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. 2003 Feb 1;31(3):935–943. doi: 10.1093/nar/gkg174

New role of zCRY and zPER2 as regulators of sub-cellular distributions of zCLOCK and zBMAL proteins

Jun Hirayama 1, Itsuki Fukuda 1, Tomoko Ishikawa 1, Yuri Kobayashi 1, Takeshi Todo 1,a
PMCID: PMC149195  PMID: 12560489

Abstract

The core oscillator that generates circadian rhythm in eukaryotes consists of transcription/translation-based autoregulatory feedback loops by which clock gene products negatively regulate their own expression. Control of the accumulation and nuclear entry of the negative regulators PER and CRY is believed to be a key step in these loops. We clarified the mutual interaction between zebrafish clock-related proteins and their sub-cellular localizations in NIH3T3 cells. Six CRYs exist in zebrafish, of which zCRY1a strongly represses zCLOCK1: zBMAL3-mediated transcription, but zCRY3 does not. We show that zCRY1a interacts with zCLOCK1 and zBMAL3, facilitating nuclear accumulation, whereas zCRY3 associates with neither one and does not influence their sub-cellular distributions. We cloned zPer2 cDNA and showed that the protein product encoded by the cDNA acts as a moderate transcriptional repressor. In our sub-cellular localization studies we also found that zPER2 interacts with the zCLOCK1:zBMAL3 heterodimer, causing its cytoplasmic retention. zCRY1a and zPER2 apparently have opposite effects on the sub-cellular distribution of zCLOCK:zBMAL heterodimer. We speculate that the opposite regulation of the sub-cellular distribution of this is associated with the different transcriptional repression abilities of zCRY1a and zPER2. zCRY1a acts as a potent transcriptional inhibitor by interacting directly with the zCLOCK:zBMAL heterodimer in the nucleus, whereas zPER2 maintains the zCLOCK:zBMAL heterodimer in the cytoplasm, resulting in transactivation repression.

INTRODUCTION

Circadian rhythms constitute a ubiquitous process that regulates various biochemical and physiological events of approximate 24 h periodicity, even in the absence of external time cues (1–3). Under natural conditions, rhythms are entrained to a 24 h day by environmental time cues, most commonly light. Timing of the clock is established in a cell-autonomous manner by a transcription/translation-based negative feedback loop. This loop consists of positive and negative elements, the positive ones being two basic helix– loop–helix, PAS domain-containing transcription factors, CLOCK and BMAL. When these transcription factors heterodimerize, they drive the transcription of such negative components of the clock genes as three Period genes (in the mouse, designated mPer1, mPer2 and mPer3) and two Cryptochrome genes (mCry1 and mCry2). The products of these clock genes then negatively regulate their own expression, thereby setting up the rhythmic oscillations of gene expression that drive the circadian clock.

To produce stable oscillations of gene expression that have a 24-h period, there must be a delay between the production and action of inhibitory clock gene products. Controlled accumulation, sub-cellular localization and degradation of clock proteins make up the important process that produces this delay. Drosophila TIMELESS (TIM) heterodimerizes with dPER, and the resulting heterodimer stabilizes dPER (4). dPER:TIM heterodimers then translocate to the nucleus where they interact with dCLOCK and CYC (Drosophila homolog of mammalian BMAL). Formation of a complex decreases dCLOCK:CYC-mediated transcription, resulting in repression of expression (5,6). Stabilization of dPER by formation of a heterodimer with TIM and subsequent nuclear translocation together constitute a key process by which the feedback loop oscillates on a 24-h time scale in Drosophila. In mammals, stabilization of PER by formation of a heterodimer with a clock protein and subsequent nuclear translocation also are important for the negative feedback loop. CRY1 and CRY2, however, are partners of the PER heterodimer instead of TIM. When mPER and mCRY proteins are translated, they form PER:CRY heterodimers that are translocated to the nucleus. In the nucleus, these proteins act as negative regulators by interacting with CLOCK and/or BMAL1 to inhibit transcription, thereby forming a negative feedback loop (7–9). Consistent with this model, mice lacking both mCry1 and 2 or both mPer1 and 2 show complete loss of circadian rhythmicity in wheel-running behavior immediately upon being placed in constant dark (10–12). The difference in the functions of these proteins is not well understood; in particular, which one is the direct factor for transcriptional repression, and how nuclear entry is regulated. Although both proteins repress the circadian promoter driven by the CLOCK/BMAL heterodimer transcription factor, PER causes only moderate repression, whereas CRY1 and CRY2 appear to be much more potent (7). In vitro transfection results show that exogenously expressed mPER2 can localize in the nucleus and that co-expression with mCRY (7) or mPER3 (13) proteins promotes its nuclear entry. Localization of endogenous proteins showed weak accumulations of mCRY1 and mCRY2 and of mPER1 and 2 in nuclei of per1-/per2- and cry1-/cry2- double mutant mice livers, respectively (14). These findings indicate that co-dependency is required between the mPER and mCRY proteins for effective nuclear translocation. Another transfection study provided contrasting evidence that mPER2 enters the nucleus in a mCRY-independent manner and shuttles between the nucleus and cytoplasm by means of a functional nuclear localization signal (NLS) and nuclear export sequences present in the protein (15). These findings suggest that the mPER and mCRY proteins enter the nucleus independently, cooperatively or both. CLOCK:BMAL-mediated transcription therefore is repressed in various ways.

