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Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School of Medicine, Chuo-ku, Kobe 650-0017, Japan

	Abstract

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Does the mammalian oscillatory protein mPER2 show the rhythm without its coding mRNA cycling? Here we answer this question by inserting a single copy of exogenous mPer2 gene to a NIH3T3 fibroblasts cell line, using Flp-In system. We generated the stable cell lines which constantly express mRNAs coding either N-terminal FLAG-tagged full length mPER2 (FLAG-mPER2(full)) or its C-terminal deleted form (FLAG-mPER2(1–1068)), which lacks the binding site to mCRY proteins, under the control of human EF-1 promoter. Although serum shock induced the rhythm of endogenous clock machinery in these cell lines, it did not initiate the rhythm of exogenously inserted FLAG-mPer2 genes at the mRNA level. In contrast, FLAG-mPER2(full) proteins showed the rhythm without their coding mRNA cycling. Since cells expressing FLAG-mPER2(1–1068) also showed the rhythm of FLAG-mPER2(1–1068) proteins, the direct binding of mCRY and mPER2 seems not necessary for this protein oscillation. This system clearly demonstrates that the intracellular endogenous clock system has an ability to modify the mPer2 gene post-transcriptionally to make mPER2 proteins oscillate without its coding mRNA cycling.

	Introduction

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Circadian rhythms have been observed in organisms from bacteria to humans. These rhythms are generated at a cellular level by a molecular oscillator, which is composed of interacting transcription/translation-based feedback loops involving a set of clock genes. In mice three Period genes (mPer1, mPer2, mPer3) have been found, and all of their mRNAs are expressed rhythmically in vivo (Albrecht et al. 1997; Shearman et al. 1997; Sun et al. 1997; Tei et al. 1997; Takumi et al. 1998a, 1989b; Damiola et al. 2000; Matsuo et al. 2003). Oscillation at the mRNA level will lead to the oscillation of the protein products which are the state variables of the intracellular circadian clock. Contrary to this speculation, recent reports suggest that post-transcriptional regulations, such as phosphorylation and ubiquitylation, contribute to the formation of rhythms of mPER proteins (Lowrey et al. 2000; Yagita et al. 2002). However, to what extent they contribute to the oscillation of mPER proteins has remained unclear.

In the present study, we tried to address whether the mammalian oscillatory protein mPER2 shows the rhythm without its coding mRNA cycling. We adopted the Flp-In system which can insert a single copy of exogenous mPer2 gene into the NIH3T3 fibroblasts cell line. This system eliminates the possible effect of a genomic site and gene dosage on gene expression: a single copy of the expression constructs is integrated in the identical genomic site among the generated cell lines. By the analyses of generated stable cell lines, here we show that exogenous mPER2 protein oscillates without its coding mRNA cycling.

	Results

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Flp-In 3T3 host cells drive the circadian expression of a clock controlled gene dbp after the serum shock

Serum shock is routinely used for the initiation of oscillation in cultured cells (Balsalobre et al. 1998; Yagita et al. 2001; Yamamoto et al. 2005), but in some cell lines, it is unsuitable for the clock oscillation study because of the lack of many of the clock components, such as COS7 (mammalian cells lacking mPer1, 2 and 3 expression), and S2 (insect cells lacking dClk expression) (Darlington et al. 1998). We selected NIH3T3 cells since it is known to have a functional circadian clock (Yamamoto et al. 2005).

The Flp-In system is schematically shown in Fig. 1A. In this system, Flp recombinase catalyzes a site-specific recombination event between the expression vector containing Flp Recombination Target (FRT) sequence, and the single FRT site integrated in Flp-In 3T3 cells, a NIH3T3 cell line containing a single copy of FRT site, to generate stable cell lines.

