To test the hypothesis that MBD2 is associated with maternally induced demethylation, we performed an in situ hybridization assay with probes for the mRNAs of a number of methylated binding proteins at day 6 postpartum. Our analysis revealed that MBD2/demethylase expression is elevated in the hippocampus at this point in time in offspring of high-LG versus low-LG mothers. A ChIP analysis with an antiMBD2/demethylase antibody demonstrates significantly increased binding of MBD2/demethylase to the exon 17 GR promoter in day6 offspring of high-LG versus low-LG mothers. We also found increased NGFIA binding to the same sequence in day-6 offspring of high-LG offspring. We then performed a NaBis mapping of the state of methylation of the exon 17 GR promoter bound to MBD2 and precipitated in the ChIP assay with antiMBD2 antibody. If MBD2 is the demethylase involved in this process or if it is part of the demethylase complex, then MBD2-bound exon 17 sequences at day 6 should be found in the process of demethylation. Indeed, most of the MBD2bound DNA was unmethylated or partially unmethylated.
