Person:

Kyritsis, Nia

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 cohesin, to locate Ds and assemble a DJH-based RC2. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG-scanning3-5; but their inactivation allows scanning to proximal VHs where additional CBEs activate rearrangement and impede scanning any further upstream5. Distal VH utilization is thought to involve diffusional RC access following large-scale Igh locus contraction6-8. Here, we test the potential of linear RAG-scanning to mediate distal VH usage in G1-arrested v-Abl-pro-B cell lines9, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably due to lack of locus contraction2,5. Through an auxin-inducible approach10, we degrade the cohesin-component Rad2110-12 or CTCF12,13 in these G1-arrested lines. 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 diffusion2. 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.

RAG endonuclease initiates IgH V(D)J recombination in pro-B cells by binding a JH-recombination signal sequence (RSS) within a recombination center (RC) and then linearly scanning upstream chromatin, presented by cohesin-mediated loop extrusion, for convergent D-RSSs1,2. Utilization of convergently-oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. RAG scanning from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5. Primary pro-B cells undergo a mechanistically-undefined VH locus contraction proposed to provide distal VHs access to the DJH-RC6-9. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both utilization of cryptic VH-locus RSSs normally in opposite orientation and RAG scanning beyond the VH locus through multiple convergent-CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary pro-B cells promotes loop extrusion-mediated RAG VH locus-scanning. We further found that expression of Wapl10, a cohesin-unloading factor, is low in primary pro-B cells versus v-Abl-transformed pro-B lines that lack contraction and RAG-scanning of the VH locus. Correspondingly, Wapl depletion in v-Abl-tranformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.