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Genes to Cells (2006) 11, 163-175. doi:10.1111/j.1365-2443.2006.00929.x
© 2006 Blackwell Publishing or its licensors
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Odd-Skipped Related 2 gene transcription is regulated by CCAAT enhancer-binding protein {delta} in mesenchymal C3H10T1/2 cells

Shinji Kawai1,*, Takahiro Kato1, Masahiro Sato2 and Atsuo Amano1

1 Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan
2 The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Odd-skipped related 2 (Osr2) gene is mouse homolog of Drosophila Odd-skipped gene involved with the pair-rule segmentation phenotype in Drosophila mutant embryos. In this study, to examine Osr2 expression regulation, the mouse Osr2 promoter region was cloned and characterized, and found to have two enhancer elements in the –1463/–1031 (distal) and –581/+3 (proximal) regions, and a repressor region (–4845/–1463, far distal). CCAAT/enhancer binding protein (C/EBP) binding sites were found in both the distal and proximal enhancer elements. Osr2 promoter activity was enhanced by C/EBP{delta}, a member of the C/EBP family, in a dose-dependent manner. Electrophoresis mobility shift assays showed that purified GST-C/EBP{delta} bound to distal (–1295/–1261) and proximal (–89/–55) C/EBP binding motifs. Chromatin immunoprecipitation demonstrated that acetylated histones H3, H4, and C/EBP{delta} in the proximal region (–280/–43), but not the distal region (–1438/–1196), indicating that the Osr2 promoter proximal region was transcriptionally activated in C3H10T1/2 cells. Our results suggest that Osr2 expression is regulated by C/EBP regulatory elements.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The odd-skipped (odd) gene, first identified as a part of Drosophila pair-rule transcription factors, contains DNA-binding C2H2-type zinc finger domains in the C-terminal half of the molecule (Coulter et al. 1990). The Drosophila odd gene is generally classified as a secondary pair-rule gene (Ingham et al. 1988), and is primarily expressed in stripes that spread in even-numbered segments and then later in narrow stripes in the center of both even- and odd-numbered segments of narrow strips (Coulter et al. 1990). Mutations in the odd gene cause pattern defects in the anterior regions of odd-numbered segments and partial substitution by mirror-image duplications in adjacent regions (Coulter & Wieschaus 1988). These defects are closely associated with altered expression levels of other segmentation genes, such as fushi tarazu (ftz), engrailed (en), and wingless (wg) (DiNardo & O’Farrell 1987). Expression of the odd gene is governed by the so-called primary pair-rule genes (eve, h, and runt) (Ingham et al. 1988). However, Berman et al. (2004) and Schroeder et al. (2004) recently demonstrated that the odd gene functioned similarly to a primary pair-rule gene.

In addition to odd, three genes in Drosophila, including brother of odd with entrails limited (bowl), sister of odd and bowl (sob), and drumstick (drm), exhibit a high degree of homology (82% for bowl, 68% for sob, and 55% for drm) throughout the zinc finger domains of the odd gene, though no significant homology is displayed outside of this domain (Hart et al. 1996). odd, bowl, sob, and drm have 4, 5, 5 and 2 zinc finger domains, respectively. Drosophila bowl is required for patterning of the hindgut and proventriculus morphogenesis (Wang & Coulter 1996), as well as for leg segmentation, and functions downstream of the Notch signaling pathway (de Celis Ibeas & Bray 2003). Drosophila drm antagonizes activity by the transcriptional regulator, lin, to control patterning and morphogenesis of the hindgut (Green et al. 2002), and Drosophila bowl acts downstream of lin, and is required for morphogenesis of both the foregut and hindgut (Johansen et al. 2003). However, the function of Drosophila sob is not well defined in Drosophila.

Homologues for odd genes have been found in Caenorhabditis elegans, Saccharomyces cerevisiae, and Xenopus laevis (Brohl et al. 1994; The C. elegans Sequencing Consortium 1998; Klein et al. 2002), and two mammalian homologs, odd-skipped related 1 (Osr1) and odd-skipped related 2 (Osr2), were subsequently cloned from both mice and humans (So & Danielian 1999; Lan et al. 2001; Katoh 2002). Mouse Osr1 has been shown to have a 65% homology with Osr2 (Lan et al. 2001); however, the tissue distribution of these mRNA/proteins appear to be different from each other. For example, mouse Osr1 is expressed in the intermediate mesoderm, limb, and branchial arch of fetuses on embryonic day (E) 9.5–12.5 (So & Danielian 1999). Further, human Osr1 is detected in fetal lungs, as well as adult colon, small intestine, prostate and testis tissues (Katoh 2002). In contrast, Osr2 is preferentially expressed in sites where epithelial-mesenchymal interactions occur during limb, tooth, and kidney development. Mouse Osr2 mRNA is abundantly expressed in the mesonephric vesicles of fetuses on E 9.25, whereas it becomes restricted to the mandibular mesenchyme immediately neighboring the maxillary processes on E 10.0. In the limb buds, Osr2 mRNA is first expressed in the central region of the forelimb mesenchyme, and then moves into the forelimb and hindlimb (Lan et al. 2001).

The expression pattern of Osr2 mRNA suggests its important role in mandibular and maxillary formation. In fact, a recent report of a targeted null mutation in the Osr2 gene demonstrated that cleft palate was induced, probably due to defects in palatal shelf growth and a delay in palatal shelf elevation (Lan et al. 2004). Palatal outgrowth is normally initiated in these mutant embryos; however, proliferation of palatal mesenchymal cells is greatly reduced in the medial halves of the downward budding palatal shelves on E 13.5 (Lan et al. 2004). This results in delayed mediolateral palatal shelves preceding palatal shelf elevation. Such growth retardation of the palate appears to be tightly associated with expression of the Osr1 gene during palate development. For example, during early palatal outgrowth, Osr1 mRNA is faintly expressed in the palatal mesenchyme, whereas by E 13.5 that expression is greatly up-regulated in the lateral halves and entirely down-regulated in the medial halves of the palatal shelves. In addition, Osr2 null mice exhibit altered gene expression patterns, including those of Osr1, Pax9 and TGFß3, during palate development (Lan et al. 2004). Therefore, Osr2 is considered likely to play a critical role in craniofacial development.

