CTCF and R-loops are boundaries of cohesin-mediated DNA looping

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome or-ganization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA loop-ing. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to co-hesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

Automated summary

This study provides direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping and organization of the three-dimensional genome. It reveals that a specific motif of CTCF blocks cohesin translocation and DNA looping, and that CTCF polarity affects the compaction of DNA by cohesin. It also suggests that RNA-DNA hybrids may play a role in forming TAD boundaries and inhibiting DNA compaction by cohesin.