The zebrafish is an attractive model with which to study the biological clock in vertebrates. The most remarkable and unique feature in the zebrafish system is the ability to respond to light. Several clock-related genes show circadian expression in zebrafish cells, including those in ex vivo peripheral tissues placed for a period of days in culture dishes and those in zebrafish-derived cell lines (16). Moreover, as the circadian clock of these cells is entrained by light (17), the zebrafish system provides a unique setting for the study of clock-related transcription. Several zebrafish homologs of clock genes have been cloned (18–22). To better understand the roles of the zCRY and zPER2 proteins in the zebrafish circadian clock, the interactions with clock-related proteins and their sub-cellular distributions after transfection were examined in cultured cells. Initially, zebrafish-derived cells were used for transfection but they had low transfection efficiencies and protein products of the transfected genes were barely detectable by immunoprecipitation. Mouse NIH3T3 cells were therefore used. The results obtained for mouse cells are consistent with those for the zebrafish cells examined. Six Cry genes (zCry1a, 1b, 2a, 2b, 3 and 4) have been cloned in zebrafish. Investigation of their in vitro functions showed that they fall into two groups: one inhibits CLOCK-BMAL-mediated transcription (repressor type CRY) and the other does not have transcription inhibitor activity (non-repressor type CRY) (19). We characterized the two zCRYs, zCRY1a and zCRY3 former and latter types of CRY, respectively. We found that zCRY1a and zPER2 function as regulators of the sub-cellular localizations of zCLOCK, zBMAL and/or the zCLOCK: zBMAl heterodimer and that they control cellular localization of the heterodimer in opposite directions. In addition, unlike in mammals, the co-expression of zCRY1a and zPER2 causes cytoplasmic retention of the zCRY:zPER2 heterodimer. These findings suggest that functions for CRY and PER2 exist that are unique to the zebrafish circadian system.

MATERIALS AND METHODS

Cloning of zebrafish Per2 cDNA

zPer2 cDNA was cloned from an adult cDNA library (purchased from Clontech) by combination of polymerase chain reaction (PCR)-based cloning strategies and cDNA library screening. Two degenerate oligonucleotides, PERL1 (5′-GGNTAYYTNCCNCCNCARGAYYT-3′) and PERR1 (5′-TTRCCNCTYATRCANTGNRANCT-3′), which encode A(V/A)(P/S)(L/A)LGYLPQDL and NGEYVTLD, respectively, were used. Amino acid sequences were well conserved between mouse and Drosophila PERs. A 200 bp fragment, amplified from cDNA synthesized from zebrafish eye total RNA, was the DNA probe used to screen the cDNA library. Sequence determination showed that that cDNA encodes a zebrafish Per2 (zPer2) gene. An EcoRI–XbaI fragment carrying full-length zPer2 cDNA was ligated to the corresponding site of pcDNA3.1, generating pcDNA-zPER2.

Plasmid construction

Deletion mutants of zPer2 were generated as follows: the PmaCI site corresponding to the 765 amino acids from the first methionine and the XhoI site located at amino acid 1293 were used to construct the deletion mutants, zPer2 (amino acid 1–764), zPer2 (amino acid 765–1400) and zPer2 (amino acid 1–1293). The EcoRI–PmaCI, PmaCI–XbaI or EcoRI–XhoI fragments derived from pcDNA-zPER2, each containing zPER2 (amino acid 1–764), zPER2 (amino acid 765–1400), and zPER2 (amino acid 1–1293), were used in the GAL4 fusion construct. To construct the deletion mutants, zPer2 (amino acid 765–1000), zPer2 (amino acid 1001–1200) and zPer2 (amino acid 1201–1400), DNAs bearing each region of zPer2 were amplified from the zPer2 cDNA containing plasmids, pGAL4-zPER2 or pcDNA-zPER2, by use of the following primers: T7 (5′-TAATACGACTCACTATAGGG-3′) and zP23000L (5′-TCTCTCTAGATCATGGAAACATGTAG-3′, XbaI site underlined), zP23000R (5′-GAGAGAATTCCAGGTGGGCAGCGCTC-3′, EcoRI site underlined) and zP23600L (5′-TCTTCTAGATCAGGCAGAGCCGGTG-3′, XbaI site underlined), and zP23600R (5′-GAGAGAATTCACATCTGGCTCCATGG-3′, EcoRI site underlined) and BGH Rev (5′-TAGAAGGCAACAGTCGAGG-3′), each amplify nucleotides 2290–3000, 3000–3600 and 3600–4200 of the zPer2 coding region.

A HindIII–EcoRI fragment bearing yeast GAL4 was excised from KS(+)-Gal4 (23) and inserted in the corresponding site of pcDNA3.1(+) to generate five GAL4 DNA binding domain (GAL4) fusions. The resulting plasmid, designated pcDNA-GAL4, was used to construct the GAL4 fusion plasmids, pGAL4-zCLOCK1, pGAL4-zPER2, pGAL4-zPER2 (amino acid 1–764), pGAL4-zPER2 (amino acid 765–1400), pGAL4-zPER2 (amino acid 1–1293), pGAL4-zPER2 (amino acid 765–1000), pGAL4-zPER2 (amino acid 1001–1200), and pGAL4-zPER2 (amino acid 1201–1400). All the inserted cDNAs are in frame to the C-terminal of GAL4.

A HindIII fragment of the activation domain of herpes simplex VP16 (VP16) excised from pCMX-VP16-N (23) and ligated to the corresponding site of Blue Script (pBS)-generated plasmid pBS-VP16. An Asp718–EcoRI fragment excised from pBS-VP16 was ligated to the corresponding site of pcDNA3.1(+), generating plasmid pcDNA-VP16. These plasmids were used to construct pVP16-zCLOCK1, pVP16-zCRY1a and pVP16-zCRY3. All the inserted cDNAs are in frame to the C-terminal of VP16.

