E-box function in a period gene repressed by light

Daniela Vallone*, Srinivas Babu Gondi*, David Whitmore†, and Nicholas S. Foulkes*‡

*Max-Planck-Institut fu¨r Entwicklungsbiologie, Spemannstrasse 35-39, Tu¨bingen, D-72076 ; and †Centre for Cell and Molecular Dynamics, Department of Anatomy and Developmental , 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 forced changes in running wheel activity (17). The precise contri- circadian timing system with the environmental day–night cycle. bution of these genes to clock entrainment by light remains unclear Light pulses that phase-shift the circadian clock also induce the (18–20). expression of period (per) genes in vertebrates. Here, we report the The E-box (CACGTG) is a key component of the circadian clock. cloning of a zebrafish per gene, zfper4, which is remarkable in being Depending on the time of day, it mediates either transcriptional repressed by light. We have developed an in vivo luciferase reporter activation or repression (10). However, this element is also the assay for this gene in cells that contain a light-entrainable clock. binding site for a multitude of other basic helix–loop–helix tran- High-definition bioluminescence traces have enabled us to accurately scription factors (21). Only a subset of E-boxes, termed circadian, measure phase-shifting of the clock by light. We have also exploited seem to represent specific binding sites for Clock–BMAL het- this model to study how four E-box elements in the zfper4 promoter erodimers (21–24). Additional sequences flanking the core hex- regulate expression. Mutagenesis reveals that the integrity of these amer as well as the presence of multiple, randomly spaced E-boxes four E-boxes is crucial for maintaining low basal expression together in a promoter region have been reported to favor circadian-clock with robust rhythmicity and repression by light. Importantly, in the regulation (25, 26). context of a minimal heterologous promoter, the E-box elements also The proven usefulness of the zebrafish for large-scale genetic direct a robust of expression that is significantly screens makes it an attractive model to study the circadian clock (27, phase-advanced compared with the original zfper4 promoter and 28). Zebrafish peripheral clocks are directly light entrainable, lacks the light-repression property. Thus, these results reveal flexibil- implying the widespread expression of a circadian photopigment in ity in the phase and light responsiveness of E-box-directed rhythmic this vertebrate (29). Zebrafish embryo-derived cell lines express a expression, depending on the promoter context. light-entrainable clock (29, 30), making them a potentially powerful in vitro model system. Sustained circadian rhythms of clock gene he use of an endogenous pacemaker or clock to anticipate expression can be established simply by exposing cultures to LD Tand thereby respond appropriately to day–night changes in cycles. This situation contrasts with mammalian cell lines such as the environment has been a highly conserved strategy through- rat-1 fibroblasts, in which only rapidly dampening rhythms enduring out evolution (1). This clock is entrained daily by environmental four or five cycles can be induced by transient treatment with timing signals, so-called such as temperature and light, various signals (31, 32). Three zebrafish per genes have been and so remains synchronized with the light–dark (LD) cycle. described to date, homologs of mper1, 2, and 3 (30, 33–35). Whereas Characteristically, under constant darkness (DD) or constant the clock regulates expression of zfper1 and 3, light activates zfper2 light (LL), the period of the clock rhythm deviates slightly from (30, 36). A blue light photoreceptor coupled to the mitogen- 24 h, and hence, it is termed circadian. This defining property is activated protein kinase pathway has been implicated in mediating thought to ensure optimal entrainment by zeitgebers (2). In light-induced expression of zfper2 (36). vertebrates, the circadian clock was originally thought to reside Here, we report the cloning of a zebrafish per gene, zfper4. Its in a small number of specialized pacemakers: the suprachias- expression in larvae and a zebrafish cell line reveals this to be an matic nucleus, the retina, and in lower vertebrates, the pineal example of a per gene that is repressed by light. By using an in vivo gland (3, 4). However, rhythmic clock gene expression was luciferase assay, we have visualized its expression in the PAC-2 cells. encountered subsequently in vivo in most cell types (5, 6) and We show that the integrity of four E-box elements within the zfper4 shown to persist in vitro (7, 8). Thus, the circadian clock seems promoter is essential