Dlxin-1, a Novel Protein That Binds Dlx5 and Regulates Its Transcriptional Function*

Yoshiko MasudaDagger , Aya SasakiDagger , Hiroshi Shibuya§, Naoto Ueno§, Kyoji IkedaDagger , and Ken WatanabeDagger

From the Dagger  Department of Geriatric Research, National Institute for Longevity Sciences, Aichi 474-8522, Japan and § Division of Morphogenesis, National Institute of Basic Biology, Aichi 444-8585, Japan

Received for publication, September 20, 2000, and in revised form, November 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dlx5, a member of the Dlx family of homeodomain proteins, plays a critical role in bone development and fracture healing. To understand the molecular mechanism underlying the transcriptional regulation by Dlx5, we performed yeast two-hybrid screening and isolated a novel protein, Dlxin-1, that binds Dlx5 and regulates its transcriptional function. Dlxin-1 cDNA encodes a 775-amino acid protein that has a partial homology with necdin at the C terminus and 25 repeats of hexapeptides (WQXPXX) in the middle region. Dlxin-1 mRNA is expressed in various adult tissues, but not the spleen, and also in osteoblastic and chondrogenic cell lines. During embryogenesis, a strong signal for Dlxin-1 mRNA was found in cell layers surrounding cartilaginous elements in bone rudiment during digit formation. Dlxin-1 binds not only Dlx5 but also Dlx7 and Msx2 and forms homomultimers in vivo. Transfection and reporter gene assays indicate that Dlxin-1 activates the transcriptional function of Dlx5. Therefore, Dlxin-1 may act as a regulator of the function of Dlx family members in bone formation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Dlx gene family, which comprises at least six mammalian homologues of the Drosophila homeodomain protein Distal-less, is expressed predominantly in forebrain, limbs, and branchial arches during fetal development (1, 2). Among this family, Dlx5 is expressed in most developing skeletal elements and is induced during the process of bone fracture healing (2-6), suggesting that it may play a crucial role in bone development and formation. Msx2 is expressed in skeletal elements and many other organs (7-9). The expression of Dlx5 mRNA, as well as Msx2, is induced by bone morphogenetic protein (5, 7, 10). It has been suggested that Dlx5 is a transcriptional activator and regulates osteoblastic functions positively (4, 5, 11, 12). In contrast, Ryoo et al. (13) have demonstrated, using a reporter gene assay, that Dlx5 is a potential negative regulator of the expression of the rat osteocalcin gene, which is specifically expressed in bone. Recently, Acampora et al. (14) and Depew et al. (15) have reported the phenotype of mice homozygous for targeted deletion of the dlx5 gene, in which the expression of the osteocalcin gene in osteoblasts was markedly increased, suggesting that Dlx5 represses the expression of the osteocalcin gene in vivo (14).

To understand the molecular mechanism(s) underlying the transcriptional regulation involving Dlx5, we attempted to identify a molecule that binds Dlx5 and regulates its function. Here we report the isolation and characterization of Dlxin-1, a novel protein that binds Dlx5 and regulates its transcriptional function. Dlxin-1 is a unique member of the necdin/melanoma-associated antigen (MAGE)1 family (16-18).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells-- KUSA/A1 (19), a mouse osteoblastic cell line, and HT1080, a human fibrosarcoma cell line, were kindly provided by Drs. A. Umezawa (Keio University, Japan) and A. Fukamizu (University of Tsukuba, Japan), respectively. A yeast strain, Y153, was a gift from Dr. S. Kato (University of Tokyo).

