Mechanisms Promoting Translocations in Editing and Switching Peripheral B Cells

Jing H Wang; Monica Gostissa; Catherine T Yan; Peter Goff; Thomas Hickernell; Erica Hansen; Simone Difilippantonio; Duane R Wesemann; Ali A Zarrin; Klaus Rajewsky; Andre Nussenzweig; Frederick W Alt

doi:10.1038/nature08159

. Author manuscript; available in PMC: 2010 Jul 20.

Published in final edited form as: Nature. 2009 Jul 9;460(7252):231–236. doi: 10.1038/nature08159

Mechanisms Promoting Translocations in Editing and Switching Peripheral B Cells

Jing H Wang ^1,^2,^3,^4,⁷, Monica Gostissa ^1,^2,^3,^4,⁷, Catherine T Yan ^1,^2,^3,^4,⁷, Peter Goff ^1,^2,^3,⁴, Thomas Hickernell ^1,^2,^3,⁴, Erica Hansen ^1,^2,^3,⁴, Simone Difilippantonio ⁵, Duane R Wesemann ^1,^2,^3,^4,⁶, Ali A Zarrin ^1,^2,^3,^4,⁸, Klaus Rajewsky ³, Andre Nussenzweig ⁵, Frederick W Alt ^1,^2,^3,⁴

PMCID: PMC2907259 NIHMSID: NIHMS212258 PMID: 19587764

Abstract

V(D)J recombination assembles immunoglobulin (Ig) heavy or light chain (IgH or IgL) variable region exons in developing bone marrow B cells, while class switch recombination (CSR) exchanges IgH constant region exons in peripheral B cells. Both processes employ DNA double strand breaks (DSBs) repaired by non-homologous end-joining (NHEJ). Errors in either V(D)J recombination or CSR can initiate chromosomal translocations, including oncogenic IgH/c-myc translocations of peripheral B cell lymphomas. Collaboration between these processes also has been proposed to initiate translocations. However, occurrence of V(D)J recombination in peripheral B cells is controversial. Here, we report that activated NHEJ-deficient splenic B cells accumulate V(D)J recombination-associated IgL chromosomal breaks, as well as CSR-associated IgH breaks, often in the same cell. Moreover, IgL breaks frequently are joined to IgH breaks to form translocations, a phenomenon associated with specific IgH/IgL co-localization. IgH and c-myc also co-localize in these cells; correspondingly, introduction of frequent c-myc DSBs robustly promotes IgH/c-myc translocations. Our studies reveal peripheral B cells that attempt secondary V(D)J recombination and elucidate a role for mechanistic factors in promoting recurrent translocations in tumors.

Recombination activating gene 1/2 (RAG) endonuclease initiates V(D)J recombination by cleaving V, D and J segments, which are joined exclusively by NHEJ to form V(D)J exons¹^,². V(D)J recombination in bone marrow (BM) pro-B cells first assembles IgH V(D)J exons leading to μ chain expression³. Subsequently, IgL VJ exons are assembled in pre-B cells, generating immature B cells that express μ plus IgL chains as surface IgM³. The two IgL families (Igκ and Igλ) are encoded in distinct loci, and primary Igκ V(D)J recombination usually precedes that of Igλ⁴. Individual B cells express either Igκ or Igλ, with about 95% of mouse IgM⁺ B cells being Igκ⁺ and the remainder Igλ⁺⁴. Newly generated BM B cells that express auto-reactive receptors can undergo tolerogenic secondary V(D)J recombination, termed receptor editing, in which they further rearrange or delete Igκ and may rearrange Igλ⁵^-⁷ (See Suppl. Fig. 1 for schematic version of these processes).

