Published online 5 February 2008

Nucleic Acids Research, 2008, Vol. 36, No. 4 e23
doi:10.1093/nar/gkn018

Detection of a circadian enhancer inthe mDbp promoter using prokaryotic
transposon vector-based strategy
Yota B. Kiyohara1,2, Keigo Nishii2, Maki Ukai-Tadenuma3, Hiroki R. Ueda3,
Yasuo Uchiyama1 and Kazuhiro Yagita1,2,*
1

Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Yamadaoka 2-2,
Suita, Osaka 565-0871, 2COE Unit of Circadian System, Nagoya University Graduate School of Science, Furo-cho,
Chikusa-ku, Nagoya 464-8602 and 3Laboratory for Systems Biology, Center for Developmental Biology, RIKEN,
2-2-3 Minatojima-minaminachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

Received October 18, 2007; Revised December 27, 2007; Accepted January 12, 2008

ABSTRACTINTRODUCTION

In mammals, the expression of 5–10% of genes
occurs with circadian fluctuation in various organs
and tissues. This cyclic transcription is thought tobe directly or indirectly regulated through circadiantranscriptional/translational feedback loops consisting of a set of clock genes. Among the clock
genes in mammals, expression of the Dbp mRNA
robustly oscillates both in vivo and in culture cells.
Here, we present circadian enhancer detection
strategy using prokaryotic transposon system. The
mDbp promoter drives reporter gene expression
in robust circadian cycles in rat-1 fibroblasts. To
identify the circadian enhancer generating thisrobust rhythm, we developed a prokaryotic transposon-based enhancer detecting vector for in vitrotransposition. Using this system, we identified astrong circadian enhancer region containing the
CATGTG sequence in the 5’ flanking region of
the mDbp gene; this enhancer region is critical forthe ability of the mDbp promoter to drive robust
oscillation in living cells. This enhancer is classified
as a CANNTG type non-canonical E-box. Thesefindings strongly suggest that CANNTG-type noncanonical E-boxes may contribute, at least in part,to the regulation of robust circadian gene expression. Furthermore, these data may help explain thewider effects of the CLOCK/BMAL1 complex incontrol of clock output genes.

The mammalian circadian clock is a highly dynamic
system that causes 5–10% of genes to be expressed cyclically; this confers near 24-h rhythmicity on behavior,
physiology and metabolic processes and allows mammals
to adjust to the time of the day (1). The suprachiasmatic
nucleus (SCN) of the hypothalamus, which is the masterclock in mammals, is entrained by light to keep pace withthe day–night cycle autonomic nerve and humoral factors
coordinate circadian clocks in peripheral tissues (2,3).
Recent studies have also shown that peripheral tissues
contain their own self-sustainable circadian clock oscillators (4). Moreover, even cultured ﬁbroblasts contain an
active circadian clock, which strikingly resembles thecentral clock in the SCN (5–8).
Rhythmic transcription of central and peripheral clock
output genes is controlled by a molecular oscillator, consisting of interlocked positive and negative transcription/translation feedback loops of a set of clock genes. BMAL1(MOP3) and CLOCK are basic-helix-loop-helix (bHLH)
PAS transcription factors that dimerize and transactivate
Period (Per1 and Per2), Cryptochrome (Cry1 and Cry2),
and an orphan nuclear receptor, Rev-Erb, via CACGTG
E-box enhancer elements. The PER and CRY proteins act
as negative elements by inhibiting the CLOCK/BMAL1
heterodimer, whereas REV-ERBa regulates Bmal1 gene
expression via ROR responsive elements (RRE) (2,3,9).
Recent reports examining genome-wide gene expression
proﬁles in the SCN and various peripheral organs haveshown that hundreds of genes are expressed under circadian regulation systems (10–12). Curiously, most of the*To whom correspondence should be addressed. Tel: +81 6 6879 3124; Fax: +81 6 6879 3129; Email: kyagita@anat1.med.osaka-u.ac.jp
Present address:
Keigo Nishii, Kowa Company, Tokyo Laboratory, Noguchi-cho 2-17-43, Higashimurayama, Tokyo 189-0022, Japan.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
ß 2008 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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e23 Nucleic Acids Research, 2008, Vol. 36, No. 4

