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

RAG endonuclease initiates V(D)J recombination in progenitor (pro)-B cells(1). Upon binding a recombination center (RC)-based J(H), RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ(H)-based RC(2). CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG-scanning(3–5); but 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 RC access 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 due to lack of locus contraction(2,5). Through an auxin-inducible approach(10), we degrade the cohesin-component Rad21(10–12) or CTCF(12,13) in these G1-arrested lines. Rad21 degradation eliminated all V(D)J recombination and RAG-scanning-associated interactions, except RC-located DQ52-to-J(H) joining in which synapsis occurs by diffusion(2). Remarkably, while CTCF degradation suppressed most CBE-based chromatin interactions, it promoted robust RC 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, down-modulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG-scanning across the 2.7Mb Igh locus.