Thinking out of the box to evaluate the performance of engineered SOX17 in pluripotency reprogramming (ATAC-Seq)
The SOX17 triple-mutant SOX17FNV is a more potent inducer of pluripotency than wild-type SOX2 for unknown features outside its DNA binding high mobility group box domain. Using an inducible reprogramming system, we verified that wild-type SOX17 is not capable to induce pluripotency but SOX17FNV outperforms SOX2 in mouse and human. In mature pluripotent stem cells, SOX17FNV can fully replace SOX2 without compromising self-renewal and pluripotency despite considerable sequence variation. Binding assays using full length proteins expressed in mammalian cells show that SOX17FNV and SOX17 co-bind OCT4 more tightly than SOX2 albeit with switched preferences for composite DNA motifs. Through the systematic analyses of domain deletions, we found that the N-terminus is dispensable for the reprogramming activity of SOX17FNV whilst the C-terminus encodes for essential as well as superfluous domains. Domains have non-additive and partially compensatory effects suggesting that versatile multivalent interaction drive transactivation and reprogramming. We define a minimal SOX17FNV (miniSOX) that can support reprogramming without loss in activity enabling payload reduction within reprogramming cassettes. The definition, functional evaluation and utilisation of potent effector domains that are optimised to pioneer cell fate transition will open up new avenues to enhance cellular reprogramming and stem cell engineering with synthetic transcription factors. Overall design: We analyzed the chromatin accessibility profiles of SOX2 and SOX17FNV rescued 2TS22C mouse ESC cell lines. Cells cultured in 2i serum/Lif medium were analyzed in this study.