Cross-platform Hi-C meta-analysis identifies functional insulators that actively block enhancer-promoter interactions.

The function of topologically associating domain (TAD) boundaries as transcriptional insulators remains a fundamental controversy in genome biology. Here, we demonstrate that bona fide Functional Insulators (FINs) should be defined dynamically by their capability to block architectural rewiring. Leveraging DeepLoop to enable robust cross-platform Hi-C analysis, we performed a meta-analysis of nine high-resolution 3D genomic datasets following acute CTCF or cohesin depletion, mapping FINs genome-wide. We show that CTCF loss triggers the reproducible formation of new enhancer-promoter loops at only a few hundred specific loci. These loops are cohesin-dependent, enriched at G-rich cis-regulatory elements, and directly drive recurrent, early-response gene activation. Multiplexed CTCF-displacement assays functionally confirmed the causal role of FINs in these localized rewiring events. Crucially, FINs reside within active euchromatin, infrequently coincide with traditional TAD boundaries, and are sensitive to WAPL depletion. Our results reveal that the genome's functional insulation is mediated by these discrete, dynamically active sites rather than static TAD boundaries.