cyclically expressed genes diﬀer from organ to organ, whichsuggests that a much larger number of genes are potential
targets of circadian control (13). Thus, each peripheral
tissue or organ cyclically expresses a speciﬁc set of genes
governing tissue-speciﬁc functions, such as cholesterol
metabolism-related genes in the liver (10). Although it isunquestionably important to study the transcriptional
regulatory network connecting the circadian clock andphysiology, the ﬁndings of micro-array studies are often
too indirect to uncover the distinct regulatory mechanisms
and pathways regulating circadian gene expression.
To further elucidate the circadian transcriptional regulatory network, we here establish an eﬀective enhancerdetecting method to identify enhancers eliciting circadiangene expression in mammalian cells using an in vitro
random transposon system and a high-throughput
luciferase-based circadian real-time monitoring system.
The expression of Dbp in the SCN, liver and cultured
ﬁbroblast cell lines oscillates very robustly(14,15). In thisstudy, we ﬁrst showed that a promoter region of the mDbp
gene, which contains 1.4 kb upstream from the transcriptional start site, generates robust circadian oscillationof luciferase bioluminescence in rat-1 and NIH3T3 ﬁbroblast cells even though no functional circadian enhancer
has been reported. Next, we used an enhancer-trap
approach based on an in vitro transposon system to eﬃciently discover unknown circadian enhancers. Using thissystem, we have identiﬁed a circadian enhancer contributing for cyclic transcriptional regulation in the 50 ﬂanking
region of the mDbp gene. This identiﬁed enhancer isclassiﬁed as a non-canonical E-box (CANNTG), which
is identical with recently reported as a new circadianenhancer of mDbp promoter (16). Moreover, our observations suggest that CLOCK/BMAL1-mediated transcriptional regulation may inﬂuence a much larger set of genes
than previously thought; these eﬀects are exerted via not
only CACGTG E-boxes, but also non-canonical
CANNTG E-boxes.
MATERIALS AND METHODS
Cell culture
Rat-1 ﬁbroblast cells (HSRRB, Osaka, Japan), NIH3T3
ﬁbroblast cells, and COS7 cells (kindly donated by Dr
Ishitani, Nagoya University) were cultured in DMEM
with 10% FBS and penicillin–streptomycin. For real-time
analysis, the medium was changed to phenol-red-free
DMEM buﬀered with HEPES at 24 h after transfection.
Construction of transposons for the enhancer trap
To construct transposons for the enhancer trap, weused the EZ::TnTM pMODTM-2<MCS> Transposon
Construction Vector (EPICENTRE Biotechnologies)(17). We used the PCR-based overlap extension method
to generate fragments carrying the minimal CMV promoter and luciferase reporter gene. First, upper fragments
carrying the minimal CMV promoter were ampliﬁed using
pCMVupKpnI (50 -CCGGGGTACCTAGGCGTGTACGGTGGGAG GCCTATATAAGC-30 ; the KpnI site isunderlined) and pCMVlowOver (50 -CCATGGTGGCAG

GCTGGATCGGTCCCGGTGTCTTCTATGG-30 ; theoverlapping section is underlined) as primers and thepTRE2 Vector (CLONTECH) as a template. Lower fragments carrying the luciferase reporter gene were ampliﬁedusing pLUCupOver (50 -GGACCGATCCAGCCTGCCA
CCATGGAAGATGCCAAAAAC-30 ; the overlapping
section is underlined) and pLUClowBamHI (50 -TTA
TTTTACTTTGGATCCTACCACATTTGTAGAGG
GC-30 ; the BamHI site is underlined) as primers andthe pGL4.11 Luciferase Reporter Vector (Promega) as
a template. The upper and lower DNA fragments wereused as templates for a second PCR reaction using
pCMVupKpnI and pLUClowBamHI as primers. The
resulting 2.2 kb PCR product was digested with KpnI and
BamHI and ligated to KpnI- and BamHI-digested
pMODTM-2<MCS> Transposon Construction Vector.
The kanamycin resistance gene was ampliﬁed by PCR
using pKmupHindIII (50 -TTCGGAAGCTTC AACAAAGCCACGTTGTGTCTC-30 ; the HindIII site is underlined) and pKmlowHindIII (50 -AGCGTAAGCTTCTGC
CAGTGTTACAACCAATTAACC-30 ; the HindIII site
is underlined) as primers and the EZ::TnTM <KAN-2>
Transposon (EPICENTRE Biotechnologies) as a template. The resulting 1.0 kb PCR product was digested
with HindIII and ligated to the HindIII-digested vector
described earlier to yield the transposon vector forthe enhancer trap. This vector was sequenced to conﬁrm
that no unexpected mutations were introduced. The
larger PshAI fragment from the transposon vector forthe enhancer trap was isolated, puriﬁed by agarose gelelectrophoresis, and used as the transposon for theenhancer trap.
Enhancer trap
The transposon for the enhancer trap was integrated into
a vector carrying the DBP promoter using EZ::TnTM
pMODTM Transposase (EPICENTRE Biotechnologies)
according to the manufacture’s instructions. One microliter of transposed sample was transformed into
Escherichia coli. DH5 alpha (Toyobo, Osaka, Japan),and plasmid DNA was prepared from 48 independentcolonies. One microgram of each plasmid was transfected
into rat-1 cells and bioluminescence was monitored.
Plasmids were sequenced to determine the insertion sitesof the transposon.
Plasmids
The Dbp promoter was obtained by PCR from C57BL/6
mouse genomic DNA using pDbpupSacI (50 -AACTCGAGCTCCACGTCCTGATAGTCTGCAC-30 ; the SacIsite is underlined) and pDbplowBglII (50 -GCTAGATCTGTACCAAGTGGGCGAGTCTC-30 ; the BglII site isunderlined) as primers. The resulting 1.4 kb PCR productwas subcloned into the pGEMT-EAZY vector (Promega)to yield the Dbp promoter vector. In the same way, themPer2 and mBmal1 promoters were obtained using theprimer sets pmPer2upKpnI (50 -AAGGTACCTCTG
CCGGCTGTGAGTTGCGCAG-30 ; the KpnI site isunderlined) and pmPer2lowXhoI (50 -TTCTCGAGACCGCTAGTCCCAGTAGCGCCG-30 ; the XhoI site