Surface IgM⁺ B cells down-regulate RAG and migrate to peripheral lymphoid tissues (e.g. spleen) where they participate in antigen-dependent responses including CSR⁸. The various sets of germline IgH constant region exons (“C_H genes”) are flanked by switch (S) regions⁹. Activation-induced cytidine deaminase (AID) initiates DSBs in Sμ and a downstream S region, which then are joined by NHEJ or, in its absence, by less efficient microhomology (MH)-mediated alternative end-joining (A-EJ)⁹^,¹⁰. Thereby, Cμ is replaced with a downstream C_H gene to effect CSR (Suppl. Fig. 1). Germinal center (GC) B cells have been argued to undergo antigen-dependent secondary V(D)J recombination, termed “receptor revision”, as a means of diversification¹¹. Like receptor editing, receptor revision is proposed to target Igκ and Igλ, but to be distinct in location and activation mechanism¹¹^,¹². However, whether or not V(D)J recombination occurs in the context of receptor revision in GC B cells has been debated¹¹^-¹⁴.

Human and mouse B lymphomas often harbor clonal translocations linking oncogenes, such as c-myc, to IgH, Igκ or Igλ¹⁵^,¹⁶. Such recurrent oncogenic translocations are thought to represent highly selected, very low frequency events. Even so, aspects of c-myc, beyond coding sequences, may increase its translocation fequency¹⁷. In this regard, loci involved in recurrent oncogenic translocations often are spatially proximal within interphase nuclei¹⁸^-²³. RAG and AID have been implicated in collaboratively initiating oncogenic translocations in human BM-derived pro-B/pre-B lymphomas²⁴^,²⁵. Many oncogenic translocations in mature B lymphomas occur during attempted CSR and involve AID-initiated breaks²⁶^-²⁹; but others result from RAG-initiated DSBs¹⁵^,³⁰^,³¹. Due to checkpoint defects, RAG-initiated IgH breaks in ATM-deficient BM pro-B cells persist and can be translocation substrates in IgM⁺ peripheral B cells³². Thus far, however, translocations have not been shown to result from RAG activity in peripheral B cells.

Xrcc4 is a critical NHEJ component². In its absence, V(D)J recombination is abrogated³³^,³⁴ and CSR impaired¹⁰^,³⁵. Conditional inactivation of LoxP-flanked Xrcc4 in p53-deficient peripheral B cells via a CD21-Cre transgene leads to recurrent “CXP” B cell lymphomas that harbor aberrant Igκ and Igλ V(D)J rearrangements, IgH CSR events, and Igλ and/or IgH/c-myc translocations³⁶. We proposed CXP tumor progenitors to be peripheral B cells that undergo secondary V(D)J recombination and CSR³⁶. To search for such putative CXP tumor progenitors, we now have analyzed splenic “CX^c/−” B cells in which Xrcc4 was peripherally inactivated but p53 was left intact to obviate B cell lymphomas.

IgH Chromosomal Breaks in Xrcc4-Deficient Splenic B Cells are AID-dependent

CX^c/− mice have normal IgM⁺ B cell numbers, as Xrcc4 is intact for primary V(D)J recombination in developing BM B cells, with inactivation starting in transitional stage peripheral B cells¹⁰^,³⁷. CX^c/− splenic B cells activated for CSR have high levels of IgH breaks on chromosome 12 due to impaired NHEJ¹⁰. While Xrcc4 deficiency is not associated with known checkpoint defects³⁴, we firmly tested AID-dependency of CX^c/− B cell IgH breaks by breeding the CX^c/− genotype onto an AID-deficient (A^−/−) background³⁸ to generate CX^c/−A^−/− mice. We stimulated CX^c/−, CX^c/−A^−/− and control (X^c/c) splenic B cells with αCD40/IL4 for 4 days to promote IgG1 CSR and assayed metaphases for IgH breaks and translocations via fluorescence in situ hybridization (FISH) with 5′ and 3′ IgH probes. While general chromosomal breaks, as expected, were largely AID-independent in activated CX^c/− splenic B cells (Suppl. Table 2), the vast majority of IgH breaks were AID-dependent (Fig. 1a and Suppl. Table 1).

