time-variation of the pixel with surrounding pixels. The cosmic
rays were detected by comparing the intensities of pixels for the temporally adjacent images. The bioluminescence of each cell
was estimated by averaging pixels within the region of interest manually defined for each cell by a custom-made software program
developed with Matlab. Remaining data processing of the obtained single-cell bioluminescence data was conducted using Mathematica (Wolfram Research).
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Construction of Plasmids
pMU2-P(Cry1)-Luc
The coding sequence of Luciferase (Luc) was amplified from pGL3 plasmid (Promega) using PCR with the following primers: Forward
primers containing PI-PspI recognition sequence: (50 -TGGCAAACAGCTATTATGGGTATTAT GGGTGAAGACGCCAAAAACATAAAG-30 , recognition sequence is indicated with italics, Hokkaido System Science), Reverse primers containing PI-SceI recognition sequence: (50 -TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGA TTTACACGGCGATCTTTCCGC-30 ). PCR product wasdigested with PI-PspI and PI-SceI (New England BioLabs) and cloned into pMU2-MCS vector (Ukai et al., 2007), and the resulting
vector was designated as pMU2-MCS-Luc.
A 1.5 kb promoter region of mouse Cry1 gene was amplified from NIH 3T3 genomic DNA using PCR with forward primers containing MluI recognition sequence (50 -ACGC GTGTAAAGATGCACATGTGGCCCTG-30 , recognition sequence is indicated with italics,
Hokkaido System Science) and reverse primers containing BglII recognition sequence (50 -AGATCTCGTCCGGAG GACACGCA
TACC-30 ). PCR product of 1.5 kb Cry1 promoter fragment was digested with MluI and BglII. The promoter fragment was cloned
into the MluI-BglII site of pMU2-MCS-Luc vector immediately upstream of Luc to obtain pMU2-P(Cry1)-Luc.
pMU2-3 3 E0 Box-P(SV40)-Luc, pMU2-3 3 D Box-P(SV40)-Luc, pMU2-3 3 RRE- P(SV40)-Luc, pMU2-P(SV40)-Luc
pGL3-3 3 CCE-P(SV40)-dLuc plasmids (Ueda et al., 2005; Ukai-Tadenuma et al., 2008) contain one of the clock-controlled elements(or CCEs; italicized): Per2 E’ box (50 -GCGCGCGCGGTCACGTTTTCCACTATGTG ACAGCGGAGG-30 ), Per3 D box (50 CCCGCGCGTTATGTAAGGTACTCG-30 ) or Bmal1 RRE (50 -AGGCAGAAAGTAGGTCAGGGACG-30 ). pGL3-P(SV40)-dLuc is
a parental plasmid. These vectors were digested with KpnI and HindIII, and the obtained 3 3 E’ box-P(SV40), 3 3 D box-P(SV40),
3 3 RRE-P(SV40), and P(SV40) promoter fragments were cloned into the KpnI-HindIII sites in a pMU2-MCS-Luc vector upstreamof Luc, and the resulting vectors were designated as pMU2-3 3 E’ box-P(SV40)-Luc, pMU2- 3 3 D box-P(SV40)-Luc, pMU2-3 3
RRE-P(SV40)-Luc and pMU2-P(SV40)-Luc, respectively.
pMU2-3 3 Cry1proD-P(SV40)-Luc and pMU2-3 3 Cry1proD mutant-P(SV40)-Luc
Oligonucleotides (Hokkaido System Science) containing three tandem repeats of wild-type or mutated Cry1proD elements were annealed and inserted into the MluI-BglII sites upstream of pMU2-P(SV40)-Luc (see above), and the resulting vectors were designatedas pMU2-3 3 Cry1proD-P(SV40)-Luc and pMU2-3 3 Cry1proD mutant- P(SV40)-Luc.
Oligonucleotide Sequences for Three Tandem Repeats of Cry1proD Element
Cry1proD Wild-Type
Forward oligonucleotide, 50 -CGCGAAACACACTTCAGAAACGTGAGGTAAACACACTTCAGAAACGTGAGGTAAACACACTTCAG
AAACGTGAGGT-30 ; reverse, 50 -GATCACCTCACGTTTCTGAAGTGTGTTTACCTCACGTTTCTGAAGTGTGTTTACCTCACGTTTCTG
AAGTGTGTTT-30 .
Cry1proD Mutant
Forward oligonucleotide, 50 -CGCGAAACACACCACCCGGCCGTGAGGTAAACACACCACCCGGCCGTGAGGTAAACACACCAC
CCGGCCGTGAGGT-30 ; reverse, 50 -GATCACCTCACGGCCGGGTGGTGTGTTTACCTCACGGCCGGGTGGTGTGTTTACCTCACGG
CCGGGTGGTGTGTTT-30 .
pMU2-Cry1
The full-length coding sequence of mouse Cry1 was amplified from pCI-Cry1 by PCR using forward primers containing I-SceI recognition sequence (50 -ATTACCCTGTTATCCCTAATGGGGTGAACGCCGTGCAC-30 , recognition sequence is indicated with italics,
Hokkaido System Science) and reverse primers containing PI-PspI recognition sequence (50 -ACCCATAATACCCATAATAGCTGTTT GCCAGTTACTGCTCTGCCGCTGG-30 ). PCR product was digested with I-SceI and PI-PspI (New England BioLabs)
and cloned into pMU2 vector (Ukai et al., 2007), and the resulting construct was designated as pMU2-Cry1. In this pMU2 vector,
Cry1 gene is regulated by a CMV promoter.
pMU2-Hlf and pMU2-Tef
The full-length coding sequences of mouse Hlf and Tef were amplified from NIH 3T3 cDNA library by PCR using forward primers containing I-SceI recognition sequence and reverse primers containing PI-PspI recognition sequence (Hokkaido System Science). PCR
product was digested with I-SceI and PI-PspI (New England BioLabs) and cloned into pMU2 vector (Ukai et al., 2007), and the resulting vectors were designated as pMU2-Hlf and pMU2-Tef. In these pMU2 vectors, genes are regulated by a CMV promoter.
Oligonucleotide Sequences for Cloning of Hlf and Tef
Cloning of Hlf
Forward primer, 50 -ATTACCCTGTTATCCCTAATGAGAAAATGTCCCGACAGCTCC-30 ; reverse, 50 -ACCCATAATACCCATAATAG
CTGTTTGCCACAGGGGCCCGTGCCTGGCCTC-30 .
Cloning of Tef
Forward primer, 50 -ATTACCCTGTTATCCCTAATGACATGCCTGAGGTCCTCAAGTCC-30 ; reverse,
50 -ACCCATAATACCCATAATAGCTGTTTGCCACAAGGGCCCGTACTTGGTCTCG-30 .
pMU2-P(Per2)-Luc
Per2 promoter was digested from pGL3-P(Per2) (Sato et al., 2006) with MluI and BglII. Promoter fragments were cloned into the MluIBglII sites of pMU2-MCS-Luc vector upstream of Luc and designated as pMU2-P(Per2)-Luc.
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pMU2-P(Cry1)-Cry1 Intron 1.03k-Luc, pMU2-P(Cry1)-Cry1 Intron 336-Luc, and pMU2-P(Cry1)-Cry1 Intron D336-Luc
We amplified the 1.03 kb and 336 base intron regions of mouse Cry1 from NIH 3T3 genomic DNA by PCR with forward and reverse
primers containing I-SceI recognition sequence (Hokkaido System Science). PCR products were cloned into pCR2.1-TOPO (Invitrogen) and designated as pCR2.1- Cry1 intron 1.03k and pCR2.1-Cry1 intron 336. We used inverse PCR to modify the Cry1 intron
sequence. For construction of pCR2.1-Cry1 intron D336, we used pCR2.1-Cry1 intron 1.03k as a template. Inverse PCR products
were treated with Mighty Cloning kit (TaKaRa) for 50 -end phosphorylation, and subsequently self-ligated. These constructs were digested with I-SceI, and pMU2-P(Cry1)-Luc plasmid (see above) was also digested with I-SceI. The three Cry1 intron fragments wereligated at the I-SceI site of pMU2-P(Cry1)-Luc, and designated as pMU2-P(Cry1)-Cry1 intron 1.03k-Luc, pMU2-P(Cry1)-Cry1 intron
336-Luc and pMU2-P(Cry1)-Cry1 intron D336 -Luc, respectively.
Oligonucleotide Sequences for Construction of Cry1 Introns
Cloning of Cry1 Intron Sequence (1.03 kb)
Forward primer, 50 -ATTACCCTGTTATCCCTAATCGAGATGGTTAAGTCGGGTCTCTAGGGTC-30 ; reverse, 50 -TAGGGATAACAGGGTAATCTTGACAGCTCAGGATCGCACTAAACGTGTG-30 .
Cloning of Cry1 Intron 336 Sequence
Forward primer, 50 -ATTACCCTGTTATCCCTAATCGAGATGGTTGTTATGACACAGTGTAGAAAC-3 0 ; reverse, 50 -TAGGGATAACAGGGTAATCTTGACAGCTCTACCTTTTACTACTATAAAAACGTAC-3 0 .
Inverse PCR of Cry1 Intron D336
Forward primer, 50 -AATTCTCGAGATAACAGTCACCAGTGGAG-30 ; reverse, 50 -AATTCTCGAGAGGTAGTGATTTCTAGAGG-30 .
pMU2-Cry1 Intron 336-P(Cry1)-Luc, pMU2-P(Cry1)-Cry1 Intron 336-Flag-Luc, and pMU2-P(Cry1)-Luc-Cry1 Intron 336
pMU2-P(Cry1)-Luc plasmid was digested with KpnI (upstream of Cry1 promoter), I-CeuI (downstream of Cry1 promoter, New
England BioLabs) or PI-SceI (downstream of coding sequence, New England BioLabs), and then blunted with T4 DNA polymerase.
pCR2.1-Cry1 intron 336 (see above) was digested with I-SceI, and blunted with T4 DNA polymerase. The Cry1 intron 336 fragment
was ligated at KpnI, I-CeuI or PI-SceI site of pMU2-P(Cry1)-Luc, and designated as pMU2-Cry1 intron 336-P(Cry1)-Luc, pMU2-P(Cry1)-Cry1 intron 336-Flag-Luc, pMU2-P(Cry1)-Luc-Cry1 intron 336, respectively.
pMU2-P(SV40)-Cry1 Intron 336-Luc, pMU2-P(SV40)-Cry1 Intron 336 R1 Deletion- Luc, pMU2-P(SV40)-Cry1 Intron 336
R2 Deletion-Luc, pMU2-P(SV40)-Cry1 Intron 336 R1,2 Deletion-Luc, pMU2-P(SV40)-Cry1 Intron 336 R1 Mutation-Luc,
pMU2- P(SV40)-Cry1 Intron 336 R2 Mutation-Luc, pMU2-P(SV40)-Cry1 Intron 336 R1,2 Mutation-Luc, pMU2-P(SV40)Cry1 Intron 336 R1 Inversion-Luc, pMU2-P(SV40)- Cry1 Intron 336 R2 Inversion-Luc, and pMU2-P(SV40)-Cry1 Intron
336 R1,2 Inversion-Luc
First, we constructed various modified Cry1 intron 336. For construction of pCR2.1-Cry1 intron 336 R1 deletion, pCR2.1-Cry1 intron
336 R2 deletion, pCR2.1-Cry1 intron 336 R1,2 deletion, pCR2.1-Cry1 intron 336 R1 mutation, pCR2.1-Cry1 intron 336 R2 mutation,
pCR2.1-Cry1 intron 336 R1,2 mutation, pCR2.1-Cry1 intron 336 R1 inversion, pCR2.1-Cry1 intron 336 R2 inversion, and pCR2.1-Cry1
intron 336 R1,2 inversion, we used pCR2.1-Cry1 intron 336 (see above) as a template. Inverse PCR products were treated with Mighty
Cloning kit (TaKaRa) for 50 -end phosphorylation, and subsequently self-ligated. These constructs and pMU2-P(SV40)-Luc (seeabove) plasmids were digested with I-SceI. The altered Cry1 intron fragments were ligated at the I-SceI site of pMU2-P(SV40)Luc, and designated as pMU2-P(SV40)-Cry1 intron 336-Luc, pMU2-P(SV40)-Cry1 intron 336 R1 deletion- Luc, pMU2-P(SV40)Cry1 intron 336 R2 deletion-Luc, pMU2-P(SV40)-Cry1 intron 336 R1,2 deletion-Luc, pMU2-P(SV40)-Cry1 intron 336 R1 mutationLuc, pMU2- P(SV401)-Cry1 intron 336 R2 mutation-Luc, pMU2-P(SV40)-Cry1 intron 336 R1,2 mutation-Luc, pMU2-P(SV40)-Cry1
intron 336 R1 inversion-Luc, pMU2-P(SV40)- Cry1 intron 336 R2 inversion-Luc, and pMU2-P(SV40)-Cry1 intron 336 R1,2 inversion-Luc, respectively.
Primer Sequence for the Inverse PCR
Cry1 Intron 336 R1 Deletion
Forward primer, 50 -GGTCATTGTGATGGGAGTATGC-30 ; reverse, 50 -CTAGTCTGTGCTAGGACAGC-30 .
Cry1 Intron 336 R2 Deletion
Forward primer, 50 -GAAACTATTTCTGTTGATTCATGTTG-30 ; reverse, 50 -GGTCATCGCTATAGCAACCAG-30 .
Cry1 Intron 336 R1,2 Deletion
Forward primer, 50 -GAAACTATTTCTGTTGATTCATGTTG-30 ; reverse, 50 -CTAGTCTGTGCTAGGACAGC-30 .
Cry1 Intron 336 R1 Mutation
Forward primer, 50 -AAAGTCCTAGCTTGTGATGGGAGTATGCTAAAC-30 ; reverse, 50 -CTAGTCTGTGCTAGGACAGC-30 .
Cry1 Intron 336 R2 Mutation
Forward primer, 50 -GAAACTATTTCTGTTGATTCATGTTG-30 ; reverse, 50 -TAAAGTCCTAGCTCGCTATAGCAACCAGTG-30 .
Cry1 Intron 336 R1 Inversion
Forward primer, 50 -TGACCTACTTTTTGTGATGGGAGTATGCTAAAC-30 ; reverse, 50 -CTAGTCTGTGCTAGGACAGC-30 .
Cry1 Intron 336 R2 Inversion
Forward primer, 50 -GAAACTATTTCTGTTGATTCATGTTG-30 ; reverse, 50 -TTGACCTACTTTTCGCTATAGCAACCAGTG-30 .
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pMU2-P(Cry1)-Cry1 Intron 336 R1 Deletion-Luc, pMU2-P(Cry1)-Cry1 Intron 336 R2 Deletion-Luc, pMU2-P(Cry1)-Cry1
Intron 336 R1,2 Deletion-Luc, pMU2- P(Cry1)-Cry1 Intron 336 R1 Mutation-Luc, pMU2-P(Cry1)-Cry1 Intron 336 R2
Mutation-Luc, pMU2-P(Cry1)-Cry1 Intron 336 R1,2 Mutation-Luc, pMU2-P(Cry1)-Cry1 Intron 336 R1 Inversion-Luc,
pMU2-P(Cry1)-Cry1 Intron 336 R2 Inversion-Luc, and pMU2-P(Cry1)-Cry1 Intron 336 R1,2 Inversion-Luc
First, the following vectors were digested with I-SceI: pCR2.1-Cry1 intron 336 R1 deletion, pCR2.1-Cry1 intron 336 R2 deletion,
pCR2.1-Cry1 intron 336 R1,2 deletion, pCR2.1- Cry1 intron 336 R1 mutation, pCR2.1-Cry1 intron 336 R2 mutation, pCR2.1-Cry1
intron 336 R1,2 mutation, pCR2.1-Cry1 intron 336 R1 inversion, pCR2.1-Cry1 intron 336 R2 inversion, and pCR2.1-Cry1 intron
336 R1,2 inversion (see above), and pMU2-P(Cry1)-Luc plasmid (see above). The altered Cry1 intron fragments were ligated at
the I-SceI site of pMU2-P(Cry1)-Luc, and designated as pMU2-P(Cry1)-Cry1 intron 336 R1 deletion- Luc, pMU2-P(Cry1)-Cry1 intron
336 R2 deletion-Luc, pMU2-P(Cry1)-Cry1 intron 336 R1,2 deletion-Luc, pMU2-P(Cry1)-Cry1 intron 336 R1 mutation-Luc, pMU2-P(Cry1)- Cry1 intron 336 R2 mutation-Luc, pMU2-P(Cry1)-Cry1 intron 336 R1,2 mutation-Luc, pMU2-P(Cry1)-Cry1 intron 336 R1 inversion-Luc, pMU2-P(Cry1)-Cry1 intron 336 R2 inversion-Luc, and pMU2-P(Cry1)-Cry1 intron 336 R1,2 inversion-Luc, respectively.
pMU2-3 3 E0 Box-P(SV40)-Cry1 Intron 336-Luc, pMU2-3 3 D Box-P(SV40)-Cry1 Intron 336-Luc, and pMU2-3 3 RRE-P(SV40)-Cry1 Intron 336-Luc
pCR2.1-Cry1 intron 336 (see above) was digested with I-SceI. pMU2-3 3 E’ box-P(SV40)-Luc, pMU2-3 3 D box-P(SV40)-Luc, andpMU2-3 3 RRE-P(SV40)-Luc plasmids (see above) were also digested with I-SceI. The Cry1 intron 336 fragment was ligated at the ISceI site of these plasmids, and designated as pMU2-3 3 E’ box-P(SV40)-Cry1 intron 336- Luc, pMU2-3 3 D box-P(SV40)-Cry1
intron 336-Luc, and pMU2-3 3 RRE-P(SV40)-Cry1 intron 336-Luc, respectively.
pMU2-P(Cry1)-Cry1
We amplified the full-length coding sequence of mouse Cry1 from pCI-Cry1 by PCR with forward primers containing PI-PspI recognition sequence (50 -TGGCAAACAGCTATTATGGGTATTATGGGTGGGGTGAACGCCGTGCAC-30 , recognition sequence is indicatedwith italics, Hokkaido System Science) and reverse primers containing PI-SceI recognition sequence (50 -TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTCAGTTACTGCTCTGCCGCTGG-30 ). PCR product was digested with PI-PspI and PI-SceI(New England BioLabs) and cloned into pMU2-P(Cry1)-Luc vector (see above) in replace of the Luc gene, and the resulting vector
was designated as pMU2-P(Cry1)-Cry1.
pMU2-P(Cry1)-Cry1 Intron 336-Cry1, pMU2-P(Cry1)-Cry1 Intron 336 R1 Deletion-Cry1, pMU2-P(Cry1)-Cry1 Intron 336
R2 Deletion-Cry1, pMU2-P(Cry1)-Cry1 Intron 336 R1,2 Deletion-Cry1, pMU2-P(Cry1)-Cry1 Intron 336 R1 MutationCry1, pMU2-P(Cry1)-Cry1 Intron 336 R2 Mutation-Cry1, pMU2-P(Cry1)-Cry1 Intron 336 R1,2 Mutation-Cry1, pMU2-P(Cry1)-Cry1 Intron 336 R1 Inversion-Cry1, pMU2- P(Cry1)-Cry1 Intron 336 R2 Inversion-Cry1, and pMU2-P(Cry1)Cry1
Intron 336 R1,2 Inversion-Cry1
The following vectors were first digested with I-SceI: pCR2.1-Cry1 intron 336, pCR2.1-Cry1 intron 336 R1 deletion, pCR2.1-Cry1
intron 336 R2 deletion, pCR2.1-Cry1 intron 336 R1,2 deletion, pCR2.1-Cry1 intron 336 R1 mutation, pCR2.1-Cry1 intron 336 R2
mutation, pCR2.1-Cry1 intron 336 R1,2 mutation, pCR2.1-Cry1 intron 336 R1 inversion, pCR2.1-Cry1 intron 336 R2 inversion, andpCR2.1-Cry1 intron 336 R1,2 inversion (see above), and pMU2-P(Cry1)-Cry1 plasmid (see above). The various Cry1 intron 336 fragments were ligated at the I-SceI site of pMU2-P(Cry1)-Cry1, and designated as pMU2-P(Cry1)-Cry1 intron 336-Cry1, pMU2-P(Cry1)Cry1 intron 336 R1 deletion-Cry1, pMU2-P(Cry1)-Cry1 intron 336 R2 deletion-Cry1, pMU2-P(Cry1)-Cry1 intron 336 R1,2 deletionCry1, pMU2-P(Cry1)- Cry1 intron 336 R1 mutation-Cry1, pMU2-P(Cry1)-Cry1 intron 336 R2 mutation-Cry1, pMU2-P(Cry1)-Cry1
intron 336 R1,2 mutation-Cry1, pMU2-P(Cry1)-Cry1 intron 336 R1 inversion-Cry1, pMU2-P(Cry1)-Cry1 intron 336 R2 inversionCry1, and pMU2- P(Cry1)-Cry1 intron 336 R1,2 inversion-Cry1, respectively.
pMU2-P(Cry1)-Cry1 Intron 336-Cry1-Rluc and pMU2-P(Cry1)-Cry1 Intron 336 R1,2 Mutation-Cry1-Rluc
First, the coding sequence of Renilla luciferase (Rluc) was amplified from phRL-SV40 plasmid (Promega) using PCR with the following
primers: Forward primers containing PI-PspI recognition sequence: (50 -TGGCAAACA GCTATTATGGGTATTATGGG
TGCTTCCAAGGTGTAC GACCCCGAGC-30 , recognition sequence is indicated with italics, Hokkaido System Science), Reverse
primers containing PI-SceI recognition sequence: (50 -TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTTACTGCTCGTTCTTCAGCACGCGC-30 ). PCR product was digested with PI-PspI and PI-SceI (New England BioLabs) and ligated at the PIPspI and PI-SceI sites of pMU2-P(Cry1)-Cry1 intron 336-Luc and pMU2-P(Cry1)-Cry1 intron 336 R1,2 mutation-Luc (see above)
to replace Luc fragment, and the resulting construct were designated as pMU2-P(Cry1)-Cry1 intron 336-Rluc and pMU2-P(Cry1)Cry1 intron 336 R1,2 mutation-Rluc.
Next, the coding sequence of Cry1 was amplified using PCR with the following primers: Forward primers containing PI-PspI recognition sequence: (50 -TGGCAAACAGCTATTATGGGTATTATGGGTGGGGTGAACGCCGTGCAC-30 ), Reverse primers containing PIPspI recognition sequence: (50 -ACCCATAATACCCATAATAGCTGTTTGCCAGTTACTGCTCTGCCGCTGG-30 ). PCR product was digested with PI-PspI and ligated at the PI-PspI site of pMU2-P(Cry1)-Cry1 intron 336-Rluc and pMU2-P(Cry1)-Cry1 intron 336 R1,2
mutation-Rluc, and the resulting construct were designated as pMU2-P(Cry1)-Cry1 intron 336-Cry1-Rluc and pMU2-P(Cry1)-Cry1
intron 336 R1,2 mutation-Cry1-Rluc.
pLV7-P(Cry1)-Cry1 Intron 336-Cry1 and pLV7-P(Cry1)-Cry1
For generation of lentiviral constructs, the Cry1 cDNA with or without 336bp of Cry1 intron sequence was first cloned into pENTR-D
entry vector (Invitrogen), and the Cry1 promoter was cloned into pENTR-50 vector (Invitrogen). The pENTR/D-cDNA and pENTR-50 promoter plasmid DNAs were then recombined with pLenti6/R4R2/V5-DEST destination vector (Invitrogen) in a Gateway
S6 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

