© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

ARTICLES

Feedback repression is required for mammalian circadianclock function
Trey K Sato1–3,9, Rikuhiro G Yamada4,9, Hideki Ukai4,9, Julie E Baggs5, Loren J Miraglia1,
Tetsuya J Kobayashi4, David K Welsh2,6,7, Steve A Kay2,5, Hiroki R Ueda4 & John B Hogenesch1,3,5,8
Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been
elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadiantranscriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediatedtranscriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1
resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant
proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data
demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenanceof circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalianclock function.

Circadian clocks have been proposed to consist of autoregulatory loops that use transcriptional feedback and regulated protein
turnover to maintain 24-h periodicity1–3. However, the universal
requirement for transcriptional feedback repression has been questioned recently by two studies showing that it is not required forcircadian rhythms in cyanobacteria4,5. Circadian feedback repression
in mammals is believed to be mediated by the CRY1 and CRY2
and PERIOD (PER1 and PER2) proteins. CRY and PER are
hypothesized to autoregulate their own expression by repressing theheterodimeric complex of the basic helix-loop-helix (bHLH) PERARNT-SIM (PAS) domain transcriptional activators CLOCK and
BMAL1, which bind to E-box elements in the CRY6 and PER7,8
promoters. Indirect support for this feedback mechanism comesfrom biochemical, molecular and genetic evidence: (i) CRY and PER
physically interact with CLOCK/BMAL1, (ii) CRY and PER repress
CLOCK/BMAL1 activity in steady-state transcriptional assays9–12
and (iii) CRY and PER genes are required for maintenance of circadianrhythms in mice13–15. However, direct evidence for the requirementof CRY-mediated repression of CLOCK/BMAL1 transcriptional
activity in the maintenance of circadian clock function has been notbeen shown.

RESULTS
Mutagenesis and functional screening of CLOCK
In order to determine the requirement of feedback repression incircadian clock function, we sought to identify CLOCK alleles thatwere insensitive to CRY1 repression but that maintained normal transcriptional activity. We generated a random library of B6,000 mutant
human CLOCK alleles. We screened clones individually in cell-based
reporter assays with wild-type BMAL1 cDNA and a PER1 promoterluciferase construct7 in the presence of cotransfected CRY1. Of theapproximately 6,000 CLOCK clones screened, three clones (Clock-1,
Clock-2 and Clock-3) reproducibly maintained threefold or greater
reporter activity in the presence or absence of CRY1 compared withwild-type CLOCK. We verified the reduced CRY1 sensitivity phenotype
by 96-well assays of activity of CLOCK mutants with increasing
amounts of CRY1 plasmid. When cotransfected with wild-type
BMAL1 and the PER1 reporter, the Clock-1, Clock-2 and Clock-3 clones
maintained noticeably greater activity than wild-type CLOCK in thepresence of 1 or 2.5 ng of CRY1 plasmid (Fig. 1a). However, cotransfection of 5 ng of CRY1 plasmid reduced the transcriptional activity ofthe mutants to a level similar to that of wild-type CLOCK, indicatingthat these clones are partially insensitive to CRY1 repression. Notably,

1Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Dr., San Diego, California 92121, USA. 2Department of Cell Biology, The Scripps
Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. 3Department of Genome Technology, The Scripps Research Institute, 5353
Parkside Drive, RF1, Jupiter, Florida 33458, USA. 4Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi,
Chuo-ku, Kobe, Hyogo, 650-0047, Japan. 5Department of Biochemistry, The Scripps Research Institute, 5353 Parkside Drive, RF1, Jupiter, Florida 33458, USA.
6Department of Psychiatry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92037, USA. 7The Veterans Administration San Diego
Healthcare System, 3350 La Jolla Village Drive, San Diego, California 92161, USA. 8Department of Neuropharmacology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, USA. 9These authors contributed equally to this work. Correspondence should be addressed to
H.R.U. (uedah-tky@umin.ac.jp) and J.B.H. (hogenesch@scripps.edu).

Received 29 August 2005; accepted 9 January 2006; published online 12 February 2006; doi:10.1038/ng1745

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140

Activity(% of WT CLOCK/BMAL1)

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E116K

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E367K
G332E

PAS

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E339K
G370D

Bmal1-1:

P840L

846

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bHLH

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Transfected CRY1 plasmid (ng)PER1-luciferase + CLOCK

G612E

f

A611T

Bmal1-2:

V601I

B

A

K407E

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H360Y

Clock-2:

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PER1-luciferase + CLOCKb
Clock-3:

BMAL1
Bmal1-5
Bmal1-6

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bHLH

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Activity(% of WT CLOCK/BMAL1)

a

B
PAS

A611T

S10L V160I:Bmal1-5

A611S

S9F

:Bmal1-6

626

A

1
bHLH

B

626

PAS

Figure 1 Mutations in CLOCK and BMAL1 confer insensitivity to CRY-mediated transcriptional repression without affecting CLOCK/BMAL1 transcriptional
activity. (a,c,e) Results of cell-based transcriptional PER1-luciferase reporter assays with mutant CLOCK and BMAL1 clones in HEK293T cells. Plasmids
expressing Flag-tagged wild-type or mutant CLOCK (a) or BMAL1 (c,e) cDNAs were transiently cotransfected with wild-type (WT) BMAL1 or CLOCK,
respectively, PER1-luciferase reporter and 0–5 ng of CRY1 plasmid. Activity is expressed as the percentage of normalized PER1-luciferase activity incells transfected with wild-type CLOCK/BMAL1 alone. Data are mean ± s.e.m. from independent experimental triplicates. (b,d,f) Domain locationsof causative mutations. Schematic locations of amino acid changes within the CLOCK PAS-B domain (b) and BMAL1 C terminus (d) that confer CRY1
desensitization are indicated. Locations of protein domains are indicated for CLOCK (bHLH, blue, amino acids 35–85; PAS domain, green, amino acids
113–377; PAS-A repeat, orange, amino acids 128–170; PAS-B repeat, yellow, amino acids 283–329) and BMAL1 (bHLH, blue, amino acids 73–126;
PAS domain, green, amino acids 148–439; PAS-A repeat, orange, amino acids 163–206; PAS-B repeat, yellow, amino acids 344–391). (f) Schematic
location of amino acid changes within the N terminus of BMAL1 that confer CLOCK/BMAL1 hyperactivity: Bmal1-5, S10L and V160I; Bmal1-6, S9F.

