Characterization of the p63/p73 hetero-tetramer using selective DARPins

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p63 is a member of the p53 protein family and plays an essential role in the development of stratified epithelial tissues such as skin. The isoform expressed in these tissues, ΔNp63α, lacks the N-terminal transactivation domain and mainly acts as a “pioneering factor” that is important for organizing the chromatin landscape in keratinocytes. Mutations in p63 result in human and mouse in severe developmental syndromes. In addition to p63, cells in the basal layer of stratified epithelial tissues as well as of pseudostratified tissues (trachea) express different isoforms of the family member p73. Both proteins, p63 and p73, form tetramers though a specialized oligomerization domain. When both proteins are present in vitro they preferentially form a hetero-tetramer with a 2:2 stoichiometry. Our previous structure determination of this hetero-tetramer has identified the molecular interactions responsible for its preferential formation. Rational design has allowed us to create mutants that either stabilize the hetero-tetramer or suppress its formation, providing the tools for further characterizing the biological function of this hetero-tetramer. While the lack of the transactivation domain in ΔNp63α impairs its transcriptional activity and makes organization of the chromatin landscape its main function, combination with transcriptionally more active p73 isoforms could be responsible for specific transcriptional programs. We now show evidence that this hetero-tetramer exists in vivo, including mouse skin. We propose to investigate its biological function by identifying which p73 isoforms are expressed in the relevant tissues, in which cells in human and mouse tissues it gets formed, where on the DNA it binds (ChIPseq), which gene expression it regulates (RNAseq) and which other factors it cooperates with (proximity labeling) in driving transcription and remodeling chromatin. As a tool for these investigations we have created a Designed Ankyrin Repeat Protein – DARPin – that binds highly selective and with sub-nM affinity to the hetero-tetramer. Experiments will be performed in cell culture of relevant cells as well as in mouse and human tissues.