PAGE 3 OF 10
is underlined), or pmBmal1upSacI (50 -GATCGAGCTCGCAGAGTCC GCAACGCAGTGG-30 ; the SacI site isunderlined) and pmBmal1lowXhoI (50 -GATCCTCGAGCGCACCCGCACTCGGATCCCG-30 ; the XhoI site isunderlined). Each promoter was then ligated into thepGL4.11 [luc2P] Vector (Promega). To generate a mutated
Dbp promoter reporter vector, a QuikChange site-directedmutagenesis kit (Stratagene) was used along with theprimers pDbpEbox-mut-up (50 -GCACGCGCAAAGC
GGTACCC TTCCCCCTTC-30 ; substituted nucleotides
are underlined) and pDbpEbox-mut-low (50 -GAAGGGGGAAGGTACCGCTTTGCGCGTGC-30 ; substituted
nucleotides are underlined). The mutated Dbp promoter
reporter vector was sequenced to conﬁrm the presence ofthe speciﬁc mutations and the absence of any unexpected
mutation. The Dbp reporter vector was digested with
KpnI and religated to generate the Dbp(-462):Luc vector.
The mutated Dbp reporter vector was digested with KpnI
and religated to generate the Dbp(-410):Luc vector.
DNA fragments carrying the non-canonical E-box fromthe Dbp promoter (416) were obtained by PCR using
pDbpNEKpnIup (50 -AAGAGGTACCCCTCTTGCCCC
AC-30 ; the KpnI site is underlined) and pDbpNEKpnIlow(50 -ATATGGTACCAATGGGAGGAGGCGCTG-30 ;the KpnI site is underlined) as primers and C57BL/6
mouse genomic DNA as template. The resulting 0.1 kb
PCR product was digested with KpnI and subcloned into
KpnI digested transposon vector for the enhancer trap,
described earlier. The reporter vector carrying the noncanonical E-box from the Dbp promoter was sequenced to
conﬁrm the presence of the expected DNA fragment andthe absence of any unexpected mutations.
Real-time monitoring of bioluminescence
The 2  105 rat-1 cells were plated in 35 mm dishes or
5  104 cells were plated in 24-well plates. For the 35 mm
dishes, rat-1 cells were transfected with 1 mg of eachconstruct using 3 ml of FUGENE 6 transfection reagent(Roche). For 24-well plate assays, cells were transfectedwith 300 ng of each construct and 0.8 ml of FUGENE 6.
After overnight culture, medium was changed to DMEM
without phenol-red and containing 100 nM luciferin
and 15 mM HEPES. After additional overnight culture,cells were reset with 100 nM dexamethasone andbioluminescence was automatically monitored using aphotomultiplier-tube-based bioluminescence monitoring
system (18).
Transcriptional assays
Luciferase reporter gene assays were performed in
NIH3T3 cells. Cells (2  105) were seeded on 6-wellplates and transfected the following day. In this study,
we used the Dual Luciferase reporter assay system(Promega). Cells in each well were transfected with 50 ng
of pGL4.11 vector containing the indicated DNA fragments as described as well as the indicated amounts ofexpression plasmids for ﬂag-tagged mClock, mBmal1 andHA-mCry1pcDNA3.1. The total amount of DNA per well
was adjusted to 1 mg by adding pcDNA3 vector as carrier.

Nucleic Acids Research, 2008, Vol. 36, No. 4 e23

Forty-eight hours after transfection, cells were harvested
and luciferase activity was determined by luminometer.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as described (19). Brieﬂy,
COS7 cells expressing ﬂag-tagged mCLOCK andmBMAL1 were washed with 5 ml of Tris-buﬀered saline(TBS) and pelleted by centrifugation at 1500  g for 5 min.
The pellet was resuspended in 1 ml of TBS, transferred
into a microtube and centrifuged for 5 s. The supernatant
was removed and the pellet was dissolved in 400 ml ofchilled Buﬀer A (10 mM HEPES pH 7.9, 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM
phenylmethylsulfonyl ﬂuoride). The cells were allowed toswell on ice for 15 min, after which 25 ml of 10% NP-40
was added. The tubes were vigorously vortexed for 10 s
and centrifuged for 30 s at 15 000 rpm. The nuclear pellet
was dissolved in 50 ml ice-cold Buﬀer C (20 mM HEPES
ph 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT and 1 mM PMSF). The nuclear extract was centrifuged at 12 000 rpm for 5 min, and 2 ml of the supernatant
was used for each reaction.
The interaction of the BMAL1 and CLOCK proteins
expressed in COS7 nuclei with the DNA fragment
containing the non-canonical E-box enhancer elementof the mDBP promoter was analyzed by EMSAs using
the LightShift Chemiluminescent EMSA Kit (Pierce,
Rockford, IL). The 30 bp DNA duplex used in the
EMSA experiments consisted of the annealed pairof oligonucleotides E-box F (50 -GCACGCGCAAAGCC
ATGTGCTTCCCCCTTC-30 ; the non-canonical E-box isunderlined), and E-box R (50 -GAAGGGGGAAGCAC
ATGGCTTTGCGCGTGC-30 ; the non-canonical E-box
is underlined). These two oligonucleotides were biotinylated at their 50 -ends to allow chemiluminescent detection.
DNA–protein binding reactions were carried out at room
temperature and consisted of nuclear extracts mixed witha reaction buﬀer composed of 10 mM Tris–HCl (pH 7.5),
50 mM KCl, 1 mM DTT, 50 ng/ml poly (dI–dC), 2.5%
glycerol, 10 mM EDTA (pH 8.0) and 0.05% NP-40.
20 fmol of biotin-labeled E-box DNA was then added to
the reaction mixture and incubated for 25 min at room
temperature. For competition experiments, a 50-foldmolar excess of unlabeled duplex DNA fragments corresponding to the E-box site (described earlier) was added to
the reaction mixture before addition of the labeledoligonucleotides. For mutant E-box probe, the annealed
pair of oligonucleotides E-box mut F (50 -GCACGCGCAAAGCGGTACCCTTCCCCCTTC-30 ; substituted
nucleotides are underlined), and E-box mut R (50 -GAA
GGGGGAAGGTACCGCTTTGCGCGTGC-30 ; substituted nucleotides are underlined) were used. Reaction
mixtures were analyzed by non-denaturing 6% polyacrylamide gel electrophoresis and transferred to Biodyne B
Nylon Membranes (PALL). The biotin-labeled DNA
probes were detected by chemiluminescence using theChemiluminescent Nucleic Acid Detection Module Kit(Pierce). The membranes were exposed to X-ray ﬁlm for
1–3 min and developed according to the manufacturer’s
instruction.

