Effective in vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding

Duyen Huynh, Philipp Hoffmeister, Tobias Friedrich, Kefan Zhang, Marek Bartkuhn, Francesca Ferrante, Benedetto Daniele Giaimo, Rhett A. Kovall, Tilman Borggrefe, Franz Oswald () and J. Christof M. Gebhardt ()
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Duyen Huynh: Ulm University
Philipp Hoffmeister: University Medical Center Ulm
Tobias Friedrich: Justus-Liebig-Universität Gießen
Kefan Zhang: Ulm University
Marek Bartkuhn: Justus-Liebig-Universität Gießen
Francesca Ferrante: Justus-Liebig-Universität Gießen
Benedetto Daniele Giaimo: Justus-Liebig-Universität Gießen
Rhett A. Kovall: University of Cincinnati College of Medicine
Tilman Borggrefe: Justus-Liebig-Universität Gießen
Franz Oswald: University Medical Center Ulm
J. Christof M. Gebhardt: Ulm University

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract Transcription factors (TFs) such as RBPJ in Notch signaling bind to specific DNA sequences to regulate transcription. How TF-DNA binding kinetics and cofactor interactions modulate gene regulation is mostly unknown. We determine the binding kinetics, transcriptional activity, and genome-wide chromatin occupation of RBPJ and mutant variants by live-cell single-molecule tracking, reporter assays, and ChIP-Seq. Importantly, the search time of RBPJ exceeds its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired RBPJ-DNA binding as in Adams-Oliver-Syndrome affect both target site association and dissociation, while impaired cofactor binding mainly alters association and unspecific binding. Moreover, our data point to the possibility that cofactor binding contributes to target site specificity. Findings for other TFs comparable to RBPJ indicate that kinetic rather than thermodynamic DNA binding stability might prevail in vivo. We propose an effective in vivo binding energy landscape of TF-DNA interactions as instructive visualization of binding kinetics and mutation-induced changes.

Date: 2025
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DOI: 10.1038/s41467-025-56515-4

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