for a low basal expression level, robust to be a fundamental property of most cells. rhythmic expression, and repression by light. Interestingly, the Many clock genes encode transcriptional regulators, which are phase of the rhythm directed by the E-boxes and its acute response components of autoregulatory feedback loops (9, 10). In verte- to light seems to be a function of the promoter context. brates, the transcription factors Clock and brain and muscle arnt- like protein (BMAL) bind as heterodimers to E-box enhancers and Materials and Methods activate the expression of other clock genes that encode transcrip- Cloning of the zfper4 Gene. The following oligonucleotides based on tional repressors: the Period (Per) and Cryptochrome (Cry) pro- the Xenopus per1 cDNA (37) were used to prime long-distance PCR teins. These repressors complex with Clock–BMAL and interfere (XL PCR kit, Perkin–Elmer) with PAC-2 DNA (38): AF250547 with transcriptional activation, thereby reducing expression of their and BE679697, 5Ј-AGTGGCTGCAGCAGTGAACAGTCT- own genes and closing the feedback loop (9, 10). GCC-3Ј (sense); and 5Ј-CCAAAGTATTTGCTGGTGTTGCT- After the original characterization of the period locus in Dro- GCTC-3Ј (antisense). The products were analyzed by Southern sophila, there was a long delay before the first vertebrate per gene blotting using an mper1 PAS domain probe (12), purified by using homolog was cloned (11, 12). Subsequently, multiple per genes were the QIAquick gel extraction kit (Qiagen, Valencia, CA), and then identified, suggesting either redundancy or specialization of func- cloned into pGemT-easy (Promega) for sequencing. RACE PCR tion 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, This paper was submitted directly (Track II) to the PNAS office. mper3 is dispensable for circadian rhythmicity (14). Both mper1 and Abbreviations: LD, light–dark; DD, constant darkness; LL, constant light; BMAL brain and mper2 are expressed with a circadian rhythm and are rapidly muscle arnt-like protein. induced in the suprachiasmatic nucleus by light pulses delivered Data deposition: The sequence reported in this paper has been deposited in the GenBank during the subjective night but not during the subjective day (6, 15, database (accession no. AY359820). 16). Also, repression of mper1 expression in the suprachiasmatic ‡To whom correspondence should be addressed. E-mail: [email protected]. nucleus has been observed during phase-shifting of the clock by © 2004 by The National Academy of Sciences of the USA

4106–4111 ͉ PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305436101 Downloaded by guest on September 28, 2021 (Marathon cDNA amplification kit, Clontech) and long-distance internal control. The quoted fold-activation and repression values PCR were used to clone the full-length zfper4 cDNA and genomic are the mean of at least three independent experiments. region. Phase–Response Curve Analysis. zfper4 promoter-luciferase reporter Promoter Reporter Constructs. The zfper4 promoter was amplified by cells were plated in 10 plates in medium supplemented with using GenomeWalker PCR (Clontech) and subcloned into luciferin. All plates were exposed for 3 days to an LD cycle; on the pGL3Basic (Promega). Two canonical and two noncanonical E- fourth day, they were individually sealed in light-proof boxes. After boxes were mutated to CTCGAG by site-directed mutagenesis 3 complete days in DD, individual plates were light pulsed for 1 or (Stratagene). Oligonucleotides consisting of four copies of the 4 h, at 3- or 4-h intervals, respectively, by using a tungsten light sequence; 5Ј-GAAGCACGTGTACTCG-3Ј (E-box, position Ϫ7) source (20 ␮W͞cm2). One control plate remained in DD. After the was cloned into pLucMCS (Stratagene) to generate 4ϫ E-box(Ϫ7). 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 Oligonucleotide Synthesis and Sequence Analysis. All oligonucleo- on the third day were then calculated. The time of onset of each light tides were synthesized by MWG Biotec (Ebersberg, Germany). pulse was expressed in circadian time (CT), where CT0 is defined Sequencing was performed by the MPI genome analysis service. as the beginning of the subjective day and CT12, the beginning of Database searches and alignments were made by using BLAST. the subjective night. The duration of one free running period is 24 Consensus transcription factor binding sites were identified by CT h. In terms of the zfper4 luciferase rhythm, CT0 is defined as 3.2 comparison with the Transfac transcription factor database. actual hours before each peak. Phase shifts were also expressed as circadian hours by multiplying actual hour times by 24͞␶ (40). Establishment of Stable Cell Lines. PAC-2 cells (29, 38) were