Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation.

Pioneer transcription factors (TF) bind nucleosome-embedded DNA motifs to activate new regulatory elements and promote differentiation. However, the complexity, binding dependencies and temporal effects of their action remain unclear. Here, we dissect how ectopic induction of the pioneer TF GATA6 triggers Primitive Endoderm (PrE) differentiation from pluripotent cells. We show that transient GATA6 binding exploits accessible regions to decommission enhancers and promote pluripotency gene silencing. Simultaneously, GATA6 targets closed chromatin and initiates extensive remodeling culminating in the establishment of fragile nucleosomes flanked by ordered nucleosome arrays and increased accessibility. This is enhanced by rapidly expressed PrE TFs (SOX17) and by pluripotency TFs repurposed for differentiation (OCT4/SOX2). Accordingly, depletion of OCT4 during GATA6 induction decreases Gata6 expression, alters GATA6 and SOX17 binding and impairs differentiation. Therefore, pioneer TFs orchestrate complex regulatory networks involving many if not all available pioneer TFs, including those required to support the original identity of differentiating cells.