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e23 Nucleic Acids Research, 2008, Vol. 36, No. 4

Chromatin immunoprecipitation (ChIP) assay and Q-PCR
NIH3T3 cells (American Type Culture Collection) weregrown in DMEM (Invitrogen) supplemented with 10%
FBS (JRH Biosciences) and antibiotics (25 U/ml penicillin,
25 mg/ml streptomycin; Invitrogen). Cells were plated at
6  105 cells in 10 cm dishes 24 h before transfection, andtransfected with two plasmids (6 mg each of FLAG tagged
BMAL1 and FLAG tagged CLOCK expression vector)
with FuGene6 transfection reagent (Roche) according to
the manufacturer’s instructions. Cells were harvested 48 h
after transfection.
ChIP assays with these transfected NIH3T3 cells were
performed as previously described (20) with the following
modiﬁcations. In this report, we used Chromatin Immunoprecipitation Assay Kits (Upstate Biotechnology), antiFlag M2 antibody (Sigma) and anti-V5 antibody (Sigma).
Protein G-Sepharose (Amersham) was used for precipitation of antibody/protein immune complexes. The resulting
precipitated DNA was quantiﬁed by Q-PCR, which was
performed according to the previously described method(21). Absolute DNA abundance was calculated using thestandard curve obtained from NIH3T3 cell’s genomic
DNA. Tbp-50 region were quantiﬁed and used as theinternal control.
Primer sequences for Q-PCR of ChIP products
P(Dbp)-forward, 50 -TGAACACTCGGCTCCTTTAAG
AG-30 ; P(Dbp)-reverse, 50 -GCGTGCTATCGGATG
CG-30 ; Act-50 -forward, 50 -CCAAGAGGCTCCCTCC
ACA-30 ; Act-50 -reverse, 50 -GTGCAAGGGAGAAAGAT
GCC-30 ; Tbp-50 -forward, 50 -GTTTGGCTAGGTTTCTGCGG-30 ; Tbp-50 -reverse, 50 -GAAAGGACCGTTAC
CTGGGC-30 .
RESULTS
Robust circadian oscillation driven by the mDbp promoter
The Dbp mRNA is expressed with a robust circadianrhythm in the SCN, peripheral tissues and cultured cells(5,22). Recent reports have suggested that a CACGTG
E-box in the second intron of the Dbp gene is likely to be
responsible for this cyclic mRNA expression (14,15).
Although the reported intronic E-box was functional in
response to CLOCK/BMAL1-mediated transcriptional
activation, it is still unknown whether this intronic
E-box is enough to generate robust circadian oscillation.
We ﬁrst focused on the 50 ﬂanking region of the mDbp
gene because many clock genes, including mPers andBmal1, have circadian enhancers near their transcription
start sites (12,23,24). We cloned the mDbp promoter
region (1332  +83) and fused it upstream of a luciferase reporter gene in the pGL4.11 vector (Promega). This
vector was then transfected into rat-1 ﬁbroblast cells, andbioluminescence was monitored. Surprisingly, the mDbp
promoter drove luciferase expression with a robust
circadian rhythm; the amplitude of this rhythm waslarger than that of the oscillation driven by the mPer2
or mBmal1 promoters (Figure 1). These results indicatethat the mDbp promoter region contains strong circadian

Figure 1. Robust circadian oscillation of mDbp:Luc-driven bioluminescence in rat-1 cells. Luciferase reporter constructs driven by the mPer2
promoter (mPer2:Luc, blue), the mBmal1 promoter (mBmal1:Luc,
yellow) and the mDbp promoter (mDbp:Luc, red) were transfected
into rat-1 ﬁbroblasts and synchronized by treatment with 100 nM
dexamethasone. Real-time bioluminescence monitoring showed that all
three promoters were able to drive robust circadian oscillation of thereporter gene. The phases of the mDbp:Luc and mPer2:Luc-driven
rhythms are quite similar, whereas the mBmal1:Luc driven rhythm is inalmost the opposite phase.

enhancer(s) that are capable of generating robust oscillation of expression.
Transposon-based enhancer detection forthe mDbp promoter
To determine the unknown circadian enhancer(s) in the1.4 kb mDbp promoter region, we developed anenhancer-detection system based on the Tn5 transposonfor in vitro tranposition. We constructed a transposoncarrying 120 bp of minimal CMV promoter fused to abioluminescence reporter gene and a kanamycin resistancegene (Figure 2A). We analyzed the CMV minimal
promoter driven bioluminescence of this trap vector
following transfection into rat-1 cells. As expected, thebioluminescence was detectable, constitutive, and showed
no circadian intensity changes during several days
(Figure 2B).
The enhancer-detecting transposon vector was randomly integrated into the plasmid carrying the mDbp
promoter in vitro, and each plasmid was sequenced todetermine the transposon insertion site. Eleven diﬀerent
clones with the transposon integrated into the Dbp
promoter region were obtained (Figure 2C). The arrows
in Figure 2C indicate the direction of reporter gene
expression. These plasmids were then transfected into
rat-1 ﬁbroblast cells and bioluminescence was monitored(Figure S1). Representative results, indicated as (a)–(e), areshown in Figure 2D. Interestingly, among these ﬁve
bioluminescence proﬁles, (a), (c) and (e) show apparent
circadian oscillation, whereas (b) and (d) show only faintbioluminescence ﬂuctuation or no apparent circadianchange (Figure 2D). These results suggest that the circadian enhancer(s) in the mDbp promoter may not be active
at long distances from the trap vector. More importantly,
these results indicate that the putative circadian enhancer islikely to be located between 700 bp (c) and 262 bp (a) of
the 50 ﬂanking region of this gene. In additon, there was no