**a, Upper:** Diagram of *IgH* FISH probes. An intact *IgH* shows co-localized red and green signals while a broken locus appears as split red and green signals. **Middle:** Example of metaphase FISH showing *IgH* breaks. **Lower:** Quantification of *IgH* abnormalities in day4 αCD40/IL4-activated control (n=6), *CX^c/−* (n=9), *CX^c/−A^−/−* (n=5) and *CX^c/−RAG^c* (n=8) splenic B cells (details in Suppl. Table 1). **b, Upper:** Diagram of *Igλ* FISH probes. Intact *Igλ* shows co-localized green and red signals, *Igλ* breaks appear as split green and red signals, either free or in translocations. **Middle:** Examples of metaphase FISH showing *Igλ* breaks (left) and an *Igλ* break and dicentric translocation (right). **Lower:** Quantification of *Igλ* abnormalities in day4 αCD40/IL4-activated control (n=11), *CX^c/−* (n=11), *CX^c/−A^−/−* (n=3), *CX^c/−RAG2^c* (n=8) splenic B cells (details in Suppl. Table 3). **c, Upper:** Diagram of *Igκ FISH* probes. *Igκ* breaks are scored similarly as *Igλ* breaks. **Middle:** Examples of metaphase FISH showing an *Igκ* break (left) and *Igκ* break and translocations (right), involving both centromeric and telomeric portions of chromosome 6. **Lower:** Quantification of *Igκ* abnormalities in day4 αCD40/IL4-activated control (n=10), *CX^c/−* (n=11), *CX^c/−A^−/−* (n=3), *CX^c/−RAG^c* (n=7) splenic B cells (details in Suppl. Table 4). **d, Upper:** Diagram of *Igλ* 3D interphase FISH Probes. **Middle:** Representative 3D interphase FISH showing intact *Igλ* (co-localization of green and red signals) and *Igλ* breaks (split green and red signals) (details in Suppl. Fig. 4). **Lower:** Quantification of *Igλ* abnormalities by 3D interphase FISH on day 0 (n=3) or day 4 (n=3) αCD40/IL4-activated splenic B cells. We could not do similar assays for *Igκ* due to the large size of this locus (greater than 3Mb). In all panels, data are presented as mean ± s.e.m. Statistical analyses were calculated by a Student's t-Test with two-tailed distribution.

RAG-dependent Igλ Breaks and Translocations in Xrcc4-deficient Splenic B Cells

We assayed activated CX^c/− splenic B cells for Igλ breaks via metaphase FISH with 5′ and 3′ Igλ probes that flank the 200kb Igλ locus on chromosome 16 (Fig. 1b). After αCD40/IL4 stimulation for 4 days, we found Igλ breaks in over 1% of CX^c/- B cells, with none in controls (Fig. 1b and Suppl. Table 3). Moreover, the Igλ breaks were frequently translocated (Fig. 1b; Suppl. Fig. 2). Metaphase FISH with BAC probes flanking Igκ revealed that 1% of activated CX^c/- B cells also harbor Igκ breaks/translocations (Fig. 1c; Suppl. Fig. 2; Suppl. Table 4). In contrast, Xrcc4-deficient embryonic stem (ES) cells lacked Igκ or Igλ abnormalities (Suppl. Table 5). To elucidate when Igλ and Igκ breaks occurred, we assayed CX^c/− splenic B cells at days 2, 3, and 4 of activation and observed both to accumulate during stimulation, with Igκ breaks kinetically preceding Igλ breaks (Suppl. Table 3, 4, and 6; Suppl. Fig. 3). We also assayed for Igλ breaks via 3D interphase FISH with 5′Igλ and 3′Igλ probes (Fig. 1d). Igλ breaks were rare in resting (day 0) CX^c/− splenic B cells, but occurred in about 1.5% of day 4 activated CX^c/- splenic B cells (Fig. 1d; Suppl. Fig 4, Table 7). We conclude that Igλ and Igκ breaks occur during expansion of activated CX^c/- splenic B cells, a conclusion supported by our findings that p53 deficiency did not markedly enhance Igλ breaks (Suppl. Table 3) and that 50% of metaphases with Igλ breaks retained the acentric chromosome 16 fragment (Fig. 1b, data not shown).

