True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy

Christophe Jung (), Peter Bandilla, Marc von Reutern, Max Schnepf, Susanne Rieder, Ulrich Unnerstall and Ulrike Gaul
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Christophe Jung: Ludwig-Maximilians-Universität München
Peter Bandilla: Ludwig-Maximilians-Universität München
Marc von Reutern: Ludwig-Maximilians-Universität München
Max Schnepf: Ludwig-Maximilians-Universität München
Susanne Rieder: Ludwig-Maximilians-Universität München
Ulrich Unnerstall: Ludwig-Maximilians-Universität München
Ulrike Gaul: Ludwig-Maximilians-Universität München

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: Abstract The complex patterns of gene expression in metazoans are controlled by selective binding of transcription factors (TFs) to regulatory DNA. To improve the quantitative understanding of this process, we have developed a novel method that uses fluorescence anisotropy measurements in a controlled delivery system to determine TF-DNA binding energies in solution with high sensitivity and throughput. Owing to its large dynamic range, the method, named high performance fluorescence anisotropy (HiP-FA), allows for reliable quantification of both weak and strong binding; binding specificities are calculated on the basis of equilibrium constant measurements for mutational DNA variants. We determine the binding preference landscapes for 26 TFs and measure high absolute affinities, but mostly lower binding specificities than reported by other methods. The revised binding preferences give rise to improved predictions of in vivo TF occupancy and enhancer expression. Our approach provides a powerful new tool for the systems-biological analysis of gene regulation.

Date: 2018
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DOI: 10.1038/s41467-018-03977-4

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