FIG. 3.

Runx2 occupancy of MMP9 promoter. (A) Nuclear extracts were prepared from ROS 17/2.8 and primary rat osteoblasts (ROB) (day 12 [matrix maturation stage] and day 20 [mineralization]). An oligonucleotide containing the Runx binding element from the mouse MMP9 promoter (−212 bp to −246 bp) was incubated with 4 μg (lanes 3, 5, and 8) or 8 μg (lanes 6 and 9) of nuclear extract. The open arrow represents the specific DNA-protein complex, and the filled arrow shows a supershift with 1 μl of Runx2 polyclonal antibody (lanes 4, 7, and 10). (B) Competition assay performed with increasing amounts (25-, 50-, and 100-fold molar excess) of either cold wild-type (WT) or Runx mutant (mt) oligonucleotide showing the specificity of the Runx2-DNA complex. (C) In vivo occupancy of Runx2 protein at the proximal MMP9 promoter, as shown by ChIP of nuclei from day 12 ROB cells. The primers used to amplify the key regulatory elements present in the proximal MMP9 promoter fragment in the ChIP assays are indicated. The arrows indicate the positions of the forward (−380) and reverse (−130) primers. The agarose gel presented is representative of three experiments and shows selective amplification of the MMP9 proximal promoter in the input and Runx2 immunoprecipitate lanes, while IgG did not show any product.