Human Molecular Genetics, 2002, Vol. 11, No. 14 1627-1636
© 2002 Oxford University Press
An evolutionarily conserved putative insulator element near the 3' boundary of the imprinted Igf2/H19 domain
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, and Department of Genetics, Graduate University for Advanced Studies, 1111 Yata, Mishima, Shizuoka 411-8540, Japan
Received February 27, 2002; Accepted May 8, 2002
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ABSTRACT |
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Igf2 and H19 are closely linked imprinted genes lying at the centromeric end of a 1 Mb imprinted domain on mouse chromosome 7. L23mrp and other genes located 3' (more centromeric) to H19 are not imprinted and do not interact with the enhancers shared by Igf2 and H19. It is therefore suggested that the intergenic region between H19 and L23mrp contains a boundary or an insulator element. We have identified a binding site for CTCF, a nuclear factor that mediates insulator activity in vertebrates, in the intergenic region. This site is conserved between human and mouse, associated with a major DNase I-hypersensitive site, and bound by CTCF in vivo. Functional assays using reporter constructs demonstrated that this element functions as an insulator in transfected cells. The findings suggest that this CTCF site contributes to the 3' boundary of this imprinted domain. Together with the findings on the differentially methylated CTCF sites 5' to H19, CTCF-dependent insulators may not only regulate but also delimit the imprinted domain.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The eukaryotic genome is partitioned into independent functional domains that are separated by DNA sequences called boundary elements or insulator elements (1,2). These elements ensure the correct expression of genes within the domain by blocking influences from regulatory elements in the neighboring domains. Thus, insulator elements are defined by their ability to block enhancer–promoter interactions when positioned between the enhancer and the promoter, or to protect transgenes from position effects. The best characterized insulators include the Su(Hw)-binding sites within the gypsy retrotransposon of Drosophila (3), the scs and scs' elements flanking the 87A7 hsp70 locus of Drosophila (4), as well as the chicken ß-globin insulators (5,6). Previously, Bell et al. (7) showed that a protein named CCCTC-binding factor (CTCF) binds to the core sequence of the chicken ß-globin insulator and that this factor plays a key role in enhancer-blocking activity. Moreover, other insulators in vertebrates also contain CTCF sites and show CTCF-dependent insulator activity (6–11).
The Igf2 and H19 genes are closely linked imprinted genes which are expressed only from the paternal and the maternal allele, respectively (12–14). Interestingly, the reciprocal imprinting of the two genes is dependent upon methylation-sensitive, CTCF-dependent insulators within the differentially methylated region (DMR) located 5' to H19 (8,9,15). These genes are located at the centromeric end of a 1 Mb imprinted domain, which contains at least 14 imprinted genes, in mouse chromosome band 7F4/F5. It is known that L23mrp (Rpl23) and other genes located 3' (more centromeric) to H19 are not imprinted. A previous study by fluorescence in situ hybridization identified a transition from asynchronous replication at H19 to synchronous replication at L23mrp (16). Moreover, it was shown that the endoderm-specific enhancers located between H19 and L23mrp interact with both Igf2 and H19 (17), but not with L23mrp (18). These findings suggest that an insulator element is present between H19 and L23mrp.
In the present study, we have looked for an insulator element within the H19/L23mrp intergenic region based on sequence homology with the known CTCF-dependent insulators. Our study identified an evolutionarily conserved CTCF-binding site that showed enhancer-blocking activity in transfected cells. The results suggest that this CTCF-dependent insulator may serve to define the 3' boundaries of this imprinted domain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of sequences that can bind CTCF in vitro
To identify insulator elements within the H19/L23mrp intergenic region, we looked for putative binding sites for CTCF. Although the sequences of the CTCF sites reported prior to this study were rather divergent, we derived a 14 bp consensus sequence for the core region (5'-CCGCNNGGNGGCAG-3') by comparing the chicken ß-globin FII sequence (7) and multiple (four in mouse and six in human) CTCF sites within the DMR of H19 (8,9). The same consensus sequence was recently derived and used by Chao et al. (19). We then scanned a 33 kb region between H19 and L23mrp (GenBank accession no. AF049091, bases 8137–41 680; GenBank accession no. AP003183, bases 8167–41 712) (Fig. 1A) for this consensus sequence with a criterion of 12 matches or more in the 14 nucleotides. As a result, we identified six candidate CTCF sites (PCT1–PCT6) on the upper strand and nine candidates (PCT7–PCT15) on the lower strand (Fig. 1A, B).