culti- Results vated as described (29). Cells were transfected with linearized plasmids; the luciferase reporter and a neomycin resistance plasmid Expression of the zfper4 Gene. By using a PCR approach based on [pcDNA3,1 His-Myc(A), Invitrogen] at a molar ratio of 7:1. Elec- the Xenopus per1 cDNA sequence, we isolated a zebrafish per gene troporation was performed at 0.29 kV, 960 ␮F, by using Gene sharing most significant homology with per1 homologs, particularly Pulser apparatus (Bio-Rad). Three days later, G-418 (GIBCO͞ within the PAS and the C-terminal PAC domain (Fig. 5, which is BRL) was added at a final concentration of 800 ␮g͞ml. During 1 published as supporting information on the PNAS web site). month of selection, the concentration was gradually reduced to 250 Initially, we used RNase protection assay analysis to examine its ␮g͞ml, and 100–200 resistant colonies per transfection were visible. expression in an adult zebrafish tissue (brain), in 6-day-old zebrafish Colonies were trypsinized and propagated as a single pool. 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 Ϯ Ϯ Ϯ In Vivo Luciferase Assay and Data Analysis. In total, 3 ϫ 104 cells per was observed (4.24 0.4, 9.47 1.2, and 5.96 0.9-fold, respec- tively), reminiscent of the described (30) zfper1 and 3 genes. A peak well were seeded into a 96-well Fluoplate (Nunc). Alternate wells occurs around lights-on and a trough occurs around the end of the were left empty to minimize interference from bioluminescence light period. We then studied its expression in PAC-2 cells under crosstalk (estimated to be 2–3% in adjacent wells). After 12 h, 0.5 various lighting conditions. After entrainment for 3 days in LD, the mM beetle luciferin, potassium salt (Promega) was added. The cells were maintained in DD or in LL for 2 days; from the beginning bioluminescence was assayed with a Topcount NXT counter (2- of the third day, they were harvested. In DD (Fig. 1D), dampened, detector model, Packard). At least six independent stable transfec- rhythmic expression (2.99 Ϯ 0.5-fold rhythm) was detected with a tions were made for each construct. Each trace shows the mean of higher basal level than observed under LD conditions (3.2 Ϯ at least two independent pools, each plated in a minimum of eight 0.7-fold higher) (Fig. 1F), suggesting circadian-clock regulation. In wells. SD was also calculated and plotted. All assays were per- LL (Fig. 1E), expression was essentially arrhythmic, with a basal formed at least three times. Each well was counted for3sat Ϸ level comparable with LD (Fig. 1F). We next investigated the acute intervals of 30 min. Plates were counted in an uninterrupted cycle, response to light in larvae raised in DD or PAC-2 cells cultured for and additional empty plates were included to adjust the counting 5 days in DD. During the first 2–3 h of light, in the PAC-2 cells, there interval. Between counting, plates were illuminated with a tungsten was no change in expression relative to DD controls (Fig. 1H), ␮ ͞ 2 light source (20 W cm ). To ensure uniform illumination, trans- whereas expression was induced in the larvae (2.7 Ϯ 0.8-fold) (Fig. parent plates were intercalated between the sample plates. The 1G). Subsequently, in both larvae and cells, expression was strongly counter was located in a thermostatically controlled dark room. repressed for the duration of the light exposure (minimum 14- and Data were imported into CHRONO (Till Roenneberg, University of 20-fold repression, respectively). This final property distinguishes Munich, Munich) and EXCEL (Microsoft) by using the ‘‘Import and our gene from previously characterized vertebrate per genes, and Analysis’’ macro (S. Kay, Scripps Research Institute). Period esti- therefore, we have termed it zfper4. mates were made by linear regression after peak finder analysis with CHRONO, measured after 2 days in DD. Single-factor ANOVA In Vivo Luciferase Reporter Assay. We next developed an in vivo statistical analysis was performed with a threshold P value for luciferase reporter assay for zfper4 expression in PAC-2 cells. We ϭ significance set at P 0.05. cloned zfper4 genomic DNA extending 3.3 kb upstream from the 5Ј end of the cDNA into a luciferase reporter construct. Its sequence Raising Adult and Larval Zebrafish and RNA Analysis. Adult zebrafish revealed two canonical E-box elements (CACGTG at positions Ϫ7 (Tu¨bingen strain) were raised according to standard methods (39). and Ϫ669) and two noncanonical E-boxes (AACGTG at positions Fertilized eggs were collected within2hoflaying,andaliquots of Ϫ156 and Ϫ172) (see Fig. 3A). Cells were stably transfected with 20 eggs were transferred into 20 ml of E3 buffer in 25-cm2 tissue this construct, and then pools of clones