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Nucleic Acids Research, 2008, Vol. 36, No. 4 e23

Figure 2. Transposon-based in vitro enhancer trap for the mDbp promoter. (A) Structure of the Tn5-based enhancer trap vector. Reduced half-life
ﬁreﬂy luciferase (Luc2P) obtained from the pGL4.11 vector (Promega) is used as a reporter driven by the CMV minimal promoter. ME indicates themosaic end sequence to transpose into the host DNA. (B) Real-time bioluminescence monitoring of rat-1 cells transfected with the enhancer trap
vector. Cells are synchronized by treatment with 100 nM dexamethasone. No rhythmic ﬂuctuation was observed over a period of 4 days. (C) The
enhancer trap vector was transposed into the 1.4 kb fragment of the mDbp promoter cloned into the pGEM T-Easy vector (Promega). After
Ampicillin and Kanamycin selection, 13 clones with integration of the trap vector into the mDbp promoter were obtained. Red arrows indicate thedirection of the luciferase gene in the trap vector. The position of integration is noted for each red arrow. Clones f and g showed integration withsome deletion. (D) Clones with trap vector integrations were transfected into rat-1 cells and analyzed by real-time bioluminescence monitoring.
Representative results from clones a to g are shown. Rat-1 cells transfected with clones a, c or e exhibited robust circadian oscillationof bioluminescence. In contrast, rat-1 cells transfected with clones b, d, f or g exhibited only faint oscillation.

strong circadian enhancer in downstream from the position of 335 bp, because only faint oscillation was seen in335 bp integrated one (Figure 2Db). Strikingly, one
transposed clone (f) in Figure 2C, lacking this region did
not show circadian ﬂuctuation of bioluminescence in rat-1
cells (Figure 2D). In addition, a similar transposition,clone (g), lacking 462 to +83 bp, also exhibited almost
no apparent circadian oscillation in bioluminescence compared to (a) or (c) (Figure 2D). This strongly suggests thatthe region between 462 and 335 bp contains the putative circadian enhancer responsible for the robust circadianoscillation of Dbp promoter driven bioluminescence.

e23 Nucleic Acids Research, 2008, Vol. 36, No. 4

PAGE 6 OF 10

The predicted circadian enhancer region in the mDbp
promoter contains a non-canonical E-box

Moreover, additional expression of mCry1 dramaticallyreduced the CLOCK/BMAL1-mediated transcriptional
activity (Figure 3B). These results are consistent with thereported features of canonical E-boxes, and suggest that
non-canonical E-box(es) in the mDbp promoter are likely
to be functional.
One CANNTG non-canonical E-box sequence was
located in the region predicted to contain the putative
circadian enhancer as shown (Figures 2 and 3A).
To determine whether this non-canonical E-box is theputative enhancer, we generated deletions of the mDbp
promoter named Dbp(462):luc and Dbp(410):luc(Figure 4A). Strikingly, transcriptional assays showedthat deletion of the region containing the non-canonicalE-box resulted in a loss of responsiveness to co-expression

Using the in vitro transposon-based enhancer-trap
method, we were able to predict a region that may containa strong circadian enhancer. Thus, the next step was to
identify the exact enhancer sequence responsible for mDbp
promoter driven circadian oscillation. Ripperger et al.
reported that CLOCK controls the circadian expression of
the mDbp gene in vivo (14). Moreover, our ﬁndings in thisstudy show that the phase of mDbp promoter driven
rhythm is similar to the rhythm driven by the mPer2
promoter and opposite from mBmal1 promoter-driven
rhythms (Figure 1). Several studies have indicated that
CLOCK/BMAL1 activate transcription via CACGTG
E-box enhancers existing in various mammalian clock
genes such as mPer1, mCry1, Rev-erb alpha and Dbp.
Recent observations have also shown that CLOCK/BMAL1 regulate not only CACGTG E-boxes, but also
CACGTT E-box like sequences (25,26). Furthermore, one
recent report suggested that a CANNTG non-canonicalE-box was able to regulate circadian expression ofprolactin (27). Although no CACGTG or CACGTT
E-boxes were seen, several CANNTG sequences exist in
the mDbp promoter region (Figure 3A). Thus, weinvestigated the possibility of CLOCK/BMAL1-mediatedregulation of the mDbp promoter region we cloned.
We performed transcriptional assays using the 1.4 kb
mDbp promoter-luciferase (Dbp:luc) reporter construct.
Co-transfection of ﬂag-tagged mClock and mBmal1 expression vectors with the Dbp:luc reporter resulted in signiﬁcant
up-regulation of luciferase activity compared to pcDNA3
empty vector co-transfection conditions (Figure 3B).

Figure 3. Transcriptional activation driven by the mDbp promoter isupregulated by CLOCK/BMAL1 co-expression. (A) Six CANNTG
non-canonical E-boxes are found within the 1.4 kb region of themDbp promoter that is able to drive robust circadian oscillation oftranscription. Each number indicates the position of a CANNTG
consensus sequence. The red-hashed box represents the location of aputative strong circadian enhancer as predicted by the enhancer-trap
assay. (B) Transcriptional assay using the mDbp:Luc reporter vector.
Co-expression of ﬂag-tagged mBmal1 and mClock results in a greaterthan 8-fold up-regulation of promoter activity. Additional expression of
HA-mCry1 dramatically suppresses mDbp promoter activity.

