Mapping of a functional recombination motif that defines isotype specificity for mu-->gamma3 switch recombination implicates NF-kappaB 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.
Kenter
Amy L
AL
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612-7344, USA. star1@uic.edu
Wuerffel
Robert
R
Dominguez
Carmen
C
Shanmugam
Ananth
A
Zhang
Hongmei
H
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D017578:Immunoglobulin Class Switching; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D007142:Immunoglobulin gamma-Chains / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D020080:Tandem Repeat Sequences; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D007132:Immunoglobulin Isotypes; D007142:Immunoglobulin gamma-Chains; D007148:Immunoglobulin mu-Chains; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D004247:DNA; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase