In vitro methylation and reporter gene assays
The IRF-4 promoter-reporter gene construct was generously provided by J. Hiscott (31). Constructs were methylated in vitro with CpG Methylase (M.Sss I) as recommended by the manufacturer (NE Biolabs) and complete methylation was checked via restriction analysis (Figure 5A). Reporter gene assays using the dual luciferase assay (Promega) were performed similar to previous reports (29). Briefly, 5 nM of the reporter construct and the transfection control construct expressing the renilla luciferase gene were transientlyco-expressed via electroporation. The control construct served as an internal reference for transfection efficiency. Forty-eight hours after transfection, luciferase activity was measured with a LB 96 P microlumat (EG&G Berthold, Bad Wildbad, Germany). IRF-4 promoter activation was quantified as a ratio of measured firefly light units (flu) relative to renilla (rlu). Each experiment was carried out at least three times.