Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity

Recent studies have shown that the protein CTCF, which plays an important role in insulation and in large-scale organization of chromatin within the eukaryotic nucleus, depends for both activities on recruitment of the cohesin complex. We show here that the interaction of CTCF with the cohesin complex involves direct contacts between the cohesin subunit SA2 and specific regions of the C-terminal tail of CTCF. All other cohesin components are recruited through their interaction with SA2. Expression in vivo of CTCF mutants lacking the C-terminal domain, or with mutations at sites within it required for SA2 binding, disrupts the normal expression profile of the imprinted genes IGF2-H19 and also results in a loss of insulation activity. Taken together, our results demonstrate that specific sites on the C terminus of CTCF are essential for cohesin binding and insulator function. The only direct interaction between CTCF and cohesin involves contact with SA2, which is external to the cohesin ring. This suggests that in recruiting cohesin to CTCF, SA2 could bind first and the ring could assemble subsequently.     

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF,WAPL, and PDS5 proteins

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome‐wide function in mediating long‐range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.