Molecular Cell 21, 201–214, January 20, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.01.005

H2AX Prevents DNA Breaks from Progressingto Chromosome Breaks and Translocations

Sonia Franco,1 Monica Gostissa,1 Shan Zha,1

David B. Lombard,1,2 Michael M. Murphy,1

Ali A. Zarrin,1 Catherine Yan,1 Suprawee Tepsuporn,1

Julio C. Morales,3 Melissa M. Adams,3 Zhenkun Lou,4

Craig H. Bassing,5 John P. Manis,6 Junjie Chen,4

Phillip B. Carpenter,3 and Frederick W. Alt1,*1Howard Hughes Medical InstituteThe Children’s HospitalDepartment of GeneticsHarvard Medical School and theCBR Institute for Biomedical ResearchBoston, Massachusetts 021152Department of PathologyBrigham and Women’s HospitalBoston, Massachusetts 021153Department of Biochemistry and Molecular BiologyUniversity of Texas Health Sciences CenterHouston, Texas 772254Department of OncologyMayo ClinicRochester, Minnesota 559055Department of Pathology and Laboratory MedicineChildren’s Hospital of Philadelphia and theUniversity of Pennsylvania School of MedicineDepartment of Cancer Biology and theAbramson Family Cancer Research InstituteUniversity of Pennsylvania School of MedicinePhiladelphia, Pennsylvania 191046Joint Program in Transfusion Medicineand Department of Laboratory Medicine

Children’s HospitalDepartment of PathologyHarvard Medical SchoolBoston, Massachusetts 02115

Summary

Histone H2AX promotes DNA double-strand break(DSB) repair and immunoglobulin heavy chain (IgH)class switch recombination (CSR) in B-lymphocytes.CSR requires activation-induced cytidine deaminase(AID) and involves joining of DSB intermediates byend joining. We find that AID-dependent IgH locuschromosome breaks occur at high frequency in pri-mary H2AX-deficient B cells activated for CSR andthat a substantial proportion of these breaks partici-pate in chromosomal translocations. Moreover, acti-vatedB cells deficient for ATM, 53BP1, orMDC1, whichinteract with H2AX during the DSB response, showsimilarly increased IgH locus breaks and transloca-tions. Thus, our findings implicate a general role forthese factors in promoting end joining and therebypreventing DSBs from progressing into chromosomalbreaks and translocations. As cellular p53 status doesnot markedly influence the frequency of such events,our results also have implications for how p53 andthe DSB response machinery cooperate to suppress

generation of lymphomas with oncogenic transloca-tions.

Introduction

H2AX is a mammalian histone H2A variant that un-dergoes rapid phosphorylation of its carboxy-terminalSQE motif by ATM and other PIKK kinases to form g-H2AX over large chromatin domains surrounding DSBs(Rogakou et al., 1999). H2AX-deficient mammalian cellshave defective chromosomal DSB repair, marked ioniz-ing radiation sensitivity, and increased genomic instabil-ity (Bassing et al., 2002; Celeste et al., 2002), yet H2AX-deficient mice have little cancer predisposition. How-ever, combined deficiency for H2AX and the p53 tumorsuppressor, which monitors DSBs via cell cycle check-points and signals either arrest or apoptosis (Vogelsteinet al., 2000), leads to dramatically increased cancer inci-dence, including T and B lineage lymphomas (Bassinget al., 2003; Celeste et al., 2003a). With respect to DSBrepair defects, H2AX-deficient mammalian cells haveimpaired homologous recombination (Celeste et al.,2002; Xie et al., 2004). However, while H2A phosphoryla-tion in yeast has been shown to facilitate DSB repair vianonhomologous DNA end joining (NHEJ) (Downs et al.,2000), there has been no direct evidence for an H2AXrole in end joining in mammalian cells.

In developing lymphocytes, the RAG endonucleasegenerates DSBs at variable (V), diversity (D), and joining(J) gene segments, which are then joined by NHEJ to ef-fect V(D)J recombination (Dudley et al., 2005). WhileH2AX deficiency does not measurably impair V(D)Jrecombination in plasmid substrates (Bassing et al.,2002; Celeste et al., 2002), a role at the chromosomallevel is indicated by the occurrence of RAG-dependentg-H2AX foci at T cell receptor loci in developing T lym-phocytes (Chen et al., 2000) and findings that combineddeficiency for H2AX and p53 promotes B lymphomaswith translocations involving the immunoglobulin heavychain (IgH) JH locus (Bassing et al., 2003; Celeste et al.,2003a). The murine IgH locus (Igh) harbors a series ofeight Igh constant region (CH) exons (CH genes), embed-ded in an approximately 200 kb region downstream ofthe V, D, and J segments. Mature B cells can exchangethe CH gene first expressed in development (Cm) fora different, downstream CH gene (e.g., Cg, C3, Ca) viathe mature B cell-specific Igh class switch recombina-tion (CSR) process. CSR requires activation-induced de-aminase (AID) (Muramatsu et al., 2000), which is thoughtto lead to generation of DSBs in large, repetitive switch(S) regions that lie upstream of individual CH genes,with joiningof twodifferentS regionsviaend joining lead-ing to replacement of Cm with a downstream CH gene tocomplete CSR (Honjo et al., 2004). Notably, g-H2AXfoci appear at the IgH locus in an AID-dependent fashion(Petersen et al., 2001), andH2AXdeficiency substantiallyimpairs chromosomal CSR (Celeste et al., 2002). Defec-tive CSR in H2AX-deficient B cells has been suggestedto reflect a role for H2AX in promoting long-range synap-ses of different S regions (Reina-San-Martin et al., 2003).*Correspondence: alt@enders.tch.harvard.edu

However, some B-lineage lymphomas in mice withmutations in H2AX and p53 harbor clonal chromosomaltranslocationswithbreakpoints in ornearS regions, sug-gesting a potential role for H2AX in suppressing inappro-priate end joining during CSR (Bassing and Alt, 2004;Bassing et al., 2003).In response to DSBs, ATM also phosphorylates other

checkpoint/repair proteins including MDC1, 53BP1, andNBS1, which specifically bind the phosphorylated SQEmotif of H2AX (Kobayashi et al., 2002; Stewart et al.,2003; Stucki et al., 2005; Ward et al., 2003a). This pro-cess results in a rapid, hierarchical assembly of theseand other factors over g-H2AX and the formation of mul-tiprotein complexes (foci) around DSBs. Although gen-eration of g-H2AX is not essential for initial recruitmentof such factors to DSBs (Celeste et al., 2003b), it is re-quired for formation of normal, extended foci (Bassinget al., 2002; Celeste et al., 2002) and may function byproviding a docking site for DSB response factorsand/or by altering chromatin structure to allow their ac-cumulation (Celeste et al., 2003b; Fernandez-Capetilloet al., 2003). Recent studies have elucidated a complexinterplay between DSB response factors around DNAbreaks. For example, 53BP1 may sense changes inchromatin structure via ability to interact with constitu-tively methylated lysine 79 (K79) of histone H3, allowingit to operate in the DSB response both upstream anddownstreamof ATMactivation (Huyen et al., 2004). How-ever, accumulation of 53BP1 within DSB-induced focidepends both on g-H2AX and MDC1 (Bekker-Jensenet al., 2005; Celeste et al., 2003b; Fernandez-Capetilloet al., 2002; Stewart et al., 2003). In this regard, MDC1appears to interactwithg-H2AXandATM tocreate a pos-itive feedback loop that propagates H2AX phosphoryla-tion from the DSB site and amplifies the damage signal(Lou et al., 2006 [this issue of Molecular Cell]).The localization of DSB response factors to DSBs and

