DeltaNp63 silencing, DNA methylation shifts and epithelial mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma [RRBS]

TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3' to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, functions as a core factor to organize super enhancers of genes essential for epidermal/craniofacial differentiation and for self-activation of DeltaNp63. To examine whether very weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair (HDR) in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, with either 'monoallelic HDR and a frameshift deletion on the other allele' or 'biallelic HDR', caused DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, replacement of KRT5 with VIM, and transcriptional suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local CpG methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha, followed by incubation with DNA methyltransferase inhibitor Zebularine. This study suggests that TAp63 is indispensable for DeltaNp63 expression by which keratinocyte-specific epigenome is maintained in SCC. Overall design: Comparison of genome DNA methylation by RRBS between parental (control) FaDu squamous cell carcinoma line and TAp63 knockout cell line derived from FaDu