these clones demonstrated similar transcriptional activities as wild-type
CLOCK in the absence of cotransfected CRY1, suggesting that thesemutations do not cause overt alterations in the heterodimerization,
nuclear localization, DNA binding and transactivation properties of themutant CLOCK/BMAL1 complex.
DNA sequencing showed that all three of the CLOCK clones had
missense mutations causing changes in amino acids C-terminal to the
PAS-B repeat but within the PAS domain16 (also called the PAC
domain; Clock-1, G332E; Clock-2, E367K; Clock-3, H360Y), in addition
to other mutations (Fig. 1b). Notably, the mutations within the PAS
domain were independently determined to be causative in the Clock-1
and Clock-2 mutants (Supplementary Fig. 1 online). In addition, theClock-E367K mutant also had approximately threefold greater activity
than the Clock-2 clone in the absence of CRY1, suggesting that theadditional mutations within Clock-2 may cause this difference. We
examined the importance of these PAS domain residues by generating
the identical mutations in the CLOCK paralogue NPAS2 (also known
as MOP4), which regulates circadian gene expression in central andperipheral tissues and is similarly repressed by CRY (refs. 11,17–19).
Corresponding mutations in the NPAS2 PAS domain reduced its
sensitivity, compared with wild-type NPAS2, to 1 and 2.5 ng of
CRY1 plasmid (Supplementary Fig. 1). Together, these results indicatethat these mutations in the PAS domains of CLOCK and NPAS2 canpartially relieve CRY1-mediated transcriptional repression.
Recently, the crystal structure of the PAS domain of the Drosophilamelanogaster PER protein was solved, and a tryptophan residue wasfound to be important for homodimerization through intermolecular
contacts with the PAS-A repeat20. As this conserved tryptophan(Trp362) in CLOCK is near the cluster of CRY1-desensitizing
mutations, a Clock-W362A site-directed mutant was tested in

NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006

PER1-luciferase reporter assays. The Clock-W362A mutant maintained
significantly greater transcriptional activity than wild-type CLOCK in
the presence of 1 and 2.5 ng of CRY1 plasmid (Supplementary Fig. 1).
Thus, these mutations near the CLOCK PAS-B repeat may affectinteractions with PAS-A repeats from other PAS proteins, such as the
PER or BMAL1 proteins.
Mutagenesis and functional screening of BMAL1
The results of the CLOCK mutagenesis screen suggested that thecorresponding residues near the BMAL1 PAS-B repeat may also be
important for CRY-mediated repression. Therefore, we generated
BMAL1 clones containing the analogous PAS domain mutations bysite-directed mutagenesis and examined their activities in PER1
reporter assays. In contrast to CLOCK, BMAL1 PAS domain mutants
had no difference in their sensitivities to CRY1 compared with wildtype BMAL1 (Supplementary Fig. 1). In order to uncover the BMAL1
residues required for sensitivity to CRY, we randomly mutagenizedplasmids expressing Flag-tagged BMAL1 and screened them with wildtype CLOCK for reduced sensitivity to CRY1 as above. Of 6,000 clones
screened, four BMAL1 clones displayed 2.5-fold or greater desensitization to CRY1. The four BMAL1 clones showed similar desensitized
activities to 0.5–2.5 ng of CRY1 plasmid as did the CLOCK PAS
domain mutants (Fig. 1c). However, in contrast to the CLOCK
mutants, these BMAL1 clones were 1.5- to twofold more active in
the absence of transfected CRY1, which may reflect partial insensitivity
to endogenous CRY, and were completely repressed by 5 ng of CRY1
plasmid. DNA sequencing showed that the four clones contained
mutations causing amino acid changes at position 611 or 612 near the
C terminus (Fig. 1d; Bmal1-1, A611S; Bmal1-2, A611T; Bmal1-3,
A611T; Bmal1-4, G612E). Notably, a single site-directed Bmal1-G612E

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Empty vector
CLOCK/BMAL1
CLOCK/Bmal1-3
CLOCK/Bmal1-4
Clock-1/BMAL1
Clock-1/Bmal1-3
Clock-1/Bmal1-4

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Clock-2/Bmal1-4

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Transfected CRY1 plasmid (ng)25PER1-luciferase

Empty vector
CLOCK/BMAL1
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Clock-3/Bmal1-4

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Activity (% of WT CLOCK/BMAL1)

d

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Transfected CRY1 plasmid (ng)PER1-luciferasecFigure 2 Coexpression of CLOCK and BMAL1
desensitized mutants confers synergistic
insensitivity to CRY1 in HEK293T cells.
(a–c) Various combinations of single and double
CRY-desensitized CLOCK and BMAL1 mutant
cDNAs were cotransfected with the PER1 reporterand 0–50 ng of CRY1 plasmid. PER1 reporter
activity alone (blue triangle) or with wild-type
CLOCK/BMAL1 (orange triangle) are also
displayed. Activity is expressed as the percentage
of normalized PER1-luciferase activity in cells
transfected with wild-type CLOCK/BMAL1 alone.
Solid lines: CLOCK and BMAL1 single mutants.
Dashed lines: double mutants. (d) Luciferase
activities were analyzed from combinations of
single and double Bmal1-3 and Clock-3 mutant
cDNAs that were cotransfected with the PER1
reporter and 0–25 ng of CRY2 plasmid. PER1
reporter activity alone (blue triangle) or withwild-type CLOCK/BMAL1 (green triangle) arealso shown. Activity is expressed as thepercentage of normalized PER1-luciferase
activity in transfection with wild-type
CLOCK/BMAL1 and no CRY1 plasmid. Data aremean ± s.e.m. determined from independent
experimental triplicates.