Plasmid Construction-- The pBIND vector was purchased from Promega (Madison, WI). Mouse Dlx5 and Msx2 cDNAs were isolated by reverse transcriptase-polymerase chain reaction, and the N-terminal domains (amino acids 2-132 for Dlx5 and 2-136 for Msx2) were inserted into pBIND, generating pBIND-Dlx5Delta C and pBIND-Msx2Delta C, respectively. For construction of the bait plasmid (pGBT- Dlx5Delta C), the Dlx5Delta C cDNA fragment was inserted into pGBT9 (CLONTECH, Palo Alto, CA). pcDNA-FLAG was constructed in pcDNA3 (HindIII/XhoI), with a DNA fragment encoding the single FLAG epitope (5'-AAG CTT GCC ACC ATG GAC TAC AAG GAT GAT GAC GAC AAA CTC GAG-3') after the initiation codon (ATG) with the Kozak leader sequence, giving rise to the N-terminal FLAG tag attachment vector. Mouse Dlx3, Dlx7, Dlx5, and Msx2 cDNAs were inserted into pcDNA-FLAG, generating pFDlx3, pFDlx7, pFDlx5, and pFMsx2, respectively. Nucleotide sequences of all polymerase chain reaction products reported here were verified by sequencing. pGL3-basic, pGL3-control, and pGL3-promoter were purchased from Promega. The potential Dlx-responsive element of the col1a1 promoter (20) was connected in tandem to the pGL3-promoter vector. Briefly, oligonucleotides corresponding to the Dlx-responsive element (DlxRE) and a sequence mutated in the core binding region (TAAT to TACG; Dlxm) were designed, and the complementary oligonucleotides were annealed and ligated. The concatemerized DNA of five repeats was purified and introduced into the BglII site on the pGL3-promoter vector, generating pDlxRE-luc and pDlxm-luc. The nucleotide sequences for the construction were as follows: Dlx5N, 5'-GGGATCCTGACAGGAGTGTTTGACAGA-3'; Dlx5Delta C, 5'-GGTCTAGATACCATTCACCATCCTCAC-3'; Msx2N, 5'-GGGATCCTGGCTTCTCCGACTAAAGGG-3'; Msx2Delta C, 5'-GGTCTAGAGGGTGCAGGTGGTGGGGCT-3'; mDlx5-F, 5'-CCTCGAGACAGGAGTGTTTGACAGAAG- 3'; mDlx5-R, 5'-GGTCTAGACTAATAAAGCGTCCCGGAGGC-3'; mMsx2-F, 5'-CCTCGAGGCTTCTCCGACTAAAGGCGG-3'; mMsx2-R, 5'-GGTCTAGATTAGGATAGATGGTACATGC-3'; mDlx3-F, 5'-GGCTCGAGAGCGGCTCCTTCGATCGCAAG-3'; mDlx3-R, 5'-GGTCTAGAGGTGGGTACTCAGTACACAGC-3'; mDlx7-F, 5'-GGCTCGAGACCTCTTTACCCTGTCCCTTC-3'; mDlx7-R, 5'-GGTCTAGACTCACATCATCTGAGGCAG-3'; DlxRE-U, 5'-GATCCTGCATTCCCTTTAATTATAGCCTCA-3'; DlxRE-R, 5'-GATCTGAGGCTATAATTAAAGGGAATGCAG-3'; Dlxm-U, 5'-GATCCTGCATTCCCTTACGTATAGCCTCA-3'; Dlxm-R, 5'-GATCTGAGGCTATACGTAAAGGGAATGCAG-3'.

Yeast Two-hybrid Screening-- Yeast two-hybrid screening was performed in accordance with the manufacturer's instructions (Matchmaker, CLONTECH). Briefly, a yeast strain, Y153, was cotransformed with pGBT-Dlx5Delta C and the mouse embryo (embryonic day 11) cDNA library constructed in pGAD10 (CLONTECH). The transformants were first selected for HIS3 gene transactivation on the basis of growth in the absence of Trp, Leu, and His and in the presence of 60 mM 3-amino-1,2,4-triazole. Colonies grown on the selection media were then selected for lacZ gene transactivation on the basis of beta -galactosidase activity in the filter assay. pLAM5' encoding GAL4 DNA-binding domain/human lamin C fusion protein was used as a negative control. Prey plasmids were recovered from beta -galactosidase-positive colonies, and sequence analysis was performed with ABI PRISMTM 310 Genetic Analyzer (PerkinElmer Life Sciences).

Trasfection-- Transfection was carried out using LipofectAMINE Plus (Life Technologies, Inc.) or FuGENE6 (Roche Diagnostics, Mannheim, Germany). pFDlx5 or pcDNA3 was introduced into P19 embryonic carcinoma cells, and stable transfectants were selected by the addition of Geneticin (400 µg/ml; Life Technologies). Colonies were taken and expanded to examine the expression of FLAG-tagged Dlx5 protein. A Dlx5-expressing P19 cell line (P19Dlx5) and a mock-transfected line (P19neo) were used in this study. There was no difference in proliferative activity and morphology between P19Dlx5 and P19neo.

Reporter Gene Assay-- The plasmids, pBIND and pG5luc, and the dual luciferase assay system were purchased from Promega. Transfected cells were lysed in Passive Lysis Buffer (Promega) and assayed for firefly and Renilla luciferase activities (Lumat LB 9507, EG & G Berthold, Bad Wildbad, Germany). Each assay was carried out at least in triplicate.

Northern Blotting and in Situ Hybridization-- Northern blotting was carried out using a 0.7-kilobase pair fragment of mouse Dlxin-1 cDNA on mouse MTN and Embryo set blot (CLONTECH). Total RNA isolated from mouse long bones, bone marrow cells in long bones, and calvaria was denatured by glyoxal/Me2SO and electrophoresed in agarose gels. The digoxigenin-labeled antisense and sense RNA probes were synthesized using T7 or Sp6 RNA polymerase (Promega) with a nucleotide mix containing digoxigenin-labeled CTP (Roche Diagnostics). Hybrid-ready tissue slides were obtained from Novagen (Madison, WI), and in situ hybridization was performed as reported previously (21).

