Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster

In this study, Amandio et al. sought to understand the functions of the highly conserved CTCF sites distributed over the Hox gene clusters. The authors produced an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border, and this demonstrated the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border.