Figure 4.
Effects of mutation and methylation of the NGFI-A response element on exon 17 GR promoter function. a, In vitro mutation of the exon 17 GR promoter was performed using primers designed for cytosine to adenine conversion within the 5′ CpG and 3′ CpG dinucleotide in the NGFI-A response element. b, NGFI-A binding of the nonmethylated exon 17 GR promoter–luciferase reporter plasmid containing either a wild-type (WT) nonmutated, 5′ CpG mutated or 3′ CpG mutated NGFI-A response element, cotransfected in the absence or presence of an NGFI-A expression plasmid (n = 4 samples per group). Mean ± SEM ROD of exon 17 sequence amplified from NGFI-A immunoprecipitated (IP) cell extract (*p < 0.02; **p < 0.001). Lanes were loaded with non-immunoprecipitated input (I), NGFI-A primary antibody immunoprecipitated (A), or non-immune IgG antibody immunoprecipitated (N) extracts. c, NGFI-A binding, to the methylated exon 17 GR promoter–luciferase reporter plasmid containing either a wild-type nonmutated, 5′ CpG mutated or 3′ CpG mutated NGFI-A response element, cotransfected in the absence or presence of an NGFI-A expression plasmid (n = 4 samples per group). Mean ± SEM ROD of exon 17 sequence amplified from NGFI-A immunoprecipitated cell extract (*p < 0.05; ***p < 0.0001). d, Mean ± SEM of luciferase expression (*p < 0.05; **p < 0.001) from the nonmethylated exon 17 GR promoter–luciferase reporter plasmid containing either a wild-type nonmutated, 5′ CpG mutated or 3′ CpG mutated NGFI-A response element, cotransfected in the absence or presence of an NGFI-A expression plasmid (n = 4 samples per group). e, Mean ± SEM of luciferase expression (**p < 0.001; ***p < 0.0002) from the methylated exon 17 GR promoter–luciferase reporter plasmid containing either a wild-type nonmutated, 5′ CpG mutated or 3′ CpG mutated NGFI-A response element, cotransfected without or with an NGFI-A expression plasmid (n = 4 samples per group).