GST Pull-down Assay-- GST and GST fusion proteins were expressed in DH5alpha , induced by 0.25 mM isopropyl-1-thio-beta -D-galactopyranoside, and purified by affinity chromatography using glutathione-Sepharose beads (Amersham Pharmacia Biotech). [35S]Methionine-labeled Dlx5 and Msx2 proteins were generated using an in vitro transcription/translation system (TNT; Promega). Aliquots were incubated with GST- or GST-Dlxin1-conjugated glutathione-Sepharose beads in binding buffer (50 mM Tris-HCl, pH 7.5, 138 mM KCl, 1 mM EDTA, 0.2% Nonidet P-40, 5% glycerol, and 5% bovine serum albumin). 35S-Labeled Dlx5 or Msx2 was synthesized as above and incubated with GST or GST-Dlxin1 deletion mutant proteins in the same binding buffer, followed by precipitation with glutathione-Sepharose beads. Bound proteins were eluted from the beads by boiling in SDS sample buffer, separated by SDS-PAGE, and visualized by autoradiography.

Immunoprecipitation and Immunoblotting-- The HA epitope-tagged Dlxin-1 expression vector (pHA-Dlxin1) and the FLAG epitope-tagged expression vectors (pFDlx3, pFDlx5, pFDlx7, pFMsx2, pF-Dlxin1) were cotransfected transiently into COS7 cells using LipofectAMINE reagent (Life Technologies). At 24 h after transfection, cells were lysed with radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) supplemented with a mixture of proteinase inhibitors (CompleteTM; Roche Diagnostics). Precleared cell lysates were subjected to immunoprecipitation with anti-FLAG M2 antibody (Sigma) following adsorption to protein G-Sepharose beads (Amersham Pharmacia Biotech). In some experiments, agarose-conjugated anti-FLAG antibody was used, with FLAG peptide as a competitor (Sigma). Bound proteins were eluted from beads by boiling in SDS sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Immunoblotting was performed using anti-FLAG M5 antibody (Sigma) or anti-HA 3F10 antibody and visibilized by ECL Plus reagents (Amersham Pharmacia Biotech).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transcriptional Activator Function of the N-terminal Domain of Dlx5-- To evaluate the transcriptional activity of Dlx5, we constructed a reporter gene assay system. The reporter construct, DlxRE, which consists of Dlx5-responsive elements and basal promoter with the luciferase gene (Fig. 1A), was introduced into Dlx5-expressing (P19Dlx5) or mock-transfected P19 cells (P19neo). As shown in Fig. 1B, a high activity of DlxRE-driven reporter was observed in P19Dlx5 cells, but not in P19neo cells. When a core sequence of DlxRE was mutated (Dlxm), reporter activity was not detected in either P19neo or P19Dlx5 cells. These results suggest that Dlx5 has a transcriptional activation function in P19 cells.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Dlx5 has transactivation function. The reporter constructs shown in A were transfected into Dlx5-expressing (P19Dlx5) or mock-transfected (P19neo) P19 cells (B). The N-terminal region of Dlx5 acts as a transcriptional activation domain (C). The GAL4-Dlx5Delta C was generated, with the N-terminal region of Dlx5 (amino acids 2-132) fused to the GAL4 DNA binding domain (GAL4DBD), as indicated in the inset. GAL4-Dlx5Delta C or GAL4 alone was transfected into 293 cells at the indicated doses (µg), with the reporter shown in the inset (pG5luc). pGL3-control, used as a nonspecific reporter instead of pG5luc, showed no GAL4-Dlx5Delta C dependent activation, indicating that the activation by GAL4-Dlx5Delta C was GAL4 element-mediated. No activation was detected when pGL3-basic, a promoterless reporter, was used.

The N-terminal domain of Dlx5 has similarities to those of Dlx2, Msx1, and Msx2 (4). To characterize the function of this shared domain, the N-terminal domain of Dlx5 (Dlx5Delta C) was fused to the GAL4 DNA-binding domain, and the transcriptional function was evaluated by reporter gene assay in 293 cells. Although GAL4 DNA-binding domain itself did not activate the transcription, the fusion construct (pBIND-Dlx5Delta C) stimulated the transcriptional activity in a dose-dependent manner (Fig. 1C). In contrast, the Msx2 fusion construct (pBIND-Msx2Delta C) did not activate, but rather suppressed, transcription (data not shown). These results suggest that the N-terminal domain of Dlx5 contains a transcriptional activation domain.

