An upstream secondary DNA motif within the IL3 insulator CTCF binding site is required for enhancer-blocking insulator activity.

CCCTC-binding Factor (CTCF) is the only known vertebrate protein that functions to organise the genome into distinct functional chromatin domains. A subset of CTCF sites also provide insulator activity and barrier activity to block inappropriate enhancer-promoter communication and spreading of histone modifications and non-coding transcription into loci where it is unwarranted. Paradoxically, other CTCF sites mediate enhancer and promoter communication, partly by supporting DNA loop extrusion within chromatin domains. Despite intensive study and abundant data, it remains poorly understood how CTCF directs these different functions. In this study we provide new data and mine published data that show that CTCF utilises zinc fingers 9-11 and an upstream DNA binding consensus sequence resembling CAGCTGTTCC to mediate high affinity binding and enhancer-blocking insulator activity. A single high affinity CTCF binding site from the IL3 locus is able block IL3 promoter activation by an upstream enhancer. Mutation of a CTGCAGCTTT sequence upstream of the CTCF core motif abolishes insulator activity. We propose that CTCF is able to confer insulator and barrier activity upon a specific subset of CTCF sites that contain the upstream DNA consensus binding motif, thereby allowing CTCF to function differently in different contexts.