Genomic Organization, Chromosomal Mapping, and Promoter Analysis of the Mouse Dentin Sialophosphoprotein (Dspp) Gene, Which Codes for Both Dentin Sialoprotein and Dentin Phosphoprotein*

Jian Q. Feng, Xianghong Luan, John Wallace, Dai Jing, Toshio OhshimaDagger , Ashok B. KulkarniDagger , Rena N. D'Souza§, Christine A. Kozak, and Mary MacDougallpar

From the University of Texas Health Science Center at San Antonio, Dental School, San Antonio, Texas 78284-7888, the Dagger  Gene Targeting Research and Core Facility, NIDR, National Institutes of Health, Bethesda, Maryland 20892, the § University of Texas Health Science Center Houston Dental Branch, Houston, Texas 78284, and  NIAID, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our laboratory has reported that two major noncollagenous dentin proteins, dentin sialoprotein and dentin phosphoprotein, are specific cleavage products of a larger precursor protein termed dentin sialophosphoprotein (MacDougall, M., Simmons, D., Luan, X., Nydegger, J., Feng, J. Q., and Gu, T. T. (1997) J. Biol. Chem. 272:835-842). To confirm our single gene hypothesis and initiate in vitro promoter studies, we have characterized the structural organization of the mouse dentin sialophosphoprotein gene. This gene has a transcription unit of ~9.4 kilobase pairs and is organized into 5 exons and 4 introns. Exon 1 contains a noncoding 5' sequence, and exon 2 contains the transcriptional start site, signal peptide, and first two amino acids of the NH2 terminus. Exons 3 and 4 contain coding information for 29 and 314 amino acids, respectively. The remainder of the coding information and the untranslated 3' region are contained in exon 5. Chromosomal mapping localized the gene to mouse chromosome 5q21 in close proximity to other dentin/bone matrix genes. Computer analysis of the promoter proximal 1.6-kilobase pair sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements of AP-1, AP-2, Msx-1, serum response elements, SP-1, and TCF-1. In vitro studies showed that the DSPP promoter is active in an odontoblast cell line, MO6-G3, with basal activity mapped to -95 bp. Two potential enhancer and suppresser elements were identified in the regions between -1447 and -791 bp and -791 and -95 bp, respectively. The structural organization of the dentin sialophosphoprotein gene confirms our finding that both dentin sialoprotein and dentin phosphoprotein are encoded by a single gene with a continuous open reading frame.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The dentin extracellular matrix (DECM)1 is the result of the cytodifferentiation of cranial neural crest-derived ectomesenchymal cells that line the tooth pulp chamber into highly specialized cells termed odontoblasts. These odontoblasts express specific genes products which form the collagenous DECM. This matrix consists of mostly type I (~86%), type I trimer, type III, type V, and type VI collagens and several noncollagenous proteins also found in bone extracellular matrix, such as osteonectin (OSN, also known as SPARC), osteocalcin, osteopontin (OPN, also known as SSP1), bone sialoprotein, and dentin matrix protein 1 (Dmp-1) (1, 2). However, two DECM proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP, also known as phosphophoryn), have been shown to be tooth specific (for review Ref. 1), being expressed by odontoblasts and transiently by ameloblasts (2-10). These noncollagenous DECM proteins are believed to be essential for initiation and control of mineralization in the transition of predentin to dentin.

DSP was first identified as a 95-kDa glycoprotein (11) with a high carbohydrate (30%) and sialic acid (10%) content accounting for 5-8% of the DECM proteins. This protein has an overall resemblance to other sialoproteins like bone sialoprotein and shares limited NH2-terminal sequence homology with other acidic phosphoproteins, OPN, Dmp-1, and bone acidic glycoprotein-75. A rat full-length cDNA DSP clone (1.2 kbp) has been reported coding for a 366-residue secreted protein with a 17-amino acid signal peptide. The calculated amino acid composition for the predicted protein was extremely close to that determined for the matrix isolated protein (5). Furthermore, two different sized genomic rat DSP clones have been isolated with one being fully characterized (5, 12). Two DSP polypeptide bands were identified by Western blot with a polyclonal DSP antibody, suggesting that more than a single DSP protein may exist (2).

DPP is the major noncollagenous dentin matrix protein which is strongly associated with the mineralization of dentin. In vivo (13) and in vitro (14, 15) data have shown that DPP is expressed by the odontoblasts and preameloblasts. After secretion, DPP is confined to the mineralized dentin layer of the matrix (3, 16). Molecular masses of DPP in numerous species have been reported: 155 kDa in bovines (7), 72 kDa in mice (3), and 90-95 kDa in rats (10, 17). The number of DPP proteins within the DECM has also varied with multiple forms being reported for rats (2, 10). Although the exact molecular size and number of DPPs is unclear, conserved high levels of aspartic acid (30-40%) and phosphoserine (38-50%) in all reported species suggest a function related to dentin mineralization. It is believed that DPP is a nucleator or modulator of hydroxyapatite crystal formation (1).

