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1 Department of Zoology and
2 Department of Neurobiochemisty, George S Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
3 Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel
4 Max-Planck-Institut fur Entwicklungsbiologie, Spemannstrasse 35-39, Tubingen, D-72076 Germany
5 Department of Biochemistry and Biological Sciences, Faculty of Chemistry, Biochemistry and Pharmacy, San Luis University, Chacabuco y Pedernera, Edif. El Barco, 2do. piso., San Luis, CP 5700 Argentina
6 Israel Oceanographic and Limmnological Research, Haifa 31080, Israel
(Requests for offprints should be addressed to Y Gothilf; Email: yoavg{at}tauex.tau.ac.il)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Melatonin is an important component of the endogenous circadian clock system in vertebrates. This hormone is typically produced during the night at two major sites: the pineal gland and the retinal photo-receptor cells (Klein et al. 1997). Pineal gland melatonin plays an endocrine role in the regulation of a variety of daily and annual physiological rhythms (Arendt 1995, Ekstrom & Meissl 1997). Retinal melatonin plays a paracrine role related to photoreceptor adaptation to darkness; it does not contribute to the large rhythms of circulating melatonin (Cahill & Hasegawa 1997).
Melatonin rhythms are generated by changes in the activity of serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT), a pineal gland- and retinal-specific enzyme (Klein et al. 1997). The increased production of melatonin during the night reflects increased AANAT activity and, in many species studied, increased aanat transcription (Ganguly et al. 2002, Falcon et al. 2003). Rhythmic aanat expression, AANAT activity, and hence melatonin production, are driven by an internal circadian clock and by external light signals (Foulkes et al. 1997, Klein et al. 1997, Li et al. 1998, Ganguly et al. 2002, Tosini & Fukuhara 2003). Moreover, E-box elements present in regulatory regions of aanats were shown to bind and mediate the activity of BMAL:CLOCK heterodimer (Chen & Baler 2000, Chong et al. 2000, Appelbaum et al. 2005). Consequently, aanat is considered a clock-controlled gene that serves as a link between the core molecular oscillator and the hormonal output signal, which in turn affects the whole organism.
In mammals, only a single aanat gene has been identified and it is expressed in both the pineal gland and retina. Like many other photoreceptor-specific genes, enhanced expression of aanat in these tissues is mediated by cone rode homeobox proteins (CRXs). A study with the crx knockout mouse revealed that CRX is essential for the expression of aanat in the pineal gland (Furukawa et al. 1999). Exclusively among vertebrates, teleost fishes have two aanats, aanat1 and aanat2 (Coon et al. 1999, Falcon et al. 2003, Zilberman-Peled et al. 2004). In zebrafish, aanat1 is expressed only in the retina while aanat2 is expressed in both the pineal gland and, at lower levels, also in the retina (Falcon et al. 2003).
In the current study, in vivo and in vitro analyses of aanat1 and aanat2 regulation by light, the circadian clock and its components were performed. The results indicate differential regulation of expression of these two related genes, highlighting that a combination of mechanisms fine-tune circadian clock-regulated outputs.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Zebrafish were raised in a light- and temperature-controlled recirculating water system under a 12 h light:12 h darkness (LD) cycle. To produce wild-type embryos, male and female zebrafish were paired in the evening, and spawning occurred within 1 h of lights on. Heterozygous TG(AANAT2:EGFP)Y8 transgenic embryos expressing enhanced green fluorescent protein (EGFP) in the pineal gland (Gothilf et al. 2002) were obtained from an outcross of adult heterozygous TG(AANAT:EGFP)Y8 and wild-type fish. Injected and un-injected wild-type and transgenic embryos were kept in a light-controlled refrigerated incubator at 28 °C as described (Appelbaum et al. 2004) and were collected at different time-points for in situ hybridization (ISH) analysis or for evaluation of EGFP expression.
Whole mount ISH
Embryos/larvae were fixed overnight in 4% paraform-aldehyde and stored in 100% methanol. Whole mount ISH was performed with digoxyginin-labeled EGFP probe at a concentration of 1 ng/µl. Detection and quantification of EGFP mRNA was as described for aanat2 (Ziv et al. 2005). Differences in the signal intensity between sampling times were determined using two-way ANOVA analysis.
