Ph.D. Graduate Seminar-Hadeel Khamis
13/01/2021 13:00


A single molecule study of protein-DNAinteractions


Bindingof transcription factors (TFs) to regulatory regions in the genome is crucialfor the initiation of transcription, and thus one of the most important layersin the regulation of gene expression. Most TFs regulate multiple genes andinduce responses that are gene-specific and vary between cell types anddevelopmental stages, suggesting that the mere binding of the TF to the DNAcannot explain this diversity of outcomes. In our work, we developed a set ofnovel optical tweezers assays to study how binding of Egr1, a TF harboring 3zinc finger motifs, is tuned by variations in the binding site sequence,sequences flanking the binding site, DNA methylation and the structure ofchromatin. Our results show that Egr1 binds to a “consensus” binding site in avery stable conformation, but variations in the core-site, as observed invivo,weaken the protein-DNA interactions, resulting in a wide spectrum of structuresand binding energies, that are also sensitive to variations in the flankingsequences. Moreover, we found that DNA methylation at the binding sitesignificantly affects the affinity of the protein and the kinetics of binding,in a sequence- and position-specific manner. Methylation changes the breathingkinetics of the DNA itself, suggesting that the methylation effect on Egr1binding is mediated by local structural changes in the DNA. Finally, we studiedEgr1 binding in the vicinity of a nucleosome that covers the binding site. Wefound that incorporation of the histone variant H2A.Z resulted in an increasein nucleosome diffusion, which increased Egr1 binding. Together, our findingsreveal novel regulatory mechanisms to fine-tune the expression of genes. 

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