Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (CH) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream CH genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction 1 . Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (γ-H2AX, also known as γ-H2afx), which facilitate DNA double-strand break (DSB) repair 2-4 , form nuclear foci at the CH region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX -/-mice. Localization of Nbs1 and γ-H2AX to the IgH locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sμ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.Correspondence and requests for materials should be addressed to A.N. (andre_nussenzweig@nih.gov) or M.C.N. (nussen@mail.rockefeller.edu).. Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Competing interests statementThe authors declare that they have no competing financial interests. To determine whether DNA repair factors associate with DSBs at the switch regions, we first examined the intracellular localization of γ-H2AX, Nbs1, Rad51 and Brca1 in activated B cells by immunofluorescence. Brca1 and Rad51 are required for homologous recombination, the Mre11-Rad50-Nbs1 complex has been implicated in both homologous recombination and non-homologous end-joining (NHEJ), and γ-H2AX is critical for recruiting these repair factors to DSBs 6 and facilitates NHEJ in Saccharomyces cerevisiae 4 . All four proteins showed diffuse nuclear staining in most of the resting B cells from C57BL/6 wild-type mice. High local concentrations of these factors (nuclear foci) were detected in a very small percentage of cells (<5%), which increased significantly when the cells were stimulated to undergo CSR in vitro with lipopolysaccharide (LPS) and interleukin (IL)-4 (Fig. 1a). After 3 days of stimulation, 37% of the B cells contained discrete Brca1 foci (12 ± 6 per cell) and 43% contained Rad51 foci (7 ± 3 per cell), consistent with previous results 7 ; the remaining cells exhibited a weak, diffuse pattern of nuclear staining (Fig. 1a). Many of the stimulated B cells also formed Nbs1 foci (32% contained, on average, 3 ± 2 per cell) and γ-H2AX foci (40% contained, on average, 4.5 ± 3 per cell). To determine which of these repair factors are co-localized in activated B cells, we performed two colour immunofluoresence experiments (Fig. 1b). Only 20% of the cells that contained Rad51 and Nbs1 (n = 687) or Brca1 and Nbs1 foci (n = 431) exhibited co-localization. In contrast, γ-H2AX foci co-localiz...

Changes in chromatin structure induced by posttranslational modifications of histones are important regulators of genomic function. Phosphorylation of histone H2AX promotes DNA repair and helps maintain genomic stability. Although B cells lacking H2AX show impaired class switch recombination (CSR), the precise role of H2AX in CSR and somatic hypermutation (SHM) has not been defined. We show that H2AX is not required for SHM, suggesting that the processing of DNA lesions leading to SHM is fundamentally different from CSR. Impaired CSR in H2AX−/− B cells is not due to alterations in switch region transcription, accessibility, or aberrant joining. In the absence of H2AX, short-range intra-switch region recombination proceeds normally while long-range inter-switch region recombination is impaired. Our results suggest a role for H2AX in regulating the higher order chromatin remodeling that facilitates switch region synapsis.

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