E-box function in a period gene repressed by light
Daniela Vallone*, Srinivas Babu Gondi*, David Whitmore†, and Nicholas S. Foulkes*‡
*Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35-39, Tübingen, D-72076 Germany; and †Centre for Cell and Molecular Dynamics,
Department of Anatomy and Developmental Biology, University College London, 21 University Street, London WC1E 6JJ, England
Edited by Jeffrey C. Hall, Brandeis University, Waltham, MA, and approved January 14, 2004 (received for review September 11, 2003)
In most organisms, light plays a key role in the synchronization of the
circadian timing system with the environmental day–night cycle.
Light pulses that phase-shift the circadian clock also induce the
expression of period (per) genes in vertebrates. Here, we report the
cloning of a zebrafish per gene, zfper4, which is remarkable in being
repressed by light. We have developed an in vivo luciferase reporter
assay for this gene in cells that contain a light-entrainable clock.
High-definition bioluminescence traces have enabled us to accurately
measure phase-shifting of the clock by light. We have also exploited
this model to study how four E-box elements in the zfper4 promoter
regulate expression. Mutagenesis reveals that the integrity of these
four E-boxes is crucial for maintaining low basal expression together
with robust rhythmicity and repression by light. Importantly, in the
context of a minimal heterologous promoter, the E-box elements also
direct a robust circadian rhythm of expression that is significantly
phase-advanced compared with the original zfper4 promoter and
lacks the light-repression property. Thus, these results reveal flexibility in the phase and light responsiveness of E-box-directed rhythmic
expression, depending on the promoter context.
T
he use of an endogenous pacemaker or clock to anticipate
and thereby respond appropriately to day–night changes in
the environment has been a highly conserved strategy throughout evolution (1). This clock is entrained daily by environmental
timing signals, so-called zeitgebers such as temperature and light,
and so remains synchronized with the light–dark (LD) cycle.
Characteristically, under constant darkness (DD) or constant
light (LL), the period of the clock rhythm deviates slightly from
24 h, and hence, it is termed circadian. This defining property is
thought to ensure optimal entrainment by zeitgebers (2). In
vertebrates, the circadian clock was originally thought to reside
in a small number of specialized pacemakers: the suprachiasmatic nucleus, the retina, and in lower vertebrates, the pineal
gland (3, 4). However, rhythmic clock gene expression was
encountered subsequently in vivo in most cell types (5, 6) and
shown to persist in vitro (7, 8). Thus, the circadian clock seems
to be a fundamental property of most cells.
Many clock genes encode transcriptional regulators, which are
components of autoregulatory feedback loops (9, 10). In vertebrates, the transcription factors Clock and brain and muscle arntlike protein (BMAL) bind as heterodimers to E-box enhancers and
activate the expression of other clock genes that encode transcriptional repressors: the Period (Per) and Cryptochrome (Cry) proteins. These repressors complex with Clock–BMAL and interfere
with transcriptional activation, thereby reducing expression of their
own genes and closing the feedback loop (9, 10).
After the original characterization of the period locus in Drosophila, there was a long delay before the first vertebrate per gene
homolog was cloned (11, 12). Subsequently, multiple per genes were
identified, suggesting either redundancy or specialization of function of the various family members (6). Three per genes have been
identified in the mouse that play distinct roles in the circadian clock
mechanism (6, 13). Whereas mper1 and mper2 seem to be essential,
mper3 is dispensable for circadian rhythmicity (14). Both mper1 and
mper2 are expressed with a circadian rhythm and are rapidly
induced in the suprachiasmatic nucleus by light pulses delivered
during the subjective night but not during the subjective day (6, 15,
16). Also, repression of mper1 expression in the suprachiasmatic
nucleus has been observed during phase-shifting of the clock by
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forced changes in running wheel activity (17). The precise contribution of these genes to clock entrainment by light remains unclear
(18–20).
The E-box (CACGTG) is a key component of the circadian clock.
Depending on the time of day, it mediates either transcriptional
activation or repression (10). However, this element is also the
binding site for a multitude of other basic helix–loop–helix transcription factors (21). Only a subset of E-boxes, termed circadian,
seem to represent specific binding sites for Clock–BMAL heterodimers (21–24). Additional sequences flanking the core hexamer as well as the presence of multiple, randomly spaced E-boxes
in a promoter region have been reported to favor circadian-clock
regulation (25, 26).
The proven usefulness of the zebrafish for large-scale genetic
screens makes it an attractive model to study the circadian clock (27,
28). Zebrafish peripheral clocks are directly light entrainable,
implying the widespread expression of a circadian photopigment in
this vertebrate (29). Zebrafish embryo-derived cell lines express a
light-entrainable clock (29, 30), making them a potentially powerful
in vitro model system. Sustained circadian rhythms of clock gene
expression can be established simply by exposing cultures to LD
cycles. This situation contrasts with mammalian cell lines such as
rat-1 fibroblasts, in which only rapidly dampening rhythms enduring
four or five cycles can be induced by transient treatment with
various signals (31, 32). Three zebrafish per genes have been
described to date, homologs of mper1, 2, and 3 (30, 33–35). Whereas
the clock regulates expression of zfper1 and 3, light activates zfper2
(30, 36). A blue light photoreceptor coupled to the mitogenactivated protein kinase pathway has been implicated in mediating
light-induced expression of zfper2 (36).
