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

Transcription factors (TFs) such as the central DNA-binding hub in Notch signal transduction, RBPJ, bind to specific DNA sequences to regulate gene transcription. How the efficiency of gene regulation depends on the TF-DNA binding kinetics and cofactor interactions is mostly unknown. We determined the DNA binding kinetics and the transcriptional activity of RBPJ and several mutant variants by live-cell single-molecule tracking and reporter assays, and measured their genome-wide chromatin occupation by ChIP-Seq. We observed that cofactor binding, in addition to DNA binding, was required for target site specificity. Importantly, the target site search time of RBPJ was longer than its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired DNA binding, e.g. by mutation K195E related to Adams-Oliver-Syndrome, modulated not only dissociation, but also association to target sites. Impaired cofactor binding mainly altered the rates of unspecific binding and target site association. For other TFs, we also observed longer search than residence times, indicating that kinetic rather than thermodynamic stability of DNA binding might be a general feature of TFs in vivo. We propose that an effective in vivo binding energy landscape of TF-DNA interactions constitutes an instructive visualization of TF-DNA binding kinetics and the changes upon mutations.