Person:

Zhang, Xuefei

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμ constant region exons (CHs) with one of six CHs lying 100–200 kb downstream1. Each CH is flanked upstream by an I promoter and long repetitive switch (S) region. Cytokines and activators induce activation-induced cytidine deaminase (AID) and I-promoter transcription, with 3′ IgH regulatory region (3′ IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3′ IgHRR interactions. Transcription through donor Sμ and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an Sμ-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining. Here we report that chromatin loop extrusion underlies the mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3′ IgHRR enhancers with the 200-kb upstream Sμ to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with Sμ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with Sμ in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

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.