Recently, our laboratory has reported the identification and characterization of a mouse 4.4-kbp cDNA clone which is homologous to DSP on its 5' region and to DPP on its 3' terminal with a single continuous open reading frame (18). We named this gene dentin sialophosphoprotein (DSPP) and mapped it to human chromosome 4 which contains the gene loci for the dentin diseases dentinogenesis imperfecta type II and dentin dysplasia type II. We proposed that DPP and DSP represent specific cleavage products of a large chimeric precursor protein which is processed. In contrast, Ritchie and Wang (19) have identified an additional open reading frame for rat DPP in a partial 2.0-kbp PCR-generated DSP cDNA clone. These authors suggest that the presence of both DSP and DPP was possibly due to an artifact generated by reverse transcription-PCR or cloning, or caused by a bicistronic gene. In addition, a partial COOH-terminal rat phosphophoryn (DPP) cDNA clone has been reported at the amino acid level only, named dentin matrix protein 2 (Dmp2), which has high homology to the COOH terminus of both the mouse DSPP and rat DPP deduced amino acid sequences (20).

Little is known regarding the transcriptional and post-transcriptional mechanisms involved in DSPP gene expression and the nature of the extrinsic or intrinsic factors that regulate it. With the objective of clarifying the single gene hypothesis, deciphering the basic structural organization, and regulation of the DSPP gene, we isolated mouse DSPP genomic clones. A 19-kbp clone containing the entire mouse DSPP gene was characterized in detail. Furthermore, we localized the transcriptional start site by a primer extension assay and determined the mouse chromosomal location by genetic mapping. Computer analysis was performed on the upstream DSPP promoter region to identify a number of potential regulatory cis-acting elements. Finally, the promoter activity of various constructs was assayed in vitro using an odontoblast cell line previously shown to express both DSP and DPP, with basal promoter activity mapped to -95 bp. Our data confirm that DPP and DSP are encoded by a single gene with a continuous open reading frame.

    MATERIALS AND METHODS
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Materials & Methods
Results
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References

Library Screening-- A mouse genomic lambda Fix II 129/SVJ library (Stratagene, La Jolla, CA) was screened using a mouse DSPP full length (4.4-kbp) clone or a partial PCR-generated DSPP611-1089 cDNA which was directionally subcloned into pGEM-5Z+ (Promega, Madison, WI). The probes were labeled with [alpha -32P]dCTP using a random primer labeling kit from Boehringer Mannheim according to supplier's instructions. Plaque lift filters were hybridized overnight in hybridization buffer containing 6 ×SSC (1 ×SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5× Denhardt's, 0.5% SDS, 200 µg/ml sonicated salmon sperm, 10 µg/ml poly(A)+, and 10 µg/ml tRNA at 68 °C. The filters were washed at 55 °C for 20 min, twice in 2 ×SSC, 0.1% SDS buffer, and once in 0.5 ×SSC, 0.1% SDS buffer.

DNA Sequence of DSPP Genomic Clone-- The isolated phage DNA clones were mapped by restriction enzyme analysis according to standard procedures (21). DNA inserts from positive DSPP clones were cut with restriction enzymes (EcoRI, SacI, BamHI, XhoI, and XmnI), and fragments were subcloned into pBluescript vector (Stratagene). The DNA sequence in both directions was determined with an ABI automatic sequencer model 372 (Applied Biosystems). Protein consensus sequences and DNA sequence alignment diagonal matrix plots were determined using MacVector 6.0 software (Kodak, Rochester, NY). The percentage of DNA sequence homology was determined using GenePro 6.1 software (Riverside Scientific Enterprises, Beinbridge Island, WA).

Primer Extension Mapping of the Transcriptional Start Site-- The transcriptional start site was mapped by primer extension using a synthetic oligonucleotide, DSPP54-37 5'-CGAGGG GACTTTGAAAAT-3' (Primer 2, Fig. 2C), in the exon 1 sequence. Total RNA was isolated from the odontoblast cell line, previously shown to express DSPP (both DSP and DPP) (22, 23). The primer extension assay was carried out using the primer extension kit from Promega according to instructions with annealing reactions carried out at 55 °C. Resulting products were electrophoresed on a 8% denaturing urea polyacrylamide gel and autoradiographed. The primer DNA sequences were used for size markers.

