In vitro binding energies capture Klf4 occupancy across the human genome

Transcription factors (TFs) regulate gene expression by binding to specific genomic loci determined by DNA sequence. Their sequence specificity is commonly summarized by a consensus binding motif. However, eukaryotic genomes contain billions of low-affinity DNA sequences to which TFs associate with a sequence-dependent binding energy. We currently lack insight into how the genomic sequence defines this spectrum of binding energies and the resulting pattern of TF localization. Here, we set out to obtain a quantitative understanding of sequence-dependent TF binding to both motif and nonmotif sequences. We achieve this by first pursuing accurate measurements of physical binding energies of human TF Krüppel-like factor 4 (Klf4) to a library of short DNA sequences in a fluorescence-anisotropy-based bulk competitive binding assay. Second, we show that the highly nonlinear sequence dependence of Klf4 binding energies can be captured by combining a linear model of binding energies with an Ising model of the coupled recognition of nucleotides by a TF. In this model, nucleotide–TF interfaces collectively switch between two states, weak binding and strong nucleotide-specific binding, and the states of neighboring interfaces are coupled with J ∼ 2 k B T . We find that this statistical mechanics model parameterized by our in vitro measurements captures Klf4 binding patterns on an individual long DNA molecule stretched in the optical tweezer, and is predictive for Klf4 occupancy across the entire human genome without additional fit parameters.