were analyzed. This culture flasks. Flasks of cells or embryos were incubated in a commonly used approach averages out the effects of clone-to-clone large-volume thermostat-controlled water bath and illuminated variability in integration sites and copy number of the plasmids. We with a tungsten light source (11 ␮W͞cm2) or maintained in DD. observed a robust rhythm of bioluminescence in LD that matched RNA extractions and RNase protection assays were as described well with the oscillation of the endogenous zfper4 transcript, a peak (8). All experiments were performed a minimum of three times, occurring around ZT3 (Fig. 2A). Remarkably robust, rhythmic and representative results are shown. Autoradiographs were luciferase expression persisted for up to 20 days without medium scanned, and band intensities were quantified by using Scion Image renewal or supplementing with additional luciferin (Fig. 2 B and C, CELL BIOLOGY software. Zfper4 expression was normalized by using the ␤-actin and data not shown). The very low transfection-to-transfection

Vallone et al. PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 ͉ 4107 Downloaded by guest on September 28, 2021 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 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)orinLL(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; ϩ 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 chiasmatic nucleus, in which only light pulses during the subjective averaging achieved by this pooling approach. night influence gene expression (6, 15, 16). Expression was then tested under various lighting conditions. We also tested the possibility that light from the luciferase After 4 days in LD, cells were transferred to DD conditions (Fig. reaction might directly influence the cellular clock. By RNase 2B). A high-amplitude rhythm of bioluminescence established in protection assay, we examined expression of the light-inducible LD continued in DD, with a free-running period (␶) of 25.19 Ϯ zfper2 gene in the reporter cells, with or without luciferin, under DD 0.21 h. The rhythm amplitude declined progressively, with peak and (34, 35). In both cases, low, stable levels of zfper2 expression were trough values tending toward intermediate values. The cells were observed (data not shown). Therefore, light emitted during the then exposed to light at the beginning of the subjective night (Fig. luciferase reaction does not significantly influence the expression of 2B). Our results showed that starting4hafterlights-on, expression light-regulated clock genes and by inference is unlikely to represent a significant zeitgeber for the clock. steadily decreased for the duration of the light period. High- amplitude rhythmic expression with a phase matching the new LD Light Regulation of the Zebrafish Cell Circadian Clock. Our results cycle was restored within one cycle, although peaks were signifi- indicate that light influences zfper4 expression by means of the cantly lower than under the original LD conditions (compare Fig. circadian clock and acute repression. Consistent with a significant 2 B and C). A switch to LL coincided with the beginning of the entraining effect of light, reversal of the phase of the LD cycle leads subjective night and lead to a pronounced attenuation of the rhythm to complete reentrainment of the zfper4 expression rhythm within and arrhythmicity by 72 h in LL (Fig. 2C). Finally, return to LD 48 h (Fig. 2D). Furthermore, exposure of the reporter cells to LD reestablished a high-amplitude expression rhythm, although again, cycles with period lengths (T) significantly longer and shorter than peak values were lower than under the original LD cycles. Inter- 24 h (30 h, 15:15 h LD; and 20 h, 10:10 h LD) leads to adjustment estingly, when DD-adapted cells were light pulsed at the beginning of the period length of the reporter rhythm to match T (Fig. 2 E and of the subjective day, we also observed down-regulation of zfper4 F). However, on return to DD conditions, both sets of cells return expression to basal levels (the trough values observed in LD; data to a free-running period length comparable with that of cells not shown). This result contrasts with mper1 and 2 in the supra- 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 white͞black 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 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305436101 Vallone et al. Downloaded by guest on September 28, 2021 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 Ϫ669 (purple trace, blue error bars), Mut Ϫ7 (green trace, pink error bars), and Mut Ϫ7͞Ϫ669 (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 Ϫ7͞Ϫ156͞Ϫ172͞Ϫ669 (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.

