Figure 7. Mutations in the Limb SHH Enhancer.

(A) Summary of interactions lost (red) or gained (blue) for different mutations in the ZRS enhancer of sonic hedgehog. Yellow circles – TFs expressed in limb during development; black dots – activators; white dots – repressors; black/white dots – TF that can be both activators or repressors.

(B) Number of interaction changes occurring with limb-expressed activators, repressors or bifunctional TFs (activators/repressors) for interactions gained or lost in ZRS enhancer mutations. p = 0.018 by Fisher's exact test.

(C) Gain of interactions detected by eY1H assays in the 105C→G mutant in the ZRS enhancer of sonic hedgehog. Blue boxes indicate positive interactions.

(D) DNA binding motifs for TFAP2A, TFAP2B and TFAP2E discriminate wild type and mutant enhancer sequences.

(E) HEK293T cells were co-transfected with enhancer fragments containing wild type (105C) or mutant (105G) sequences cloned upstream of a Firefly luciferase reporter vector, and the indicated TF expression vectors. Relative luminescence activity is plotted as fold change compared to cells co-transfected with control vector expressing GFP. Experiments were performed four times in 3-6 replicates. Average relative luminescence activity ± SEM is plotted. *p<0.05 by Student's t-test.

See also Table S5.