X-chromosomal Amelogenin Gene Research Articles

Physical dissection of the CCAAT/enhancer-binding protein α in regulating the mouse amelogenin gene

The amelogenin gene is tightly regulated at the temporal and spatial level in accord with the developmental requirement for tooth formation. Previous studies have shown that CCAAT/enhancer-binding protein α (C/EBPα) is a transactivator of the mouse X-chromosomal amelogenin gene. C/EBPα contains four highly conserved regions (CR) named CR1, CR2, CR3, and CR4. Transient transfection assays showed that CR2 in isolation had an exceptional capacity to enhance transcription from the 2.3 kb mouse amelogenin promoter. The remaining conserved regions of C/EBPα, either in isolation or in selected combinations, were less effective in amelogenin transactivation than the full length C/EBPα. Msx2 has previously been shown to antagonize C/EBPα through protein–protein interactions with C/EBPα, and the carboxyl-terminus of Msx2 is required for protein–protein interactions. Co-immunoprecipitation analyses identified that the carboxyl-terminal domain (residues 218–359) of C/EBPα is required for the C/EBPα–Msx2 protein–protein interactions.

NF-Y and CCAAT/Enhancer-binding Protein α Synergistically Activate the Mouse Amelogenin Gene

Amelogenin is the major protein component of the forming enamel matrix. In situ hybridization revealed a periodicity for amelogenin mRNA hybridization signals ranging from low to high transcript abundance on serial sections of developing mouse teeth. This in vivo observation led us to examine the amelogenin promoter for the activity of transcription factor(s) that account for this expression aspect of the regulation for the amelogenin gene. We have previously shown that CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a potent transactivator of the mouse X-chromosomal amelogenin gene acting at the C/EBPalpha cis-element located in the -70/+52 minimal promoter. The minimal promoter contains a reversed CCAAT box (-58/-54) that is four base pairs downstream from the C/EBPalpha binding site. Similar to the C/EBPalpha binding site, the integrity of the reversed CCAAT box is also required for maintaining the activity of the basal promoter. We therefore focused on transcription factors that interact with the reversed CCAAT box. Using electrophoretic mobility shift assays we demonstrated that NF-Y was directly bound to this reversed CCAAT site. Co-transfection of C/EBPalpha and NF-Y synergistically increased the promoter activity. In contrast, increased expression of NF-Y alone had only marginal effects on the promoter. A dominant-negative DNA binding-deficient NF-Y mutant (NF-YAm29) dramatically decreased the promoter activity both in the absence or presence of exogenous expression of C/EBPalpha. We identified protein-protein interactions between C/EBPalpha and NF-Y by a co-immunoprecipitation analysis. These results suggest that C/EBPalpha and NF-Y synergistically activate the mouse amelogenin gene and can contribute to its physiological regulation during amelogenesis.

X-linked amelogenesis imperfecta may result from decreased formation of tyrosine rich amelogenin peptide (TRAP)

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine for an adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N-terminal to the cleavage site for enamel matrix metalloproteinase-20 (MMP-20) in amelogenin. MMP-20 releases the tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate at which MMP-20 hydrolyses mutated amelogenin relative to unmutated amelogenin. A full-length recombinant human amelogenin and a mutated amelogenin with a substitution of proline by threonine were expressed and purified by ammonium sulphate precipitation and reverse phase HPLC. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the recombinant proteins, which resulted in fragments with a mass predicted for TRAP. The proteolytic site was also modelled as substrates by two synthetic peptides, SYGYEPMGGWLHHQ and SYGYETMGGWLHHQ, selected from residues 36 to 49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. These two peptides were labelled at their N- and C-termini respectively by using rhodamine and biotin. After digestion with MMP-20, the truncated peptides were separated by avidin-labelled magnetic Dynal beads and were identified by mass spectrometry. These results demonstrated that both oligopeptides were cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. Enzyme kinetics showed that the k cat/ K m of rMMP-20 against the unmutated amelogenin peptide was 21 times greater than that against the mutated peptide. This study suggests that the reduced rate of TRAP formation by a single amino acid substitution alters enamel matrix hydrolysis by MMP-20, which may result in amelogenesis imperfecta.