The high definition of our bioluminescence rhythms facilitates Ϫ172͞Ϫ669) (Fig. 3C) lead to a further increase in expression accurate measurement of phase-shifts. We, therefore, used these levels, with a consequent reduction in rhythm amplitude and cells to quantify systematically how light pulses delivered during the repression by light as well as a considerable increase in SD of the subjective day and night phase shift the clock (40). We delivered 1- trace through the entire experiment. Statistical analysis reveals that or 4-h light pulses to DD-adapted cells at 3- or 4-h intervals, the highly significant difference between peak and trough in the respectively, throughout the subjective day and night, and then WT construct (P ϭ 4.5 ϫ 10Ϫ16) was reduced in the Mut Ϫ7͞ phase-shifts were measured after 3 days in DD. These shifts were Ϫ156͞Ϫ172͞Ϫ669 construct (P ϭ 0.01). Furthermore, the high then plotted as a function of the time of the light pulse to generate significance of the repression by light for the WT construct (P ϭ a phase–response curve. The phase-shifting properties of 1- and 4-h 1.6 ϫ 10Ϫ7) was lost in this mutant (P ϭ 0.07). The presence of light pulses were comparable (Fig. 2G) with very large phase-shifts residual rhythmicity for this construct suggests the contribution of induced by light pulses during the early subjective night (40). additional elements within the zfper4 promoter to this regulation. However, these results indicate that the presence of all four E-boxes Analysis of E-Box Function in the zfper4 Promoter. We next explored is necessary for a low, stable basal expression level, robust rhythmic which enhancers within the zfper4 promoter mediate clock regula- expression, and repression by light. tion and repression by light. We initially focused on the four E-box Finally, to determine whether the E-box-directed regulation was elements given their predicted importance within the circadian influenced by other promoter elements, we generated three het- clock (9, 10). We mutated single or multiple E-boxes (Fig. 3A) and erologous promoter constructs where four copies of each E-box then tested the expression of these constructs in stably transfected present in the zfper4 promoter were cloned upstream of a TATA PAC-2 cell pools in LD, then DD followed by a light pulse. box element and a luciferase reporter. These constructs were stably Mutation of the Ϫ669 canonical E-box (Mut Ϫ669) resulted in transfected, and their expression patterns were compared with rhythmic expression, dampening in DD and repression by light those of the zfper4 promoter constructs [Fig. 4A; data from the Ϫ7 comparable with that of the WT construct (Fig. 3B). Mutating the canonical E-box, 4ϫE-box(Ϫ7)]. For all constructs in LD, a rhyth- Ϫ7 canonical E-box alone and in combination with Ϫ669 (Mut Ϫ7 mic expression pattern was observed that persisted in DD. How- and Mut Ϫ7͞Ϫ669) resulted in a significant increase in basal ever, surprisingly this rhythm was phase-advanced by6hcompared expression and accompanying decrease in the rhythm amplitude with the zfper4 promoter (Fig. 4A). In addition, expression of the and repression by light, when expressed as fold induction and 4ϫE-box reporter constructs was not repressed by light and under repression, respectively (Fig. 3 D and E). However, mutation of all LL, rhythmic expression dampened, with peak levels remaining CELL BIOLOGY consensus and nonconsensus E-box elements (Mut Ϫ7͞Ϫ156͞ constant and trough levels progressively increasing (Fig. 4B). Sub-

Vallone et al. PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 ͉ 4109 Downloaded by guest on September 28, 2021 Fig. 4. Expression of 4ϫE-box(Ϫ7) heterologous promoter construct. (A) Comparison of the bioluminescence profiles of cells stably transfected with the zfper4 (blue trace) and 4ϫE-box(Ϫ7) promoter constructs (green trace). After two LD cycles (12:12), cells were transferred to DD. (B) The 4ϫE-box(Ϫ7) cells after two LD cycles were transferred to LL. (C) Comparison of 4ϫE-box(Ϫ7) 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 observed representing the so-called dead zone (40). These prop- thymidine kinase or SV40 minimal promoter sequences did not erties are consistent with our results showing that the clock entrains alter these properties, although the basal levels of expression were rapidly to large phase shifts in the entraining LD cycle. Further- increased (data not shown). Interestingly, the phase differences more, it synchronizes with LD cycles with a broad range of period between the zfper4 and 4ϫE-box promoter rhythms are a function lengths. Thus, our results implicate light as a strong zeitgeber for of T of the entraining LD cycle (Fig. 4 C and D). Thus, where T ϭ this cellular clock. Therefore, one may anticipate the importance of 20 h, the 4ϫE-box rhythm is phase-advanced by only 2.9 (Ϯ0.3) h direct light entrainment for peripheral clocks in the context of the (Fig. 4C), whereas where T ϭ 30 h, the phase advance is 11.3 zebrafish circadian system. (Ϯ0.49) h (Fig. 4D). This result seems to be the consequence of the We have analyzed the zfper4 promoter by stably transfecting phase of the zfper4 promoter rhythm being locked so that its peak luciferase promoter reporter constructs into PAC-2 cells and then occurs 2–4 h after lights-on, whereas the 4ϫE-box peak shifts from testing for regulation by light and the endogenous clock. This the beginning of the light period (T ϭ 20) to the middle of the dark approach has the major advantage that regulation by physiological period (T ϭ 30). These results imply a significant contribution of the levels of endogenous factors is tested. Performing promoter analysis local promoter environment to the phase and light responsiveness with transgenic animals offers similar advantages, but it has the of E-box-generated expression rhythms. drawback of being far more time consuming. Many previous studies exploring transcriptional regulation in the clock have been based on Discussion transient transfection