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To determine which one of the candidate sequences can bind CTCF in vitro, electrophoretic mobility shift assays (EMSAs) were carried out with 84 bp duplex probes containing the sequences (Table 1). When the probes were incubated with nuclear proteins from 12.5 dpc mouse embryo, all probes formed at least one, but usually more, complexes (Fig. 1C). Among the complexes, a major complex formed with PCT4, PCT12 and PCT6/14 (PCT6 and PCT14 were located close each other; Fig. 2A), respectively, showed the same mobility as the CTCF complex formed with the m1 probe from the H19 DMR (8). The three complexes were all super-shifted with polyclonal anti-CTCF antibodies (Fig. 1C). Also, formation of the complexes was greatly diminished by competition with excess unlabeled FII fragments but not with Sp1 consensus duplexes (Fig. 1D). Binding of CTCF to the three probes, but not to the others, was confirmed by EMSA with an in vitro transcribed/translated CTCF protein, although the affinity of PCT4 to the synthesized protein was significantly lower than that of the others (data not shown, but see Fig. 5). These findings show that the PCT4, PCT12 and PCT6/14 sequences can specifically bind CTCF in vitro. A second complex formed with the PCT4 probe probably contain Sp1, because formation of this complex was inhibited by addition of unlabeled Sp1 consensus duplexes or unlabeled fragments of FII (which has one Sp1 site) as competitors (Fig. 1D).
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Cross-species conservation of the putative CTCF sites
To further assess the significance of the four potential CTCF sites (PCT4, PCT6, PCT12 and PCT14), we asked whether these sites are conserved through evolution. When we examined the human H19 region (GenBank accession no. AF087017, bases 10 988–40 560; GenBank accession no. AC004556, bases 15 016–20 800) for these sequences, two sites corresponding to PCT12 and PCT14 were present at orthologous positions (Fig. 2A). The other two potential CTCF sites as well as the 11 non-CTCF-binding sites were not conserved.
To examine whether the conserved human sequences can bind CTCF, we carried out EMSA with a nuclear extract from 12.5 dpc mouse embryo. The major complexes formed with the human probes (hPCT12 and hPCT14; Table 1) showed the same mobility as the CTCF complex formed with the m1 probe (data not shown). These complexes were competed with excess unlabeled FII fragments but not with Sp1 consensus duplexes (Fig. 2B). Furthermore, the complexes were super-shifted with anti-CTCF antibodies (Fig. 2C). Thus, the two conserved sequences (PCT12 and PCT14) from both human and mouse were capable of binding CTCF in vitro.
DNase I hypersensitivity at PCT12
The known CTCF-dependent insulators, such as the chicken ß-globin insulator (6,7) and mouse H19 DMR insulator (20,21), are all associated with DNase I-hypersensitive sites in chromatin. We therefore examined the DNase I sensitivity of the mouse PCT4, PCT12 and PCT6/14 regions. We first tested a 7.7 kb region containing both PCT4 and PCT12 (Fig. 3A). Nuclei isolated from 12.5 dpc mouse embryo were treated with increasing concentrations of DNase I, and the DNA was purified and analyzed by Southern blotting. End fragments from the 7.7 kb EcoRV region were used as probes (Fig. 3A, top). The study revealed three hypersensitive sites (Fig. 3A, bottom): one located at, or very close to PCT12 and the others located in the middle and at the 3' end of the second exon of the muscle-specific transcription unit Nctc1 (22). No hypersensitive site was detected at or around PCT4 (Fig. 3A, bottom).
