1247 Runx2 Regulates FGF3 Expression Through Direct Binding and Promoter Occupancy

Saturday, March 24, 2012: 9:45 a.m. - 11 a.m.
Presentation Type: Poster Session
M.F. MORELAND1, H. RASHID1, H. CHEN1, A. SAROF1, S. GUTIERREZ2, and A. JAVED1, 1Oral and Maxillofacial Surgery, University of Alabama, Birmingham, AL, 2University of Concepcion, Concepcion, Chile
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.
This abstract is based on research that was funded entirely or partially by an outside source: R01AG030228 T32DE017607

Keywords: Cell biology, Gene expression, Mineralization, Molecular biology and Oral biology