140

Activity (% of WT CLOCK/BMAL1)

Activity (% of WT CLOCK/BMAL1)

b

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0

Activity (% of WT CLOCK/BMAL1)© 2006 Nature Publishing Group http://www.nature.com/naturegeneticsa

Empty vector
CLOCK/BMAL1
CLOCK/Bmal1-3
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Transfected CRY1 plasmid (ng)Transfected CRY2 plasmid (ng)PER1-luciferase

PER1-luciferase

mutant phenocopied the CRY1-desensitized activity of Bmal1-4(Supplementary Fig. 1), which contained an additional mutation
outside of the C terminus. These results confirm that, although
CLOCK uses the PAS domain for responsiveness to CRY1, aminoacid residues within BMAL1 C terminus are important for CRY1mediated repression.
Identification of hyperactive BMAL1 mutants
In addition to identifying CRY1-desensitized mutants, we isolated two
gain-of-function BMAL1 clones that showed 2- to 2.5-fold greater
Anti-Flag IP

aCLOCK allele

–

WT

WT WT

1

Bmal1-3
2

3 WT 1

2

Anti-MYC IPb

CRY1
BMAL1 allele

activity in the absence of CRY1 but were normally repressed by CRY1
(Fig. 1e). DNA sequencing detected missense mutations resulting inchanges at the N terminus upstream of the bHLH region of BMAL1(Fig. 1f; Bmal1-5, S10L; Bmal1-6, S9F). BMAL1 is known to bephosphorylated by casein kinase Ie21, which has a prominent role
in regulating activities within the core circadian feedback loop22.
To address the possibility that these mutations altered the phosphorylation state of these serine residues, we performed site-directedmutagenesis to change Ser9 or Ser10 to alanine, mimicking
the unphosphorylated residue, or to glutamate, mimicking theBmal1-4
3

WT 1

2

3

Flag-BMAL1

MYC

MYC-CRY1

CLOCK allele

WT –

WT

BMAL1 allele

WT –

WT 3

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Clock-2

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CLOCK

Flag-BMAL1
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10 11 12

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Flag-PER2

Figure 3 Mutations in the CLOCK PAS domain and BMAL1 C terminus
abrogate interactions between the CLOCK/BMAL1 complex and transcriptional
repressors CRY1 and PER2. (a–c) Native coimmunoprecipitations (co-IPs)
were performed on cell extracts from HEK293T cells transiently transfectedwith plasmids expressing epitope-tagged circadian components. (a) Flagtagged wild-type or mutant BMAL1 protein was coexpressed with or without
CRY1 and various untagged CLOCK alleles and precipitated with anti-Flag.
Copurified proteins were visualized by protein blotting with antibodies to Flag
or CLOCK. (b) Co-IPs were performed with anti-MYC on extracts from cells
coexpressing MYC-tagged CRY1, combinations of various Flag-tagged CLOCK
and BMAL1 CRY-desensitized alleles and Flag-tagged PER2. Anti-Flagand CRY1 antibodies were used in protein blotting. (c) Flag-PER2 wascoprecipitated (using anti-Flag) from cells coexpressing additional
combinations of untagged CLOCK and BMAL1 alleles. Protein blottingwith anti-Flag, anti-CLOCK and anti-BMAL1 was used to identify
copurified proteins.

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ARTICLES
the double mutants showed synergistic insensitivity to CRY2 (Fig. 2d).
In sum, the synergistic, rather than additive, insensitivity of these
double mutants suggest that the CLOCK/BMAL1 complex acts as thetarget of CRY-mediated repression.