Ultrastructure of Forming Enamel in Mouse Bearing a Transgene That Disrupts the Amelogenin Self-Assembly Domains

The mouse X-chromosomal amelogenin gene promoter was used to drive the expression of mutated amelogenin proteins in vivo. Two different transgenic mouse lines based on deletions to either the amino-terminal (A-domain deletions) or to the carboxyl-region (B-domain deletions) were bred. In the molars of newborn A-domain deleted transgenic mice the formation of the initial layer of aprismatic enamel was delayed. There were severe structural alterations in the enamel of incisors of newborn mice bearing the A-domain deletion which were not apparent in animals bearing the B-domain deletion. In the A-domain-deleted animals, stippled material accumulated throughout the entire thickness of the forming enamel apparently causing a disruption of the normal rod-to-inter-rod relationship. This stippled material was likened to and interpreted as being groupings of amelogenin nanospheres. In the B-domain-deleted animals the stippled material was detected only in minute defects of the forming enamel. These data suggest significant differences in nanosphere assembly properties for animals bearing either the A-domain or the B-domain-deleted transgene. The present in vivo experimental approach suggests that at early stages of enamel formation, the A-domain plays a greater role than does the B-domain in amelogenin self-assembly, and consequently in enamel architecture and structure.

Amelogenin-deficient Mice Display an Amelogenesis Imperfecta Phenotype

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

Functional Antagonism between Msx2 and CCAAT/Enhancer-binding Protein α in Regulating the Mouse Amelogenin Gene Expression Is Mediated by Protein-Protein Interaction

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBPalpha) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBPalpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBPalpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBPalpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBPalpha. Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression.

Mutational analysis of X-linked amelogenesis imperfecta in multiple families

Seven mutations in the amelogenin gene are associated with X-linked amelogenesis imperfecta. These mutations can produce reductions in the amount of enamel and the degree of mineralization. Two families have been identified from western North Carolina exhibiting features of amelogenesis imperfecta, characterized by brown enamel in affected males and interposed vertical bands of normal appearing and brown enamel in presumably heterozygous females. Mutational analysis reveals a C–A mutation in exon 6 at codon 41 of the X-chromosomal amelogenin gene, resulting in a pro–thr change in all individuals having the amelogenesis imperfecta phenotype. This mutation was previously reported in a family with X-linked hypomaturation amelogenesis imperfecta. There is no known relationship between any of the three families but the presence of similar phenotypes and common mutations suggests they may be distantly related. For individuals from all three families, the haplotype for six highly polymorphic loci flanking the amelogenin gene was determined. A common haplotype was demonstrated among two of the three families, suggesting that the mutation may have been inherited from a common ancestor. The finding that the third family had a distinct haplotype may indicate that the C–A mutation at codon 41 represents a mutational hotspot that occurs with greater frequency than other known amelogenin gene mutations. The phenotype resulting from this mutation was highly consistent in affected male members of the same family and between families.

Regulation of Amelogenin Gene Expression

The X-chromosomal amelogenin gene is expressed at a high level by ameloblast cells within the enamel organ for a short time during tooth development. Therefore, expression is both tooth specific and developmentally regulated. A Y-chromosomal amelogenin gene is also active in human and cow, but has not been detected in mouse. Genes and/or cDNAs have been cloned for mouse, human, cow, rat, pig, opossum, and hamster, and analyses have indicated that coding and upstream regions are conserved across species. Alternative splicing is extensive and produces as many as 9 mRNAs from the 7 exon murine gene, resulting from single and multiple exon skipping and alternate 3' site selection within exon 6. The pattern of alternative splicing varies both between species and during development, which is expected to result in some diversity through varying complements of amelogenin proteins associated with this highly conserved gene.