Mouse Genetic Mapping of DSPP Gene-- The chromosomal location of the gene encoding DSPP was determined by analysis of two sets of multilocus crosses: (NFS/N or C58/J × Mus musculus musculus) × M. m. musculus (24) and (NFS/N × Mus spretus) × M. spretus or C58/J (25). DNAs from the progeny of these crosses have been typed for >1000 markers distributed over the mouse genome including the chromosome 5 markers Afp (alpha -fetoprotein), Gro1 (GRO1 onogene), Ibsp (bone sialoprotein), Spp1 (secreted phosphoprotein 1, also known as osteopontin) and Pdeb (phosphodiesterase beta ) which were typed as described previously (26). D5Mit9 (Chr 5 DNA marker, MIT-9) was typed using the primers and conditions described by Dietrich et al. (27). The partial DSPP cDNA probe was labeled as described previously. Linkage was determined using the program LOCUS designed by C. E. Buckler (NIAID, National Institutes of Health, Bethesda, MD). The percent recombination and standard errors were determined according to the method of Green (28), and the gene order was determined by minimizing recombinants.

DSPP Promoter Plasmid Construction-- Since no appropriate restriction enzyme sites existed within exon 1 of the mouse DSPP gene, a PCR-amplified product, corresponding to the sequence between nucleotides -1447 and +54 of exon 1, was generated with a set of synthesized primers: DSPP 1-S-1447-1430 (5'-GAATTCTAGGACAAGCAG-3'), and DSPP 2-AS37-54 (5'-CGAGGGGACTT TGAAAAT-3'). The PCR reaction was 3 min at 94 °C for 1 cycle, 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C for 35 cycles followed by 10 min at 72 °C. The amplified 1.5-kbp fragment was first cloned into the pCRII vector using TA cloning kit (InVitrogen, San Diego, CA) and then released with HindIII on the 3' side and various restriction enzymes on the 5' side to produce the 5 different constructs, and recloned into pGL3 luciferase (LUC) basic expression vector (Promega). Correct orientation of the all inserts with respect to the pGL3 LUC vector was verified by DNA sequencing. The pGL3 LUC-1447-+54-kbp plasmid consisted of pGL3 LUC vector with a 1.5-kbp EcoRI and HindIII fragment of the DSPP gene. The pGL3LUC-1243-+54 plasmid was similarly generated from a 1.3-kbp BglII and HindIII fragment, the pGL3LUC-791-+54 plasmid from an 845-bp XhoI and HindIII fragment, the pGL3LUC-510-+54 plasmid from a 650-bp XmnI and HindIII fragment, and the pGL3LUC-95-+54-bp plasmid from a 149-bp BamHI and HindIII fragment. All the various plasmid constructs contained part of the exon 1 noncoding region.

Transfection and Dual Luciferase Assay-- An established odontoblast cell line MO6-G3 (20) was used as the recipient cells for transient transfection assays. After electroporation (320 V; capacitance, 960 mFD), the cells were divided into aliquots, replated in a 6-well plate, and cultured for 72 h in alpha -minimal essential medium (Life Technologies, Inc., Gaithersburg, MD) with 10% fetal calf serum (Life Technologies, Inc.)(29). The cell lysates were assayed for dual luciferase activity according to the manufacturer's procedure (Promega). In this dual luciferase system, DSPP promoter fragments were linked to the firefly luciferase gene while the co-transfected renilla luciferase gene was driven by the SV40 promoter. The second reporter gene provided an internal control by which each value within an experimental set could be normalized. Since both enzymes used different substrates, dual luciferase measurements were performed using TD-20/20 (Promega) in a single tube, with firefly luciferase assayed first followed by renilla luciferase. The promoter activity was reflected by the ratio of firefly/renilla luciferase for each construct.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning and Sequencing of Mouse DSPP Gene-- Seven clones were isolated from 2 × 106 plaques of mouse 129/SVJ genomic library using mouse DSPP cDNA probes (18). These overlapping clones spanned 35 kbp of the Dspp locus (Fig. 1). One 19-kbp clone (lambda 1) contained 5 exons, 4 introns, and ~1.6-kbp 5'- and ~8-kbp 3'-flanking regions. This clone was utilized for the characterization of the DSPP gene. A diagram of the lambda 1 clone with exon organization is shown in Fig. 1. The ~9.4-kbp transcription unit and ~1.6 kbp of the 5'-flanking region were sequenced. The nucleotide sequence of mouse DSPP and deduced amino acid sequence of coding exons (934 residues) are shown in Fig. 2A. Full DNA sequence of the intron regions can be found in the Genbank/EMBL Data Bank submission. DNA sequence of the coding and untranslated regions of DSPP are identical with those of the mouse DSPP cDNA except for a single T initially reported as a G in the cDNA sequence at nucleotide 2888 (18). This nucleotide change results in the shortening of the coding region by 6 amino acids (18). Fig. 2B shows the DNA sequence of 1628 bp of the 5'-flanking region and candidate DNA response elements located upstream of the first exon. Oligonucleotide primers used in the primer extension experiments and promoter constructs are also indicated in Fig. 2.