In vivo transient expression assays of aanat2 promoter constructs
In vivo transient expression assays of promoter–EGFP constructs were performed by microinjection of zebrafish embryos as described (Appelbaum et al. 2004). Each construct (see below and Fig. 3A
) contained the aanat2 promoter with various deletions and mutations, the EGFP reporter gene and a 257 bp enhancer termed pineal restrictive downstream module (PRDM) (Appelbaum et al. 2004). The PRDM was included because it was shown to be required for the pineal-specific expression of aanat2 (Appelbaum et al. 2004). Each construct was tested in more than a hundred embryos and the expression patterns, pineal specific, ectopic or both, as illustrated in previous studies (Appelbaum et al. 2004, 2005), were monitored. Statistical differences in the expression pattern were determined by 
2 analysis.
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– 1320 AANAT2-EGFP-PRDM
A 1320 bp fragment was PCR amplified using AANAT2-EGFP-PRDM (Appelbaum et al. 2004; Fig. 3A
) as a template and sets of primers containing SalI and BamHI restriction sites. The PCR product was double digested with SalI and BamHI and ligated into SalI/BamHI-cut AANAT2-EGFP-PRDM, in place of the original promoter (GenBank accession number AF494081
[GenBank]
), yielding a 1320 bp promoter with a 343 bp 5' deletion.
– 502 AANAT2-EGFP-PRDM
A construct with 502 bp of aanat2 promoter, i.e. a 1161 bp 5' deletion, was generated as described above.
– 254 AANAT2-EGFP-PRDM
1409 bp of the 5' region of the aanat2 promoter were deleted, yielding a 254 bp promoter. AANAT2-EGFP-PRDM was digested with PstI to produce two fragments, 1415 and 4742 bp; the latter was religated, yielding the desired construct.
AANAT2(
-1237/-300)-EGFP-PRDM
A fragment from the center of the 1663 bp aanat2 promoter was removed, yielding a 726 bp promoter. AANAT2-EGFP-PRDM was digested with SpeI to produce two fragments, 937 and 5220 bp; the latter was re-ligated, yielding the desired construct.
AANAT2(-1237/-300)-EGFP-PRDM and E-box mutation
The two E-boxes within the 726 bp promoter, located at positions 1472–1477 and 84–89 upstream to the transcription start site, were each mutated. For each E-box, two complementary primers containing the desired mutation were utilized to introduce the mutations (CACGTG to CTCGAG) into AANAT2(-1237/-300)-EGFP-PRDM, using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) as instructed by the manufacturer. Colonies with the desired mutation were selected on the basis of a PmlI and XhoI digestion pattern and sequencing. The resulting constructs were named AANAT2(-1237/-300)-ME1-EGFP-PRDM and AANAT2(-1237/-300)-ME2-EGFP-PRDM (Fig. 3A).
AANAT2(-1237/-300)-MP-EGFP-PRDM
The three photoreceptor conserved elements (PCE) (TAATT) within the 726 bp promoter, originally located at positions –282 (PCE3), –1591 (PCE2) and –1605 (PCE1), were mutated. For each PCE, two complementary primers containing the desired mutation were sequentially utilized to introduce the mutations (TAATT was changed to TCGAG, GATCT and GGGCC for PCE1, PCE2 and PCE3 respectively) into AANAT2(-1237/-300)-EGFP-PRDM, as described above, to obtain a triple PCE mutation construct. Colonies with the desired mutation were selected on the basis of XhoI, BglII and ApaI digestion pattern for PCE1, PCE2 and PCE3 respectively and sequencing.
Real-time PCR quantification of aanat mRNAs in the eyes
Expression of zebrafish, aanat1, aanat2 and glyceraldehyde 3-phosphate dehydrogenase (g3pdh) in the eyes throughout the 24-h cycle was determined at the transcript level using quantitative PCR assays. Five adult fishes were killed every 4 h. Fish were anesthetized in tricane, decapitated, the eyes removed and total RNA was extracted using TriPure RNA isolation reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The mRNA (200 ng) was reverse transcribed using Oligo(dT) primer and MMLV reverse transcriptase (Promega, Madison, WI, USA). Transcript levels were determined by real-time PCR using the GeneAmp 5700 PCR thermocycler (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Triplicate first-strand cDNA aliquots (2µl) from each sample served as templates in PCR using master mix, SYBR Green I fluorescent dye (Applied Biosystems) and 200 nM gene-specific primers. Amplification reactions were carried out under the following conditions: incubation at 50 °C for 2 min, then 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The copy number in unknown samples was determined by comparing threshold cycles values with those of recombinant plasmid standards (0·1–100 pmol) containing the cDNA inserts and normalized to the amount of g3pdh mRNA in each sample. Data were log transformed to achieve normality for statistical analysis. The significance of the effects of the photoperiodic condition (LD and constant darkness (DD), the time of day (ZT) and interactions of these variables on the levels of each mRNA was determined by ANOVA with repeated measures. Specific comparisons were performed using Tukey’s post hoc tests.
