Effect of methylation on transcription factor binding to the <i>Ins2</i> promoter.

<p>(A) Increasing amounts of NIT-1 nuclear extract was bound to methylated or unmethylated mouse <i>Ins2</i> promoter CRE oligonucleotide in an Electrophoretic Mobility Shift Assay (EMSA). There was a dose-dependent increase in binding to both the methylated and unmethylated CRE with increasing amounts of NIT-1 cell nuclear extract. (B) The binding of NIT-1 nuclear proteins to one nanomolar of either labeled methylated (mCRE) or unmethylated (CRE) was competed with increasing concentrations of unlabeled CRE or mCRE. The figure is one representative of four experiments. (C) Chromatin immunoprecipitation (ChIP) analysis of the endogenous mouse <i>Ins2</i> gene promoter in NIT-1 cells. Genomic DNA was immunoprecipitated with anti-CREB, anti-ATF2, anti-MECP2, and control IgG. The results indicate that CREB and ATF2, but not MeCP2, are normally associated with the endogenous insulin promoter. (D) Methylated or mock-methylated pGL4.10<i>Ins2</i>500bp (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006953#pone-0006953-g003" target="_blank">Fig. 2A</a>) was transfected into NIT-1 cells and then ChIP was carried out with anti-CREB, anti-ATF2, and anti-MeCP2 antibodies. Methylation of the <i>Ins2</i> promoter decreases the binding of CREB and ATF2, but increases the binding of MeCP2.