The effect of methylation on the transcriptional activity of the erythropoietin promoter and 5′-UTR.

The minimal inducible erythropoietin promoter (P117) was methylated or mock methylated, then ligated into unmethylated pGL2 (pGL2-P117) or pGL2 with the unmethylated erythropoietin enhancer (pGL2-P117+E126). The 5′-UTR was methylated or mock methylated and ligated in its natural position into unmethylated pGL2-P117+E126, creating pGL2-P117 + 5′-UTR+E126. Reporter vectors were introduced into Hep3B cells, and luciferase assays were performed. Relative light units are expressed as raw light units divided by mU/min of β-galactosidase activity. (error bars) ±1 SD. (A) pGL2-P117 (n = 4). (B) pGL2-P117+E126 (n = 4). (C) pGL2-P117 + 5′-UTR+E126 (n = 5). U, unmethylated; M, methylated; Nox, normoxia; Hyp, hypoxia.