Isolation of a Molecule That Binds to the N-terminal Domain of Dlx5-- In an attempt to search for a protein(s) that associates with the N-terminal domain of Dlx5 and regulates its function, yeast two-hybrid screening was performed. From screening of the mouse embryo (embryonic day 11) cDNA library, seven independent and overlapping clones were obtained on the basis of selection for histidine requirement for growth and lacZ expression (Fig. 2A). We found, by BLAST search (22), that several expressed sequence tag sequences were identical with the isolated clones, and the 5'-nucleotide sequence was determined by 5'-rapid amplification of cDNA ends. As shown in Fig. 2B, the amino acid sequence predicted from the full-length cDNA reveals a novel protein of 775 amino acids, and the predicted molecular mass of the protein is 85.7 kDa. We propose that the novel protein be called Dlxin-1, for Dlx interaction. Dlxin-1 has a partial similarity to necdin (23) and MAGEs at the C-terminal region, as shown in Fig. 3A. A putative human counterpart of mouse Dlxin-1 has been reported as MAGE-D1 (18). In the data base, SNERG-1 (GenBankTM accession number AF274043) from rat testis showed a high homology (96%) to mouse Dlxin-1, suggesting that SNERG-1 is a rat orthologue of mouse Dlxin-1 (Fig. 3B). There is no significant hydrophobic stretch corresponding to a signal sequence or transmembrane domain, suggesting that Dlxin-1 is an intracellular protein. Notably, 25 hexapeptide repeats in tandem were found in the middle part of the protein, with a consensus sequence of WQXPXX (Fig. 3C). The shortest clone (clone 16) isolated in the two-hybrid screening encodes 18 out of the 25 repeats, and the sequence was included in all of the overlapping clones, suggesting that the WQXPXX repeat region represents the binding site of the N-terminal domain of Dlx5 (Fig. 2A). No significant homology to any protein sequences was found in the N-terminal part of Dlxin-1 by searching data bases.



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 2.   Primary structure of Dlxin-1. Schematic presentation of the primary structure of Dlxin-1 and the clones isolated by yeast two-hybrid screening (A). Tandem repeats of WQXPXX (in black) are located in the middle portion of Dlxin-1. The necdin homology region is indicated by a gray box. The scale bar in the right top of the figure corresponds to 100 amino acids. The clones isolated are indicated below the full-length Dlxin-1. The domain encoded by the shortest clone, clone 16, corresponds to a putative Dlx5-binding domain. B, the nucleotide and deduced amino acid sequences of Dlxin-1. The boxed sequence is encoded by clone 16 (see above). The underlined sequence indicates a potential polyadenylation signal (AATAAA). The nucleotide sequence of mouse Dlxin-1 has been submitted to GenBankTM/EMBL/DDBJ with accession number AB029448.



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 3.   Alignment of Dlxin-1 related proteins and the WQXPXX repeat. A, necdin homology domain. The amino acid sequences of the necdin domain were aligned. The boxes at the top of each row indicated the identity: all (black) or three (gray) of the four sequences are matched. The sequence data were obtained from GenBankTM as accession numbers D76440, AAC23618, and U10686, for mouse necdin, human MAGE-B3, and human MAGE-11, respectively. The numbers correspond to the amino acid number in each protein. B, Dlxin-1 homologues. The deduced amino acid sequence of Dlxin-1 is compared with those of human MAGE-D1 and a rat sequence showing high similarity submitted to GenBankTM with the accession number AF274043, with identities of 91 and 96%, respectively. C, the WQXPXX repeats of Dlxin-1. The tandem hexapeptide repeats are aligned. 22, 16, and 21 residues out of 25 repeats are tryptophan, glutamine, and proline in the position of the each repeat, respectively, showing a consensus.

Expression of Dlxin-1 mRNA-- To determine the expression of Dlxin-1 mRNA in various tissues and cell lines, Northern blot analyses were performed. Most adult mouse tissues were found to express a 3.0-kilobase pair transcript of Dlxin-1, except for the spleen (Fig. 4A). As Dlx5 has been reported to be expressed predominantly in skeletal elements (1, 14, 15), Dlxin-1 mRNA was detected in bone, but not in bone marrow, suggesting that Dlxin-1 is expressed in adherent cells rather than hematopoietic cells in bone. The Dlxin-1 mRNA was expressed at embryonic stages (embryonic days 7-17 (ED 7-17)) in mouse development (Fig. 4A). Dlxin-1 was also widely expressed in various human and mouse cell lines, including osteoblastic (KUSA/A1 and MC3T3-E1) and chondrogenic (ATDC5) cells (data not shown).



View larger version (102K):
[in this window]
[in a new window]
 
Fig. 4.   Expression of Dlxin-1 mRNA. A, mouse embryo MTN blot (left) and mouse MTN blot (middle panel) were used for Northern blot analyses. Total RNA (5 µg) from mouse long bones, bone marrow, and calvaria were blotted and hybridized. ED, embryonic day. A single band of Dlxin-1 transcript was estimated as 3.0 kilobase pairs in length. B, in situ hybridization. DIG-labeled Dlxin-1 RNA probe in antisense (left) or sense (right) orientation was hybridized to adjacent parasagittal sections of an embryonic day 15 mouse embryo. Bar, 0.1 mm.

Next, we performed in situ hybridization to localize the expression of the Dlxin-1 gene in embryonic and adult tissues. The expression detected by the antisense probe for Dlxin-1 mRNA was ubiquitous in tissues on mouse embryo days 13 and 15 (not shown). Notably, the strongest signal was detected in the cell layers surrounding cartilaginous elements in bone rudiment during embryonic digit formation (Fig. 4B).

