Methylation suppresses the activity of both the mouse and human insulin gene promoters.

<p>(A) A diagram of the three luciferase reporter plasmids used to test the effect of methylation on insulin promoter activity. PGL4.10<i>Ins2</i>500bp contains bases −480 to +15 (three CpG sites) of the <i>Ins2</i> gene, pGL4.10<i>Ins2</i>MP contains bases −169– +15 bp (no CpG sites), and, pGL4.10<i>INS</i>421bp contains bases −385 to +24 of the human <i>INS</i> gene (nine CpG sites). The positions of CpG dinucleotides are represented with “lollipop” markers and the positions of primers for the ChIP experiments by arrows. (B) Methylated (white bar) or mock-methylated (black bar) human <i>INS</i> promoter constructs were transfected into NIT-1 insulinoma cells 48 hours prior to the luciferase assay. The data were normalized with co-transfected control vector and are the average and standard error from three separate experiments of eight replicates each. Data are presented as relative expression compared with the non-methylated, non-stimulated sample. * = p<0.0001 (C) Methylated (white bars) or mock-methylated (black bars) mouse <i>Ins2</i> promoter constructs were transfected into NIT-1 insulinoma cells and stimulated for four hours with 2.8 mM glucose (Stimulation -) or with 16.7 mM glucose and 10 µM forskolin (Stimulation +). The data were scaled to the unmethylated, unstimulated control. The results are the average and standard error from three separate experiments of eight replicates each. * = p<0.0001 compared with control. (D) The minimal <i>Ins2</i> promoter construct was treated and analyzed as in 1C. There was no significant effect of methylation or stimulation on minimal promoter activity (one-way ANOVA p = 0.9928). (E) The mouse <i>Ins2</i> promoter construct was methylated individually at each CpG site (white bars) or mock-methylated (black bar) and then transfected and analyzed as in 1B. The results are the average and standard error from three to five experiments of eight replicates each. * = p<0.0001 compared with control.