Figure 4.

Dose-dependent repression of BORIS promoter activity by DNA methylation. The BORIS 5' promoter region insert was methylated in vitro with no enzyme (mock), HpaII and HhaI, or SssI methylases, then was re-ligated into pGL3-Basic and transfected into cell lines. (A) Confirmation of the methylation status of the BORIS promoter. Following in vitro methylation reactions, BORIS promoter fragments were digested with either HpaII (a methylation-sensitive restriction enzyme) or McrBC (a methylation-specific restriction enzyme). (B) Dual luciferase activity assay measurement of differentially methylated BORIS promoter constructs following transfection into 293, RKO, and HCT116 cell lines. The mean values from triplicate data points are plotted, along with error bars corresponding to 1 SD. Data from each cell line are normalized to the mock-methylated control of the same cell line. (C) Activity of BORIS promoter constructs following transfection into wild-type and DNMT1-/-, 3b-/- HCT116 cells. To illustrate differences in basal activity of the mock methylated constructs in the two cell lines, luciferase values were not normalized to mock-methylated control samples in this instance.