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We next analyzed the DNase I sensitivity of the PCT6/14 region. Two probes (PPm1 and BlP1) derived from the ends of a 4 kb PstI fragment containing PCT6/14 were used (Fig. 3B, top). Two hypersensitive sites were detected and mapped at both ends of CS9 (Fig. 3B, bottom), which is a mesoderm-specific enhancer (23). However, we did not observe any DNase I-hypersensitive site associated with PCT6/14 (Fig. 3B, bottom). These results suggested that PCT12 may be the only CTCF-dependent insulator that works in vivo.
In vivo binding of CTCF to PCT12
To know whether PCT12 and other potential CTCF sites are bound by CTCF in vivo, we carried out chromatin immunoprecipitation (ChIP) assays with nuclei isolated from 12.5 dpc mouse embryo. We treated the nuclei with formaldehyde to crosslink protein with DNA, fragmented the chromatin by sonication, and carried out immunoprecipitation with anti-CTCF antibodies. By PCR using specific primers, we examined whether PCT12 and the other two potential CTCF sites are co-immunoprecipitated with CTCF. As shown in Fig. 4, the PCT12 region, as well as the positive control (m3) from the H19 DMR, was greatly enriched in the anti-CTCF immunoprecipitates. In contrast, PCT4, PCT6/14 and a negative control region (H19 exon 5) showed no or little enrichment, if any. The results clearly demonstrated that PCT12 is bound by CTCF in the chromatin of mouse embryo.
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Methylation prevents CTCF from binding to PCT6/14
It is known that, if CpG dinucleotides within the recognition sequence for CTCF are methylated, the factor cannot bind to the target. While PCT12 contained no CpG, PCT4, PCT6 and PCT14 each contained one CpG. We therefore examined the methylation status of the three sites in 14.5 dpc mouse embryo by bisulfite genomic sequencing. The results showed that all sites were highly methylated: methylation was observed in 17 out of 19 sequenced clones for PCT6 (89%), 16 out of 19 for PCT14 (84%) and 10 out of 11 for PCT4 (91%). This raised the possibility that the lack of CTCF binding to PCT4 and PCT6/14 could be due to methylation of the target sequences. To see whether methylation of these sequences inhibits CTCF binding, we carried out EMSA with in vitro methylated PCT4 and PCT6/14 probes. Complex formation with in vitro transcribed/translated CTCF protein was greatly diminished by methylation of the PCT6/14 probe, while it was minimally affected by methylation of the PCT4 probe (Fig. 5). The reason for the lack of in vivo binding of CTCF to PCT4 is yet to be determined.
