Fundamental Roles of Chromatin Loop Extrusion in Antibody Class Switching

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμconstant region exons (C(H)s) with one of 6 C(H)s lying 100-200kb downstream(1). Each C(H) is flanked upstream by an I-promoter and long repetitive switch (S) region(1). 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 interactions(3-8). 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 motifs(9-11). 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 CSR(12). AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining(11). Here, we report that chromatin loop extrusion underlies the "unprecedented" mechanism(11) 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.