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Fig. 1.   Orientation, partial restriction enzyme maps, and gene structure of the mouse DSPP genomic clones. The enlarged region of the mouse DSPP genomic clone lambda 1 shows ~1.6 kbp of the 5'-flanking region and a small region (~100 bp) of the 3'-flanking area. The mouse DSPP gene transcription unit is~ 9.4 kbp and contains 4 coding exons (black-square), labeled exons 2 though 5, and 2 noncoding exons, labeled exons 1 and 5. The exons with homology to rat DSP cDNA and genomic clones (5, 12) and rat DPP cDNA clones (19) are indicated as filled boxes. B, BamH1; R, EcoRI; Bg, BglII; S, SacI; X, XhoI; Xm, XmnI; K, KpnI.


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Fig. 2.   DNA sequence of the mouse DSPP gene and 5' promoter region. A, DNA sequence of the mouse DSPP gene and the predicted amino acid sequences within the coding exons. The sequences of the exons and intron borders are present. The numbers on the right show the position of nucleotide sequence, and the bold numbers indicate the location of amino acid sequence of the coding region. The end of the transcription unit was estimated based on a ~4.4-kbp mRNA transcript size and polyadenylation signal (AATAAA) site. The three potential polyadenylation signal sites are marked by bold type. B, DSPP 5'-flanking region and potential response elements of the DSPP promoter. The DNA sequence of the 1.6-kbp flanking region and exon 1 of the mouse DSPP gene are shown. Nucleotides are numbered on the right with +1 corresponding to the transcription start site of the promoter. The potential response elements of SP-1, serum response elements (SRE), Msx-1, TCF-1, AP-1, and AP-2 are underlined and labeled. Primers 1 (-1347 to -1330) and 2 (+37 to +54) are indicated with dotted lines below the DNA sequence; these were used for generation of the promoter constructs. Primer 1 was also utilized in the primer extension studies for analysis of the transcription start site.

Intron-Exon Organization-- As shown in Table I, the conserved 5'-dinucleotide sequence GT is contained in all four introns of mouse DSPP gene. All AG- consensus dinucleotide sequences on 5' donor regions are conserved except in exon 2 which has the sequence CG. These dinucleotide sequences are putative splice sites implicated in the primary transcript splicing (30).

                              
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Table I
Exon-intron organization

Mapping of the Transcriptional Start Site-- Primer extension analysis was performed to map the mouse DSPP gene transcription start site. Total RNA from a stable, previously characterized mouse odontoblast cell line, MO6-G3, and a DSPP 254-37 primer (Fig. 2B) corresponding to nucleotides in exon 1 were used for this assay. As shown in Fig. 3, a clear extended fragment was obtained with the odontoblast-derived RNA but not with tRNA. Based on this 54-bp extended band, the size of exon 1 is estimated as 67 bp which is in agreement with the size of the longest mouse DSPP cDNA clone isolated (18).


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Fig. 3.   The mouse DSPP gene primer extension assay. The total RNA prepared from the odontoblast cell line MO6-G3 (right lane) and tRNA (left lane) were used with primer 2 (Fig. 2B). An extended fragment of 54 bp was obtained from odontoblast total RNA, while no fragment was detected using mouse tRNA.

Mouse DSPP Gene Codes for Both DSP and DPP-- DNA sequence homology diagonal plots between the mouse DSPP gene, rat DSP cDNA (5, 12), and rat DPP cDNA (19) using MacVector software are shown in Fig. 4. These data show (Fig. 4, A and B) diagonal lines as expected when sequences are aligned throughout the sequence. It is clear that the DNA sequence of rat DSP clone is homologous (>80%) to mouse DSPP gene exons 1, 2, 3, and 4 and the most 5'-portion of exon 5 (Fig. 4, A and C). The rat DPP is similar (~70% homology) to the remainder of the mouse DSPP gene exon 5 (Fig. 4, B and C). In the plot against the rat DPP clone (Fig. 4B), numerous diagonal lines occur off the central line, appearing as a black box, due to the region of extensive internal homology (repetitive serine-aspartic acid region) within DPP. These data confirm that DSP and DPP are encoded from a single gene.


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Fig. 4.   DNA sequence diagonal homology plot of the mouse DSPP gene with rat DSP and DPP cDNA clones. In A, the central diagonal line represents the homology between the sequence of the rat DSP (5, 12) and that of mouse DSPP gene exons 2, 3, and 4 and the beginning of exon 5. In B, the central diagonal line represents the homology between the sequence of the rat DPP (19) and that of the remainder of mouse DSPP exon 5. The black box area is produced by the tight clustering of small parallel lines representing the region of extensive internal homology of the DPP/DSPP highly repetitive sequence region (18). C, shows a diagram of the structural localization of both DSP and DPP cDNA clones within the mouse DSPP gene.

