ATAC-seq of squamous cell carcinoma cells varying in FAK status

Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression. Overall design: To identify which transcription factors may be candidate mediators of FAK-dependent gene expression, we used ATAC-seq to analyze active (accessible) chromatin in FAK-deficient SCC cells or cells expressing different FAK mutant proteins. Specifically, we compared SCC cells in which FAK had been depleted by Cre-lox-mediated Ptk2 (which encodes FAK) gene deletion (FAK-/- cells) with the same cells re-expressing wild-type FAK (FAK-WT-expressing cells) or FAK mutants that were impaired in nuclear localization (FAK-nls-expressing cells) or deficient in kinase activity (FAK-kd-expressing cells) (cell lines generated previously in Serrels et al., 2012 and Serrels et al., 2015). These permitted the identification of FAK-dependent, FAK nuclear localization-dependent and FAK kinase-dependent alterations in chromatin accessibility and transcription factor motif enrichment in accessible regions of chromatin across the genome.