their accumulation in g-H2AX foci has been suggestedto function in cell cycle checkpoint responses, recruit-ment/activation of repair proteins, and/or tethering bro-ken DNA ends prior to repair (reviewed in Bassing andAlt [2004]). In this context, mice deficient for ATM,53BP1, NBS1, MDC1, and H2AX share a number of phe-notypes potentially related to the defective DSB re-sponse, including genomic instability, DNA repair de-fects, and radiation sensitivity. Moreover, defects inMDC1, ATM, 53BP1, and NBS1, like H2AX deficiency,all lead to impaired CSR, albeit to varying degrees(Kracker et al., 2005; Lumsden et al., 2004; Maniset al., 2004; Reina-San-Martin et al., 2004, 2005; Wardet al., 2004; Lou et al., 2006). The requirement for all ofthese DSB response factors for normal CSR raises thepossibility that they may have overlapping functions inthe synapses or joining phase of this process (Reina-San-Martin et al., 2003; Manis et al., 2004; Bassing andAlt, 2004; Fernandez-Capetillo et al., 2004), as outlinedabove for H2AX.Experimental systems in which molecular events on

both sides of a defined chromosomal DSB can be char-acterized in a temporal and spatial manner have yieldednovel insights into the function of the yeast H2AX homo-log (H2A), including the role of cohesin recruitment viaphosphorylated H2A in DSB repair via homologousrecombination (reviewed in van Attikum and Gasser

[2005]). However, as many repair factors that function-ally interact with g-H2AX in mammalian cells lack clearyeast homologs, it is unclear how far such studies canbe generalized. As a high percentage of activated B cellsundergo CSR over a few cell divisions, we theorized thatthis system could provide a useful experimental meansof tracking the fate of DNA ends on each side of a re-gion-specific DSB within a population of mammaliancells. Herein, we employ this approach to demonstratethat, in a process independent of p53, H2AX suppressesthe progression of AID-dependent DSBs at the IgH locusto chromosomal breaks and translocations. Moreover,we find that ATM, 53BP1, and MDC1 have similar func-tions in suppressing chromosomal breaks and translo-cations during CSR.

Results

Frequent Chromosome and Chromatid Breaksin H2AX2/2 B Cells Activated for CSRTreatment of cultured splenic IgM+ B cells with specificactivators (such as LPS or a-CD40) plus cytokines(such as IL-4) activates the cells to divide, upregulateAID expression, and, after several cell divisions over 3or 4 days, induce DSBs in targeted S regions and un-dergo CSR (Rush et al., 2005). Under such conditions,20%–30% or more of wild-type (wt) B cells may undergoCSR, as evidenced by surface expression of down-stream Igh isotypes such as IgG1 (see Figure S1 in theSupplemental Data available with this article online).The mouse IgH locus spans several megabases in thetelomeric portion of chromosome 12 with the variableregion gene (VH) segments lying closest to the telomereand the CH genes, which undergo CSR, lying more cen-tromeric (Figure 1A, left) (Riblet, 2004). Thus, fluores-cence in situ hybridization (FISH) analyses with probesthat recognize the telomeres themselves or sequencesjust upstream of the VH domain, along with a probe thatrecognizes sequences immediately downstream of theCH genes, allows testing for chromosomal breaks ortranslocations specifically within Igh in B cells activatedfor CSR (Figures 1A and 2A).

We first employed spectral karyotyping (SKY) anda FISH assay that combines DAPI staining with a telo-mere-specific peptide nucleic acid (PNA) probe (T-FISH)to assay metaphase chromosomes for instability in wtand H2AX2/2 B cells activated for CSR. Normal mousemetaphase chromosomes have four telomeres, withone at each end of the two chromatids, capping theirlong (q) and very short (p) arms (Figure 1A). Becausethe shortest mouse telomere is about 10 kbp and our as-say is sensitive to telomere signals below 1 kbp, we usu-ally detect the expected four telomere signals in everychromosome of a normal mouse metaphase. Loss ofa telomere signal on both chromatids of a given arm(a chromosome break) is consistent with replication ofa lesion that occurs in a prereplicative cell cycle phase,whereas loss of a telomere on a single chromatid ofa given arm (a chromatid break) most commonly repre-sents a postreplicative lesion.

Consistent with earlier findings on an independent lineof H2AX-deficient mice (Celeste et al., 2002), we find thatCSR in H2AX2/2 B cells is reduced to levels that are20%–50% those of wt (Figure S1). Strikingly, however,

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we further observed dramatically increased levels ofchromosomal aberrations in LPS- or a-CD40/IL-4-stim-ulated H2AX2/2 B cells, as compared to H2AX+/2 or wtcontrols (Table 1, Figure S2). These general chromo-

somal aberrations were nearly equally divided into chro-mosome and chromatid breaks (Table 1, Figure S2K),indicating that they represent defective repair of DSBsintroduced throughout the cell cycle. Overall, the levels

Figure 1. Sequential Telomere/Igh FISH in H2AX-Deficient B Cells Activated for CSR

(A) Schematic of themouse Igh and FISHprobes used for sequential telomere FISH (TTAGGGprobe) and FISHwith aBACspanning the 30Igh region(left diagram). The cytogenetic readout for an intact Igh or a chromosome break at Igh is illustrated on the right.(B and C) Partial metaphase spread of an H2AX2/2 B cell after 3 days of stimulation with a-CD40/IL-4. Loss of telomere signal (left panel, yellowarrow) with intact 30Igh BAC signal (middle panel, yellow arrow) indicates a chromosome break between the 30 end of Igh and the 12q telomere.A normal chromosome 12 with both telomere signals (left panel, white arrow) and 30Igh BAC signal (middle panel, white arrow) is shown for com-parison. An overlaywith diagrams is shown on the right panel. An example of ametaphasewith Ighbreaks at both chromosome 12s is shown in (C).(D) Quantification of the relative contribution of Igh breaks to total chromosome aberrations in LPS- or a-CD40/IL-4-stimulated H2AX2/2 B cellsor concanavalin A-stimulated H2AX2/2 T cells. Bars represent total chromosome or chromatid breaks as determined by telomere-FISH. AfterFISH with a 30Igh BAC, they were further classified as labeled with the 30Igh BAC near the break (suggesting a break within Igh, in black) ornot labeled with the 30Igh BAC (suggesting a break elsewhere, in white).

