Figure 6. Effect of methylation of the 5′ upstream region on Sox4 promoter activity.

(A) Sox4/pCpGL harboring the 1.8-kb 5′ upstream region of Sox4 (see Figure 5B) was methylated in vitro using SssI methylase. Methylation of the plasmid was assessed by digesting the unmethylated and methylated plasmids with BstUI and AclI. The methylated plasmids were resistant to digestion (lanes 2 and 4), whereas the unmethylated plasmids (lanes 3 and 5) released expected digestion products. CpG85 is one of only two sites in Sox4/pCpGL that can be digested by AclI. The figure clearly indicates that CpG85 is fully methylated, as evidenced by the absence of the 1.2-kb digestion product from the methylated plasmid (lane 4) when compared with that derived from digestion of the unmethylated plasmid (lane 5). (B) Normalized promoter activities of the methylated and unmethylated plasmids after transfection into murine embryonic maxillary mesenchymal cells are presented. The relative promoter activity of the unmethylated plasmid was set at 100%. The experiment was repeated four times using duplicates and values are presented as mean ± standard deviation (*p < 0.0001).

M: Methylated; Mr: Marker; U: Unmethylated.