Dlxin-1 Binds to Dlx5 in Vivo-- Although the clones for Dlxin-1 were isolated by interaction trap with Dlx5, the protein interaction remained to be confirmed in vitro and in mammalian cells. In the GST pull-down assay, in vitro translated Dlx5 coprecipitated with GST-Dlxin-1, indicating that Dlxin-1 associates with Dlx5 in vitro, probably via direct binding (data not shown). Interestingly, Msx2, another member of the Dlx/Msx homeodomain protein family, also bound to Dlxin-1 (data not shown).

Next, the association of Dlx5 with Dlxin-1 was determined in COS7 cells. As shown in Fig. 5, FLAG-tagged Dlx5 protein coprecipitated with HA-tagged Dlxin-1. Dlx7, Msx2, and FLAG-tagged Dlxin-1 also coprecipitated with HA-tagged Dlxin-1. These results indicate that Dlxin-1 associates not only with Dlx5 but also with Dlx7 and Msx2 and forms multimers in vivo.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Dlxin-1 binds to Dlx/Msx homeodomain proteins in vivo. HA-tagged Dlxin-1 (HA-Dlxin1) was coimmunoprecipitated with FLAG-tagged Dlx5 (F-Dlx5), Dlx7 (F-Dlx7), Msx2 (F-Msx2), and Dlxin-1 (F-Dlxin1), but not when the FLAG peptide was included in the precipitation (+).

Dlxin-1 Activates Dlx5Delta C-mediated Transcriptional Activity-- To determine whether and how the binding of Dlxin-1 to Dlx5 modulates the transcriptional activity of Dlx5, we employed a GAL4-dependent transcriptional activation assay in HT1080 cells, which express a low level of Dlxin-1 mRNA. As shown in Fig. 6, Dlxin-1 stimulated the reporter activity through GAL4-Dlx5Delta C, but not GAL4, dose-dependently, suggesting that Dlxin-1 augments the transcription function of the N-terminal domain of Dlx5. At higher a dose of Dlxin-1, the stimulation was attenuated, but the basal activity by Dlx5Delta C remained, raising the possibility that the ratio between the amounts of the Dlxin-1 and Dlx5 complexes is important in transcriptional regulation.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of Dlxin-1 on Dlx5Delta C-mediated transcriptional activity. pFDlxin-1 at the indicated doses (µg) was cotransfected into HT1080 cells, with GAL4-Dlx5Delta C or GAL4 only and the reporter construct, pG5-luc.

Dlxin-1 Plays a Role in Dlx5-dependent Transcription-- Finally, to test the hypothesis that Dlxin-1 stimulates Dlx5-dependent transcription, a reporter gene assay was carried out using a Dlx5/Dlx5-responsive element reporter system. The Dlx5-binding domain of Dlxin-1 (Dlxin-DlxBD, clone 16, in Fig. 7A) was transfected into P19Dlx5 cells, which express a relatively high level of Dlxin-1 mRNA, and reporter activity was then measured. The reporter activity was reduced to the basal level by the addition of Dlxin-DlxBD, whereas cotransfection of full-length Dlxin-1 (Dlxin-FL) showed little effect (Fig. 7B). It is suggested that Dlxin-1 is required for maximal Dlx5-dependent transcriptional activation in P19 cells.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 7.   Dlxin-1 is required for Dlx5-dependent transcription. The schematic diagram (A) shows expression vectors for the full-length of Dlxin-1 (Dlxin-FL) and the Dlx-binding domain of the protein (Dlxin-DlxBD), which represents to the region encoded by clone 16 (see Fig. 2). Either construct (1 µg each) was cotransfected with pDlxRE-luc into Dlx5-expressing P19 (P19Dlx5) or mock-transfected P19 (P19neo) cells, and the luciferase activity was measured.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dlxin-1, a Novel Dlx5-binding Protein-- In this paper, we describe the identification of a novel Dlx5-binding protein, named Dlxin-1. When compared with MAGE-D1, a human orthologue of Dlxin-1, the N-terminal section, flanking the WQXPXX repeats, is longer in mouse Dlxin-1, raising the possibility that the initiation codon of mouse Dlxin-1 is located downstream of the predicted most 5' ATG codon in the reading frame. However, a purified polyclonal antibody raised against GST-Dlxin1-C reacted with endogenous 90-kDa proteins (data not shown) corresponding to the molecular mass of the transfected, epitope-tagged Dlxin-1. Although there remains the possibility that Dlxin-1 is subjected to some post-translational modification, we predict the open reading frame to code for 775 amino acids.

Dlxin-1 shows similarities to MAGE/necdin family proteins. Necdin, one of the most characterized proteins in the family, is a postmitotic neuron-specific nuclear protein (23). Necdin associates with E2F1 and mimics the function of Rb protein, thereby regulating cell cycle progression (24, 25). We could not detect any Rb-like activity or binding to E2F1 of Dlxin-1 (data not shown). We isolated a RING finger protein that binds to the necdin homology domain of Dlxin-1.2 It is therefore suggested that this domain, structurally similar to necdin, is involved in protein-protein interaction. Translated regions of MAGE/necdin family proteins are encoded by a single exon (17), whereas the coding sequence of the MAGE/necdin homologous domain is split into at least three exons in the mouse Dlxin-1 gene,2 which are unique to the Dlxin-1 gene among known MAGE/necdin family members. Thus, Dlxin-1 gene may be evolutionarily distant from other MAGE/necdin family proteins.

