Antibody class switching is mediated by somatic recombination between switch regions of the immunoglobulin heavy chain gene locus. Targeting of recombination to particular switch regions is strictly regulated by cytokines through the induction of switch transcripts starting 5′ of the repetitive switch regions. However, switch transcription as such is not sufficient to target switch recombination. This has been shown in mutant mice, in which the I-exon and its promoter upstream of the switch region were replaced with heterologous promoters. Here we show that, in the murine germline targeted replacement of the endogenous γ1 promoter, I-exon, and I-exon splice donor site by heterologous promoter and splice donor sites directs switch recombination in activated B lymphocytes constitutively to the γ1 switch region. In contrast, switch recombination to IgG1 is inhibited in mutant mice, in which the replacement does not include the heterologous splice donor site. Our data unequivocally demonstrate that targeting of switch recombination to IgG1 in vivo requires processing of the Iγ1 switch transcripts. Either the processing machinery or the processed transcripts are involved in class switch recombination.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by predisposition to hematopoietic malignancy, cell-cycle checkpoint defects, and ionizing radiation sensitivity. NBS is caused by a hypomorphic mutation of the NBS1 gene, encoding nibrin, which forms a protein complex with Mre11 and Rad50, both involved in DNA repair. Nibrin localizes to chromosomal sites of class switching, and B cells from NBS patients show an enhanced presence of microhomologies at the sites of switch recombination. Because nibrin is crucial for embryonic survival, direct demonstration by targeted deletion that nibrin functions in class switch recombination has been lacking. Here, we show by cell-type-specific conditional inactivation of Nbn, the murine homologue of NBS1, that nibrin plays a role in the repair of ␥-irradiation damage, maintenance of chromosomal stability, and the recombination of Ig constant region genes in B lymphocytes.Nijmegen breakage syndrome ͉ B lymphocytes ͉ DNA repair

Ig class switch recombination occurs in B lymphocytes upon activation, and is targeted to distinct switch (S) regions by cytokine-mediated induction of switch transcripts spanning the entire S region and the adjacent constant region gene segments. Using a novel type of switch recombination substrate, constructed according to the intron-exon structure of the IgH locus, but with heterologous elements, we here have tested the structural requirements for targeting and the kinetics of switch recombination in activated primary murine B cells. When transfected at various times after activation, up to 10% of the transfected B cells perform recombination of the substrate within 12 h. Switch recombination in primary B cells is restricted to the first 72 h after onset of activation, then rapidly decreases to background levels, as obtained in plasmacytoma cells or with substrates carrying no S region sequences. In terms of structural requirements, switch recombination is targeted to any transcription unit that contains an intronic S region and depends on processing of the primary transcript by splicing.

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