Dspp Genetic Mapping-- The mouse locus Dspp was mapped using a 479 bp cDNA fragment as a hybridization probe to type DNAs from two sets of genetic crosses. HpaI digestion generated fragments of 25 kbp in NFS/N and C58/J and 14.5 kbp in M. m. musculus. HindIII digestion produced fragments of 7.4 kbp in NFS/N and C58/J and 7.0 in M. spretus. Inheritance of the polymorphic fragments was followed in the progeny of both crosses and compared with that of >1000 markers previously typed and mapped. Dspp was linked to markers on chromosome 5 and mapped between Afp and Pdeb as shown in Fig. 5. In the Afp-Dspp interval, 13 recombinants were identified of which 11 were also typed for D9Mit9. Only 2 of these recombinants were recombinant with Dspp, suggesting the D5Mit9 can be placed 1.4 cM proximal to Dspp. The closest linkage was observed between Dspp and Ibsp with no recombinants identified in 159 mice, and between Dspp and Spp1 with no recombinants in 103 mice, indicating that at the upper limit of the 95% confidence level, Dspp is within 1.87 cM of Ibsp and 2.87 cM of Spp1.


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Fig. 5.   Genetic map location of Dspp on mouse chromosome 5. The recombination fractions between adjacent loci are give on the right of the map with the first fraction representing data from the M. m. musculus crosses and the second representing data form the M. spretus crosses. Numbers in parentheses, percentage of recombination and standard recombinant between Afp and Dspp; 11 were recombinant in the Afp-D5Mit9 interval and 2 in the D5Mit9-Dspp interval. Spp1 was typed only in the M. spretus crosses. The map locations for the human genes are given to the left of the map.

Analysis of Proximal Promoter Sequence Region-- The mouse DSPP gene contains no TATA box in the region adjacent to the transcription start site but does contain a GC-rich stretch of 22 nucleotides that lies between -143 and -165 (Fig. 2B). A perfect SP1 binding site GGGCG is located in this region. It is believed that genes lacking the TATA box may rely on these GC-rich sites and that proteins bound to them to initiate transcription.

There are six potential binding sites for the Msx-1 homeobox gene. Recent data have shown that Msx-1 is essential for tooth development, since the gene knockout results in an arrest of molar development (31). Interestingly, both Msx-1 (32) and the DSPP contain four TCF-1 binding sites, suggesting potential regulation by members of the ets family of transcriptional factors (33). Four potential serum response elements in the promoter region may indicate possible regulation of DSPP by growth factors identified in serum, such as platelet-derived growth factor and epidermal growth factor.

Mouse DSPP Promoter Activity in MO6-G3 Odontoblast Cell Line-- An established mouse odontoblast cell line was selected for analysis of mouse DSPP promoter activity, since both DSP and DPP have been shown at the transcriptional and translational levels to be expressed by odontoblasts using in situ hybridization and immunohistochemistry (22). After 72 h of transfection with the five DSPP-luciferase reporter gene constructs (Fig. 5A), the cells were harvested and the luciferase activity was determined. As indicated in Fig. 5B, pGL3-791-+54 plasmid had the lowest luciferase activity while pGL3-95-+54 plasmid had the highest activity. This initial analysis of the promoter activity suggests that potential transcriptional or translational enhancers exist in the region between -1447 and -791. Also, the data suggest that potential suppressor elements are located in the region between -791 and -95 bp, since the promoter activity is increased more than 2-fold after removal of this region. In addition, strong promoter activity of the -95-bp fragment indicates that this fragment may contain the basal control elements for expression of DSPP.


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Fig. 6.   Maps of the five mouse DSPP 5'-flanking-luciferase constructs and promoter activity in the MO6-G3 cell line. A, five fragments of the mouse DSPP 5'-flanking region containing 54 bp of exon 1 sequence were separately inserted into pGL3 luciferase plasmid as described under "Materials and Methods." The closed box indicates the noncoding portion of exon 1, and the LUC box represents the luciferase reporter gene. The values are percentages of luciferase activity relative to that of pGL3-1447 (100%) and represent the average of five independent assays. B, the actual values of different promoter constructs transiently transfected in the odontoblast cell line. Luciferase activity was normalized by co-transfected renilla luciferase activity driven by the SV40 promoter (see "Materials and Methods" for details).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we have isolated genomic clones of the mouse DSPP gene and examined its DNA sequence to support the hypothesis that DSP and DPP are cleavage products of a large chimeric protein encoded by a single gene. The genomic organization (exon/intron) has been defined by DNA sequence analysis of the entire 9394 nucleotides of the mouse DSPP transcriptional unit. The original reported 940-amino acid open reading frame has been shortened to 934 amino acids due to a single base correction and is contained in 4 exons. In addition, the 5' flanking region has been examined to lay the foundation for understanding the cellular factors and signaling pathways that may regulate transcription of this gene during odontogenesis. More than 1.6 kbp of the DSPP 5'-flanking region has been sequenced and analyzed. This is the first reported promoter sequence of a tooth-specific dentin matrix protein.