The WQXPXX repeats in the middle of Dlxin-1 molecule are unique to this molecule and show no homology with any sequence in the data base, other than MAGE-D1 and SNERG-1, human and rat orthologues of Dlxin-1, respectively. Although some variations were observed, the repeats are highly conserved between species. Interestingly, the domain for binding to the Dlx/Msx proteins is mapped in the tandem repeats. It has been shown that the Dlx and Msx homeodomain proteins form homo- or heterodimers through their homeodomain, thereby regulating each other's functions. The N-terminal domain of Dlx5, which shares a weak homology with other Dlx/Msx family proteins, is a primary binding site for Dlxin-1, because the domain, but not the homeodomain, was used for the bait in isolating Dlxin-1 by the two-hybrid screening. Tryptophan is included in many protein motifs for molecular recognition, like the WW domain and WD40 repeat proteins (26-28). It is tempting to speculate that Dlxin-1 binds to multiple proteins via the WQXPXX repeats, due to the presence of multiple tryptophan residues in tandem. In light of the recent identification of Miz1 (29) and MINT (30) that bind to Msx2 via proline-rich domains, it is also possible that proline residues are involved in the interaction between Dlxin-1 and Msx2 and/or Dlx homeodomain proteins.

Expression of Dlxin-1-- Dlxin-1 mRNA was ubiquitously expressed in many tissues and also during embryonic development, with some variation in the expression level depending on cell or tissue types. In hematopoietic tissues, such as spleen and bone marrow, little expression was detected, suggesting that Dlxin-1 expression is more pronounced in adherent cells. Dlx5 is predominantly expressed in skeletal elements and osteoblasts (1, 14, 15), in which Dlxin-1 is also expressed. It is suggested that Dlxin-1 may play a role in Dlx5-mediated osteoblastic function and that Dlxin-1 associates not only with Dlx5 but also other proteins, to regulate the function in tissues and cells other than bones. Notably, it has been reported that a deletion in the dlx5/dlx6 locus causes the split hand/split foot malformation in humans (31). It has also been described that digit abnormality is observed in mice that are deficient in both msx1 and msx2 genes. Considering the strong expression of Dlxin-1 in the digit (Fig. 4B), tissues surrounding chondrocytes and the chondrocytes, it is suggested that Dlxin-1 may be involved in digit formation activity in collaboration with Dlx/Msx homeodomain proteins.

It has been reported that the expression of Dlx5 mRNA, as well as Msx2, is up-regulated by bone morphogenetic protein. However, the expression level of the Dlxin-1 gene did not significantly change following treatment with bone morphogenetic protein 4 or transforming growth factor beta  in MC3T3-E1 and ST2 cells, while treatment of P19 cells with forskolin slightly decreased the expression of Dlxin-1 mRNA (data not shown). It is speculated that the expression of Dlxin-1 may be regulated during cell differentiation rather than being constitutive.

Dlxin-1 Is a Transcriptional Regulator-- It has been reported that Dlx5 is a positive regulator of mouse osteocalcin gene and osteoblastic functions (4, 5, 20). In this study, we demonstrated that the N-terminal region of Dlx5 bears an intrinsic transcription activation domain. It has recently been reported, however, that expression of the osteocalcin gene was greatly increased in the osteoblasts of dlx5 knockout mice, suggesting that Dlx5 is a negative regulator of osteocalcin gene expression in vivo. There are some possibilities that may explain this apparent inconsistency. First, the dose of Dlx5 may be critical for transcriptional regulation. The homeodomain proteins form dimers to act on DNA (4). Liu et al. (32) demonstrated that the dosage of Msx2 affects the number of proliferative osteoblasts. In this study, we showed that Dlxin-1 activates Dlx5-dependent transcription in a dose-related manner. Thus, not only the ratio between Msx and Dlx family proteins, but also the ratio between the homeodomain proteins and Dlxin-1 may be important in transcriptional regulation. Second, Dlx5 has two modes of transcriptional function, one through direct binding to DNA and the other by protein-protein interaction without binding to DNA. Newberry et al. (33) suggested that Dlx5 antagonizes the transcriptional repression by Msx2 to maintain the basal promoter activity (33). Thus, Dlx5 may be involved in the basal activity of the osteocalcin promoter and function as a potential inhibitor for a transcription factor(s) that stimulates the expression of the osteocalcin gene in a later phase of osteoblastic differentiation. Third, the function may be regulated by alternative form(s). It has been reported that multiple Dlx5 transcripts generated by alternative splicing exist in mouse brain (34). We have found a splice variant form of Dlx5, delta Dlx5, in mouse osteoblast cell lines.3 This variant form codes for only the N-terminal domain of Dlx5 and lacks both the homeodomain and the C-terminal region. Although the physiological function of delta Dlx5 is not known at the present time, there is the possibility that the splice variant interferes with binding between Dlx5 and Dlxin-1.

