Fundamental roles of chromatin loop extrusion in antibody class switching

Abstract

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμconstant region exons (CHs) with one of 6 CHs lying 100-200kb downstream1. Each CH is flanked upstream by an I-promoter and long repetitive switch (S) region1. Cytokines/activators induce Activation-Induced Cytidine Deaminase (AID)2 and I-promoter transcription, with 3’IgH regulatory region (3’IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3’IgHRR interactions3-8. Transcription through donor Sμ and an activated downstream acceptor S region targets AID-generated deamination lesions at, potentially, any of 100s of individual S region deamination motifs9-11. General DNA repair pathways convert these lesions to DSBs and join an Sμ upstream DSB-end to an acceptor S region downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here, we report that chromatin loop extrusion underlies the "unprecedented" mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3’IgHRR enhancers with the 200kb upstream Sμ to generate a CSR center ("CSRC"). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic sub-domains 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, while converting adjacent downstream sequences into an ectopic S region by, respectively, inhibiting or promoting their dynamic alignment with Sμ in the CSRC. 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.