DOI: 10.5061/dryad.mkkwh716k
title: Data from: Effective In vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding

description: 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 in vivo binding energy landscape of TF-DNA interactions as
 instructive visualization of binding kinetics and mutation-induced
 changes.
description: # 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 measured in HeLa wildtype are labeled as: * HT-RBPJ-WT = HaloTag-RBPJ wildtype * HT-RBPJ-R_H = HaloTag-RBPJ R218H mutant (DNA binding deficient mutant) * HT-RBPJ-K_E = HaloTag-RBPJ K195E mutant (DNA binding deficient mutant) * HT-RBPJ-KRS_EHD = HaloTag-RBPJ K195E, R218H, S221D mutant (DNA binding deficient mutant) * HT-RBPJ FL_AA = HaloTag-RBPJ F261A, L388A mutant (Cofactor binding deficient mutant) * HT-RBPJ-RFL_HAA = HaloTag-RBPJ R218H, F261A, L388A (DNA and Cofactor binding deficient mutant) HT-RBPJ-WT and KRS/EHD molecules were measured in two different HeLa backgrounds and are labeled as: * S-KO = SHARP-knockout HeLa cells * RBPJ-KO = RBPJ-knockout HeLa cells The .mat files contain the variable 'batch'. This Matlab 'struct' array contains the data of one single-molecule movie. The .mat files were created with the open source single-molecule tracking software TrackIt ([https://gitlab.com/GebhardtLab/TrackIt(opens in new window)](https://gitlab.com/GebhardtLab/TrackIt)) using the 'File &gt; Save batch file as...' function and merged using the 'File &gt; Merge multiple batch files' function. The workflow starting from tracking to quantitative analysis can be visualized and repeated by running the 'TrackIt_v1_5_1.m' file followed by pressing 'File &gt; Load batch file'. Alternatively, the batches can be loaded into TrackIts data analysis tool by running the 'data_analysis_tool.m' file followed by pressing 'Load batch .mat file(s)'