Ferniani, Marco (2020) Transcriptional control orchestrated by p63 in skin disease. [Tesi di dottorato]

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The p63 gene encodes a master regulator of epidermal development. Mutations or alteration of its expression can lead to different pathological phenotypes. In particular, p63 is often overexpressed or its gene is amplified in squamous cell carcinomas, behaving as a pro-oncogenic factor and facilitating tumor formation. In a first part of this thesis, we focused the attention on gene regulation downstream of p63 in cutaneous squamous cell carcinoma (cSCC). We find that p63 regulates a large number of genes crucial for cell proliferation, and that its ablation impairs cell proliferation, clonogenicity and tumor-sphere formation. To identify p63 protein partners in search for therapeutic targets for skin disease, we identified novel interactors by LC-MS/MS. Among them, we focused on PARP1. PARP1 is an enzyme that add Poly-ADP-Ribose (PAR) moieties onto target proteins and regulates different cellular processes including DNA repair and transcriptional regulation. To investigate whether PARP1 and p63 coordinately regulate global patterns of gene expression, either PARP1 or p63 were depleted in SCC cells followed by RNA-seq. Correlation analyses of the transcriptomic profile under basal condition, demonstrate a strong genetic interaction between p63 and PARP1 regulating a largely overlapping set of genes involved in mitosis and cell cycle progression. In the second part of my thesis, we investigated heterozygous mutations in p63 gene that can cause different rare genetic disorders with partially overlapping phenotypes, such as Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndrome, Ectodermal Ectrodactyly Cleft-lip palate syndrome (EEC), and Acro–dermato–ungual–lacrimal–tooth syndrome (ADULT) syndrome. Using the fibroblast conversion to Keratinocytes like cells (iKC) assay, we investigate the different molecular mechanisms affecting p63 involved in skin genetic diseases. In particular, we identified that AEC-associated p63 mutations lead to thermodynamic protein destabilization, misfolding, and aggregation abolishing the DNA binding and the disruption of the aggregation leads to rescue of the p63 transcriptional activity. In contrast, we also characterize some EEC and ADULT mutations that can partially affect transcriptional activity with different mechanisms involving binding to the DNA.

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