[論文レビュー] In vitro binding energies capture Klf4 occupancy across the human genome

Anne Schwager, Jonas Neipel+5 more|arXiv (Cornell University)|Jan 22, 2026

Kruppel-like factors research被引用数 0

ひとこと要約

tldr: The study measures human Klf4 binding energies in vitro and shows that a linear-energy plus Ising-model framework captures Klf4 binding across motif and non-motif sequences, enabling genome-wide occupancy predictions without extra fitting.

ABSTRACT

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 non-motif sequences. We achieve this by first pursuing accurate measurements of physical binding energies of the human TF Klf4 to a library of short DNA sequences in a fluorescence-anisotropy-based bulk competitive binding assay. Second, we show that the highly non-linear 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. We find that this statistical mechanics model parametrized by our in vitro measurements captures Klf4 binding patterns on individual long DNA molecules stretched in the optical tweezer, and is predictive for Klf4 occupancy across the entire human genome without additional fit parameters.

研究の動機と目的

• Quantify the sequence-dependent binding energy landscape of Klf4 to short DNA sequences in vitro.
• Demonstrate how binding energy nonlinearity can be captured by a simple statistical model.
• Test whether the in vitro energy model predicts Klf4 occupancy on long DNA molecules and across the human genome without additional fitting.
• Explore how well the model generalizes from motif-containing to non-motif sequences.

提案手法

• Measure physical binding energies of human Klf4 to a library of short DNA sequences using a fluorescence-anisotropy-based bulk competitive binding assay.
• Characterize the highly non-linear dependence of Klf4 binding energies on sequence.
• Combine a linear model of binding energies with an Ising model to capture coupled nucleotide recognition by Klf4.
• Validate the model against single-miber long DNA molecules stretched in optical tweezers.
• Test genome-wide occupancy predictions using the in vitro-derived model without additional fit parameters.

実験結果

リサーチクエスチョン

• RQ1 How well do in vitro measured binding energies reproduce Klf4 occupancy patterns on long DNA molecules?
• RQ2 Can a linear energy model augmented by an Ising-type nucleotide interaction capture Klf4 sequence specificity across motif and non-motif DNA sequences?
• RQ3 Is the in vitro energy model predictive of Klf4 genome-wide occupancy without further parameter fitting?

主な発見

• The non-linear sequence dependence of Klf4 binding energies is captured by a linear energy model plus an Ising model of nucleotide recognition.
• Model parameters inferred from in vitro measurements reproduce Klf4 binding patterns on long DNA in optical tweezers.
• The combined model predicts Klf4 occupancy across the human genome without additional fit parameters.
• The approach links measurable binding energies to genome-wide localization patterns for a vertebrate TF.
• The work provides a quantitative bridge between in vitro energetics and in vivo-like occupancy patterns.