Fig. 3. A modular promoter element contains closely linked sites that mediate activation and Dpp-dependent repression. (A) Schematic showing L12 and derivatives. The red oval marks the SMM site containing C rather than A/G at position 2. The triangle in L12+Spacer marks the location of the insert. (B) Lysates from S2 cells transfected as indicated were used to gel-shift an oligo containing the SMM site. Lane 1 contains probe alone. The presence of Mad/Med results in a slower mobility complex (lane 2) that is further retarded by anti-Flag (lane 3) or Myc-ShnCT (lane 4). The Shn/Mad/Med complex is supershifted by incubation with anti-Myc (lane 5). (C) Wild-type (WT) or mutant (M) SMM oligos were incubated with GST-Mad or Med. Both proteins bind wild-type (lanes 3, 7) but not the mutant site (lanes 4, 8). Excess wild-type (lanes 5, 9), but not mutant, oligos (lanes 6, 10) block Mad/Med binding. L12^M containing the mutant SMM site is ubiquitously expressed in (D) wing discs and (E) embryos. (F-I) Expression patterns of L12 derivatives in wing discs. (F) L12-a drives laterally restricted expression. (G) L12-b, which lacks the SMM site, is derepressed medially. (H) A 187 bp L12-d fragment drives brk-like expression, indicating the presence of closely linked SMM and activator sites. (I) Insertion of a 380 bp spacer between the SMM site and activator sequences (L12+Spacer) results in broad expression.