Figure 4. The predicted circadian enhancer region is essential
for robust circadian oscillation driven by the mDbp promoter.
(A) Deletions of the mDbp promoter: Dbp(462):Luc contains thewhole predicted enhancer region, Dbp(410):Luc contains only apart of the predicted enhancer region. (B) Transcriptional assay using
these reporter constructs. Both Dbp:Luc and Dbp(462):Luc arestrongly up-regulated (16-fold and 19-fold induction, respectively) by
co-expression of the CLOCK/BMAL1 proteins, whereas the
Dbp(410):Luc reporter is induced only about 2-fold induction in
response to CLOCK/BMAL1 co-expression. (C) The Dbp(410):Lucdriven bioluminescence rhythm exhibits a dampened oscillation of very
low amplitude; in contrast, both Dbp:Luc and Dbp(462):Luc drive
robust circadian oscillation of bioluminescence.

PAGE 7 OF 10of the CLOCK/BMAL1 proteins (Figure 4B). More
importantly, real-time bioluminescence monitoring
showed that the circadian oscillation of Dbp(410):luc
was dramatically reduced, whereas Dbp(462):luc exhibited robust circadian oscillation similar to that of Dbp:luc(Figure 4C). These results indicate that the predictedcircadian enhancer region containing a non-canonicalE-box (CATGTG) was actually responsible for transcriptional activation by the CLOCK/BMAL1 heterodimer
and for exhibiting robust circadian oscillation of bioluminescence reporter expression. The weak bioluminescence oscillation observed in Dbp(410):luc transfected
cells may be elicited through another non-canonical E-box
existing in the position of 155 bp. In addition, an RRE
compatible sequence (indicated in Figure 3) in mDbp
promoter may be not signiﬁcantly functional, because thephase of Dbp(410):luc was still similar to the bioluminescence rhythm driven by other two Dbp promoter
luciferase reporter vectors.

Nucleic Acids Research, 2008, Vol. 36, No. 4 e23

Identification of the non-canonical E-box as
an enhancer contributing for robust circadianoscillation driven by the mDbp promoter

Transcriptional assays were performed to examine
the eﬀects of CLOCK/BMAL1 on this DNA fragment.
The relative luciferase activity of En + pCMVmin:luc was
up-regulated after CLOCK/BMAL1 co-expression, andthis transcriptional activity was suppressed completely by
the additional expression of mCRY1 (Figure 6A right).
In contrast, no response was observed inform a CMV
minimal promoter luciferase reporter without the predicted enhancer DNA fragment (Figure 6A left). Realtime bioluminescence monitoring demonstrated that thepredicted enhancer region had the ability to generate
circadian gene expression in living rat-1 ﬁbroblast cells(Figure 6B). These results indicated that the non-canonicalE-box contained in this enhancer region functioned as acircadian enhancer. Thus, we next conﬁrmed the directbinding of the CLOCK/BMAL1 complex to this noncanonical E-box sequence using electrophoretic gelmobility shift assays (EMSA). When a biotin-labeled
30-bp oligo DNA probe containing the non-canonicalE-box (CATGTG) sequence was incubated with nuclear
extract from COS7 cells transfected with expression
vectors for mClock and mBmal1, a shifted band wasobserved (Figure 6C lanes 2 and 3). The speciﬁc band

To determine whether the non-canonical-E-box(CATGTG: 416 bp from the putative transcription
start site of the Dbp promoter) located in the predictedcircadian enhancer region is actually functional, we
generated a mutation of this non-canonical-E-box in the
Dbp:luc reporter [Dbp(mut):luc] using site-directed mutagenesis. The non-canonical E-box CATGTG was substituted with the sequence GGTACC, and transcriptional
assays using the mutated promoter construct were
performed to examine its responsiveness to CLOCK/BMAL1. The [Dbp(mut):luc] promoter was induced
1.6-fold by CLOCK/BMAL1 co-expression, whereas thewild-type Dbp:luc promoter was induced 13.5-fold(Figure 5A). Subsequently, real-time bioluminescencemonitoring was performed using the wild-type andmutated Dbp promoters. Dbp(mut):luc driven bioluminescence showed very weak circadian oscillation, but theamplitude of this oscillation was dramatically lower thanthat of Dbp:luc-driven bioluminescence oscillation(Figure 5B). These ﬁndings are consistent with the results
obtained from deletion of this predicted enhancer region(Figure 4C). Therefore, these results strongly suggest thatthe non-canonical E-box located in the predicted circadianenhancer region was responsible for driving robust circadian transcriptional oscillation from the Dbp promoter.
Although mutation of CATGTG E-box sequence resulted
in the dramatic damp of promoter driven circadian oscillation, slight circadian rhythm of bioluminescence still
remained. This suggested that other weak circadianenhancer(s) in part may contribute the transcriptional
rhythm of Dbp gene.
Finally, we investigated the ability of the predictedenhancer region alone to generate exact circadian oscillation of gene expression. We subcloned a 127 bp DNA
fragment, containing 466 to 340 bp of the mDbp
promoter, upstream of a CMV minimal promoter driven
luciferase reporter construct (En + pCMVmin:luc).

