The BMAL1 C terminus regulates the circadiantranscription feedback loop
Yota B. Kiyohara*†, Sayaka Tagao*†, Filippo Tamanini‡, Akira Morita*, Yukiko Sugisawa*, Maya Yasuda*,
Iori Yamanaka*, Hiroki R. Ueda§, Gijsbertus T. J. van der Horst‡, Takao Kondo¶, and Kazuhiro Yagita*储
*Center of Excellence Unit of Circadian Systems and ¶Division of Molecular Genetics, Department of Biological Sciences, Nagoya University Graduate
School of Science, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; ‡MGC, Department of Cell Biology and Genetics, Erasmus University Medical
Center, P.O. Box 1738, 3000 DR, Rotterdam, The Netherlands; and §Laboratory for Systems Biology, Center for Developmental Biology, RIKEN,
2-2-3 Minatojima-minaminachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Edited by Joseph S. Takahashi, Northwestern University, Evanston, IL, and approved May 11, 2006 (received for review February 20, 2006)

The circadian clock is driven by cell-autonomous transcription兾translation feedback loops. The BMAL1 transcription factor is anindispensable component of the positive arm of this molecularoscillator in mammals. Here, we present a molecular genetic
screening assay for mutant circadian clock proteins that is based onreal-time circadian rhythm monitoring in cultured fibroblasts. By
using this assay, we identified a domain in the extreme C terminusof BMAL1 that plays an essential role in the rhythmic control of
E-box-mediated circadian transcription. Remarkably, the last 43 aaof BMAL1 are required for transcriptional activation, as well as for
association with the circadian transcriptional repressor CRYPTOCHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associatewith mPER2 (another protein of the negative feedback loop),suggesting that an additional repression mechanism may converge
on the N terminus. Taken together, these results suggest that the
C-terminal region of BMAL1 is involved in determining the balance
between circadian transcriptional activation and suppression.
circadian clock 兩 real-time monitorT

