Abstract
DNA G-quadruplexes (G4s), formed by guanine-rich sequences in mammalian genomes, are non-canonical structures implicated in gene regulation. However, their strand-specific genomic distribution and mechanistic roles in transcription remain poorly understood. Here, we report a strand-specific G4 sequencing (ssG4-seq) method for global profiling of G4 structures across multiple mammalian genomes. This method faithfully recapitulates known G4 structures and identifies thousands of previously unannotated G4s in human K562 cells. Remarkably, over 95% of G4s are located at enhancers and promoters across species, with promoters containing dual-strand G4s exhibiting significantly stronger transcriptional activation compared to those with single-strand G4s. Mechanistically, we identify SP1 as a potent G4 reader that facilitates transcription by modulating enhancer-promoter chromatin looping. Furthermore, we demonstrate that cancer-associated mutations can destabilize G4 structures, impair SP1-mediated chromatin interactions, and contribute to tumorigenesis. Our study demonstrates the power of ssG4-seq in elucidating G4 functions in gene regulation and disease.