FIGURE 5.

Glucocorticoid receptor is recruited to a functional GRE located in the intron of the KLF13 gene in vitro and in vivo. A, schematic representation of the KLF13 gene. Sequence alignment of the consensus GRE and GRE identified within the KLF13 intron of the mouse and human genes. HL-1 cells were treated with vehicle-control (white bars) or 100 nm Dex (black bars) for 3 h, and ChIP assays were performed with equivalent amounts of rabbit IgG or rabbit anti-GR antibody. Coimmunoprecipitated DNA was analyzed by RT-PCR using primers to the KLF13 promoter, the KLF13 intron (Int-KLF13) and a functional GRE previously identified in the promoter of the glucocorticoid-regulated gene leucine zipper (GILZ) (left panel). Adrenalectomized C57BL/6 male mice were injected with vehicle (Veh, white bars) or 1 mg/kg of Dex (black bars). Six h after this treatment hearts were harvested. Coimmunoprecipitated DNA was analyzed by RT-PCR using primers to the KLF13 intron (Int-KLF13) and GILZ promoter GRE (right panel). Results are plotted as a function of input DNA. B and C, HL-1 cells were transfected with the following luciferase reporter plasmids: control vector (empty vector), KLF13 promoter, intron-KLF13 (Int-KLF13), and mutant-KLF13 (Mut-KLF13). Twenty-four h after transfection cells were treated for 18 h with vehicle control (white bars), 100 nm Dex (black bars), 1 μm RU486 (striped bars) or both 1 μm RU486 and 100 nm Dex (cross-hatched bars). Cells were harvested and luciferase activity was measured. Data represent ± S.E. for four independent experiments and n = 5–10 mice per group. *, p < 0.05; **, p < 0.01.