The FLAG fragment was amplified by PCR with a set of partially complementing primers FLAG-R (5′-GAGACAATTGCGCCACCATGGACTACAAAG-3′, MunI site underlined) and FLAG-L (5′-GAGAATTCCTTGTCGTCATCG TCTTTGTAG-3′, EcoRI site underlined), digested with MunI and EcoRI, then ligated to the EcoRI site of pcDNA3.1(+). The resulting plasmid, designated pcDNA-FLAG, was used to construct pFLAG-zCLOCK1, pFLAG-zBMAL3 and pFLAG-zPER2 (amino acid 1–1296). All the inserted cDNAs are in frame to the C-terminal of FLAG.

An EcoRI–XhoI fragment of pcDNA-zPER2 was inserted in the corresponding sites of pcDNA-HA and pcDNA-V5, generating respectively pHA-zPER2 (amino acid 1–1293) and pV5-zPER2 (amino acid 1–1293). Nucleotides 3600– 4197 of the zPer2 coding region were amplified by PCR with the primers zP23600R and zP24197L (5′-GCGCCTCGAG GGTGTCTGGACCGG-3′, XboI site underlined), then inserted in the XhoI sites of pHA-zPER2 (amino acid 1–1293) and pV5-zPER2 (amino acid 1–1293), which generated pHA-zPER2 and pV5-zPER2. Both the inserted cDNAs are in frame to the N-terminal of HA or V5.

The dual expression plasmid pCRY1aPER2, designed to express both zCRY1a-HA and zPER2-V5 from each of two CMV promoters, was constructed as follows. A fragment having both the CMV promoter and zCRY1a-HA was excised from pHA-zCRY1a and ligated to pKSCX-EGFP (24). The resulting plasmid was named pKSCX-zCRY1aHA. The SwaI fragment was excised from pKSCX-zCRY1aHA and ligated to the SmaI site of pV5-zPER2, generating pCRY1aPER2.

The dual expression plasmid pCLOCK1BMAL3, designed to express both VP16-zCLOCK1 and FLAG-zBMAL3 from each of two CMV promoters, was constructed as follows. A fragment bearing both the CMV promoter and FLAG-zBMAL3 was excised from pFLAG-zBMAL3 and ligated to pKSCX-EGFP, generating pKSCX-FLAGzBMAL3. The SwaI fragment was excised from pKSCX-FLAGzBMAL3 and ligated to the StuI site of VP16-zCLOCK1, generating pCLOCK1BMAL3.

The other plasmids used in this study have been described elsewhere (19,21).

Cells, transfection, and the luciferase assay

NIH3T3 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 5% calf bovine serum. The day before transfection NIH3T3 cells were plated in a 12-well plate at 1.5 × 105 cells per well then transected the next day with 10 ng of firefly luciferase reporter plasmid, 20 ng of sea-pansy luciferase reporter plasmid [pRL-SV40 (Promega)] and 200 ng of the expression plasmids (indicated in each figure) by means of Lipofectamine-Plus according to the manufacturer’s instructions (Invitrogen). As the reporter plasmid, the E-box element and its flanking sequences within the promoter/enhancer region of mouse vasopressin and five GAL4-binding sites were cloned into the pGL-basic vector (Promega) in the luciferase-reporter and two-hybrid assays (designated pGL-mAVP and pGL-5G), respectively. Total amounts of the expression plasmids were adjusted by adding the pcDNA3.1 vector as the carrier. Preparation of the cell lysates and dual luciferase assays, using the dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega), were done 48 h after transfection. Firefly and sea-pansy luciferase activities were quantified in a luminometer, firefly luciferase activity being normalized for transfection efficiency based on sea-pansy luciferase activity. All experiments were done three times.

Co-immunoprecipitation

Co-immunoprecipitation was done as described previously (7) with some modification. NIH3T3 cells were seeded in 10 cm dishes and transfected the following day with the expression plasmids described. The cells were washed twice with phosphate-buffered saline (PBS) 48 h after transfection, homogenized in binding buffer (150 mM NaCl, 5 mM EDTA, 0.5% NP-40, and 50 mM Tris–HCl pH 7.5) containing protease inhibitor cocktail tablets, then clarified by centrifugation for 10 min at 10 000 g. Total protein (30 ng) from the supernatant was incubated for 1 h at 4°C with 15 µl of protein A/G agarose beads (Santa Cruz), after which the material was centrifuged. The supernatant was incubated for 3 h at 4°C with anti-VP16 rabbit monoclonal antibodies (Santa Cruz) or anti-V5 antibodies mouse monoclonal antibody (Invitrogen), and 15 µl of protein A/G agarose beads, after which the beads were washed three times with binding buffer, boiled in sodium dodecyl sulfate (SDS) sample buffer and centrifuged. The supernatant was separated by SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by western blotting as described hereafter.

Western blot analysis

Total protein (10 µg), extracted from the cells as described previously, was separated by SDS–PAGE in 10% gel and transferred electrophoretically to a nitrocellulose membrane (Hybond-ECL, Amersham Pharmacia Biotech). The membrane was blocked with 5% non-fat milk, first incubated with mouse anti-HA antibody (Santa Cruz), mouse anti-V5 antibody or rabbit anti-VP16 antibody for 1 h at room temperature, then with peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Santa Cruz), and developed with the ECL western blotting detection system (Amersham Pharmacia Biotech).