Here, we report the cloning of a zebrafish per gene, zfper4. Its
expression in larvae and a zebrafish cell line reveals this to be an
example of a per gene that is repressed by light. By using an in vivo
luciferase assay, we have visualized its expression in the PAC-2 cells.
We show that the integrity of four E-box elements within the zfper4
promoter is essential for a low basal expression level, robust
rhythmic expression, and repression by light. Interestingly, the
phase of the rhythm directed by the E-boxes and its acute response
to light seems to be a function of the promoter context.
Materials and Methods
Cloning of the zfper4 Gene. The following oligonucleotides based on
the Xenopus per1 cDNA (37) were used to prime long-distance PCR
(XL PCR kit, Perkin–Elmer) with PAC-2 DNA (38): AF250547
and BE679697, 5⬘-AGTGGCTGCAGCAGTGAACAGTCTGCC-3⬘ (sense); and 5⬘-CCAAAGTATTTGCTGGTGTTGCTGCTC-3⬘ (antisense). The products were analyzed by Southern
blotting using an mper1 PAS domain probe (12), purified by using
the QIAquick gel extraction kit (Qiagen, Valencia, CA), and then
cloned into pGemT-easy (Promega) for sequencing. RACE PCR
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: LD, light– dark; DD, constant darkness; LL, constant light; BMAL brain and
muscle arnt-like protein.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. AY359820).
‡To
whom correspondence should be addressed. E-mail: nix@tuebingen.mpg.de.
© 2004 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0305436101
Promoter Reporter Constructs. The zfper4 promoter was amplified by
using GenomeWalker PCR (Clontech) and subcloned into
pGL3Basic (Promega). Two canonical and two noncanonical Eboxes were mutated to CTCGAG by site-directed mutagenesis
(Stratagene). Oligonucleotides consisting of four copies of the
sequence; 5⬘-GAAGCACGTGTACTCG-3⬘ (E-box, position ⫺7)
was cloned into pLucMCS (Stratagene) to generate 4⫻ E-box(⫺7).
Oligonucleotide Synthesis and Sequence Analysis. All oligonucleo-
tides were synthesized by MWG Biotec (Ebersberg, Germany).
Sequencing was performed by the MPI genome analysis service.
Database searches and alignments were made by using BLAST.
Consensus transcription factor binding sites were identified by
comparison with the Transfac transcription factor database.
Establishment of Stable Cell Lines. PAC-2 cells (29, 38) were cultivated as described (29). Cells were transfected with linearized
plasmids; the luciferase reporter and a neomycin resistance plasmid
[pcDNA3,1 His-Myc(A), Invitrogen] at a molar ratio of 7:1. Electroporation was performed at 0.29 kV, 960 ␮F, by using Gene
Pulser apparatus (Bio-Rad). Three days later, G-418 (GIBCO兾
BRL) was added at a final concentration of 800 ␮g兾ml. During 1
month of selection, the concentration was gradually reduced to 250
␮g兾ml, and 100–200 resistant colonies per transfection were visible.
Colonies were trypsinized and propagated as a single pool.
In Vivo Luciferase Assay and Data Analysis. In total, 3 ⫻ 104 cells per
well were seeded into a 96-well Fluoplate (Nunc). Alternate wells
were left empty to minimize interference from bioluminescence
crosstalk (estimated to be 2–3% in adjacent wells). After 12 h, 0.5
mM beetle luciferin, potassium salt (Promega) was added. The
bioluminescence was assayed with a Topcount NXT counter (2detector model, Packard). At least six independent stable transfections were made for each construct. Each trace shows the mean of
at least two independent pools, each plated in a minimum of eight
wells. SD was also calculated and plotted. All assays were performed at least three times. Each well was counted for 3 s at
intervals of ⬇30 min. Plates were counted in an uninterrupted cycle,
and additional empty plates were included to adjust the counting
interval. Between counting, plates were illuminated with a tungsten
light source (20 ␮W兾cm2). To ensure uniform illumination, transparent plates were intercalated between the sample plates. The
counter was located in a thermostatically controlled dark room.
Data were imported into CHRONO (Till Roenneberg, University of
Munich, Munich) and EXCEL (Microsoft) by using the ‘‘Import and
Analysis’’ macro (S. Kay, Scripps Research Institute). Period estimates were made by linear regression after peak finder analysis with
CHRONO, measured after 2 days in DD. Single-factor ANOVA
statistical analysis was performed with a threshold P value for
significance set at P ⫽ 0.05.