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of general chromosomal aberrations in H2AX-deficientB cells were at least as high as those observed in conca-navalin A-activated H2AX2/2 T lymphocytes assayed inparallel (Bassing et al., 2003; Celeste et al., 2003a) (Fig-ures S2A and S2B, Table 1). Finally, we sequentially an-alyzed these metaphase spreads via T-FISH (red signal)and then via FISH with a BAC (30Igh BAC) that specifi-cally recognizes sequences just beyond themost down-stream CH (Ca) gene (green signal) and thus would notbe altered or deleted byCSR (Figure 1A). These analysesrevealed that approximately one-third of all chromo-some, but not chromatid, breaks detected by T-FISHin H2AX2/2 B cells had a 30Igh locus BAC signal nearthe breakpoint (Figures 1B–1D, Table S1), suggesting

that a sizable percentage of chromosome breaks inH2AX2/2 B cells occurred within the IgH locus.

Frequent Chromosome Breaks at Igh in H2AX2/2 BCells Activated for CSRTo firmly establish and quantify apparent Igh chromo-some breaks in activated H2AX2/2 B cells, we per-formed two-color FISHwith the same 30IghBAC (labeledfor red signal), plus a second BAC (labeled for green sig-nal) that spans the region immediately telomeric to theVH locus (termed 50Igh BAC) (Figure 2A). The 50IgH BAClies just 50 of the most telomeric VH segment of the Ighlocus and, therefore, is not deleted or otherwise alteredby V(D)J recombination. In normal cells, both copies of

Table 1. T-FISH Analysis of DSB Repair-Deficient B and T Cells

GenotypeNumberof Mice Mitogen

Number ofMetaphasesAnalyzed

Number ofMetaphaseswith Aberrations(%)

Number of Cytogenetic Aberrations (%)

Number ofChromosomeBreaks

Number ofChromatidBreaks

Numberof OtherAberrations

Numberof TotalAberrations (%)

H2AX+/+ 3 LPS 90 1 (1.1%) 1 0 0 1 (1.1%)3 a-CD40/IL-4 90 2 (2.2%) 2 0 0 2 (2.2%)3 a-CD40 90 7 (7.8%) 3 0 4 7 (7.8%)3 Con A 90 1 (1.1%) 1 0 0 1 (1.1%)

H2AX+/2 3 LPS 90 4 (4.4%) 2 2 0 4 (4.4%)3 a-CD40/IL-4 90 2 (2.2%) 2 0 0 2 (2.2%)2 a-CD40 60 2 (3.3%) 2 1 0 3 (5%)3 Con A 91 (7.6%) 5 8 1 14 (15.2%)

H2AX2/2 3 LPS 259 62 (26.0%) 40 28 17 85 (35.3%)3 a-CD40/IL-4 130 50 (36.9%) 32 24 21 77 (61.2%)1 a-CD40 30 10 (33.3%) 6 9 2 17 (56.7%)3 Con A 90 31(23.3%) 23 9 9 41 (31.5%)

H2AX+/+/AID+/+ 2 LPS 100 4 (4%) 4 0 0 4 (4%)2 a-CD40/IL-4 100 2 (2%) 2 1 0 3 (3%)

H2AX+/+/AID2/2 2 LPS 100 2 (2%) 0 2 0 2 (2%)2 a-CD40/IL-4 100 0 (0%) 0 0 0 0 (0%)

H2AX2/2/AID+/+ 2 LPS 100 17 (17%) 19 1 0 20 (20%)2 a-CD40/IL-4 100 32 (32%) 28 6 2 36 (32%)

H2AX-/-/AID-/- 2 LPS 100 17 (17%) 13 8 0 21 (21%)2 a-CD40/IL-4 100 22 (22%) 17 9 2 28 (28%)

H2AX2/2/p532/2 2 LPS 60 2 (3.3%) 2 0 0 2 (3.3%)2 a-CD40/IL-4 60 4 (6.7%) 4 1 0 5 (8.3%)2 a-CD40 60 1 (1.7%) 1 0 0 1 (1.7%)2 Con A 60 1 (1.7%) 1 0 0 1 (1.7%)

H2AX2/2/p532/2 2 LPS 60 16 (26.7%) 15 3 5 23 (38.3%)2 a-CD40/IL-4 60 28 (46.7%) 27 9 6 42 (70.0%)2 a-CD40 60 18 (30%) 11 9 10 30 (50%)2 Con A 60 17 (28.3%) 13 5 6 24 (40.0%)

53BP1+/+ 2 LPS 80 3 (3.7%) 3 0 0 3 (3.7%)2 a-CD40/Il-4 80 4 (5.0%) 3 0 1 4 (5.0%)2 a-CD40 80 1 (1.2%) 1 0 0 1 (1.2%)

53BP12/2 5 LPS 170 23 (13.5%) 22 3 2 27(15.9%)5 a-CD40/Il-4 170 45 (26.5%) 43 6 8 57 (33.5%)5 a-CD40 170 8 (4.7%) 8 2 0 10 (5.9%)

ATM+/+ 2 LPS 60 2 (3.3%) 2 0 0 2 (3.3%)2 a-CD40/Il-4 60 4 (6.6%) 2 2 0 4 (6.6%)2 a-CD40 60 3 (5%) 1 1 1 3 (5%)2 Con A 60 1 (1.6%) 0 0 1 1 (1.6%)

ATM2/2 2 LPS 60 15 (25%) 15 1 1 17 (28.3%)2 a-CD40/Il-4 60 30 (50%) 41 8 6 55 (91%)2 a-CD40 60 14 (23.2) 21 6 1 28 (46.6%)2 Con A 60 17 (28.3%) 16 4 4 24 (40%)

MDC1+/+ 5 LPS 132 1 (0.8%) 1 0 0 1 (0.8%)MDC1+/2 1 LPS 30 0 (0%) 1 0 0 1 (3.3%)MDC12/2 7 LPS 240 44 (18.3%) 23 10 16 49 (20.4%)

Metaphases of B and T cells activated with the indicated mitogens were hybridized with a telomere probe, counterstained with DAPI, and an-alyzed for chromosome aberrations.

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chromosome 12 show red and green signals in the telo-meric region (diagrammed as ‘‘Intact Igh’’; Figure 2A;Table 2). These signals are visualized as two sets ofdots when the chromatids are well separated and some-times as a single set of dots when they are not well sep-arated (diagrammed in Figures 2B and 2C, respectively).Chromosome 12 breaks within the IgH locus result in lib-eration of a small telomeric portion of chromosome 12,containing the 50IghBAC, from the body of chromosome12. Moreover, in such IgH locus breaks, the 30Igh BACwill lie very near the breakpoint (Figure 2A; diagrammedas ‘‘Chromosome break at Igh’’). In such a situation, the

small telomeric fragment could be present elsewhere inthe metaphase or lost; in either case, we refer to a meta-phase harboring a chromosome with a 30Igh signal andlacking an adjacent 50Igh signal as having a ‘‘split IghBAC signal,’’ which represents our readout for a breakwithin Igh.

