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

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Schaepe JM, Fries T, Doughty BR, Crocker OJ, Hinks MM, Marklund E, Greenleaf WJ

bioRxiv - (-) - [2025-01-30; online 2025-01-30]

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 Krüppel-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.

PubMed 39975040

DOI 10.1101/2025.01.27.635162

Crossref 10.1101/2025.01.27.635162

pmc: PMC11838358
pii: 2025.01.27.635162