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.