Strikingly, we detected split Igh BAC signals in about10% of the a-CD40/IL-4-stimulated H2AX2/2 B cellmetaphases after 72 hr stimulation and in about 20% af-ter 96 hr stimulation, a time when more B cells have un-dergoneCSR (Figures 2B–2E, Table 2).We also detectedsplit BACsignals inup to10%ofLPS-stimulatedH2AX2/2

Figure 2. Chromosomal IgH Locus Breaks inH2AX-Deficient B Cells Activated for CSR

(A) Schematic of mouse Igh and BAC probesused for two-color Igh FISH (left diagram).The cytogenetic readout for an intact Igh ora chromosome break at Igh is illustrated onthe right.(B and C) Partial metaphase spread of an a-CD40/IL-4-stimulated B cell after two-colorIgh FISH, showing split BAC signals (30Igh,red, yellow arrow; 50Igh, green, white arrow).A normal chromosome 12 with adjacent30Igh (red) and 50Igh (green) signals is shownfor comparison (yellow arrowheads). An ex-ample of a partial metaphase with Igh breaksat the two chromosomes 12 is shown in (C).(D) Quantification of split signals for 30Igh and50Igh BACs on wt, H2AX+/2, and H2AX2/2 Bcells stimulated with a-CD40/IL-4 for 3 days.Bars represent the mean and standard devia-tion of three mice per genotype.(E) Time course of the frequency of split sig-nals for 30Igh and 50Igh BACs on H2AX2/2

metaphases at day 3 and day 4 of stimulationwith a-CD40/IL-4. Bars represent averageand standard deviation of two H2AX2/2 miceat each time point.

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B cell metaphases (Table 2). As controls, we rarely de-tected split Igh BAC signals in activated wt or H2AX+/2

B cell metaphases and failed to detect any in activatedH2AX2/2 T cells, despite their similar levels of overall ge-nomic instability (Tables 1 and 2). Moreover, nearly two-thirds of the H2AX2/2 B cell metaphases with a split IghBAC signal retained a small fragment that harbored the50Igh sequences, presumably the liberated telomericfragment of chromosome 12 (e.g., Figures 2B and 2C).Attesting to the sensitivity and specificity of the assay,other than these small fragments, we essentially neverobserved any chromosomes that harbored the 50IghBAC in the absence of the 30Igh BAC in either mutant orcontrol samples. CSR often occurs on both allelic chro-mosomes (Dudley et al., 2005); correspondingly, a sub-stantial percentage (15%) of activated H2AX2/2 B cellswith split Igh BAC signals showed split signals on bothchromosomes (Figure 2C and data not shown). Thisdata is in accord with the T- FISH findings, which alsoshowed frequent metaphases with two chromosomesharboring the 30Igh BAC on their long arms in the ab-sence of a telomere signal (Figure 1C). Finally, as sug-gested by T-FISH (see above), split Igh BAC signalswere associated exclusively with chromosome and notchromatid breaks (Figure 2 and data not shown), inmarked contrast to general chromosomal aberrations,which occurred as both chromosome and chromatidbreaks (Table 1). Thus, the DNA lesions that led to the

Igh chromosomal breaks likely occurred primarily ina prereplicative stage of the cell cycle, in agreementwith previous conclusions based on use of g-H2AX focias amarker for IghDSBs (Petersen et al., 2001). We con-clude that a large proportion of H2AX2/2 B cells acti-vated to undergo CSR generate lesions that result in un-repaired breaks within the IgH locus, potentiallycontributing to their CSR defect.

Chromosome Breaks at Igh Result from AID ActivityTomore directly test whether IgH locus chromosome 12breaks in activated H2AX2/2 B cells resulted from at-tempted CSR, we took two different approaches. First,we tested H2AX2/2 B cells activated with a-CD40 inthe absence of IL-4 (or other cytokines), which inducessubstantial proliferation but generally induces onlylow-level CSR (Rush et al., 2005). In these experiments,we found a similar high frequency of general chromo-somal aberrations in H2AX2/2 B cells treated with a-CD40 alone as in those treated with a-CD40 plus IL-4(Figure 3A, Table 1) but found very few IgH locus-spe-cific DSBs with a-CD40 treatment alone (Figure 3B, Ta-ble 2). Second, we generated H2AX2/2/AID2/2 B cellsby breeding H2AX+/2 and AID+/2 (Muramatsu et al.,2000) mice. AID deficiency does not affect B cell activa-tion or induction of S region transcription but completelyblocks CSR (Muramatsu et al., 2000) (Figure 3C), mostlikely by eliminating ability to generate S region DSBs

Table 2. Frequency of Igh Breaks in DSB Repair/CSR-Deficient B Cells Undergoing CSR

Genotype Time in Culture

Percent Metaphases with Igh Breaks (%1 chrom 12/%2 chrom12)

LPS a-CD40/ IL-4 a-CD40 Con A

Control Mice

Wild-Type 72 hr 1 (1/0) 0 (0/0) n.d. 0 (0/0)H2AX+/2 72 hr 0 (0/0) 1 (1/0) n.d. 1 (1/0)p532/2 72 hr 0 (0/0) 1 (1/0) 0 (0/0) n.d.AID2/2 72 hr 0 (0/0) 0 (0/0) n.d. n.d.AID2/2 72 hr 0 (0/0) 0 (0/0) n.d. n.d.

Mice Deficient for DSB Response Factors

H2AX2/2 72 hr 2 (2/0) 9 (6/3) n.d. 0 (0/0)H2AX2/2 72 hr 7 (7/0) 9 (8/1) 3 (3/0) 0 (0/0)H2AX2/2 72 hr 3 (3/0) 10 (10/0) n.d. n.d.H2AX2/2 72 hr 7 (7/0) 20 (18/1) n.d. n.d.H2AX2/2 72 hr 6 (5/1) 10 (8/2) 0 (0/0) n.d.H2AX2/2 72 hr 2 (2/0) 7 (6/1) 0 (0/0) n.d.H2AX2/2 96 hr 10 (8/2) 16 (16/0) 0 (0/0) n.d.H2AX2/2 96 hr 8 (8/0) 21 (17/4) 3 (3/0) n.d.H2AX2/2/p532/2 72 hr 4 (3/1) 17 (16/1) 2 (2/0) n.d.H2AX2/2/p532/2 96 hr 10 (9/1) 31 (24/7) 2 (2/0) n.d.H2AX2/2/AID2/2 72 hr 0 (0/0) 0 (0/0) n.d. n.d.H2AX2/2/AID2/2 72 hr 0 (0/0) 0 (0/0) n.d. n.d.53BP12/2 72 hr 2 (2/0) 10 (7/3) 1 (1/0) n.d.53BP12/2 96 hr 8 (6/2) 26 (24/2) 1 (1/0) n.d.53BP12/2 96 hr 6 (4/2) 22 (19/3) 4 (4/0) n.d.53BP12/2 96 hr 1 (1/0) 10 (10/0) 0 (0/0) n.d.53BP12/2 96 hr 2 (2/0) 5 (4/1) 1 (1/1) n.d.ATM2/2 96 hr 16 (13/3) 22 (19/3) 8 (7/1)a n.d.ATM2/2 96 hr 10 (9/1) 21 (15/6) 10 (9/1)a n.d.MDC12/2 72 hr 6 (6/0) n.d. n.d. n.d.MDC12/2 72 hr 6 (6/0) n.d. n.d. n.d.