An Amelogenin Gene Mutation Associated with X-linked Amelogenesis Imperfecta

A kindred was described with X-linked hypo-maturation Amelogenesis Imperfecta, in which the teeth of the propositus had mottled yellowish white enamel and his mother had vertical bands of opaque white and translucent enamel (Sauk et al., Am. J. Hum. Genet., 24, 267, 1972). Members of this family were contacted to obtain blood samples, in order to search for a mutation within the coding region of the X-chromosomal amelogenin gene. Primers were designed to amplify by the polymerase chain reaction (PCR) the coding regions: PCR products were sequenced directly, or cloned and sequenced. A point mutation was identified in the sequence encoding the 8th amino acid (pro > thr) of exon-6 of three affected family members. Two unaffected sisters of the propositus each had the normal DNA sequence as described by Salido et al. (Am. J. Hum. Genet., 50, 303, 1992). This proline is conserved in human X and Y chromosomal amelogenin genes, as well as in cow, pig, mouse, rat and opossum amelogenins, and is located within a highly conserved region of the protein. This mutation is significant in that it localizes a domain in the amelogenin protein predicted to be required for normal enamel formation. This study was supported by NIH grants DE10149 (CWG) and DE08239 (JR).

An amelogenin gene defect associated with human X-linked amelogenesis imperfecta

Dental enamel is a product of ameloblast cells, which secrete a mineralizing organic matrix, composed primarily of amelogenin proteins. The amelogenins are thought to be crucial for development of normal, highly mineralized enamel. The X-chromosomal amelogenin gene is a candidate gene for those cases of amelogenesis imperfecta, resulting in defective enamel, in which inheritance is X-linked. In this report, a kindred is described that has a C to A mutation resulting in a pro to thr change in exon 6 of the X-chromosomal amelogenin gene in three affected individuals, a change not found in unaffected members of the kindred. The proline that is changed by the mutation is conserved in amelogenin genes from all species examined to date.

Analysis of the regulatory region of the bovine X-chromosomal amelogenin gene.

The amelogenin proteins, which are crucial for normal enamel mineral formation, are secreted by ameloblasts during development of tooth enamel. In order to better understand the mechanisms involved in regulation of expression of the amelogenin genes, the bovine X-chromosomal amelogenin gene was cloned and a 3.5 KB fragment upstream of exon 1 was inserted into a beta galactosidase (beta gal) expression vector for production of transgenic mice. When tissues from these mice were treated with Xgal, a substrate for beta gal, only ameloblasts and some of the adjacent stratum intermedium cells contained blue stain. To obtain further information concerning regulation of expression, the 3.5 KB amelogenin gene fragment was evaluated in transfection experiments. Nonoverlapping 1.9 and 1.5 KB fragments of the upstream region were subcloned separately into a vector that contains the SV40 promoter and the CAT reporter gene. Each amelogenin gene fragment was able to suppress CAT activity driven by the heterologous SV40 promoter in transfected HeLa cells. We theorize that each of these gene fragments contains regulatory elements important for the tissue-specific and developmentally-regulated pattern of expression of the X-chromosomal amelogenin gene.

Regulation of amelogenin gene expression during tooth development

The amelogenins are the predominant matrix proteins in developing enamel and are crucial for proper enamel mineralization. Transgenic mice were constructed in order to identify the segment of the amelogenin gene required for specific expression in enamel organ cells. A 3.5 kb fragment of the bovine X-chromosomal amelogenin gene that includes a TATA box, the transcription initiation site, and 32 bp of exon 1 was linked to the beta galactosidase gene and injected into fertilized mouse eggs. Newborn transgene positive mice expressed beta galactosidase activity in developing teeth treated with the chromogenic substrate Xgal. Foci of ameloblasts were positive in newborn mice; stain intensity and number of positive ameloblasts increased in 1-day and 2-day postnatal mice. Some of the adjacent stratum intermedium cells also were positive in the later stages. Targeting of the transgene to the enamel organ was specific; the only other cells observed to be positive were macrophages, which have endogenous beta galactosidase activity.