Accepted_test

In eukaryotes, replication domains coincide with topologically associating domains, which are established in a CTCF-dependent manner during the G1 phase of the cell cycle. We argue that G-quadruplexes (G4s), noncanonical nucleic acid structures observed in both G1 and S phases, may contribute to the establishment of chromatin domains and their subsequent dynamics that determine the accessibility of replication origins according to a recent model of replication initiation. These arguments are based on the enrichment of G4 sites at chromatin domain boundaries and intra-domain regulatory regions, including the hotspots of enhancer-promoter interactions, such as super-enhancers (SEs). However, little direct evidence for the involvement of G4s in chromatin organization or remodeling has been reported. To partially fill this gap, we verified three steps of these processes, namely CTCF recruitment to G4s, nucleosome exclusion around G4s, and phase separation at nucleosome-depleted G4-rich SEs. The representative G4s of CTCF-bound sites and SEs were determined based on chromatin immunoprecipitation and sequencing data. Their interactions with CTCF and the SE marker BRD4 were assessed using microscale thermophoresis or surface plasmon resonance-based methods. Nucleosome exclusion around G4s in a simplified chromatin model was confirmed by the electrophoretic method. Phase separation in BRD4 solutions containing G4 DNA was visualized by microscopic imaging. Our results confirm that stable G4s facilitate CTCF recruitment and support efficient demarcation of topologically associating domains/replication domains with G4-rich boundaries. Our results also suggest the importance of G4s for the dynamics of transcriptionally active chromatin domains, namely phase-separation-dependent enhancer-promoter communication.