Functional dissection of the Sox9–Kcnj2 locus identifies nonessential and instructive roles of TAD architecture

Abstract

The genome is organized in three-dimensional units called topologically associating domains (TADs), through a process dependent on the cooperative action of cohesin and the DNA-binding factor CTCF. Genomic rearrangements of TADs have been shown to cause gene misexpression and disease, but genome-wide depletion of CTCF has no drastic effects on transcription. Here, we investigate TAD function in vivo in mouse limb buds at the Sox9– Kcnj2 locus. We show that the removal of all major CTCF sites at the boundary and within the TAD resulted in a fusion of neighboring TADs, without major effects on gene expression. Gene misexpression and disease phenotypes, however, were achieved by redirecting regulatory activity through inversions and/or the repositioning of boundaries. Thus, TAD structures provide robustness and precision but are not essential for developmental gene regulation. Aberrant disease-related gene activation is not induced by a mere loss of insulation but requires CTCF-dependent redirection of enhancer–promoter contacts. Removal of boundary and intra-TAD CTCF-binding sites at the Sox9– Kcnj2 locus in mice leads to TAD fusion but no major changes in gene expression. Gene misexpression and disease phenotypes were obtained through inversions and/or repositioning of TAD boundaries.