CCAAT/enhancer binding proteins (C/EBPs) consist of six known basic leucine zipper transcription factors, namely C/EBP{alpha}, ß (also known as NF-IL6, IL-6DBP, LAP, CRP2, AGP/EBP, NF-M, or ApC/EBP), {gamma} (Ig/EBP), {delta} (CELF, CRP3, NF-IL6ß, RcC/EBP2), {varepsilon} (CRP1), and {zeta}[CHOP-10 (C/EBP homologous protein) or GADD153]. These proteins are involved in various biological systems including lineage commitment, cell growth, adipocyte differentiation, sugar metabolism, natural immunity, hematopoiesis, and liver development (reviewed in Lekstrom-Himes & Xanthopoulos 1998). For example, C/EBP{delta}-deficient mice exhibit neurologic defects and defective lipid storage, and C/EBP{delta} is known to cooperate with C/EBPß in adipocyte differentiation (Tanaka et al. 1997), while inflammatory irritation induces the expression of C/EBP{delta} (Alam et al. 1992). In addition, C/EBP{delta} is abundantly expressed in the liver, lungs, and small intestines in mice (Lekstrom-Himes & Xanthopoulos 1998), and known to be activated by phosphorylation during IL-1-induced signal transduction pathway (Lacorte et al. 1997).

The expression pattern of Osr2 has been well documented, as described above; however, the mechanism underlying regulation of its expression remains to be elucidated. To assess Osr2 expression regulation, we cloned the 5'-flanking DNA sequence (spanning 4845 bp) of the mouse Osr2 gene. Functional analysis of this region revealed that C/EBP{delta} was able to enhance Osr2 promoter activity. This report is the first known direct evidence that C/EBP{delta} proteins have the ability to induce Osr2 expression.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Molecular cloning of the 5'-flanking region of the Osr2 gene and putative transcription factor binding sites

A BAC clone containing the Osr2 gene, termed RP24-335P21, was identified by a BLAST search and shown to contain a 4970-bp fragment of the Osr2 5'-flanking region (DDBJ/EMBL/GENBANK accession number AB210138). The 5'-flanking region of the Osr2 gene was amplified by PCR with oligo-primers using RP24-335P21 as a template, as described in the Experimental procedures section. The putative transcription factor binding sites on the 5'-flanking region of Osr2 gene are identified by TFSEARCH. In Fig. 1A, the nucleotide sequence of the proximal region of the 5'-flanking region of Osr2 gene is shown. Although there was a GC-rich region found around nucleotides –40 of the Osr2 gene, a canonical TATA box was not found in the area spanning nucleotides –1 to –581. Sequence analysis of the 5'-flanking region revealed the presence of multiple potential transcription factor binding sites. In the distal region (nucleotides –4845 to –581), there were two C/EBP-binding sequences, one each for the GATA-binding factor 1 (GATA-1) and GATA-2 sequences, as well as one activator protein 1 (AP-1) binding site, one myeloid zinc finger protein 1 (MZF1) sequence, two sex-determining region Y gene product (SRY) sequences, one acute myeloid leukemia gene 1a (AML1a)/PEBP2{alpha}B/CBFA2 sequence, one homeobox gene (CdxA) sequence, two Nkx-2 sequences, and one Sox5 sequence. The proximal 5'-flanking region (nucleotides –581 to –1) had five SRY sequences, one heat shock factor 2 (HSF2) sequence, one October-1 sequence, one Nkx-2 sequence, three CdxA sequences, one GATA-2 and one GATA-1 sequence, two MZF1 sequences, one AP-1 site, one Ikaros 2 (Ik-2) sequence, one C/EBP sequence, one hepatic nuclear factor 3ß (HNF-3ß) sequence, and one stimulating protein 1 (Sp-1) sequence. Notably, the 5'-flanking region of the Osr2 gene had two long runs of CA repeats, which were a 26-bp repeat in nucleotides –3573 to –3548 (CAR1) and a 46-bp repeat in nucleotides –2494 to –2449 (CAR2).


Figure 1
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  		Figure 1  The 5'-flanking proximal region of the mouse Osr2 gene. (A) Nucleic acids sequence of the 5'-flanking proximal region of the mouse Osr2 gene and transcription factor binding motifs. The TSS (+1) of the mouse Osr2 gene was determined by 5'-RACE analysis using total RNA from C3H10T1/2 cells. The first exon region is shown in bold. The numbers shown to the left are relative to the putative start sites of Osr2 mRNA. Consensus sequences for the transcription factor sites are boxed or shadowed. The transcription factor binding motifs were predicted using TFSEARCH. The threshold score was set at 90.0. AAGCTT and CTGCAG denoted by double-underlines indicate HindIII and PstI sites, respectively. (B) Comparison of mouse and human Osr2 promoter. The mouse Osr2 promoter and human Osr2 promoter in the human BAC clone of chromosome 8 (RP24-335P21) were aligned using the BLAST 2 SEQUENCES program. Overall conserved regions exhibiting more than 80% homology between mouse and human are shown with boxes; S1, –3004/–2958 (46 bp); S2, –2583/–2545 (38 bp); S3, –1610/–1475 (135 bp); S4, –1288/–1217 (71 bp); and S5, 803/–1 (803 bp). E1 indicates exon 1. Several putative transcription factor binding motifs (including E47, AML1a, C/EBP, HSF2, SRY, AP-1, C/EBP, HNF-3ß, and Sp-1) identified by a search of the mouse Osr2 promoter region using TFSEARCH were found in the human sequences.

