Mapping of a Functional Recombination Motif that Defines Isotype Specificity for μ→γ3 Switch Recombination Implicates NF-κB p50 as the Isotype-specific Switching Factor

Ig class switch recombination (CSR) requires expression of activation-induced deaminase (AID) and production of germline transcripts to target S regions for recombination. However, the mechanism of CSR remains unclear. Here we show that an extrachromosomal S plasmid assay is AID dependent and that a single consensus repeat is both necessary and sufficient for isotype-specific CSR. Transfected switch substrates specific for mu-->gamma3 and mu-->gamma1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An Sgamma3/Sgamma1 substrate containing only three Sgamma3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for mu-->gamma3 CSR. This functional recombination motif colocalized with a binding site for NF-kappaB p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based mu-->gamma3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Smu and Sgamma3 relative to wild-type cells, indicating a function for p50 in the mechanics of CSR. We note a striking parallel between the affects of p50 and Msh2 deficiency on Smu/Sgamma3 junctions. The data suggest that p50 may be the isotype-specific factor in mu-->gamma3 CSR and epistatic with Msh2.