Figure 5. Mutation of the non-canonical E-box localized in thepredicted circadian enhancer region results in loss of robust circadianoscillation. (A) Substitution of the CATGTG non-canonical E-box to
GGTACC results in loss of CLOCK/BMAL1-mediated transactivationof the Dbp:Luc reporter. Mutated Dbp:Luc is named Dbp(mut):Luc.
Relative luciferase activity is reported as mean + SEM (B) Real-time
bioluminescence analysis using wild-type Dbp:Luc (three blue lines) andDbp(mut):Luc (three red lines) shows a dramatic dampening ofcircadian oscillation in the case of the mutant. Each line represents
one 35-mm dish transfected with Dbp:Luc or Dbp(mut):Luc. Cells weresynchronized by treatment with 100 nM dexamethasone and bioluminescence monitoring was performed using the same photomultiplier.

e23 Nucleic Acids Research, 2008, Vol. 36, No. 4

PAGE 8 OF 10

Figure 7. BMAL1/CLOCK binds the endogenous non-canonical E-box
region of 50 ﬂank end of the Dbp gene in NIH3T3 cells. Chromatin
immunoprecipitation (ChIP) was performed to analyze the binding of
BMAL1/CLOCK with the endogenous non-canonical E-box identiﬁed inthis study. (A) The position of identiﬁed non-canonical E-box is indicatedas underline (NC E-box). Arrows below the DNA sequences indicate theprimers [P(DBP)-F and P(DBP)-R] to detect co-immunoprecipitatedtarget Dbp promoter region. (B) ChIP is performed using ﬂag-tagged
BMAL1/CLOCK expressed NIH3T3 cells. Quantitative PCR (Q-PCR)
using P(DBP)-F and P(DBP)-R primers revealed that ampliﬁed signals
from the precipitants with anti-Flag antibody are signiﬁcantly stronger
than the signals ampliﬁed from the precipitants using anti-V5 antibody as
a control (left two columns). As a negative control, Q-PCR using speciﬁcprimers for beta-actin promoter (Act50 ) shows that ampliﬁed signals fromprecipitant samples using anti-ﬂag or anti-V5 antibodies both are low andno signiﬁcant diﬀerence between them (right two columns). Error bars
indicate standard deviation (SD).

Figure 6. The isolated predicted circadian enhancer region containing thenon-canonical E-box elicits distinct circadian oscillation. (A) The isolated
predicted circadian enhancer region of the mDbp promoter was subcloned
into the pCMVmin:Luc reporter vector, and strong up-regulation oftranscriptional activity was observed after co-expression of CLOCK/BMAL1. CLOCK/BMAL1-mediated transcriptional activity was dramatically reduced by the additional expression of HA-mCRY1. In contrast,
the pCMVmin:Luc vector without the non-canonical E-box showedno response to co-expression of CLOCK/BMAL1 and HA-mCRY1.
(B) Real-time bioluminescence monitoring of pCMVmin:Luc with orwithout the non-canonical E-box. The pCMVmin:Luc vector exhibitedno cyclic ﬂuctuation of bioluminescence over 3 days; in contrast, the
En+pCMVmin:Luc vector, which contains the predicted circadianenhancer elicited apparent circadian oscillation of bioluminescence(red line). (C) Electrophoretic gel mobility shift assays (EMSA) indicate
direct binding of the CLOCK/BMAL1 complex to the CATGTG noncanonical E-box located in the predicted circadian enhancer region of themDbp promoter. Shifted bands are detected when labeled non-canonicalE-box probe is incubated with nuclear extract from CLOCK/BMAL1
expressing COS7 cells (lanes 2 and 3); incubation with an excess of
unlabeled probe results in dramatic reduction of the shifted bands (lane 4).
An excess of unlabeled mutated non-canonical E-box probe (CATGTG to
GGTACC) does not reduce the shifted band (lane 5). Asterisks indicate
non-speciﬁc bands.

was abolished by a 50-fold excess of unlabeled probe(Figure 6C lane 4); in contrast, it was not abolished by
a 50-fold excess of unlabeled probe containing a mutation of the non-canonical-E-box sequence from CATGTG
to GGTACC (Figure 6C lane 5). These results indicatethat the CLOCK/BMAL1 complex directly binds the
CATGTG non-canonical E-box located in the predictedenhancer region of the mDbp promoter.
Interaction of BMAL1/CLOCK with Dbp promoter around
identified non-canonical E-box region in NIH3T3 cells
To conﬁrm the identiﬁed non-canonical E-box actuallyfunctioned in living cells, we performed chromatin
immunoprecipitation (ChIP) using ﬂag-tagged BMAL1
and CLOCK expressed NIH3T3 cells. Co-precipitatedgenomic DNA fragments by anti-ﬂag immunoprecipitation were analyzed by quantitative PCR (Q-PCR) method
to detect the identiﬁed non-canonical E-box region.
Position of primer sequences for Q-PCR and the noncanonical E-box of Dbp promoter region are presented in
Figure 7A. Q-PCR analysis revealed that the signiﬁcantrecruitment of ﬂag-tagged BMAL1 and CLOCK aroundthe non-canonical E-box region of Dbp promoter[P(DBP)] was observed, comparing with the beta-actin
promoter region (Act50 ) as a negative control (Figure 7B).

PAGE 9 OF 10

ChIP using anti-V5 antibody for ﬂag-BMAL1 and ﬂagCLOCK expressed NIH3T3 showed no signiﬁcant diﬀerence between P(DBP) targeted and Act50 -targeted Q-PCR(Figure 7B). These results conﬁrmed that BMAL1/
CLOCK actually bound the 50 ﬂank region of Dbp genearound the identiﬁed non-canonical E-box.

