G4 quadruplex landscape and its regulation revealed by a new antibody capture method

Currently, the identification of G4 quadruplexes in vivo depends on a published and widely used G4-ChIP protocol (PMID: 29470465) that uses the anti-G4 antibody to captures the quadruplexes like regular chromatin. This method is challenged by some false positives and false negatives in G4-quadruplex capture. We have developed an antibody capture G4-ChIP (AbC G4-ChIP) method to overcome these challenges. The AbC G4-ChIP achieves (i) minimized in vitro artifacts during the incubation steps, and (ii) capture of G4-quadruplexes which are not protein-bound. We have applied the AbC G4-ChIP to study the regulatory effect of a CGG-repeat binding protein CGGBP1 on G4-quadruplex formation. The AbC G4-ChIP identifies inter-strand G/C-skew as a sequence property associated with CGGBP1-regulated G4-quadruplexes. Overall design: The antibody capture G4-ChIP (AbC G4-ChIP) employs permeabilization of fixed cells in presence of a buffer (Tris-KCl-Triton X100) such that 1H6 antibody could capture endogenous DNA G4-quadruplexes. The antibody-G4-quadruplex interaction is then crosslinked and pulled down. The AbC G4-ChIP ensures (i) minimization of the presence of in vitro G4-quadruplex artifacts (as false positives) formed during the assay, and (ii) loss of endogenous-formed G4-quadruplex (as false negatives) due to melting during crosslinking. The AbC G4-ChIP profile has been compared against both in vitro and conventional G4-ChIP profiles. The regulatory effect of CGG-repeat binding protein CGGBP1 has been studied in presence and absence of endogenous levels of CGGBP1. The functional relevance of genome-wide profile of CGGBP1-dependent G4-quadruplex regions has been analyzed.