Immunofluorescence

Cells (3 × 105) were seeded on glass coverslips in 6-well dishes and transfected the following day (as described above) with 1 µg of total DNA per well. Forty-eight hours after transfection, the cells were washed twice with PBS, fixed first with –20°C acetone (5 min) then with –20°C methanol (5 min), washed, and blocked for 30 min at 37°C in 1% bovine serum albumin (BSA)/0.1% Triton X-100 in PBS. Anti-HA antibody, anti-V5 antibody, anti-FLAG antibody (Stratagene) or anti-VP16 antibody was diluted in 0.5% BSA in PBS then incubated with the cells for 1 h at 37°C. Next the cells were washed three times with 0.1% Triton in 10% PBS then incubated with fluorescein isothiocyanate (FITC) (Santa Cruz) and/or the Cy3-conjugated secondary antibody (Jackson Immuno Research Laboratories, Inc.) for 1 h at 37°C, after which they were washed, their nuclei stained with 4′,6′-diamidino-2-phenylindole (DAPI), and the cells mounted for fluorescence microscopy.

RESULTS

Both zCRY1a and zPER2 inhibit CLOCK:BMAL-mediated transcription but they inhibit it in a different manner

The effects of zCRYs and zPER2 on zCLOCK:zBMAL-mediated transcription were examined by means of a luciferase reporter gene assay. A mouse vasopressin (mAVP) E-box element was the reporter construct. Co-expression of zCLOCK1 with zBMAL3 strongly stimulated transcription from the mAVP E-box element (Fig. 1A, lane 2). zCRY1a efficiently inhibited the zCLOCK1-zBMAL3 mediated transactivation, whereas zCRY3 did not (Fig. 1A, lanes 3–8), as reported previously (19). On the other hand, zPER2 caused a moderate transcriptional inhibition. zCRY1a inhibited CLOCK:BMAL-mediated transcription almost completely by transfection of 50 ng DNA, whereas zPER2 caused a 60% inhibition by transfection of 700 ng DNA, indicative that zCRY1a and zPER2 inhibit CLOCK:BMAL-mediated transcription in a different manner. Next zCRY1a and zPER2 were co-expressed and their effects on the CLOCK:BMAL-mediated transcription were examined. To exclude the possibility that either one of the added DNAs, zPER2 or zCRY, alone would be expressed in the transfected cells, we constructed a dual expression plasmid, pCRY1aPER2, designed to express both zCRY1a-HA and zPER2-V5 simultaneously, and performed the same assay. When pCRY1aPER2 was used, zCLOCK1:zBMAL3-induced transactivation was completely repressed by transfection of 200 ng of DNA (Fig. 1A, lane 15), however transfection of 50 ng DNA caused only 40% inhibition (lane 13). Co-expression of zPER2 did not show any additive effects, and on the contrary slightly inhibited the activity of zCRY1a. Western blots of cell lysates transfected with pCRY1aPER2 showed expression of zCRY1a-HA and zPER2-V5 at detectable levels and both proteins co-immunoprecipitated (Fig. 1B). Absence of any additive effect of zPER2 co-expression therefore was neither due to failure of the expression of the transfected genes nor to lack of interaction between the expressed proteins. To know why zPER2 co-expression did not show any obvious effect on the zCRY’s inhibition ability of CLOCK:BMAL-mediated transcription, we examined the interactions between zCRYs, zPER2, zCLOCK1 and zBMAL3 and their sub-cellular localizations.

Figure 1.

Figure 1

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zCRY1a and zPER2 inhibit CLOCK:BMAL-mediated transcription in a different manner. (A) The transcription-inhibiting ability of each of zCRY and zPER2 proteins examined by a luciferase reporter gene assay. The pGL-mAVP reporter plasmid was co-transfected with the expression vectors shown. Transactivations of the reporter plasmid were examined. Values are means ± SEM of three independent experiments. In each experiment, luciferase activity of the zCLOCK1:zBMAL3-containing sample was adjusted to 100%. (B) Physical interaction of zCRY1a-HA and zPER2-V5. zPER2-V5 was immunoprecipitated from NIH3T3 cell extracts, transfected with 4 µg of the pCRY1aPER2 vector by means of the anti-V5 antibody, after which one-half of the precipitate underwent immunoblot analysis with anti-V5 (zPER2, lane 2) and anti-HA (zCRY1a, lane 4). Immunoblotting of the total cell lysate with anti-V5 antibody (zPER2, lane 1) or anti-HA antibody (zCRY1a, lane 3) confirms that both zPER2-V5 and zCRY1a-HA proteins are expressed in pCRY1aPER2-transfected cells.

Different abilities of zCRY1a and 3 to interact with other clock proteins

The mammalian two-hybrid assay was used to examine the abilities of zCRY1a and 3 to interact with each of three other clock proteins: zCLOCK1, zBMAL3 and zPER2. In that assay, one of the zCRYs or zPER2 which fused to the GAL4 DNA-binding domain (GAL4) was co-expressed with each clock protein fused to the VP16 transactivation domain (VP16) in the NIH3T3 cells. If GAL4-zCRY or GAL4-zPER2 interacts functionally with VP16-fused protein, VP16 would be recruited to the vicinity of the promoter and should cause transactivation. As shown in Figure 2A, when GAL4-zCRY1a was co-expressed with VP16-zCLOCK1 (lane 6) or VP16-zBMAL3 (lane 7) transcription was marked. In contrast, when GAL4-zCRY3 was co-expressed with VP16-zCLOCK1 or VP16-zBMAL3 no transactivation occurred (lanes 8 and 9).

Figure 2.

Figure 2

Figure 2

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zCRY1a and 3 have different affinities for the other clock components. (A) Mammalian two-hybrid assay results of each zCRY’s interaction with zCLOCK1 or zBMAL3. VP16-zCLOCK1 or VP16-zBMAL3 was co-transfected with GAL4, GAL4-zCRY1a or GAL4-zCRY3. Effects on pGL-5G reporter plasmid transactivation were assayed. (B) Schematic representation of zPER2 protein showing the positions of bHLH, PAS, CLD and NLD and the constructs of six deletion mutants. Numbers indicate the amino acid residues in the protein of origin. (C) The zCRY binding domain of zPER2. GAL4-zPER2, or each deletion mutant that fused to GAL4 as described in (B), was co-transfected with VP16-zCRY1a or VP16-zCRY3. Effects on pGL-5G reporter plasmid transactivation were assayed. Relative luciferase activity is shown as the fold increase compared with activity produced with the 10 ng of pGL-5G reporter plasmid (set at 1). Values are means ± SEM of three independent experiments.

