IER5 is required for Notch-mediated induction of squamous cell differentiation [H3K27ac ChIP-seq]

Conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. IER5 is a direct Notch-regulated gene and required for Notch-induced differentiation of squamous carcinoma cells. To create a squamous cell carcinoma (SCC) model in which GSI (ɣ-secretase inhibitor) washout activates NOTCH1, we engineered a cDNA encoding a mutated truncated form of NOTCH1, ΔEGF-L1596H, that cannot respond to ligand and that has a point substitution in its negative regulatory region that produces ligand-independent, ɣ-secretase-dependent Notch activation. ΔEGF-L1596H was transduced into human SCC cell line IC8 that have biallelic inactivating mutation in NOTCH1. GSI washout was performed for 1,2 and 4 hours to activate the Notch signaling pathway in IC8-ΔEGF-L1596H cells.