 
Comparison of Osr2 promoter in mouse and human genes

We also performed a BLAST search against human Osr2 cDNA (GENBANK accession number NM_054049 [GenBank] ) and identified the BAC clone of human chromosome 8 (RP24-335P21) containing the Osr2 genomic 5'-flanking region. Overall conserved regions between mice and humans are shown in Fig. 1B. In the distal 5'-flanking region, only four short regions corresponding to nucleotides –3004/–2958 (46 bp), –2583/–2545 (38 bp), –1610/–1475 (135 bp), and –1288/–1217 (71 bp) exhibited more than 80% homology between mice and humans. Notably, the latter three regions contained E47, AML1a, and C/EBP sequences, respectively. In the proximal 5'-flanking region between nucleotides –803 to –1, there was 84% homology between mice and humans. Several putative transcription factor binding sites identified in the mouse Osr2 sequence were also found in the human sequence, which included HSF2, SRY (three sites), AP-1, C/EBP, HNF-3ß, and Sp-1 sequences. These findings suggest that the proximal region is essential for Osr2 gene function.

Determination of transcription start site (TSS) of Osr2 gene

Prior to determining the TSS of the Osr2 gene, we searched for Osr2 mRNA-high expressing cells using the previously established cell lines C3H10T1/2, C2C12, and MC3T3-E1. RT-PCR (Fig. 2A), and quantitative real-time PCR (Fig. 2B) results showed that C3H10T1/2 cells expressed Osr2 mRNA highly, whereas the transcript was expressed in low amounts in C2C12 and MC3T3-E1 cells. Thus, we decided to use total RNA obtained from C3H10T1/2 cells in an RLM-RACE assay. After cloning the RACE products, 14 independent clones were randomly chosen and subjected to sequencing analysis. Two of these clones exhibited the longest position, and the 5' end of the insert was designated as TSS +1 (Fig. 1A). The other clones exhibited shorter positions than the previous two clones; +103 for three clones, +211 for three clones, +291 for one clone, and +303 for one clone. It should be noted that the proximal regions lacked canonical TATA, whereas they contained CCAAT consensus and GC-rich sequences around the TSS.


Figure 2
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  		Figure 2  Expression analysis of Osr2 in cultured cell lines. (A) Detection of Osr2 mRNA was performed by RT-PCR. Arrows indicate the expected bands (0.9 and 0.8 kb) obtained after 25 or 30 cycles of PCR. These two bands correspond to splicing variants. ß-Actin was used as the internal control for reporting RNA quality. (B) Quantification of Osr2 mRNA was determined by real-time PCR. The RT-PCR products were quantified by LightCycler with SYBR Green. Expression levels of mRNA are shown as relative cycle number normalized by the cycle number of GAPDH.

 
Promoter activity of 5'-flanking region of Osr2 gene

To determine promoter activity in the isolated 5'-flanking region of Osr2 gene, we constructed six luciferase reporter vectors carrying various lengths of the 5'-flanking sequences (Fig. 3). These constructs were transiently transfected into an Osr2 mRNA-high expressing cell line, C3H10T1/2, and then luciferase activity was measured. As shown in Fig. 3, the construct (parental construct) carrying a 4845-bp 5'-flanking region of the Osr2 gene exhibited luciferase activity approximately five-fold higher than the promoter-less pGL3-basic vector. Removal of the sequence corresponding to nucleotides –4845 to –1463 from the parental construct resulted in a dramatic increase in luciferase activity, implicating the presence of a suppressor element within a region spanning nucleotides –4845 to –1463. Sequential deletion of the regions corresponding to nucleotides –1463/–1031 and nucleotides –581/+3 caused a drastic decrease in promoter activity, though the construct carrying the region corresponding to nucleotides +3/+125 retained slight promoter activity. These findings suggest that the regions corresponding to nucleotides –1463/–1031 and –581/+3 contain enhancer elements for Osr2 mRNA expression.


Figure 3
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  		Figure 3  Promoter activity and serial deletion analysis of 5'-flanking region of mouse Osr2 gene. The serially deleted 5'-flanking regions were ligated to the luciferase reporter vector, promoter-less pGL3-basic (expressed as Luc), and the resulting vectors were assayed for luciferase activity using C3H10T1/2 cells. Constructs are indicated on the left of the diagram. Bold lines in each construct indicate various lengths of the 5'-flanking region starting from position +125. The arrow indicates the position of TSS and Ex1 shows Exon 1. The results are expressed as the mean ± standard deviation and indicated as fold induction over the pGL3-basic vector. The experiments were done in triplicate and repeated at least twice. *Significant (P < 0.05) induction vs. pGL3-basic vector and significant (P < 0.05) induction between the next-bar neighbor are indicated.

 
Effects of forced expression of C/EBP{delta} on expression of the Osr2 gene

In our previous study, it was suggested that expression of the Osr2 gene might be regulated through binding of several of the transcription factors listed in Fig. 1. To test this possibility, some available transcription factors such as Cbfa1 (which binds to the AML1a site), E12 and E47 (both of which bind to the E47 sequence), and C/EBPs (including C/EBP{delta}) expression vectors were co-transfected with the parental luciferase expression construct in C3H10T1/2 cells. Cbfa1 slightly suppressed the Osr2 promoter activity, whereas E12 and E47 had no effect on luciferase activity (data not shown). In Fig. 4A, C/EBP sites in the mouse Osr2 promoter are listed. Among the three C/EBPs tested, C/EBP{delta} was the most potent to induce increasing Osr2 promoter activity (Fig. 4B). Based on these findings, we examined the regulatory mechanism of Osr2 expression by C/EBP{delta}. The activity of the Osr2 promoter was enhanced when the parental luciferase expression construct was co-transfected with a C/EBP{delta}-expressing vector into C3H10T1/2 cells (Fig. 4C). This enhancement was further accelerated by increasing the amount of parental luciferase expression construct added to the cells. To identify the sites that can exert the function of C/EBP in the Osr2 promoter, luciferase expression constructs carrying sequentially deleted 5'-flanking regions of the mouse Osr2 gene (as in Fig. 3) were co-transfected with a C/EBP{delta}-expressing vector into C3H10T1/2 cells. As shown in Fig. 4D, removal of the region from nucleotides –4845 to –1463, which contained one C/EBP sequence, did not affect the C/EBP{delta}-induced Osr2 promoter activity. Removal of another region from nucleotides –1031 to –581, which had no C/EBP sequence, was less effective. Deletion of the region from nucleotides –581 to +3, which had one C/EBP site, dramatically reduced Osr2 promoter activity, indicating that the most proximal C/EBP site is essential for enhanced expression of Osr2 mRNA. Unexpectedly, elimination of the region corresponding to nucleotides –1463/–1031 increased luciferase activity, suggesting that distal C/EBP site is spatially concealed by upstream DNA structure or that C/EBP{delta} also functions as a transcriptional repressor through binding to distal region. Induction by C/EBP{delta} was also detected in pPs construct without putative C/EBP site, suggesting that this region contains unidentified or irregular C/EBP binding site (Fig. 4D). Mutagenesis of proximal C/EBP binding site significantly decreased promoter activity, while mutation in distal site stayed slight reduction (Fig. 4E). These findings suggest that C/EBP{delta} directly regulates the expression of Osr2 mRNA.