PCT12 has an insulator activity
Among the candidates that we identified in our initial bioinformatic screen, the best candidate left for a CTCF-dependent insulator was PCT12. We therefore examined its enhancer-blocking activity by a colony assay. Various constructs shown in Fig. 6 were respectively transfected into Hep3B cells, and their enhancer-blocking activity was assayed by counting the number of G418-resistant colonies. When the H19 DMR insulator (a control fragment), which has four CTCF sites, was inserted between the promoter and the enhancer (pHNIE), the colony number decreased to approximately 10%. We then tested a 1.5 kb XhoI–PmaCI fragment containing PCT12 (pHNPXE) and observed that the colony number decreased to approximately 40%. This suggested that PCT12 indeed has an enhancer-blocking activity. A similar reduction in colony number was observed in pHNXPE, which was identical to pHNPXE except that the fragment was inserted in the opposite direction. Thus, the enhancer-blocking activity was independent of the orientation of the fragment. To exclude the possibility that the reduced colony number was due to a silencer activity, we relocated the fragment to a position 3' to the enhancer (pHNEPX). This construct affected the colony number only minimally indicating that the fragment possesses little silencer activity. Lastly, to determine whether the insulator activity was dependent on the CTCF site, base substitutions were introduced at all 14 nucleotides of the PCT12 sequence (pHNME), which abolished CTCF binding. The result suggested that the insulator activity of the PCT12 fragment is largely (>60%) dependent upon the CTCF site, but some other sequences within the fragment could also contribute to the insulator activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have identified a putative CTCF-dependent insulator in the H19/L23mrp intergenic region based on sequence homology with known CTCF-binding sites. This sequence element (PCT12) is conserved between human and mouse, associated with a DNase I-hypersensitive site, bound by CTCF in vivo, and exhibited an insulator activity in transfected cells. These results demonstrate that a sequence-based approach is effective in identifying CTCF-dependent insulators, despite the view that the factor binds to a diverse range of sequences by combinatorial use of the 11 zinc fingers (24). The existence of this element in the region is consistent with the transition from asynchronous replication at H19 to synchronous replication at L23mrp (16) and with the lack of interaction of the endoderm-specific H19 enhancers with the L23mrp promoter (18). Although elucidation of the exact function of this element awaits germline deletion in mice, our data suggests that it is an important component of the 3' (centromeric) boundary of the imprinted Igf2/H19 domain.
Previous studies showed that the reciprocal imprinting of Igf2 and H19 is regulated by a cluster of methylation-sensitive CTCF-dependent insulators in the DMR located 5' to H19 (8,9,15) and the multiple tissue-specific enhancers located 3' to H19 (17,23,25). It is proposed that the methylation status of the DMR determines the activity of the insulators and decides which gene is to be activated by the 3' enhancers on each parental chromosome. The present study suggests that H19 also has a CTCF-dependent, but methylation-insensitive, insulator in the 3' flanking region. This domain structure (Fig. 1A) is reminiscent of the chicken ß-globin domain, which is flanked by a CTCF-dependent insulator on each side (6,7). Thus, CTCF-dependent insulators may be a common component of the domain boundaries in the vertebrate genome.
The imprinted domain defined by the putative insulator includes a non-imprinted transcription unit, Nctc1 (22) (Fig. 1A). This transcription unit is active only in adult skeletal muscle. Some of the genes located in this 1 Mb imprinted domain escape imprinting, with a common feature of not being associated with CpG islands (e.g. Tssc6 and Th). The lack of a CpG island associated with Nctc1 may be the reason why this transcription unit escapes imprinting even though it is located on the imprinted side of the element.
We previously identified multiple tissue-specific enhancers in the H19/L23mrp intergenic region (23). Among these, CS9, which is a mesoderm-specific enhancer, is located more 3' to the putative insulator (Fig. 1A). Other studies involving BAC and YAC transgenes showed that additional enhancers for expression in the heart, kidney and lung should be present further 3' (26,27). These findings pose a potential problem that the far 3' enhancers must act on Igf2 and H19 over this element. We consider the following possibilities. Firstly, despite our transfection data, the putative insulator could act in an orientation-dependent manner in the genomic context and/or in specific tissues. There are examples of such an orientation-dependent insulator (28,29). Secondly, there is evidence that enhancer-blocking activity of an insulator is dependent upon enhancer–promoter combination (30,31), and thus certain enhancer–promoter pairs may not be blocked by CTCF-dependent insulators. Thirdly, a promoter targeting sequence (PTS) (32), which can override the activity of an insulator to mediate interactions between genes and enhancers, could be present in a region 3' to the CTCF site. Then the more 3' enhancers should be able to activate Igf2 and H19 through this element.
In addition to PCT12, this region could contain other insulator elements as well. For example, one putative CTCF site that we identified in this study was conserved between human and mouse (PCT14). Although this site was heavily methylated and lacked in vivo evidence for CTCF binding in whole 14.5 dpc embryo, it could display insulator activity in a restricted tissue or at a specific developmental time. Also, there may be sites that show little homology with the consensus sequence but can in fact bind CTCF. Finally, insulators that interact with unknown factors may be present.
