FIGURE 5.

CpG methylation attenuates HIF-2α binding to the MMP13 promoter. ChIP assays were performed using cell lysates of C28/I2 cells transfected with methylated (Meth−) or non-methylated (Meth+) WT −214/+14-bp MMP13 promoter constructs and expression vectors encoding HA-tagged HIF-2α (A) or FLAG-tagged ELF3 (B). At 24 h after transfection, chromatin was cross-linked and enzymatically sheared, and after reverse cross-linking of the DNA-protein complexes, the precleared lysates were incubated with antibodies against HA, FLAG, or normal IgG overnight at 4 °C. The HA-HIF-2α and FLAG-ELF3 binding to the human MMP13 promoter region was analyzed by real time PCR, utilizing PCR primers bracketing the transfected reporter vector boundaries. All data are shown as means ± S.E. and represented as fold-change versus IgG (*, p < 0.05; ***, p < 0.001).