R-loop associated G-quadruplexes reinforce CTCF interactions

We report that three-stranded R-loop structures facilitate CTCF binding genome-wide through the formation of G-quadruplex (G4) structures. Genome-wide assays of CTCF and R-loop formation under various treatment conditions reveal that R-loop removal and G4 stabilization can both alter CTCF occupancy, and these changes are associated with alterations in gene expression and chromatin organization. CUT&RUN, MapR, RNA-Seq, ChIP, and Hi-C was performed on E14 and EN52.9.1 mouse embryonic stem cells. Cells were untreated, treated with 150 U Rnase H for 1 hour during CUT&RUN and MapR protocol, or treated with auxin, pyridostatin, PhenDC3, and/or actinomycin D prior to genomics experiments.