aanat–luciferase constructs and expression vectors for in vitro analyses
pGL3–498 AANAT2
The proximal aanat2 promoter was placed upstream of a luciferase reporter gene in the pGL3 vector (Promega). A fragment containing 498 bp of 5'-flanking region and the 122 bp 5' UTR of the aanat2 promoter was PCR amplified using AANAT2-EGFP-PRDM as a template and sets of primers containing NheI and BglII restriction sites. The PCR product was double digested with NheI and BglII and ligated into NheI/BglII-cut pGL3.
pGL3–784 NAT1
A fragment containing 784 bp of 5'-flanking region and the 92 bp 5' UTR of the aanat1 promoter was placed upstream of a luciferase reporter gene, as described for pGL3–498 AANAT2.
Cytomegalovirus (CMV) promoter-driven mouse CLOCK (mCLOCK) and hamster BMAL1 (hBMAL1) expression vectors were generously provided by Drs N Gekakis and C Weitz, Harvard University, MA, USA. Zebrafish orthodentical homeobox 5 (OTX5) expression vector (CMV driven) was generously provided by Dr Jennifer Liang, Case Western Reserve University, Cleveland, OH, USA.
Establishment of stable PAC-2 cell lines
PAC-2 cells were cultivated as previously described (Whitmore et al. 2000, Vallone et al. 2004). Cells were transfected with linearized plasmids; the aanat–luciferase constructs (see above) 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 µF, using a Gene Pulser apparatus (Bio-Rad). Three days later, G-418 (GIBCO/BRL) was added at a final concentration of 800 µg/ml. During 1 month of selection, the concentration was gradually reduced to 250 µg/ml and 100–200 resistant colonies per transfection were obtained. Colonies were trypsinized and propagated as a single pool.
Luciferase assay in stable PAC-2 cell lines
In total, 3 x 104 cells per well were seeded into a 96-well Fluoplate (Nalge Nunc, Rochester, NY, USA). 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 (two-detector model; Packard). At least six independent stable transfections were made for each construct. 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. Between counting, plates were illuminated with a tungsten light source (20µW/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 (T Roenneberg, University of Munich, Munich, Germany) and EXCEL (Microsoft) using the ‘Import and Analysis’ macro (S Kay, Scripps Research Institute, CA, USA). Period estimates were made by linear regression after peak finder analysis with CHRONO, measured under DD.
In vitro transient transfection assays in NIH-3T3 cells
NIH-3T3 cells (ATCC, CRL-1658) were plated and transfected with 10 ng luciferase reporter vector driven by aanat1 (pGL3–784 AANAT1) or aanat2 (pGL3–498 AANAT2) promoters and 0·75 µg of a 1:1:1 expression vector mix (hBMAL1/mCLOCK/OTX5) or empty vector pcDNA (as described in Appelbaum et al. 2005). Differences in transfection efficiency were taken into account by measuring the enzyme activity generated by a co-transfected thymidine kinase promoter-driven Renilla luciferase plasmid (0·5 ng). Firefly and Renilla luciferase enzyme activities were measured using the Stop and Glo kit (Promega) following the manufacturer’s instructions. Data were subjected to two-way ANOVA analysis. Results are the means of at least three independent experiments each performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Manual and computer analysis of the 5' regulatory regions of the two genes revealed several putative transcription factor binding sites, some of which are shared by both promoters (Fig. 1). Two perfect E-box elements (CACGTG) were found in both promoters at approximately the same position upstream of the transcription start sites; nucleotides 84–89 and 1472–1477 in aanat2 (Gothilf et al. 2002) and nucleotides 120–125 and 1447–1452 in aanat1 (Fig. 1). Multiple copies of the PCE (TAATT/C) were present in both promoters; four PCEs in the aanat2 and 18 PCEs in the aanat1 promoter (Fig. 1). This element is known to mediate photoreceptor-specific expression by recruiting CRX and OTX5 in mice and zebrafish respectively (Chen et al. 1997, Li et al. 1998, Furukawa et al. 1999, Gamse et al. 2002, Appelbaum et al. 2005).