The immunoprecipitation data showed that zCRY1a and zPER2 form a stable complex (Fig. 1B). The interactions between zCRYs and zPER2 were also examined by the two-hybrid assay (Fig. 2C). When VP16-zCRY1a or 3 was co-expressed with GAL4-zPER2, both induced transactivation although the transactivation induced by zCRY3 was weaker than that by zCRY1a. These findings show that zCRY1a interacts with zPER2, zCLOCK1 and zBMAL3 whereas zCRY3 only with zPER2.

Determination of the binding domain of zPER2 to zCRY

zPER2 deletion mutants were constructed to determine the CRY-binding domain of zPER2 (Fig. 2B). The ability of each mutant to bind zCRY1a or 3 was determined by the mammalian two-hybrid assay. Intact zPER2 and all mutants bearing the C-terminal domain showed transactivation when co-transfected with each of the zCRYs (Fig. 2C, lanes 2, 4, 7, 9, 11 and 14), whereas deletion mutants lacking that domain showed weak or no transactivation (lanes 5, 6, 8, 10, 12, 13 and 15), indicative that the C-terminal region of zPER2 (amino acid 1201–1400) is important for its binding to zCRYs. Except for the interaction between the N-terminal region of zPER2 and zCRY1a (lanes 3 and 8), this is consistent with the findings for mammalian PER2 (15,25–27).

zCRY1a, zPER2, zCLOCK1 and zBMAL3 form a stable complex

Next we examined whether zPER2 interacts with zCLOCK1, zBMAL3 or zCLOCK1:zBMAL3 heterodimer by immunoprecipitation assay (Fig. 3). When zPER2-V5 was co-expressed with zCLOCK1-VP16 or with zBMAL3-VP16, zPER2 alone was detected in the anti-V5 precipitated materials (Fig. 3A, lanes 1 and 2). However, when zPER-HA, zCLOCK-VP16, and zBMAL-HA were co-expressed, all three proteins were immunoprecipitated by the use of anti-VP16 antibody (Fig. 3B, lane 1). These results indicate that zPER2 does not form a complex either with zCLOCK1 or zBMAL3, but does with zCLOCK1:zBMAL3 heterodimer.

Figure 3.

Figure 3

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zCRY1a, zPER2 and the zCLOCK1:zBMAL3 heterodimer form multimeric complexes. (A) zPER2 does not interact with zCLOCK1 or zBMAL3. zPER2-V5 was immunoprecipitated from cell lysates of NIH3T3 cells transiently co-expressing zPER2-V5 and either VP16-zCLOCK1 or VP16-zBMAL3. Immunoprecipitated materials were analyzed by immunoblotting with anti-VP16 antibody [zCLOCK1 (lane 1) or zBMAL3 (lane 2), top] or anti-V5 antibody (zPER2, middle). Analyses of the total cell lysates done with anti-VP16 antibody are shown at the bottom, confirming the presence of VP16-zCLOCK1 or VP16-zBMAL3. (B) Multimeric complexes composed of zCRY1a, zPER2, zCLOCK1 and zBMAL3 were detected by a co-immunoprecipitation assay. VP16-zCLOCK1, zPER2-HA, and zBMAL3-HA or VP16-zCLOCK1, zCRY1a-HA, zPER2-HA and zBMAL3-HA were co-expressed in NIH3T3 cells. The cell lysates underwent immunoprecipitation with the anti-VP16 antibody. Immunoprecipitated materials were analyzed by immunoblotting with anti-HA antibody (zCRY1a, zPER2 and zBMAL3, top) or anti-VP16 antibody (zCLOCK1, middle). Analyses of the total cell lysates done with anti-HA antibody are shown at the bottom, confirming the presence of the three proteins, zCRY1a-HA, zPER2-HA and zBMAL3-HA.

We next undertook co-expression of four proteins: zCRY1a, zPER2, zCLOCK1 and zBMAL3. When the four proteins were co-expressed, they were immunoprecipitated by the use of anti-VP-16 antibody (Fig. 3B, lane 2), which is indicative that zCRY1a does not disrupt zPER2-zCLOCK1: zBMAL3 complex but forms a ternary zCRY1a-zPER2-zCLOCK1:zBMAL3 complex.

zCRY1a facilitates nuclear accumulation of zCLOCK1, zBMAL3 and zCLOCK1:zBMAL3 heterodimer

A recent in vivo study found that in mouse liver a CRY deficiency alters the sub-cellular distribution of both the CLOCK and BMAL1 proteins (14). Conceivably, zCRY affects the sub-cellular location of both zCLOCK1 and zBMAL3 in cultured cells. We therefore used the imunofluorescence of epitope-tagged proteins to examine the effect of the co-expression of zCRY1a or 3 on the sub-cellular localization of zCLOCK1 or zBMAL3. The location was the cytoplasm (N<C), both the cytoplasm and nucleus (N=C) or the nucleus (N>C).

First, each of the zCRYs, zBMAL3 and zCLOCK1 was expressed singly in NIH3T3 cells (Fig. 4). Interestingly, zCRY1a and 3 had different sub-cellular localizations. zCRY1a was distributed in the nucleus (Fig. 4A and B, lanes 1–3). zCRY3 was distributed mainly in the cytoplasm (Fig. 4B, lanes 10–12). zBMAL3 and zCLOCK1 also had different sub-cellular distributions (Fig. 4A and C). zBMAL3 was detected in the nucleus and in both the cytoplasm and nucleus (Fig. 4C, lanes 1–3), whereas zCLOCK1 was distributed only in the cytoplasm (Fig. 4C, lanes 10–12).