Two-color Igh FISH was performed on metaphase spreads of the indicated genotypes stimulated with the indicated mitogens; each data pointrepresents the frequency of Igh BAC split signals in 100 metaphases.a Expression of surface IgG1, IgG2b, and IgG3 above background was observed in these cultures by FACs analysis at 96 hr.

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(Catalan et al., 2003; Rush et al., 2004; Schrader et al.,2005; Wuerffel et al., 1997). Strikingly, while H2AX2/2/AID2/2 B cells showed similar numbers and types ofgeneral chromosomal aberrations as H2AX2/2 B cells(Figure 3D, Table 1), they lacked detectable IgH locus-specific chromosome 12 breaks at the resolution ofour assay (Figure 3E, Table 2). Therefore, the vast major-ity of Igh chromosomal breaks in activated H2AX2/2 Bcells depend on activation treatments that induce CSRand are strictly dependent on AID. The fact that theseIgh chromosomal breaks are strictly AID dependent,coupled with the additional fact that B cells activatedin culture do not undergo somatic hypermutation of vari-able region exons (the other known AID-dependent pro-cess), strongly implies that they were initiated within Sregions via attempted CSR.

AID-Dependent Chromosomal Igh Breaks AreFrequent Participants in TranslocationsIn many human and mouse B cell lymphomas, onco-genes are activated via fusion to the IgH locus, with Sregions at translocation junctions (Kuppers and Dalla-

Favera, 2001). Strikingly, chromosome 12 translocationsthat contained the 30Igh BAC occurred in over 20% ofactivated H2AX2/2 B cell metaphases that containedsplit Igh BAC signals (Figures 4A–4G and Figure 4I, leftbar). Dicentric chromosomes, which can lead to escalat-ing genomic instability via breakage-fusion-bridge (BFB)cycles (Mills et al., 2003), represented about 70% of alltranslocations in activated H2AX2/2 B cells (Figures4A–4C; Figure 4G; Figure 4J, left bar). In addition, two-color IgH locus FISH combined with chromosome-specific paints revealed that dicentric chromosome 12derivatives containing the 30 but not 50 region of the IgHlocus represented a substantial proportion of dicentricsin H2AX2/2 B cells (Figure 4G, right panels, and data notshown), likely promoted by the frequent breakage ofboth chromosome12s at Ighduring attemptedCSR (Fig-ures 1C and 2C). Finally, these analyses also revealedtranslocations between chromosome 12 and otherchromosomes in which we could clearly locate the split30Igh BAC signal at or near the translocation junction(e.g., Figure 4G, left panels). Based on mouse B celllymphoma models, one study concluded that S region

Figure 3. AID Is Required for Generation of High-Frequency Chromosome 12 Breaks within the IgH Locus in H2AX2/2 B Cells

(A) H2AX2/2 and control H2AX+/+ and H2AX+/2Bcells were stimulatedwith a-CD40 alone or in combination with IL-4 for 3 days and the frequencyof total chromosomal breaks measured by telomere FISH (average and standard deviation of three mice).(B) Frequency of Igh-specific DSBs onH2AX2/2Bcells stimulatedwith a-CD40 alone or in combinationwith IL-4 quantified by two-color Igh FISHat days 3 and 4 of in vitro culture (average and standard deviation of two mice).(C–E) H2AX2/2/AID2/2, H2AX2/2, AID2/2, and wt B cells were stimulated with a-CD40/IL-4 for 3 days and the expression of surface IgG1 (sIgG1)assessed by flow cytometry (FACs). A representative experiment is shown. Similar data was obtained at 96 hr and for LPS-stimulated cells (datanot shown). Metaphase spreads from these cultures were used for quantification of general and Igh-specific chromosome breaks by telomereFISH (D) and two-color Igh FISH (E), respectively. Bars represent average and standard deviation of two independent stimulations.

Function of DSB Response Proteins in IgH CSR207

Figure 4. Chromosomal IgH Locus Breaks in H2AX-Deficient B Cells Participate in Frequent Translocations in a p53-Independent Manner

(A–F) Examples of chromosomal translocations involving a chromosome break at Igh in H2AX2/2 B cells stimulated with a-CD40/IL-4 for 3 days.The involvement of a chromosome 12 with a break at Igh in the translocation is indicated by the presence of 30Igh BAC signal (red) and theabsence of adjacent 50Igh BAC signal (green). The translocation partner can be another centric fragment (A–C), acentric fragments (D), or twoother chromosomes simultaneously (E and F). A normal chromosome 12 with an intact Igh (adjacent red and green signals) is shown in (A) forcomparison (white arrow). The normal chromosome and one dicentric also are illustrated in (A).(G) Combined two-color Igh FISH and chromosome 12 paint on activated H2AX2/2 B cells. Translocations of chromosome 12 to a non-12 chro-mosome (T1) or between the two homologous 12 chromosomes (T2) were observed.(H) Quantification of total chromosome breaks by T-FISH in p532/2, H2AX2/2, and H2AX2/2/p532/2 B cells stimulated with LPS, a-CD40, ora-CD40/IL-4 for 3 days. Parallel analyses of concanavalin A-stimulated T cells are shown for comparison. Bars represent average and standarddeviation of two mice per genotype.

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translocations to c-myc were strictly AID dependent(Ramiro et al., 2004), while another suggested they couldbeAID independent (Unniramanet al., 2004). Notably,wedid not detect Igh translocations in H2AX2/2/AID2/2 Bcells, despite their substantial level of general genomicinstability, clearly demonstrating that the vast majorityof Igh translocations in H2AX2/2 B cells are AID depen-dent. We further conclude that the frequent Igh translo-cations in H2AX2/2 B cells are unlikely to involve selec-tion, since they are nonclonal and likely occurreddownstream of AID-induced DSBs at S regions, whichoccur only after a few division cycles subsequent toB cell activation (Rush et al., 2005).