Physical interactions between mutant CLOCK/BMAL1 and CRY
Three possible biochemical mechanisms can explain how the mutant
CLOCK/BMAL1 heterodimers could overcome CRY repression:
enhanced expression or stability of mutant proteins, enhanced formation of mutant complexes, or reduced interactions between mutant
CLOCK/BMAL1 complexes and CRY proteins. Protein blotting of
epitope-tagged CLOCK and BMAL1 proteins coexpressed in HEK293T
cells did not show any significant differences in the mutant protein
levels relative to the wild-type forms (Supplementary Fig. 3 online).
To determine if the CRY-desensitized mutants show enhanced heterodimer formation in the presence of CRY, we performed native
coimmunoprecipitations with antibodies against Flag (anti-Flag) on
HEK293T cell extracts containing Flag-tagged wild-type or mutant
BMAL1 and untagged wild-type or mutant CLOCK. Protein blotting
showed that wild-type CLOCK copurified with wild-type Flag-BMAL1
but did not copurify with the Flag epitope alone (Fig. 3a). In contrast,
for single- or double-mutant combinations of CLOCK and BMAL1,
there were moderately greater levels of mutant CLOCK/BMAL1
heterodimers than in the wild-type combination (Fig. 3a). This was
similar to the increased amounts of wild-type CLOCK that thehyperactive Bmal1-5 or Bmal1-6 proteins bound relative to wild-type
BMAL1 in the absence of cotransfected CRY1 (Supplementary Fig. 3).
Notably, the interactions between CLOCK and BMAL1 seemed to be
disrupted in the presence of CRY1 (Supplementary Fig. 3) and thus
may represent a mechanism contributing to CRY repression.
Previous studies have shown that CRY canphysically interact with the CLOCK/BMAL1
complex10,11,23 and that this interaction is
PER2-dLuc
PER2-dLuc
important for CRY-mediated repression6. To
a 300
b 300
CLOCK/BMAL1
CLOCK/BMAL1
examinethe interactions with CRY, wild-type
CLOCK/Bmal1-3
Clock-1/Bmal1-3
250
250
CLOCK/Bmal1-4
Clock-1/Bmal1-4
and mutant Flag-tagged CLOCK/BMAL1/
Clock-1/BMAL1
Clock-2/Bmal1-3
Clock-2/BMAL1
200
200
Clock-2/Bmal1-4
Clock-3/BMAL1
PER2 complexes were affinity purified with
Clock-3/Bmal1-3
Clock-3/Bmal1-4
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150
MYC-tagged CRY1 by coimmunoprecipitation. MYC-tagged CRY1 appeared to bind
100
100
substantially less CLOCK and Bmal1 single50
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and double-mutant complexes (Fig. 3b)
0
0
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compared with the wild-type complex
Time (h)Time (h)(Fig. 3b) but maintained direct interactions
with PER2, as expected9,11. Notably, coimBMAL1-dLuc
BMAL1-dLuc
c 500
d 500
munoprecipitations of Flag-PER2 showed
CLOCK/BMAL1
CLOCK/BMAL1
Clock-1/Bmal1-3
CLOCK/Bmal1-3that the CRY-desensitizing mutations in
400
400
Clock-1/Bmal1-4
CLOCK/Bmal1-4
Clock-2/Bmal1-3
Clock-1/BMAL1
CLOCK also blocked interactions with PER2
Clock-2/Bmal1-4
Clock-2/BMAL1
Clock-3/Bmal1-3
300
300
Clock-3/BMAL1
Clock-3/Bmal1-4
when coexpressed with either wild-type or200
200
mutant BMAL1 (Bmal1-4, Fig. 3c, andBmal1-3, data not shown). However, interac100
100
tions with wild-type CLOCK and mutant
0
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BMAL1 heterodimers were unaffected
0
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0
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(Fig. 3c and data not shown), supporting
Time (h)Time (h)the earlier notion that the CLOCK PAS
Figure 4 Coexpression of CLOCK/BMAL1 mutant heterodimers that are insensitive to CRY repression
domain may mediate interactions with PER.
ablates circadian E-box and RORE activities in NIH3T3 cells. Plasmids expressing Flag-tagged CLOCK
Together, these results suggest that the CRY
and BMAL1 alleles were transiently cotransfected with the PER2-destabilized luciferase (dLuc) reporter
insensitivity phenotypes resulted from reduplasmid into NIH3T3 cells. (a,b) PER2 promoter activities in NIH3T3 cells transfected with single (a)ced interactions between mutant CLOCK/or double (b) CRY1-insensitive CLOCK and BMAL1 mutants were monitored over 5 d. (c,d) BMAL1
BMAL1 complexes and the CRY/PER represpromoter activities in NIH3T3 cells transfected with single (c) or double (d) CRY-insensitive mutants
sor proteins, as well as moderately enhancedof CLOCK and BMAL1 were monitored over 6 d. All reporter activities were normalized such that the
CLOCK/BMAL1 heterodimer formation.
median wild-type luciferase activity over the time-course was 100%.
Mean activity (% of WT)

Mean activity (% of WT)
Mean activity (% of WT)

Molecular analyses of CLOCK/BMAL1 double mutants
The identification of CLOCK and BMAL1 mutants partially insensitive
to CRY suggested that the double CLOCK/BMAL1 mutant complex
may show greater insensitivity. This hypothesis was tested by examining the activities of various combinations of CLOCK and BMAL1
mutants in the presence of 0 to 50 ng of CRY1 plasmid in PER1luciferase reporter assays. With higher amounts (10–50 ng) of CRY1
plasmid, the activity of the CLOCK and BMAL1 single mutants(Fig. 2a–c) was reduced to a basal level, equivalent to that of thereporter (Fig. 2a–c). In contrast, CLOCK/BMAL1 double mutants
showed marked and synergistic desensitization to high levels of CRY1
plasmid (Fig. 2a–c). Even with 25 ng of cotransfected CRY1 plasmid,
all CLOCK/BMAL1 double mutants retained approximately 50% ofreporter activation compared with wild-type CLOCK/BMAL1 lacking
CRY1 plasmid. This synergistic insensitivity to CRY1 repression was
also seen with NPAS2/BMAL1 and Clock-W362A/Bmal1-3 double
mutants (Supplementary Fig. 2 online) but was not when Bmal1-5
or Bmal1-6 hyperactive alleles were coexpressed with mutant Clock-3(Supplementary Fig. 2). Thus, BMAL1 hyperactivity alone did notaccount for synergistic insensitivity to CRY1.
To determine if these mutations also block repression by mammalian Cryptochromes in general, we also examined Clock-3/Bmal1-3
double mutants for sensitivity to CRY2. Notably, single Clock-3 andBmal1-3 mutants were desensitized to low amounts of CRY2, whereas

Mean activity (% of WT)© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

phosphorylated form. Single- or double-alanine and glutamate
exchanges in the BMAL1 N terminus as well as deletion of the first
11 N-terminal amino acids (Supplementary Fig. 1) all resulted inenhanced PER1 reporter activity, suggesting that phosphorylation
states of Ser9 and Ser10 do not modulate BMAL1 activity.