Raising Adult and Larval Zebrafish and RNA Analysis. Adult zebrafish
(Tübingen strain) were raised according to standard methods (39).
Fertilized eggs were collected within 2 h of laying, and aliquots of
20 eggs were transferred into 20 ml of E3 buffer in 25-cm2 tissue
culture flasks. Flasks of cells or embryos were incubated in a
large-volume thermostat-controlled water bath and illuminated
with a tungsten light source (11 ␮W兾cm2) or maintained in DD.
RNA extractions and RNase protection assays were as described
(8). All experiments were performed a minimum of three times,
and representative results are shown. Autoradiographs were
scanned, and band intensities were quantified by using Scion Image
software. Zfper4 expression was normalized by using the ␤-actin
Vallone et al.
internal control. The quoted fold-activation and repression values
are the mean of at least three independent experiments.
Phase–Response Curve Analysis. zfper4 promoter-luciferase reporter
cells were plated in 10 plates in medium supplemented with
luciferin. All plates were exposed for 3 days to an LD cycle; on the
fourth day, they were individually sealed in light-proof boxes. After
3 complete days in DD, individual plates were light pulsed for 1 or
4 h, at 3- or 4-h intervals, respectively, by using a tungsten light
source (20 ␮W兾cm2). One control plate remained in DD. After the
final light pulse, all plates were counted for 3 days in DD. Stable
phase-shifts for each light-treated plate relative to the DD control
on the third day were then calculated. The time of onset of each light
pulse was expressed in circadian time (CT), where CT0 is defined
as the beginning of the subjective day and CT12, the beginning of
the subjective night. The duration of one free running period is 24
CT h. In terms of the zfper4 luciferase rhythm, CT0 is defined as 3.2
actual hours before each peak. Phase shifts were also expressed as
circadian hours by multiplying actual hour times by 24兾␶ (40).
Results
Expression of the zfper4 Gene. By using a PCR approach based on
the Xenopus per1 cDNA sequence, we isolated a zebrafish per gene
sharing most significant homology with per1 homologs, particularly
within the PAS and the C-terminal PAC domain (Fig. 5, which is
published as supporting information on the PNAS web site).
Initially, we used RNase protection assay analysis to examine its
expression in an adult zebrafish tissue (brain), in 6-day-old zebrafish
larvae, and in the embryo-derived PAC-2 cell line maintained in LD
(Fig. 1 A–C). In each case, a high-amplitude rhythm of expression
was observed (4.24 ⫾ 0.4, 9.47 ⫾ 1.2, and 5.96 ⫾ 0.9-fold, respectively), reminiscent of the described (30) zfper1 and 3 genes. A peak
occurs around lights-on and a trough occurs around the end of the
light period. We then studied its expression in PAC-2 cells under
various lighting conditions. After entrainment for 3 days in LD, the
cells were maintained in DD or in LL for 2 days; from the beginning
of the third day, they were harvested. In DD (Fig. 1D), dampened,
rhythmic expression (2.99 ⫾ 0.5-fold rhythm) was detected with a
higher basal level than observed under LD conditions (3.2 ⫾
0.7-fold higher) (Fig. 1F), suggesting circadian-clock regulation. In
LL (Fig. 1E), expression was essentially arrhythmic, with a basal
level comparable with LD (Fig. 1F). We next investigated the acute
response to light in larvae raised in DD or PAC-2 cells cultured for
5 days in DD. During the first 2–3 h of light, in the PAC-2 cells, there
was no change in expression relative to DD controls (Fig. 1H),
whereas expression was induced in the larvae (2.7 ⫾ 0.8-fold) (Fig.
1G). Subsequently, in both larvae and cells, expression was strongly
repressed for the duration of the light exposure (minimum 14- and
20-fold repression, respectively). This final property distinguishes
our gene from previously characterized vertebrate per genes, and
therefore, we have termed it zfper4.
In Vivo Luciferase Reporter Assay. We next developed an in vivo
luciferase reporter assay for zfper4 expression in PAC-2 cells. We
cloned zfper4 genomic DNA extending 3.3 kb upstream from the 5⬘
end of the cDNA into a luciferase reporter construct. Its sequence
revealed two canonical E-box elements (CACGTG at positions ⫺7
and ⫺669) and two noncanonical E-boxes (AACGTG at positions
⫺156 and ⫺172) (see Fig. 3A). Cells were stably transfected with
this construct, and then pools of clones were analyzed. This
commonly used approach averages out the effects of clone-to-clone
variability in integration sites and copy number of the plasmids. We
observed a robust rhythm of bioluminescence in LD that matched
well with the oscillation of the endogenous zfper4 transcript, a peak
occurring around ZT3 (Fig. 2A). Remarkably robust, rhythmic
luciferase expression persisted for up to 20 days without medium
renewal or supplementing with additional luciferin (Fig. 2 B and C,
and data not shown). The very low transfection-to-transfection
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(Marathon cDNA amplification kit, Clontech) and long-distance
PCR were used to clone the full-length zfper4 cDNA and genomic
region.
