CTCF directly binds G-quadruplex structures to regulate genome topology and gene expression

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 a proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in genomic regulators of nucleosome remodeling, paraspeckle assembly, RNA splicing, 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 developing new G4 mapping tools, we define a comprehensive catalog of genomic G4s and investigate their association with CTCF binding. Using genetic reconstitution of mouse embryonic stem cells with a G4-specific CTCF mutant, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Interestingly, our studies reveal a subset of G4-linked chromatin loop anchors that form "persistent loops" which are retained even upon CTCF depletion. Collectively, our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.