View larger version (22K): [in this window] [in a new window]   Figure 1  Flp-In system and rhythmic characterization of FRT integrated NIH3T3 cells. (A) Schematic representation of the Flp-In Flag-mPer2 system adopted in this study. A site-specific recombination event occurs between the FRT sequence of FLAG-mPer2 expression vector and the single FRT site in Flp-In 3T3 cells, with a co-transfection of the FLAG-mPer2 expression vector containing the FRT and Flp recombinase encoding vector to the host cell. A single copy of FLAG-mPer2 expression vector is integrated to produce a stable cell line. (B) Rhythmic nature of dbp mRNA after the serum shock in Flp-In 3T3 host cells. Quantitative data were shown in lower panel. Signal intensities of dbp was normalized with those of gapdh, the value before the application of serum shock being 100.

Generation of stable Flp-In cell lines constitutively expressing FLAG-mPer2(full) or the C-terminal deleted form FLAG-mPer2(1–1068)

We generated Flp-In 3T3 cells constitutively over-expressing mPer2. In addition to the full length mPER2 (mPER2(full)), we used a C-terminal deleted mutant (mPER2(1–1068)) which lacks the ability to bind mouse CRYPTOCHROME (mCRY) proteins (Fig. 2A) (Miyazaki et al. 2001; Akashi et al. 2002; Yagita et al. 2002). We selected this mutant to examine the effect of the disturbance of the heterodimerization of mCRY-mPER2 on rhythm formation, since mCRY proteins are the most powerful component constituting the negative limbs of the circadian oscillatory loop. We established stable cell lines either expressing N-terminal FLAG-tagged mPER2(full) (FLAG-mPER2(full)) or N-terminal FLAG-tagged mPER2(1–1068) (FLAG-mPER2(1–1068)) using the Flp-In system. Since we made the C-terminal-deleted-mutant expression vector by single nucleotide insertion to the FLAG-mPer2(full) expressing vector, the predicted mRNAs were virtually the same size. Indeed, the FLAG-mPer2(full) and its mutant mRNAs displayed nearly equal electrophoretic mobility (Fig. 2B left), whereas the expressed protein gives a truncated form (Fig. 2B right).

View larger version (27K): [in this window] [in a new window]   Figure 2  Stable Flp-In cell lines constitutively expressing each FLAG-mPER2(full) or the C-terminal deleted mutant FLAG-mPER2(1–1068). (A) Description of domain deleted in FLAG-mPER2(1–1068) protein. Both FLAG-mPER2(full) and FLAG-mPER2(1–1068) proteins are FLAG-tagged at their N-terminal and possess PAS domain and casein kinase I binding domain. FLAG-mPER2(1–1068) protein lost C-terminal domain to which mCRY proteins bind. (B) (left) Northern blot of FLAG-mPer2(full) and FLAG-mPer2(1–1068) mRNA expression in stable cell lines. Both mRNAs displayed an equal mobility since they differ in only a single nucleotide; (right) Western blot analysis of immunoprecipitated FLAG-mPER2(full) and FLAG-mPER2(1–1068) proteins expressed in stably transfected Flp-In cell lines. A truncated form of FLAG-mPER2 protein was observed for FLAG-mPER2(1–1068) expressing cell line. Electrophoretic mobility of both FLAG-mPER2(full) and FLAG-mPER2(1–1068) proteins were altered to more rapidly migrating forms by phosphatase treatment, indicating the phosphorylation of the proteins in the cells.

Both stable Flp-In cell lines retain the ability to drive the internal circadian clock after the serum shock

As expected from the characteristics of the human EF-1 promoter controlling the gene expression, FLAG-mPer2(full) and FLAG-mPer2(1–1068) mRNAs were constitutively expressed in each cell line even after the serum shock (Fig. 3A,B). In contrast, an endogenous clock controlled protein dbp mRNA showed a circadian gene expression in both cell lines after the serum shock (Fig. 3A,B), suggesting the cells possess the ability to drive the internal circadian clock.

