Open AccessSingle molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation
Author(s) -
Hadeel Khamis,
Sergei Rudnizky,
Philippa Melamed,
Ariel Kaplan
Publication year - 2021
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkab843
Subject(s) - biology , methylation , dna methylation , transcription factor , transcription (linguistics) , sequence motif , dna , binding site , dna binding site , cpg site , genetics , promoter , microbiology and biotechnology , gene , gene expression , linguistics , philosophy
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.