Nonreciprocal and Conditional Cooperativity Directs the Pioneer Activity of Pluripotency Transcription Factors

ABSTRACT Cooperative binding of transcription factors (TFs) to chromatin orchestrates gene expression programming and cell fate specification. However the biophysical principles of TF cooperativity remain incompletely understood. Here we use single-molecule fluorescence microscopy to study the partnership between Sox2 and Oct4, two core members of the pluripotency gene regulatory network. We find that Sox2’s pioneer activity (the ability to target DNA inside nucleosomes) is strongly affected by the translational and rotational positioning of its binding motif, while Oct4 can access nucleosomal sites with equal capacities. Furthermore, the Sox2-Oct4 pair displays nonreciprocal cooperativity, with Oct4 modulating Sox2’s binding to the nucleosome but not vice versa. Such cooperativity is conditional upon the composite motif residing at specific nucleosomal locations. These results reveal that pioneer factors possess distinct properties of nucleosome targeting and suggest that the same set of TFs may differentially regulate transcriptional activity in a gene-specific manner on the basis of their motif positioning in the nucleosomal context.