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

Using an auxin-inducible approach, the authors show that downmodulation of CTCF activity promotes cohesin-driven RAG endonuclease scanning, and thus V(D)J recombination, across theIghlocus. The RAG endonuclease initiatesIghlocus 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-scaleIghlocus contraction(6-8). Here we test the potential of linear RAG scanning to mediate distal V(H)usage in G1-arrestedv-Ablpro-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-MbIghlocus.