Figure 4
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  		Figure 4  Regulation of Osr2 promoter activity by C/EBP{delta}. (A) Schematic representation of potential C/EBP transcription factor binding motifs in mouse Osr2 promoter. The C/EBP site between –1529 and –1097 is referred to as the Distal C/EBP site and that between –647 and –64 as the Proximal C/EBP site. (B) Luciferase activity in C3H10T1/2 cells transfected with a construct carrying the Osr2 promoter (4845 bp)-luciferase reporter gene (pBm), plus various C/EBPs, {alpha}, ß, {delta} (100 ng) expression vector or empty pcDNA3 vector lacking C/EBP cDNAs (ctrl). Luciferase activity was measured at 48 h after transfection. The results are indicated as fold induction over control transfection with an empty vector. *Significant (P < 0.05) induction by C/EBPß and C/EBP{delta} is indicated. (C) Luciferase activity on pBm plus various amounts (ng) of the C/EBP{delta} expression vector (pC/EBP{delta}). Significant (P < 0.05) induction by C/EBP{delta} is indicated with an asterisk. (D) The reporter constructs carrying serial deleted Osr2 promoter shown in Figure 3 were co-transfected without ({square}) or with (100 ng; {blacksquare}) the pC/EBP{delta} vector. The results are indicated as fold induction over the pGL3-basic vector. *Significant (P < 0.05) induction vs. with pBm/C/EBP{delta} expression vector is indicated. (E) Luciferase activity on the Osr2 promoter (1463 bp)-luciferase reporter (pXh), or a construct carrying mutation at distal or proximal C/EBP binding site of pXh. The distal C/EBP binding site GTTTTACCAAAGA was mutated to GTTTTAttgAAGA (mut1), and proximal site TGCTATCCAAACA was changed to TGCTATttgAACA (mut2). The results are indicated as fold induction over pGL3-basic vector. Significant (P < 0.05) reduction by mutation is indicated with an asterisk.

 
C/EBP{delta} interacts with C/EBP binding elements in Osr2 promoter (EMSA)

We next investigated whether C/EBP can bind to its putative consensus site in the Osr2 promoter region by EMSA using GST-C/EBP{delta}. Incubation of GST-C/EBP{delta} with a DIG-labeled oligonucleotide probe corresponding to the C/EBP binding site at the distal (–1463/–1031) or proximal (–581/+3) region resulted in generation of specific complexes, which were clearly detected by shifting of the bands (Fig. 5). The shifts were completely abolished by the addition of 200-fold excess amounts of unlabeled oligonucleotides corresponding to both C/EBP binding sites (Fig. 5). Addition of anti-human C/EBP{delta} antibodies (Active Motif, Carlsbad, CA, USA) to the mixture of GST-C/EBP{delta} and oligonucleotides resulted in the super-shift pattern. These results confirmed that C/EBP{delta} is able to bind to the C/EBP consensus site in the Osr2 promoter.


Figure 5
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  		Figure 5  Binding of C/EBP{delta} to Osr2 promoter. Electrophoretic mobility shift assay performed using both strands of oligonucleotides in two regions, the distal site (–1295/–1261) and proximal site (–89/–55), both of which included the C/EBP binding motifs (as shown in Figure 4A). The GST-C/EBP{delta} was incubated with or without DIG-labeled oligonucleotide. In the first and fifth lanes, no GST-C/EBP{delta} was added. In the second and sixth lanes, the GST-C/EBP{delta} incubated with DIG-labeled oligonucleotide was loaded. Note the complex formation between the GST-C/EBP{delta} and DIG-labeled probe (indicated by an arrowhead). The open-arrowhead indicates supershifted complexes with antibodies against C/EBP{delta} ({alpha}C/EBP{delta}, third and sixh lanes). Addition of 100-fold excess amounts of unlabeled oligonucleotide to a mixture of GST-C/EBP{delta} and DIG-labeled oligonucleotide reduced the complex formation between the GST-C/EBP{delta} and DIG-labeled probe (shown in lanes 4 and 8).

 
Histones H3 and H4 acetylated in proximal Osr2 promoter region (ChIP)

We also investigated whether histones are acetylated around C/EBP binding sites. A ChIP assay was performed with C3H10T1/2 cells using antibodies against acetylated histone H3 and histone H4 to examine the possible association of acetylated histone with portions of the distal (–1438/–1196) and proximal (–280/–43) Osr2 promoters. Primers were designed to amplify the 243-bp fragment containing the distal portion or the 238-bp fragment containing the proximal portion of the promoter that encompassed the C/EBP binding site (Fig. 4A). As shown in Fig. 6A, antibodies against acetylated histone H3 and histone H4 precipitated fragments from the proximal, but not distal, Osr2 promoter region. These results indicate that the acetylation of histones H3 and H4 within the proximal Osr2 promoter region frequently occurs in C3H10T1/2 cells.