Previously, we hypothesized that DSP and DPP are protein cleavage products originating from a single gene based on sequence homology of a mouse DSPP cDNA with DSP and DPP and the presence of a continuous open reading frame (18). To confirm our single gene hypothesis, we have isolated and sequenced the mouse DSPP gene. As shown in Fig. 4, the sequence of rat DSP cDNA (5) is homologous to that of exons 1, 2, 3, and 4 and the 5'-region of exon 5, while the rat DPP cDNA (19) is similar to that of the 3' region of exon 5. A full-length rat DSPP cDNA (~4.4 kbp) has been cloned and sequenced.2 The 5' region of this clone is identical with that of rat DSP cDNA (5), and the 3' region is the same as that of the partial rat DPP (19) and dentin matrix protein 2 (20) cDNAs. The single DSPP gene is further substantiated by Southern blot analysis for the chromosomal mapping studies in both mice (Fig. 5) and humans (18), which revealed single hybridization DNA fragments. Moreover, in situ hybridization experiments have demonstrated that probes for DSP, DPP, and DSPP have identical patterns of expression in odontoblasts and preameloblasts during tooth development in vivo (9). Lastly, the structural organization of the rat DSP gene (12) is very similar to that of the mouse DSPP gene except the rat DSP gene is missing most of the last major exon. Thus, we conclude that there is only one gene, DSPP, which codes for both DSP and DPP.

To date, four different but related cDNAs from either rat or mouse tooth cDNA libraries for DPP and DSP have been reported (5, 18-20). Each cDNA clone is supported by Northern analysis indicating the presence of multiple transcript(s), with the largest transcript at~ 4.4-5 kbp. The minor variance of mRNA transcripts size determinations may be due to estimation differences based on various reference markers or possibly species differences. The deduced amino acids from all of these clones are largely in agreement with amino acid composition and NH2-terminal protein sequence for DSP and DPP. Furthermore, there is high homology to the nucleotide/amino acid sequence of the mouse DSPP cDNA and genomic clones.

However, some evidence still remains that could be interpreted as possibly supporting multiple DPP genes, alternative splicing, or multiple promoters. For example, the presence of multiple hybridization bands reported in all Northern blots and the fact that DPP accounts for as much as 50% of noncollagenous DECM proteins while DSP accounts for only 5-8%. Multiple transcripts identified on Northern blots have been shown to be due in part by the use of multiple polyadenylation signals, since three polyadenylation signal sites have been identified in the mouse DSPP cDNA clone (18) and gene (Fig. 2A). At this time, there is some limited evidence showing possible alternative splicing within the coding exons.3 However, there is no substantial evidence for the presence of multiple promoters. Therefore, it is most likely that the unequal proportion of DSP and DPP in the DECM is due to stability differences between the two polypeptide cleavage products, suggesting that DSP is less stable or more easily degraded. Immunohistochemistry using region-specific antibodies to several enamel proteins, amelogenin, ameloblastin, and enamelin, has shown that the cleavage products of these proteins containing the NH2 terminus are found in higher abundance than those containing the COOH terminus (34-36).

Genetic mapping studies have place Dspp within a cluster of genes expressed during dentin and/or bone formation located on mouse chromosome 5q21. These genes, Ibsp, Spp1, and Dmp1, are all contained within a 1-cM interval of the long arm of mouse chromosome 5 (37). Recently, dentin matrix protein 2 has been mapped to this same region of chromosome 5, being linked to Dmp1. This gene was mapped using a partial (~2500 bp, 245 amino acids) rat cDNA that was isolated using affinity to a phosphophoryn (DPP) antibody. Therefore, Dmp2 may in fact be a partial DSPP clone based on its high homology at the amino acid level to the COOH terminus of DPP or represent another new related gene in this gene cluster. This region of mouse chromosome 5 has been shown to share homology with human 4q21. In fact, the human DSPP gene has been mapped by fluorescence in situ hybridization to human chromosome 4 band 4q21.3 (38). More refined mapping has placed the gene in relationship to sequence-tagged sites on the physical map of human chromosome 4 between markers D4S567 and D4S1292 (39). This study revealed that the DSPP and DMP1 loci are within a maximum distance of 110 kbp. The order of these dentin/bone "mineralizing-matrix" genes was determined to be DSPP-DMP1-IBSP-SPP1. These data are consistent with the mouse genetic mapping data in this study showing that Dspp is linked to both Ibsp and Spp1. Human mapping studies place the DSPP gene within the defined critical regions of two dentin genetic diseases, dentinogenesis imperfecta type II and dentin dysplasia type II (39). Therefore, DSPP is a strong candidate gene for both these diseases.

