Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

Abstract

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule 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 on the function of transcription factors.

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor

“…This is a particular issue for the determination of binding kinetics, which are highly sensitive to concentration variations between measurements. Methods have been put forward to perform binding experiments in parallel, including spatially separating substrates, employing multichannel microfluidic cells, or combining sm-TIRF with in situ single-molecule DNA sequencing . However, both the complexity of existing methods and their limitations in the number of binding substrates and applicable experimental conditions prompted us to develop a novel approach.…”

The role and interaction between transcription factor NAC‐NOR and DNA demethylase SlDML2 in the biosynthesis of tomato fruit flavor volatiles

“…Given a highly complex process for the interaction of TFs with their binding sites in DNA, methylation of the sequence further increases the complexity of the system. Recently, new approaches have been developed to quantitatively measure the contribution of DNA methylation on the interaction with TFs, indicating that DNA methylation state powerfully modulates binding and dissociation kinetics (Sabatucci et al ., 2020; Khamis et al ., 2021).…”