Disease-related p63 DBD mutations impair DNA binding by distinct mechanisms and varying degree [ChIP-seq]

The transcription factor p63 shares a high sequence identity with the tumour suppressor p53 which manifests itself in high structural similarity and preference for DNA sequences. While mutations in the DNA binding domain of p53 cause cancer, mutations in the p63 DNA binding domain result in developmental syndromes affecting lip/palate, skin and limbs. For p53 the effect of mutations has been studied in great detail, enabling the identification of several mechanisms by which mutations inhibit high affinity binding of p53 to DNA. In this study we provide a similar detailed investigation of all currently known mutations in the p63 DNA binding domain by measuring the transcriptional activity, DNA binding affinity, zinc binding capacity and thermodynamic stability. Some of the mutations we have further characterized with respect to their ability to convert human dermal fibroblasts into induced keratinocytes, to elicit changes in the transcriptional program similar to those observed by wild type p63, and to bind regulatory regions in the genome. The data demonstrate that no mutation globally destabilizes and unfolds the domain. Several mutations affect the DNA binding affinity by disturbing the interaction between the DNA binding domains, thereby disrupting p63 ability to bind DNA as a tetramer cooperatively. These mutations retain partial DNA binding capacity which correlates with a milder patient phenotype.