contains a number of canonical and noncanonical NGFI-A recognition elements (Fig. 3a). We wondered whether, as in the rat17, NGFI-A could regulate gene transcription through the NR3C1 promoter and whether this effect might be influenced by the methylation status of the promoter. We used a transient transfection assay in human HEK293 cells to examine transcriptional activity of a NR3C1 promoter ligated to a promoter-less firefly luciferase expression vector (pGEM-LUC, Promega; Fig. 3a) in the presence or absence of ectopic NGFI-A expression. The use of HEK293 cells allowed us to concurrently transfect a number of expression vectors with high efficiency. The absence of plasmid replication during the transient transfection assay precludes the loss of methylation via passive demethylation27.