Fig. 1. RNase protection assay analysis of zfper4 (410-nt protected fragment, amino acid positions 88 –225) and ␤-actin (282-nt protected fragment) was
performed with RNA harvested at the indicated zeitgeber times (ZT0 is lights-on, ZT12 is lights-off) or CT. (A) Whole brain from adult zebrafish maintained in
LD. (B) Six-day-old larvae raised in LD. (C) PAC-2 cells were maintained in LD for 5 days. Cells were entrained in LD and harvested in DD (D) or in LL (E). (F) Peak
and trough samples from the LD, DD, and LL sets of PAC-2 cells (C–E) assayed together to allow comparison of the relative expression levels. (G) Larvae raised
for 6 days in DD and then exposed to light for the indicated times (hours; ⫹ Light). Control larvae remained in DD and were harvested in parallel. (H) Analysis
of PAC-2 cells equivalent to G.
variation observed in the bioluminescence traces validates the
averaging achieved by this pooling approach.
Expression was then tested under various lighting conditions.
After 4 days in LD, cells were transferred to DD conditions (Fig.
2B). A high-amplitude rhythm of bioluminescence established in
LD continued in DD, with a free-running period (␶) of 25.19 ⫾
0.21 h. The rhythm amplitude declined progressively, with peak and
trough values tending toward intermediate values. The cells were
then exposed to light at the beginning of the subjective night (Fig.
2B). Our results showed that starting 4 h after lights-on, expression
steadily decreased for the duration of the light period. Highamplitude rhythmic expression with a phase matching the new LD
cycle was restored within one cycle, although peaks were significantly lower than under the original LD conditions (compare Fig.
2 B and C). A switch to LL coincided with the beginning of the
subjective night and lead to a pronounced attenuation of the rhythm
and arrhythmicity by 72 h in LL (Fig. 2C). Finally, return to LD
reestablished a high-amplitude expression rhythm, although again,
peak values were lower than under the original LD cycles. Interestingly, when DD-adapted cells were light pulsed at the beginning
of the subjective day, we also observed down-regulation of zfper4
expression to basal levels (the trough values observed in LD; data
not shown). This result contrasts with mper1 and 2 in the supra-
chiasmatic nucleus, in which only light pulses during the subjective
night influence gene expression (6, 15, 16).
We also tested the possibility that light from the luciferase
reaction might directly influence the cellular clock. By RNase
protection assay, we examined expression of the light-inducible
zfper2 gene in the reporter cells, with or without luciferin, under DD
(34, 35). In both cases, low, stable levels of zfper2 expression were
observed (data not shown). Therefore, light emitted during the
luciferase reaction does not significantly influence the expression of
light-regulated clock genes and by inference is unlikely to represent
a significant zeitgeber for the clock.
Light Regulation of the Zebrafish Cell Circadian Clock. Our results
indicate that light influences zfper4 expression by means of the
circadian clock and acute repression. Consistent with a significant
entraining effect of light, reversal of the phase of the LD cycle leads
to complete reentrainment of the zfper4 expression rhythm within
48 h (Fig. 2D). Furthermore, exposure of the reporter cells to LD
cycles with period lengths (T) significantly longer and shorter than
24 h (30 h, 15:15 h LD; and 20 h, 10:10 h LD) leads to adjustment
of the period length of the reporter rhythm to match T (Fig. 2 E and
F). However, on return to DD conditions, both sets of cells return
to a free-running period length comparable with that of cells
adapted to LD (12:12) conditions.
Fig. 2. (A) Bioluminescence assay of pools of stably transfected zfper4 luciferase reporter cells maintained for 3 days in LD. Bioluminescence is plotted on the
y axis (counts per second) and hours on the x axis (time 0 indicates the beginning of assay). For each point, error bars represent the SD. A white兾black bar shows
the light and dark periods. (B) Cells maintained for two cycles in LD and then transferred to DD before a light pulse. (C) Immediately after the experiment shown
in B, cells were returned to LD for 2 days and then remained in LL before being returned to LD. (D) Cells entrained in LD were then subjected to a reversal of
the phase of the LD cycle (indicated by arrowhead) and were monitored for an additional 60 h. (E) Cells were entrained for four 30-h LD cycles (15:15) before
being assayed for an additional three cycles in LD. They were then transferred to DD. (F) Equivalent experiment with cells entrained to a 20-h LD cycle (10:10).
(G) Phase–response curve analysis of PAC-2 cells for 1- or 4-h light pulses. Phase shifts are plotted on the y axis (negative and positive values correspond to phase
delays and advances, respectively). The time of the onset of each light pulse is plotted on the x axis. Calculations were based on data obtained from 16
independent culture wells per plate for each of four independent experiments. Mean phase shifts are plotted together with error bars indicating SD.
4108 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0305436101
Vallone et al.
