Nibrin Functions in Ig Class-Switch Recombination

Nibrin functions in Ig class-switch recombination

Sven Kracker*, Yvonne Bergmann*, Ilja Demuth†‡, Pierre-Olivier Frappart§, Gabriele Hildebrand†, Rainer Christine¶, Zhao-Qi Wang§, Karl Sperling†, Martin Digweed†ʈ, and Andreas Radbruch*ʈ**††

*German Rheumatism Research Center, Schumannstrasse 21-22, 10117 Berlin, Germany; †Institute of Human Genetics, Charite´Medical University, Augustenburger Platz 1, 13353 Berlin, Germany; §International Agency for Research on Cancer, Lyon 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France; ¶Amaxa GmbH, Nattermannallee 1, 50829 Cologne, Germany; and **Experimental Rheumatology, Charite´, Humboldt University, Schumannstrasse 21-22, 10117 Berlin, Germany

Communicated by Klaus Rajewsky, Harvard Medical School, Boston, MA, December 9, 2004 (received for review July 16, 2004) Nijmegen breakage syndrome (NBS) is a rare autosomal recessive Materials and Methods disorder characterized by predisposition to hematopoietic malig- Animals. Mice with a targeted mutation of the Nbn gene of nancy, cell-cycle checkpoint defects, and ionizing radiation sensi- 129͞Sv mice were generated as described in detail elsewhere tivity. NBS is caused by a hypomorphic mutation of the NBS1 gene, (14), by insertion of lox P sequences upstream and downstream encoding nibrin, which forms a protein complex with Mre11 and of exon 6 of Nbn (Nbnlox-6) in E 14.1 ES cells, injection of Rad50, both involved in DNA repair. Nibrin localizes to chromo- NbnWT/lox-6 and NbnWT/⌬6 E 14.1 cells, the later being obtained somal sites of class switching, and B cells from NBS patients show from the former by transient expression of Cre (pMC-Cre), into an enhanced presence of microhomologies at the sites of switch C57BL͞6 blastocysts and mating of the resulting chimera to ⌬6/⌬6 recombination. Because nibrin is crucial for embryonic survival, 129͞Sv mice. Offspring with Nbn genotypes was completely lox-6/⌬6 direct demonstration by targeted deletion that nibrin functions in absent when Nbn mice were interbred, indicating that Nbn class switch recombination has been lacking. Here, we show by is functionally inactivated by deletion of exon 6 (14). For Cre-mediated deletion of exon 6 of Nbn in Nbnlox-6/lox-6 B cell-type-speciﬁc conditional inactivation of Nbn, the murine ho- lox-6/lox-6 mologue of NBS1, that nibrin plays a role in the repair of ␥-irra- lymphocytes, Nbn mice were crossed with heterozygous CD19-Cre mice of C57BL͞6 background, a kind gift of K. Rajewsky diation damage, maintenance of chromosomal stability, and the (Harvard Medical School, Boston) (15). In all experiments, het- recombination of Ig constant region genes in B lymphocytes. erozygous NbnWT littermates served as controls. Animals were maintained and bred in specific-pathogen-free facilities. ͉ ͉ Nijmegen breakage syndrome B lymphocytes DNA repair CD19-Cre efficiency was determined by quantitative multiplex PCR, specific for the floxed exon 6, deleted exon 6 and WT Nbn he nuclear complex of Mre11, Rad50, and nibrin (also known alleles, as described elsewhere in detail (14), using the primers Tas Nbs1 or p95) is relevant for chromosomal stability, meiotic flox-forward (5Ј-GCTTGGCTCAAGTAGTACTG-3Ј), del-͞WT- recombination, and telomere maintenance in eukaryotic cells (1–3), forward (5Ј-ATAAGACAGTCACCAC-3Ј), and the correspond- and the targeted inactivation of Mre11 (4), Rad50 (5), or nibrin (3) ing, fluorescein-conjugated reverse primer (5Ј-fluorescein- causes embryonic lethality in the mouse. In humans, two related TTATGTCACTGAGGACCTCC-3Ј). PCR products were chromosome instability disorders, Nijmegen breakage syndrome separated on a 10% polyacrylamide gel, and quantified in a vistra (NBS) and Ataxia-Telangiectasia-like disorder (ATLD) are caused FluorImager SI (Amersham Pharmacia Bioscience, Freiburg, Ger- by mutation of the genes encoding nibrin and Mre11, respectively. many) and IMAGE QUANT software (Amersham Pharmacia Bio- More than 90% of NBS patients are homozygous for a 5-bp deletion science). in exon 6 of the NBS1 gene. This mutation leads to a premature Deletion of Nbn Exon 6 by tat-Cre-Fusion Protein in Vitro. Recombi- termination of translation that results in a 27-kDa amino-terminal nant tat-Cre fusion protein was purified from extracts of Escherichia peptide and a 70-kDa carboxyl-terminal peptide because of an coli bacteria transformed with a vector encoding a His-tat-nuclear alternative initiation of translation (6). Because the mutations in the localization sequence-Cre fusion protein as described (16), except NBS patients and of MRE11 in ATLD are hypomorphic, it is that all buffers contained 500 mM NaCl. The vector was a kind gift difficult to deduce the exact role of nibrin and Mre11 in DNA repair of F. Edenhofer (University of Bonn, Bonn) and K. Rajewsky. The (7). NBS patients are characterized by predisposition to hemato- purified tat-Cre protein was stored in 500 mM NaCl͞20 mM Hepes poietic malignancy, cell-cycle checkpoint defects, and ionizing (pH 7.4) at 80 ␮MatϪ70°C. For Cre-loxP-mediated deletion of radiation sensitivity of fibroblasts and lymphoblastoid cells. A exon 6 of Nbn in vitro,2.5ϫ 106 B lymphocytes of genotypes as characteristic, variable deficiency of serum IgG and IgA with indicated, purified by magnetic depletion of CD43ϩ cells from normal IgM levels is observed (8, 9). Individual S␮–S␣ switch- spleen cells (see below), were incubated for1hat37°C in 1 ml of recombination junctions of Ig class-switched B lymphocytes from serum-free RPMI medium 1640, with 1.5 ␮M tat-Cre fusion protein NBS and ATLD patients show a preponderance of microhomolo- added. For control, (untreated) cells were incubated alike, adding gies at the site of recombination (9, 10). This observation could be 0.5 M NaCl͞20 mM Hepes (pH 7.4) instead of the tat-Cre stock explained by the direct involvement of nibrin in the recombination solution. Cells were then washed with PBS with 0.5% BSA and of Ig constant region genes, by impaired survival or proliferation of activated (see below). activated B lymphocytes, or by impaired T cell help. In two murine models for NBS with hypomorphic mutations, in which Nbn exons Activation of B Lymphocytes in Vitro and by Cytometric Analysis. B 2and3orNbn exons 4 and 5 are replaced by a neo gene, the total lymphocytes were isolated from murine spleens by magnetic IgG levels in the serum are indistinguishable from normal controls

