Systematic dissection of sequence features affecting binding specificity of a pioneer factor reveals binding synergy between FOXA1 and AP-1

Despite the unique ability of pioneer factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called chromatin immunoprecipitation with integrated synthetic oligonucleotides (ChIP-ISO) to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with in vitro and neural network analyses, we find that (1) FOXA1 binding is strongly affected by co-binding transcription factors (TFs) AP-1 and CEBPB; (2) FOXA1 and AP-1 show binding cooperativity in vitro; (3) FOXA1’s binding is determined more by local sequences than chromatin context, including eu-/heterochromatin; and (4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.