A proteomics screen uncovers CTCF as a key G-quadruplex binding protein [CUT&Tag]

DNA G-quadruplexes (G4s) are non-B form secondary DNA structures that are highly conserved across evolution. G4 structures occupy key regulatory sites in the mammalian genomes and are implicated in several cellular processes. However, the mechanisms by which G4s contribute to distinct facets of genome function are not well understood. Here, we conduct an unbiased proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in key genomic regulators involved in regulation of nucleosome remodeling, paraspeckle assembly, RNA splicing, transcriptional elongation, and 3D genome organization. Among the most prominent hits, we identify the genomic architectural protein, CTCF as one of the strongest G4 binders. Building on this discovery, we perform extensive biochemical validation of CTCF-G4 interaction and identify CTCF mutants, with pronounced affinity for G4s over its consensus DNA motif. By implementing well-established approaches and newly developed G4 mapping tools, we define a comprehensive catalog of genomic G4s and study their association with CTCF binding. Furthermore, using genetic reconstitution studies with specific CTCF mutants, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.