(11, 12); however, a defect in the T cell-dependent B cell activation Abbreviations: NBS, Nijmegen breakage syndrome; LPS, lipopolysaccharide; CFSE, carboxy- is observed (11). Nevertheless, an argument in favor of a direct ﬂuorescein diacetate succinimidyl ester. involvement of nibrin in switch recombination is the colocalization ‡Present address: Benaroya Research Institute at Virginia Mason, Seattle, WA 98101. of nibrin foci with the IgH locus in activated B lymphocytes (13). ʈM.D. and A.R. contributed equally to this work. Here, we show by conditional inactivation of Nbn in murine ††To whom correspondence should be addressed at: Deutsches Rheuma-Forschungszen- B lymphocytes, that nibrin plays a role in Ig class-switch trum, Schumannstrasse 20-21, 10117 Berlin, Germany. E-mail: [email protected]. recombination. © 2005 by The National Academy of Sciences of the USA

1584–1589 ͉ PNAS ͉ February 1, 2005 ͉ vol. 102 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409191102 Downloaded by guest on September 23, 2021 depletion of other cells with CD43-specific magnetic microbeads Analysis of Nibrin Expression. Whole-cell extracts were prepared (Miltenyi Biotec, Bergisch Gladbach, Germany). When indi- from Histopaque-purified cells (Sigma-Aldrich, Munich) at differ- cated, the cells were then pretreated with tat-Cre fusion protein ent time points of culture and total cell lysates equivalent to 5 ϫ 105 as described above. cells were loaded onto a 10% SDS-polyacrylamide gel followed by For activation of splenic B cells for antibody class switching, the electrophoresis and immunoblotting, as described (19). Nibrin and cells were stimulated with bacterial lipopolysaccharide (LPS; 40 Mre11 proteins were detected with rabbit antibodies specific for the ␮g͞ml, Sigma–Aldrich, St. Louis) or LPS and IL-4 (X63-IL4 carboxyl terminus of Nbs1 (nibrin 15 CR) (20) and Mre11-specific supernatant, 20 ng͞ml final concentration of IL-4) as described rabbit antibodies (Novus Biologicals, Littleton, CO). Rabbit anti- (17). Cell division was tracked according to loss of carboxyfluores- bodies were visualized on the blot with horseradish peroxidase- cein diacetate succinimidyl ester (CFSE) label (17). Cells were conjugated goat anti-rabbit Fab-fragment, and ECL detection stained immediately after magnetic purification with 1 ␮M CFSE reagents (Amersham Pharmacia Bioscience). (Molecular Probes) in PBS for 2 min and 45 sec at room temperature. Cell numbers were determined by counting Trypan blue- Analysis of Switch Recombination Junctions. Genomic DNA was prepared from day 3 LPS͞IL-4 culture after proteinase K digestion. stained cells in a Neubauer chamber, or cytometrically, by refer- ␮ ␥ encing their frequencies to defined numbers of TrueCount beads S -S 1 junctions were amplified, cloned, and sequenced as described in refs. 21 and 22. The junctions were either amplified by (Becton Dickinson). Viability was determined cytometrically ac- using an Expand long template PCR system (Roche) with primers cording to light scatter and exclusion of propidium iodide (see and conditions as described in ref. 22 and cloned with a TOPO-TA below). cloning kit (Invitrogen) before sequencing, or as described in ref. 21 B lymphocytes in spleens of transgenic mice were analyzed with a nested PCR by using homemade Taq-polymerase and cytometrically by staining spleen cells with anti-B220 conjugated to cloning into pBluescript after digestion with EcoRI. Switch junc- Cy5 (CD45R and RA3.6B2) and anti-IgM conjugated to phyco- tions were identified by a BLAST search. erythrin (R6–60.2, Pharmingen). To determine the frequencies of ͞ ⌬ activated B cells expressing IgG1, CFSE-labeled, LPS IL4- Karyotyping. Nbnlox-6/ 6 B cells were incubated with either tat-Cre stimulated B cells were harvested on day 3 of culture and stained or with dialysis buffer as control and stimulated with LPS͞IL-4. On for expression of IgG1 on the cell surface, with digoxigenin-labeled day 3 of culture, cells were irradiated as indicated and cultured monoclonal rat anti-mouse IgG1 [0.5 mg͞ml;akindgiftofM. further for3hat37°C.Fortheanalysis of chromosomal breaks, 60 Assenmacher (Miltenyi Biotech) and H. Schliemann (German ng͞ml colcemid was then added (KaryoMax colcemid solution, Rheumatism Research Center)] and anti-digoxigenin Fab conju- Gibco͞BRL), and the cells