Following the binding and dissociation kinetics of unlabeled transcription factors at the single molecule level

Already from the seminal work of Jacob and Monod, a central regulatory role was recognized for the equilibrium occupancy of response elements by transcription factors (TFs), which depends on their concentration, affinity, and, in eukaryotes, the chromatin accessibility of their binding sites. However, recent studies have highlighted the importance of transient TF–DNA interactions, and indicate that the kinetics of TF binding and dissociation play an important role in regulating transcription in vivo. Their characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule optical-tweezers assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein’s binding properties, and demonstrate how the proposed approach can provide new insights into the function of transcription factors.