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). CRE has been shown to mediate the norepinephrine/cAMP-induced aanat expression in the rat pineal gland (Baler et al. 1997). In addition, an activator protein-1 (AP-1)-like binding site (Angel et al. 1987), a key element implicated in cell proliferation and speciation (Angel & Karin 1991), was centered at position –271 in aanat1 and –224 and –307 in aanat2. Clock-controlled regulation of the pineal aanat2 promoter
To determine, in vivo, whether the clock-controlled aanat2 mRNA rhythm reflects rhythmic promoter activity, EGFP mRNA levels in the pineal gland of TG(AANAT2:EGFP)Y8 transgenic zebrafish embryos (Gothilf et al. 2002) were monitored throughout the 24-h cycle. TG(AANAT2:EGFP)Y8 heterozygous embryos were kept under LD during the first 2 days of development. Under these photoperiodic conditions, the oscillator that drives rhythmic expression of pineal aanat2 is functional (Gothilf et al. 1999, Ziv et al. 2005). On the third day of development, embryos were transferred to DD or kept under normal LD, collected at 3-h intervals for 36 h (39–75 hours post fertilization), and subjected to whole mount ISH analysis for EGFP mRNA. Under both LD and DD, embryos exhibited significant (P < 0·001) day/night differences in EGFP mRNA expression (Fig. 2), indicative of rhythmic activity of the aanat2 promoter.
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The aanat2 promoter was analyzed in vivo by microinjection of promoter–EGFP constructs into zebrafish zygotes followed by monitoring of fluorescence. Similar in vivo analysis of the aanat1 promoter resulted in occasional ectopic EGFP expression but with no retinal expression, either because of late differentiation of the retina or the absence of elements in the cloned promoter that are essential for retinal expression.
Injection of the AANAT2-EGFP-PRDM construct, which contained the entire promoter, resulted in EGFP expression in 39% of injected embryos. Among these EGFP-positive embryos, a signal in the pineal gland was observed in 87% and most exhibited a restricted signal. Ectopic expression of EGFP was observed in only 22% of EGFP-positive embryos (Fig. 3B), confirming previous results (Appelbaum et al. 2004, 2005). Constructs with 5' promoter deletions, –1320AANAT2-EGFP-PRDM, –502 AANAT2-EGFP-PRDM and –254 AANAT2-EGFP-PRDM (Fig. 3A), generated significantly (P < 0·01) lower pineal expression and higher ectopic expression (Fig. 3B). Injection of AANAT2(-1237/-300)-EGFP-PRDM (Fig. 3A), which contains 726 bp of the proximal and distal promoter and lacks a region of repetitive DANA sequences (Izsvak et al. 1996), resulted in high levels of pineal EGFP expression (82%) and reduced ectopic expression (30%), similar to the wild-type construct (AANAT2-EGFP-PRDM; Fig. 3B). Since these promoter regions include E-box and PCE elements, the function of these elements was tested by injection of mutated constructs. Injection of E-box-mutated constructs (AANAT2(-1237/-300)-ME1-EGFP-PRDM and AANAT2(-1237/-300)-ME2-EGFP-PRDM; Fig. 3) significantly (P < 0·001) reduced the pineal gland-specific expression and increased ectopic expression as compared with wild-type and the 726 bp promoters. Among the EGFP-expressing embryos, only 26% and 30% showed pineal expression and 97% and 98% showed ectopic expression for ME1 and ME2 respectively (Fig. 3B). The triple PCE mutation had an even stronger effect. Injection of AANAT2(-1237/-300)-MP-EGFP-PRDM (Fig. 3A) resulted in ectopic expression in all EGFP-expressing embryos; 8% also demonstrated pineal expression (Fig. 3B). These results indicated that E-boxes and PCEs, distributed in the proximal and distal promoter, are essential for accurate transcription of aanat2.
Differential expression of aanats in the zebrafish retina
The daily expression pattern of aanat1 and aanat2 in the eyes was determined throughout the 24-h cycle. This analysis revealed a significant (P < 0·01) effect of ZT on the mRNA levels of both aanats when fish were kept under normal LD cycles; both genes exhibited a daily rhythm (Fig. 4). Interestingly, under these conditions aanat2 mRNA levels peaked at ZT10 and started to decline at ZT18, while aanat1 mRNA levels peaked at ZT14 and started to decline at ZT22. These results suggested that under LD the two genes exhibit a rhythmic expression pattern with different phase.