were incubated for another 1 h. Meta- gated to Cy5 (Roche, Mannheim, Germany). To determine the phase spreads were prepared after hypotonic shock and stained frequencies of activated B cells expressing IgG3, CFSE-labeled, with Giemsa by using standard technology (23). For assignment of LPS-activated B cells of day 3 cultures were stained for expression chromosome breaks to individual chromosomes, metaphase of IgG3 on the cell surface with biotinylated rat anti-murine IgG3 spreads were prepared without colcemid, the chromosomes were (R40–82) (Pharmingen) and streptavidin-Cy5 (Roche). Cells were partially digested with trypsin, and stained with Giemsa (23). analyzed by using a FACSCalibur cytometer (Becton Dickinson, Identification of chromosomes according to Giemsa-banding pat- Heidelberg) and CELLQUEST and FCS-EXPRESS software. Viable terns was performed by following procedures in ref. 23. cells were gated according to forward and sideward light scatter, and exclusion of propidium iodide (1 ␮g͞ml in PBS͞BSA). All Quantification of I␮-C␮ and I␥1-C␥1 Transcripts. Total RNA was stainings were performed in the presence of 50 ␮g͞ml rat mono- prepared with RNeasy (Qiagen, Hilden, Germany) from B cells clonal antibody against mouse CD16͞32 (2.4G2). activated for 2 days with LPS͞IL-4, and transcribed into cDNA with a 1:1 ratio mixture of oligo(dT) and random hexamer primers Irradiation. To determine irradiation sensitivity, Nbnlox-6/⌬6 and (TaqMan reverse transcription reagent; Applied Biosystems). The lox-6/WT cDNA was used for real-time PCR (LightCycler, FastStart DNA Nbn B cells were labeled with CFSE and treated with tat-Cre IMMUNOLOGY or control buffer, stimulated for 3 days with LPS͞IL-4, harvested, master SYBR green I kit, Roche Diagnostics, Penzberg, Germany). ␮ ␥ aliquoted, and ␥-irradiated, as indicated, in RPMI medium 1640 Specificity of the , 1, and GAPDH primers (22) was confirmed by analysis of dissociation curves. The efficiency of amplification with 10% FCS on ice, in a Cs-137 irradiator (BIOBEAM 2000, ␮ ␮ ␥ ␥ MCP Medical International, Berlin). The cells were cultured for per round of PCR was 1.92, 1.95 and 1.96 for I -C ,I 1-C 1, and another 2 days in their original culture supernatant supplemented GAPDH, respectively. with another volume of fresh RPMI medium 1640 with 10% FCS Detection of ␥-H2AX Foci. Activated B cells were fixed for 10 min at before their numbers and CFSE label were analyzed cytometrically. room temperature in PBS with 4% formaldehyde, washed twice in PBS, and permeabelized with 0.1%Triton X-100 in PBS for 5 min Detection of Deletion of Exon 6 of Nbn in Individual B Cells. Individual on ice. The cells were washed again and blocked with 0.1% B cells were labeled as described above and sorted by fluorescence- BSA͞0.1% Triton X-100͞PBS for 20 min at 37°C. ␥-H2AX foci activated single-cell sorting on a Cytomation MoFlow sorter (Cy- were stained with ␥-H2AX-specific rabbit antibodies (Trevigen, tomation, Freiburg, Germany) into wells of 96-well plates (Eppen- Gaithersburg, MD) for1hat37°CandthenwithCy2-conjugated dorf). For detection of both floxed and deleted exon 6 Nbn genes goat anti-rabbit IgG (Dianova, Hamburg) for 1 h at 37°C. DNA was by nested single-cell PCR, as described earlier (18), the primer stained with DAPI, 1 ␮M, in PBS for 5Ј on ice. The cells were Ј Ј combinations nbs-up1: 5 -TGCTGGCTATGTGAAGACTA-3 , cytocentrifuged onto slides and analyzed by fluorescence micros- Ј Ј nbs-down2: 5 -CTTCCAATAGCTGGTTCATC-3 , and nbs- copy (Zeiss Axioplan, Oberkochen, Germany). down4: 5Ј-CCTGGGATGAAAGTGTGTTC-3Ј were used for an initial amplification of genomic DNA. Floxed Nbn exon 6 was then Results amplified specifically with the primers nbs-up2 (5Ј-GATTCGT- Nibrin-Deficient, Activated B Lymphocytes Show Increased Sensitivity GAATGTAGTGCTG-3Ј) and nbs-down1 (5Ј-AGTGACTGAT- to ␥-Irradiation. Disruption of exon 6 of the Nbn gene by insertion ACCAAAAGGG-3Ј). Nbn genes with deleted exon 6 were ampli- of a neomycin-resistance gene (Nbnins6) leads to embryonic lethality fied with the primers nbs-up2 (5Ј-GATTCGTGAATG- (20). Here, we have analyzed mice with targeted insertion of LoxP TAGTGCTG-3Ј) and nbs-down3 (5Ј-AATACAGTGACTCCTG- sites flanking exon 6 of the Nbn gene (Nbnlox-6), allowing Cre- GAGG-3Ј). mediated conditional inactivation of Nbn in the germ-line or