recombination reaction to generate lentiviral expression constructs. Vector construction, lentivirus production, and infection into
Cry1/:Cry2/:Per2Luc cells were done as previously described (Liu et al., 2008; Liu et al., 2007). Infected cells were selected with blasticidin
for 3-7 days prior to bioluminescence recording on a LumiCycle (Actimetrics).
pMU2-2 3 RRE-P(Cry1)-Cry1
We used inverse PCR to fuse two tandem repeats of Bmal1 RRE element to P(Cry1). For construction, we used pMU2-P(Cry1)-Cry1(see above) as a template with the following primers: Forward primers: (50 -AGGCAGAAAG TAGGTCAGGGACGCTGTTGGGAAGGGCGATCGGGTACC-30 , Hokkaido System Science), Reverse primers: (50 -CGTCCCTGACCTACTTTCTGCCTTTGCGCAGCCTGAATGGCGAATGGG-30 ). Inverse PCR products were treated with Mighty Cloning kit (TaKaRa) for 50 -end phosphorylation, andsubsequently self-ligated, and designated as pMU2-2 3 RRE-P(Cry1)-Cry1.
pMU2-P(SV40)-Cry2
We amplified the full-length coding sequence of mouse Cry2 from pCI-Cry2 by PCR with forward primers containing PI-PspI recognition sequence (50 -TGGCAAACAGCTATTATGGGTATTATGGGTGCGGCGGCTGCTGTGGTG-30 , recognition sequence is indicatedwith italics, Hokkaido System Science) and reverse primers containing PI-SceI recognition sequence (50 -TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTCAGGAGTCCTTGCTTGCTG-30 ). PCR product was digested with PI-PspI and PI-SceI (NewEngland BioLabs) and cloned into pMU2-P(SV40)-Luc vector (see above) instead of Luc gene, and the resulting construct was designated as pMU2-P(SV40)-Cry2.
pMU2-2 3 D Box-P(SV40)-Luc, pMU2-1 3 D Box-P(SV40)-Luc, pMU2-2 3 Cry1proD- P(SV40)-Luc, and pMU2-1 3Cry1proD-P(SV40)-Luc
Oligonucleotides (Hokkaido System Science) containing one or two tandem repeats of Per3 D box or Cry1proD elements were annealed, and inserted into the MluI-BglII site upstream of SV40 basic promoter of pMU2-P(SV40)-Luc (see above), and the resulting
constructs were designated as pMU2-2 3 D box-P(SV40)-Luc, pMU2-1 3 D box-P(SV40)-Luc, pMU2- 2 3 Cry1proD-P(SV40)-Luc,
pMU2-1 3 Cry1proD-P(SV40)-Luc.
Oligonucleotide Sequences for One or Two Tandem Repeats of D Box or Cry1proD Element
One D Box Element
Forward oligonucleotide, 50 -CGCGCCCGCGCGTTATGTAAGGTACTCG-30 ; reverse, 50 -GATCCGAGTACCTTACATAACGCGC
GGG-30 .
Two Repeats of D Box Element
Forward oligonucleotide, 50 -CGCGCCCGCGCGTTATGTAAGGTACTCGCCCGCGCGTTATGTAAGGTACTCG-30 ; reverse, 50 -GATCCGAGTACCTTACATAACGCGCGGGCGAGTACCTTACATAACGCGCGGG-3 0 .
One Time of Cry1proD Element
Forward oligonucleotide, 50 -CGCGAAACACACTTCAGAAACGTGAGGT-30 ; reverse, 50 -GATCACCTCACGTTTCTGAAGTGTG
TTT-30 .
Two Repeats of Cry1proD Element
Forward oligonucleotide, 50 -CGCGAAACACACTTCAGAAACGTGAGGTAAACACACTTCAGAAACGTGAGGT-30 ; reverse, 50 -GATC
ACCTCACGTTTCTGAAGTGTGTTTACCTCACGTTTCTGAAGTGTGTTT-3 0 .
pMU2-2 3 D Box-P(SV40)-Cry1 Intron 336-Luc, pMU2-1 3 D Box-P(SV40)-Cry1 Intron 336-Luc, pMU2-3 3 Cry1proD-P(SV40)-Cry1 Intron 336-Luc, pMU2-2 3 Cry1proD-P(SV40)-Cry1 Intron 336-Luc, and pMU2-1 3 Cry1proD-P(SV40)Cry1 Intron 336-Luc
pCR2.1-Cry1 intron 336 (see above) was digested with I-SceI. pMU2-2 3 D box-P(SV40)-Luc, pMU2-1 3 D box-P(SV40)-Luc, pMU23 3 Cry1proD-Luc, pMU2-2 3 Cry1proD-P(SV40)-Luc, pMU2-1 3 Cry1proD-P(SV40)-Luc plasmids (see above) were also digested
with I-SceI. Cry1 intron 336 fragment was ligated at the I-SceI site of these plasmids, and designated as pMU2-2 3 D box-P(SV40)Cry1 intron 336-Luc, pMU2-1 3 D box-P(SV40)-Cry1 intron 336-Luc, pMU2-3 3 Cry1proD-P(SV40)- Cry1 intron 336-Luc, pMU2-2 3Cry1proD-P(SV40)-Cry1 intron 336-Luc and pMU2- 1 3 Cry1proD-P(SV40)-Cry1 intron 336-Luc, respectively.
pMU2-P(SV40)-Cry1 Intron 336-Cry1, pMU2-3 3 D Box-P(SV40)-Cry1 Intron 336-Cry1, pMU2-2 3 D Box-P(SV40)-Cry1
Intron 336-Cry1, pMU2-1 3 D Box-P(SV40)-Cry1 Intron 336-Cry1, pMU2-3 3 Cry1proD-P(SV40)-Cry1 Intron 336-Cry1,
pMU2-2 3 Cry1proD-P(SV40)-Cry1 Intron 336-Cry1, and pMU2-1 3 Cry1proD-P(SV40)-Cry1 Intron 336-Cry1
pMU2-P(Cry1)-Cry1 (see above) was digested with PI-PspI and PI-SceI, and the Cry1 fragment was cloned into pMU2-P(SV40)-Cry1
intron 336-Luc, pMU2-3 3 D box-P(SV40)-Cry1 intron 336-Luc, pMU2-2 3 D box-P(SV40)- Cry1 intron 336-Luc, pMU2-1 3 D box-P(SV40)-Cry1 intron 336-Luc, pMU2-3 3 Cry1proD-P(SV40)-Cry1 intron 336-Luc, pMU2-2 3 Cry1proD-P(SV40)-Cry1 intron 336-Luc,
pMU2-1 3 Cry1proD-P(SV40)-Cry1 intron 336-Luc vector (see above) in replace of the Luc gene, and the resulting constructs were
designated as pMU2- P(SV40)-Cry1 intron 336-Cry1, pMU2-3 3 D box-P(SV40)-Cry1 intron 336-Cry1, pMU2-2 3 D box-P(SV40)Cry1 intron 336-Cry1, pMU2-1 3 D box-P(SV40)-Cry1 intron 336-Cry1, pMU2-3 3 Cry1proD-P(SV40)-Cry1 intron 336-Cry1,
pMU2-2 3 Cry1proD- P(SV40)-Cry1 intron 336-Cry1 and pMU2-1 3 Cry1proD-P(SV40)-Cry1 intron 336-Cry1, respectively.
pMU2-P(TKms)-Cry1 and pMU2-P(TKms)-Cry1 Intron 336-Cry1
TKms promoter fragment (short sequence of TK promoter) (gift from Dr. H. Ukai, 50 -GTCCCAGGT CCACTTCGCATATTAAGGTG
ACGCGTGTGGCCTCGAA-30 ) was cloned into the KpnI-HindIII sites of pMU2-P(SV40)-Cry1 and pMU2-P(SV40)-Cry1 intron
Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S7

