Thermodynamic principles link in vitro transcription factor affinities to single-molecule chromatin states in cells

The molecular details that drive TF binding and the establishment of accessible chromatin are not understood from first principles - including how TFs scan DNA, how affinity is modulated by local sequence and motif grammars, and how kinetics drive TF binding. Here, we quantitatively compare in vitro and in vivo TF binding for the mammalian erythroid Kruppel-like factor (eKLF/KLF1). We perform high-throughput measurements of binding rates and affinities in vitro and single-molecule footprinting to measure TF and nucleosome occupancies in cells. We find that motif recognition probability drives KLF1 affinity and that in vitro and in nuclei measures of residence time agree on the minutes timescale. We use thermodynamic models to explain how nucleosome competition alters KLF1 occupancy across motif grammars. Lastly, a linear model captures how motif-flanking sequence drives variability in affinities and, paired with thermodynamic models, accurately predicts in vivo occupancy.