DNA and Protein Determinants for p63 Transcriptional Activity

The transcription factor p63 is critical for development of the epidermis and other epithelial-related structures. Mutations in the DNA-binding domain of p63 lead to a range of human developmental disorders characterized by symptoms such as ectrodactyly, painful skin dysplasia, and cleft lip and palate. The TP63 gene is also amplified in squamous cell carcinomas, contributing to tumor pathogenesis. While the importance of p63 in human health is well established, the mechanisms underlying its control of an essential gene network remain unclear. TP63 encodes multiple isoforms with distinct N- and C-terminal domains. The ∆Np63α isoform is directly linked to epidermal development and maintenance, but the physiological and molecular roles of other p63 isoforms remain unexplored. Here, we use massively parallel reporter assays to identify key DNA sequence and co-factor requirements for p63 activity at cis-regulatory elements. These data reveal insight into how ∆Np63α serves as a context-dependent activator and repressor. We also found that two C-terminal β isoforms are constitutive activators, Thus, the same DNA elements support multiple p63-dependent activities, depending on isoform and co-factor availability. We then identified that the ∆Np63β isoform controls a distinct gene regulatory network and induces enhancer-associated chromatin modifications reflecting increased transcriptional activity. These novel activities depend on distinct domains in the β -specific C-terminus. Finally, we examined the unique 14 amino acid ∆N domain and found it functions as a novel transactivation domain (TAD) with characteristics consistent with known TADs. These results reveal key context into how p63 functions to regulate its indispensable gene network and controls myriad physiological processes in human development. Our data provide insight into differential activities of p63 isoforms, how the local sequence content and cellular context influence p63-dependent behaviors, and how switching between isoforms with distinct N-terminal TADs controls a range of behaviors in the p63-dependent gene regulatory network.