Large-scale genomic reorganizations of topological domains (TADs) at the HoxD locus (4C-Seq)

The transcriptional activation of Hoxd genes during mammalian limb development involves dynamic interactions with the two Topologically Associating Domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior Hoxd genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts. To assess the structure-function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH. We challenged the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel the long-range contacts. We report multi-partite associations between Hoxd genes and up to three enhancers and show that breaking the native chromatin topology leads to the remodelling of TAD structure. Our results reveal that the re-composition of TADs architectures after severe genomic re-arrangements depends on a boundary-selection mechanism that uses CTCF-mediated gating of long-range contacts in combination with genomic distance and, to a certain extent, sequence specificity. Overall design: 4C-seq analysis of Hoxd13 and Island-II 3D interactions in proximal and distal limbs from E12.5 wt or mutant embryos