Combinatorial pioneer transcription factor binding reinforces bivalent epigenetic states to preserve lineage fidelity

Combinatorial binding of transcription factors (TFs) is central to eukaryotic gene regulation, providing regulatory specificity and robustness to cell fate control. However, its impact on epigenetic regulation remains poorly understood. Here, we show that the pioneer TFs GATA6, EOMES, and SOX17 cooperate with the zinc finger TF PRDM1 to recruit Polycomb Repressive Complexes (PRCs) and establish enhancers marked by H3K4me1 and PRC-associated histone modifications during endoderm development. Increasing the number and diversity of pioneer TFs bound at enhancers drives synergistic nucleosome remodeling and promotes the formation of “hyper-bivalent” enhancers that reinforce repression of alternative-lineage programs. Together, our findings demonstrate that combinatorial pioneer TF binding creates locally accessible regions that facilitate recruitment of not only active but also PRC-associated epigenetic regulators to preserve lineage fidelity during development.