he mammalian circadian clock is a highly dynamic system thatgenerates periodic fluctuations in the mRNA expression levelsof hundreds of genes to confer near 24-h rhythmicity to behavior,
physiology, and metabolic processes, thereby allowing mammals to
anticipate the momentum of the day (1). The master clock resides
in the suprachiasmatic nuclei (SCN) of the brain and, in turn,
synchronizes circadian clocks in peripheral tissues (2). Even fibroblasts in culture contain an active circadian clock that has the same
genetic makeup of the central clock in the SCN (3–6). To keep pacewith the day–night cycle, the SCN clock, but not peripheral clocks,
are entrained by light.
Circadian rhythms are generated by a molecular oscillator thatconsists of intertwined positive and negative transcription兾translation feedback loops involving a set of clock genes (7) andclock-controlled output genes that link the oscillator to clockcontrolled processes (8). BMAL1 (MOP3) and CLOCK are basic
helix–loop–helix PAS transcription factors that heterodimerize and(by means of binding to E-box promoter elements) transactivate the
Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes andan orphan nuclear receptor Rev-Erb␣ core oscillator gene. Subsequently, PER and CRY proteins act as negative elements by
inhibiting the activity of the CLOCK兾BMAL1 heterodimer,whereas REV-ERB␣ negatively regulates Bmal1 gene expression(1, 9). The above feedback mechanism is supported by biochemical,
molecular, and genetic evidence; however, formal proof of its
requirement in the maintenance of circadian clock oscillations has
not been shown thus far.
Genetic ablation of mBmal1 results in complete disruption of the
mammalian circadian clock at the behavioral and molecular levels(10). However, except for the PAS elements, which are required for
association with CLOCK (11), relatively little is known aboutprotein domains that regulate BMAL1 function. A recent study
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revealed that constitutive high expression of BMAL1 protein in
Rev-Erb␣-disrupted mice still allows robust circadian molecular andbehavioral rhythms (12). Moreover, it has been shown that sumoylation of BMAL1 influences posttranscriptional features of
BMAL1 and that this process is CLOCK-dependent (13). Taken
together, these results suggest that posttranslational modification of
BMAL1 is an important prerequisite for its performance in thecircadian oscillator.
To understand the mechanism of BMAL1 function in the clock,we generated several BMAL1 mutants and analyzed their effect on
circadian clock performance in mammalian cells. We show that the
C terminus of BMAL1 is involved in both positive and negative
regulation of transcriptional activity, which in turn is mandatory formolecular clock oscillation in the living cell.
Results and Discussion
To dissect the role of BMAL1 in circadian clock performance, we
decided to perform a random mutagenesis-based structure兾function analysis of this protein in mammalian cells undergoing
multiple cycles of clock oscillation. To monitor circadian oscillations in real-time mode, we used mPer2 or mBmal1 promoterdriven luciferase reporter vectors (mPer2:Luc and mBmal1:Luc,
respectively). After transient expression of mPer2:Luc (ormBmal1:Luc) in Rat-1 cells for 48 h, followed by synchronization ofindividual cellular clocks with 100 nM dexamethasone (14, 15), we
could detect clear rhythmic expression of the reporter gene for fivecycles with a periodicity of ⬇21 h (Fig. 1A). First, we validated thissystem for use as a BMAL1 mutant screening tool by analyzing theeffect of constitutive overexpression of Flag-tagged WT mBMAL1(WT-BMAL1) on circadian oscillations. After dexamethasone
synchronization, WT-BMAL1-overexpressing Rat-1 fibroblasts areable to produce a robust circadian rhythm with a period andamplitude comparable to cells transfected with an empty vector(Fig. 1B). Thus, constant high levels of BMAL1 are well tolerated
by the molecular oscillator, which contrasts with the severe impact
of constitutive overexpression of circadian clock components of thenegative limb of the oscillator (i.e., mCRY1 and mPER2) (16, 17).
In line with the observation that Rev-Erb␣⫺/⫺ mice (constitutively
expressing Bma1) maintain behavioral and molecular rhythms (12),this finding indicates that mammalian core oscillator function does
not require cyclic transcription of Bmal1. Consistent with the above
observations, circadian bioluminescence rhythms were also notattenuated after cotransfection of WT-Bmal1 and WT-Clock expression vectors (Fig. 5, which is published as supporting information on the PNAS web site).
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CFP, cyan fluorescent protein; HA, hemagglutinin; YFP, yellow fluorescentprotein.
†Y.B.K. and S.T. contributed equally to this work.
储To whom correspondence should be addressed. E-mail: kyagita@bio.nagoya-u.ac.jp.

© 2006 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0601416103

Next, we performed a molecular genetic screen to identify
protein domains that are critical for BMAL1 function. To this end,
we generated an expression library of random mutant (Flag-tagged)
BMAL1 proteins by using a commercially available Tn5 transposon-based insertion system that introduces 19 aa in-frame (18). In
this way, we obtained 30 in-frame protein mutants, named FlagBMAL1-Bm1 to Flag-BMAL1-Bm30, that were sequenced todetermine the position of the 19-aa insertion. Then, mutant
BMAL1 proteins were overexpressed in Rat-1 cells and cotransfected with mPer2:Luc (or mBmal1:Luc) clock reporter genes,
followed by continuous and quantifiable monitoring of circadianclock parameters (e.g., period length and amplitude). We identified
two mutant BMAL1 proteins (Flag-BMAL1-Bm29 and -Bm30,
carrying insertions after Ala-599 and Leu-606 of mBMAL1, respectively; Fig. 1D) that impaired circadian rhythmicity in a dominant negative manner, as evident from the severe dampening ofmPer2:Luc-driven bioluminescence rhythms (Fig. 1C).
In parallel, we tested the capacity of these 30 mutant BMAL1
proteins to drive E-box-mediated transcription in the NIH 3T3
cell-based transcription activation assay. Interestingly, the second
screen uncovered more mutant proteins with a clear molecular
phenotype than the real-time assay. For example, BMAL1 mutant
proteins with insertions in the basic helix–loop–helix (FlagBMAL1-Bm7兾8兾9兾10) and PAS-B domains (Flag-BMAL1Bm19兾20兾21) exhibited a reduced transcriptional activation capacity (possibly due to a lack of association with CLOCK), yet they did
not disturb the performance of the molecular oscillator in thereal-time assay (Fig. 1D). In addition, we isolated three BMAL1
mutant proteins that showed 2-fold greater activity in the presenceof coexpressed CLOCK (as compared with WT BMAL1), suggesting that those are gain-of-function BMAL1 mutants. In this case,insertions were located upstream and downstream of the basic
helix–loop–helix region (-Bm5 and -Bm12, respectively) and downstream of the PAS-A domain (-Bm13). Importantly, however, we
identified Flag-BMAL1-Bm29 and -Bm30 (shown in the previous
experiment to have a dominant negative effect on the circadian coreoscillator) as the two mutant BMAL1 proteins that failed to drive
Kiyohara et al.