A 1627-bp 5'-flanking region of DSPP was sequenced, and several potential regulatory elements have been identified by their sequence homology to known transcription factor-binding sites. Five DSPP 5'-flanking region-luciferase reporter gene constructs with different size deletions were transfected into an established odontoblast cell line, MO6-G3, to begin to define regions of the promoter that are important for transcriptional regulation. Strong promoter activity of the -95-bp 5'-flanking region in odontoblasts suggests that this region may contain full basal promoter activity. Two weak enhancers between -1447 and -791 and two suppresser regions between -791 and -95 were identified. The potential significance of these regions in vitro and analysis of transgenic mice harboring -1447/+54 fragment are currently under investigation. These observations are critical for future studies in vivo to determine the mechanisms of DSPP tooth-specific regulation during odontoblast and ameloblast cytodifferentiation.

In summary, we have isolated mouse genomic clones for the DSPP gene and shown conclusively that a single gene codes for the two major dentin matrix proteins, DSP and DPP. Chromosomal mapping localized this gene to mouse chromosome 5q21. This investigation has built a molecular basis for future in vivo loss or gain of gene function studies. In addition, the in vitro characterization of the DSPP promoter in odontoblasts has facilitated future transgenic mouse studies.

    ACKNOWLEDGEMENTS

Technical support on the mouse genetic crosses by M. Charlene Adamson at the National Institute Health is acknowledged. We also thank Jason Nydegger, Darrin Simmons, and TingTing Gu for their general assistance on this project.

    FOOTNOTES

* This work is supported by NIDR Grants DE 11658 (M. M.) and DE 09875 (M. M.).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(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJOO2141.

par To whom correspondence and requests for reprints should be addressed: University of Texas Health Science Center at San Antonio, Dental School, Dept. of Pediatric Dentistry, 7703 Floyd Curl Dr., San Antonio, TX 78284-7888. Tel.: 210-567-3542; Fax: 210-567-6603; E-mail: MacDougall{at}UTHSCSA.edu.

1 The abbreviations used are: DECM, dentin extracellular matrix; DMP1, dentin matrix protein 1; DPP, dentin phosphoprotein; DSP, dentin sialoprotein; DSPP, dentin sialophosphoprotein; PCR, polymerase chain reaction; SPP1, secreted phosphoprotein 1; LUC, luciferase; bp, base pair; kbp, kilobase pair.