336-Cry1 vectors (see above) in replace of P(SV40) promoter, and designated as pMU2- P(TKms)-Cry1 and pMU2-P(TKms)-Cry1
intron336-Cry1.
pMU2-P(SV40)-Cry1 Intron 336-Cry1-Rluc
First, the coding sequence of Renilla luciferase (Rluc) was amplified from phRL-SV40 plasmid (Promega) using PCR with the following
primers: Forward primers containing PI-PspI recognition sequence: (50 -T GGCAAACAGCTATTATGGGTATTATGG
GTGCTTCCAAGGTGTACGACCCCGAGC-30 , recognition sequence is indicated with italics, Hokkaido System Science), Reverse
primers containing PI-SceI recognition sequence: (50 -TGCCATTTCATTACC TCTTTCTCCGCACCCGACATAGATTTACTGCTCGTTCTTCAGCACGCGC-30 ). PCR product was digested with PI-PspI and PI-SceI (New England BioLabs) and ligated at the PIPspI and PI-SceI sites of pMU2-P(SV40)-Cry1 intron 336-Luc (see above) to replace Luc fragment, and the resulting constructwere designated as pMU2-P(SV40)-Cry1 intron 336-Rluc.
Next, the coding sequence of Cry1 was amplified using PCR with the following primers: Forward primers containing PI-PspI recognition sequence: (50 -TGG CAAACAGCTATTATGGGTATTATGGGTGGGGTGAACGCCGTGCAC-30 ), Reverse primers containing PIPspI recognition sequence: (50 -ACCCATAATACCCATAATAG CTGTTT GCCAGTTACTGCTCTGCCGCTGG-30 ). PCR product wasdigested with PI-PspI and ligated at the PI-PspI site of pMU2-P(SV40)-Cry1 intron 336-Rluc, and the resulting construct were designated as pMU2-P(SV40)-Cry1 intron 336-Cry1-Rluc.
pMU2-P(SV40)-Cry1 Intron 336 R1,2 Mutation-Cry1
pCR2.1-Cry1 intron 336 R1,2 mutation (see above) was digested with I-SceI. The Cry1 intron 336 R1,2 mutation fragment was ligatedat the I-SceI site of pMU2-P(SV40)-Cry1 intron 336-Cry1 plasmid (see above) to replace the Cry1 intron 336 fragment, and the resulting construct was designated as pMU2-P(SV40)-Cry1 intron 336 R1,2 mutation-Cry1.

