G-quadruplex landscape and its regulation revealed by a new antibody capture method

Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggests that these structures form at a large number of regions with no such canonical G4-forming signatures. New functions, such as regulation of gene expression regulation and replication have been attributed to the genomic G4s. Many G4-binding proteins have been described, but their roles in formation and maintenance of G4s remain unknown. The representation of G4s not bound to proteins in these genomic landscapes remains unaddressed. We have developed a simple variation of the conventional G4-chromatin immunoprecipitation (G4-ChIP) assay to capture G4s in the cells using the 1H6 antibody. A comparison with the conventional G4-ChIP and in vitro DNA pull-down assays shows that our method minimizes false G4 detection. We use this method along with a transfected or in vitro-added control DNA (containing canonical G4-forming sequences) to study the G4 regulatory effects of the CGG repeat-binding protein CGGBP1. We present a reliable G4 landscape and apply the new protocol to discover a functionally relevant regulation of G4s by CGGBP1. Our results show that sharp interstrand transitions of G/C-skew, rich in binding sites for CTCF and CGGBP1, form G4s. Thus, our method demonstrates a sequence property-specific constraints of the nuclear environment that mitigates G4 formation.