To test the hypothesis that NGFIA targets demethylation to GR exon 17 promoter we resorted to nonneuronal cell line HEK293. Here, we can isolate the direct effect of NGFIA from other neuron-specific events that might confound the interpretation of data from the hippocampal cultures. By comparing the fate of a transiently transfected methylated GR exon 17 promoter-luciferase vector in the presence and absence of NGFIA we could better determine the specific effects of NGFIA on demethylation. Whereas in vitro methylated GR exon 17 promoter-luciferase vector remains methylated in HEK293 cells, coexpression of NGFIA results in active demethylation of a significant fraction of the transf ected plasmids. To demonstrate that this DNA demethylation requires direct contact between NGFIA and its recognition element, we performed site-directed mutagenesis of the two CGs included in the NGFIA recognition element. Our preliminary results suggest that these manipulations abolished the ability of NGFIA to activate and demethylate the GR exon 17 promoter. These experiments provide a molecular mechanism on how demethylation is triggered to specific sequences. The outstanding question is to determine how NGFIA triggers demethylation
