Removing an acid residue is necessary and sufficient to turn SOX17 into a pluripotency factor

SOX17 directs the differentiation of the extraembryonic endoderm and acts as a human germline specifier. The replacement of an acidic residue at position 57 with a basic residue found in SOX2 turns SOX17 into a pluripotency factor. Here we systematically interrogated how mutations at this critical position affect the cellular reprogramming activity of SOX17. We found that most mutations turn SOX17 into a pluripotency factor regardless of their biophysical properties. The mutation to an aspartate allows the SOX17E57D protein to maintain a self-renewing endodermal state. Only the glutamate found in the wild-type protein can effectively block a SOX17/OCT4 dimer from binding composite DNA elements found in pluripotency enhancers. Insights into how modifications of an ultra-conserved residue affect functions of developmental transcription factor provide avenues to advance cell fate engineering. Overall design: To investigate the effects SOX17E57 variants have on maintaining pluripotency, SOX2 or the SOX17E57 variants were introduced into engineered Tet-Off Sox2 mESCs (also known as the 2TS22C cell line) using lentiviruses. Cells were cultured in mouse serum/LIF/2i medium for ten passages prior to analysis.