E-box-mediated transcription. Thus, the C terminus of BMAL1
must contain a domain (or domains) required for transcription
activation, the disruption of which causes a dominant negative
effect on core oscillator function.
To obtain more insight into the role of the C-terminal regionof BMAL1 in controlling the generation of circadian rhythmicity, we generated by in vitro mutagenesis three BMAL1 ‘‘deletion’’ constructs in which amino acid Glu-447, Leu-554, orIle-584 of mBMAL1 was substituted by a stop codon (Fig. 2A).
Western blot analysis of cell lysates from COS7 cells transiently
expressing Flag-BMAL1(E447X), Flag-BMAL1(L554X), or
Flag-BMAL1(I584X) revealed proteins of expected molecularweight at WT amounts (Fig. 6A, which is published as supporting
information on the PNAS web site). In contrast to WT FlagBMAL1, all three mutant BMAL1 proteins remained inactive in
the E-box-mediated transcription assay (Fig. 2B). These results
support a previous study in which the C-terminal region of BMAL1
was shown to be required for its transcriptional activation function(19). Importantly, similar to Flag-BMAL1-Bm29 (Fig. 7, which is
published as supporting information on the PNAS web site), they
inhibited WT Flag-BMAL1兾CLOCK-driven transcription activation in a dose-dependent manner, pointing to a dominant negative
effect of deletion of the C terminus on the transcription activation
property of BMAL1 (Fig. 2C).
We next investigated whether these deletion mutant proteins also
affected real-time core oscillations in Rat-1 cells. Overexpression of
Flag-BMAL1(L554X) dramatically blunted mPer2:Luc- andBmal1:Luc-mediated luminescence rhythms in a dose-dependent(dominant negative) manner, but, importantly, whereas mPer2:Luc
expression levels dropped to basal values (Fig. 2D), Bmal1:Luc
luminescence levels were markedly elevated (Fig. 2E). These results
fit well with the recently reported mechanism of transcriptional
regulation of Bmal1 expression (12), which proposes that the
CLOCK兾BMAL1 heterodimer up-regulates transcription of the
Rev-Erb␣ gene, causing REV-ERB␣-mediated suppression of
Bmal1 transcription through REV-ERB␣ response element enhancer elements in the Bmal1 promoter. Similar results were
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NEUROSCIENCE

Fig. 1. Screening for functional protein domains in BMAL1. (A) Bioluminescence oscillations of dexamethasonesynchronized Rat-1 cells expressing themPer2:Luc reporter demonstrate the reproducibility of the system. (B) Effect ofoverexpression of WT Flag-mBmal1 on
mPer2:Luc rhythms (red lines). As a control, we used cells transfected with theempty pcDNA3 vector (blue line). (C)Functional screen for mutant BMAL1
proteins (containing random transposon-based in-frame insertions of 19 aa)that affect circadian core oscillations.
Different colors indicate different proteins. Note the severely impaired
mPer2:Luc rhythm obtained with FlagBMAL1-Bm29, which is indicated in red
and marked by an arrow. (D) Functional
screen for mutant BMAL1 proteins (containing random transposon-based inframe insertions of 19 aa) that affecttranscription activation. Upper showstranscription activity of WT Flag-mBmal1(black bar) and Flag-mBmal1-Bm1 to
Flag-mBmal1-Bm30 (gray bars). Baseline
luciferase activity is indicated by theopen bar. Each bar indicates the mean ofthree independent samples, and error
bars indicate the SEM. Lower shows the
positions of the 19-aa insertions.