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), indicating that a retinal circadian oscillator controls expression of aanat2 mRNA. In contrast, aanat1 mRNA levels were similar throughout the 24-h cycle, except for the last time-point, indicating that rhythmic aanat1 expression was lost when fish were transferred into DD. These results suggested that different transcriptional regulatory mechanisms might drive expression of these two related genes within the same tissue. Clock-controlled regulation of the two aanat promoters
The activities of aanat1 and aanat2 promoters were tested by stable transfection in the PAC-2 zebrafish cell line, which contains a light entrainable clock (Vallone et al. 2004). Cells were stably transfected with the constructs pGL3–784 AANAT1 and pGL3–498 AANAT2 in which luciferase is driven by promoters that contain one E-box. Expression was tested by exposure of the transfected cells to an LD cycle followed by DD and then reversed light:darkness cycles (DL).
The aanat2 promoter drove a rhythm of expression under LD conditions with a peak of luciferase activity at ZT20·4 ± 1·47 as determined by peakfinder analysis (Fig. 5). This rhythmic expression was also maintained in DD with a free-running period of 25·19 ± 0·2 h and an accompanying rise in the basal expression levels (Fig. 5). When cells were then subjected to DL conditions, the phase of the rhythm gradually shifted to match the new light:darkness cycle but was still not completely entrained following three DL cycles. These results were consistent with previous observations on the entrainment of expression rhythms directed by E-box enhancer elements in PAC-2 cells (Vallone et al. 2004). The aanat1 promoter showed a lower amplitude and less robust activity rhythm; however, in LD and DD, the timing of increases in aanat1 expression resembled that of the aanat2 peaks of expression. Thus, these stable luciferase reporter assays indicate that, in the context of PAC-2 cells, the aanat2 and possibly the aanat1 promoter is controlled by the endogenous, light-entrainable circadian oscillator.
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Previous results indicated that OTX5 and BMAL/CLOCK synergistically bind PCEs and E-box elements respectively, located within the PRDM region (Appelbaum et al. 2005). In order to test, in vitro, the action of BMAL/CLOCK and OTX5 on the PCE- and E-box-containing aanat promoters, the pGL3–784 AANAT1 and pGL3–498 AANAT2 promoter–reporter constructs were co-transfected with either an empty vector (pcDNA) or mixtures of mBMAL/hCLOCK and OTX5 expression vectors into NIH-3T3 cells. Co-transfection of the promoter–reporter constructs with OTX5 or BMAL/CLOCK alone did not increase reporter gene expression over control levels (Fig. 6). Nevertheless, co-transfection with both hBMAL/mCLOCK and OTX5 resulted in a > 2-fold and > 3·4-fold increase in luciferase activity, indicating that BMAL/CLOCK and OTX5, possibly through the E-box and PCE elements, cooperatively enhance the activity of aanat promoters.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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AANAT activity is regulated at the transcriptional, post-transcriptional and post-translational levels (Ganguly et al. 2002, Klein et al. 2002, Falcon et al. 2003, Kim et al. 2005). The current study focuses on the transcriptional regulation of this clock-controlled gene. Strong support for the involvement of the core oscillator comes from the rhythmic activity of both zebrafish aanat promoters in the zebrafish PAC-2 cell line. Moreover, the rhythms generated by the aanat2 promoter have similar characteristics to those generated by a heterologous promoter construct containing multimerized E-boxes (Vallone et al. 2004). Rhythmic transcription of zebrafish aanats is regulated in part by the interaction of clock proteins BMAL/CLOCK and the homeobox protein OTX5. This has been shown in vitro using NIH-3T3 cells where aanat promoter activities were enhanced by co-transfection of BMAL/CLOCK:OTX5 (Fig. 6), and was further confirmed in vivo by demonstrating that their putative binding sites in the promoter, the E-box and PCE elements respectively, are important for enhanced pineal expression (Fig. 3).
The effect of BMAL/CLOCK/OTX5 on aanat1 promoter activity was higher than for the aanat2 promoter when tested in NIH-3T3 cells (Fig. 6). A reasonable explanation for these differences could be the larger number of PCE sites in the tested aanat1 promoter. These promoter differences are also reflected in the retinal expression levels of the two genes (Fig. 4). An abundance of PCE sites occurs in other photo-receptor specific genes including chicken and rat aanat, rat pineal night-specific ATPase (pina) and zebrafish exorhodopsin (exorh) ( Li et al. 1998, Chen & Baler 2000, Chong et al. 2000, Asaoka et al. 2002). In aanat2, additional PCEs are found in a downstream regulatory region, PRDM. This region, in conjunction with the promoter, has been shown to be important for enhanced pineal expression (Appelbaum et al. 2004). The current study confirms this observation and extends it by identifying PCE-containing regions and E-boxes as promoter elements that are important for this function. Although PRDM can drive enhanced pineal expression of the aanat1 promoter (Appelbaum et al. 2004), it is as yet not known what drives retinal-specific expression of aanat1. Taken together, functional analyses of the aanat promoters in the current study link the transcriptional machinery of the core oscillator with the regulation of clock-controlled genes in photoreceptors.
