DETERMINATION AND EXPRESSION OF AMELOGENIN GENE PRODUCTS

University Of Southern California, Los Angeles CA

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Abstract

DESCRIPTION (Adapted from the applicant's Abstract): During organ formation, molecular mechanisms operate to establish pattern formation including the determination of cell identity and hence the types of genes expressed in the differentiated state. The developing tooth is a well established model for examining patterning mechanisms. Reciprocal-, instructive-epithelial-mesenchymal interactions result in the expression of growth factors and/or transcription factors used by cells of the ectoderm or the mesoderm to provide signals for cell determination and differentiation. A unique feature of tooth organogenesis is the differentiation of the ectodermally-derived ameloblasts in which a hierarchical cascade of molecular signals culminates in the determination of oral epithelial cells to the ameloblast phenotype. Only differentiated ameloblasts express amelogenin, in a highly regulated spatial-and temporal fashion reflecting pattern information gained during organogenesis. A 2263 nucleotide (nt) piece of DNA from the murine X-chromosome is responsible for sequence specific regulation of amelogenin gene expression. The applicant and his colleagues have engineered mice to express a transgene construct formed by the luciferase reporter gene and the amelogenin promoter. Mice express the transgene in a manner that recapitulates the temporal- and spatial-regulation of the canonical amelogenin gene. This promoter contains the DNA sites that bind factors forming the molecular basis for the cell responding to the hierarchical clues provided during normal tooth development. Enamel organ cell lines have been created that maintain gene expression profiles consistent with the ameloblast phenotype and when transfected with the 2263nt-luciferase construct, high levels of luciferase expression results, suggesting that these cells biosynthesize the factors required for regulating amelogenin gene expression. The factors, and their interactions with DNA, that are responsible for this complex level of amelogenin expression, will be identified. The hypothesis to be tested is that amelogenin gene regulation is dependent upon a cascade of signals provided during instructive secondary germ layer interaction serving to pattern oral epithelium to the ameloblast phenotype. These interactions provide the molecular clues in the biochemical form of transcription factors or growth factors that bind in a sequence specific manner to regulatory domains contained within the amelogenin gene promoter. Specific Aim 1 is to establish the minimal promoter necessary to convey accurate spatial and temporal regulation for amelogenin gene expression. Specific Aim 2 is to identify the factors that bind DNA to activate or repress amelogenin gene expression. Specific Aim 3 will perturb the molecular clues acting as critical components of the hierarchical cascade for amelogenin gene regulation, using gain-, or loss of function transgenic mice and to follow the developmental consequences of these changes in cell determination during odontogenesis.