Dual native G-quadruplex folding is associated with chromatin looping at the MYC locus.

DNA can adopt noncanonical four-stranded structures known as G-quadruplexes (G4s), which are enriched at regulatory regions and implicated in transcriptional control. However, the process by which native G4 folding contributes to enhancer-promoter communication and three-dimensional genome organization remains poorly understood. Here, we investigated the relationship between G4 folding, chromatin state and long-range regulatory interactions by integrating chromatin state maps, DNA methylation profiles, and BG4 ChIP-seq data across two closely related human keratinocyte models. To assess long-range regulatory contacts directly, we applied locus-specific chromosome conformation capture (3C-qPCR) at endogenous regulatory loci. Genome-wide analyses revealed that both folded and unfolded G4 loci, operationally defined by BG4 detection, are enriched at transcription start sites and active enhancer regions. However, unfolded G4s preferentially associate with weak enhancers, consistent with a poised regulatory state. Focusing on the endogenous MYC-PVT1 locus, enhancer-promoter contacts were detectable only in the cellular context where folded G4s were present at both the promoter and distal enhancers, independently of active histone marks alone. Together, these findings position this dual G4 folding as a genomic feature associated with enhancer-promoter communication and three-dimensional regulatory architecture.