In addition to the apparent common clock regulation, the results of the current study suggest that phase differences exist in the rhythmic expression of retinal aanat1 and aanat2. A somewhat similar pattern was also found in pike; retinal aanat1 mRNA peaks 6 h before that of pineal aanat2 and this phase-lag persists under constant conditions, indicating differences between retinal and pineal clock regulation in the pike (Coon et al. 1999). Here, however, we show that these differences occur within the same tissue, the retina. Furthermore, in vivo, aanat2 expression in pineal gland and retina is primarily, but not only, controlled by the circadian oscillator (Gothilf et al. 1999, Gamse et al. 2002, Ziv et al. 2005; Fig. 4) while aanat1 is, at least in part, light regulated as was suggested for trout aanat1 (Mizusawa et al. 2000, Besseau et al. 2005). These differences may indicate a gene-specific regulation within the retinal photoreceptors, or a different spatial expression pattern; the two aanats are expressed in different subsets of retinal cells. The apparent contradiction between the in vivo and in vitro expression pattern of aanat1 may reflect regulatory differences between retinal photoreceptor cells and the PAC-2 cell line; although PAC-2 cells contain a light-entrainable circadian oscillator, the phototransduction mechanism may well be distinct from that present in the retina.
Gene duplication, commonly seen in teleost fish, is the result of a whole genome duplication that is predicted to have happened at the time when fish emerged from the vertebrate lineage. Functional subdivision may occur when two genes, formerly served by a single ancestral gene, are expressed in separate subsets of cells (Amores et al. 1998, Force et al. 1999). This may be the case with fish aanat; the ancestral gene might have been expressed in all photoreceptor cells, as seen in chickens and mammals, while subsequent mutations in the duplicated genes subdivided their function and profile of expression to the pineal gland (aanat2) and retina (aanat1), as seen in trout, pike and seabream (Coon et al. 1999, Benyassi et al. 2000, Zilberman-Peled et al. 2004). Since in zebrafish both aanat genes are co-expressed in the retina, it is likely that they are required for different retinal functions (Zilberman-Peled et al. 2006). This possibility is reflected in their differential tissue distribution and in their differential regulation by the circadian clock and light in the retina (Fig. 4).
Retinal aanat/melatonin is generally associated with visual adaptation to darkness (Cahill & Hasegawa 1997). However, in several fish species retinal melatonin levels were found to be high during the day (Falcon et al. 2003, Zilberman-Peled et al. 2006). In frog retina, a poor correlation between aanat activity and melatonin production was found (Delgado et al. 1993). Moreover, in monkey and rat retina, aanat is expressed in the inner nuclear layer while there is no evidence in support of melatonin synthesis in this region (Coon et al. 2002, Liu et al. 2004). These cases in which aanat activity is not associated with darkness or with melatonin production suggest an additional role for aanat in the retina. Such a role may be the acetylation of arylalkylamines other than serotonin and/or synthesis of products other than melatonin (Falcon et al. 2003, Klein 2004, Zilberman-Peled et al. 2004, 2006).
Several experimental systems were used in the current study to explore and dissect the differential regulation of two related clock-controlled genes in photoreceptors. Future studies using these complementary approaches promise to provide important new insights into the links between the core molecular oscillator and rhythmic behavioral and physiological outputs.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Angel P & Karin M 1991 The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochimica et Biophysica Acta 1072 129–157.[Medline]
Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P & Karin M 1987 Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated transacting factor. Cell 49 729–739.[CrossRef][Web of Science][Medline]
Appelbaum L, Toyama R, Dawid IB, Klein DC, Baler R & Gothilf Y 2004 Zebrafish serotonin-N-acetyltransferase-2 gene regulation: pineal-restrictive downstream module contains a functional E-box and three photoreceptor conserved elements. Molecular Endocrinology 18 1210–1221.
Appelbaum L, Anzulovich A, Baler R & Gothilf Y 2005 Homeobox-clock protein interaction in zebrafish: a shared mechanism for pineal-specific and circadian gene expression. Journal of Biological Chemistry 280 11544–11551.
Arendt J 1995 Melatonin and the Mammalian Pineal Gland. London: Chapman and Hall.
