Xu, W.; Cui, J.; Zhang, S.; LU, L.; Liu, X.; Li, Y.; Jin, F.

CTCF forms the boundaries of topologically associating domains (TADs) but their insulating function in transcription is controversial. Here we report that functional insulators (FINs) shall be determined not only by their ability to form static loops or boundaries, but also by the dynamic context if their flanking sequences form new loops upon boundary loss. We performed a meta-analysis of nine independent Hi-C and micro-C datasets upon acute CTCF- or cohesin protein-depletion and found that only at several hundred loci, newly gained enhancers-promoters (E-P) loops can be reproducibly observed upon CTCF depletion. The new E-P loops are cohesin-dependent and associated with recurrent gene activation. These findings allow us to map FINs and define their direct target genes genome-wide. FINs are mostly present in euchromatin, do not overlap with TAD boundaries, and can be perturbed by Wapl-depletion or CTCF-displacement. We therefore conclude that FINs, but not TAD boundaries, are bona fide insulators.