In vivo binding free energy landscape reveals kinetic control of transcription factor function

Transcription factors such as RBPJ in Notch signal transduction bind to specific DNA sequences and initiate either repression or activation of genes. Which sites they select and how often and long they bind affects the efficiency of gene regulation. To resolve the underpinnings of RBPJ-DNA binding, we determined the in vivo binding free energy landscape of RBPJ using live-cell single-molecule tracking and genome-wide chromatin immunoprecipitation. Importantly, DNA binding of RBPJ was thermodynamically unstable in vivo and instead governed by the binding kinetics: Cofactors contributed to target site specificity by tuning both association and dissociation of unspecific binding, while mutation K195E underlying Adams-Oliver-Syndrome destabilized specific DNA binding by mainly altering the association rate. We showed thermodynamic instability in vivo also for other transcription factors, indicating that kinetic rather than thermodynamic control of DNA binding might be a general feature of transcription factors in vivo. ### Competing Interest Statement The authors have declared no competing interest.