Figure 4.

Sequential 5′-deletion analysis of the Klf4 promoter. (A) Schematic representing the putative functional elements in the mouse Klf4 promoter within the region −1014/+545 (base pairs are enumerated from the transcriptional start site). Sp1, specificity protein 1; Sp3, specificity protein 3; SF1, steroidogenic factor 1; CREB, cAMP responsive element binding protein; C/EBPβ, CCAAT enhancer binding protein β. The length and region of the deletion constructs that it spans are represented by the arrows. (B) Preovulatory GCs were transfected with pGL2-basic vectors containing −1014/, −715/, −402/, or −126/+545 bp of the mouse Klf4 5′-flanking region (designated as pKlf4-1014/luc, pKlf4-715/luc, pKlf4-402/luc, and pKlf4-126/luc, respectively) (1 μg). At 24 h after transfection, cells were treated with or without LH (200 ng/mL) for 3 h. Cell lysates were assayed for luciferase, expressed as relative light units (RLU), and normalized to Renilla luciferase activity. Cells transfected with the pKlf4-1014/luc 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, ^‡ p < 0.01 vs. LH treated pKlf4-1014/luc.