Fig. 2. Dominant negative effect of Cterminally truncated BMAL1. (A) Schematic
representation of the BMAL1 protein (including the positions of 19-aa insertions in
Flag-BMAL1-Bm29 and -Bm30) and threemutant proteins lacking the C-terminal region as a result of the introduction of stopcodons. (B) NIH 3T3 cell-based promoter
assay for CLOCK兾BMAL1 transcriptional activity using the 2xE-box-pGL3 reporter construct, Flag-Clock, and WT and mutant
Flag-Bmal1 constructs (as indicated). Relative luciferase activities are calculated as aseveralfold induction from the basal expression level. Each bar indicates the meanof three independent samples, and error
bars indicate the SEM. (C) Same as B exceptthat WT Flag-Bmal1 is coexpressed with FlagBmal1(E447X), Flag-Bmal1(L554X), or FlagBmal1(I584X) constructs (amounts as indicated). (D) Effect of overexpression of WT
Flag-mBmal1 and Flag-mBmal1(L554X) on
mPer2:Luc rhythms in dexamethasone-synchronized Rat-1 cells. Each line represents
one dish, and all dishes presented were simultaneously analyzed by using the same
photomultiplier. (E) Effect of overexpressionof Flag-mBmal1(L554X) on mBmal1:Luc
rhythms in dexamethasone-synchronizedRat-1 cells.

obtained when Flag-BMAL1(L554X) was replaced by FlagBMAL1(E447X) or Flag-BMAL1(I584X) (data not shown).
Taken together, these data indicate that the C-terminal 43 aa of
BMAL1 protein contain a domain that is essential for mammalian
circadian oscillator performance and that, consistent with recent
reports that show interaction of p300兾CREB-binding protein coactivators by means of the C-terminal region of BMAL1 (19–21),
this domain must be involved in transcription activation.
Next, we investigated whether the impaired transcription activation properties of the C-terminally truncated BMAL1 proteinsoriginated from an inability to physically interact with CLOCK orfrom improper subcellular localization of the heterodimer. In acoimmunoprecipitation experiment, Flag-tagged mutant BMAL1
proteins pulled down cyan fluorescent protein (CFP)-tagged
CLOCK equally as well as WT Flag-tagged BMAL1 (Fig. 3A),
indicating that the C terminus is not involved in CLOCK兾BMAL1
heterodimerization. Furthermore, transient COS7 cell expression
studies revealed nuclear localization of Flag-BMAL1(E447X),
Flag-BMAL1(L554X), and Flag-BMAL1(I584X) (Fig. 3B Upper),suggesting that the C terminus neither contains nuclear localization
sequences nor facilitates nuclear localization of BMAL1 by meansof cotransport with other proteins. Transiently expressed CLOCK
displays a nucleocytoplasmic distribution and becomes completely
nuclear after coexpression with BMAL1 (11). In line with theirretained ability to bind CLOCK, the truncated BMAL1 proteinscaused complete nuclear accumulation of CLOCK (Fig. 3B Lower).
Taken together, these results indicate that nuclear accumulation of
BMAL1, as well as its interaction with CLOCK, does not involve
the BMAL1 C terminus and that, as a consequence, defective
CLOCK binding and improper nuclear localization are not responsible for the dominant negative behavior of the truncated BMAL1
proteins in the assays reported above.
Other proteins known to interact in vivo with CLOCK and
BMAL1 are mPER2 and mCRY1 proteins. These interactions, aswell as mPER and mCRY association, are believed to be important
10076 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0601416103

