Binding of XWBSCR11 and FoxH1 to the gsc promoter. (A) Bacterially expressed GST-XWBSCR11 R4–5 and GST-FoxH1 FH were used for footprint analysis of the −309 gsc promoter. XWBSCR11 R4–5 bound to the promoter primarily in the 17-bp DE, and FoxH1 FH bound to the 3′ end of the previously identified proximal element (PE). The PE has been shown previously to respond to Xtwin/Xsia-mediated Wnt signals. (B) XWBSCR11 and Xenopus FoxH1 synergistically activate transcription of the DE(6×)gsc/Luc reporter. Injection of either VP16-XWBSCR11 or a Myc-tagged Fast1 alone did not activate the DE(6x)gsc/Luc reporter. However, coexpression of Myc-tagged FoxH1 together with VP16-XWBSCR11 resulted in significant reporter gene activation, reaching a maximum induction level of 54-fold. The synergistic effect of FoxH1 and XWBSCR11 was not affected by the presence of Smad2 or Smad3. (C) Bacterially expressed GST-XWBSCR11-R4-5, GST-FoxH1, and various GST-Smads were used to perform an EMSA with DE oligonucleotides. (Arrows) Presumed ternary complexes among these proteins and DNA.