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

Oncotarget 2024 March 15
Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, the formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggest that these structures form at a large number of regions with no such canonical G4-forming signatures. Many G4-binding proteins have been described with no evidence for any protein that binds to and stabilizes G4s. It remains unknown what fraction of G4s formed in human cells are protein-bound. The G4-chromatin immunoprecipitation (G4-ChIP) method hitherto employed to describe G4 landscapes preferentially reports G4s that get crosslinked to proteins in their proximity. Our current understanding of the G4 landscape is biased against representation of G4s which escape crosslinking as they are not stabilized by protein-binding and presumably transient. We report a protocol that captures G4s from the cells efficiently without any bias as well as eliminates the detection of G4s formed artifactually on crosslinked sheared chromatin post-fixation. We discover that G4s form sparingly at SINEs. An application of this method shows that depletion of a repeat-binding protein CGGBP1 enhances net G4 capture at CGGBP1-dependent CTCF-binding sites and regions of sharp interstrand G/C-skew transitions. Thus, we present an improved method for G4 landscape determination and by applying it we show that sequence property-specific constraints of the nuclear environment mitigate G4 formation.

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