Human antibody heavy chain variable region exon interacts with 3' regulatory region to form a stable somatic hypermutation center

Abstract

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) of immunoglobulin heavy chain (HC) variable region exons in germinal center (GC) B cells, allowing selection of affinity-matured B cell receptors. The V(D)J exon location is privileged for SHM targeting of diverse sequences within it. In analogy to the related HC class switch recombination center, we proposed that cohesin-mediated loop extrusion juxtaposes widely separated enhancers, including 3 Igh regulatory region ("3RR"), with the V(D)J exon in GC B cells to establish a SHM-center (SHM-C) "privileged" for AID access. To test this hypothesis, we employed a human RAMOS GC B lymphoma cell SHM model with inducible AID expression in which we targeted an unmutated mouse HC V(D)J exon in place of its human counterpart. Activation of AID expression in this cycling RAMOS line for 10 days led to robust SHM accumulation in the mouse V(D)J exon with a pattern similar to that in mouse GC B cells. In this model, the mouse V(D)J exon was indeed juxtaposed to the two human 3RRs and other elements consistent with SHM-C formation. While deletion of individual 3RRs had little effect, deletion of both greatly reduced SHM, but left V(D)J exon transcription unabated. Moreover, the SHM-C and SHM accumulation remained intact over a 10-day period in viably G1-arrested and viably G1-arrested RAD21 (cohesin)-depleted versions of the model. We discuss implications for SHM-C structure and function, its long-term functional stability in the absence of the loop extrusion process proposed to assemble it, and for 3RR SHM functions beyond transcriptional activation. Significance StatementUpon encountering pathogens, B cells enter specialized lymphoid structures known as germinal centers (GCs). GC B cells express activation-induced cytidine deaminase (AID) that initiates somatic hypermutation (SHM) of the portion of antibody genes that encodes antigen recognition. B cells in which mutations increase antibody affinity for activating pathogen are selected to enhance antibody responses. We proposed SHM occurs in a chromosomal structure in the antibody gene locus termed a SHM-center (SHM-C). By studying SHM in a human B cell lymphoma model, we provide direct evidence for existence of a SHM-C assembled from different functional components normally separated by considerable distances. We also find that the SHM-C is surprisingly stable and continues to function for many days in non-dividing cells.