Ci is required for maintenance of Hh-responsive gene expression. (A) Wild-type expression of wg mRNA, stage 11. (B) wg mRNA in heat-shocked Hs-hh embryo. (C) Expression of Wg protein in a Df(4)M62f mutant embryo, stage 11. (D) Statistical analysis of number (N) and relative percents of observed phenotypes, indicates that ci is required for Hh signal transduction.

Ci acts epistatically to Hh. (A, D, G, and J) wg mRNA expression patterns, in late stage 10 embryos: (A) wild type, (D) hh^GS1 mutant, (G) ci^D mutant, and (J) hh^GS1 ci^D double mutant. (B, E, H, and K) gsb mRNA expression patterns late stage 10 embryos: (B) wild type, (E) hh^GS1 mutant, (H) ci^D mutant, and (K) hh^GS1 ci^D double mutant. (C, F, I, and L) ptc mRNA expression patterns, stage 11 embryos: (C) wild type, (F) hh^GS1 mutant, (I) ci^D mutant, and (L) hh^GS1 ci^D double mutant.

Sequence-specific DNA binding of GST-Ci. P, free BS-1 DNA; C, GST-Ci/BS-1 complex; S, supershifted complex. Lane 1, BS-1 (TGGGTGGTC) alone. Lanes 2–5, GST-Ci/BS-1 complex with 0, 10, 100, and 500 ng competing unlabeled BS-1. Lanes 6–9, GST-Ci/BS-1 complex with 0, 10, 100, and 500 ng competing unlabeled BS-2 (TGGGTAGTC). Lanes 10 and 11, GST-Ci/BS-1 complex supershifted with 1 and 2 μl GST antiserum.

Binding of Ci to wg promoter is required for transcriptional activation. (A) There are 10 putative Ci binding sites (defined as at least 5 of 9 match with BS-1) in the wg promoter/enhancer region. wg-LucΔS deletes the distal 1 kb of enhancer. wg-LucΔB deletes the distal 2 kb. Δwg-Luc and Δwg*-Luc eliminate all but the distal 1 kb of wg promoter and are fused to the tk minimal promoter. Δwg*-Luc contains mutations in the four putative Ci binding sites. The sequences of Ci binding sites 1–4 in Δwg-Luc are TGGCTGCTC, AGCGTGGAC, TGCGTGAAC, and GACCTCCCA, respectively. Underlined letters indicate mutagenized bases in Δwg*-Luc. The complete sequence for the wg promoter is available from GenBank. (B) Ci activates transcription from the wg promoter. Schneider line 2 (S2) cells were cotransfected with the indicated amounts of mt-Ci expression vector and 50 ng wg-Luc (shaded bars) or tk-Luc (open bars). Luciferase activity is normalized relative to β-gal activity as an internal control for transfection efficiency. Fold activation is presented as the increase in luciferase activity relative to the value from transfections lacking mt-Ci. Duplicate samples varied by less than 18%. Basal levels of luciferase units for reporter constructs in the absence of mt-Ci were 30,000 for wg-Luc and 5,000 for tk-Luc. (C) Deleting distal regions within the wg promoter/enhancer results in reduced activation by Ci. Cotransfection of varying concentrations of mt-Ci, as specified, with 200 ng wg-LucΔS (open bars), or with 200 ng wg-LucΔB (striped bars). Basal levels of luciferase units for reporter constructs in the absence of mt-Ci were 45,000 for wg-LucΔS and 30,000 for wg-LucΔB. (D) Transcriptional activation of Δwg-Luc is reduced when Ci binding sites are mutagenized. Cotransfection of mt-Ci with Δwg-Luc (shaded bars) or Δwg*-Luc (open bars) resulted in a 90% reduction in the ability to activate luciferase expression. Basal levels of luciferase units for reporter constructs in the absence of mt-Ci were 400 for Δwg-Luc and 500 for Δwg*-Luc.

GST-Ci binds wild-type wg promoter fragments more efficiently than mutagenized fragments. P, free DNA; C, GST-Ci/DNA complex. (A) Electrophoretic mobility shifts of wg promoter DNA fragment 1, containing Ci binding sites 1 and 2. Lanes 1–4, wild-type fragment 1. Lanes 5–8, mutagenized fragment 1. Lane 1, no GST-Ci; lanes 2–4, with GST-Ci and 0, 100, or 500 ng competing cold BS-1, respectively. (B) EMSA of wg promoter DNA fragment 2, containing Ci binding sites 3 and 4. Lanes 1–4, wild-type fragment 2. Lanes 5–8, mutagenized fragment 2. Lane 1, no GST-Ci; lanes 2–4, with GST-Ci and 0, 100, or 500 ng unlabeled BS-1 competitor, respectively; lane 5, no GST-Ci; lanes 6–8, with GST-Ci and 0, 100, or 500 ng unlabeled BS-1 competitor.