To test AID involvement, we assayed for Igκ and Igλ breaks in day 4 αCD40/IL4-activated CX^c/-A^-/- B cells and found a comparable frequency as in CX^c/− B cells (Fig. 1b,c, Suppl. Table 3 and 4, Suppl. Fig. 5). Similar to earlier studies³²^,³⁹, we found only very low RAG expression in activated normal and CX^c/− splenic B cells (data not shown). To further assess RAG involvement, we bred a LoxP-flanked Rag2 conditional allele (RAG^c/c)⁴⁰ into the CX^c/− genotype to generate CX^c/−RAG^c/c or CX^c/−RAG^c/− (“CX^c/−RAG^c”) mice. Upon activation, the RAG conditional allele was largely deleted in day 3 and 4 activated CX^c/−RAG^c cells (Suppl. Fig. 6). While IgH break frequency was comparable between CX^c/− and CX^c/− RAG^c B cells, Igλ break frequency was significantly reduced in CX^c/−RAG^c B cells (Fig. 1a,b, Suppl. Table 1 and 3). Thus, in activated CX^c/− splenic B cells, IgH breaks are AID-dependent and RAG-independent; while Igλ breaks are AID-independent and mostly RAG-dependent. Igκ breaks were not significantly reduced in activated CX^c/−RAG^c B cells (Fig. 1c, Suppl. Table 4), suggesting they either are not initiated by AID or RAG or their earlier kinetic onset allows accumulation before RAG activity is eliminated.

RAG and AID Collaborate in Generating High Frequency IgH/Igλ Translocations

We employed sequential FISH to ask if IgH, Igκ or Igλ breaks occurred simultaneously in CX^c/− B cell metaphases. Analyses of over 2000 day 4 αCD40/IL4-activated CX^c/− B cell metaphases revealed none had both Igκ and Igλ breaks (Suppl. Fig. 7). However, analyses with a Jκ-Cκ probe showed that nearly 50% of metaphases with a broken Igλ had deleted Jκ-Cκ on one or both alleles (Suppl. Fig.8), similar to secondary V(D)J recombination events in CXP B lymphomas³⁶. We found one Igκ/IgH translocation in over 2000 activated CX^c/− B cell metaphases, consistent with a high frequency but at levels just below ready cytogenetic measurement (Suppl. Fig. 9a). Nearly 60% of metaphases with Igλ breaks also had IgH breaks and/or translocations and about 20% of these retained both centric and acentric portions of chromosome 12 and 16 (Suppl. Fig. 7; Suppl Fig. 9b,c; not shown), suggesting attempted V(D)J recombination and CSR in the same or successive cell cycles. In this regard, combined FISH with Igλ and IgH probes and chromosome paints revealed that 30% of Igλ translocations involved IgH (e.g. Fig. 2a; Suppl. Fig. 9b,c). As many IgH/Igλ translocations resulted in dicentrics with 3′Igλ and 3′ IgH probes juxtaposed (Suppl. Fig. 9b), we performed FISH with these probes simultaneously, which revealed AID-dependent IgH/Igλ translocations in about 0.2% of CX^c/− B cells (Fig. 2a, Suppl. Table 8, Fig. 10). We conclude that unrepaired RAG-dependent Igλ breaks in activated CX^c/− splenic B cells are frequently fused to AID-dependent IgH breaks in the same cell to form chromosome 12/16 translocations.

**a, Top left:** Diagram showing *3′Igλ* probe (green) on chromosome 16 and *3′IgH* probe (red) on chromosome 12. **Bottom left:** Representative *Igλ/IgH* translocation showing green and red signals juxtaposed on a dicentric chromosome (yellow arrow). **Right**: Quantification of *IgH/Igλ* translocations in day4 αCD40/IL4-activated control (n=2) or CX^c/− (n=4) B cells analyzed by metaphase FISH (details in Suppl. Table 8). Data are presented as mean ± std. b, PCR-isolated *Igh/Igλ* translocation junctions from day4 activated CX^c/− B cells (n=3) (primers indicated by horizontal black arrows). Junctional sequences are shown in Suppl. Fig. 11. A vertical green arrow indicates breakpoints. For a given translocation, the same number is used to indicate the corresponding *IgH* and *Igλ* breakpoints, with the *IgH* breakpoint denoted by a (') symbol.