assays. However, such studies may be mis- Here, we describe an example of a vertebrate period gene that is leading because overexpression of a candidate regulator may drive repressed by light and shares significant homology with per1. physiologically nonrelevant interactions. Here, we demonstrate in a Expression of a zebrafish per1 homolog has been described, vertebrate cell culture model that a functional circadian clock drives although sequence data were not presented (30). Rhythmic rhythmic expression by means of E-box elements in the context of expression was documented in LD and DD conditions and a a minimal heterologous promoter. This result is consistent with transient induction was observed in response to a light pulse (30). current models for the circadian clock; however, these rhythms are Given that the per gene documented here is repressed strongly 6-h phase-advanced compared with the zfper4 promoter. It is by light in both larvae and PAC-2 cells, we believe that it most therefore clear that E-boxes can direct rhythmic expression with likely represents a homolog that we have named zfper4. The significant differences in phase depending on their promoter con- preceding, relatively weak induction of zfper4 expression ob- text. Furthermore, the phase relationship of the E-box and pro- served only in larvae may indicate cell-type specificity in this moter rhythms varies depending on the period length of the light response. The existence of more than three per genes in entraining LD cycle. This observation points to light also playing a zebrafish could be anticipated because many mammalian genes key role in determining the phase of the promoter rhythms. have been reported to have two paralogues in zebrafish as the Mutational analysis has demonstrated that the E-boxes contrib- result of a whole-genome duplication during the evolution of the ute to maintaining a low level of promoter expression. This result teleost lineage (41). Furthermore, six cryptochrome genes have is surprising because they would be predicted to bind Clock͞BMAL been described in zebrafish, suggesting additional complexity in and, thus, function as enhancers. Indeed, mutation of circadian zebrafish clock gene families (28, 42). E-boxes has been documented (25, 43) to reduce expression levels We present phase–response curve data for the phase-shifting in vivo. We have also implicated these elements in robust rhythmic effects of light on a cell culture clock. We demonstrate that the expression and down-regulation by light. Interestingly, in the con- PAC-2 clock shows a typical high-amplitude phase–response curve text of a heterologous promoter, these E-box elements direct (type 0). Maximum phase shifts are obtained with light pulses rhythmic expression that is not repressed by light pulses, implying delivered during the early subjective night. In addition, at the that the local promoter environment might determine their func- beginning of the early subjective day, only small phase delays are tion. It is noteworthy that none of the four E-boxes corresponds to

4110 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305436101 Vallone et al. Downloaded by guest on September 28, 2021 the optimal binding sites for mammalian Clock͞BMAL (22). It will ideal also for studying light-input pathways. We have established be interesting to test whether the additional zebrafish Clock and PAC-2 luciferase reporter cell lines that significantly increase the BMAL homologs bind differentially to these elements and, thereby, value of this culture system. The high definition of the biolumines- confer light-responsive, rhythmic expression with low basal levels cence data obtained may be explained by the emission of light from (44). a static monolayer of uniformly expressing cells. Furthermore, the The repression of zfper4 expression after exposure to light occurs viability of these cells during long periods at confluence, the only after a delay of 4 h. This result suggests earlier induction of a stability of luciferin in the culture medium, and the ease with which repressor factor. Expression of the zfper2 gene is induced within the it can diffuse into the cells are all likely to contribute to the stability first2hafterlightexposure (30, 36). Given the role of Per proteins of the luciferase signal. The ability to maintain reproducible, in the circadian timing mechanism, it is tempting to speculate that high-amplitude bioluminescence rhythms over long time periods light-induced zfper2 may down-regulate zfper4 expression by means contrasts with the transient, dampening rhythms described for of the E-box elements. Per proteins seem to function in combina- mammalian cell lines (32). Furthermore, the growth of these cells tion with Crys to repress Clock:BMAL heterodimer activation. In at room temperature in atmospheric CO2, the use of a 96-well plate the chicken pineal, light has been shown to acutely induce Cry format, and a high-throughput automated scintillation counter to expression (45). However, the lack of repression of the 4ϫE-box perform the luciferase assay make these cells ideal for large-scale heterologous promoter constructs by light would tend to argue analysis. against this. Alternatively, light may induce expression of other transcriptional repressors that bind to distinct enhancer elements We thank T. Roenneberg, M. Merrow, K. Tamai, and all laboratory and then interact with E-box-bound factors in the context of the members for helpful discussions and T. Roenneberg for adapting CHRONO for Topcount files. D.V. and S.B.G. were supported by the Max promoter (46). Planck Society. N.S.F. was supported by Centre National de la Recher- Zebrafish cell lines offer many advantages for studying the che Scientifique and Max Planck funding; D.W. was supported by funds vertebrate circadian clock. They express a functional clock that can from the Biotechnology and Biological Sciences Research Council and be entrained by direct light exposure (29). For this reason, they are the Wellcome Trust.