Figure 6
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  		Figure 6  Chromatin immunoprecipitation analysis. (A) Chromatin was immunoprecipitated with antibodies against acetylated histone H3 ({alpha}acH3) or acetylated histone H4 ({alpha}acH4), as well as with control rabbit IgG. The DNA extracts were amplified using pairs of primers (each of which corresponded to a distal or proximal portion of the mouse Osr2 promoter) indicated by arrows in Fig. 1B. Input indicates that the DNA extract sample was amplified without being immunoprecipitated. Immunoprecipitation with {alpha}acH3 and {alpha}acH4 resulted in generation of bands derived from amplification of the proximal, but not distal, portion of the mouse Osr2 promoter. (B) Chromatin was immunoprecipitated with antibodies against C/EBP{delta} ({alpha}C/EBP{delta}) or C/EBPß ({alpha}C/EBPß), as well as with control rabbit IgG. The DNA extracts were also amplified using same pairs of primers above. Immunoprecipitation with {alpha}C/EBP{delta} resulted in generation of bands derived from amplification of the proximal, but not distal, portion of the mouse Osr2 promoter. {alpha}C/EBPß did not precipitate any fragments.

 
C/EBP{delta} interacts with proximal C/EBP binding elements in Osr2 promoter

We further investigated whether C/EBP{delta} endogenously binds C/EBP binding sites by ChIP assay. Antibodies against C/EBP{delta} and C/EBPß were used in C3H10T1/2 cells to examine the potential association of C/EBPs with portions of the distal (–1438/–1196) and proximal (–280/–43) Osr2 promoters. As shown in Fig. 6B, antibodies against C/EBP{delta} precipitated the fragments from the proximal, but not the distal Osr2 promoter region. Antibodies against C/EBPß did not precipitate any fragments. These results indicate that the C/EBP{delta} directly and endogenously interact with the proximal Osr2 promoter region in C3H10T1/2 cells.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We cloned and characterized the mouse Osr2 promoter region, which contained GC-rich sequences and a CCAAT sequence with a C/EBP consensus motif, but lacked a TATA box. Multiple transcription factor binding sites were also found in this region. Further, a transient expression assay using constructs carrying the luciferase reporter gene fused with various lengths of the Osr2 promoter demonstrated the presence of enhancer elements in the –1463/–1031 and –581/+3 regions. Remarkably, the function of these enhancer elements appeared to be regulated by a far-upstream repressor region (–4845/–1463). Osr2 promoter activity was shown to be enhanced by expression of C/EBP{delta} in C3H10T1/2 cells and EMSA revealed that nuclear factors bound to the –89/–55 region, suggesting direct involvement of the C/EBP{delta} protein in regulation of Osr2 mRNA expression. In addition, the presence of acetylated histones H3 and H4 in the proximal region (–280/–43) suggests that this region is active in C3H10T1/2 cells in a transcriptional manner. The present data suggest that expression of mouse Osr2 mRNA is regulated by at least one of several transcription factors, particularly C/EBP{delta}, which is known to be essential for mammalian development.

Several putative transcription factor binding sites, including SRY, HSF2, Oct-1, Nkx-2, CdxA, GATA-1/2, MZF1, AP-1, Ik-2, C/EBP, and HNF-3ß, have been found in experiments using cloned mouse Osr2 promoter. Among these sites, 3 putative binding sites for C/EBP are known to exist in the far-distal, distal, and proximal regions of the Osr2 promoter. C/EBP{delta}, a protein first characterized as an acute phase inflammatory response protein (Chen & Liao 1993), is known to be rapidly induced by a variety of extracellular stimuli including growth hormone, insulin, interferon (IFN)-{gamma}, IL-1, IL-6, lipopolysaccharide (LPS), tumor necrosis factor (TNF)-{alpha}, dexamethasone, norepinephrine and glutamate (Lekstrom-Himes & Xanthopoulos 1998). C/EBP{delta} is expressed in osteoblasts (Billiard et al. 2001), lung epithelial cells (Lekstrom-Himes & Xanthopoulos 1998) and mammary glands (Gigliotti & DeWille 1999) while Osr2 mRNA is expressed in the limbs, teeth, kidneys and mandibular and maxillary tissues of developing fetuses (Lan et al. 2001). Previously, we reported that Osr2 mRNA is expressed in mesenchymal cells, as well as lung, kidney, muscle and testis tissues of adult mice (Kawai et al. 2005), while it was also shown in osteoblastic cell lines in the present study. Further study is required to determine whether the extracellular stimuli regulate Osr2 mRNA expression via C/EBP{delta}. CCAAT box-binding factors including C/EBP, as well as NF-Y (nuclear factor-Y)/CBF (CCAAT-binding factor) and NF-I (nuclear factor-I)/CTF (CCAAT-transcription factor). In a review of previous supershift EMSA experiments with anti-NF-Y antibodies and competition experiments with Y box oligonucleotides, Mantovani (1999) reported that NF-Y/CBF is a major binding factor for the CCAAT binding site. Therefore, it is highly possible that NF-Y/CBF regulates Osr2 mRNA expression through competitive binding of C/EBP to the target binding sites on the Osr2 promoter. Interestingly, the Osr2 promoter contains several putative SRY binding motifs, while the SRY gene denotes the sex-determining region in the Y chromosome and is important for male sex determination (Goodfellow & Lovell-Badge 1993). Although the role of Osr2 in sex determination remains unclear, the fact that it is expressed strongly in the testes supports a possible role of Osr2 in sex determination.

CA repeats are frequently observed in non-coding regions, including the intron and 5'/3'-flanking regions, of a gene and believed to influence the chromatin configuration, such as nucleosome organization, recombination, and gene expression (Tripathi & Brahmachari 1991). CA repeat polymorphism is found in several promoters, such as insulin-like growth factor (IGF)-I (Rietveld et al. 2003), GLC1A (GLAUCOMA 1, OPEN ANGLE, A) (Sjostrand et al. 2002), and Type I {alpha}2 collagen (Dietzsch & Parker 1999). Several studies have also suggested that CA repeat polymorphism is closely associated with susceptibility to a disease (Sjostrand et al. 2002; Tsuchiya et al. 2005). Sequencing analysis of the 5'-flanking region of the mouse Osr2 gene demonstrated that CAR1 and CAR2 contain 26- and 46-bp CA repeats, respectively. This result is in contrast with previously published data regarding the mouse Osr2 gene that CAR1 and CAR2 contained 26- and 44-bp CA repeats, respectively. Such a discrepancy suggests the presence of CA repeat polymorphism in the Osr2 promoter, which led us to suppose a correlation between polymorphism in the CA repeat of the Osr2 promoter and some syndrome, as has been demonstrated for other gene promoters.