Kracker et al. PNAS ͉ February 1, 2005 ͉ vol. 102 ͉ no. 5 ͉ 1585 Downloaded by guest on September 23, 2021 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 LPS͞IL4- activated Nbnlox-6/⌬6 B lymphocytes, not treated or treated with tat-Cre in vitro, at the indicated time points after activation. Cell extracts of 5 ϫ 105 cells each were submitted to Western blot analysis, as described in Materials and Methods.

somatic cells (Nbn⌬6;Fig.1A–C). Offspring with Nbn⌬6/⌬6 geno- 1 as compared with Mre 11, and was nearly absent on days 2 and types was completely absent when Nbnlox-6/⌬6 mice were interbred, 3 (Fig. 1C). As expected, tat-Cre-mediated inactivation of Nbn indicating that Nbn is functionally inactivated by deletion of exon in activated B cells leads to an increased sensitivity of the cells 6 (14). to ␥-irradiation-induced DNA damage on day 3 of culture, To determine the role of Nbn in activation and differentiation reflected by dose-dependent impairment of proliferation (Fig. of B cells, naı¨ve B lymphocytes were isolated from the spleens 2A) and an enhanced chromosomal instability (Fig. 2B). How- of Nbnlox-6/⌬6 mice, and labeled with CFSE, to track the prolif- ever, the spontaneous chromosomal instability observed is not as erative history of switched cells. Nbn exon 6 was deleted from the drastic as observed by Reina-San-Martin et al. (25). floxed alleles by incubation of the cells with tat-Cre fusion protein (16). The cells were then activated with bacterial LPS for Ig Class Switching Is Impaired by Inactivation of Nbn. In the absence 3 days, in the presence or absence of IL-4, to target switch of intentional irradiation, tat-Cre-mediated conditional inactivation recombination to IgG1 or IgG3, respectively (24). The efficiency of Nbn in LPS- or LPS͞IL-4-activated B cells did not impair their of tat-Cre-mediated deletion of Nbn exon 6 was determined by survival, as indicated by the numbers of viable cells between days single-cell PCR analysis for the deleted versus the floxed allele. 1, 2, and 3 of culture (Fig. 5, which is published as supporting In Ͼ75% of Nbnlox-6/⌬6 B cells, exon 6 was deleted from the information on the PNAS web site) and the frequencies of dead floxed allele 3 days after tat-Cre treatment (Fig. 1B). Without cells according to propidium iodide staining (Table 3 and Fig. 6A, tat-Cre, Nbn exon 6 was detectable in Ͼ95% of the cells, which are published as supporting information on the PNAS web indicating the efficiency of the single-cell PCR. On the protein site), nor was proliferation changed detectably, as measured by loss level, expression of p95͞nibrin was significantly reduced on day of CFSE-staining (Fig. 3A). Inactivation of Nbn did affect antibody

Fig. 2. Radiation sensitivity and chromosomal instability of nibrin-deﬁcient B lymphocytes. (A) Radiation sensitivity of Nbnlox-6/⌬6 and NbnWT/⌬6 B lymphocytes after deletion of exon 6 of Nbn by tat-Cre in vitro. Cells were labeled with CFSE and activated with LPS͞IL4 for 3 days. Cells were then not irradiated intentionally (black line), or ␥-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 LPS͞IL4-activated Nbnlox-6/⌬6 B lymphocytes after treatment with tat-Cre in vitro.Onday3of culture, metaphase spreads were prepared from cells 3 h after ␥-irradiation (1 Gy). Metaphases were Giemsa-stained and scored for aberrant chromosome numbers and apparent chromosomal breaks (ϪCre͞ ϪGy, 102 metaphases analyzed; ϪCre͞ϩGy, 59 metaphases; ϩCre͞ϪGy, 236 metaphases; ϩCre͞ϩGy, 138 metaphases).

