Figure 5. Runx2 Regulates the Lef1 p2 Promoter.

A. Schematic of the 5' portion of murine Lef1. Wildtype (wt) and a mutant (mt) p2 promoter wherein the Runx2 site is eliminated were cloned into the pGL2 luciferase plasmid and used in D–E. Transcriptional and translational start sites are indicated by the thin and thick arrows, respectively. B–C. Runx2 binds a consensus-binding element in the Lef1 p2 promoter as determined by EMSA (B) and chromatin immunoprecipitation (C). D. The p2 promoter (−447-+35) is active in C3H10T1/2 cells when placed in the forward (F) but not reverse (R) orientation. E. Runx2 activates the p2 promoter in a concentration-dependent manner in C3H10T1/2 cells. F. Mutating the Runx2 binding element prevents Runx2-dependent activation of the p2 promoter. G. Primary osteoblasts derived from wildtype (WT) and Runx2^−/− mice were differentiated in osteogenic media for 34 days. RNA was collected for quantitative PCR. All data were normalized to murine actin. Lef1ΔN transcript levels were determined by differential quantitative real-time PCR as described in Figure 2.