Supplementary Figure 10: A 5-bp point mutation within the p63 binding site disrupts p63-mediated regulation of TFAP2C expression.

a, The p63 binding site was mutated using CRISPR–Cas9 and homology-directed repair techniques to disrupt p63 binding (p63BSPM). The two overlapping p63 motifs within the 520-bp BSKO region were targeted with two gRNAs to mutate the four highlighted nucleotides (*)—the most highly conserved nucleotides within the canonical predicted p63 binding motif. The chromatogram depicts the sequence of the BSPM cell line and the mutated base pairs are indicated by asterisks. An EcoRI site was engineered in at the fifth highlighted base pair and this new sequence does not contain a predicted p63 binding motif. b, Disruption of the p63 binding site leads to a significant increase in TFAP2C expression similar to the levels seen in day 7 p63BSKO cells. Relative pixel intensity was calculated from three independent images and error bars represent s.e.m. (***P < 0.005; NS, not significant). Scale bar, 20 μm. c, qRT–PCR reveals that TFAP2C mRNA levels are significantly increased in the p63BSPM line (***P < 0.005) relative to day 7 p63WT cells. Error bars represent s.e.m., n = 3. d, ChIP–qPCR for p63 at the TFAP2C locus shows that disruption of the p63 binding site (p63BS) results in a significant decrease in p63 binding (***P = 0.00014) at this region (n = 2). This decrease in binding results in a loss of TFAP2C regulation. The graph depicts signal relative to input and error bars represent s.e.m.