Figure 4.

Figure 4

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zCRY1a facilitates nuclear accumulation of zCLOCK1 and zBMAL3. (A) Sub-cellular localization of one zebrafish clock protein transiently transfected in NIH3T3 cells with or without the other protein. Representative examples of fluorescent cells. HA-tagged zCRYs and FLAG-tagged zCLOCK1 or zBMAL3 were stained respectively with a combination of anti-HA antibody and FITC-conjugated secondary antibody (green) or anti-FLAG antibody and Cy3-conjugated secondary antibody (red). Nuclei were made visible with DAPI (blue). (B) Quantitative analysis of the sub-cellular localization of each zCRY in the absence or presence of co- expressed zCLOCK1 or zBMAL3. In each experiment 150–250 cells were evaluated for nuclear (N>C, black bars), nuclear–cytoplasmic (N=C, gray bars) and cytoplasmic (N<C, white bars) fluorescence. Values shown are representative of three independent experiments. (C) Quantitative analysis of the sub-cellular localizations of zCLOCK1 and zBMAL3 in the absence or presence of each co-expressed zCRY, as described in (B).

zCLOCK1 and zBMAL3 act as transcriptional regulator by forming heterodimer. We then examined whether the sub-cellular distributions of the two proteins change when they are expressed in the same cell and then form heterodimer. For this purpose, a dual expression plasmid, pCLOCK1BMAL3, designed to express VP16-zCLOCK1 and FLAG-zBMAL3 simultaneously, was constructed. VP16-zCLOCK1 and FLAG-zBMAL3 were efficiently expressed as a functional heterodimer by pCLOCK1BMAL3 because transfection of the pCLOCK1BMAL3 plasmid markedly induced transcription from the reporter gene (data not shown). When zCLOCK1 and zBMAL3 were expressed in the same cell, the sub-cellular location of zCLOCK1 changed from the cytoplasm to the nucleus (Fig. 5, lanes 4–6).

Figure 5.

Figure 5

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zCRY1a promotes nuclear accumulation of the zCLOCK1:zBMAL3 heterodimer. Sub-cellular localizations of zCLOCK1 and zBMAL3 expressed by the dual expression plasmid pCLOCK1BMAL3 with or without HA-tagged zCRY1a or 3 in NIH3T3 cells were determined. Quantitative analysis of the sub-cellular localizations of zCLOCK1 and zBMAL3 expressed by pCLOCK1BMAL3 in the absence or presence of each zCRY was done as in Figure 4. VP16-tagged zCLOCK1 and FLAG-tagged zBMAL3, respectively, were stained with a combination of anti-VP16 antibody and FITC-conjugated secondary antibody and anti-FLAG antibody and Cy3-conjugated secondary antibody. Each HA-tagged zCRY was stained with a combination of rabbit anti-HA antibody and FITC- conjugated secondary antibody or mouse anti-HA antibody and Cy3- conjugated secondary antibody. Nuclei were stained with DAPI.

Next we examined the effect of the co-expression of zCRY1a or 3 on the sub-cellular localization of zCLOCK1 or zBMAL3. Interestingly, when zCRY1a was co-transfected with zBMAL3 or zCLOCK1, it promoted the nuclear accumulation of both zBMAL3 (Fig. 4C, lanes 4–6) and zCLOCK1 (Fig. 4C, lanes 13–15). In particular, the sub-cellular localization of zCLOCK1 changed markedly from the cytoplasm to the nucleus. The effect of each zCRY on the sub-cellular distribution of the zCLOCK1:zBMAL3 heterodimer was examined. By the use of pCLOCK1BMAL3 together with a plasmid to express a cry gene, three proteins could be expressed at the same time. The tendency for nuclear localization of the heterodimer was greatly enhanced by co-expression with zCRY1a. When zCLOCK1 and zBMAL3 were expressed together with zCRY1a, almost all the co-expressed proteins were in the nucleus (Fig. 5, lanes 7–18). In contrast, the sub-cellular locations of zBMAL3 and zCLOCK1 were not affected by co-expression with zCRY3 (Fig. 4C, lanes 7–9 and 16–18, and Fig. 5, lanes 19–30).

zPER2 traps zCRY1a in the cytoplasm

In cultured mammalian cells, mammalian CRYs interact with mammalian PERs to facilitate nuclear translocation (7,25). As shown in Figure 2C, zCRY1a and 3 interact with zPER2, therefore, the effect of each zCRY on the sub-cellular localization of zPER2 was examined. When expressed singly, zPER2 localized mainly in the cytoplasm (Fig. 6A and B, lanes 1–3). Unlike mammalian PER2, the sub-cellular localization of zPER2 was not affected by co-expression with zCRY (Fig. 6A, lanes 7–9 and 16–18). Surprisingly, co-expression with zPER2 caused cytoplasmic retention of zCRY1a (Fig. 6A, lanes 10–12). The effect of the co-expression of zPER2, which lacks the C-terminal, on the sub-cellular localization of zCRY1a was also examined (Fig. 6B). Deletion of that region did not change the sub-cellular localization of zPER2 (Fig. 6B, lanes 1–3). Nor was the sub-cellular distribution of zCRY1a affected by co-expression with zPER2 lacking the C-terminal (Fig. 6B, lanes 10–12). zCRY3 co-expressed with the intact or C-terminal-lacking zPER2 remained cytoplasmic (Fig. 6A and B, lanes 19–21). These findings and the fact that the C-terminal domain of zPER2 is the zCRY binding site (Fig. 2B and C) suggest that zPER2 causes cytoplasmic trapping of zCRY1a by direct interaction with it via its C-terminal domain.