According to the bioinformatics provided by NCBI Entrez for a single nucleotide polymorphism (SNP), two SNPs exist in the 5'- and 3'-non-coding regions of the mouse Osr2 gene. In humans, two (rs985794 and rs3019295) of ten SNPs are known to exist in the promoter region corresponding to the distal promoter region of the mouse Osr2 gene. The significance of the presence of SNPs in the promoter region remains to be elucidated. Interestingly, Debeer et al. (2002) investigated the possible relationship between a polymorphism of the Osr2 gene and generation of certain syndromes, and concluded that the Osr2 gene may be responsible for autosomal recessive syndrome of mental retardation, distal limb deficiencies, oral involvement, and renal defects (OMIM 246560 [OMIM] ). Although they did not assess the possible presence of mutations in the Osr2 promoter region, no mutation was found in the coding region or splice juncture regions of the Osr2 gene in patients carrying the syndrome.

In the present study, we observed that histones H3 and H4 were acetylated in the proximal Osr2 promoter region. Generally, the chromatin structure is remodeled via histone modification, in which transcription is activated (Aalfs & Kingston 2000). When transcription factor binds to the target gene promoter, recruitment of coactivators with histone acetyl transferase (HAT) activity commences and those coactivators subsequently acetylate histones. Such acetylated histones induce remodeling of chromatin by chromatin remodeling factors. Subsequently, transcription is exerted under participation of basic transcription factor and RNA polymerase. Using a ChIP assay, we determined that histones H3 and H4 were acetylated around the proximal Osr2 promoter region containing C/EBP binding sites in normally cultured C3H10T1/2 cells. We considered it likely that C/EBPs or other transcription factors in C3H10T1/2 cells bind to the Osr2 promoter and recruit HAT coactivators such as CBP/p300 acetylate histones, after which the acetylated histones induce transcription of the Osr2 gene by eliciting chromatin remodeling.

The TSS database is available at http://dbtss.hgc.jp/index.html. In the present study, we could not assign a TSS for the Osr2 gene at one site in spite of employing a more accurate and efficient method for mapping the CAP site, the so-called oligo-capping method of mRNA. However, we located a new TSS, which appears to be the longest so far examined, at 66 bp upstream from the assigned data in the database. In the region immediately upstream of the newly identified TSS, there are a CCAAT box (around –80) and GC box (around –47 to –34). Such characteristics are found in many TATA-less promoters, which generally possess typical motifs including a CCAAT box (–60 to –100) and GC island (–30 to –50). The discrepancy regarding assignment of the CAP site between our data and the database may be due to use of a different source of mRNA, or another property (carrying multi-TSS) of the TATA- and CCAAT-less promoter itself.

In summary, we cloned the mouse Osr2 promoter region and found that Osr2 mRNA expression is regulated by C/EBP{delta}. Our findings should be helpful for understanding the role of Osr2 in vivo and in vitro.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cloning of 5'-flanking region of mouse Osr2 gene

First, we performed a BLAST search for mouse Osr2 cDNA (GENBANK accession number NM_054049 [GenBank] ) and found that GENBANK accession number AC116596 (BAC clone of mouse chromosome 15, RP24-335P21) contained the 5'-flanking region of Osr2 gene. The 5 kb 5'-flanking region of the Osr2 gene was amplified by a PCR assay using mouse genomic DNA with high fidelity LA (long and accurate) Taq polymerase (Takara Bio Inc., Shiga, Japan). The primers used were 5'-AGCGAGAAAGCCTGAGCTCTGCAGGGAAGT-3' (forward) and 5'-CGGATCCCACCCTCCGGCTGCGAGCGCGCT-3' (reverse; underlined bases indicate BamHI site used later for cloning). The amplified product was cloned into a pGEM-T easy vector (Promega Corporation, Madison, WI, USA). DNA sequencing was performed using BigDye cycle sequencing and read on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The sequence of the construct was verified by comparing with that in the mouse genome database.

Search for transcription factor binding sites

We performed a transcription factor binding motif search with TFSEARCH (Akiyama, Y., http://www.cbrc.jp/research/db/TFSEARCHJ.html), based on the TRANSFAC database (Wingender et al. 1996). Matrix similarity scores were set at 90.0, except when another value was indicated.

Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR

Total RNA from cells was prepared using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) and reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen). The primer set used for detection of the Osr2 transcript was 5'-CACCATGGGGAGCAAGGCCTTGCCAGCT-3' (forward; underlined bases indicate start codon) and 5'-TCAGGCTGTGCCGCCGCAGATCGC-3' (reverse; underlined bases indicate stop codon). The PCR conditions were 25 or 30 cycles at 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 1 min, performed with Taq PCR Master Mix (QIAGEN, Valencia, CA, USA). The product (about 0.8 kb) was resolved by electrophoresis on a 1.5% agarose gel. The primer set for ß-Actin mRNA was 5'-TGGAATCCTGTGGCATCCATGAAAC-3' (forward) and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3' (reverse), with the same PCR conditions as above, except with an annealing temperature of 57 °C and 25 PCR cycles.