DISCUSSION
In this study, we have presented that we developed atransposon-based in vitro enhancer trapping method,which is a new strategy to identify unknown enhancers
contributing to circadian gene expression in living cells.
Using this method, we determined that the CATGTG
sequence, which can be classiﬁed as a CANNTG
non-canonical E-box, at position 416 bp in the mDbp
promoter functioned as a strong circadian enhancer toelicit robust oscillation of mDbp promoter driven luciferase bioluminescence. These results were compatible withrecent study showing that this non-canonical E-box
actually functioned as a circadian enhance in vivo (16).
Transposon-based enhancer detection system
In this study, we developed a Tn5 transposon-basedin vitro enhancer detection vector to identify unknown
circadian enhancer elements. The Tn5 transposon system
was originally developed for mutagenesis of bacterial
genes in vitro and in vivo (17). However, this system is also
useful for examining mammalian genes in vitro when thetarget DNA fragments are subcloned into a cloning
vector. Therefore, we used this transposon system to try
to trap putative enhancer elements. Moreover, using shorthalf-life ﬁreﬂy luciferase (from pGL4.11, Promega), wewere able to analyze real-time transcriptional activity forseveral days following transfection of this trap vector into
rat-1 cells. Combining the newly generated enhancer trap
vector with high throughput real-time bioluminescencemonitoring allowed us to detect a highly dynamicallycontrolled circadian enhancer element. As shown in
Figure 2, some of the transposons integrated into themDbp promoter exhibited apparent circadian bioluminescence cycles, whereas others did not (Figure 2). This
indicated that the circadian enhancer in the mDbp promoter must localized on the up-stream of the luciferase,
and allowed us to identify the CATGTG non-canonicalE-box as a strong circadian enhancer driving robust
circadian oscillation.
Another interesting point of this study is why thesecircadian enhancers exhibit ‘positional eﬀect’ or ‘distanceeﬀect’. In general, it is known that an enhancer shows its
function without any relation of distance and direction.
However, our strategy was actually useful to ﬁnd out the
non-canonical E-box enhancer. Although we have not yet
had exact answer for this question, one speculation is thatthe position of BMAL1/CLOCK E-box may be importantfor expressing its function. Because in many clock genes
having E-box on their promoter, the E-boxes reside neartranscriptional start sight in many case. The exact answer
should be waited for further studies.

Nucleic Acids Research, 2008, Vol. 36, No. 4 e23This study used transient transfections to analyze thereal-time bioluminescence driven by the enhancer detection vector. We were able to predict the circadianenhancer region of the mDbp promoter based on theamplitudes of reporter oscillation observed in trap vector
integrated clones; this suggests that our in vitro enhancer
trap strategy is a powerful and convenient method to
identify unknown enhancers. Furthermore, much microarray data has recently become available in public
databases. Using these data, in vitro enhancer trapping
may become a much more eﬀective way to understand theregulatory mechanisms of gene expression.
A non-canonical E-box as a strong circadian enhancer
Here, we have identiﬁed a CATGTG non-canonical E-box
as a strong circadian enhancer driving an mDbp promoter
luciferase reporter with clear circadian rhythm. One report
recently suggested that a CANNTG non-canonical E-box(CATTTG) in the prolactin promoter is regulated by the
CLOCK/BMAL1 complex (27). These results showedthat both CACGTG canonical E-boxes and CANNTG
non-canonical E-boxes could be important circadianenhancers. On the other hand, our ﬁndings raise a newquestion. The CANNTG sequence is mathematically
predicted to appear once every 256 bp in the genome.
This means that circadian enhancers may exist widely inthe promoters and intronic regions of many genes. Storch
et al. reported that hundreds of genes are cyclically
expressed in liver and heart; however, most of these genes
do not overlap in both organs (11). Other microarraystudies have supported the idea of divergent circadiangene expression in a tissue-speciﬁc manner (10,12). Thesestudies suggest the possibility that a large number of genes
have the potential to exhibit circadian expression underappropriate conditions (13). In addition, our data showedthat weakly but apparent circadian oscillation of bioluminescence was still remained even this non-canonicalE-box enhancer was mutated (Figure 5B). These results
may suggest that other weakly functioned unknown
circadian enhancers still existed on the mDbp promoter
region or other mechanism (for example, chromatin
remodeling) would contribute for circadian gene expression. Another important and interesting question arisingfrom this study is how the circadian system selectively
regulates gene expression. Tissue-speciﬁc circadian gene
expression may be controlled by a combination ofenhancer and tissue-speciﬁc regulatory factors, such as
the genome sequence around the non-canonical E-boxes
or chromatin regulatory mechanisms. Further studies are
required to address these questions.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Sayaka Tagao, Akira Morita and Yukiko
Sugisawa for technical support. This work is supported inpart by a Grant-in-Aid for Scientiﬁc Research from thee23 Nucleic Acids Research, 2008, Vol. 36, No. 4

Ministry of Education, Culture, Sports, Science andTechnology of Japan (16021237 and 17650092 to K.Y.,
Genome Network Project to H.R.U.); an intramural
Grant-in-Aid from CDB (to H.R.U.); SAKURA Grant
from JSPS (K.Y.); Grant-in-Aid from KANAE Medical
Foundation (K.Y.); a Leading Project for Biosimulation(K.Y. and H.R.U.). Funding to pay the Open Accesspublication charges for this article was provided by
Grant-in-Aid from Ministry of Education, Culture,
Sports, Science and Technology of Japan (to YU andKY 16GS0315).
Conﬂict of interest statement. None declared.
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