1586 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409191102 Kracker et al. Downloaded by guest on September 23, 2021 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 Nbnlox-6/⌬6 B lymphocytes analyzed on day 3 of LPS͞IL-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 Nbnlox-6/⌬6 (white bars) and NbnWT/lox-6 (black bars) B cells switched to IgG1 upon activation by LPS͞IL-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 ϭ 6 for Nbnlox-6/⌬6 and n ϭ 4 NbnWT/lox-6)orLPS͞IL4 cultures (n ϭ 7 for Nbnlox-6/⌬6 and n ϭ 5 NbnWT/lox-6; P Ͻ 0.01, Mann–Whitney U test). Flow cytometric analysis of surface IgG1 (C) and surface IgG3 expression by CFSE-labeled Nbnlox-6/⌬6 B lymphocytes (D) treated with tat-Cre (white bars) or not treated (black bars), after 3 days of LPS͞IL-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 LPS͞IL4 or LPS cultures. (E) Efficiency of depletion of exon 6 of Nbn in vivo, in CD19-Cre Nbnlox-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 Nbnlox-6/lox-6 (solid line) and CD19-Cre Nbnlox-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 Nbnlox-6/lox-6 (white bars) and CD19-Cre Nbnlox-6/WT B lymphocytes (black bars) stimulated with LPS͞IL-4 for 3 days. The frequency of IgG1ϩ 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 LPS͞IL4 cultures. IMMUNOLOGY class switching, in that the frequencies of activated B cells that had location of breakpoints within the switch regions (Fig. 7, which is switched to IgG3 or IgG1, respectively, were reduced by Ͼ50% in published as supporting information on the PNAS web site) nor the cells of all generations in a given stimulation on day 3 (Fig. 3 B–D). frequency and average length of microhomologies and insertions at The similar reduction in switched cells over all generations suggests the switch-recombination sites (Table 1) differed between tat-Cre that nibrin is directly involved in the process of switch recombina- treated and nontreated cells. The frequencies of mutations in the tion, rather than in the survival of switched cells. In support of this vicinity of switch junctions (Ϯ 10 bp) was higher in tat-Cre-treated hypothesis, on day 3 of LPS͞IL-4 culture, Ϸ80% of tat-Cre-treated Nbnlox-6/ins6 (3.3 ϫ 10Ϫ2) than in nontreated cells (1.8 ϫ 10Ϫ2) but Nbnlox-6/⌬6 B cells that had switched to IgG1 had deleted exon 6 of was also not statistically significant (P ϭ 0.108). Nbn (data not shown). In an attempt to delete the Nbn gene before the onset of Sequences of switch recombination breakpoints, isolated from activation of B cells, Nbn was inactivated early in B cell ontogeny tat-Cre-treated Nbnlox-6/ins6 populations, show no statistically signif- in vivo by CD19-Cre-mediated deletion of exon 6 in CD19-Cre͞ icant difference when compared with breakpoints of switched Nbnlox-6/lox-6 and CD19-Cre͞Nbnlox-6/WT B cells, and splenic B cells Nbnlox-6/ins6 cells not treated with tat-Cre (Table 1). Neither the from such mice were activated with LPS or LPS͞IL-4 in vitro.

Table 1. S␮–S␥1 switch recombination junctions of tat-Cre-treated Nbnlox-6͞ins6 cells Percentage of junctions Average length Average length with Ն 2-bp of perfect of imperfect Percentage of junctions Average length Cells homology* match, bp match,† bp with Ն 1-bp insertion of insertion, bp

Nbnins6͞lox-6 (n ϭ 19) 63 2.3 3.4 11 1.5 Nbnins6͞lox-6, tat-Cre-treated (n ϭ 41) 46 2.9 3 24 1.2

*Percentages of perfect matches over Ն 2 bp: 47% of Nbn versus 22% of Nbn, tat-Cre-treated. †Percentage of perfect matches over Ն 2 bp matches, one mismatch.

Kracker et al. PNAS ͉ February 1, 2005 ͉ vol. 102 ͉ no. 5 ͉ 1587 Downloaded by guest on September 23, 2021 Table 2. Induction of switch transcripts Mouse I␮–C␮ transcripts versus I␥1–C␥1 transcripts versus Culture Id. Genotype GAPDH transcripts* GAPDH transcripts* Percent IgG1 cells†

43 CD19-Cre NbnWT/lox-6 24.3 4.1 9.4 I 44 CD19-Cre Nbnlox-6/ins6 20.7 4.1 3.2 42 CD19-Cre Nbnlox-6/lox-6 21.8 4.3 4.3 31 CD19-Cre NbnWT/ins6 14.6 2.4 27.3 II 35 CD19-Cre Nbnlox-6/ins6 19.4 7.2 7.9 33 CD19-Cre Nbnlox-6/lox-6 16.7 4.3 4.2 51 CD19-Cre NbnWT/lox-6 23.3 10.0 4.8 III 52 CD19-Cre Nbnlox-6/ins6 21.9 8.4 2.5 46 CD19-Cre Nbnlox-6/lox-6 22.1 7.5 1.8

Id., identiﬁcation. *RT-PCR analysis was performed on day 2 after LPS͞IL-4 activation. †IgG1 analysis was performed on day 3 after LPS͞IL-4 activation.