Asaoka Y, Mano H, Kojima D & Fukada Y 2002 Pineal expression-promoting element (PIPE), a cis-acting element, directs pineal-specific gene expression in zebrafish. PNAS 99 15456–15461.
Baler R, Convington S & Klein DC 1997 The rat arylalkylamine N-acetyltransferase gene promoter. cAMP activation via a cAMP-responsive element-CCAAT complex. Journal of Biological Chemistry 272 6979–6985.
Benyassi A, Schwartz C, Coon SL, Klein DC & Falcon J 2000 Melatonin synthesis: arylalkylamine N-acetyltransferases in trout retina and pineal organ are different. Neuroreport 11 255–258.[Web of Science][Medline]
Besseau L, Benyassi A, Moller M, Coon SL, Weller JL, Boeuf G, Klein DC & Falcon J 2005 Melatonin pathway: breaking the ‘high-at-night’ rule in trout retina. Experimental Eye Research 82 620–627.[CrossRef][Web of Science][Medline]
Cahill GM & Hasegawa M 1997 Circadian oscillators in vertebrate retinal photoreceptor cells. Biological Signals 6 191–200.[Web of Science][Medline]
Chen S, Wang QL, Nie Z, Sun H, Lennon G, Copeland NG, Gilbert DJ, Jenkins NA & Zack DJ 1997 Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19 1017–1030.[CrossRef][Web of Science][Medline]
Chen W & Baler R 2000 The rat arylalkylamine N-acetyltransferase E-box: differential use in a master vs. a slave oscillator. Molecular Brain Research 81 43–50.[Medline]
Cheng Y, Bullock CM, Li C, Lee AG, Bermak JC, Belluzzi J, Weaver DR, Leslie FM & Zhou Q 2002 Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature 417 405–410.[CrossRef][Medline]
Chong NW, Bernard M & Klein DC 2000 Characterization of the chicken serotonin N-acetyltransferase gene. Activation via clock gene heterodimer/E box interaction. Journal of Biological Chemistry 275 32991–83299.
Coon SL, Begay V, Deurloo D, Falcon J & Klein DC 1999 Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish. Journal of Biological Chemistry 274 9076–9082.
Coon SL, Del Olmo E, Young WS & Klein DC 2002 Melatonin synthesis enzymes in Macaca mulatta: focus on arylalkylamine N-acetyltransferase (EC 2·3·1·87). Journal of Clinical Endocrinology and Metabolism 87 4699–4706.
Delgado MJ, Alonso-Gomez AL, Gancedo B, de Pedro N, Valenciano AI & Alonso-Bedate M 1993 Serotonin N-acetyltransferase (NAT) activity and melatonin levels in the frog retina are not correlated during the seasonal cycle. General and Comparative Endocrinology 92 143–150.[CrossRef][Web of Science][Medline]
Ekstrom P & Meissl H 1997 The pineal organ of teleost fishes. Reviews in Fish Biology and Fisheries 7 199–284.[CrossRef][Web of Science]
Falcon J, Gothilf Y, Coon SL, Boeuf G & Klein DC 2003 Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina. Journal of Neuroendocrinology 15 378–382.[CrossRef][Web of Science][Medline]
Force A, Lynch M, Pickett FB, Amores A, Yan YL & Postlethwait J 1999 Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151 1531–1545.
Foulkes NS, Whitmore D & Sassone-Corsi P 1997 Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Biology of the Cell 89 487–494.[CrossRef][Web of Science][Medline]
Furukawa T, Morrow EM, Li T, Davis FC & Cepko CL 1999 Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nature Genetics 23 466–470.[CrossRef][Web of Science][Medline]
Gamse JT, Shen YC, Thisse C, Thisse B, Raymond PA, Halpern ME & Liang JO 2002 Otx5 regulates genes that show circadian expression in the zebrafish pineal complex. Nature Genetics 30 117–121.[CrossRef][Web of Science][Medline]
Ganguly S, Coon SL & Klein DC 2002 Control of melatonin synthesis in the mammalian pineal gland: the critical role of serotonin acetylation. Cell and Tissue Research 309 127–137.[CrossRef][Web of Science][Medline]
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS & Weitz CJ 1998 Role of the CLOCK protein in the mammalian circadian mechanism. Science 280 1564–1569.
Gothilf Y, Coon SL, Toyama R, Chitnis A, Namboodiri MA & Klein DC 1999 Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function. Endocrinology 140 4895–4903.