2 X. Luan, F. Q. Feng, and M. MacDougall, unpublished data.

3 M. MacDougall, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Linde, A., and Goldberg, M. (1993) Crit. Rev. Oral Biol. Med. 4, 679-728[Abstract]
  2. Butler, W. T., and Ritchie, H. H. (1995) Int. J. Dev. Biol. 39, 169-179[Medline] [Order article via Infotrieve]
  3. MacDougall, M., Zeichner-David, M., and Slavkin, H. C. (1985) Biochem. J. 232, 493-500[Medline] [Order article via Infotrieve]
  4. Gorter de Vries, I., Quartier, E., Van Steirteghem, A., Boute, P., Coomans, D., and Wisse, E. (1986) Arch. Oral Biol. 31, 57-66[Medline] [Order article via Infotrieve]
  5. Ritchie, H. H., Hou, H., Veis, A., and Butler, W. T. (1994) J. Biol. Chem. 269, 3698-3702[Abstract/Free Full Text]
  6. Butler, W. T., Bhown, M., D'Souza, R. N., Farach-Carson, M. C., Happonen, R. P., Schrohenloher, R. E., Seyer, J. M., Somerman, M. J., Foster, R. A., Tomana, M., and VanDijk, S. (1992) Matrix 12, 343-351[Medline] [Order article via Infotrieve]
  7. Stetler-Stevenson, W., and Veis, A. (1983) Biochemistry 22, 4326-4335[Medline] [Order article via Infotrieve]
  8. MacDougall, Nydegger J, Gu TT, Simmons D, Luan X, Cavender A, and D'Souza RN. (1998) Conn. Tissue Res., in press
  9. D'Souza, R. N., Cavender, A., Sunavala, G., Alverez, J., Ohshima, T., Kulkarni, A. B., and MacDougall, M. J. (1997) J. Bone Miner. Res. 12, 2040-2049[Medline] [Order article via Infotrieve]
  10. Butler, W. T., Bhown, M., Dimuzio, M. T., Cothran, W. C., and Linde, A. (1983) Arch. Biochem. Biophys. 225, 178-186[Medline] [Order article via Infotrieve]
  11. Butler, W. T., Bhown, M., Dimuzio, M. T., and Linde, A. (1981) Coll. Relat. Res. 1, 187-199[Medline] [Order article via Infotrieve]
  12. Ritchie, H., Pinero, G. L., Hou, H., and Butler, W. T. (1995) Connect. Tissue Res. 33, 73-79[Medline] [Order article via Infotrieve]
  13. Weinstock, M., and Leblond, C. P. (1973) J. Cell Biol. 56, 838-845[Free Full Text]
  14. Dimuzio, M. T., and Veis, A. (1978) J. Biol. Chem. 253, 6845-6852[Medline] [Order article via Infotrieve]
  15. Munksgaard, E. C., Richardson, W. S., and Butler, W. T. (1978) Arch. Oral Biol. 23, 583-585[Medline] [Order article via Infotrieve]
  16. Gorter de Vries, I., and Wisse, E. (1989) Arch. Oral. Biol. 31, 57-66
  17. Sabsay, B., Stetler-Stevenson, W. G., Lechner, J. H., and Veis, A. (1991) Biochem J. 276, 699-707[Medline] [Order article via Infotrieve]
  18. MacDougall, M., Simmons, D., Luan, X., Nydegger, J., Feng, J., and Gu, T. T. (1997) J. Biol. Chem. 272, 835-842[Abstract/Free Full Text]
  19. Ritchie, H. H., and Wang, L.-H. (1996) J. Biol. Chem. 271, 21695-21698[Abstract/Free Full Text]
  20. George, A., Bannon, L., Sabsay, B., Dillon, J. W., Malone, J., Veis, A., Jenkins, N. A., Gilbert, D. J., and Copeland, N. G. (1996) J. Biol. Chem. 271, 32869-32873[Abstract/Free Full Text]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. MacDougall, M., Thiemann, F., Ta, H., Hsu, P., Chen, L. S., and Snead, M. (1995) Connect. Tissue Res. 33, 97-103[Medline] [Order article via Infotrieve]
  23. MacDougall, M. (1996) Proceedings of the International Conference on Dentin/Pulp Complex 1995 and the International Meeting on Clinical Topics of Dentin/Pulp Complex, Quintessence International, pp. 116-123
  24. Kozak, C. A., Peyser, M., Krall, M., Mariano, T. M., Kumar, C. S., Pestka, S., and Mock, B. A. (1990) Genomics 8, 519-524[Medline] [Order article via Infotrieve]
  25. Adamson, M. C., Silver, J., and Kozak, C. A. (1991) Virology 183, 778-781[Medline] [Order article via Infotrieve]
  26. Crobsy, A. H., Lyu, M. S., Lin, K., McBride, O. W., Kerr, J. M., Aplin, H. M., Fisher, L. W., Young, M. F., and Kozak, C. A. (1996) Mamm. Genome 7, 149-151[CrossRef][Medline] [Order article via Infotrieve]
  27. Dietrich, W., Katz, H., Lincoln, S. E., Shin, H.-S., Friedman, J., Dracopoli, N. C., and Lander, E. S. (1992) Genetics 131, 423-447[Abstract/Free Full Text]
  28. Green, E. L. (1981) Genetics and Probability in Animal Breeding Experiments., Oxford University Press, New York
  29. Feng, J. Q., Chen, D., Cooney, A. J., Tsai, M.-J., Harris, M. A., Tsai, S. Y., Feng, M., Mundy, G. R., and Harris, S. E. (1995) J. Biol. Chem. 270, 28364-28373[Abstract/Free Full Text]
  30. Breathnch, R., Benoist, D., O'Hare, K., Gannon, F., and Chambon, P. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4853-4857[Abstract]
  31. Satokata, I., and Maas, R. (1994) Nat. Genet. 6, 348-356[Medline] [Order article via Infotrieve]
  32. Janknecht, R., and Hunter, T. (1997) J. Biol. Chem. 272, 4219-4224[Abstract/Free Full Text]
  33. Kuzuoka, M., Takahashi, T., Guron, C., and Raghow, R. (1994) Genomics 21, 85-91[CrossRef][Medline] [Order article via Infotrieve]
  34. Uchida, T., Murakami, C., Wakida, K., Dohi, N., Iwai, Y., Simmer, J. P., Fukae, M., Satoda, T., and Takahashi, O. (1997) Eur. J. Oral Sci. 105, in press
  35. Hu, C.-C., Simmer, J. P., Bartlett, J. D., Qian, Q., Zhang, C., Ryu, O. H., Xue, J., Fukae, M., Uchida, T., and MacDougall, M. J. (1998) Connect. Tissue Res., in press
  36. Uchida, T., Tanabe, T., Fukae, M., Shimizu, M., Yamada, M., Miake, K., and Kobayashi, S. (1991) Histochemistry 96, 129-138[Medline] [Order article via Infotrieve]
  37. Kozak, C. A., and Stephenson, D. (1997) Mamm. Genome 7, S80-S99[CrossRef][Medline] [Order article via Infotrieve]
  38. MacDougall, M., Simmons, D., Luan, X., Gu, T. T., and DuPont, B. R. (1998) Cytogenet. Cell Genet., in press
  39. MacDougall, M. (1997) Eur. J. Oral Sci. 105, in press


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