Figure 7.

Loss of LH-induced Klf4 promoter activity by mutation of the ^{−698/−688}Sp1 binding site. (A) Site-directed mutants were prepared from pKlf4-715/luc and were designated M1 (pKlf4Δ^−698/688Sp1/luc), M2 (pKlf4Δ^−660/652Sp1/luc), and M3 (pKlf4Δ^−541/536Sp1/luc) as shown on the upper panel with the mutated region underlined. Preovulatory GCs were transiently transfected with either the wild-type Klf4 luciferase reporter (WT) or mutant versions in which one of the Sp1 elements was mutated (M1, M2, or M3) (1 μg/well). At 24 h after transfection, cells were treated with (closed bar) or without LH (200 ng/mL) (open bar) for 3 h. Cell lysates were assayed for luciferase, expressed as relative light units (RLU), and normalized to Renilla luciferase activity in transfected cells. Cells transfected with wild-type construct, without any treatment, constituted the control group (CT). Values are fold-changes relative to the control, and are means ± SDs of four independent experiments, each performed in triplicate. * p < 0.05 vs. CT; ^† p < 0.05 vs. CT+LH. (B) The Sp1 binding sites in the −715/−500 bp region of the Klf4 promoter is aligned to homologous regions of the mice, rats, and humans. The conserved Sp1 site is boxed with numbers indicating the relative position of the beginning of the Sp1 site.