The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we use domain swapping and mutagenesis to study Oct4’s reprogramming ability, identifying a redox-sensitive DNA binding domain cysteine residue (Cys48) as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs), but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression and aberrant differentiation. Pou5f1C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1C48S(Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

This raw dataset includes:

1)    Reprogramming data. GFP+ colonies with iPSC morphology were counted by microscopic observation. Viral titer data was collected in parallel. Titer was obtained using p24 levels or conserved viral RNA sequences.

2)    ESC western blot quantification data. Intensities were collected and normalized using Image J.

3)    EMSA quantification data. Intensities were collected and normalized using Image J.

4)    Parental and Oct4^C48S mutant ESC pluripotent marker expression in the pluripotent state and in RA differentiation. Intensities were collected and normalized using Image J.

5)    Parental and Oct4C48S mutant differential gene expression in the pluripotent state and in RA differentiation. mRNA expression levels were obtained using RNA sequencing, and normalized using methods described in teh materials and methods section of the Shen et al. manuscript.

6)    Parental and Oct4C48S mutant ESC teratoma formation. Differentiated structures were scored in the blanded manner by a clinical pathologist.

7)    Oct4^C48S mouse line generation and defects in the homozygous Jky/Jky mouse. Genotypes were obtained using endpoint PCR.