for proper performance of the circadian feedback loops (22).
Therefore, we investigated the physical interactions of mutant
BMAL1 proteins with mPER2 and mCRY1. Coimmunoprecipitation experiments revealed that mPER2 protein physically associates with Flag-BMAL1(E447X) and Flag-BMAL1(L554X) and,accordingly, does not require the C-terminal region of BMAL1(Fig. 3C). Instead, the PER2-binding site of BMAL1 must reside in
the N-terminal half of BMAL1, including the PAS domain. A
different situation is encountered for mCRY1, where antihemagglutinin (HA) antibodies hardly pull down WT FlagBMAL1 or Flag-CLOCK from lysates containing either coexpressed HA-mCRY1 and Flag-BMAL1 or HA-mCRY1 and FlagCLOCK. However, coexpression of HA-mCRY1 with WT FlagBMAL1 and Flag-CLOCK resulted in distinct coprecipitation of
the latter two proteins with HA-mCRY1 (Fig. 3D). Importantly,
when WT Flag-BMAL1 was replaced by Flag-BMAL1(L554X),
coimmunoprecipitation of (truncated) BMAL1 and CLOCK with
HA-mCRY1 was dramatically reduced (Fig. 3E). Similarly, insertion mutants Bm29 and Bm30 hardly interacted with HA-mCRY1
in the presence of CLOCK protein (Fig. 8, which is published as
supporting information on the PNAS web site). In line with arecently published study by Sato et al. (23), these results strongly
indicate that the C-terminal region of BMAL1 is critical for thebinding of mCRY1 to the CLOCK兾BMAL1 complex.
These findings prompted us to speculate that the C-terminal
region of BMAL1 is the interface for activation as well as for(mCRY-mediated) suppression of E-box gene transcription. To test
the above concept, we generated two additional deletion BMAL1
mutants named Flag-BMAL1(E608X) and Flag-BMAL1(F619X),
which lack 19 and 8 aa of the C terminus, respectively (Figs. 4A and
6B). Interestingly, in a transcription activation assay using 2xE-boxdriven luciferase reporter, transcriptional activity was gradually
recovered by these mutant proteins as compared with FlagBMAL1(L554X) (Fig. 4B). Particularly, Flag-BMAL1(F619X)兾
CLOCK heterodimers exhibited transactivation activity up to
Kiyohara et al.

Fig. 3. Role of the C terminus of BMAL1
in the binding of other clock proteins. (A)Immunoprecipitation (IP) studies showing physical interaction of Flag-mCLOCK
with WT mBMAL1 or mutant FlagBMAL1(E447X), Flag-BMAL1(L554X), andFlag-BMAL1(I584X). Proteins were expressedin COS7 cells, precipitated from cell lysates with
anti-FlagM2monoclonalantibodyconjugatedto agarose beads, and analyzed on Western
blots by using anti-CFP and anti-Flag antibodies. The Western blot of the total lysate served
as a control. (B) Immunofluorescence imagesof COS7 cells expressing CFP-clock and YFPBMAL1(E447X), YFP-BMAL1(L554X), or YFPBMAL1(I584X). Upper shows YFP-BMAL1 mutantproteins,andLowershowsCFP-CLOCK.(C)Immunoprecipitation studies showing physicalinteraction of mPER2 with WT Flag-BMAL1
protein or Flag-BMAL1(E447X) and FlagBMAL1(L554X). Procedures were as describedin A except that anti-mPER2 antibodies wereused to detect mPER2. (D) Immunoprecipitation study showing physical interaction of the
Flag-BMAL1兾Flag-CLOCK complex with HAmCRY1. Procedures were as described in A
except that anti-HA antibodies were used toprecipitate and detect mCRY1. (E) Immunoprecipitation study showing impaired physical interactionbetweenHA-mCRY1andFlag-BMAL1(L554X)兾Flag-CLOCK. Procedures were as
described D.

Kiyohara et al.