View larger version (61K): [in this window] [in a new window]   Figure 3  mRNA and protein analyses of the stable Flp-In cell lines expressing either FLAG-mPer2(full) or FLAG-mPer2(1–1068). (A,B) Serum shock induced mRNA expression in cell lines stably expressing either (A) FLAG-mPer2(full) or (B) FLAG-mPer2(1–1068). Note dbp mRNA showed the circadian expression in both cell lines with a constant expression of inserted FLAG-mPer2(full) or FLAG-mPer2(1–1068). Quantitative results shown are mean ± standard error of means in three independent experiments. Signal intensities of dbp and FLAG-mPer2 were normalized with those of gapdh, the value before the application of serum shock being 100. (C,D) Rhythmic expression of FLAG-mPER2(full) and FLAG-mPER2(1–1068) proteins in the stable cell lines after serum shock. Total cell lysates of stable cell lines were collected at the indicated time after serum shock and immunoprecipitated with anti-FLAG M2 antibody. After SDS-PAGE, FLAG-mPER2 proteins were detected with anti-mPER2 (RY360) antibody. Lower panel shows immunoblot analysis of total cell lysates collected at the indicated time. Actin was immunoblotted as a control of protein quantity subjected to immunoprecipitation procedure. There was a tendency that FLAG-mPER2(1–1068) signals was higher than those of FLAG-mPER2(full). Note FLAG-mPER2(full) protein showed circadian oscillation (C) despite the lack of its coding mRNA cycling (A). FLAG-mPER2(1–1068), which lost its ability to bind mCRY proteins, also displayed circadian rhythm (D) with its mRNA being constant (B). Quantitative results shown are mean ± standard error of means in three independent experiments. Signal intensities of FLAG-mPER2 were normalized with those of ACTIN, the value 6 h after the application of serum shock being 100.

FLAG-mPER2 proteins show circadian oscillation without mRNA cycling

Finally, using these stable cell lines we investigated whether mPER2 proteins show the rhythms without mRNA cycling. In FLAG-mPer2(full) expressing cell lines, serum-shock could induce the rhythm of FLAG-mPER2(full) protein (Fig. 3C), although no rhythm was detected at the mRNA level (Fig. 3A). This indicates that the cell clock directly regulates mPER2 protein oscillation without its mRNA cycling. The truncation of C-terminus of mPER2 did not affect the rhythm generation ability since FLAG-mPER2(1–1068) protein showed the rhythmical change in the Flp-In cells after serum shock, which was similar to FLAG-mPER2(full) protein (Fig. 3D).

	Discussion

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In the present study, we adopted the Flp-In system to generate stably transfected NIH3T3 fibroblasts cell lines expressing FLAG-mPer2(full) and FLAG-mPer2(1–1068). Flp-In system enables the insertion of a single copy of the exogenous gene in a site-specific manner, and this eliminates the possible effect of the genomic site and gene dosage on gene expression.

In our previous study (Yamamoto et al. 2005), we utilized the Tet-off system to generate stably transfected NIH3T3 fibroblasts cell lines, in which the expression of exogenous mPer2 was controlled through a tetracycline-regulatable transcription factor. We showed that mPER2 protein oscillates despite the absence of cycling mPer2 mRNA level. However, we could not discriminate the mPER2 proteins derived from the endogenous mPer2 gene and those from the exogenous gene. Moreover, it was somewhat difficult to regulate the expression of the exogenous mPer2 gene strictly at a certain level, because multi copies were integrated at random sites. The relatively high expression of exogenously integrated mPer2 by the Tet-off system sometimes impairs the endogenous rhythm.

Here, we used the human EF-1 promoter to constantly express the exogenously integrated FLAG-mPer2 genes even after the serum shock. The expressed levels of FLAG-mPer2(full) and FLAG-mPer2(1–1068) did not interfere with the endogenous circadian molecular loop in the cells. Thus, in this Flp-In system, the expression profiles of integrated gene products, FLAG-mPER2(full) and FLAG-mPER2(1–1068) proteins, reflect the regulation by the internal cell clock which was still ticking after the gene insertion.

