Data from: Effective In vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding
Data files
Jan 15, 2025 version files 574.43 MB
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README.md
2.46 KB
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Single_molecule_data.zip
574.43 MB
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 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.
README: Effective In vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding
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)) using the 'File > Save batch file as...' function and merged using the 'File > 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 > 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)'