Regulation of Enamelin Tissue-specific Expression

Enamelin is an extracellular enamel protein with unique functional roles during enamel formation. Enamelin expression initiates in pre-ameloblasts, continues throughout the secretory stage and ends in the beginning of the maturation stage. It is not expressed in other cell types. This restricted spatial and temporal pattern of expression is critical for the completion of amelogenesis, and perturbation of this expression results in abnormal enamel in mice and humans. Objectives: To characterize the enamelin 5' cis-regulatory promoter region and to identify trans-acting factors that control tissue-specific expression of enamelin. Methods: DNA constructs covering different parts of the enamelin 5' regulatory region have been generated by PCR and subcloned into a LacZ or Luc reporter gene vector for generation of Enam-LacZ transgenic mice and in vitro promoter assays, respectively. The final constructs were verified by DNA sequencing and tested for promoter activity in vitro using LS8 ameloblast-like cells (Malcolm Snead, University of Southern California). Sequence analysis, transient transfection experiments and gel shift assays were undertaken to elucidate the role of potential trans-acting factors on enamelin expression. The developmental pattern of transcription factors that may control enamelin expression was also investigated by immunohistochemistry. Results: Analysis of enamelin promoter driven reporter activity in vivo and in vitro resulted in identification of regions of the Enam promoter that may control tissue-specific expression. Sequence analysis and transfection assays suggested that Runx2 and Dlx/Msx transcription factors might play a role in regulating enamelin expression. Dlx3 in particular was expressed in secretory ameloblasts concomitant with enamelin expression. Conclusion: Our study provides novel insights into factors influencing the developmental and tissue-specific expression of enamelin within the enamel-forming ameloblasts. This study is supported by NIDCR grant DE011301.