The high definition of our bioluminescence rhythms facilitates
accurate measurement of phase-shifts. We, therefore, used these
cells to quantify systematically how light pulses delivered during the
subjective day and night phase shift the clock (40). We delivered 1or 4-h light pulses to DD-adapted cells at 3- or 4-h intervals,
respectively, throughout the subjective day and night, and then
phase-shifts were measured after 3 days in DD. These shifts were
then plotted as a function of the time of the light pulse to generate
a phase–response curve. The phase-shifting properties of 1- and 4-h
light pulses were comparable (Fig. 2G) with very large phase-shifts
induced by light pulses during the early subjective night (40).
Analysis of E-Box Function in the zfper4 Promoter. We next explored
which enhancers within the zfper4 promoter mediate clock regulation and repression by light. We initially focused on the four E-box
elements given their predicted importance within the circadian
clock (9, 10). We mutated single or multiple E-boxes (Fig. 3A) and
then tested the expression of these constructs in stably transfected
PAC-2 cell pools in LD, then DD followed by a light pulse.
Mutation of the ⫺669 canonical E-box (Mut ⫺669) resulted in
rhythmic expression, dampening in DD and repression by light
comparable with that of the WT construct (Fig. 3B). Mutating the
⫺7 canonical E-box alone and in combination with ⫺669 (Mut ⫺7
and Mut ⫺7兾⫺669) resulted in a significant increase in basal
expression and accompanying decrease in the rhythm amplitude
and repression by light, when expressed as fold induction and
repression, respectively (Fig. 3 D and E). However, mutation of all
consensus and nonconsensus E-box elements (Mut ⫺7兾⫺156兾
Vallone et al.
⫺172兾⫺669) (Fig. 3C) lead to a further increase in expression
levels, with a consequent reduction in rhythm amplitude and
repression by light as well as a considerable increase in SD of the
trace through the entire experiment. Statistical analysis reveals that
the highly significant difference between peak and trough in the
WT construct (P ⫽ 4.5 ⫻ 10⫺16) was reduced in the Mut ⫺7兾
⫺156兾⫺172兾⫺669 construct (P ⫽ 0.01). Furthermore, the high
significance of the repression by light for the WT construct (P ⫽
1.6 ⫻ 10⫺7) was lost in this mutant (P ⫽ 0.07). The presence of
residual rhythmicity for this construct suggests the contribution of
additional elements within the zfper4 promoter to this regulation.
However, these results indicate that the presence of all four E-boxes
is necessary for a low, stable basal expression level, robust rhythmic
expression, and repression by light.
Finally, to determine whether the E-box-directed regulation was
influenced by other promoter elements, we generated three heterologous promoter constructs where four copies of each E-box
present in the zfper4 promoter were cloned upstream of a TATA
box element and a luciferase reporter. These constructs were stably
transfected, and their expression patterns were compared with
those of the zfper4 promoter constructs [Fig. 4A; data from the ⫺7
canonical E-box, 4⫻E-box(⫺7)]. For all constructs in LD, a rhythmic expression pattern was observed that persisted in DD. However, surprisingly this rhythm was phase-advanced by 6 h compared
with the zfper4 promoter (Fig. 4A). In addition, expression of the
4⫻E-box reporter constructs was not repressed by light and under
LL, rhythmic expression dampened, with peak levels remaining
constant and trough levels progressively increasing (Fig. 4B). SubPNAS 兩 March 23, 2004 兩 vol. 101 兩 no. 12 兩 4109
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Fig. 3. Mutational analysis of zfper4 promoter E-boxes. (A) Schematic representation of the WT and mutated constructs. Boxes denote the two consensus
E-boxes, and the closely spaced ellipses denote the noncanonical E-boxes. The positions of the elements relative to the transcription start site (arrowhead) are
indicated. Mutation of the E-boxes into CTCGAG is shown by a cross. (B) Luciferase assay of cell pools transfected with WT (black trace, red error bars), Mut ⫺669
(purple trace, blue error bars), Mut ⫺7 (green trace, pink error bars), and Mut ⫺7兾⫺669 (dark red trace, black error bars) in LD, followed by DD and then a light
pulse. (C) Equivalent analysis of cells transfected with WT (black trace) and Mut ⫺7兾⫺156兾⫺172兾⫺669 (blue trace). (D) Quantification of rhythm amplitude in
LD on days 2 and 3 of assay for each construct in terms of fold induction. Vertical bars indicate SD. (E) Quantification of acute repression by light. The
bioluminescence values at the beginning and end of the 12-h light pulse were measured and plotted as fold repression for each construct.