The Tumor Suppressor p53 Exerts Its FunctionDownstream of the Initial Translocation EventPrior studies suggested that p53 deficiency may berequired to observe chromosomal instability in H2AX-deficient B cells activated for CSR (Celeste et al., 2003a;Reina-San-Martin et al., 2003). However, our current re-sults document dramatic chromosomal instability in Bcells deficient for H2AX alone. To assay for any markedeffects of p53 deficiency on genomic instability in acti-vated B cells, we compared general and Igh instabilityin H2AX+/+/p532/2, H2AX2/2/p532/2, and H2AX2/2/p53+/+ B cells. Notably, H2AX+/+/p532/2 B cells, whichare impaired for multiple checkpoints (Iliakis et al.,2003), appeared similar to wt controls in lacking chro-mosome 12 breaks or other types of genomic instability(Figure 4H, Table 1). Moreover, H2AX2/2/p532/2 B cellsdid not show markedly increased genomic instabilitywith respect to frequency or type of aberrations as com-pared to H2AX2/2 B cells (Tables 1 and 2, Figures 4H–4J). Thus, as p53 deficiency does not cause detectablegenomic instability in B cells activated for CSR, potentialcheckpoint deficiencies per se do not appear to accountfor general or IgH locus genomic instability in activatedH2AX2/2 cells. While the defective G2/M response tolow-level DNA damage associated with H2AX deficiency(Fernandez-Capetillo et al., 2002) conceivably couldaugment ability to observe broken IgH loci at meta-phase, we note that chromosome breaks persist intometaphase in NHEJ-deficient mouse embryonic fibro-blasts that have no checkpoint defects (Sekiguchiet al., 2001). We conclude that p53 deficiency is notrequired for the appearance of dramatic genomic insta-bility, including frequent chromosome breaks and trans-locations at Igh, in H2AX-deficient B cells activated forCSR in culture.

Increased IgH Locus Chromosomal Breaksand Translocations in 53BP1-, MDC1-,and ATM-Deficient B CellsWe reasoned that factors that interact with g-H2AX inthe context of DSBs and which are required for normalCSR also might function to prevent AID-dependent IgHlocus DSBs from progressing to chromosomal breaks.

To explore this possibility, we performed two-color IghFISH on activated B cells from 53BP1-, ATM-, andMDC1-deficient mice. Strikingly, similar to H2AX2/2 Bcells, a substantial proportion (up to 20% or more) ofmetaphases from LPS- or a-CD40/IL-4-stimulatedATM2/2 and 53BP12/2 B cells harbored Igh split BACsignals; in addition, levels of Igh split BAC signals wereincreased in a-CD40/IL-4-stimulated cells versus thosetreated with a-CD40 alone (Table 2). Likewise, ina more limited set of experiments, about 6% of meta-phases from LPS-activated MDC1-deficient B cellsshowed Igh split BAC signals (Table 2). We note thatall of these results are highly significant, as IgH locusbreaks in similarly stimulated wt cells were extremelyrare, as assayed either by the two-color Igh FISH orT-FISH (Table 2). We also characterized, via T-FISH,general chromosomal instability in 53BP1-, ATM-, orMDC1-deficient B cells activated for CSR. Significantly,LPS- and a-CD40/IL-4-activated B cells of all three ge-notypes displayed dramatically increased levels of chro-mosomal aberrations compared to wt littermate con-trols (Table 1). However, while the overall level ofchromosomal aberrations in ATM2/2 B cells was muchgreater than that contributed by Igh instability alone,the overall level in activated 53BP12/2 B cells mostlycould be attributed to Igh chromosomal breaks(Tables 1 and 2). Correspondingly, a-CD40-stimulated53BP12/2Bcells showed relatively low levelsof chromo-somal aberrations compared to H2AX2/2 or ATM2/2

B cells (Table 1), in accord with findings that 53BP12/2

T cells show limited chromosomal instability (Moraleset al., 2006). We employed two-color FISH to analyzemetaphases from activated ATM2/2 and 53BP12/2 Bcells for translocations involving the IgH locus. As for ac-tivated H2AX2/2 B cells, we found a significant propor-tion (about 25%) of Igh split BAC signals in activatedATM2/2 B cell metaphases in the context of transloca-tions, with a still substantial, but somewhat lower, per-centage (13%) in activated 53BP1-deficient B cells(Figure 5A), indicating that a substantial proportion ofbroken IgH loci enter into chromosomal translocationsin all three mutant backgrounds. Moreover, dicentricchromosomes containing split Igh BAC signals wereabundant, occurring in a similarly high proportion (about15%) of activated ATM2/2B cell metaphases as in thoseof activated H2AX2/2 B cells and in about 5% of acti-vated 53BP12/2 B cell metaphases (Figure 5B). To iden-tify Igh translocation partners, we performed SKY onactivated ATM2/2 B cell metaphases. Analysis of 20 di-centrics revealed that 11 involved chromosome 12 andthat about half of those (five of 11) were chromosome12 to chromosome 12 dicentrics (Table S2, Figure 5C).In the limited samples analyzed, non-12 partners repre-sented various other chromosomes (Table S2). Finally,reprobing these same metaphases with 30Igh and 50IghBACs revealed split Igh BAC signals in most transloca-tions involving chromosome 12 (Figure 5C, Table S2).We conclude that, like H2AX2/2 B cells, ATM2/2 or

(I) After two-color Igh FISH, each Igh break was further classified as either a free untranslocated end (examples in Figure 2) or a translocated end(examples in Figures 4A–4G). Comparative analyses for 102 Ighbreaks inH2AX2/2Bcells and 76 Ighbreaks inH2AX2/2/p532/2Bcells are shown.(J) Relative frequency of dicentrics to all observed chromosomal translocations involving Igh breaks in H2AX2/2 and H2AX2/2/p532/2 B cells(n = 24 and n = 10 translocations, respectively).

Function of DSB Response Proteins in IgH CSR209

53BP12/2 B cells activated for CSR frequently generatechromosomal breaks at the IgH locus that lead to trans-locations.

Discussion

H2AX Promotes End Joining in the Context of CSRWe demonstrate that mammalian H2AX prevents AID-dependent DNA breaks in the IgH locus from progress-ing into chromosome breaks in primary B lymphocytesactivated for CSR. During CSR, multiple AID-dependentDSBs may occur within Sm, some of which can be joinedto each other to yield internal Sm deletions, rather thanbeing joined to downstream S region DSBs to yieldCSR (Dudley et al., 2002; Reina-San-Martin et al., 2003).Based on normal internal Sm deletions inH2AX2/2B cells,prior studies suggested that their CSR defects might beassociatedwith impaired long-range synapses of two dif-ferent S regions (Reina-San-Martin et al., 2003). While our

studies do not address this postulated role, we find thatAID-dependent Igh chromosome breaks occur in a largeenough proportion of CSR-activated H2AX-deficient Bcells to contribute to their CSR defect. We cannot distin-guishwhether AID-dependent Igh chromosomebreaks inH2AX2/2 B cells occur during attempted joining of twodifferent S regions, joining of DSBs within a single S re-gion, or both. We note that the residual CSR in H2AX-de-ficient B cells indicates that end joining is not abrogated.Based on the apparently high level of Sm internal dele-tions, DSBs have been proposed to occur more often inSm than in downstream S regions, perhaps to drive CSRto downstream S regions (Dudley et al., 2002; Chaudhuriand Alt, 2004). If so, failure to join only a subset of SmDSBs could dissociate the V(D)J exon from the CH exons,lead to chromosome breaks, and thereby contribute todefective CSR. The structure of CSR junctions clearlyimplicates NHEJ and/or a highly related pathway in theprocess (Dunnick et al., 1993), a notion supported by the

Figure 5. Role of ATM and 53BP1 in Suppressing Igh Chromosomal Breaks and Translocations