Bioinformatics Search for Potential Clock-Controlled Elements
We comprehensively examined conserved regions in the Cry1 promoter sequence and the 1st intron sequence for potential clock
controlled elements using a Bioinformatics approach. Genomic sequence data and evolutionary conserved region data were obtained from UCSC Genome Browser (Rhead et al., 2010; Siepel et al., 2005). There are 5 conserved regions in Cry1’s promoterand 23 regions in the 1st intron sequence. The first and second most closely resembling D box sequences were found in Cry1
promoter by using position weight matrix analysis with MA0025 of JASPAR database (Bryne et al., 2008). Two consensus RRE
sequences were found in the 1st intron by regular expression analysis of Perl script language.

Rhythmicity, Period Length, Phase, and Relative Amplitude Analysis of Real-Time Bioluminescence Data
The rhythmicity and period length of the promoter activity for each reporter were determined as described previously (Sato et al.,2006), with a slight change. Briefly, Bioluminescence time-series data beginning 21 hr after forskolin stimulation were used for analysis in order to distinguish endogenous circadian oscillation from acute effects of stimulation. Bioluminescence data were detrended
using the trend curve calculated by smoothing spline method. The smoothing parameter for this calculation was set such that its
frequency-response was 33% at the frequency of about 2 cycles (42 hr) of the typical circadian period (21 hr) observed in fibroblasts
transfected with an empty vector at 30 C. Then, autocorrelation of the detrended bioluminescence time-series data was calculated
within the range of 15-36 hr to determine the circadian period of oscillation. Statistical significance (p = 0, most significant to 1, leastsignificant) of circadian oscillation was evaluated by comparing the strongest autocorrelation of the detrended
pﬃﬃﬃﬃ data within the rangeof 15-36 hr against that of white-noise (theoretical value for the autocorrelation of white noise is 1=N ± 1= N, mean ± SD, where N is
the number of time points). To calculate the phase, a cosine wave with the period corresponding to the maximum autocorrelation in
the range, was fitted to the detrended bioluminescence data. The calculated phase was scaled to the phase in 24 hr period, for phasecomparison of different period waves.
To calculate the relative amplitude of bioluminescence data, the moving average of the absolute value of the detrended bioluminescence was calculated first. The window size of the moving average was set to half of the estimated period. Then, the moving averaged data was smoothened by the smoothing spline method resulting in the amplitude trend. The amplitude trend was then divided
by the trend curve of the original time-series at each time point to be used as the relative amplitude.

Periodogram and Significance Test
For drawing periodogram, time-series data of bioluminescence was detrended by subtracting trend curve. The detrended timeseries was divided by standard deviation for normalization. The trend curve was estimated by smoothing spline method as describedin the previous sub-section. Then, inner-products of the detrended time-series with cosine and sine waves were evaluated. Before
taking inner-product, the cosine and sine waves were scaled so that their norm was 1. Amplitude of time-series at each period
component was evaluated as square-root of sum-of-squared those two inner-products. The amplitudes are plotted against periodsof the sine and cosine waves varying with 0.1 hr interval from 5 to 34 hr. For statistical test of 11 hr periodicity, amplitudes of theharmonic periods in the range of 814 hr were extracted. The sum-of-squared amplitude of those harmonic periods was comparedagainst chi-square distribution for statistical test.
S8 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

