Modeling G4s in chromatin context confirms partial nucleosome exclusion and reveals nucleosome-disrupting effects of the least selective G4 ligands

G-quadruplexes (G4s) are gaining increasing attention as possible regulators of chromatin packaging, and robust approaches to their studies in pseudo-native context are much needed. Here, we designed a simple in vitro model of G4-prone genomic DNA and employed it to elucidate the impact of G4s and G4 -stabilizing ligands on nucleosome occupancy. We obtained two 226-bp dsDNA constructs composed of the strong nucleosome positioning sequence and an internucleosomal DNA-imitating tail. The tail was G4-free in the control construct and harbored a strong (stable) G4 motif in the construct of interest. An additional weak (semi-stable) G4 motif was found within the canonical nucleosome positioning sequence. Both G4s were confirmed by optical methods and 1H NMR spectroscopy. Electrophoretic mobility assays showed that the weak G4 motif did not obstruct nucleosome assembly, while the strong G4 motif in the tail sequence diminished nucleosome yield. Atomic force microscopy data and molecular modeling confirmed that the strong G4 was maintained in the tail of the correctly assembled nucleosome structure. Using both in vitro and in silico models, we probed three known G4 ligands and detected nucleosome-disrupting effects of the least selective ligand. Our results are in line with the negative correlation between stable G4s and nucleosome density, support G4 tolerance between regularly posi-tioned nucleosomes, and highlight the importance of considering chromatin context when targeting genomic G4s.(c) 2022 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Automated summary

G-quadruplexes (G4s) are potential regulators of chromatin packaging, and a new in vitro model was designed to study their impact on nucleosome occupancy. Stable G4 structures were found to decrease nucleosome formation, supporting the negative correlation between stable G4s and nucleosome density. This study emphasizes the importance of considering chromatin context when targeting genomic G4s.