The putative insulator may have relevance to the phenotype of the mouse mutant called minute (Mnt), which exhibits dwarfism only upon paternal transmission. A molecular analysis of this mutation revealed that it involved an inversion of a region from immediately 5' of the 3' insulator to several megabases 3' (33). It is tempting to speculate that the removal of not only the tissue-specific enhancers but also this element leads to deterioration of the expression of nearby genes, including Igf2 (33), resulting in the phenotype. Another recent study showed that a targeted deletion of a region between the endoderm-specific enhancers (CS3 and CS4) and L23mrp, including mesoderm-specific enhancers and the element, did not affect the expression of L23mrp (25). This again suggests that other insulator elements, which are not deleted in these mice, may be present in the region. Further studies are needed to reveal all the sequence elements that contribute to the boundary function of this intergenic region.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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EMSA
Preparation of nuclear extracts and EMSA was carried out as described previously (23). CTCF protein was synthesized by a coupled in vitro transcription/translation reaction using a TNT T7 Quick system (Promega) according to the manufacturer's protocol. For super-shift assays, the reaction mix was combined with 2 µl of anti-CTCF antibody (Upstate biotechnology) and incubated for 1.5 h at 4°C before the addition of radiolabeled probes. The sequences of the probes are shown in Table 1. Methylation of the probes was carried out using SssI methylase (New England Biolabs) according to the supplier's instructions. The methylation reaction was monitored by digestion of the probes with a methylation-sensitive restriction enzyme AciI (CCGC). Oligonucleotide duplexes used as competitors were as follows: FII, 5'-TGT AAT TAC GTC CCT CCC CCG CTA GGG GGC AGC AGC GAG CCG C-3'; Sp1, 5'-ATT CGA TCG GGG CGG GGC GAG C-3'.
DNase I-hypersensitive site assay
Isolation of cell nuclei from 12.5 dpc mouse embryos and mapping of DNase I-hypersensitive sites were performed as described in (34).
ChIP assay
Nuclei from 12.5 dpc mouse embryos were isolated as described in (34). ChIP assay was performed according to the protocol supplied by Upstate Biotechnology with some modifications. To crosslink proteins to DNA, formaldehyde was added to the nuclear suspension (3x106 nuclei) to a final concentration of 1%, and incubated at 25°C for 10 min. Two microliters of anti-CTCF antibody (Upstate Biotechnology) was added and the mixture was incubated at room temperature for 1 h. The antibody-binding reaction was followed by an addition of 60 µl of salmon sperm DNA/protein A agarose slurry (Upstate Biotechnology) and a further incubation at room temperature for 1 h. PCR was carried out for 25 cycles with [
-32P]dCTP. The PCR products were separated by PAGE and visualized by autoradiography. The primers used were as follows: ChIP4-1 (PCT4, up), 5'-AAA AGG TGC CCA TCT TGA TGG CTG-3'; ChIP4-2 (PCT4, down), 5'-TTT CTG ACT CTC CTG ATA CCA TGT-3'; ChIP12-1 (PCT12, up), 5'-GGT GGA GGA AGG CGC CAT GTG G-3'; ChIP12-2 (PCT12, down), 5'-CTG ACT TCA GGA GGG TCT GGG ACT-3'; ChIP6/14-1 (PCT6/14, up), 5'-TAG AAT CAC TCC AAC TGG CAT GTC-3'; ChIP6/14-2 (PCT6/14, down), 5'-TAA TAC CAG CTA CAT GAG ATC CTG-3'; m3-s (H19 DMR m3, up), 5'-CTG TTA TGT GCA ACA AGG GAA-3'; m3-a (H19 DMR m3, down), 5'-GGT CTT ACC AGC CAC TGA-3'; OLG-1 (H19 exon 5, up), 5'-GTG AAG CTG AAA GAA CAG ATG GTG-3'; OLG-5 (H19 exon 5, down), 5'-AAG CAC ACG GCC ACA CCC AGT-3'.