Since we made the C-terminal-deleted-mutant expression vector by a single nucleotide insertion to the FLAG-mPer2(full) expressing vector, the difference in mRNA stability and translational efficiency among the FLAG-mPer2(full) and the FLAG-mPer2(1–1068) genes will be minimum. Moreover, because of the recombination events in integrating the exogenous genes, the genetic background of established cell lines was identical. Thus, it is safe to conclude that the post-transcriptional events will lead to the changes in the accumulation of these gene products. From the fact that the accumulation levels of FLAG-mPer2 mRNA were constant, the translation of FLAG-mPer2 mRNA and/or the degradation of FLAG-mPER2 proteins themselves must be regulated in a rhythmic manner in clock ticking cells.

Since mCRY proteins were shown to suppress the ubiquitylation of mPER2 (Yagita et al. 2002), we speculate that mCRY-binding-lacking FLAG-mPER2(1–1068) protein might be fragile, and easily lose the oscillation. However, FLAG-mPER2(1–1068) proteins showed rhythmical change in the Flp-In cells after serum shock. This indicates that mPER2 protein oscillation mediated by the cell clock does not need the direct binding of mCRY proteins to mPER2 proteins. It is also possible that the indirect binding of mCRY to FLAG-mPER2(1–1068), with the help of other proteins, such as mPER1, is sufficient for these regulation, and then contributes to the protein oscillation.

Although the detailed mechanism regulating this process remains to be addressed, this study suggests that the intracellular endogenous clock system possesses the ability to modify the mPer2 gene post-transcriptionally to make mPER2 proteins oscillate without its coding mRNA cycling.

	Experimental procedures

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Plasmids

N-terminal FLAG-tagged mPer2 fragments were obtained by PCR with 5'-GCGGTACCATGGATTACAAGGATGACGACGATAAGATGAATGGATACGTGGAC-3' and 5'- GTGGTGTAGCTGTGGAACACACTG-3' as primers, and C-terminal fragments were excised from mPer2-pTRE2 (Yamamoto et al. 2005). These fragments were cloned into pEF5/FRT/V5/D-TOPO (Invitrogen, Carlsbad, CA, USA), and designated FLAG-mPer2(full)/pEF5/FRT. Sequences of the inserts were verified by DNA sequencing.

Site-directed mutagenesis

FLAG-mPer2(1–1068)/pEF5/FRT were generated by introducing a stop codon in the C-terminal portion of FLAG-mPER2(full) coding sequence. A single base pair was inserted to C-terminal fragment of FLAG-mPer2(full)/pEF5/FRT using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Mutagenesis primers were 5'-TGGCTCAGCCCTGTCTAGATAGCGGGGCATCCGCCACCTC-3' and its complementary strand (inserted nucleotide is indicated as a bold letter). The mutated fragment was excised by restriction enzyme and exchanged with the corresponding region of FLAG-mPer2(full)/pEF5/FRT vector. Presence of the expected mutation was confirmed by DNA sequencing.

Cell culture and generation of cell lines

Flp-In 3T3 cells, NIH3T3 fibroblast cells containing a single copy of integrated FRT sequence, were purchased from Invitrogen. The cells were maintained in Dulbecco's modified eagle's medium (Nacali Tesque, Kyoto, Japan) supplemented with 10% Donor Calf Serum (Gibco)(DMEM/10%DCS). To generate Flp-In cell lines stably expressing FLAG-mPer2(full) and FLAG-mPer2(1–1068) under the control of human EF-1 promoter, Flp-In 3T3 cells were plated on 60 mm dishes and transfected with 1.8 µg of the Flp-In recombinase-encoding pOG44 vector and either 0.2 µg of FLAG-mPer2(full)/pEF5/FRT or FLAG-mPer2(1–1068)/pEF5/FRT. After 2 days, we started selection with 200 µg/mL HygroGold (Invivogen) for 10 days. Colonies were picked up, expanded and assayed for expression of appropriate mRNAs and proteins. Established cell lines were maintained in DMEM/10%DCS containing 50 µg/mL HygroGold. Serum shock was performed as follows: Cells were cultured for 3-4 days in DMEM/10%DCS containing 50 µg/mL HygroGold to reach confluence. Twelve hours before serum shock, the medium was replaced with DMEM/5%DCS. At time = 0, the medium was replaced with DMEM with 50% Horse Serum (Gibco), and after an hour this medium was replaced again with serum-free DMEM and cells were cultured for the indicate time.

