The developing mammalian dentition provides a valuable model system for investigating specific gene regulation, morphogenesis and mineralization. Ameloblastin, a recently cloned tooth-specific gene, displays a unique and specific developmental expression pattern in the enamel organ epithelium of developing teeth. The applicant has previously described the full-length cDNA sequence, localization, and chromosomal mapping of rat ameloblastin. In humans, ameloblastin maps to chromosome 4q21 in a locus that is linked to an autosomal dominant form of amelogenesis imperfecta. Thus, ameloblastin is a candidate gene for an inherited defect in humans. The long term goals of this project are to isolate and characterize the regulatory elements that direct cell-specific expression of ameloblastin, and to study the role of ameloblastin in enamel formation. These objectives are based on the hypothesis that cell-specific expression of ameloblastin depends on unique cis and trans-acting elements that function to specify tooth organogenesis and that ameloblastin is an integral factor in the synthesis and function of enamel. Murine genomic clones have been isolated and will be sequenced. The 5'-flanking DNA region will be ligated to reporter gene constructs for the identification and characterization of promoter elements that direct transcription of ameloblastin in an ameloblast-like cell line. Because few well characterized ameloblast cell lines exist, new methods to analyze gene function in teeth will be developed. Adenovirus-mediated infections will deliver ameloblastin promoter constructs to molar tooth germ organ cultures. Initial studies will identify DNA elements in the promoter that confer basal transcription activity by investigating the activity of 5'-deletion mutants in a functional assay. Once DNA loci are identified, the function of these putative regulatory regions will be confirmed using oligonucleotide mutagenesis of the positive constructs. DNA-protein interactions will be analyzed by mobility shift assays in cells, and by in vivo footprinting in organ cultures. The molecular studies on the nature of the regulatory elements of the ameloblastin gene will provide the tools necessary to investigate fundamental questions regarding the biology of the gene product. The function of ameloblastin in enamel formation will be probed by loss of function studies in organ culture and by analysis of in vitro hydroxyapatite binding properties. These studies will help define the genetic hierarchies involved in amelogenesis, will lead to a better understanding of the role of ameloblastin in the pathophysiology of enamel dysplasias, and contribute towards the basic understanding of cell-specific transcriptional mechanisms.
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