Frequencies and absolute numbers of splenic B cells were not cytes. It acts downstream of switch transcript-induced targeting of affected by CD19-mediated inactivation of Nbn (Fig. 8, which is switch regions (26, 27) and activation-induced cytidine deaminase published as supporting information on the PNAS web site). More (AID)-induced DNA breaks in the targeted switch regions (28). than 80% of the Nbn alleles of CD19-Cre͞Nbnlox-6/lox-6 B lymphocytes had been inactivated in vivo, corresponding to at least 60% of cells deficient for Nbn (Fig. 3E). On day 3 of culture, 70% of the floxed Nbn alleles of IgG1ϩ cells were deleted, as compared with 90% of IgG1Ϫ B cells (data not shown). When compared with CD19-Cre͞Nbnlox-6/WT B cells, CD19-Cre͞Nbnlox-6/lox-6 cells showed diminished proliferative capacity (Fig. 3F), and cell counts were Ϸ30–50% lower on day 3 of stimulation (Table 3). The frequencies of cells expressing IgG1 were consistently reduced by 50–70% in all generations of the activated B cells (Fig. 3G), confirming the role of nibrin in switch recombination.

Inactivation of Nbn Affects Late but Not Early Stages of Switch Recom- bination. B lymphocytes isolated from CD19-Cre͞NbnWT/ins6, CD19-Cre͞NbnWT/lox-6, CD19-Cre͞Nbnlox-6/lox-6, and CD19-Cre͞ Nbnlox-6/ins6 mice were activated with LPS͞IL-4, and after 48 h, the I␮–C␮ and I␥1–C␥1 switch transcripts were quantified by RT-PCR to determine the initial targeting of the S␮ and S␥1 switch regions for switch recombination. Activated B cells of all genotypes showed no detectable differences in expression of switch transcripts (Table 2). The subsequent induction of DNA breaks in switch regions of activated B cells is reflected by the formation of ␥-H2AX foci (13). This step was not impaired by inactivation of Nbn. Forty eight hours after onset of activation, Ͼ22% of CD19-Cre͞Nbnlox6/WT B cells showed one or two ␥-H2AX foci, as compared with 8% after 24 h, and 5% after 72 h. Less than 6% of the cells had three to seven foci at all times (Fig. 4). Of CD19-Cre͞Nbnlox6/lox6 B cells, 18% after 24 h, 20% after 48 h, and 16% after 72 h showed one or two ␥-H2AX foci, and 11% after 24 h, 15% after 48 h, and 6% after 72 h expressed three to seven foci, indicating an overall higher and persistent presence of DNA double-strand breaks, presumably, not only in switch regions but also in B cells lacking functional nibrin. Of 56 metaphase spreads derived from day 3 LPS͞IL4-activated CD19-Cre͞Nbnlox-6/lox-6 B cells, 8 metaphase spreads showed identifiable chromosome breaks. Four of the metaphase spreads could be attributed by differential Giemsa staining to the telomeric end of chromosome 12, i.e., the IgH locus (Fig. 9, which is published as supporting information on the PNAS web site). Metaphase spreads from activated Nbnlox-6/⌬6 B cells had not shown chromosome breaks at detectable frequencies Fig. 4. Generation and maintenance of ␥-H2AX foci in conditionally Nbn- ␥ (Fig. 2B). inactivated, activated B lymphocytes. -H2AX foci were stained with a speciﬁc antibody in CD19-Cre Nbnlox6/lox6 (black bars) and CD19-Cre Nbnlox6/WT B ͞ Discussion 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 micros- The present data strongly suggest that nibrin is directly involved in copy. (WT͞24 h, 976 cells; WT͞48 h, 213 cells; WT͞72 h, 379 cells; lox6͞24 h, 253 the process of class-switch recombination of activated B lympho- cells; lox6͞48 h, 169 cells; lox6͞72 h, 352 cells.)

