Molecular basis of CTCF binding polarity in genome folding

Abstract

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.

CTCF loss has limited effects on global genome architecture in Drosophila despite critical regulatory functions

“…Therefore, despite profound divergence, the fly CTCF N-terminus interacts directly with cohesin in vitro. This interaction was suggested to impart directionality to CTCF-dependent boundaries in mammalian cells 10 , 39 , but we find that CTCF has at best a very weak preference to establish directional boundaries (Supplementary Fig. 2g ) consistent with a previous study 2 .…”

“…A region in the N-terminus of human CTCF directly interacts with cohesin and stabilizes cohesin on DNA 10 , 39 , partly explaining how human CTCF forms CD boundaries. Vertebrate and fly CTCF N-termini are highly diverged, yet a 10 amino acid residue stretch in CTCF’s N-terminus that binds to cohesin in human cells is present at a similar distance from the zinc finger domain in fly CTCF 10 (boxed in Supplementary Fig.…”

“…Indeed, previous in silico simulations 6 and experiments affecting loop-extrusion processivity across CTCF-dependent boundaries in human cells 7 , 9 , 10 described CDs with weaker corner interactions more similar to domains observed in flies. The N-terminus of DNA-bound mammalian CTCF may stall or stabilize cohesin by directly interacting with cohesin subunits and regulators 10 , 39 , 52 , 53 via binding interfaces that are not all conserved in fly orthologs (Supplementary Fig. 2f ).…”

Multi-Dimensional Gene Regulation in Innate and Adaptive Lymphocytes: A View From Regulomes

“…Within the nucleus, the stretch of one-meter long DNA is segregated into active (euchromatin) and inactive (heterochromatin) territories, which are spatially organized into individual regulatory domains, designated topologically associating domains (TADs) (5,151,152). TADs are formed via an extrusion process mediated by a cohesin ring and blocked by two convergently orientated CCCTC-binding factor (CTCF) sites (Figures 2A, C) (2,133,134,(153)(154)(155)(156)(157)(158). CTCF is a chromatin FIGURE 2 | A model of rapid gene induction in NK cells through higher-order chromatin architecture and remodeling.…”

“…Hi-C plots allow for visualization of threedimensional TADs and sub-TADs, which form during the cohesin-mediated loop extrusion process. Looping can occur between two convergently oriented CCCTCbinding factor (CTCF) sites, using a cohesin ring that extrudes DNAs as shown in (A) (2,(133)(134)(135).…”