Nibrin functions in Ig class-switch recombination

Abstract

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 gamma-irradiation damage, maintenance of chromosomal stability, and the recombination of Ig constant region genes in B lymphocytes.

Fig. 1.

Generation of nibrin-deficient B lymphocytes. (A) Schematic representation of the targeted Nbn gene locus and the protein structure of nibrin. (B) Efficiency of tat-Cre-mediated Nbn exon 6 deletion. Nested single-cell PCR detects the deleted allele and the floxed exon 6 Nbn allele. Data represent the mean and SD of five individual cultures. (C) Expression of nibrin and Mre11 proteins in populations of LPS/IL4-activated Nbn^lox-6/Δ6 B lymphocytes, not treated or treated with tat-Cre in vitro, at the indicated time points after activation. Cell extracts of 5 × 10⁵ cells each were submitted to Western blot analysis, as described in Materials and Methods.

Fig. 2.

Radiation sensitivity and chromosomal instability of nibrin-deficient B lymphocytes. (A) Radiation sensitivity of Nbn^lox-6/Δ6 and Nbn^WT/Δ6 B lymphocytes after deletion of exon 6 of Nbn by tat-Cre in vitro. Cells were labeled with CFSE and activated with LPS/IL4 for 3 days. Cells were then not irradiated intentionally (black line), or γ-irradiated with 1.5 Gy (blue) or 3.6 Gy (green line). Two days later, proliferation and survival of the cells was analyzed cytometrically. Proliferative history of surviving cells was determined according to loss of CFSE label and numbers of surviving cells relative to reference beads (TrueCount, Becton Dickinson), standardizing the numbers of recorded viable cells not stained for propidium iodide. (B) Chromosomal instability of LPS/IL4-activated Nbn^lox-6/Δ6 B lymphocytes after treatment with tat-Cre in vitro. On day 3 of culture, metaphase spreads were prepared from cells 3 h after γ-irradiation (1 Gy). Metaphases were Giemsa-stained and scored for aberrant chromosome numbers and apparent chromosomal breaks (-Cre/-Gy, 102 metaphases analyzed; -Cre/+Gy, 59 metaphases; +Cre/-Gy, 236 metaphases; +Cre/+Gy, 138 metaphases).

Fig. 3.

Antibody class-switch recombination in B lymphocytes conditionally inactivated for Nbn in vitro or in vivo. (A) Proliferation according to loss of CFSE label of Nbn^lox-6/Δ6 B lymphocytes analyzed on day 3 of LPS/IL-4 activation, and treated with tat-Cre (solid line) or not treated (shaded line) in vitro, before the onset of activation. Data shown are from a representative experiment. (B) Comparison of Nbn^lox-6/Δ6 (white bars) and Nbn^WT/lox-6 (black bars) B cells switched to IgG1 upon activation by LPS/IL-4, or switched to IgG3 upon activation by LPS, in tat-Cre treated relative to cultures not treated with tat-Cre. Data represent the mean and SD of individual LPS (n = 6 for Nbn^lox-6/Δ6 and n = 4 Nbn^WT/lox-6)orLPS/IL4 cultures (n = 7 for Nbn^lox-6/Δ6 and n = 5 Nbn^WT/lox-6; P < 0.01, Mann–Whitney U test). Flow cytometric analysis of surface IgG1 (C) and surface IgG3 expression by CFSE-labeled Nbn^lox-6/Δ6 B lymphocytes (D) treated with tat-Cre (white bars) or not treated (black bars), after 3 days of LPS/IL-4 and LPS stimulation, respectively. The frequencies of IgG1- or IgG3-expressing cells are shown separately for each population that has undergone the indicated number of cell divisions since the onset of activation, according to CFSE staining. Data represent the mean and SE of four individual LPS/IL4 or LPS cultures. (E) Efficiency of depletion of exon 6 of Nbn in vivo, in CD19-Cre Nbn^lox-6/lox-6 B lymphocytes, detected by quantitative PCR specific for the deleted and nondeleted floxed Nbn alleles. (Data from three mice are represented.) (F) Proliferation of CD19-Cre Nbn^lox-6/lox-6 (solid line) and CD19-Cre Nbn^lox-6/WT B lymphocytes (shaded line) after activation with LPS for 3 days, according to loss of CFSE label. (G) Flow cytometric analysis of surface IgG1 expression on CFSE-labeled CD19-Cre Nbn^lox-6/lox-6 (white bars) and CD19-Cre Nbn^lox-6/WT B lymphocytes (black bars) stimulated with LPS/IL-4 for 3 days. The frequency of IgG1⁺ cells is shown for each population that had undergone the indicated number of cell divisions since the onset of activation, according to loss of CFSE label. Data represent the mean and SE of four individual LPS/IL4 cultures.

Fig. 4.

Generation and maintenance of γ-H2AX foci in conditionally Nbn-inactivated, activated B lymphocytes. γ-H2AX foci were stained with a specific antibody in CD19-Cre Nbn^lox6/lox6 (black bars) and CD19-Cre Nbn^lox6/WT B lymphocytes (white bars) after 24, 48, and 72 h of LPS/IL-4 activation. The samples were double-blinded and foci were scored by fluorescence microscopy. (WT/24 h, 976 cells; WT/48 h, 213 cells; WT/72 h, 379 cells; lox6/24 h, 253 cells; lox6/48 h, 169 cells; lox6/72 h, 352 cells.)