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CLOCK/BMAL1

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Real-time analysis of circadian gene expression
The prevailing transcriptional feedback model predicts that impairment of CRY-mediated repression should have marked effects on
circadian expression of the PERIOD genes. This notion is supported
by in vivo observations that expression of PER1 and PER2 isconstitutively elevated in Cry1/Cry2 double knockout mice14,24. To
determine whether these mutations in CLOCK and BMAL1 cause
phenotypic changes in circadian gene expression, we performed realtime bioluminescence assays in an oscillating mammalian cell
model25,26. This approach allows for a continuous and quantifiable
report of circadian promoter activity from the same transfected cells
over multiple days and enables statistical evaluation of importantparameters of circadian clock function such as period length andamplitude. Mouse NIH3T3 fibroblasts were transfected with plasmids
containing destabilized luciferase driven by the PER2 or SV40 promoters along with various combinations of BMAL1 and CLOCK
alleles, stimulated with forskolin to synchronize circadian rhythmicity,and then were monitored in real-time for circadian luciferase activity(Fig. 4a,b and Supplementary Fig. 4 online). Cotransfection of wildtype CLOCK and BMAL1 did not substantially alter rhythmicity
compared with cells transfected with empty vector, as their period
lengths were 21.4 h ± 0.4 h (mean ± s.d., Supplementary Fig. 4). In
contrast, transfection of either of the single CLOCK or BMAL1 mutant
alleles resulted in substantial impairment of circadian rhythmicity
after one or two cycles of oscillations when compared with wild-type

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© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

10

Clock-1/Bmal1-3

b 100
Activity (% of maximum)

Activity (% of maximum)

a 100

50

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70 Autocorrelation

Figure 5 Coexpression of CLOCK/BMAL1
mutant heterodimers impairs circadianrhythmicity in individual cells. (a–d) PER2luciferase reporter activities from individual
NIH3T3 cells (n ¼ 133) transfected with Flagtagged wild-type CLOCK/BMAL1 (a,c) or doublemutant Clock-1/Bmal1-3 (b,d) were monitored
over 3 d. Reporter activities from each wild-type(a) or double-mutant (b) cell were normalized
such that the maximum bioluminescence value
was set to 100% for each panel. The mean
reporter activity for all analyzed single cells at
each time point is indicated by a black line.
Normalized bioluminescence activities fromeach wild-type (c) or double-mutant (d) cellwere detrended and ranked according tocorresponding significance by autocorrelation(upper panels) or COSOPT (lower panels).
Autocorrelation and MMC-b values for each cellare depicted to the right of each heat map from0 (bottom, dark blue) to 1 (top, white). Red andgreen denote high and low normalized reporter
activities, respectively. Results shown are
representative of duplicate experiments.

CLOCK/BMAL1 (Fig. 4a and Supplementary
Fig. 4). Notably, cotransfection of CRY-insensitive mutant CLOCK and BMAL1 together
resulted in the loss of circadian PER2 promoter activity (Fig. 4b and Supplementary
Fig. 4). Thus, transcriptional repression of
10–1
CLOCK/BMAL1 by CRY is required for
10–2
10–3
circadian E-box activity.
–4
10
In addition to PER and CRY, rhythmic
expression of BMAL1 mRNA is also under
50 60 70 MMC-β
circadian clock regulation12. However, theBMAL1 promoter does not have E-box sites
and instead contains retinoic acid–relatedorphan nuclear receptor binding elements (RORE)25,27, whose activities are reciprocally controlled by the rhythmically expressed transcriptional repressor REV-ERBa27 and activator Rora28,29. As an additional
test for circadian clock function, the effects of mutant CLOCK and
BMAL1 on rhythmic RORE activity were examined by real-time assays
with a BMAL1-destabilized luciferase (BMAL1-dLuc) reporter. Similar
to results with the PER2-dLuc reporter, transfection of single CLOCK
or BMAL1 mutants resulted in the decreased amplitude of cycling of
BMAL1-dLuc activity compared with wild-type CLOCK/BMAL1(Fig. 4c and Supplementary Fig. 4). Moreover, this decrease in cycling
amplitude was further exacerbated upon cotransfection of the double
mutant heterodimer (Fig. 4d and Supplementary Fig. 4). These results
are probably due to misexpression of endogenous REV-ERBa, which isregulated by CLOCK/BMAL1 and CRY27 through E-box elements in
the REV-ERBa promoter. Consistent with this, cotransfection of wildtype CLOCK/BMAL1 inhibited steady-state BMAL1 promoter activity,
whereas additional cotransfection of CRY1 caused activation (Supplementary Fig. 5 online). In contrast, CRY-mediated activationof the BMAL1 reporter was abrogated when cotransfected with the
CLOCK/BMAL1 double mutants (Supplementary Fig. 5 and data notshown). These results indicate that transcriptional repression of
CLOCK/BMAL1 by CRY is also required for circadian BMAL1 expression through ROR elements, which is dependent upon transcriptional, translational and post-translational actions of endogenous
cellular factors.

