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

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 determined 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 exceeded its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired RBPJ-DNA binding as in Adams-Oliver-Syndrome affected both target site association and dissociation, while impaired cofactor binding mainly altered 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 ..., , , # Effective In vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding

[https://doi.org/10.5061/dryad.mkkwh716k](https://doi.org/10.5061/dryad.mkkwh716k)

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

Single molecule data

The folder '`Single molecule data`' contains .mat files which include single-molecule tracking data used in the manuscript Huynh et al., Effective In vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding

It contains subfolders labeled with respective illumination scheme or analysis names used in the publication:

* Diffusion = 11.7 ms continuous movies used to determine diffusion coefficients and bound fractions
* Time-lapse = time-lapse microscopy for residence time and bound fractions

The subfolders contain .mat files in Matlab format, each labeled by the illumination scheme (either Diffusion or Time-lapse) and the variant that has been measured.

RBPJ variants ...