1. Pittendrigh, C. S. (1993) Annu. Rev. Physiol. 55, 16–54. 25. McDonald, M. J., Rosbash, M. & Emery, P. (2001) Mol. Cell. Biol. 21, 2. Roenneberg, T., Daan, S. & Merrow, M. (2003) J. Biol. Rhythms 18, 183–194. 1207–1217. 3. Klein, D. M., Moore, R. Y. & Reppert, S. M. (1991) Suprachiasmatic Nucleus: 26. Ripperger, J. A., Shearman, L. P., Reppert, S. M. & Schibler, U. (2000) Genes The Mind’s Clock (Oxford Univ. Press, New York). Dev. 14, 679–689. 4. Menaker, M., Moreira, L. F. & Tosini, G. (1997) Braz. J. Med. Biol. Res. 30, 27. Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., 305–313. Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P., et 5. King, D. P., Zhao, Y., Sangoram, A. M., Wilsbacher, L. D., Tanaka, M., al. (1996) Development (Cambridge, U.K.) 123, 1–36. Antoch, M. P., Steeves, T. D., Vitaterna, M. H., Kornhauser, J. M., Lowrey, 28. Cahill, G. M. (2002) Cell Tissue Res. 309, 27–34. 89, P. L., et al. (1997) Cell 641–653. 29. Whitmore, D., Foulkes, N. S. & Sassone-Corsi, P. (2000) Nature 404, 87–91. 6. Zylka, M. J., Shearman, L. P., Weaver, D. R. & Reppert, S. M. (1998) Neuron 30. Pando, M. P., Pinchak, A. B., Cermakian, N. & Sassone-Corsi, P. (2001) Proc. 20, 1103–1110. Natl. Acad. Sci. USA 98, 10178–10183. 7. Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block, 31. Balsalobre, A., Damiola, F. & Schibler, U. (1998) Cell 93, 929–937. G. D., Sakaki, Y., Menaker, M. & Tei, H. (2000) Science 288, 682–685. 32. Izumo, M., Johnson, C. H. & Yamazaki, S. (2003) Proc. Natl. Acad. Sci. USA 8. Whitmore, D., Foulkes, N. S., Strahle, U. & Sassone-Corsi, P. (1998) Nat. Neurosci. 1, 701–707. 100, 16089–16094. 9. Lowrey, P. L. & Takahashi, J. S. (2000) Annu. Rev. Genet. 34, 533–562. 33. Delaunay, F., Thisse, C., Marchand, O., Laudet, V. & Thisse, B. (2000) Science 10. Reppert, S. M. & Weaver, D. R. (2001) Annu. Rev. Physiol. 63, 647–676. 289, 297–300. 11. Konopka, R. J. & Benzer, S. (1971) Proc. Natl. Acad. Sci. USA 68, 2112–2116. 34. Hirayama, J., Fukuda, I., Ishikawa, T., Kobayashi, Y. & Todo, T. (2003) Nucleic 12. Tei, H., Okamura, H., Shigeyoshi, Y., Fukuhara, C., Ozawa, R., Hirose, M. & Acids Res. 31, 935–943. Sakaki, Y. (1997) Nature 389, 512–516. 35. Delaunay, F., Thisse, C., Thisse, B. & Laudet, V. (2003) Gene Expression 13. Bae, K., Jin, X., Maywood, E. S., Hastings, M. H., Reppert, S. M. & Weaver, Patterns 3, 319–324. D. R. (2001) Neuron 30, 525–536. 