(A) Fate of broken Igh ends (free untranslocated versus translocated to another chromosome) in H2AX2/2, ATM2/2, and 53BP12/2 activatedB cells. The total number of Igh breaks studied for each genotype is indicated.(B) Comparative frequency of dicentrics with a breakpoint at Igh in H2AX2/2, ATM2/2, and 53BP12/2 activated B cells. The total number of meta-phases analyzed for each genotype is indicated.(C) Two representative examples of sequential SKY and two-color Igh FISH on a-CD40/IL-4-stimulated ATM2/2 B cells. Chromosome 12 dicen-trics were observed frequently (top row, left and middle panel, green arrows; see Table S1 for quantification). Reprobing of metaphases with30Igh and 50Igh BACs confirmed their CSR-related origin (split 30Igh and 50Igh BAC signals; top right panel, green arrow). A telomeric fragmentcontaining the 50Igh BAC signal was present in the metaphase and is indicated by a red arrow in the DAPI (top right), spectral (middle), and IghFISH (right) images. Translocations to chromosomes other than the homologous 12 appeared random; a t(3, 12) is shown as an example in thebottom images (green arrows).

Molecular Cell210

dependenceofCSRonNHEJ factors (Pan-Hammarstromet al., 2005; Reina-San-Martin et al., 2003). In this regard,we observe similar IgH locus chromosome breaks inNHEJ-deficientBcellsactivated forCSR(ourunpublisheddata). Therefore, our studies provide the first direct evi-dence that mammalian H2AX impacts on this form ofDSB repair.

Potential Roles of H2AX in the Joining Phase of CSRH2AX might influence CSR end joining by several con-ceivable mechanisms. In yeast, phosphorylated H2A in-fluences NHEJ by altering chromatin structure at DSBs(Downs et al., 2000) and influences HR via a processthat brings cohesins to DSBs to tether sister chromatids(van Attikum and Gasser, 2005). In this context, our find-ings are consistent with an ‘‘anchoring’’ model that pro-poses that DSB-dependent phosphorylation of H2AXand other repair/checkpoint factors, including 53BP1and MDC1, by ATM or related PIKK kinases leads to for-mation of complexes that alter chromatin structure andprevent DSBs from turning into chromosomal breaks,thereby facilitating proper end joining and suppressingtranslocations (Bassing and Alt, 2004; Rogakou et al.,2000). One prediction of this general model is thatDSBs introduced during CSR would turn into chromo-some breaks at high frequency in H2AX-deficient cells,and our findings show this to be the case. Another pre-diction is that DSBs in two different chromosomes inH2AX-deficient cells could often dissociate and be inap-propriately joined to cause translocations, a predictiondemonstrated by our finding that IgH locus breaks onboth copies of chromosome 12 in individual H2AX-defi-cient B cells often are joined to form dicentric chromo-some 12 translocations. A final prediction of this modelis that impaired CSR inmice deficient for ATM (Lumsdenet al., 2004; Reina-San-Martin et al., 2004), 53BP1 (Maniset al., 2004; Ward et al., 2004), or MDC1 (Lou et al., 2006)would be similarly associated with chromosome 12breaks and Igh translocations, a prediction that wehave confirmed.The common phenotype of deficiencies for H2AX,

ATM, 53BP1, and MDC1 with respect to generation ofIgH locus chromosome breaks and translocations in Bcells activated for CSR implies that they serve commonfunctions. Given that deficiency for an NHEJ factorshows a similar phenotype (our unpublished data), onecommon functionmay involve promotion of NHEJ. How-ever, there are some apparently distinct aspects of the53BP1-deficient phenotype that are noteworthy. First,while 53BP1 deficiency results in roughly similar levelsof Igh chromosomal breaks as H2AX or ATM deficiency,it leads to a nearly complete abrogation of CSR (Maniset al., 2004; Ward et al., 2004). In addition, unlike ATMor H2AX deficiency, 53BP1 deficiency does not lead todramatic general chromosomal instability, as most ob-served instability in activated B cells is associated withIgh chromosome breaks. Correspondingly, there is littlespontaneous genomic instability in 53BP1-deficientMEFs or T cells (Morales et al., 2003, 2006; Ward et al.,2003b). Thus, these findings are consistent with a morespecialized role of 53BP1 in CSR-associated DSBsand/or more effective elimination of cells with othertypes of DSBs in the 53BP1-deficient versus ATM- orH2AX-deficient backgrounds, with the latter possibility

being supported by an increased chromosomal instabil-ity in T cells deficient in both 53BP1 and p53 (Moraleset al., 2006). However, it also is conceivable that 53BP1has additional roles in CSR, perhaps upstream of theDSB, via its ability to sensechromatin structural changes(Huyen et al., 2004). Finally, if more extensive studiesconfirm that MDC1 has as great an effect on IgH locuschromosomal stability as deficiencies for the other fac-tors, its requirement for propagatingH2AXphosphoryla-tion by ATMand allowing extended focus formation (Louet al., 2006) might suggest that putative tethering func-tions of H2AX would occur by more short-range mecha-nisms.

Does H2AX Promote End Joining Beyond CSR?We have been able to follow AID-dependent Igh DSBsbecause they occur at high frequency. Thus, the ques-tion arises as to how applicable our findings are to gen-eral DSBs. In this regard, H2AX-deficient B cells, T cells,MEFs, and embryonic stem cells all show marked in-creases in general genomic instability including chro-mosome breaks and translocations. However, whilethe IgH locus chromosome breaks associated withCSR appear to be initiated in a prereplicative cell cyclephase, those that lead to general instability, at least asobserved in H2AX-deficient B cells, appear to occurover a broader portion of the cell cycle. On the otherhand, while V(D)J recombination employs a DSB, therehas been no measured effect of H2AX deficiency onthis process (Bassing et al., 2002; Celeste et al., 2002).In this context, the ability of RAG to hold ends togetherin a postcleavage synaptic complex and coordinatetheir repair via NHEJ (Gellert, 2002) might largely preventRAG-induced DSBs from progressing into chromo-somal breaks in H2AX-deficient cells, although H2AXmay function in further stabilizing such DSBs (Bassingand Alt, 2004). Support for an H2AX role in V(D)J recom-bination comes from the finding that H2AX/p53-defi-cient mice often develop pro-B lymphomas with compli-cons resulting from translocations that fuse the IgH JHlocus to sequences downstream of c-myc (Bassinget al., 2003; Celeste et al., 2003a) and that these H2AX/p53-deficient complicons are similar in structure toRAG-dependent complicons in NHEJ/p53-deficientmice (Bassing and Alt, 2004). Finally, it remains possiblethat subtle defects in joining of RAG-initiated DSBscould contribute to the lower lymphocyte numbers inH2AX2/2 mice (Celeste et al., 2002).