1588 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409191102 Kracker et al. Downloaded by guest on September 23, 2021 The nibrin͞Mre11͞Rad50 complex could be targeted to sites of recombination, or whether alternative, nibrin-independent mech- action of AID by means of replication protein A, which can bind to anisms do exist. The extended microhomologies observed at class Mre11 (29), and to AID (30). So far, the exact function of nibrin in switch junctions of NBS patients (9, 10) could be interpreted as class-switch recombination is not clear. Because nibrin by itself has evidence in favor of alternate pathways, i.e., homology-directed no apparent enzymatic activity (7, 31), it has been speculated that recombination, most likely mediated by the mismatch repair system. it may be required to assemble Mre11 and Rad50 in the nucleus, Even there, the nibrin͞Mre11͞Rad50 complex could be involved, targeting them to staggered DNA ends of switch regions, and because direct interaction of Mre11 and Mlh1 has been reported perhaps, modulating their enzymatic activity (7, 31, 32). In vitro,the (36). Here, we do not find extended microhomologies in switch lox-6/ins6 nibrin͞Mre11͞Rad50 complex can bind to single-stranded or dou- recombination junctions of switched, tat-Cre-treated Nbn ble-stranded DNA, tether DNA ends (33), and process DNA as a cells, suggesting that these microhomologies might, rather, reflect 3Ј-5Ј exonuclease, endonuclease, and helicase (34). In class-switch the hypomorphic action of mutated nibrin in switching NBS B cells ͞ ͞ than the action of a mismatch-repair system. The observed persis- recombination, the nibrin Mre11 Rad50 complex may be involved ␥ in modification, realignment, and ligation of switch-region breaks, tence of -H2AX foci and chromosome 12 breaks in activated acting in concert with the Ku70͞80͞DNA-Pkc and the DNA- nibrin-deficient B cells support the notion that nibrin is required for efficient repair of nonhomologous DNA break recombination of ligase4͞XRCC4 complexes (35). class-switch regions. Conditional inactivation of Nbn in B cells activated in vitro and Ͼ In addition to the important role that nibrin plays in class-switch in vivo reduces the frequencies of switched cells by 50%. This recombination, it is also crucial for the survival of B lymphocytes. effect is direct, rather than indirect, because the survival of the This role did not affect our analysis of switch recombination, which activated B cells is not affected, as for tat-Cre-mediated Nbn was focused on short-lived plasma blasts and performed genera- inactivation, or only slightly affected, as for CD19-Cre-mediated tion-wise, but retarded proliferation and impaired survival of in inactivation. The survival of switched B cells as reflected by vitro-activated CD19-Cre͞Nbnlox-6/lox-6 B cells was observed. This propidium iodide uptake is not affected in either system. Further finding was not surprising, because Ͼ20% of activated B cells show evidence that proliferation and survival of switched cells is not obvious chromosome breaks on day 3 of culture, breaks that are selectively impaired is provided by the observation that the reduc- apparently not repaired efficiently, as reflected by their persistence tion in frequencies and numbers of switched cells was similar for all through mitosis and by the persistence of ␥-H2AX foci in activated generations of Nbn-inactivated B cells at the time of analysis. As B cells. The important role of nibrin in the repair of class-switch with tat-Cre-mediated inactivation of Nbn in vitro, CD19-Cre- recombination breaks, as demonstrated here, may be related to the mediated deletion of Nbn exon 6 in vivo resulted in an Ϸ50% preponderance of B cell lymphoma among NBS patients, Ϸ40% of reduction in the frequencies of switched cells. This result makes it whom develop this malignancy in early childhood (37). unlikely that all of the remaining switched cells had used residual functional nibrin for switch recombination although this result We thank F. Edenhofer for providing the His-tat-nuclear localization cannot formally be excluded for some cells. Because of technical sequence-Cre fusion protein expression plasmid, K. Rajewsky for pro- limitations of the systems used here for conditional inactivation of viding CD19-Cre mice, and Farah Hatam, Hyun-Dong Chang, Katharina Hein, and Uwe Niesner for discussion. We also thank L. Reiners- the Nbn gene, which does not generate homogeneous populations Schramm, H. Schliemann, T. Kaiser, K. Raba, and J. Radszewski for of Nbn inactivated B cells, the present results give only a minimum excellent technical assistance, and B. Reina-San-Martin, M. C. Nussen- estimate of the contribution of nibrin to switch recombination. It zweig, A. Nussenzweig, and S. Difilippantonio for discussions. This work remains to be shown whether nibrin is critical for successful switch was supported by SFB 577 of the Deutsche Forschungsgemeinschaft.

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