Phase Vector and Addition of Waves
The addition of multiple trigonometric waves with same frequency results in a trigonometric wave of same frequency with a novel
phase. This can be shown as follows.
Assume we have two wave functions f1 ðtÞ; f2 ðtÞ:f1 ðtÞ = A1 cosðqðtÞ + f1 Þ;
f2 ðtÞ = A2 cosðqðtÞ + f2 Þ
where A1 and A2 are amplitudes, f1 and f2 are phases of corresponding waves, qðtÞ is angle which depends on time: t.
Then the summed function fc ðtÞ of these two waves is derived by using formulae of ‘‘angle sum and difference identities’’ and‘‘linear combination’’:fc ðtÞ = Ac cosðqðtÞ + fc Þ
where
Ac =

qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
22
ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ + ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ ;ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ;cosðfc Þ = qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
22
ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ + ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ
ðA1 sinðf1 Þ + A2 sinðf2 ÞÞsinðfc Þ = qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ2 + ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ2
On the other hand, the two waves can be represented as phase vectors where the phase and amplitude of a wave are represented
by the direction and length of a vector, respectively. In Figure S7B, wave1 and wave2 are respectively represented as red and bluephase vectors. Coordinates of the two phase vectors: P1 and P2 are ðA1 cosðf1 Þ; A1 sinðf1 ÞÞ and ðA2 cosðf2 Þ; A2 sinðf2 ÞÞ; summed
phase vector: Pc ’s coordinate is ðA1 cosðf1 Þ + A2 cosðf2 Þ; A1 sinðf1 Þ + A2 sinðf2 ÞÞ. In this phase vector, Pc represents the wave
qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
00
with amplitude Ac = ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ2 + ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ2 and phase fc such that 0
ðA1 cosðf1 Þ + A2 cosðf2 ÞÞcos fc = qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
22
ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ + ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ
 0
ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ
sin fc = qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ:
22
ðA1 cosðf1 Þ + A2 cosðf2 ÞÞ + ðA1 sinðf1 Þ + A2 sinðf2 ÞÞ00This means Ac = Ac and fc = fc , i.e., the summed phase vector represents the wave of fc ðtÞ.
Period of Negative Feedback Loop with Time Delay
There is an excellent review which succinctly described basic properties of negative feedback loop with time delay (Novak andTyson, 2008). Here, we adopt their mathematical model, and present the relation between the oscillation period and the time delay
by supplementing their derivation.
The model of the delayed negative feedback loop in the review can be interpreted in terms of delayed repression onto its own transcriptional regulatory element (Figure S7C). The mathematical description of the model:
dYðtÞ
Kdp
YðtÞ
= k1 S p
p  k 2 ET
dt
Km + YðtÞ
Kd + Yðt  tÞ(1)

can be regarded as, Y(t) is the concentration of the repressor protein at time t, S is the activator concentration, Kd is the dissociation
constant for the repressor protein with the regulatory element, ET is the concentration of a protease that degrades the repressor
protein, Km is Michaelis constant of the degradation reaction, k1 and k2 are the synthesis and the degradation rate of the repressor
protein respectively, p is Hill coefficient, and t is the time delay.
In numerical simulations of the model (1) with parameters: k1 = 1; k2 = 1; S = 2; Kd = 1; ET = 1; Km = 0:1; p = 2; t = 1; 3; or 6, we
see that the period of oscillation becomes longer as the time delay becomes larger (Figure S7D). Note that under a critical value
Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S9

of time delay t which is 1<t<3 in this case, the model is stable and does not display sustained oscillation. Here, we first intend to findthe constraint on the time delay for a sustained oscillation, and then we seek to approximately describe the relation between the oscillation period and the time delay.
The model Equation (1) can be formatted as
n o
n o
YðtÞ
YðtÞ
d
Kd
Kd
Kd
k1 S
1
n o
=
n
op 
Km
dt
k2 ET
k2 ET 1 + YðttÞ+ YðtÞ
Kd

Kd

Kd

tÞ = YðtÞ=Kd and bt = ðk2 ET =Kd Þt, we obtain a dimensionless form of (1)By introducing variables s = k1 S=k2 ET ,k = Km =Kd ,yð b
dyð b
tÞ
s
yð b
tÞ

=
:
p
db
t
1 + fyð btb
tÞg
k + yð btÞ

(3)

The properties of this equation can be analyzed around the fixed points (steady points). The fixed points can be obtained as in thecase of non-delayed differential equation, because yð b
tÞ = yð btb
tÞ at fixed points. A fixed point y0 is a positive real root of the equationy p + 1  ðs  1Þy  sk = 0(4)which is given by setting dyð b
tÞ=d bt = 0 in (3).
To investigate a solution around the fixed point, the Equation (3) is linearized around y0 by Taylor expansion

  

dyð b
tÞ
spy0p1   b bky bt  y0= 
2 y t  t  y 0 ðk + y0 Þ2dbt
1 + yp(5)0bAssuming yð b
tÞ = y0 + el t in (5), the characteristic equation is obtained
spy0p1 lbt
k
=0
l+
 e +
p 2
ðk
+
y0 Þ2
1 + y0(6)This Equation (6) states that the real part of l is negative when there is no time delay: b
t = 0, because
spy0p1kl=  <0 2
p 2
ðk + y0 Þ
1 + y0(7)

t = 0.
Therefore, the solution of the model around y0 is stable for b
Let’s assume there is a critical value of bt at which the sign of the real part of l changes from negative to positive as bt increases. At
the critical bt, the real part of l is 0, and the model loses its stability (Hopf bifurcation). To find the critical bt, we substitute l = iu into (7),and obtain  
spy0p1 kiu + 
t  isin ubt += 0:2 cos ub
2ðk + y0 Þ
1 + y0p(8)

In order for (8) to hold, the real and imaginary parts of (8) must be 0. By introducing variables r = k=ðk + y0 Þ2 , f = spy0p1 =ð1 + y0p Þ2 ,and dealing the real and the imaginary part separately, we obtain Real : fcos ub
t = r(9) 
Imaginary : fsin ub
t =u

(10)

Squaring each (9) and (10) and summing them result in
r2 + u2 = f2

(11)

And dividing (10) by (9) yieldsbt=

S10 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

 arctan uru(12)By deleting u from (11) and (12) we arrive at the equationrﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
 2
farctan 
1r
brﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
t=

r

f
r

2

(13)

1

This equation gives the critical value of bt . With a larger time delay than the critical bt , the model displays a sustained oscillation(Figure S7E, F top panels) (Novak and Tyson, 2008).
The period of oscillation with time delay near the critical bt can be roughly estimated by (9):u=arccosð  r=fÞand period = 2p=u
b
t

(14)

These are summarized asperiod =2pbt
arccosðr=fÞ

(15)

By using this equation with (13), we can visualize combinations of variables which can generate oscillation along with the associated period (Figures S7E and S7F top panels). Note that there is a proportional relation between the oscillation period and the timedelay in this model, and the coefficient is in range of 24:
period = ðconstant of 2 4Þ bt

(16)This is because that f and r are both positive and f>r (i.e., f=r>1) according to (11), therefore 1<  r=f<0. This meansthat p=2<arccosðr=fÞ<p in (14). We should emphasize again that the period estimation by (15) deviates from actual values or estimation by numerical simulation, and the deviation becomes larger as bt becomes larger. This is because the Equation (15) strictly
holds only on the critical bt (Figure S7E, F bottom panels).
We used xpp-aut (http://www.math.pitt.edu/bard /xpp/xpp.html) for numerical simulation of the model (1).
Statistics and Significance Test for Correlation
For evaluation of correlation, linear fit of a first-order-polynomial was performed by the least square method. Statistical significancewas evaluated by Pearson’s correlation. Analysis was performed using Microsoft Excel or R version 2.8.1.
SUPPLEMENTAL REFERENCES
Bryne, J.C., Valen, E., Tang, M.H., Marstrand, T., Winther, O., da Piedade, I., Krogh, A., Lenhard, B., and Sandelin, A. (2008). JASPAR, the open access databaseof transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res. 36, D102–D106.
Kumaki, Y., Ukai-Tadenuma, M., Uno, K.-D., Nishio, J., Masumoto, K.-h., Nagano, M., Komori, T., Shigeyoshi, Y., Hogenesch, J.B., and Ueda, H.R. (2008). Analysis and synthesis of high-amplitude Cis-elements in the mammalian circadian clock. Proc. Natl. Acad. Sci. USA 105, 14946–14951.
McCarthy, E.V., Baggs, J.E., Geskes, J.M., Hogenesch, J.B., and Green, C.B. (2009). Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression. Mol. Cell. Biol. 29, 5465–5476.
Rhead, B., Karolchik, D., Kuhn, R.M., Hinrichs, A.S., Zweig, A.S., Fujita, P.A., Diekhans, M., Smith, K.E., Rosenbloom, K.R., Raney, B.J., et al. (2010). The UCSC
Genome Browser database: update 2010. Nucleic Acids Res. 38, D613–D619.
Schmutz, I., Ripperger, J.A., Baeriswyl-Aebischer, S., and Albrecht, U. (2010). The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors. Genes Dev. 24, 345–357.
Siepel, A., Bejerano, G., Pedersen, J.S., Hinrichs, A.S., Hou, M., Rosenbloom, K., Clawson, H., Spieth, J., Hillier, L.W., Richards, S., et al. (2005). Evolutionarily
conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050.
Wuarin, J., and Schibler, U. (1990). Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm. Cell 63, 1257–1266.