A real-time PCR assay was performed using a LightCycler system (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the manufacturer's instructions. The reaction was carried out with QIAGEN QuantiTect SYBR Green PCR Master Mix in a 20-µL volume containing 0.5-µM of Osr2 primers. A typical protocol, which took approximately 15 min to complete, included a 30-second denaturation step, followed by 35 cycles with a denaturation for 5 s at 95 °C, annealing for 5 s at 58 °C, and extension for 5 s at 72 °C. The extension periods varied with the specific primers, depending on the length of the product (~1 s/25 bp). An amplification curve was generated after analyzing the raw data and adjusting the threshold cycle (Ct) value, and a standard curve was acquired to calculate the unknown quantity. We tested each sample 3 times. The expression levels of mRNA are indicated as the relative cycle number normalized by the cycle number of GAPDH. The primer set for GAPDH mRNA was 5'-GTCTTCACCACCATGGAGAAG-3' (forward) and 5'-GCCAAAGTCATCCATGACAAC-3' (reverse). Each procedure was repeated at least 3 times to assess the reproducibility.

RNA ligase-mediated and oligo-capping rapid amplification cDNA ends (RLM-RACE)

To identify the transcription start site (TSS) of Osr2 mRNA, an RLM-RACE reaction was performed with a commercial GeneRacer kit from Invitrogen, according to the manufacturer's protocol (Maruyama & Sugano 1994). The gene specific primers for Osr2 were 5'-TGAATTCTCAGGCTGTGCCGCCGCAGATCG-3' and 5'-CGACGCGTTCACAATCTCCCTCGGGAGGGC-3' (1276–1298 and 941–959 of Osr2 sequence, GENBANK accession number NM_054049 [GenBank] ). Following cloning of the PCR products into a pCR4Blunt-TOPO vector (Invitrogen), independent colonies were chosen randomly and subjected to sequencing analysis.

Construction of promoter-reporter expression vectors

The 5'-flanking region of the Osr2 gene in a pCR4Blunt-TOPO vector isolated by digestion with BamHI was subcloned into the BglII site of a pGL3-basic firefly luciferase reporter vector (Promega) that carried neither the eukaryotic promoter nor its enhancer. The resulting plasmid was termed pGL3/Osr2. Serial deletions of the 5'-flanking region of the Osr2 gene were carried out by self-ligation after enzymatic digestion of pGL3/Osr2. The restriction enzyme sites used for construction of the serially deleted promoter regions are shown in Fig. 3, with a total of six promoter-luciferase reporter expression vectors constructed. Point mutation in distal or proximal C/EBP binding site of pXh was performed by QuickChange Site-Directed Mutagenesis System according to the manufacturer's protocol (Stratagene, La Jolla, CA, USA). The mutation primers for distal and proximal C/EBP binding sites were 5'-CTAGTCCTGAATTACGTTTTAttgAAGAAACGAAAATTAATATTC-3' and 5'-CCTTTAAGACTCAGTTGCTATttgAACACAAGTAAACAGAGTGGA-3', respectively. Lower cases, ttg, in primers indicated mutated nucleotide sequences to the change of authentic CCA sequence.

Cell culture, transfection, and luciferase activity assay

A mesenchymal cell line C3H10T1/2 (RCB0247), myoblastic cell line C2C12 (RCB0987), and osteoblastic cell line MC3T3-E1 (RCB1126) were purchased from Riken Bioresource Center (Tsukuba, Ibaragi, Japan). A COS-7 cell line was obtained from Cell Resource Center for Biomedical Research, Tohoku University, Japan. C3H10T1/2 and MC3T3-E1 cells were maintained in {alpha}-modified Eagle's medium ({alpha}-MEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin and 50 mg/mL streptomycin. C2C12 and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin, and 50 mg/mL streptomycin.

One day before transfection, cells were plated in 24-well culture plates (Iwaki, Asahi Techno Glass, Chiba, Japan) at a density of 4 x 104 per well. The promoter firefly luciferase reporter vector, 0.4 µg, was transiently transfected using FuGENE6 transfection reagent (1.2 µL/well; Roche Diagnostics). A pRL-CMV vector (20 ng; Invitrogen), in which expression of Renilla luciferase was driven by the CMV promoter, was concomitantly transfected as an internal control. About 48 h after transfection, cells were lyzed with a passive-lysis buffer and both luciferase activities were measured using a Dual Luciferase Reporter Assay System (Promega). The promoter firefly luciferase activity was normalized by Renilla luciferase activity. The experiments were done in triplicate and repeated at least twice.

Effects of C/EBP{delta} expression on Osr2 promoter activity

C/EBPß and C/EBP{delta} genes were a kind gift from Dr Shizuo Akira (Osaka University), C/EBP{alpha} was from Dr Ormond A. MacDougald (University of Michigan Medical School, Ann Arbor, MI, USA). For co-transfection, cells were treated as previously described and transfected with a mixture of 1.2 µL of FuGENE6, 100 ng of the Osr2 promoter-reporter vector, 20 ng of pRL-CMV, and different amounts (from 25 to 200 ng) of C/EBP{delta} expression vector (pC/EBP{delta}). The total amount of plasmid was adjusted to 200 ng by adding an empty vector, pcDNA3 (Invitrogen). The following procedures were the same as previously described. The experiments were done in triplicate and repeated at least twice.

Electrophoresis mobility shift assay (EMSA)

