CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells 1 . Upon binding a recombination centre-based J H , RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ H -based recombination centre 2 . CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning 3 – 5 ; however, their inactivation allows scanning to proximal V H s, where additional CBEs activate rearrangement and impede scanning any further upstream 5 . Distal V H utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction 6 – 8 . Here we test the potential of linear RAG scanning to mediate distal V H usage in G1-arrested v-Abl pro-B cell lines 9 , which undergo robust D-to-J H but little V H -to-DJ H rearrangements, presumably owing to lack of locus contraction 2 , 5 . Through an auxin-inducible approach 10 , we degraded the cohesin component RAD21 10 – 12 or CTCF 12 , 13 in these G1-arrested lines. Degradation of RAD21 eliminated all V(D)J recombination and interactions associated with RAG scanning, except for reecombination centre-located DQ52-to-J H joining, in which synapsis occurs by diffusion 2 . Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust V H -to-DJ H joining of, distal V H s, with patterns similar to those of ‘locus-contracted’ primary pro-B cells. Thus, downmodulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG scanning across the 2.7-Mb Igh locus.