To elucidate the functional role of the Dlx3 homeodomain protein during early development and epidermal differentiation, we are currently in the process of determining the target genes and the Dlx3 interacting factors necessary to exert the transcription regulatory function. Utilizing a retroviral system we have transduced keratinocytes with a recombinant retrovirus expressing an mRNA encoding FLAG-Dlx3 (tagged with FLAG epitope). Identification of Dlx3 interacting proteins is being performed by affinity chromatography followed by mass spectrometry. Initial studies of transduced cells showed a delayed growth when compared to untransduced cells. A detectable difference in morphology was also noted among the cell lines. Furthermore, biochemical characterization of a mutant form of Dlx3 that in humans is associated with the autosomal dominant Tricho-Dento-Osseous (TDO) syndrome is also being performed. TDO syndrome is characterized by defects in ectodermal derivatives such as hair and teeth and craniofacial bone abnormalities. Recent findings show that Dlx3 is also expressed in the developing bone, and the expression is retained in trabecular bone of the adult skeleton. Stratification of the epidermis commences during embryonic development and is a process that continues to occur throughout the life of the organism. The process entails the outward movement of the proliferative basal cells that are adjacent to the basement membrane toward the surface of the skin. The stratification is concurrent with a Ca++-dependent differentiation process and the layer-specific expression of structural and enzymatic markers, with the basal proliferative cell differentiating first to a spinous cell, then to a granular cell, to ultimately terminally differentiate as a cornified, anucleated cell. A central role in the transduction of Ca++ signals is played by members of the EF-hand containing Ca++-binding proteins. The functionality of the EF-hand motifs may contribute to specificity in the interaction with the target molecules. Thus, it is through the binding of Ca++ by the EF-motifs that these proteins are able to bind or liberate target interacting proteins and in this way modulate their functions. We have identified novel Ca++-binding proteins, which we have termed Scarf and Scarf2, which have homology to Calmodulin. The open reading frames encode proteins that contain conserved EF-hand motifs. Identification of Scarf target proteins is being performed by immunoprecipitation assays followed by mass spectrometry. Identification of target proteins will aid in understanding the role of Scarf during keratinocyte differentiation. Concurrent analysis of the highly homologous gene, Scarf2, has shown that it is closely linked to Scarf in the genomic region of murine chromosome 13. Analysis of the Scarf2 transcript and protein, have determined a highly overlapping pattern of expression between Scarf and Scarf2. Studies are ongoing to determine the functional Ca++-binding ability of Scarf and Scarf2 and how this correlates to their function in vivo.
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