Oct4 redox sensitivity potentiates iPSC reprogramming.

The transcription factor Oct4/Pou5f1 is a key component of the regulatory circuitry governing pluripotency. Oct4 is also widely used to generate induced pluripotent stem cells (iPSCs) from somatic cells. These observations provide compelling rationale to understand Oct4's functions. Here we used domain swapping and mutagenesis to compare Oct4's reprogramming activity with the paralog Oct1/Pou2f1, identifying a DNA binding domain cysteine residue (Cys48) as a key determinant of both reprogramming and differentiation. In combination with the Oct4 N-terminus, Oct1 S48C is sufficient to confer strong reprogramming activity. Conversely, Oct4 C48S strongly reduces reprogramming potential. We find that Oct4 C48S confers DNA binding sensitivity to oxidative stress. Further, C48S sensitizes the protein to oxidative stress-mediated ubiquitylation and degradation. Engineering a Pou5f1 C48S point mutation in mouse embryonic stem cells (ESCs) has little effect on undifferentiated cells, but upon retinoic acid (RA)-mediated differentiation causes retention of Oct4 expression, decreased proliferation and increased apoptosis. Pou5f1 C48S ESCs also contribute poorly to adult somatic tissues. Collectively, the data support a model in which Oct4 redox sensing serves as a positive reprogramming determinant during one or more steps promoted by Oct4 downregulation during iPSC generation.