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

Abstract

The molecular details governing transcription factor (TF) binding and the formation of accessible chromatin are not yet quantitatively understood - including how sequence context modulates affinity, how TFs search DNA, the kinetics of TF occupancy, and how motif grammars coordinate binding. To resolve these questions for a human TF, erythroid Kruppel-like factor (eKLF/KLF1), we quantitatively compare, in high throughput, in vitro TF binding rates and affinities with in vivo single molecule TF and nucleosome occupancies across engineered DNA sequences. We find that 40-fold flanking sequence effects on affinity are consistent with distal flanks tuning TF search parameters and captured by a linear energy model. Motif recognition probability, rather than time in the bound state, drives affinity changes, and in vitro and in nuclei measurements exhibit consistent, minutes-long TF residence times. Finally, pairing in vitro biophysical parameters with thermodynamic models accurately predicts in vivo single-molecule chromatin states for unseen motif grammars. HighlightsO_LIKLF1:DNA binding is consistent with a three state binding model wherein the probability of recognizing the motif from a nonspecifically-bound state drives affinity. C_LIO_LISubstantial effects from proximal and distal motif-flanking sequence on KLF1 binding affinity are captured by an extended PWM model. C_LIO_LIKLF minutes-long residence time inferred from single-molecule footprinting in nuclei is consistent with in vitro measurement. C_LIO_LIIn vitro binding energies combined with thermodynamic models predict in vivo, single-molecule chromatin configurations across motif grammars and sequence contexts. C_LI