FIG 3.

KLF15 and the GR cooperate to stimulate IEtu1 promoter activity. (A) Schematic of IEtu1 promoter and location of the TATA box, TAATGARAT motif, and the two GREs. Numbers in parentheses indicate the genomic location of the first nucleotide of each motif. (B) Schematic of the 280-bp fragment that contains the IEtu1 GREs and a KLF-like motif. This fragment was cloned upstream of the minimal SV40 early promoter in the luciferase vector (pGL3-promoter vector; Promega). (C) Nucleotide sequence of motifs in the IEtu1 GREs and mutations that were prepared. The mutations in GRE1 and GRE2 were previously shown to disrupt transactivation by the GR in transient-transfection studies (13). (D) Neuro-2A cells were treated with 2% stripped fetal calf serum 24 h prior to transfection. Neuro-2A cells were then transfected with the designated IEtu1 GRE plasmid (0.5 μg of DNA) and, where indicated, a plasmid that expresses the mouse GR protein (1.0 μg of DNA) and/or KLF15 (0.5 μg of DNA). To maintain the same amount of DNA in each sample, empty vector was included in certain samples. Designated cultures were then treated with water-soluble DEX (10 μM; Sigma) at 24 h after transfection. At 48 h after transfection, cells were harvested, and protein lysate was subjected to a dual-luciferase assay as described in the Materials and Methods section. Levels of promoter activity in the empty luciferase vector (pGL3-promoter vector) were normalized to a value of 1, and fold activation values for other samples are presented. The results are the average of three independent experiments, and error bars denote the standard errors. The double asterisks denote a significant difference (P < 0.05) between results for IEtu1 GREs relative to those for all mutant constructs following transfection with GR plus KLF15 and treatment with DEX, as determined by the Student t test. The single asterisk denotes a significant difference (P < 0.05) between results for the ΔKLF mutant relative to results for the other mutant constructs (ΔGRE1, ΔGRE1ΔKLF, and Δ2×GREΔKLF) when they are cotransfected with GR plus KLF15 and treated with DEX, as determined by the Student t test.