VOLUME 38 [ NUMBER 3 [ MARCH 2006 NATURE GENETICS© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

ARTICLES
Role of circadian feedback repression in single cells
The arrhythmic PER2 expression seen from a population of cells
expressing the double mutant CLOCK/BMAL1 complex may be dueto the disruption of oscillator function or a lack of synchrony between
individual rhythmic cells. In order to address these possibilities,
quantitative imaging of PER2-luciferase reporter activity was measured from individual NIH3T3 fibroblasts by an approach similar tothat used in analyzing BMAL1 reporter rhythms from single cells30. As
with the whole-well assays, the median reporter activity for the
population of imaged individual fibroblasts coexpressing wild-type
CLOCK/BMAL1 oscillated rhythmically (Fig. 5a). In contrast, thepopulation of individual Clock-1/Bmal1-3 mutant cells (Fig. 5b) was
visibly arrhythmic. Individual reporter activities from single wild-type
cells were rhythmic (Fig. 5c and Supplementary Fig. 6 online), as
expected, whereas individual Clock-1/Bmal1-3 double mutant cells
showed arrhythmic reporter activities (Fig. 5d and Supplementary
Fig. 7 online). These differences in activity patterns were evaluated by
autocorrelation (see Methods) and COSOPT31 analyses, two independent statistical methods that score the circadian rhythmicity ofexperimental time-course data. Reporter activity traces from eachwild-type or double mutant cell were assigned autocorrelation and
COSOPT (Multiple Measures Corrected minus b, MMC-b) significance scores (Fig. 5c,d). Statistically significant differences between
wild-type (n ¼ 133) and double mutant (n ¼ 133) cells were observed
from the individually assigned autocorrelation (Wilcoxon test, P ¼ 6.8
 10–11) and MMC-b (Wilcoxon test, P ¼ 1.1  10–13) scores. Thus,
both visual inspection and statistical analyses indicate that the double
mutant cells differ from rhythmic wild-type cells. We also observed
this arrhythmic behavior in individual Clock-3/Bmal1-4 double
mutant cells (Supplementary Fig. 8 online). Therefore, the arrhythmicity seen upon expression of double CLOCK/BMAL1 mutants inpopulation studies was a reflection of the activity of mutant complexesin single cells rather than desynchrony of individual cellular oscillators.
DISCUSSION
The current molecular model for the mammalian circadian clock
includes transcriptional feedback repression by CRY and PER proteins3,32. Evidence for this mechanism is based on observations that
CLOCK/BMAL1 activates CRY and PER gene expression, whose
protein products, in turn, repress CLOCK/BMAL1 activity in transienttransfection assays. Repression is believed to be mediated throughphysical interactions, as CRY associates with CLOCK/BMAL1 incoimmunoprecipitation and yeast two-hybrid assays. Finally, both
Cry1/Cry2 and Per1/Per2 double knockout mice are behaviorally
arrhythmic. While this evidence is strongly supportive for such amechanism, functional requirement for feedback repression in the
mammalian circadian clock has not been previously uncovered. Proofof this necessity is of import, as recent studies of the cyanobacterial
clock found that circadian oscillations in protein phosphorylation canbe maintained in the absence of transcriptional feedback repressionin vivo5 and with purified proteins in vitro4. Therefore, we sought toformally test the requirement of CRY-mediated transcriptional feedback repression in the mammalian circadian clock by developing andimplementing a new, unbiased cellular genetics approach that uses
robust mutagenesis techniques and mammalian cell–based screening.
Using this approach, we identified an allelic series of CLOCK and
BMAL1 clones that were uncoupled from CRY-mediated repression.
Coexpression of these mutants in fibroblasts disrupted the rhythmic
activities of both E-box and ROR elements, the two primary transcription factor binding sites used by the circadian oscillator26. These
data provide direct evidence that CRY-mediated feedback repression

NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006of the CLOCK/BMAL1 complex is required for mammalian clockfunction. Notably, some oscillatory properties of the mammalian
circadian clock, such as protein phosphorylation, may remain intact
upon uncoupling of transcriptional feedback repression. Although it islikely that the mammalian clock is governed by a combination ofboth transcriptional and nontranscriptional feedback mechanisms,
any residual circadian properties that remain upon uncouplingof transcriptional feedback are insufficient to maintain circadiantranscriptional output and molecular clock function.
Finally, we predict that application of cellular genetics technology
will have a significant impact on mammalian biology as similar
approaches have had on prokaryotic and yeast biology.
METHODS
Mutant generation and screening protocols are provided in Supplementary
Methods online.
Cell-based reporter assays. Luciferase reporter assays were performed intriplicate wells of 96-well white Co-Star bioassay plates (Corning). Total
plasmid DNA (750 ng) was incubated with 150 ml of serum-free Dulbecco’s
modified Eagle’s minimal essential medium (DMEM; GIBCO) and 2.25 ml
FuGENE6 for 30 min in microcentrifuge tubes. For the PER1 reporter assays,
we used the following amounts of plasmid DNA per well: 25 ng pGL3-mPER1,
50 ng pCMV-mBMAL1, 120 ng pCMV-hCLOCK or hNPAS2 and 5 ng pCMV-bGALACTOSIDASE. In assays with CRY1 or CRY2, indicated amounts of pCMVmCRY plasmid were compensated with empty pCMV-Sport6 vector to total
50 ng. HEK293T cells were cultured to 80% confluence at 37 1C with 5% CO2
and were harvested in DMEM/20% fetal bovine serum (FBS; GIBCO)/0.2 mM
nonessential amino acids (NEAA; GIBCO)/2 penicillin-streptomycin-glutamine (PSG; GIBCO) at 8  105 cells ml–1. We dispensed 50 ml of harvested cells
into individual wells in triplicate of the 96-well bioassay plate. After 30 min,
50 ml of plasmid DNA/DMEM/FuGENE6 mix was dispensed into the appropriate well with cells and incubated at 37 1C (5% CO2) for 24 h. Luciferase andb-galactosidase activities were analyzed with Dual Light Kit (Tropix) and
Acquest reader (LJL Biosystems) according the manufacturers’ specifications.
Ratios of luciferase:b-galactosidase activities from technical triplicates wereaveraged and normalized as the percent of luciferase:b-galactosidase activity
ratios with wild-type CLOCK and BMAL1 plasmids in the absence of CRY. Final
percentage activities were calculated from three independent experiments.
Native coimmunoprecipitations and protein blotting. We seeded 6  106
HEK293T cells on 10-cm Petri dishes with 7 ml DMEM/10% FBS/0.1 mM
NEAA/1 PSG and incubated them at 37 1C/5% CO2 for 20 h. Cells
were then transfected with 6 mg of total plasmid DNA in 100 ml of serum-free
DMEM and 18 ml of FuGENE6. Twenty-four hours post-transfection, cells were
washed and harvested with Dulbecco’s phosphate buffered saline (DPBS,
GIBCO) and pelleted by centrifugation. Cell pellets were solubilized in 1 ml
TGED buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 5% (vol/vol)glycerol, 0.5 mM DTT), complete protease inhibitors (Roche Applied Sciences)and 0.5% Triton X-100. Soluble lysate was cleared by centrifugation for 15 min
at 13,000g at 4 1C. Six microliters of anti-Flag (Sigma-Aldrich) or anti-MYC(Pierce Biotechnology) conjugated to Sepharose beads were added to 1 ml ofcleared lysate and incubated for 2 h at 4 1C with agitation. Bound proteins werethen washed twice with TGED + 0.5% Triton, twice with TGED + 1%
Triton, three times with TGED at 250 mM NaCl + 1% Triton and twicewith TGED. Bound proteins were eluted with 50 ml of Laemmeli’s Reducing
Sample Buffer (Biorad Laboratories), heated to 42 1C for 5 min and resolved
by SDS-PAGE electrophoresis (Biorad Laboratories). Copurified proteins
were identified by protein blotting with rabbit polyclonal antibodies to
Flag (Sigma-Aldrich), CLOCK (Affinity BioReagents), BMAL1 (Affinity BioReagents) or mCRY1 (a gift from S. Panda, The Salk Institute, La Jolla,
California). For comparison of protein expression from cotransfected CLOCK
and BMAL1 cDNAs, equal amounts of cleared cell lysates were processed with1 sample buffer and resolved by protein blotting with anti-Flag or appropriate
antibodies. Results of co-IP and protein blotting experiments were independently repeated three times.

317

© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

ARTICLES
Additional plasmid construction. All site-directed mutations were made with
the QuikChange Mutagenesis kit (Stratagene). Wild-type and mutant untagged
pCMV-BMAL1, CLOCK, NPAS2, CRY1 and CRY2 constructs were generated bycloning the coding sequences for each gene into the pCMV-Sport6 vector(Invitrogen) by PCR and standard recombinant DNA techniques. The
Flag-mPER2 plasmid was constructed by PCR amplification and cloning into
the SpeI and NotI restriction sites of pFlag. MYC-tagged CRY1 and BMAL1
constructs were generated by PCR amplification with primers containing
in-frame EcoRI and XhoI restriction sites and cloning into the identicallydigested pTag3C vector (Stratagene). The PER2-dLuc and PER2-Luc reporters
were generated by cloning the mouse PER2 promoter (–219 to +76 nucleotides
from transcriptional start site) into pGL3-dLuc25 and pGL3-Basic (Promega),
respectively. The BMAL1-dLuc reporter was generated by cloning the mouse
BMAL1 promoter from pGL3-BMAL1 (ref. 28) into the dLuc plasmid. The DNA
sequences of all constructs generated in this study were verified. The pGL3PmPER1 reporter7 and Rora28 plasmids are described elsewhere.
Real-time whole-well circadian reporter assays. Whole-well, real-time circadian assays were performed as previously described26 with the following
modifications. NIH3T3 cells (American Type Culture Collection) were grownin DMEM supplemented with 10% FBS (JRH Biosciences) and antibiotics(25 units ml–1 penicillin, 25 mg ml–1 streptomycin; GIBCO). Cells were plated
at 5  104 cells per well in 24-well plates 24 h before transfection. Cells weretransfected with FuGENE6 according to the manufacturer’s instructions. Cells
in each well were transfected with 0.32 mg (total) of plasmid (0.08 mg of PER2,
BMAL1 or SV40-promoter reporter plasmids and 0.24 mg of pEGFP-N3 or
pFlag-BMAL1/CLOCK variants). After 72 h, medium in each well was replacedwith 500 ml of culture medium (DMEM/10% FBS) supplemented with 10 mM
HEPES (pH 7.2), 0.1 mM luciferin (Promega), antibiotics and 0.01 mM
forskolin (Nacalai Tesque). Bioluminescence was measured with photomultiplier tube (PMT) detector assemblies (Hamamatsu Photonics). The modules
and cultures were maintained in a darkroom at 30 1C and interfaced withcomputers for continuous data acquisition. Photons were counted for 2 min at
24-min intervals.
Real-time single-cell bioluminescence imaging. We plated 1  105 NIH3T3
fibroblasts on 35-mm dishes 24 h before transfection. Plated fibroblasts werethen transfected for 72 h with 0.6 mg pGL3-PER2 and 1 mg each of CLOCK or
BMAL1 alleles and 3 ml FuGENE6 and were stimulated for 2 h with 0.01 mM
forskolin. For Flag-tagged wild-type CLOCK/BMAL1 and Clock-1/Bmal1-3transfections, medium was replaced with 2 ml fresh medium containing
luciferin as described for whole-well assays. We sealed the 35-mm culture
dishes with cover slips, placed them on the stage of a luminescence microscope(Olympus) and incubated them at 30 1C (Olympus). Bioluminescence wasimaged by an Olympus 10 objective and transmitted to a cooled CCD camera(ORCA-AG C4742-80-12AG, Hamamatsu Photonics) mounted on the bottom
port of the microscope. We used 4  4 binning of the 336  256 pixel array forsingle-cell measurement. Time-lapse images were collected at 30-min intervals
with 25-min exposures by a computer using an image analysis program(AquaCosmos, Hamamatsu Photonics). The protocol for analysis of
Clock-3/Bmal1-4 single cells is provided in Supplementary Methods.
Image and data processing for single-cell imaging. Images were analyzed
using MetaMorph (Universal Imaging) and corrected for bias and dark current
by subtracting a background image. Luminescence intensity was measured
within a region of interest defined manually for each cell. The position of theregion was adjusted, if necessary, to accommodate cell movement. Cosmic ray
artifacts were removed by comparing the bioluminescence intensity with thecorresponding value calculated from temporally adjacent images. When bioluminescence intensity of a cell at a certain time point was 50% greater than the
average of those of the temporally adjacent data, the value was replaced with theaverage of the adjacent data. In some experiments, cosmic ray artifacts were
removed by pixel-wise minimization of temporally adjacent images. Remaining
data processing of extracted single-cell bioluminescence intensity data wasconducted with Mathematica (Wolfram Research).
Rhythmicity, period length and amplitude analysis of real-time bioluminescence data. Bioluminescence time-series data beginning 21 h after forskolin

