FIGURE 6.

Identification of regulatory elements important for TGF-β-induced transactivation of the MIXL1 promoter. A, schematic representation of HNF4α-binding motifs in the Smad2/3 and HNF4α binding region of the MIXL1 promoter. Promoter reporters with mutations in their HNF4α-binding motifs are shown in the lower panel. B, activation of the MIXL1 gene promoter by TGF-β and effects of mutations in putative HNF4α-binding motifs. HepG2 cells were transfected with the MIXL1 promoter and its mutants and treated with TGF-β for 24 h. C, conserved Smad-binding elements (SBEs) of the MIXL1 promoter. Four Smad-binding elements that were conserved between mouse and human (SBE 1–4) are shown with their relative positions from the transcription start site. Nucleotide sequences of Smad-binding elements and their mutations used in D are also shown. WT, wild-type; mut, mutant. D, effect of mutations in Smad-binding elements on TGF-β-induced transcriptional activity of MIXL1 promoter. Cells were treated as in B, and luciferase activities were determined. *, p < 0.05 compared with WT without TGF-β; **, p < 0.05 compared with SBE4 mutant, without TGF-β; n.s., not significant compared with WT and SBE2 mutant, without TGF-β; error bars, S.D.