Figure 6.

Figure 6

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zPER2 traps zCRY1 in the cytoplasm. Quantitative analyses of the sub-cellular localizations of zPER2 (A) and zPER2 (amino acid 1–1293) (B) in the absence and presence of each co-expressed zCRY, as described in Figure 4B. V5-tagged zPER2 or FLAG-tagged zPER2 (amino acid 1–1293) was stained with a combination of anti-V5 (zPER2) or anti-FLAG [zPER2 (amino acid 1–1293)] antibody and Cy3-conjugated secondary antibody. Each HA-tagged zCRY was stained as described in Figure 5A. Nuclei were made visible with DAPI.

zPER2 tends to induce the cytoplasmic distribution of the zCLOCK1:zBMAL3 heterodimer

Whether zPER2 affects the sub-cellular locations of zCLOCK1 and zBMAL3 was examined. When zCLOCK1 or zBMAL3 was co-expressed with zPER2, no change in the sub-cellular distribution of zCLOCK1, zBMAL3 or zPER2 occurred (Fig. 7A). However zPER2 had an effect on the sub-cellular distributions of these proteins when they formed heterodimers. When zCLOCK1 and zBMAL3 both were co-expressed with zPER2, the cell population in which zCLOCK1 and zBMAL3 were present in the nucleus disappeared, and that present in the cytoplasm increased markedly (Fig. 7B, lanes 7–18). These results are consistent with the immunoprecipitation data (Fig. 3) and are indicative that zPER2 tends to induce the cytoplasmic distribution of zCLOCK1:zBMAL3 heterodimer.

Figure 7.

Figure 7

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zPER2 induces the cytoplasmic distribution of the zCLOCK1:zBMAL3 heterodimer. (A) Quantitative analysis of the sub- cellular localization of zCLOCK1 or zBMAL3 with or without zPER2 was done as described in Figure 4B. (B) Quantitative analysis of the sub- cellular localizations of zCLOCK1 and zBMAL3 expressed by pCLOCK1BMAL3 in the absence or presence of both zCRY1a and zPER2 was done as described in Figure 4B. VP16-tagged zCLOCK1 and FLAG-tagged zBMAL3 were stained as described in Figure 4A. HA- and V5-tagged zPER2 proteins, respectively, were stained with combinations of anti-HA antibody and FITC-conjugated secondary antibody and anti-V5 antibody and Cy3-conjugated secondary antibody. Nuclei were stained with DAPI. VP16-zCLOCK1, FLAG-zBMAL3, zCRY1a-HA and zPER2-V5 were expressed together in NIH3T3 cells by the co-transfection of pCLOCK1BMAL3 with pCRY1aPER2 vectors. Sub-cellular distributions were determined by double staining with a combination of zCLOCK1 (anti-VP16 antibody) and zPER2 (anti-V5 antibody) or with zBMAL3 (anti-FLAG antibody) and zCRY1a (anti-HA antibody).

We next undertook co-expression of four proteins: zCRY1a, zPER2, zCLOCK1 and zBMAL3. Co-expression of zCRY1a in addition to zPER2, zCLOCK1 and zBMAL3 changed the distributions of the proteins; the cell population having the nuclear distribution increased and that having cytoplasmic distribution decreased (Fig. 7B, lanes 19–30). When zCRY1a, zPER2, zCLOCK1 and zBMAL3 were co-expressed, all four proteins form a ternary complex (Fig. 3). Taken together, these findings show that zCRY1a facilitates nuclear accumulation of zCLOCK1:zBMAL3 heterodimer, whereas zPER2 promotes its cytoplasmic distribution.

DISCUSSION

Previous circadian mechanism models have shown that control of the nuclear accumulation of circadian regulators is an important process in the transcriptional/translational negative feedback loop (28). CRYs and PERs constitute an important type of circadian regulator in mammalian cells (7,10–12), therefore their nuclear entry and subsequent degradation by an unknown factor or their nuclear export must be a key factor in establishing the rhythmic oscillation of gene expression. We identified a novel function of zebrafish CRY and PER2. They regulate the sub-cellular localizations of other circadian regulators, the zCLOCK and zBMAL proteins. Moreover, these two proteins were shown to control sub-cellular localization of the zCLOCK:zBMAL heterodimer in opposite directions (Figs 5 and 7B).

These opposite effects of the proteins on the nuclear-cytoplasmic distribution of the zCLOCK:zBMAL heterodimer may be due to different transcriptional inhibition abilities. In the reporter assay (Fig. 1A), zCRY1a and zPER2 repressed CLOCK:BMAL-mediated transcription differently; zCRY1a almost completely inhibited transcription, whereas zPER2 produced only moderate repression. A sub-cellular localization experiment (Fig. 5) showed that when zCRY1a is co-expressed with the zCLOCK1:zBMAL3 heterodimer, all three proteins accumulate in the nucleus, consistent with our recent report that zCRY1a forms a stable complex with CRY-CLOCK-BMAL-E-box-containing DNA (21). zCRY1a therefore is believed to act as a transcriptional repressor that interacts directly with CLOCK and/or BMAL1 in the nucleus, as do mCRYs (7,8,14). In contrast, zPER2 promotes cytoplasmic retention of the zCLOCK1:zBMAL3 heterodimer (Fig. 7B, lanes 7–18). Conceivably, this zPER2-induced cytoplasmic retention of the heterodimer results in repression of zCLOCK:zBMAL-mediated transcription (Fig. 1A, lanes 9–12). The significance of the protein’s nucleo-cytoplasmic localization control has been described for the cell cycle regulator cdc2-cyclin B1 complex. Translocation of the complex to the nucleus in late G2 initiates cell progression from G2 to mitosis. 14-3-3 Sigma, a protein that regulates cellular activity, sequesters the complex in the cytoplasm, maintaining G2 arrest (29). It is tempting to speculate that zPER2 inhibits zCLOCK:zBMAL-induced transcription via a similar mechanism. Interestingly, when zCRY1a, zPER2 and the zCLOCK:zBMAL3 heterodimer were co-expressed, the four proteins co-localized both in the nucleus (N>C) and cytoplasm (N<C). This suggests another zCRY1a-mediated mechanism for transcriptional repression, in which zCRY1a, working together with zPER2, keeps the zCLOCK:zBMAL heterodimer in the cytoplasm.