mentary data are in complete agreement with the idea that the
C-terminal region of BMAL1 acts as a sensor domain for CRYdependent transcriptional repression and thus has an essential rolein cyclic regulation of transcriptional activity.
Concluding Remarks
We have shown here that the C-terminal 43 aa of BMAL1 containa domain that is required for transcription activation as well as forinhibition of this activity by the mCRY1 protein. Deletion of thisdomain does not abolish mPER2 binding, which suggests thattranscription suppression by mCRY and mPER proteins in thenegative limb of the core oscillator uses different interfaces.
Whereas constitutive high expression of BMAL1 does not perturb the circadian core oscillator (as shown in this study and in ref.
12), deletion of the mCRY1-binding domain in a transcription
activation-proficient BMAL1 protein abolishes circadian oscillations. Apparently, periodic inhibition of transactivation capacity of
BMAL1 by mCRY1, rather than cyclic expression of BMAL1, ismandatory for preservation of circadian oscillations. The dual roleof the BMAL1 C terminus suggests the presence of a ‘‘switch’’mechanism that allows the BMAL1兾CLOCK heterodimer to control the balance between activation of transcription by coactivator
function, such as p300兾CREB-binding protein (‘‘on’’ mode), andrepression by mCRY1 (‘‘off’’ mode) (Fig. 4E and ref. 23). Because
the C terminus contains (putative) casein kinase I␧ (amino acids
517–526) and mitogen-activated protein kinase (amino acids 525–
530, 531–535, and 596–603) phosphorylation sites (24), this periodic on兾off status might be achieved through cyclic phosphorylation, controlling transcription activation potential (25) and mCRY1
binding. Although constitutive high overexpression of mCRY1 andmPER2 results in severe impairment of cellular circadian clock
oscillations, constitutive low to moderate levels of both proteins in
Rat-1 or NIH 3T3 cells still allows the clock to oscillate with normal
PNAS 兩 June 27, 2006 兩 vol. 103 兩 no. 26 兩 10077

NEUROSCIENCE⬇70% of WT Flag-BMAL1兾CLOCK activity, which was used as acontrol (Fig. 4B). Immunoprecipitation studies showed that interaction of both Flag-BMAL1(F619X)兾CLOCK and FlagBMAL1(E608X)兾CLOCK complexes with HA-mCRY1 were still
extremely weak (Fig. 4C). Thus, Flag-BMAL1(F619X) appeared tomaintain transcription activation capacity, although it lost thecapability to bind and thus be inhibited by mCRY1. To test theconsequence of this mutation for circadian core oscillator performance, we overexpressed Flag-BMAL1(L554X), FlagBMAL1(E608X), or Flag-BMAL1(F619X) with either themPer2:Luc or mBmal1:Luc reporter in Rat-1 cells. Each of thetruncated BMAL1 proteins severely impaired oscillations, as wasevident from the extremely low amplitudes of bioluminescencerhythms (Fig. 4D). Importantly, as could be predicted for a dominant negative form of BMAL1 that lacks transcription activation
potential, we noticed an obvious correlation between the level of(flattened) mPer2:Luc expression (Fig. 4D Upper) and transcription
activation capacity of the mutant BMAL1 protein (Fig. 4B). In linewith the level of E-box gene expression (including the Bmal1
transcription repressor Rev-Erb␣ and visualized by the mPer2:Luc
reporter), an inverse correlation is seen between Bmal1:Luc expression and transcription activation capacity of the mutant
BMAL1 protein (Fig. 4D Lower). Most important, however, is ourobservation that transactivation of E-box genes by BMAL1 apparently is not sufficient to generate a robust circadian oscillation of themolecular clock as long as the protein cannot be inhibited by
mCRY1.
Recently, Sato et al. (23) performed a large-scale screening study
with panels of mutant BMAL1 and CLOCK proteins to identify
amino acids that are critical for the biological function of these
proteins. Amino acid substitutions at position 611 or 612 of the
BMAL1 protein were shown to result in a reduced sensitivity of theprotein to CRY1-mediated transcription repression. Our comple-