EMSA was performed as described earlier (Senear & Brenowitz 1991), with some modifications. Glutathione S-transferase (GST)-C/EBP{delta} fusion protein expression vector (GST-C/EBP{delta}) was constructed by inserting the full-length C/EBP{delta} coding region into pGEX-6P1 (Amersham Biosciences Corporation, Piscataway, NJ, USA), and the GST fusion proteins were purified by standard procedures (Smith & Johnson 1988). Briefly, the vector was constructed following PCR-amplification and cloning of the full-length C/EBP{delta} coding region, and transformed into competent Escherichia coli TOP10 (Invitrogen). These recombinant strains were grown in LB broth containing 100 µg/mL ampicillin at 37 °C with shaking until mid log-phase, and expression of the targeted proteins were induced by addition of 1 mM isopropyl-ß- D-galactopyranoside (IPTG). After 3 h incubation, the bacterial cells were harvested by centrifugation, resuspended in 0.5 mL of sonication buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol [DTT], and 1% Triton X-100), and were disrupted by sonication. Cellular debris was removed by centrifugation, and proteins were purified from the supernatants by batch method (Glutathione Sepharose 4B; Amersham Biosciences) according to the manufacturer's instruction (Amersham Biosciences). The protein amount was determined using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as the standard. EMSA was performed using the digoxigenin (DIG) Gel Shift Kit system (Roche Diagnostics) as follows. Both strands of oligonucleotides, 5'-TCCTGAATTACGTTTTACCAAAGAAACGAAAATTA-3' (distal, –1295/–1261) and 5'-CTCAGTTGCTATCCAAACACAAGTAAACAGAGTGG-3' (proximal, –89/–55), were synthesized. These oligonucleotides were end-labeled using terminal deoxynucleotidyl transferase (Roche Diagnostics). The fusion protein (50–75 ng) was preincubated with or without competitor DNA in 20 µL of buffer I containing 20 mM HEPES, pH 7.6, 1 mM EDTA, 10 mM NH2SO4, 1 mM DTT, 2% Tween 20, 30 mM KCl, and 50 µg/mL poly[d(I-C)] (Roche Diagnostics) at room temperature for 15 min. Then, approximately 0.8 ng of a DIG-labeled probe was added and incubation was continued for an additional 30 min at room temperature. For the antibodies supershift assay, specific rabbit antibodies against human C/EBP{delta} (2 µL, Active Motif Inc.) were incubated with the fusion protein in buffer I for 30 min at room temperature, prior to addition of the probe. The reaction products were electrophoretically separated on 6.0% polyacrylamide gels in 0.5 x TBE at 100 V for about 2 h. Following electrophoretic separation, the oligonucleotide-protein complexes were transferred by electroblotting on to nylon membranes, positively charged (Roche Diagnostics). The DIG-labeled probes were detected by an enzyme immunoassay using alkaline phosphatase-labeled anti-DIG antibodies and the supplied chemiluminescent substrate CSPD (disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricycle [3.3.1.1] decan)-4-yl) phenyl phosphate, Roche Diagnostics). The chemiluminescent signals were visualized using a VersaDoc 5000 imaging system and Quantity One 1-D analysis software (Bio-Rad).

Chromatin immunoprecipitation (ChIP) assay

A ChIP assay was performed as described earlier (Das et al. 2004), with some modifications. Cells were grown to 95% confluence in {alpha}-MEM supplemented with 10% FBS for at least 3 days. Approximately 106 cells were washed twice with PBS and histones or C/EBP{delta} were cross-linked to DNA with 1% formaldehyde at room temperature for 10 min. The cross-linking reaction was stopped by adding 0.15 M glycine to the cross-linking mixture. The cells were then centrifuged for 5 min at 3000 r.p.m and washed with 1 mL of ice-cold PBS containing 2% BSA, then re-suspended in 0.2 mL of SDS lysis buffer (containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 1x protease inhibitor cocktail; Sigma-Aldrich, St Louis, MO, USA) and sonicated 3 times for 10 s each at the maximum setting (Handy Sonic, Tomy Seiko, Tokyo, Japan). After the cell lysate was centrifuged for 10 min, the supernatants were collected and diluted in ice-cold buffer [containing 1.1% Triton X-100, 0.11% sodium deoxycholate (SDC), 167 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 1x protease inhibitor cocktail]. The mixture was then precleared by incubation for 2 h at 4 °C with a 50% Protein A/G PLUS-agarose slurry (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a solution containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% SDC, and 100 µg/mL sheared salmon sperm DNA. Immunoprecipitation was performed overnight at 4 °C with anti-acetylated histone H3 (Upstate, Lake Placid, NY, USA), acetylated histone H4 (Upstate), C/EBP{delta} (Active Motif), C/EBPß (Santa Cruz), or control rabbit IgG (Santa Cruz) antibodies. After immunoprecipitation, a 50% Protein A/G PLUS-agarose slurry was added and the incubation was continued for another 2 h. The precipitates were washed sequentially for 5 min each in buffer I (containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1% SDC), buffer II (containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1% SDC), and buffer III (containing 10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, and 0.5% SDC), then washed twice with TE buffer, suspended in a solution containing 10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM EDTA and 0.5% SDS, and finally heated at 65 °C for at least 4 h to reverse the DNA cross-linked by formaldehyde. The eluates were treated with 20 µg/mL RNase A (QIAGEN) for 30 min and subsequently with 50 µg/mL Proteinase K (QIAGEN) at 55 °C for 1 h. The solutions were then extracted with phenol and ethanol-precipitated. DNA was dissolved in TE buffer. For PCR, 1 µL from a 20-µL DNA extraction and 25 cycles of amplification were used. PCR using primers flanking the distal site (–1438 to –1414: forward 5'-CAACTCTTCCTAGGCGTTCTCCTG-3' and –1196 to –1172: reverse 5'-AGACACTACGGAAACGATCCCATT-3') produced a 243-bp DNA fragment, and proximal (–280 to –257: forward 5'-AAATGCCCTTTGGACTTCTCTGC-3' and –43 to –19: reverse 5'-CCCGCTAATGGTCCACTCTGTTTA-3') produced a 238-bp DNA fragment.

Statistical analysis

Values are presented as the mean ± standard deviation (SD) of results of separate experiments. They were compared using Student's t-test. Values of P < 0.05 were considered to indicate significant differences. All analyses were conducted using JMP IN version 5 software (SAS Campus Drive, Cary, NC, USA).


   Acknowledgements
 
We thank Dr Shizuo Akira (Osaka University, Osaka, Japan) for kind gifts of C/EBPß and C/EBP{delta} genes. We thank Dr Ormond A. MacDougald (University of Michigan Medical School, Ann Arbor, MI) for kind gifts of C/EBP{alpha} gene. We thank Ms. Saeko Tanimura for expert technical assistance on the determination of TSS. This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (No. 16044229). This work was a part of the 21st Century COE program entitled ‘Origination of Frontier BioDentistry’ at Osaka University Graduate School of Dentistry, supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yoshinori Ohsumi

* Correspondence: E-mail: skawai{at}dent.osaka-u.ac.jp


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 Discussion
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Received: 10 August 2005
Accepted: 14 November 2005





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