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 cells¹. 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 centre². CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning^3-5; however, their inactivation allows scanning to proximal V_Hs, where additional CBEs activate rearrangement and impede scanning any further upstream⁵. 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 lines⁹, 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 approach¹⁰, 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 diffusion². 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_Hs, 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.