P53 Functions Downstream of Translocationsto Suppress H2AX-Deficient B LymphomasWe show that H2AX deficiency per se is sufficient togenerate a large pool of activated B cells that harborAID-dependent chromosomal translocations involvingthe IgH locus. Yet, H2AX-deficientmice are notmarkedlycancer prone and, in particular, have not been found tobe susceptible to B cell lymphomas. On the other hand,H2AX2/2p532/2 mice are dramatically cancer prone,and most characterized H2AX2/2p532/2 tumors haveclonal translocations, including B lineage lymphomasthat harbor Igh translocations that lead to c-myc activa-tion (Celeste et al., 2003a; Bassing et al., 2003). Togetherwith our current results, the latter finding supports thenotion that defective checkpoints associated with p53

Function of DSB Response Proteins in IgH CSR211

deficiency may not be required for generation of poten-tially oncogenic translocations, but rather to allow ex-pansion of cells harboring them. For example, p53 defi-ciency may permit cells to continue cycling in thepresence of chromosome breaks and other forms of ge-nomic instability (Mills et al., 2003) and/or allowactivatedoncogene (e.g., c-myc) expression from translocationswithout inducing apoptosis (Hemann et al., 2005).

Experimental Procedures

MiceWe used previously described H2AX-deficient mice (Bassing et al.,2003), 53BP1 (Morales et al., 2003), ATM (Borghesani et al., 2000),and MDC1 (Lou et al., 2006). Mice doubly deficient for H2AX andp53 (Donehower et al., 1992) or for H2AX and AID (Muramatsuet al., 2000) and their control littermates were generated by breed-ing.

Mature B and T Cell Purification and CulturesSplenocyte single-cell suspensions were prepared and fractionatedinto CD43+ and CD432 populations as described (Manis et al., 2004).For B cell stimulation, the CD432 fraction was incubated with the Bcell mitogens bacterial lypopolysaccharide (LPS, 20 mg/mL; Sigma),anti-CD40 antibody (a-CD40, 1 mg/mL, BDPharmingen) and interleu-kin 4 (IL-4, 20 ng/mL, R&D Systems). For T cell stimulation, theCD43+ fraction was incubated with concanavalin A (2.5 mg/mL,Sigma).

Assessment of CSR by Flow CytometryStimulated B cells (0.5 million) were washed with 2% FCS/PBS;stained with antibodies labeled with Cy-Chrome (B220), fluorescein(IgG2b, IgG1), or phycoerithrin (IgE and IgG3) for 15 min; and ana-lyzed in a FACSCAN using Cellquest (Becton Dickinson) or Flo-Jo(Tree Star) software. At least 10,000 events of live lymphoid cellswere recorded.

FISH ProbesWe detected the 30 end of the IgH locus using BAC199 (a gift of Bar-bara Birshtein, Albert Einstein College of Medicine, New York) andthe 50 end using BAC207 (a gift of Peter Brodeur, Tufts University,Boston). FITC-conjugated paints for mouse chromosomes 12 werepurchased from Applied Spectral Imaging. For telomere staining,a Cy3-conjugated (TTAGGG)3 PNA probe was purchased fromApplied Biosystems.

PNA FISH of TTAGGG SequencesLymphocytes were incubated in colcemid (KaryoMAX, GIBCO),swollen in prewarmed 30mM sodium citrate for 25min at 37ºC, fixedin methanol/acetic acid (3/1), and air dried on slides overnight. Afterpepsin digestion (1 mg/ml, 10 min at 37ºC), slides were denatured at80ºC for 3 min, hybridized with a Cy3-labeled PNA telomeric probe(Cy3-[TTAGGG]3) in 70% formamide at RT for 2 hr, washed, dehy-drated, and mounted in Vectashield with DAPI (Vector Laboratories,Burlingame, California). Metaphase images were captured usinga Nikon Eclipse microscope equipped with a CCD camera (AppliedSpectral Imaging, Carlsbad, California) and a 633 objective lens.For cytogenetic analysis, at least 30 metaphases of each genotypewere scored for chromosomal aberrations. After image recording,selected slides were washed in 2XSSC, dehydrated through serialethanols, and reprobed with BACs as described below.

Two-Color Fluorescence In Situ HybridizationBACs were labeled with either biotine (Biotin-Nick Translation Mix,Roche) or digoxigenin (Dig-Nick Translation Mix, Roche), as permanufacturer’s instructions. Two hundred nanograms of BAC DNAwas precipitated with mouse Cot1 DNA (Invitrogen; ratio of BACDNA to Cot1 DNA, 1:20), resuspended in 15 ml of hybridization solu-tion (50% formamide, 2X SSC, 10% dextran sulfate, 0.15% SDS),and codenatured on slides for 5 min at 76ºC. Slides were then incu-bated at 37ºC for 16 hr, washed in 50% formamide/2X SSC for 5 mintwice at 45ºC and 2X SSC for 5min twice at 45ºC, and incubated withavidin-Cy3 and antidigoxigenin-FITC (Roche, 1:250 dilution) in

2XSSC/0.05% Tween-20 for 1 hr at RT. After three washes in2XSSC/0.05% Tween-20, slides were mounted in Vectashield withDAPI (Vector Laboratories). Metaphase images were captured usinga Nikon Eclipse microscope equipped with a CCD camera (AppliedSpectral Imaging, Carlsbad, California) and a 633 objective lens.One hundred metaphases were analyzed for each sample. Afterimage recording, selected slides were dehydrated through serialethanols and reprobed with fluorochrome-conjugated paints tomouse chromosome 12, following manufacturer’s instructions.

Spectral Karyotype AnalysisMetaphase spreads were hybridized using a SkyPaint DNA Kit(Applied Spectral Imaging), following manufacturer’s instructions.Spectral images were captured and analyzed using an interferome-ter and software from Applied Spectral Imaging. Selected slideswere dehydrated through serial ethanols, and two-color FISH wasperformed as described above.

Supplemental DataSupplemental Data include two figures and two tables and can befound with this article online at http://www.molecule.org/cgi/content/full/21/2/201/DC1/.

Acknowledgments

We thank Charles Lee, Hwei-Ling Cheng, Tiffany Borjeson, andAlyssa Riley for technical advice; Barbara Haskell for technical sup-port; and all Alt lab members for helpful discussions. This work wassupported by NIH Grants PO1CA092625-05 and 2PO1AI031541-15(to F.W.A.), GM65812 (to P.B.C.), and RO1 CA89239 and CA92312(to J.C.); a Long-Term Fellowship of the EuropeanMolecular BiologyOrganization (to S.F.); a Fellowship of the Leukemia and LymphomaSociety (to S.Z.); and a NIA/NIHKO8 Award (to D.B.L.). Z.L. is the re-cipient of a DOD Breast Cancer Postdoctoral Fellowship (DAMD17-03-1-0610). C.H.B. was a Lymphoma Research Foundation Fellowand is a Pew Scholar in the Biomedical Sciences. J.C. is a recipientof DOD Breast Cancer Career Development Award (DAMD17-02-1-0472). F.W.A. is a Howard Hughes Medical Institute investigator.

Received: November 12, 2005Revised: December 16, 2005Accepted: January 5, 2006Published: January 19, 2006

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