Fig. 4. Expression of 4⫻E-box(⫺7) heterologous promoter construct. (A) Comparison of the bioluminescence profiles of cells stably transfected with the zfper4
(blue trace) and 4⫻E-box(⫺7) promoter constructs (green trace). After two LD cycles (12:12), cells were transferred to DD. (B) The 4⫻E-box(⫺7) cells after two
LD cycles were transferred to LL. (C) Comparison of 4⫻E-box(⫺7) and zfper4 promoter luciferase rhythms under 20-h LD cycles (10:10) and (D) 30-h LD cycles
(15:15). In both C and D, cells were entrained for four LD cycles before starting the assay and then experienced an additional three cycles.
stitution of the TATA element in the heterologous promoter with
thymidine kinase or SV40 minimal promoter sequences did not
alter these properties, although the basal levels of expression were
increased (data not shown). Interestingly, the phase differences
between the zfper4 and 4⫻E-box promoter rhythms are a function
of T of the entraining LD cycle (Fig. 4 C and D). Thus, where T ⫽
20 h, the 4⫻E-box rhythm is phase-advanced by only 2.9 (⫾0.3) h
(Fig. 4C), whereas where T ⫽ 30 h, the phase advance is 11.3
(⫾0.49) h (Fig. 4D). This result seems to be the consequence of the
phase of the zfper4 promoter rhythm being locked so that its peak
occurs 2–4 h after lights-on, whereas the 4⫻E-box peak shifts from
the beginning of the light period (T ⫽ 20) to the middle of the dark
period (T ⫽ 30). These results imply a significant contribution of the
local promoter environment to the phase and light responsiveness
of E-box-generated expression rhythms.
Discussion
Here, we describe an example of a vertebrate period gene that is
repressed by light and shares significant homology with per1.
Expression of a zebrafish per1 homolog has been described,
although sequence data were not presented (30). Rhythmic
expression was documented in LD and DD conditions and a
transient induction was observed in response to a light pulse (30).
Given that the per gene documented here is repressed strongly
by light in both larvae and PAC-2 cells, we believe that it most
likely represents a homolog that we have named zfper4. The
preceding, relatively weak induction of zfper4 expression observed only in larvae may indicate cell-type specificity in this
light response. The existence of more than three per genes in
zebrafish could be anticipated because many mammalian genes
have been reported to have two paralogues in zebrafish as the
result of a whole-genome duplication during the evolution of the
teleost lineage (41). Furthermore, six cryptochrome genes have
been described in zebrafish, suggesting additional complexity in
zebrafish clock gene families (28, 42).
We present phase–response curve data for the phase-shifting
effects of light on a cell culture clock. We demonstrate that the
PAC-2 clock shows a typical high-amplitude phase–response curve
(type 0). Maximum phase shifts are obtained with light pulses
delivered during the early subjective night. In addition, at the
beginning of the early subjective day, only small phase delays are
4110 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0305436101
observed representing the so-called dead zone (40). These properties are consistent with our results showing that the clock entrains
rapidly to large phase shifts in the entraining LD cycle. Furthermore, it synchronizes with LD cycles with a broad range of period
lengths. Thus, our results implicate light as a strong zeitgeber for
this cellular clock. Therefore, one may anticipate the importance of
direct light entrainment for peripheral clocks in the context of the
zebrafish circadian system.
We have analyzed the zfper4 promoter by stably transfecting
luciferase promoter reporter constructs into PAC-2 cells and then
testing for regulation by light and the endogenous clock. This
approach has the major advantage that regulation by physiological
levels of endogenous factors is tested. Performing promoter analysis
with transgenic animals offers similar advantages, but it has the
drawback of being far more time consuming. Many previous studies
exploring transcriptional regulation in the clock have been based on
transient transfection assays. However, such studies may be misleading because overexpression of a candidate regulator may drive
physiologically nonrelevant interactions. Here, we demonstrate in a
vertebrate cell culture model that a functional circadian clock drives
rhythmic expression by means of E-box elements in the context of
a minimal heterologous promoter. This result is consistent with
current models for the circadian clock; however, these rhythms are
6-h phase-advanced compared with the zfper4 promoter. It is
therefore clear that E-boxes can direct rhythmic expression with
significant differences in phase depending on their promoter context. Furthermore, the phase relationship of the E-box and promoter rhythms varies depending on the period length of the
entraining LD cycle. This observation points to light also playing a
key role in determining the phase of the promoter rhythms.
Mutational analysis has demonstrated that the E-boxes contribute to maintaining a low level of promoter expression. This result
is surprising because they would be predicted to bind Clock兾BMAL
and, thus, function as enhancers. Indeed, mutation of circadian
E-boxes has been documented (25, 43) to reduce expression levels
in vivo. We have also implicated these elements in robust rhythmic
expression and down-regulation by light. Interestingly, in the context of a heterologous promoter, these E-box elements direct
rhythmic expression that is not repressed by light pulses, implying
that the local promoter environment might determine their function. It is noteworthy that none of the four E-boxes corresponds to
Vallone et al.
the optimal binding sites for mammalian Clock兾BMAL (22). It will
be interesting to test whether the additional zebrafish Clock and
BMAL homologs bind differentially to these elements and, thereby,
confer light-responsive, rhythmic expression with low basal levels
(44).
