Xenbase Image ID: 132738

Fig. 4. Identification of Pax3 and Zic1 binding sites on Snail1/2 promoters in vitro (A) Induction of snail2 between stage 11.5 and 14 is strongly reduced by Pax3 knock-down, but only mildly affected by Zic1 knock-down. (B) The increase in snail1 transcription between stage 11.5 and 14 is blocked by both Pax3 and Zic1 knock-down. (C) Location of the ECRs studied here (yellow boxes), containing Pax3 or Zic1 putative binding site (B.S.), in the genomic sequence upstream of snail1 and snail2 transcription start site (TSS). (D) Pax3 binds specifically to the motif identified in the snail2 promoter ECR: the electrophoretic mobility of a radiolabeled oligonucleotide containing the putative Pax3 B.S. (Pax3BS) is shifted in the presence of Pax3-transfected cell extract, but not with the non-specific (N.S) control (i.e. GFP-transfected cell extract). Intensity of the shift is reduced when the Pax3 binding site is mutated (mut. Pax3BS). In the presence of a specific anti-Pax3 antibody, we detect a mobility supershift, indicating that the Pax3 protein is indeed responsible for the observed shift. (E) Zic1 binds specifically to the motif identified in the snail1 ECR: the electrophoretic mobility shift detected in the presence of Zic1-transfected cell extract can be competed by increasing doses of non-labeled oligonucleotide (Zic1BS), but only weakly by the mutated oligonucleotide (mut. Zic1BS). Image published in: Plouhinec JL et al. (2014) Copyright © 2014. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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