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S11

E'-box-P(SV40)

P(Cry1)
4000000

6000000

2000000

4000000

1000000

2000000

3000000
2000000
1000000

0

1

2

3

4

0

5

0

1

2

B

4

0

5

0

1

2

9000000

1200000

3

4

5

Day

Cry1proD-mut-P(SV40)

E'-box-P(SV40)

D-box-P(SV40)

4000000

7500000

3000000

6000000

5000000

8000002000000
3000000

2500000

400000

0

1

2

3

4

0

5

0

1

2

0

5

0

1

2

Day

3

4

0

5

0

1

2

Day

3
Day

10
5

D-P(SV40)

Cry1proD-P(SV40)

Cry1proD-mut-P(SV40)

P(SV40)P(SV40)-+ -+ -+ -+Relative Luc Activity

42-+ -+ -+ -+S12 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

6
42-+ -+ -+ -+E'-P(SV40)

P(SV40)6

0
DBP + HLF

8

0
HLF

Relative Luc Activity

2

8

P(SV40)4

Cry1proD-P(SV40)-+ -+ -+ -+E'-P(SV40)

6E'-P(SV40)

2

0
TEF

Relative Luc Activity

80
DBP + TEF

4

P(SV40)Cry1proD-P(SV40)

D-P(SV40)-+ -+ -+ -+E'-P(SV40)

0
DBP

6

Cry1proD-P(SV40)2

8Cry1proD-P(SV40)

Relative Luc Activity

4

P(SV40)E'-P(SV40)

9
6
3

0
DBP + TEF + HLF-+ -+ -+ -+P(SV40)-+-+- - ++

Cry1proD-P(SV40)-+-+- - ++

Cry1proD-P(SV40)-+-+- - ++

D-P(SV40)-+-+- - ++

D-P(SV40)-+-+- - ++

D-P(SV40)

Relative Luc Activity

Relative Luc Activity

4

15

0
BMAL1/CLOCK
CRY1

Relative Luc Activity

3

D-P(SV40)

0

1000000

D-P(SV40)

Bioluminescence (counts/min)

Cry1proD-P(SV40)
1600000

Day

D

3
Day

E'-P(SV40)

0

Day

C

D-box-P(SV40)

3000000

E'-P(SV40)

Bioluminescence (counts/min)

A

4

5

Figure S1. Related to Figure 1(A) Transcriptional activities of P(Cry1) (left), E’ box-P(SV40) (middle) and D box-P(SV40) (right). Raw data of bioluminescence time-series corresponding to
Figure 1A are shown (n = 3).
(B) Transcriptional activities of Cry1proD-P(SV40) (left), E’ box-P(SV40) (middle) and D box-P(SV40) (right). Raw data of bioluminescence time-series corresponding to Figure 1D are shown (n = 3).
(C) The responsiveness of E’ box, D box, Cry1proD, mutated Cry1proD, and SV40 promoters to clock factors. Reporters were constructed as in Figure 1F.
Regulatory elements were activated by co-transfected E/E’ box activator BMAL1/CLOCK and repressed by E/E’ box repressor CRY1. Relative Luciferase activity
for each promoter was scaled so that the activity with no transcriptional regulation is normalized to 1.
(D) The responsiveness of E’ box, D box, Cry1proD, and SV40 promoters to clock factors. Regulatory elements were activated by co-transfected D box activators
DBP, TEF or HLF, and their combinations DBP + TEF, DBP + HLF, or DBP + TEF + HLF. Relative Luciferase activity for each promoter was scaled so that theactivity with no transcriptional regulation is normalized to 1. Error bars represent SD (n = 3). Data are representative of two independent experiments (A–D).

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S13

A

P(Cry1)-Cry1 intron 1.03k

Bioluminescence (counts/min)

P(Cry1)1500000
6000000

P(Cry1)-Cry1 intron Δ336

6000000

3000000

4000000

2000000

2000000

1000000

1000000
30000000500000
0
0

1

2

3

4

0
0

5

1

2

Day

B

3

4

5

0
0

1

2

Day

2500000

3

43000000

1

2

3

4

5

Day

P(Cry1)-Cry1 intron 336-Flag-Luc

4000000
2000000

1500000
1000000

3000000
2000000

1000000

5000001000000
0

0
0

1

2

3
Day

4

0

5

0

1

2

3

4

5

0

1

2

Day

Bioluminescence (counts/min)

5

P(Cry1)-Flag-Luc-Cry1 intron 336-polyA
2000000

8000001500000
1000000

400000500000
0

0
1

2

3

4

0

5

1

2

Hlf

0.0015

3

4

5

Day

Day

Dbp

4

2500000

1200000

00.00093Day

P(Cry1)-Flag-Cry1 intron 336-Luc

mRNA/Gapdh

05000000

2000000

C

5

Day

Cry1 intron 336-P(Cry1)-Flag-Luc

P(Cry1)
Bioluminescence (counts/min)

P(Cry1)-Cry1 intron 336

2000000

9000000

Tef

0.000250.00020.00060.001

0.00030.00050.000150.00010.000050ZT(h) 0 4 8 12 16 20

wild type0 4 8 12 16 20

Dbp-/-:Tef-/-:Hlf-/-

0

0 4 8 12 16 20

0 4 8 12 16 20

wild typeDbp-/-:Tef-/-:Hlf-/-

0

0 4 8 12 16 20

0 4 8 12 16 20

wild typeDbp-/-:Tef-/-:Hlf-/-

Figure S2. Related to Figure 2(A) The Cry1 promoter was combined with the 1.03 kbp of Cry1 intron sequence, 336 bp of Cry1 intron sequence, or Cry1 intron D336 deletion mutant sequence togenerate composite promoters, which were fused with a Luciferase reporter gene to generate reporter constructs. The reporter was then transiently transfected
into NIH 3T3 cells followed by real-time bioluminescence recording. Raw data of bioluminescence time-series corresponding to Figure 2B are shown (n = 3).
(B) Transcriptional activity of Cry1 promoter with 336 bp of Cry1 intron sequence inserted into upstream or downstream of the Cry1 promoter, and within or
downstream of the Luciferase reporter coding sequence. Raw data of bioluminescence time-series corresponding to Figure 2C are shown (n = 3). Data arerepresentative of two independent experiments (A and B).
(C) Circadian expression profiles of Dbp, Tef and Hlf in vivo. RNA from liver of wild-type (gray) or Dbp/:Tef/:Hlf/ (orange) mice was prepared at 4 hr intervals
from mice held in a 12 hr light-12 hr dark cycle (LD 12:12). Relative mRNA levels of each gene were measured by qPCR assay. All RNA samples were normalized to
Gapdh mRNA accumulation. ZT: Zeitgeber time. Mean and SEM from two pools of three mice each per time point. The representative data from three independent experiments was shown.

S14 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

B
P(SV40)-Cry1 intron 336
6000002000000
4000001000000

200000

17.58
0
1

2

3

4

5

0

1

2

10000017.28

0mutation

1

2

3

4

600000400000400000200000200000

17.77

0
0

5

800000600000

17.14
0
0

1

2

3

4

5

7500005000001

2

3

4

deletion0505000005000004000004000003000003000000200000200000100000100000

0

0
0

1

2

3

4

5

500000400000400000300000300000

1

2

3

4

5

0

1

2

3

4

5

0

1

2

3

4

0
0

1

2

100000

17.62
0

Day

1

2

3
Day

4

5

3

4

5

0

1

2

0

17.74
0

1

2

3
Day

4

5

3

4

5

Day

R2

6000000

6000000

4000000

4000000

2000000

2000000

R1,2
4000000
3000000
2000000
1000000

0

0

0

0

5

20000010000017.30

05000002000002500000R14000002000002500000

2500000

R1,2

600000mutation

2000005000000
5000000

Day

4000003000007500000

5

R25000000

Bioluminescence (counts/min)

4

1

2

3

4

5

2500000
2000000

0

1

2

3

4

5

4000000

4000000

3000000

3000000

2000000

2000000

1000000

1000000

0

1

2

3

4

5

0

1

2

3

4

5

0

1

2

3
Day

4

5

1500000
1000000
5000000

00

inversiondeletion

Bioluminescence (counts/min)R1

inversion

3

P(Cry1)-Cry1 intron 336
7500000

Day

Bioluminescence (counts/min)

0

Bioluminescence (counts/min)

0P(Cry1)

10000000

8000003000000

Day

Bioluminescence (counts/min)

Bioluminescence (counts/min)

P(SV40)

Bioluminescence (counts/min)

Bioluminescence (counts/min)

A

1

2

3

4

5

6000000

0

0

1

2

3

4

5

4000000

4000000

4000000

3000000

3000000

2000000

2000000

1000000

1000000

2000000

0

0

1

2

3
Day

4

5

0

0

1

2

3
Day

4

5

0

Figure S3. Related to Figure 3(A) Transcriptional activities of various P(SV40)-Cry1 intron 336-Luc constructs. Raw data of bioluminescence time-series corresponding to Figure 3C are shown.
In the independent measurement for the strength of intronic RREs, we examined the amplitude of circadian oscillations expressed by the constructs in reporterrhythm assays. Bioluminescence levels from the control reporter construct, P(SV40)-Cry1 intron 336-Luc harboring an SV40 basic promoter and the intact 336 bpof the Cry1 intron sequence, were rhythmic and peaked at CT17.58 ± 0.52. As expected, circadian oscillation was not readily detectable for constructs with R1,2
deletion or R1,2 mutation. Most other mutant constructs (except R2 mutation) displayed significant circadian oscillations in bioluminescence, with phases not too
different from the control, all peaking around CT17-18. Average phase of circadian oscillation is shown in each panel if the rhythmicity is significant.
(B) Transcriptional activities of various P(Cry1)-Cry1 intron 336-Luc constructs. To examine how intronic RRE mutation affects phase delay, we replaced the SV40
basic promoter in the P(SV40)-Cry1 intron 336-Luc vector with the Cry1 promoter, P(Cry1), to obtain an array of reporter constructs, namely P(Cry1)-Cry1 intron
336-Luc, each carrying different intronic RRE mutations as described in Figure 3E. Individual constructs were transfected into NIH 3T3 cells and bioluminescencelevels in the transfected cells were monitored in real time, from which the phases of circadian oscillation were obtained (see also Figure 3E and Table S1). Raw
data of bioluminescence time-series corresponding to Figure 3E are shown. Data are representative of two independent experiments (A and B).