The repression of zfper4 expression after exposure to light occurs
only after a delay of 4 h. This result suggests earlier induction of a
repressor factor. Expression of the zfper2 gene is induced within the
first 2 h after light exposure (30, 36). Given the role of Per proteins
in the circadian timing mechanism, it is tempting to speculate that
light-induced zfper2 may down-regulate zfper4 expression by means
of the E-box elements. Per proteins seem to function in combination with Crys to repress Clock:BMAL heterodimer activation. In
the chicken pineal, light has been shown to acutely induce Cry
expression (45). However, the lack of repression of the 4⫻E-box
heterologous promoter constructs by light would tend to argue
against this. Alternatively, light may induce expression of other
transcriptional repressors that bind to distinct enhancer elements
and then interact with E-box-bound factors in the context of the
promoter (46).
Zebrafish cell lines offer many advantages for studying the
vertebrate circadian clock. They express a functional clock that can
be entrained by direct light exposure (29). For this reason, they are
ideal also for studying light-input pathways. We have established
PAC-2 luciferase reporter cell lines that significantly increase the
value of this culture system. The high definition of the bioluminescence data obtained may be explained by the emission of light from
a static monolayer of uniformly expressing cells. Furthermore, the
viability of these cells during long periods at confluence, the
stability of luciferin in the culture medium, and the ease with which
it can diffuse into the cells are all likely to contribute to the stability
of the luciferase signal. The ability to maintain reproducible,
high-amplitude bioluminescence rhythms over long time periods
contrasts with the transient, dampening rhythms described for
mammalian cell lines (32). Furthermore, the growth of these cells
at room temperature in atmospheric CO2, the use of a 96-well plate
format, and a high-throughput automated scintillation counter to
perform the luciferase assay make these cells ideal for large-scale
analysis.
1. Pittendrigh, C. S. (1993) Annu. Rev. Physiol. 55, 16–54.
2. Roenneberg, T., Daan, S. & Merrow, M. (2003) J. Biol. Rhythms 18, 183–194.
3. Klein, D. M., Moore, R. Y. & Reppert, S. M. (1991) Suprachiasmatic Nucleus:
The Mind’s Clock (Oxford Univ. Press, New York).
4. Menaker, M., Moreira, L. F. & Tosini, G. (1997) Braz. J. Med. Biol. Res. 30,
305–313.
5. King, D. P., Zhao, Y., Sangoram, A. M., Wilsbacher, L. D., Tanaka, M.,
Antoch, M. P., Steeves, T. D., Vitaterna, M. H., Kornhauser, J. M., Lowrey,
P. L., et al. (1997) Cell 89, 641–653.
6. Zylka, M. J., Shearman, L. P., Weaver, D. R. & Reppert, S. M. (1998) Neuron
20, 1103–1110.
7. Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block,
G. D., Sakaki, Y., Menaker, M. & Tei, H. (2000) Science 288, 682–685.
8. Whitmore, D., Foulkes, N. S., Strahle, U. & Sassone-Corsi, P. (1998) Nat.
Neurosci. 1, 701–707.
9. Lowrey, P. L. & Takahashi, J. S. (2000) Annu. Rev. Genet. 34, 533–562.
10. Reppert, S. M. & Weaver, D. R. (2001) Annu. Rev. Physiol. 63, 647–676.
11. Konopka, R. J. & Benzer, S. (1971) Proc. Natl. Acad. Sci. USA 68, 2112–2116.
12. Tei, H., Okamura, H., Shigeyoshi, Y., Fukuhara, C., Ozawa, R., Hirose, M. &
Sakaki, Y. (1997) Nature 389, 512–516.
13. Bae, K., Jin, X., Maywood, E. S., Hastings, M. H., Reppert, S. M. & Weaver,
D. R. (2001) Neuron 30, 525–536.
14. Shearman, L. P., Jin, X., Lee, C., Reppert, S. M. & Weaver, D. R. (2000) Mol.
Cell. Biol. 20, 6269–6275.
15. Shigeyoshi, Y., Taguchi, K., Yamamoto, S., Takekida, S., Yan, L., Tei, H.,
Moriya, T., Shibata, S., Loros, J. J., Dunlap, J. C. & Okamura, H. (1997) Cell
91, 1043–1053.
16. Shearman, L. P., Zylka, M. J., Weaver, D. R., Kolakowski, L. F., Jr., & Reppert,
S. M. (1997) Neuron 19, 1261–1269.
17. Maywood, E. S., Mrosovsky, N., Field, M. D. & Hastings, M. H. (1999) Proc.
Natl. Acad. Sci. USA 96, 15211–15216.
18. Bae, K. & Weaver, D. R. (2003) J. Biol. Rhythms 18, 123–133.
19. Albrecht, U., Zheng, B., Larkin, D., Sun, Z. S. & Lee, C. C. (2001) J. Biol.
Rhythms 16, 100–104.
