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. 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.