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

RAG endonuclease initiates V(D)J recombination in progenitor (pro)-B cells1. Upon binding a recombination center (RC)-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin2-10, to locate Ds and assemble a DJH-based RC11. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG-scanning12-15; but their inactivation allows scanning to proximal VHs where additional CBEs activate rearrangement and impede scanning any further upstream15, 16. Distal VH utilization is thought to involve diffusional RC access following large-scale Igh locus contraction17-23. Here, we test the potential of linear RAG-scanning to mediate distal VH usage in G1-arrested, v-Abl-pro-B cell lines24, 25, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably due to lack of locus contraction11, 15. Through an auxin-inducible approach26, 27, we degrade the cohesin-component Rad214, 7, 27 or CTCF7, 9 in these G1-arrested lines, which maintain substantial viability throughout four-day experiments. Rad21 degradation eliminated all V(D)J recombination and RAG-scanning-associated interactions, except RC-located DQ52-to-JH joining in which synapsis occurs by diffusion11. Remarkably, while CTCF degradation suppressed most CBE-based chromatin interactions, it promoted robust RC interactions with, and robust VH-to-DJH joining of, distal VHs, 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.