20. Cermakian, N., Monaco, L., Pando, M. P., Dierich, A. & Sassone-Corsi, P.
(2001) EMBO J. 20, 3967–3974.
21. Munoz, E., Brewer, M. & Baler, R. (2002) J. Biol. Chem. 277, 36009–36017.
22. Hogenesch, J. B., Gu, Y. Z., Jain, S. & Bradfield, C. A. (1998) Proc. Natl. Acad.
Sci. USA 95, 5474–5479.
23. Gekakis, N., Staknis, D., Nguyen, H. B., Davis, F. C., Wilsbacher, L. D., King,
D. P., Takahashi, J. S. & Weitz, C. J. (1998) Science 280, 1564–1569.
24. Darlington, T. K., Wager-Smith, K., Ceriani, M. F., Staknis, D., Gekakis, N.,
Steeves, T. D., Weitz, C. J., Takahashi, J. S. & Kay, S. A. (1998) Science 280,
1599–1603.
25. McDonald, M. J., Rosbash, M. & Emery, P. (2001) Mol. Cell. Biol. 21,
1207–1217.
26. Ripperger, J. A., Shearman, L. P., Reppert, S. M. & Schibler, U. (2000) Genes
Dev. 14, 679–689.
27. Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M.,
Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P., et
al. (1996) Development (Cambridge, U.K.) 123, 1–36.
28. Cahill, G. M. (2002) Cell Tissue Res. 309, 27–34.
29. Whitmore, D., Foulkes, N. S. & Sassone-Corsi, P. (2000) Nature 404, 87–91.
30. Pando, M. P., Pinchak, A. B., Cermakian, N. & Sassone-Corsi, P. (2001) Proc.
Natl. Acad. Sci. USA 98, 10178–10183.
31. Balsalobre, A., Damiola, F. & Schibler, U. (1998) Cell 93, 929–937.
32. Izumo, M., Johnson, C. H. & Yamazaki, S. (2003) Proc. Natl. Acad. Sci. USA
100, 16089–16094.
33. Delaunay, F., Thisse, C., Marchand, O., Laudet, V. & Thisse, B. (2000) Science
289, 297–300.
34. Hirayama, J., Fukuda, I., Ishikawa, T., Kobayashi, Y. & Todo, T. (2003) Nucleic
Acids Res. 31, 935–943.
35. Delaunay, F., Thisse, C., Thisse, B. & Laudet, V. (2003) Gene Expression
Patterns 3, 319–324.
36. Cermakian, N., Pando, M. P., Thompson, C. L., Pinchak, A. B., Selby, C. P.,
Gutierrez, L., Wells, D. E., Cahill, G. M., Sancar, A. & Sassone-Corsi, P. (2002)
Curr. Biol. 12, 844–848.
37. Zhuang, M., Wang, Y., Steenhard, B. M. & Besharse, J. C. (2000) Brain Res.
Mol. Brain Res. 82, 52–64.
38. Lin, S., Gaiano, N., Culp, P., Burns, J. C., Friedmann, T., Yee, J. K. & Hopkins,
N. (1994) Science 265, 666–669.
39. Dekens, M. P., Santoriello, C., Vallone, D., Grassi, G., Whitmore, D. &
Foulkes, N. S. (2003) Curr. Biol. 13, 2051–2057.
40. Johnson, C. H. (1999) Chronobiol. Int. 16, 711–743.
41. Postlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A., Brownlie,
A., Donovan, A., Egan, E. S., Force, A., Gong, Z., et al. (1998) Nat. Genet. 18,
345–349.
42. Kobayashi, Y., Ishikawa, T., Hirayama, J., Daiyasu, H., Kanai, S., Toh, H.,
Fukuda, I., Tsujimura, T., Terada, N., Kamei, Y., et al. (2000) Genes Cells 5,
725–738.
43. Hao, H., Allen, D. L. & Hardin, P. E. (1997) Mol. Cell. Biol. 17, 3687–3693.
44. Ishikawa, T., Hirayama, J., Kobayashi, Y. & Todo, T. (2002) Genes Cells 7,
1073–1086.
45. Yamamoto, K., Okano, T. & Fukada, Y. (2001) Neurosci. Lett. 313, 13–16.
46. Doi, M., Nakajima, Y., Okano, T. & Fukada, Y. (2001) Proc. Natl. Acad. Sci.
USA 98, 8089–8094.
CELL BIOLOGY
We thank T. Roenneberg, M. Merrow, K. Tamai, and all laboratory
members for helpful discussions and T. Roenneberg for adapting
CHRONO for Topcount files. D.V. and S.B.G. were supported by the Max
Planck Society. N.S.F. was supported by Centre National de la Recherche Scientifique and Max Planck funding; D.W. was supported by funds
from the Biotechnology and Biological Sciences Research Council and
the Wellcome Trust.
Vallone et al.
PNAS 兩 March 23, 2004 兩 vol. 101 兩 no. 12 兩 4111