We confirmed that DNA methylation inhibits the ability of NGFIA to activate the exon 17 promoter using a transient cotransfection assay in HEK293 cells. The HEK293 cells are not of neural origin and thus allow us to measure the transcriptional consequences of interaction of NGFIA with either a methylated or nonmethylated version of the GR exon 17 promoter per se, independent of the complications associated with other neuronal signals. We used transfection technology to introduce into the HEK cells (i) a viral vector containing the NGFIA gene, to produce a intracellular signal usually inactive in HEK cells; and (ii) an exon 17-luciferase reporter construct. This genomic construct that included the exon 17 promoter sequence fused with a luciferase reporter gene (the level of the easily measured luciferase activity is used as a measure of exon 17 promoter activity). Cotransfection of the NGFIA expression vector significantly increases luciferase activity; however, this effect is dramatically reduced if the CpG dinucleotides within the exon 17 sequence are methylated. Moreover, the effect of NGFIA on transcription through an exon 17-luciferase reporter construct was almost