Northern blot analysis

Cultured cells were washed 3 times with ice-cold PBS and harvested in 1 mL TRIzol reagent (Invitrogen). These samples were frozen and stored at –70 °C until the extraction of whole cell RNA. For the assay of circadian gene expression, samples harvested just before serum shock were indicated as time = 0. Twelve micrograms of total RNA was electrophoresed in a 1.2% agarose gel containing 2% formaldehyde, transferred to Byodyne A membrane (PALL Biosupport, New York, NY, USA) and hybridized with probes. We used the total coding region of mouse dbp (GENBANK accession U29762 [GenBank] ), 1–878 bp (GENBANK accession NM011066) of mPer2 cDNA, and gapdh (Clontech, Palo Alto, CA, USA) as templates to detect dbp, exogenously expressed mPer2 and gapdh mRNA, respectively.

For estimating the oscillation of cellular rhythm, endogenous mPer2 can also be a marker. In Northern blotting, endogenous mPer2 mRNA is approximately 7 kb (Albrecht et al. 1997; Shearman et al. 1997; Takumi et al. 1998a), which is distinguishable from approximately 4.6 kb exogenous FLAG-mPer2 mRNAs. In our present Flp-In cell line, clear staining of endogenous mPer2 mRNA above the bands of FLAG-mPer2 mRNAs was detected just after serum shock (data not shown). However, in later time points, we could hardly detect the endogenous mPer2 mRNAs because of the low levels of expression.

Probes were labeled with ³²P-deoxycitidine triphosphate using Prime-It II Random Primer Labeling Kits (Stratagene). Hybridization was performed at 42 °C for 16 h, and membranes were washed twice in 0.2xSSC/0.1% SDS at 60 °C for 1 h each. Membranes were exposed to an imaging plate and analyzed by BAS 5000 (Fuji Film, Tokyo, Japan).

Immunoprecipitation

Stably transfected cells plated on 90 mm dishes were harvested with 700 µL of lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 50 mM NaF, 100 µM Na₃VO₄, complete protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany)). After centrifugation, lysates were incubated at 4 °C for 2 h with anti-FLAG M2 monoclonal antibody (Sigma). After incubating with Protein G agarose beads for 2 h at 4 °C, beads were washed with lysis buffer. Samples were subjected to SDS-PAGE. For dephosphorylation experiments, we used lysis buffer without NaF and Na₃VO₄, and prior to electrophoresis, beads containing immunoprecipitated proteins were washed 3 times with BAP buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM MgCl₂). Then the beads were incubated in 30 µL BAP buffer with or without 2.5 units of bacterial alkaline phosphatase (BAP) (TaKaRa, Japan) for 1 h at 37 °C. Immunoblot analysis was performed using anti-mPER2 antibody (RY360, Yanaihara Institute; raised against mPER2 (66–83)) and anti-Actin (sc-1616, Santa Cruz) as primary antibodies. As secondary antibodies anti-rabbit Ig HRP-linked antibody (Amersham) and anti-goat IgG HRP-linked antibody (Santa Cruz) were used. Chemiluminescence was performed using Western Blotting Luminol Reagent (Santa Cruz), and analyzed by LAS-1000 (Fuji Film).

	Acknowledgements

 
This work was supported by Scientific Grants from the Ministry of Health, Welfare and Labor, The Special Coordination Funds and The Scientific Grants of the 21st Century COE Program from Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Yanaihara Institute Inc (Fujinomiya) for the production of anti-mPER2 serum.

	Footnotes

 
Communicated by: Kozo Kaibuchi

^a Present address: Department of Biological Science, Nagoya University Graduate School of Science, Nagoya 464–8602, Japan

^* Correspondence: E-mail: okamurah{at}kobe-u.ac.jp

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