Our results show that Dlxin-1 binds to Msx2 more strongly than to Dlx5 and other Dlx family proteins. It could be partially explained by the lower expression of Dlx5, in comparison with that of Msx2. Although a weak similarity has been suggested to exist between the N-terminal regions of Msx and Dlx family proteins (4), it is surprising that most Dlx/Msx family proteins show binding to Dlxin-1. Thus, Dlxin-1 may be a common regulator for transcriptional function, mediated by the Dlx/Msx homeodomain proteins.

We failed to detect any transactivation of Dlxin-1 itself using GAL4-mediated transcriptional assays (data not shown). The overexpression of Dlxin-1 reduced the maximal transcriptional activation without affecting the basal level of activation in Dlx5Delta C- or Dlx5-dependent transcription (Fig. 6). These findings suggest that Dlxin-1 may bridge or stabilize the complex of Dlx5 and coactivators rather than being directly involved in transcriptional activation of Dlx5. However, it remains to be determined whether Dlxin-1 has any histone acetylase or chromatin-remodeling activities.


    ACKNOWLEDGEMENTS

We thank Drs. A. Umezawa (Keio University), A. Fukamizu (University of Tsukuba), and S. Kato (University of Tokyo) for providing cell lines and yeast strain. We also appreciate useful information on necdin from Dr. K. Yoshikawa (Osaka University). We thank Drs. A. Matsuura and N. Motoyama for critical comments and Dr. R. Thornhill for proofreading.


    FOOTNOTES

* This work was supported by the Research Grant for Longevity Sciences from the Ministry of Health and Welfare of Japan (to K. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB029448.

To whom correspondence should be addressed: Dept. of Geriatric Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan. E-mail: kwatanab@nils.go.jp.

Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008590200

2 A. Sasaki and K. Watanabe, unpublished data.

3 Y. Masuda, A. Sasaki, and K. Watanabe, unpublished data.


    ABBREVIATIONS

The abbreviations used are: MAGE, melanoma-associated antigen; DlxRE, Dlx-responsive element; GST, glutathione S-transferase; HA, hemagglutinin.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Simeone, A., Acampora, D., Pannese, M., D'Esposito, M., Stornaiuolo, A., Gulisanp, M., Mallamaci, A., Kastury, K., Druck, T., Huebner, K., and Boncinelli, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2250-2254[Abstract]
2. Qiu, M., Bulfone, A., Ghattas, I., Meneses, J. J., Christensen, L., Sharpe, P. T., Presley, R., Pedersenn, R. A., and Rubenstein, J. L. R. (1997) Dev. Biol. 185, 165-184[CrossRef][Medline] [Order article via Infotrieve]
3. Chen, X., Li, X., Wabg, W., and Lufkin, T. (1996) Ann. N. Y. Acad. Sci. 785, 38-47[Medline] [Order article via Infotrieve]
4. Zhang, H., Hu, G., Wang, H., Sciavolino, P., Iler, N., Shen, M. M., and Abate-Shen, C. (1997) Mol. Cell. Biol. 17, 2920-2932[Abstract]
5. Miyama, K., Yamada, G., Yamamoto, T. S., Takagi, C., Miyado, K., Sakai, M., Ueno, N., and Shibuya, H. (1999) Dev. Biol. 208, 123-133[CrossRef][Medline] [Order article via Infotrieve]
6. Yaoita, H., Orimo, H., Shirai, Y., and Shimada, T. (2000) J. Bone Miner. Metab. 18, 63-70[CrossRef][Medline] [Order article via Infotrieve]
7. Maas, R., Chen, Y. P., Bei, M., Woo, I., and Satokata, I. (1996) Ann. N. Y. Acad. Sci. 785, 171-181[Medline] [Order article via Infotrieve]
8. Catron, K. M., Wang, H., Hu, G., Shen, M. M., and Abate-Shen, C. (1996) Mech. Dev. 55, 185-199[CrossRef][Medline] [Order article via Infotrieve]
9. Satokata, I., Ma, L., Ohshima, H., Bei, M., Woo, I., Nishizawa, K., Maeda, T., Takano, Y., Uchiyama, M., Heaney, S., Peters, H., Tang, Z., Maxson, R., and Maas, R. (2000) Nat. Genet. 24, 391-395[CrossRef][Medline] [Order article via Infotrieve]
10. Vainio, S., Karavanova, I., Jowett, A., and Thesleff, I. (1993) Cell 75, 45-58[Medline] [Order article via Infotrieve]
11. Newberry, E. P., Latifi, T., and Towler, D. A. (1998) Biochemistry 37, 16360-16368[CrossRef][Medline] [Order article via Infotrieve]
12. Benson, M. D., Bargeon, J. L., Xiao, G., Thomas, P. E., Kim, A., Cui, Y., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 13907-13917[Abstract/Free Full Text]
13. Ryoo, H. M., Hoffman, H. M., Beumer, T., Frenkel, B., Towler, D. A., Stein, G. S., Stein, J. L., van Wijnen, A. J., and Lian, J. B. (1997) Mol. Endocrinol. 11, 1681-1694[Abstract/Free Full Text]
14. Acampora, D., Merlo, G. R., Paleari, L., Zerega, B., Postiglione, M. P., Mantero, S., Bober, E., Barbieri, O., Simeone, A., and Levi, G. (1999) Development 126, 3795-3809[Abstract/Free Full Text]
15. Depew, M. J., Liu, J. K., Presley, R., Meneses, J. J., Pedersen, R. A., and Rubenstein, J. L. R. (1999) Development 126, 3831-3846[Abstract/Free Full Text]
16. van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De Plaen, E, van den Eynde, B., Knuth, A., and Boon, T. (1991) Science 254, 1643-1647[Medline] [Order article via Infotrieve]
17. De Plaen, E., Arden, K., Traversari, C., Gaforio, J. J., Szikora, J. P., De Smet, C., Brasseur, F., van der Bruggen, P., Lethe, B., Lurquin, C., Brasseur, R., Chomez, P., De Backer, O., Cavenee, W., and Boon, T. (1994) Immunogenetics 40, 360-369[Medline] [Order article via Infotrieve]
18. Pold, M., Zhou, J., Chen, G. L., Hall, J. M., Vescio, R. A., and Berenson, J. R. (1999) Genomics 59, 161-167[CrossRef][Medline] [Order article via Infotrieve]
19. Umezawa, A., Maruyama, T., Segawa, K., Shadduck, R. K., Waheed, A., and Hata, J. (1992) J. Cell. Physiol. 151, 197-205[Medline] [Order article via Infotrieve]
20. Dodig, M., Kronenberg, M. S., Bedalov, A., Kream, B. E., Gronowicz, G., Clark, S. H., Mack, K., Liu, Y. H., Maxon, R., Pan, Z. Z., Upholt, W. B., Rowe, D. W., and Lichtler, A. C. (1996) J. Biol. Chem. 271, 16422-16429[Abstract/Free Full Text]
21. Watanabe, K., Yamada, H., and Yamaguchi, Y. (1995) J. Cell Biol. 130, 1207-1218[Abstract]
22. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
23. Maruyama, K., Usami, M., Aizawa, T., and Yoshikawa, K. (1991) Biochem. Biophys. Res. Commun. 178, 291-296[Medline] [Order article via Infotrieve]
24. Taniura, H., Taniguchi, N., Hara, M., and Yoshikawa, K. (1998) J. Biol. Chem. 273, 720-728[Abstract/Free Full Text]
25. Taniura, H., Matsumoto, K., and Yoshikawa, K. (1999) J. Biol. Chem. 274, 16242-16248[Abstract/Free Full Text]
26. Sudol, M., Chen, H. I., Bougeret, C., Einbond, A., and Bork, P. (1995) FEBS Lett. 369, 67-71[CrossRef][Medline] [Order article via Infotrieve]
27. Neer, E. J., Schmidt, C. J., Nambudripad, R., and Smith, T. F. (1994) Nature 371, 297-300[CrossRef][Medline] [Order article via Infotrieve]
28. Smith, T. F., Gaitatzes, C., Saxena, K., and Neer, E. J. (1999) Trends Biochem. Sci. 24, 181-185[CrossRef][Medline] [Order article via Infotrieve]
29. Wu, L., Wu, H., Ma, L., Sangiorgi, F., Wu, N., Bell, J. R., Lyons, G. E., and Maxson, R. (1997) Mech. Dev. 65, 3-17[CrossRef][Medline] [Order article via Infotrieve]
30. Newberry, E. P., Latifi, T., and Towler, D. A. (1999) Biochemistry 38, 10678-10690[CrossRef][Medline] [Order article via Infotrieve]
31. Crackower, M. A., Scherer, S. W., Rommens, J. M., Hui, C. C., Poorkaj, P., Soder, S., Cobben, J. M., Hudgins, L., Evans, J. P., and Tsui, L.-C. (1996) Hum. Mol. Genet. 5, 571-579[Abstract/Free Full Text]
32. Liu, Y. H., Tang, Z., Kundu, R. K., Wu, L., Luo, W., Zhu, D., Sangiorgi, F., Snead, M. L., and Maxon, R. E., Jr. (1999) Dev. Biol. 205, 260-274[CrossRef][Medline] [Order article via Infotrieve]
33. Newberry, E. P., Latifi, T., Battaile, J. T., and Towler, D. A. (1997) Biochemistry 36, 10451-10462[CrossRef][Medline] [Order article via Infotrieve]
34. Yang, L., Zhang, H., Hu, G., Wang, H., Abate-Shen, C., and Shen, M. M. (1998) J. Neurosci. 18, 8322-8330[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.