Cooperative FOXA1-HNF4A binding emerges from motif spacing and nucleosome architecture

The pioneer factor hypothesis posits that specialized transcription factors access nucleosomal DNA to enable binding of secondary factors, implying a hierarchical mechanism of chromatin opening. However, recent evidence indicates that cooperative binding between pioneer and non-pioneer factors can occur in a context-dependent manner. Here we define the sequence and chromatin features that specify cooperative binding between FOXA1 and HNF4A. Using dual-induction of FOXA1 and HNF4A in naive cells, which lack endogenous expression of either factor, we identify a class of genomic sites that require both factors for binding and reside in initially inaccessible chromatin. A dual-head convolutional neural network finds no novel composite motif at these sites, instead implicating motif arrangement as the distinguishing feature. Cooperative loci are enriched for FOXA1-HNF4A motif pairs spaced 15-60 bp apart, a soft-syntax grammar consistent with nucleosome-mediated cooperativity. This spacing signature recurs at endogenously co-bound sites in HepG2 cells and primary fetal hepatocytes. Using a multiplexed in vitro nucleosome-binding assay (Pioneer-seq), we show that FOXA1 binds nucleosomal DNA more strongly than HNF4A across positions, and that cooperative binding strength on natural nucleosome sequences is predicted primarily by the position of the FOXA1 motif relative to the nucleosome dyad. Together, these results support a model in which positioned nucleosomes act as scaffolds that facilitate cooperative transcription factor binding through spatial motif organization, rather than a barrier one factor clears for the other.