Figure 7. Proximal promoter activity repressed by an increased DNA methylation level.

(a) Treatment of four luciferase constructs with methylase SssI induced a significant reduction of MUPCDH promoter activity in vitro. The luciferase activity following SssI methylase treatment is shown as a percentage of that of untreated constructs. (b) The luciferase activity of pGL3-19 wild-type and mutant constructs was assayed following treatment with SssI methylase. The ratio of Renilla luciferase to Firefly luciferase was calculated for each experiment and the values from triplicate experiments were averaged. The mean values of each test construct were normalized to the activity of the empty vector. (c,d) Chromatin immunoprecipitation-quantitative reverse transcription polymerase chain reaction was used to assay for RNA polymerase II, AP-2α, and histone modification marks in untreated and 5-aza-2′-deoxycytidine-treated (5 μM) WT9-7 cystic epithelial cells. Bars represent the mean of the normalized values with error bars indicating the range. Each experiment was performed in triplicate. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.