Gothilf Y, Toyama R, Coon SL, Du SJ, Dawid IB & Klein DC 2002 Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish. Developmental Dynamics 225 241–249.[CrossRef][Web of Science][Medline]
Izsvak Z, Ivics Z, Garcia-Estefania D, Fahrenkrug SC & Hackett PB 1996 DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio). PNAS 93 1077–1081.
Jin X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ & Reppert SM 1999 A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96 57–68.[CrossRef][Web of Science][Medline]
Kim TD, Kim JS, Kim JH, Myung J, Chae HD, Woo KC, Jang SK, Koh DS & Kim KT 2005 Rhythmic serotonin N-acetyltransferase mRNA degradation is essential for the maintenance of its circadian oscillation. Molecular and Cellular Biology 25 3232–3246.
Klein DC 2004 The 2004 Aschoff/Pittendrigh lecture: Theory of the origin of the pineal gland – a tale of conflict and resolution. Journal of Biological Rhythms 19 264–279.
Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA, Zatz M, Iuvone PM, Rodriguez IR, Bégay V, Falcón J, Cahill GM, Cassone VM & Baler R 1997 The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland. Recent Progress in Hormone Research 52 307–357.[Web of Science][Medline]
Klein DC, Ganguly S, Coon S, Weller JL, Obsil T, Hickman A & Dyda F 2002 14–13–3 proteins and photoneuroendocrine transduction: role in controlling the daily rhythm in melatonin. Biochemical Society Transactions 30 365–373.[CrossRef][Web of Science][Medline]
Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, Hastings MH & Reppert SM 1999 mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98 193–205.[CrossRef][Web of Science][Medline]
Li X, Chen S, Wang Q, Zack DJ, Snyder SH & Borjigin J 1998 A pineal regulatory element (PIRE) mediates transactivation by the pineal/retina-specific transcription factor CRX. PNAS 95 1876–1881.
Liu C, Fukuhara C, Wessel JH, Iuvone PM & Tosini G 2004 Localization of Aa-nat mRNA in the rat retina by fluorescence, in situ hybridization and laser capture microdissection. Cell and Tissue Research 315 197–201.[CrossRef][Web of Science][Medline]
Mizusawa K, Iigo M, Masuda T & Aida K 2000 Photic regulation of arylalkylamine N-acetyltransferase 1 mRNA in trout retina. Neuroreport 11 3473–3477.[Web of Science][Medline]
Pittendrigh CS 1993 Temporal organization: reflection of a Darwinian clock-watcher. Annual Review of Physiology 55 16–54.[Web of Science][Medline]
Reppert SM & Weaver DR 2000 Comparing clockworks: mouse versus fly. Journal of Biological Rhythms 15 357–364.
Reppert SM & Weaver DR 2001 Molecular analysis of mammalian circadian rhythms. Annual Review of Physiology 63 647–676.[CrossRef][Web of Science][Medline]
Ripperger JA, Shearman LP, Reppert SM & Schibler U 2000 CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes and Development 14 679–689.
Tosini G & Fukuhara C 2003 Photic and circadian regulation of retinal melatonin in mammals. Journal of Neuroendocrinology 15 364–369.[CrossRef][Web of Science][Medline]
Vallone D, Gondi SB, Whitmore D & Foulkes NS 2004 E-box function in a period gene repressed by light. PNAS 101 4106–4111.
Whitmore D, Foulkes NS & Sassone-Corsi P 2000 Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature 404 87–91.[CrossRef][Medline]
Zilberman-Peled B, Benhar I, Coon SL, Ron B & Gothilf Y 2004 Duality of serotonin-N-acetyltransferase in the gilthead seabream (Sparus aurata): molecular cloning and characterization of recombinant enzymes. General and Comparative Endocrinology 138 139–147.[CrossRef][Web of Science][Medline]
Zilberman-Peled B, Ron B, Gross A, Finberg JP & Gothilf Y 2006 A possible new role for fish retinal serotonin-N-acetyltransferase-1 (AANAT1): Dopamine metabolism. Brain Research. In Press.
Ziv L, Levkovitz S, Toyama R, Falcon J & Gothilf Y 2005 Functional development of the zebrafish pineal gland: light-induced expression of Period2 is required for onset of the circadian clock. Journal of Neuroendocrinology 17 314–320.[CrossRef][Web of Science][Medline]
Received in final form 2 December 2005
Accepted 9 January 2006
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) and half remained under DD conditions (•) and were collected every 3 h for 36 h. Zebrafish EGFP mRNA levels were monitored by whole mount ISH analyses. Each value represents the mean±S.E. optical density of pineal signals (n =12).
