The Variable CTCF Site from Drosophila melanogaster Ubx Gene is Redundant and Has no Insulator Activity

CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the surrounding chromatin. One such variable CTCF site (vCTCF) was found in the intron of the Ubx gene, in close proximity to the BRE and abx enhancers. CTCF binds to the variable site in tissues where Ubx gene is active, suggesting that the vCTCF site plays a role in facilitating contacts between the Ubx promoter and its enhancers. Using CRISPR/Cas9 and attP/attB site-specific integration methods, we investigated the functional role of vCTCF and showed that it is not required for normal Drosophila development. Furthermore, a 2161-bp fragment containing vCTCF does not function as an effective insulator when substituted for the Fab-7 boundary in the Bithorax complex. Our results suggest that vCTCF function is redundant in the regulation of Ubx.