?Np63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes

?Np63, a master regulator of epithelial biology, is involved in regulating epithelial stem cell function, maintaining the integrity of stratified epithelial cells and committing epidermal cells to the differentiation program. To this end, ?Np63 exploits several direct mechanisms. Here, we elucidated a novel mechanism whereby ?Np63 efficiently sustains the expression of epidermal differentiation genes. We show that ?Np63 interacts with Senataxin (SETX), an RNA/DNA helicase able to resolve the R-loop intermediates over the GC-rich termination sites of coding genes. Notably, we found that SETX and ?Np63 co-regulate a subset of genes involved in the early step of the keratinocyte differentiation program. At the molecular level, SETX physically binds the p63 DNA binding motifs present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus promoting efficient transcriptional termination and gene expression. Remarkably, SETX loss affects the activation of the proper epidermal differentiation program in vitro and impacts epidermal layer stratification in organotypic human skin. Furthermore, we found that SETX is mutated or downmodulated in SCC and SETX gene mutation is a negative prognostic factor for cutaneous SCC patient survival. Collectively, our results unveil SETX as a novel molecular player of skin homeostasis, potentially involved in hyperproliferative skin disorders. Overall design: RNA profiles of the human head and neck squamous cell carcinoma cell line A253 and the immortalized human keratinocytes (Ker-CT) transfected with siRNA targeting p63 (sip63), SETX (siSETX) or a nonrelevant mRNA (scramble, SCR) were generated by deep sequencing using Illumina Novaseq. We analyzed three biological replicates for each conditions. qRT–PCR validation was performed using SYBR Green assays.