In mammals, CRY and PER form heterodimers, which are redistributed from the cytoplasm to the nucleus. Unlike mammalian PER2, zPER2 localizes in the cytoplasm and is not translocated to the nucleus when co-expressed with zCRY (Fig. 6A and B, lanes 7–9). Instead, it is retained in the cytoplasm and causes cytoplasmic retention of zCRY (Fig. 6A, lanes 10–12). This unique sub-cellular localization of zPER2 must be due to the malfunction of its NLS. A recent in vitro study showed that in mammalian cells CRY interacts with PER2 (rat PER2), rendering its NLS (residues 778–794) functional to translocate the CRY:PER2 complex to the nucleus and that the elimination of NLS from PER2 results in cytoplasmic retention of the CRY:PER2 complex (25). The corresponding NLS (residues 876–892) of zPER2 has little similarity to the NLSs of mammalian PER2 proteins. We speculate that zPER2 NLS is not functional, which leads to cytoplasmic trapping of the zCRY:zPER2 complex as with mammalian PER2 lacking its NLS. A recent study reported that zPER2 functions in light entrainment pathways (20). Identification of the in vivo role of the zPER2-mediated cytoplasmic retention of zCRY should provide a clearer understanding of the input mechanism in the zebrafish circadian system.

We showed that zCRY1a interacts with zCLOCK1, zBMAL3 and the zCLOCK1:zBMAL3 heterodimer, thereby facilitating their nuclear accumulations (Figs 2, 4 and 5). As zCRY1a is a potent transcriptional inhibitor (Fig. 1A) (19), the translocation of the positive component (CLOCK and BMAL) to the nucleus by the negative component (CRY) is logically inconsistent. Possibly, the transcriptional repressor activity of zCRY1a is blocked by some unknown factor and only when that factor is inactivated does zCRY1a work as a transcriptional repressor. Alternatively, certain intracellular conditions may regulate zCRY1a repressor activity. Recent Arabidopsis and Drosophila studies showed that light induces a conformational change in CRY by means of an intramolecular or intermolecular redox reaction that is interceded by flavin adenine dinucleotide, resulting in the active CRY form (30–32). Although the transcriptional inhibition reaction by CRY is reported to be independent of light (8,33), the fact that mammalian CRYs are flavoproteins (34,35), as are those of Arabidopsis and Drosophila, indicates that their transcriptional repression may be regulated by the cellular redox state.

At least six kinds of CRYs are present in zebrafish cells. They are classified as repressor (zCRY1a, 1b, 2a and 2b) and non-repressor (zCRY3 and 4) type CRYs (19). The functional differences in these zCRYs have yet to be clarified. Here we have shown the difference between the former, zCRY1a, and latter, zCRY3, types. zCRY2b, a repressor type CRY, is similar to zCRY1a in its sub-cellular localization and interaction with the CLOCK:BMAL heterodimer (data not shown), indicative that repressor-type CRYs have these features in common. Differences in their expression patterns and strengths of interaction with other clock-related proteins may be important factors in clock regulation in zebrafish cells. Another interesting issue is identification of the photoreceptor. In a recent report, zCRY1b and zCRY3 have been pointed to be good candidates for the photoreceptor because their expressions do not oscillate in the light-responsive zebrafish culture cell line (36). The data on zCRY3 given here are not incompatible with the prediction that zCRY3 is the photoreceptor. What is the actual photoreceptor for entrainment in light-responsive zebrafish cells, however, remains a question.

Multiple Clock and Bmal genes exist in the zebrafish; three Clock genes (zClock1, 2 and 3) and three Bmal genes (zBmal1, 2 and 3) (18,21). Each pair-wise combination of these zCLOCKs and zBMALs activates transcription from the E-box-containing promoter, but the extent of transactivation differs (21). Conceivably, zCRYs and zPER2 regulate zCLOCK:zBMAL-mediated transcription by controlling the nucleo-cytoplasmic distribution of each of the zCLOCKs, zBMALs or the zCLOCK:zBMAL heterodimers thereby changing their combinations. This is done by masking the activity of CRY in order to anchor the heterodimer in the nucleus or by masking the PER2 domain that facilitates cytoplasmic distribution. Further study of the proteins associated with CRY and/or PER, which regulate their interactions with the CLOCK:BMAL heterodimer as well as nuclear-cytoplasmic shuttling, should clarify the molecular mechanism of transcriptional repression which produces the circadian oscillation of gene expression.

Acknowledgments

ACKNOWLEDGEMENTS

We are grateful to Y. Agata who provided the mammalian two-hybrid vectors. This research was supported by grants from the ministry of Education, Science, Sports and Culture of Japan [Grants-in-Aid for Scientific Research on Priority Area (A) 12050225 and (C) 13206034] and by the ‘Ground-based Research Announcement for Space Utilization’ promoted by the Japan Space Forum.

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