FIG 5.

KLF6 regulates placental development and cooperates with PPARγ, RXRα, and LCoR in Muc1 induction. (A) Extracts of 293T cells cotransfected with the indicated combinations of Flag-LCoR and KLF6 were resolved without IP (Input, middle) or after IP with anti-Flag Ab beads (top, bottom) and probed using Abs against Flag (top) or KLF6 (middle, bottom). (B) Putative SP1/KLF-binding sites (orange boxes) within the minimal PPARγ-responsive sequences of the Muc1 promoter. Sequences designated K1, K2, and K3 delineate the alterations of the three mutant KLF/SP motifs analyzed in the experiments whose results are shown in panel H. The blue box marks the previously determined proximal PPRE (9). (C and D) Midsections of WT (C) and Klf6-null placentas at E9.5 (D) were stained with hematoxylin and eosin. Both WT and Klf6-null placentas have undergone allantoic (Al) fusion, but the Klf-null placenta is completely devoid of the vascular labyrinth (La in panel C; none in panel D) and exhibits aberrant expansion of the spongiotrophoblast (Sp) and, particularly, the trophoblast giant cell (TGC) layer. Ch, chorion; de, decidua. (E, F) Normalized RLU in CV1 cells transfected with pCMX-βGAL and RXRα (all), as well as PPARγ, LCoR, and/or KLF6 where indicated, and treated with 1 μM Rosi as marked, along with either Muc1-luc (E) or Ldhb-luc (F). (G) Normalized RLU in CV1 cells transfected with pCMX-βGAL, Muc1-luc, RXRα and PPARγ (all), with or without LCoR, as labeled, in the presence or absence of 1 μM Rosi, as marked, and a plasmid expressing the indicated control, scramble shRNA (Scr). or one of three CV1 Klf6-specific shRNA molecules. Inset, RT-qPCR analysis of endogenous Klf6 in CV1 cells transfected with a GFP-expressing vector along with a control or Klf6-specific shRNA construct, as indicated, and enriched to approximately 50 to 60% via 8 days of selection with puromycin. (H) Normalized RLU in CV1 cells transfected with pCMX-βGAL, Muc1-luc, and RXRα (all), as well as PPARγ and/or LCoR, as labeled, and treated with 1 μM Rosi, alone or with 1 μM LG268, as marked, in the absence or presence of KLF6. (I) Normalized RLU in CV1 cells transfected with pCMX-βGAL, RXRα, and PPARγ and treated with 1 μM Rosi (all), as well as LCoR and/or KLF6, as labeled, and Muc1 promoter variants carrying the indicated single and combined KLF/SP motif mutations. (J) Normalized RLU in CV1 cells transfected with pCMX-βGAL, Muc1-luc, and RXRα (all), as well as PPARγ and/or LCoR, as labeled, with or without 1 μM Rosi, as marked, and the indicated members of the SP/KLF family. Bars and error bars show mean values and SE.