318

stimulation were used for analysis in order to distinguish endogenous circadianoscillation from acute effects of stimulation. Bioluminescence data weredetrended by using the trend curve calculated by the smoothing spline
method33. The smoothing parameter for this calculation was set such that its
frequency-response was 50% at a frequency of about two cycles (42 h) of thetypical circadian period (B21 h) observed in fibroblasts transfected with empty
vector at 30 1C. Then, autocorrelation of the detrended bioluminescence timeseries data was calculated within the range of 16–28 h to determine thecircadian period of oscillation. Statistical significance (with 0 as most significant and 1 as least significant) of circadian oscillation was evaluated bycomparing the strongest autocorrelation of the detrended data within therange of 16–28 h against that of white noise
pﬃﬃﬃﬃ (theoretical value for the
autocorrelation of white noise is –1/N ± 1/ N (mean ± s.d.), where N is
the number of time points). COSOPT calculations were performed as previously described31 with period length parameters of 16–28 h.
The effects on amplitude of cycling by coexpression of CRY-insensitive
mutants on circadian oscillation in whole-well assays were determined bymeasuring the amplitude difference (percentage variation) between maximum
and minimum bioluminescence relative to wild-type CLOCK/BMAL1 within
24–48 h from forskolin stimulation. In some circumstances, real-time recordings were omitted when peak and trough points from the luminescence traces
could not be clearly identified.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
This research was supported by the Novartis Research Foundation (L.J.M. and
J.B.H.), a Rena and Victor Damone Postdoctoral Fellowship from the American
Cancer Society (T.K.S.), US National Institute of Health (NIH) grants (D.K.W.
and S.A.K.), Scripps Florida (T.K.S., J.E.B., and J.B.H.), RIKEN Center for
Developmental Biology (H.R.U.), NIH/Silvio O. Conti Center for Neurosciencegrant P50 MH074924-01 (J.E.B. and J.H.), RIKEN Strategic Programs (H.U. and
H.R.U.), New Energy and Industrial Technology Organization (NEDO) Scientific
Research grant (H.R.U.) and Scientific Research grant and Genome Network
Project grant from the Japanese Ministry of Education, Culture, Sports, Scienceand Technology (H.R.U.). This is manuscript number 17558-CB of The Scripps
Research Institute. We thank N. Gekakis, S. Reppert, C. Joazeiro, A. Curtis and
G. FitzGerald for plasmids; S. Panda for anti-mCRY1; J. Zhang and T. Orth for
robotics support; T. Kondo for high-throughput monitoring systems; M. UkaiTadenuma, J. Cartzendafner and J. Geskes for technical support and S. Panda,
R. Van Gelder, T. Reyes, M. Pletcher, K. Hayes, B. Miller, M. Conkright and
M. Givens for critical reading of the manuscript.
AUTHOR CONTRIBUTION STATEMENT
T.K.S. and J.B.H. developed the mutagenesis screening concept. R.G.Y., H.U., and
H.R.U. developed the high through-put real-time assay concept. T.K.S. developed,
constructed and screened the mutant libraries, performed the steady-state reporterand biochemical analyses, and created the related figures. R.G.Y. developed thestatistical method specific for whole-well and single cell real-time bioluminescence
experiments, analyzed the data and created the related figures. H.U. developed thehigh throughput real-time assays and performed the related experiments. J.E.B.
generated data for Figure 2a, the Clock-W362A mutant, Supplementary Figure 6
and performed additional steady-state reporter and biochemical analyses. L.J.M.
developed the screening protocol and constructed and screened the mutant
libraries. T.J.K. and H.U. performed single-cell imaging experiments, analyzed
the data and generated related figures. D.K.W. and S.A.K. performed single-cell
imaging experiments and analysis. H.R.U. developed and analyzed all real-timeassays. R.G.Y., H.U., T.J.K. and H.R.U. wrote the sections relevant to real-timeassays. T.K.S. and J.B.H. wrote the remaining sections. All authors discussed theresults and commented on the manuscript text.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturegenetics
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/

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