Runx2 Regulates FGF3 Expression Through Direct Binding and Promoter Occupancy

Runx2 is critical for bone development and required for epithelial-mesenchymal interactions that regulate odontogenesis. Tooth development arrest at cap-stage in Runx2-knockout mice. Fibroblast growth factors (FGF) are important regulators of embryonic development and tooth formation. FGF3 plays an essential role during dental mesenchymal-epithelial morphogenesis. FGF3-signaling is impaired in Runx2-null mice, suggesting this may be a downstream-target gene of Runx2. Objective: Define the role of Runx2 in transcriptional-regulation of FGF-3 gene. Methods: Biochemical, molecular, and cellular approaches were used to assess Runx2-mediated transcription of FGF3-gene. Results: We cloned the mouse -1.0kb-FGF3-promoter, containing seven Runx-binding sites, upstream of the luciferase-reporter. To define Runx2 contribution, we also cloned deletion mutants that contained 5, 2 or no Runx-binding sites. We initially tested the basal-activity of the FGF3-promoters in epithelial cells. Removing 180bp of the distal sequences (-0.8kb FGF3-promoter) from the full-length promoter caused 8-fold increased activity, suggesting existence of suppressor-sequences in this region. Additional 450bp deletion resulted in a 2-fold decrease in promoter-activity. This data suggests presence of activator sequences between -0.8kb and -0.4kb. Further removal of 140bp causes significant loss in basal promoter-activity. Similar pattern of basal promoter-activity was noted in mesenchymal cells. To identify Runx2-mediated transcription of the FGF3-pomoter, we performed co-transfection experiments. We observed a robust activation (10-fold) of full-length promoter by Runx2. Runx2-mediated enhanced promoter-activity was also observed with the deletion mutants carrying five and two Runx-binding motifs but not with -0.2kb promoter that lacks any Runx-binding sites. These data suggest that Runx2 activation of FGF3-promoter require Runx-binding motifs. We next confirmed binding of Runx2 with some of the selected Runx-sites by EMSA. In-vivo occupancy of these sites by Runx2 was confirmed by chromatin-immunoprecipitation assay. Finally we established that Runx2 regulates transcription of the endogenous FGF3-gene by utilizing Runx2-reconstitution cell model. Conclusion: Runx2 directly induces transcription of the FGF3-gene.