Fig. 4. Detailed analysis of the BMAL1 C
terminus for circadian clock function. (A)Schematic representation of two additional
C-terminally truncated BMAL1 mutant proteins (blue) generated by introduction ofstop codons. (B) NIH 3T3 cell-based promoter
assay for CLOCK兾BMAL1 transcriptional activity using the 2xE-box-pGL3 reporter construct, Flag-mClock, and WT and mutant
Flag-Bmal1 constructs (as indicated). Relative
luciferase activities are calculated as a severalfold induction from the basal expression
level. Each bar indicates the mean of three
independent samples, and error bars indicate the SEM. (C) Immunoprecipitation study
showing lack of physical interaction between HA-mCRY1 and Flag-BMAL1(F619X)兾
Flag-CLOCK. Procedures were as described in
the legend of Fig. 3D. (D) Effect of overexpression of WT Flag-BMAL1(WT) (black),
Flag-BMAL1(L554X) (red), Flag-BMAL1(E608X) (blue), and Flag-BMAL1(F619X) (yellow) on mPer2:Luc (Upper) and mBmal1:Luc(Lower) reporter-driven bioluminescencerhythms in dexamethasone-synchronizedRat-1 cells. (E) Schematic model depicting
protein–protein interactions for balanced
regulation of transcription activation by coactivators and transcription suppression by
the mPER2 and兾or mCRY1 components of
the negative limb of the mammalian circadian core oscillator.

period length (16, 17). Although research on the function andkinetics of cyclic posttranslational modifications of positive andnegative factors is far from complete, it is tempting to speculate
that, as with the cyanobacterial oscillator (26, 27), cyclic posttranslational modification, rather than cyclic gene expression, is the
major requisite for mammalian core oscillator function and thattranscriptional regulation of clock genes only serves to donate
robustness to the system.
Materials and Methods
Plasmids and Cloning. Flag-tagged mBmal1 and mClock pcDNA3.1
expression constructs were kindly provided by T. Todo (Kyoto
University, Kyoto). For generation of Flag-BMAL1(E447X),(L554X), (I584X), (E608X), and (F619X) constructs, we mutagenized the Flag-mBmal1 cDNA by using the QuikChange Mutagenesis Kit (Stratagene) and the following primer pairs: E447X
forward, 5⬘-GTT TTA GCC AAT GTC CTG TAA GGC GGGGAC CC-3⬘; E447X reverse, 5⬘-GG GTC CCC GCC TTA CAG
GAC ATT GGC TAA AAC-3⬘; L554X forward, 5⬘-CT TCC ACTGGA CTA T AA CCA GGG CAG GCT C-3⬘; L554X reverse, 5⬘-GAGC CTG CCC TGG TTA TAG TCC AGT GGA AG-3⬘; L584X
forward, 5⬘-GGC ATC GAT ATG TAA GAT AAC GAC CAA
GG-3⬘; I584X reverse, 5⬘-CCT TGG TCG TTA TCT TAC ATA
TCG ATG CC-3⬘; E608X forward, 5⬘-ATC ATG AGC CTC TTGTAA GCA GAT GCG GGG-3⬘; E608X reverse, 5⬘-CCC CGC
ATC TGC TTA CAA GAG GCT CAT GAT-3⬘; F619X forward,
5⬘-GGC CCC GTT GAC TAA AGT GAC TTG CCA TGG-3⬘;
F619X reverse, 5⬘-CCA TGG CAA GTC ACT TTA GTC AAC
GGG GCC-3⬘ (italicized bases indicate substituted nucleotides).
Mutated Flag-mBmal1 constructs were sequenced to confirm the10078 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0601416103

presence of the specific mutations and that no other unexpected
mutations were introduced.
The mPer2 promoter fragment (423 bp) was obtained from
C57BL兾6 mouse genomic DNA by PCR using forward primer
5⬘-AA GGT ACC TCT GCC GGC TGT GAG TTG CGC AG-3⬘
and reverse primer 5⬘-TT CTC GAG ACC GCT AGT CCC AGT
AGC GCC G-3⬘, containing the KpnI and XhoI sites, respectively.
Similarly, a mBmal1 promoter fragment (530 bp) was amplified byusing primers reported in ref. 6. These promoter regions were thencloned into pGL4.11 vector (Promega).
To generate CFP-mClock and yellow fluorescent protein (YFP)mBmal1 expression vectors, the ORFs of mClock and mBmal1 weredigested with BamHI兾XhoI from the respective Flag-tagged constructs and ligated into BglII兾SalI-digested pECFP-C1 andpEYFP-C1 (BD Clontech).
Cell Culture. Rat-1 fibroblast (Health Science Research Resources

