Identification of Pro-Hyp response element in the proximal region of the Runx2 distal P1 promoter via Foxg1 and Foxo1
Schematic illustration of the luciferase expression vectors containing different regions of the Runx2 distal P1 promoter (A). Boldface nucleotides represent the wildtype Runx2-binding sequence (WT). The solid underlined region represents the wildtype Fox consensus sequence (WT). The uppercase nucleotides represent the mutations (MT) introduced into the pGL3 control vector. The MC3T3-E1 cells were transfected with 0.1 µg of pGL3-Runx2 distal P1 promoter DNA and 1 ng pNL DNA. After 48 h, the cells were treated with Pro-Hyp for 48 h, as indicated (B–F). Luciferase activity was measured and normalized to the activity of NanoLuc luciferase. Luciferase assay was used to identify the Pro-Hyp response element in the proximal region of the Runx2 distal P1 promoter (B). The effect of the mutations of the Fox core sequence in the proximal region of the Runx2 distal P1 promoter on the induction of Runx2 distal P1 promoter activity by Pro-Hyp was tested (C,D). The effect of the mutations in the proximal region of the Runx2 distal P1 promoter on the promotion of the Runx2 distal P1 promoter activity by Pro-Hyp was tested (E,F). Significance was calculated using the Student’s t test (B) and Tukey’s post hoc test (C–F); the mean error bars represent the standard deviation. For clarity, not all the significant differences are indicated. Each data point is the mean ± S.D. of four independent assays. *P<0.05.