Fig. 4. Sox9 directly activates Snail2 promoter. (A) Activation of Snail2 promoter by Sox9 in neural plate explants. D1.2-Luciferase reporter gene and other `crest-specific' transcription factors were electroporated into neural plates, and cultured for 20 hours without BMP4. Only Sox9 can activate the reporter. Results are shown as fold-induction compared with the result obtained from explants, transfected with D1.2-Luciferase and empty pyDF30 plasmid. (B) Sox9 binds to the flanking sequence of E-box2. EMSA was performed to determine the binding site of Sox9 on Snail2 promoter. The sequence and the position of the probe and competitors are shown below. E-box2 is indicated by an open box. The Sox9 binding to the E-box probe is interfered by the addition of competitor 1, 3 and 5, but not by competitor 2 or 4. (C) The flanking sequence of E-box2 is required for the Snail2 promoter activation by Sox9. When the C-rich sequence next to the E-box2 in the D0.1 reporter is mutated (Em3), activation level of the promoter by Sox9 (+) is significantly decreased, compared with the wild-type D0.1. (D) Sox9 and Snail2 synergistically activate D0.1-Luciferase promoter. D0.1-Luciferase was co-transfected with Sox9 or Snail2 (or both) into NIH3T3 cells. Results are shown as a fold-induction compared with the result obtained from cells transfected with D0.1-Luciferase and an empty pyDF30.