Bisulfite methylation assay
Genomic DNA (2 µg) from 14.5 dpc mouse embryos was digested overnight with StuI restriction enzyme. Bisulfite treatment was carried out as described by Paulin et al. (35). The bisulfite-treated DNA was amplified by PCR. The PCR products were subcloned into pBluescript plasmids and sequenced by the Big Dye Terminator Cycle Sequencing Kit and ABI PRISM 377 Sequencer (Perkin Elmer). The primers used were as follows: bisP1 (PCT6/14, upper strand, up), 5'-TTG GTA TGT TAG TTG GTT TTG GTG ATG GG-3'; bisP2 (PCT6/14, upper strand, down), 5'-ACC TAA CTC CTA TCC TCA ATC CCA ATA AAT-3'; bisP3 (PCT6/14, lower strand, up), 5'-ATA ACT CTT CCA AAA CCC TAA CCA TCC TAA-3'; bisP4 (PCT6/14, lower strand, down), 5'-GAG ATT TTG GTT GGT AGA AGA ATA ATA GTA G-3'; bisP5 (PCT4, upper strand, up), 5'-ATT TTG ATG GTT GGT TTT GTT AGG GGT AAA-3'; bisP6 (PCT4, upper strand, down), 5'-TCC TTT CTA ACT CTC CTA ATA CCA TAT AAA-3'.
Colony assay
The reporter plasmid pHN consisted of a neomycin-resistance (neo) gene driven by the H19 promoter (-818 bp to +6 bp) and a 1.8 kb AatII–HindIII fragment containing the H19 DMR insulator. This insulator should prevent influence from the adjacent regions. The plasmid pHNE was made by linking the 2.5 kb NsiI–BglII fragment containing the endoderm-specific enhancers (the CS3/4 region) to pHN. Test fragments were excised from the cosmid cDH2 (22) by appropriate restriction enzymes, treated with T4 DNA polymerase and subcloned into the pBluescript plasmids at the EcoRV site. The fragments were liberated by double digestion with XhoI and SpeI and inserted into the XhoI/SpeI sites of pHNE between the neo gene and the enhancers. A test fragment for pHNME was generated by introducing base substitutions into the CTCF consensus sequence at PCT12 (from 5'-CTG CCC CCT TTA GG-3' to 5'-AGT AAA AAG GGC TT-3') of a plasmid clone carrying the 1.5 kb XhoI–PmaCI fragment by a PCR-mediated mutagenesis method described by Imai et al. (36). The primers used were as follows: 12mut-1 (up), 5'-TTT TAC TTA GAG GAG CAA GCA TGC CCA-3'; 12mut-2 (down), 5'-AGG GCT TTA GCC CAA GGC TCA GAA CCA-3'. To make pHNEPX, the 1.5 kb XhoI–PmaCI fragment was ligated to the 3' end of the enhancer fragment and brought into pHN. The reporter constructs were linearized with MluI, and 0.2 pmol of each construct was transfected into 1.2x106 Hep3B cells using Lipofectamine Plus (Gibco BRL), together with 0.2 pmol of a plasmid encoding a hygromycin-resistance gene linearized with XhoI. After 48 h, cells were replated into two separate plates and selected by G418 (800 µg/ml) and hygromycin-B (250 µg/ml), respectively. Colonies were counted after 2 weeks of selection. The number of G418-resistant colonies was corrected for transfection efficiency based on the number of hygromycin-resistant colonies and normalized to that obtained with pHNE.
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ACKNOWLEDGEMENTS |
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We are grateful to Hisao Shirohzu and Takashi Sado for their advice and discussion. K.I. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. This work was supported by grants from the Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +81 559 816799; Fax: +81 559 816800; Email: hisasaki{at}lab.nig.ac.jp
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