Oct4 is a gatekeeper of epithelial identity by regulating cytoskeletal organization in skin keratinocytes.

Oct4 is a pioneer transcription factor regulating pluripotency. However, it is not well known whether Oct4 has an impact on epidermal cells. We generated OCT4 knockout clonal cell lines using immortalized human skin keratinocytes to identify a functional role for the protein. Here, we report that Oct4-deficient cells transitioned into a mesenchymal-like phenotype with enlarged size and shape, exhibited accelerated migratory behavior, decreased adhesion, and appeared arrested at the G2/M cell cycle checkpoint. Oct4 absence had a profound impact on cortical actin organization, with loss of microfilaments from the cell membrane, increased puncta deposition in the cytoplasm, and stress fiber formation. E-cadherin, β-catenin, and ZO1 were almost absent from cell-cell contacts, while fibronectin deposition was markedly increased in the extracellular matrix (ECM). Mapping of the transcriptional and chromatin profiles of Oct4-deficient cells revealed that Oct4 controls the levels of cytoskeletal, ECM, and differentiation-related genes, whereas epithelial identity is preserved through transcriptional and non-transcriptional mechanisms.