Figure 3.

DNA methylation affects NGFI-A binding to and activation of an exon 1₇ GR promoter–luciferase reporter construct. a, Physical map of the exon 1₇ GR promoter–luciferase reporter construct. b, Physical map of the pEF6/V5–His–NGFI-A expression vector. c, Western blot analysis of NGFI-A expression in HEK 293 cells 72 h after transfection with the pEF6/NGFI-A/V5–His plasmid. The top shows the level of V5 immunoreactivity with the ectopically expressed NGFI-A protein. The middle shows the endogenous and exogenous levels of NGFI-A immunoreactivity. Because of the strong affinity of the NGFI-A antibody for the ectopically expressed NGFI-A protein, the signal for the endogenous NGFI-A protein could not be detected on the same film. Therefore, although the samples were run on and transferred from the same gel, the nitrocellulose membrane was cut so that one piece contained the untransfected and V5 empty vector transfected samples, and the other piece contained the sample transfected with V5 vector expressing NGFI-A. To detect a signal for the endogenous NGFI-A protein, the membrane was exposed to film for 10 min, whereas to detect the exogenous NGFI-A protein, the membrane was exposed to film for <20 s. The bottom shows the level of β-actin immunoreactivity to control for equal loading. d, Effects of ectopic NGFI-A expression on exon 1₇ GR promoter function. Mean ± SEM of luciferase expression (**p < 0.001) from the nonmethylated and methylated exon 1₇ GR promoter–luciferase reporter plasmid cotransfected without or with an NGFI-A expression plasmid (n = 4 samples per group). NGFI-A binding to either the nonmethylated or methylated exon 1₇ GR promoter–luciferase reporter plasmid cotransfected without or with an NGFI-A expression plasmid in HEK 293 cells was determined by ChIP analysis (n = 4 samples per group). Lanes were loaded with non-immunoprecipitated input (I), NGFI-A primary antibody immunoprecipitated (A), or non-immune IgG antibody immunoprecipitated (N) extracts. e, Representative Southern blots of the amplified exon 1₇ GR promoter region from NGFI-A immunoprecipitated (IP) HEK 293 cells (194 bp band). f, Mean ± SEM ROD of exon 1₇ sequence amplified from NGFI-A immunoprecipitated cell extract normalized to the input values (*p < 0.05; **p < 0.001; n = 4 samples per group).