Functional dissection of regulatory landscapes reveals non-essential and instructive roles of TADs in regulating gene expression [Capture Hi-C-seq]

The genome is organized in megabase­sized three-dimensional units, called Topologically Associated Domains (TADs), that are separated by boundaries. TADs bring distant cis-regulatory elements into proximity, facilitated by the cooperative action of cohesin and the DNA binding factor CTCF. However, how TADs and their boundaries impinge on enhancer function remains an open question. Here, we investigate TAD function in vivo in mice at the Sox9/Kcnj locus. We find that TADs are formed by a redundant system of CTCF sites requiring the removal of all major sites within the TAD and at the boundary for two neighboring TADs to fuse. TAD fusion resulted in a low degree of regulatory spread from the Sox9 to the Kcnj TAD, but no major changes in gene expression, indicating that TAD structures provide robustness and precision, but are not essential for developmental gene regulation. Gene misexpression and consecutive disease phenotypes, however, were attained by re-directing regulatory activity through inversions and/or the re-positioning of boundaries. Thus, efficient re-wiring of enhancer promoter interaction and aberrant disease causing gene activation is not induced by a mere loss of insulation but requires the re-direction of contacts. We used Capture Hi-C enriching a 6 Mb region at the Sox9locus in Mus musculus to study the effects of CRISPR/Cas9-induced CTCF-site deletions, insertions, and inversions on the genomic landscape. We analysed wildtype and homozygous mutant limb buds (from 9 mutants) as singleton (limb buds of 3-8 embryos for each condition), as it was done in similar studies (See Franke et la., Nature, 2016 or Bianco et al., Nat. Gen. 2018). Embryonic E12.5 limb tissue was used.