Figure 6

Non-promoter association of Runx2 regulates novel targets Adamts4 and Crabp2 . (A) Increasing Runx2 enrichment was observed in the first intron (peak A, boxed region) and last exon (peak B, boxed region) of the Adamts4 locus during osteogenic differentiation. (B) Adamts4 mRNA levels (normalized to Hprt1) were significantly upregulated (P < 0.05) in differentiating MC3T3-E1 cells (days 9 to 28) when compared to proliferating cells (day 0). (C) Runx2 knockdown (shRunx2) decreases Adamts4 expression, compared to a scrambled shRNA (Scr) control (**P < 0.05). (D) DNA sequences identical to peak regions A and B were cloned into individual pGL2-SV40-Luc reporters and relative luciferase activity was measured in transfected MC3T3-E1 cells and significant increases and decreases (*P < 0.01) in luciferase activity were observed for peak A and B reporters, respectively. (E) Increasing Runx2 enrichment was observed up- (peak C) and downstream (peaks D and E) of the Crabp2 locus during osteogenic differentiation. (F) Crabp2 expression increases during differentiation. Knockdown of Runx2 (by shRunx2) significantly reduces (**P < 0.05) the endogenous expression of Crabp2 (right panel). (G) DNA sequences identical to peak regions C, D and E were cloned into individual pGL2-SV40-Luc reporters and relative luciferase activity was measured in transfected MC3T3-E1 cells at days 0 and 7 (*P < 0.01). (H) Runx2 knockdown by inducible shRNA results in a significant decrease (**P < 0.05) of luciferase activity mediated by peak C and E regions and a downward trend (P = 0.08) in luciferase activity regulated by peak D region. Statistical significance for all experiments was determined by Student’s t-test from mean ± SEM from three biological replicates.