The Journal of Experimental Medicine
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Published 1 March 2004. doi:10.1084/jem.20031935
Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 199, Number 5, 617-627
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Mapping of a Functional Recombination Motif that Defines Isotype Specificity for µ->{gamma}3 Switch Recombination Implicates NF-{kappa}B p50 as the Isotype-specific Switching Factor

Amy L. Kenter, Robert Wuerffel, Carmen Dominguez, Ananth Shanmugam, and Hongmei Zhang

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612

Address correspondence to Amy L. Kenter, Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612-7344. Phone: (312) 996-5293; Fax: (312) 996-6415; email: star1{at}uic.edu

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 µ->{gamma}3 and µ->{gamma}1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An S{gamma}3/S{gamma}1 substrate containing only three S{gamma}3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for µ->{gamma}3 CSR. This functional recombination motif colocalized with a binding site for NF-{kappa}B p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based µ->{gamma}3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Sµ and S{gamma}3 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 Sµ/S{gamma}3 junctions. The data suggest that p50 may be the isotype-specific factor in µ->{gamma}3 CSR and epistatic with Msh2.

Key Words: AID • B lymphocyte • immunoglobulin • NF-{kappa}B p50 • class switch

The online version of this article contains supplemental material.

A. Shanmugam's present address is Box 8134, Dept. of Psychiatry, Washington University, 660 S. Euclid Ave., Saint Louis, MO 63110-1010.

Abbreviations used in this study: {alpha}{delta}dex, anti–{delta} dextran; AID, activation-induced deaminase; CSR, class switch recombination; DC, Digestion circularization; DSB, double strand break; FRM, functional recombination motif; gt, germline transcription; MMR, mismatch repair; nAChR, nonrearranging acetylcholine receptor; RRL, relative recombination level; TR, tandem repeat.


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