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S15

Bioluminescence

without Cry1 intron 336

Cry1 intron 336

500

1500000

180000400

120000500000

0

1

2

3

4

5

0

3000000

3000002000000

2000001000000

100000

0

0

0

1

2

3

4

5

1800000

0

1

2

3

4

0

1

2

3

4

0

2

3
Day

4

5

300 25
8

10

12

0

14

200

5

10

15

20

25

30

35

RRE
2

3
Day

4

5

400

50
25
8

10

12

10

15

20

25

30

35

25

30

35

25

30

35

75
50

0

14

5

10

15

20

25

30

35

0

8

10

5

0

10

12

15

14

20

500
100

100

75

400 755050

25300 25
8

10

12

0

14

200

8

10

12

14

100

100
0

14

300 25

0

0

0
1

75

100

200

5

0

100

300

12

500 100

200

400

0

0

200

240000

10

8

100

0

500

80000

1

50

25

0

300

100

400 7550

100

320000

0

75

0

0

5

160000

0

100

200

400

Cry1 intron 336
500

500 100

1200000

600000

300

0

5

D-box

0

60000

Amplitude (A.U.)

1000000

E'-box

240000
Amplitude (A.U.)

2000000

Amplitude (A.U.)

E'-boxD-box

Bioluminescence (counts/min)
Bioluminescence (counts/min)

RRE

Bioluminescence (counts/min)without Cry1 intron 336

Periodogram

5

10

15 20 25
Period (h)

30

35

0

0

5

10

15

20

Period (h)Figure S4. Related to Figure 4(Left) Transcriptional activities of E’ box, D box or RRE promoter with or without the 336 bp of Cry1 intron sequence (‘‘Cry1 intron 336’’). Raw data of bioluminescence time-series corresponding to Figure 4A are shown (n = 3). Data are representative of two independent experiments. (Right) Periodograms of time-series
data presented on the left. Amplitude of corresponding period component of time-series data is plotted. Periods ranging 814 hr are zoomed in and displayed in
insets. Notably, only a composite promoter with E’ box and Cry1 intron 336 induced significant amplitude within this range (p < 0.01). Data are representative of
two independent experiments.

S16 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

A

P(Cry1)Cry1 intron 336Cry1

0.15 μg

Bioluminescence(counts/min)

RLUC Activity (counts/sec)

18000

12000

P(Cry1)Cry1 intron 336 R1,2 mutation-Cry1

20000000

20000000

15000000

15000000

10000000

10000000
5000000

5000000
0

6000

0
0

1

2

3 4
Day

5

6

0

P(Cry1)Cry1 intron 336Cry1-RlucC

0.15 μg

Bioluminescence(counts/min)

0.45 μg

Bioluminescence(counts/min)

1.5 μgP(Cry1)-Cry1 intron 336-Cry1-Rluc

1.5 μg

0.6

0.3

0.2

0.3

P(Cry1)-Cry1
Bioluminescence(counts/min)

1

2

3

4

5

0

6

12000000

12000000

8000000

8000000

4000000

0

1

2

3

4

5

6

0

1

2

3

4

5

6

0

1

2

3 4
Day

5

6

4000000
0

1

2

3

4

5

0

6

10000000

10000000

7500000

7500000

5000000

5000000

2500000

2500000
0

1

2

3 4
Day

5

0

6

y = -5E-06x +10.54
r2= 0.0018
0.3

02500000

8000000
6000000

4000000

4000000

0

1

2

3
Day

4

5

0

period: 26.28
6000000
4000000
2000000

0

1

2

3
Day

4

5

0

0

1

2

3
Day

4

5

P(Cry1)-Cry1 intron 336-Cry1
P(SV40)-Cry2

P(SV40)-Cry2
20000000

period: 26.24

12000000

period: ---15000000

8000000

RRE-P(Cry1)-Cry1

period: 26.83

P(Cry1)-Cry1 intron 336-Cry1
12000000

7500000

8000000

2000000

2000000

5000000

Baseline ofP(Cry1)-Cry1 intron 336-Luc

P(Cry1)-Cry1 intron 336-Cry1

period: ---no Cry1 intron
wild type Cry1 intron 336
R1 deletion
R2 deletion
R1,2 deletion
R1 mutation
R2 mutation
R1,2 mutation
R1 inversion
R2 inversion
R1,2 inversion

0.0

6000000

0

Bioluminescence(counts/min)

016000000

0.4

8000000

pLV7-P(Cry1)-Cry1 intron 336-Cry1
pLV7-P(Cry1)-Cry1
pLV7-P(mCMV)-EGFP

10000000

16000000

Relative amplitude ofP(Cry1)-Cry1 intron 336-LucD

65000000
00.6

y = -14.79x + 4.23
r2= 0.12

0.0
0.1

5

15000000

10000000

0.9

Relative amplitude of
rescued oscillation

Relative amplitude of
rescued oscillation

3 4
Day

20000000

0

0.9

E

2

25000000

20000000

0Bioluminescence(counts/min)

0.45 μgP(Cry1)-Cry1 intron 336 R1,2 mutation-Cry1-Rluc

P(Cry1)-Cry1 intron 336-Cry1-Rluc

P(Cry1)-Cry1 intron 336 R1,2 mutation-Cry1-Rluc

P(Cry1)-Cry1 intron 336-Cry1-Rluc

P(Cry1)-Cry1 intron 336 R1,2 mutation-Cry1-Rluc0.15 μg1

P(Cry1)Cry1 intron 336 R1,2 mutation-Cry1-Rluc

30000000

0B

period: 25.38

8000000

10000000
400000004000000

5000000
0

1

2

3 4
Day

5

6

0

0

1

2

3 4
Day

5

6

0

0

1

2

3 4
Day

5

6

Figure S5. Related to Figure 5(A) Circadian oscillations in Cry1/:Cry2/ cells rescued by Cry1-Renilla luciferase (Rluc) fusion gene. To confirm that the observed rescue capability was
independent of the CRY1 protein level, we employed a CRY1-Rluc (Renilla Luciferase) fusion protein in which the protein expression level in Cry1/:Cry2/ cells
can be quantitatively measured through a bioluminescence assay (McCarthy et al., 2009). We generated Cry1 rescue constructs, in which Cry1-Rluc is driven by
the combination of the Cry1 promoter with either wild-type or mutated Cry1 intron sequence. Cry1/:Cry2/ cells were transiently co-transfected with P(Per2)dLuc, and various amounts (up to 10-fold) of Cry1-Rluc driven by P(Cry1) with wild-type or R1,2 mutation of 336bp of Cry1 intron sequence. The RLUC activities ofeach sample were measured (left). P(Per2)-dLuc reporter activity in transfected Cry1/:Cry2/ cells expressing either Cry1 (top) or Cry1-Rluc (bottom) wasrecorded (n = 3). Cry1 expression was under the control of either a wild-type (middle) or R1,2 mutation (right) of 336bp of Cry1 intron sequence. The results
indicate that the differences in protein expression level among the constructs are relatively smaller compared to those in DNA amounts used in transfection.

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S17

Nevertheless, all three tested DNA amounts rescued circadian rhythms and only the constructs containing wild-type intron sequence were able to reliably rescue
rhythmicity with comparable amplitude. Error bars represent SD (n = 3).
(B) Representative records of bioluminescence rhythms in Cry1/:Cry2/:Per2Luc cells through cellular genetic complementation. Lentiviral expression vectors
carrying Cry1 under the control of P(Cry1) in the presence or absence of the 336 bp of Cry1 intron sequence were introduced into Cry1/:Cry2/:Per2Luc cells.
GFP was used as a negative control. Bioluminescence expression was recorded on a LumiCycle device (Actimetrics Inc.) and representative plots using baselinesubtracted bioluminescence data are shown. The results indicate that the rescue capability was also independent of vector type or method of DNA delivery. Meanand SD (error bar) of two independent experiments are shown (each experiment contains three samples; n = 3 unless otherwise indicated in Table S1).
(C) Rescue capability does not correlate with either amplitude (left) or basal (right) bioluminescence levels of P(Cry1)-Cry1 intron 336-Luc. The relative amplitudesof circadian oscillations in Cry1/:Cry2/ cells rescued by P(Cry1)-Cry1 intron 336-Cry1 are plotted against the relative amplitude (left) and the baseline (right) ofvarious P(Cry1)-Cry1 intron 336-Luc activity presented in Figure S3B. Mean and SD (error bar) of two independent experiments are shown (each experimentcontains three samples; n = 3).
(D) Circadian oscillations in Cry1/:Cry2/ cells rescued with Cry1 driven by 2 tandem copies of RRE which were fused to P(Cry1). Cry1/:Cry2/ cells weretransiently co-transfected with P(Per2)-dLuc and Cry1 gene driven by P(Cry1) (left), P(Cry1) and Cry1 intron 336 sequence (middle), or RRE (from Bmal1) fused toP(Cry1) (right).
(E) Circadian rhythmicity in Cry1/:Cry2/ cells rescued with Cry1 and Cry2. The Cry1-rescued Cry1/:Cry2/ cells (a Cry2 knockout, in essence) showeda rather long period length of 27 hr (see also Figure 5A and Table S3), which is consistent with previous reports showing that Cry2/ single knockout cells
display long periods compared to wild-type cells (24-25 hr) (Liu et al., 2007). Since Cry2 expression does not show robust circadian oscillation in SCN or any ofthe examined peripheral tissues (Kume et al., 1999), we hypothesized that we could achieve double rescue by introducing Cry2 under a constitutive promoter in
Cry1-rescued Cry1/:Cry2/ cells. To test this, Cry1/:Cry2/ cells were transiently co-transfected with P(Per2)-dLuc, and P(Cry1)-Cry1 intron 336-Cry1(left), P(SV40)-Cry2 (middle), or both (right). The period of Cry1/:Cry2/ cells rescued with both Cry1 and Cry2 was 25.38 ± 0.06 hr (see also Table S3), which
was significantly shorter than Cry1 rescue alone (p < 0.05, Welch’s t test) and close to the natural period of WT cells. These results support the notion that genetic
complementation of Cry1 in Cry1/:Cry2/ cells recapitulates the circadian phenotype in Cry2 single knockout cells, thus phenotypically validating the Cry1
rescue assay. Data are representative of two independent experiments (A–E).

S18 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.

1000000

1000000

2

3Day

2× element

4000000

3000000

2000000

2000000

2000000

1000000

1000000

1000000

1

2

3

4

0

5

0

1

2

3

4

0

5

1250000

1250000

1250000

1000000

1000000

1000000

750000750000750000500000500000500000250000250000250000

0

0

1

2

3
Day

4

0

5

1

12000000

period: --5000000

3
Day

4

0

5

0

1

2

3

4

5

6

0

period: 27.02

0

1

2

3

4

5

6

12000000

period: ---period: 30.99
8000000

8000000
4000000

4000000

0

0

0

1

2

3

4

5

6

10000000

0

1

2

3

4

5

6

10000000

period: 30.65
7500000

5000000

5000000

2500000

2500000
0

1

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3 4
Day

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0

0

1

0

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2

3

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Day

0

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200000500000
0

4

5

4

5

0

1

2

3× element

600000300000

0

3
Day

4

0

5

0

1

2

2× element

600000

0

1

2

3

4

0

5

3
Day

0

1

2

3

4

0

5

400000400000300000300000300000200000200000200000100000100000
0

1

2

3
Day

4

0

5

1

2

3
Day

4

26

1× element

0

1

2

3

4

5

0

5

0

1

2

3
Day

4

5

10000000

5000000
5000000

2500000
0

0
1

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12500000

0

1

2

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4

5

6

15000000

10000000
10000000

7500000
5000000

5000000

2500000
0

0
0

1

0

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4

5

Phase-delay ofcomposite promoters (h)15000000

7500000

0

y = 1.07x + 27.07
r2= 0.82

5

P(SV40)P(Cry1)
3×D-box-P(SV40)
2×D-box-P(SV40)
1×D-box-P(SV40)
3×Cry1proD-P(SV40)
2×Cry1proD-P(SV40)
1×Cry1proD-P(SV40)

P(SV40)Cry1 intron 336Cry1-RLuc

10000000

2500

28

1000000P(Cry1)Cry1 intron 336Cry1-RLuc

5000

30

300000400000046000003000007500

0period: --7500000

1× elementE

6000000

1200000002+ Cry1 intron 336- Cry1 intron 336

10000000

001000000

54000000

3000000

0

Period ofrescued oscillation (h)Bioluminescence (counts/min)

4

P(SV40)400000

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1

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6

P(SV40)P(Cry1)Cry1 intron 336- Cry1 intron 336Cry1-RLuc
Cry1-RLuc

1

P(Cry1)1500000

0.3 μg00.2 μg0

55Bioluminescence (counts/min)

4

40.15 μg3Day3Bioluminescence (counts/min) Bioluminescence (counts/min)

2

2

P(SV40)Cry1proD-P(SV40) D-box-P(SV40)

5000001

1

0.2 μg

1000000

500000001500000

10000000+ Cry1 intron 336

050.15 μg

40.3 μg3

P(SV40)Cry1 intron 336Cry1-RLuc

2

P(Cry1)

0.2 μg10.15 μg

00.3 μg01500000

3000000

0C
32

P(Cry1)Cry1 intron 336Cry1-RLuc

P(Cry1)

Bioluminescence (counts/min)
Bioluminescence (counts/min)

P(SV40)P(TKms)2000000

B

RRE-P(SV40)

3000000

2000000

3× element

4000000

D

Bioluminescence (counts/min)

E'-box-P(SV40)

3000000

RLUC Activity (counts/sec)

Bioluminescence (counts/min)Bioluminescence (counts/min)
Bioluminescence (counts/min) Bioluminescence (counts/min)

Cry1proD-P(SV40) D-box-P(SV40)

A

0.3 μg0.2 μg0.15 μg
0.3 μg0.2 μg0.15 μg
0

1

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Day

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Normalized Bioluminescence
Low
High
-1
0
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Day

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6

Figure S6. Related to Figure 6(A) Raw data of bioluminescence time-series corresponding to heatmaps in Figure 6A.
(B) Raw data of bioluminescence time-series corresponding to heatmaps in Figure 6B.
(C) Prolonged phase-delay of Cry1 expression correlates with lengthened period length of rescued oscillations. The period lengths of rescued oscillations areplotted against the phase delay exhibited by various composite promoters relative to P(Cry1)-Cry1 intron 336-Luc activity. Data here and in Figure 6E are similarand each represents an independent experiment.
(D) Circadian oscillations in Cry1/:Cry2/ cells rescued by Cry1 driven by a TK minimal promoter P(TKms). Cry1/:Cry2/ cells were co-transfected with P(Per2)-dLuc reporter plasmid and Cry1 cDNA driven by P(Cry1), P(SV40), or P(TKms) in the absence (left) or presence (right) of 336bp of Cry1 intron sequence. P(Per2)-dLuc reporter activity in transfected Cry1/:Cry2/ cells was recorded. Period lengths are indicated in each panel.
(E) Circadian oscillations in Cry1/:Cry2/ cells rescued by Cry1-Renilla luciferase (Rluc) fusion gene. To confirm that CRY1 protein level was not responsible forthe changes in rescued period, we employed CRY1-Rluc fusion protein and generated Cry1 rescue constructs, in which Cry1-Rluc is driven by the combination of
the wild-type Cry1 intron sequence with either Cry1 promoter or SV40 promoter. Cry1/:Cry2/ cells were transiently co-transfected with P(Per2)-dLuc, andvarious amounts (up to 2 fold) of Cry1-Rluc driven by P(Cry1) or P(SV40) with wild-type of 336bp of Cry1 intron sequence. The RLUC activities of each sample were
measured (left panel). The construct containing SV40 promoter exhibited circadian oscillations that are 4 hr longer than the construct containing Cry1 promoter,irrespective of the different DNA amounts and the resulting different CRY1 protein levels. Error bars represent SD (n = 3). P(Per2)-dLuc reporter activity intransfected Cry1/:Cry2/ cells expressing Cry1-Rluc was recorded (middle panels, n = 3). Cry1-Rluc expression was under the control of either P(Cry1) orP(SV40). The orange boxes indicate the similar amount of Cry1-Rluc expression. Heatmaps represent average promoter activities from three independent
samples (right heatmaps). Raw data was detrended for baseline and amplitude, and then scaled into a range of 1 to 1. Data are representative of two independent experiments (A–E).

Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc. S19

AC

morning-time activator / night-time repressor

day-time activator / morning-time repressor

night-time activator / day-time repressor

0

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r2= 0.99

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10morning-time activator / night-time repressor
day-time activator / morning-time repressor
night-time activator / day-time repressor
morning-time repressor / night-time activator
day-time repressor / morning-time activatornight-time repressor / day-time activator

Figure S7. Phase Vector and Addition of Waves, Period of Negative Feedback Loop with Time Delay, Related to Figure 7 and Referenced in
Extended Experimental Procedures(A) A phase vector model recapitulates novel phases emerged from the combination of a transcriptional activator and a repressor. Plotted in the polar coordinate
are phase vectors of transcriptional activators (green arrow) and repressors (solid red arrow) driven by morning-time (E’ box), day-time (D box), or night-time (RRE)elements. The phase vector of the repressor is 180 rotated to represent its repression effect (dashed red arrow). Vector sum of activator- and repressor-vectors
is represented by dashed blue arrow. Predicted phase vector is represented by solid blue arrow, which took into account the time-delay associated withtranscription & translation of the regulator and Luciferase proteins (which was estimated in the previous report; 4 hr (Ukai-Tadenuma et al., 2008)). Measured
phase vectors emerged from combinatorial regulation (orange arrow) are consistent with predicted phase vectors (solid blue arrow), exhibiting significant
correlation (bottom panel). See also Table S4. Error bars represent SD (n = 3).
(B) Phase vector and addition of waves. The component waves f1 and f2 are displayed as phase vectors P1 and P2 . The length and direction of phase vectors
represent phase and amplitude of corresponding waves. Pc is the summed phase vector of P1 and P2 . See the sub-section ‘Phase vector and addition of waves’in Extended Experimental Procedures.
(C–F) Period of negative feedback loop with time delay.
(C) A simple negative feedback loop with time delay is schematically illustrated. The expression of repressor is driven by a regulatory element, and the expressed
repressor suppresses the element’s activity with time delay.
(D) Numerical simulation of the model illustrated in (C). The period of oscillation increases as time delay (t) increases. Under a critical value of time delay, oscillation
dampens.
(E and F) Constraints on two variables (bt, S) for generating oscillation under certain values of synthesis and turnover rate (k1 = 1 and k2 = 1 respectively), theconcentration of protease (ET = 1), dissociation constant (Kd = 1) and Michaelis constant (Km = 0:1).These graphs were drawn at two different Hill coefficients(p): p = 1 on (E), and p = 2 on (F). In the area of dimensionless time delay (bt) and the concentration of activator (S) above the curves, the model displays oscillation
with period indicated by colored rectangles (green to red: short to long period, black: dampened or no sustained oscillation). The proportional relation ofoscillation period with time delay (bt) is illustrated on bottom panels at the value of S = 2. The orange lines were calculated by the equation derived in the text
(Equation (14, 15)). The blue lines were calculated by the numerical simulations. The period of numerical simulation was measured as difference of two neighboring peak times. (See also Extended Experimental Procedures.)

S20 Cell 144, 268–281, January 21, 2011 ª2011 Elsevier Inc.