Bank, Osaka), NIH 3T3, and COS7 (kindly donated by T. Ishitani,
Nagoya University) cells were cultured in DMEM with 10% FBS
and penicillin-streptomycin.
Transposon-Based Bmal1 Mutant Library. Random insertions of 19 aa

were introduced in the BMAL1 protein by using the EZ-Tn5
In-Frame Linker Insertion Kit (Epicentre Technologies, Madison,
WI). Briefly, a kanamycin resistance gene containing transposon
was incubated with EZ-Tn5 transposase and Flag-mBal1
pcDNA3.1. Transposon-containing Flag-mBmal1 colonies wereselected on kanamycin-containing LB plates and harvested in LB
medium. After plasmid extraction using the QIAfilter Plasmid Maxikit (Qiagen, Valencia, CA), DNA was digested with NotI restriction
Kiyohara et al.

Real-Time Circadian Rhythm Monitoring. The mechanics of thebioluminescence detection system used to analyze the circadianrhythm are described in ref. 28. Rat-1 cells were cultured in 10%
FBS and penicillin-streptomycin-containing medium. Cells wereplated in 35-mm dishes (2 ⫻ 105 cells per dish) or 24-well plates (5 ⫻104 cells per well). Cells were cotransfected with the Bmal1 or
mPer2 luciferase reporter construct (250 or 200 ng for 35-mm dishes
and 24-well plates, respectively), Flag-mBmal1 mutant construct(750 or 200 ng for 35-mm dishes and 24-well plates, respectively),and FuGENE 6 transcription reagent (3 or 0.8 ␮l for 35-mm dishes
and 24-well plates, respectively, Roche, Indianapolis, IN). Afterovernight culture, medium was changed to luciferine (100 nM) andHepes (15 mM) containing DMEM without phenol-red. After
additional overnight culture, cells were synchronized by treatment
with 100 nM dexamethasone and set on the turntable of a homemade real-time monitoring machine (28).
Western Blot Analysis and Immunoprecipitation. Cells were washedthree times with ice-cold PBS and harvested in 50 ␮l of SDS sample
buffer (125 mM Tris䡠HCl, pH 6.8兾2% SDS兾10% glycerol兾0.05%
bromophenol blue兾1 ␮M PMSF兾50 ␮M NaF兾100 ␮M NaVO3兾40
␮M DTT). As primary antibodies, we used rabbit anti-GFP polyclonal antibody (1:2,000, MBL, Nagoya, Japan), rabbit anti-mPER2
polyclonal antibody (1:1,000, Alpha Diagnostic International, San
Antonio, TX), and anti-FLAG M2 mouse monoclonal antibody(1:500, Sigma). As secondary antibody, we used horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies(1:2,000, Jackson ImmunoResearch). Chemiluminescence detection was performed by using ECL Western Blotting Luminol
Reagent (Amersham Pharmacia).
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Tris䡠HCl, pH 7.5兾150 mM NaCl兾1% Nonidet P-40兾10% glycerol兾1
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Cells were analyzed with an Axiovert 200M fluorescent microscope(Zeiss).
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Nagoya University Graduate School of Science, and the Netherlands
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PNAS 兩 June 27, 2006 兩 vol. 103 兩 no. 26 兩 10079

NEUROSCIENCE

enzyme to remove the kanamycin resistance marker, self-ligated,
and transformed in Escherichia coli. The mutant Flag-mBmal1
constructs were sequenced by the following primers: BML1 forward, 5⬘-GAC CAG AGA ATG GAC ATT T-3⬘; BML1 reverse,
5⬘-CCT TGC ATT CTT GAT CCT TC-3⬘; BML2 forward, 5⬘-GTA
CCA ACA TGC AAT GCA ATG-3⬘; BML3 forward, 5⬘-CTG
CAT CCA AAA GAT ATT GCC-3⬘; BML4 forward, 5⬘-CTG
GAC GAA GAC AAT GAG CC-3⬘; BML5 forward, 5⬘-C GCAGAA TGT CAC AGG CAA G-3⬘; BML6 forward, 5⬘-GGA GCA
GGA AAA ATA GGT CG-3⬘; BML7 forward, 5⬘-CC ACT GGA
CTA TTA CCA GGG-3⬘.