36. Cermakian, N., Pando, M. P., Thompson, C. L., Pinchak, A. B., Selby, C. P., 14. Shearman, L. P., Jin, X., Lee, C., Reppert, S. M. & Weaver, D. R. (2000) Mol. Gutierrez, L., Wells, D. E., Cahill, G. M., Sancar, A. & Sassone-Corsi, P. (2002) Cell. Biol. 20, 6269–6275. Curr. Biol. 12, 844–848. 15. Shigeyoshi, Y., Taguchi, K., Yamamoto, S., Takekida, S., Yan, L., Tei, H., 37. Zhuang, M., Wang, Y., Steenhard, B. M. & Besharse, J. C. (2000) Brain Res. Moriya, T., Shibata, S., Loros, J. J., Dunlap, J. C. & Okamura, H. (1997) Cell Mol. Brain Res. 82, 52–64. 91, 1043–1053. 38. Lin, S., Gaiano, N., Culp, P., Burns, J. C., Friedmann, T., Yee, J. K. & Hopkins, 16. Shearman, L. P., Zylka, M. J., Weaver, D. R., Kolakowski, L. F., Jr., & Reppert, N. (1994) Science 265, 666–669. S. M. (1997) Neuron 19, 1261–1269. 39. Dekens, M. P., Santoriello, C., Vallone, D., Grassi, G., Whitmore, D. & 17. Maywood, E. S., Mrosovsky, N., Field, M. D. & Hastings, M. H. (1999) Proc. Foulkes, N. S. (2003) Curr. Biol. 13, 2051–2057. 96, Natl. Acad. Sci. USA 15211–15216. 40. Johnson, C. H. (1999) Chronobiol. Int. 16, 711–743. 18. Bae, K. & Weaver, D. R. (2003) J. Biol. Rhythms 18, 123–133. 41. Postlethwait, J. H., Yan, Y. L., Gates, M. A., Horne, S., Amores, A., Brownlie, 19. Albrecht, U., Zheng, B., Larkin, D., Sun, Z. S. & Lee, C. C. (2001) J. Biol. A., Donovan, A., Egan, E. S., Force, A., Gong, Z., et al. (1998) Nat. Genet. 18, Rhythms 16, 100–104. 345–349. 20. Cermakian, N., Monaco, L., Pando, M. P., Dierich, A. & Sassone-Corsi, P. (2001) EMBO J. 20, 3967–3974. 42. Kobayashi, Y., Ishikawa, T., Hirayama, J., Daiyasu, H., Kanai, S., Toh, H., 21. Munoz, E., Brewer, M. & Baler, R. (2002) J. Biol. Chem. 277, 36009–36017. Fukuda, I., Tsujimura, T., Terada, N., Kamei, Y., et al. (2000) Genes Cells 5, 22. Hogenesch, J. B., Gu, Y. Z., Jain, S. & Bradfield, C. A. (1998) Proc. Natl. Acad. 725–738. Sci. USA 95, 5474–5479. 43. Hao, H., Allen, D. L. & Hardin, P. E. (1997) Mol. Cell. Biol. 17, 3687–3693. 23. Gekakis, N., Staknis, D., Nguyen, H. B., Davis, F. C., Wilsbacher, L. D., King, 44. Ishikawa, T., Hirayama, J., Kobayashi, Y. & Todo, T. (2002) Genes Cells 7, D. P., Takahashi, J. S. & Weitz, C. J. (1998) Science 280, 1564–1569. 1073–1086. 24. Darlington, T. K., Wager-Smith, K., Ceriani, M. F., Staknis, D., Gekakis, N., 45. Yamamoto, K., Okano, T. & Fukada, Y. (2001) Neurosci. Lett. 313, 13–16. Steeves, T. D., Weitz, C. J., Takahashi, J. S. & Kay, S. A. (1998) Science 280, 46. Doi, M., Nakajima, Y., Okano, T. & Fukada, Y. (2001) Proc. Natl. Acad. Sci. 1599–1603. USA 98, 8089–8094. CELL BIOLOGY

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