We isolated IgH/Igλ translocation junctions from CX^c/− B cells via PCR (Suppl. Fig. 10), and found most fused Sμ to sequences downstream of Jλ1/Jλ3 V(D)J recombination signal sequences (Fig.2b, Suppl. Fig.11). Consistent with AID-initiated IgH breaks joined to RAG-initiated Igλ breaks, point mutations and other alterations were observed in IgH- but not Igλ-derived junctional sequences (Suppl. Fig.11). Consistent with RAG-initiated breaks resolved in the absence of NHEJ, Igλ junctions were at variable distances downstream of Jλ1 and Jλ3. Finally, most IgH/Igλ junctions contained microhomologies indicative of A-EJ (Suppl. Fig. 11). We conclude that, in activated splenic CX^c/− B cells, A-EJ joins RAG-induced Igλ breaks to AID-initiated IgH breaks at high frequency.

Cell-type Specific and Focal Co-localization of IgH and Igλ in B Cell Interphase Nuclei

3D interphase FISH with 3′IgH and 3′Igλ probes revealed co-localization of the loci (≤0.5μm apart) in about 14% of resting (day 0) and 7-8% of day 3.5 αC40/IL4-activated control and CX^c/- splenic B cells (Fig. 3a-d; Suppl. Table 9,10). As there are no IgH or Igλ breaks in resting B cells (Figs. 1a, d), and AID-initiated breaks begin at day 2⁴¹, we conclude IgH and Igλ co-localize before and after DSB induction and that Xrcc4 deficiency does not alter this association. To assess cell-type specificity, we assayed wt thymocyte and ES cell interphase nuclei and found only low-level IgH/Igλ co-localization (Fig. 3c, Suppl. Table 9). To examine specificity of the IgH/Igλ association within chromosome 16, we tested co-localization of IgH with two control loci (C2 and K10), which map, respectively, about 15Mb telomeric or centromeric to Igλ (Fig. 3b). IgH/C2 co-localization was at background levels in resting and activated B cells and thymocytes, while IgH/K10 co-localization occurred at substantially lower levels than IgH/Igλ co-localization (Fig. 3c, Suppl. Table 11). Therefore, IgH and Igλ co-localization is cell-type specific and focal on chromosome 16 with respect to Igλ. Notably, IgH and Igκ also specifically and focally, at least with respect to Igκ, co-localize in about 5% of splenic B cells (Suppl. Fig.12, Table 12).

**a, Top:** Diagram showing 3′*IgH* (green) and 3′*Igλ* (red) probes used for 3D interphase FISH. **Bottom:** Representative co-localization of *IgH/Igλ* in day 0 control and *CX^c/−* B cell interphase nuclei. b, Schematic map of *Igλ, C2* and *K10* BAC probes on chromosome 16. c, Quantification of co-localization of *IgH-Igλ, IgH-C2*, or *IgH-K10* loci in nuclei of day 0 control and *CX^c/−* splenic B cells and in nuclei of thymocytes (details in Suppl. Tables 9 and 11). d, Quantification of co-localization of *IgH-Igλ*, *IgH-C2*, or *IgH-K10* loci in day3.5-activated control or *CX^c/−* peripheral B cells (details in Suppl. Table 10). At least three mice were analyzed per data set; data are presented as mean ± s.e.m.

The c-myc DSB Frequency is Rate-limiting for IgH/c-myc Translocations

Given that CXP tumors routinely have IgH/c-myc translocations³⁶, we tested for IgH/c-myc co-localization in B cell nuclei via 3D interphase FISH (Fig. 4b). Approximately, 4-6% of resting, 15′ activated and 3.5 day activated control or CX^c/- B cell nuclei had co-localized IgH/c-myc signals (Fig. 4b,c; Suppl. Table 13), which were specific as IgH and c-myc did not co-localize in ES cells (Fig. 4c; Suppl. Table 13). While c-myc breaks and IgH/c-myc translocations were too infrequent to detect via FISH (Suppl. Table 14), PCR revealed an approximately 5-fold increase in IgH/c-myc translocations in activated CX^c/− B cells over low (<1×10^-6/cell) control levels (Fig. 4a; Suppl. Fig.13 and Table 15). Based on frequent IgH breaks and IgH/c-myc co-localization, we hypothesized c-myc breaks to be rate-limiting for IgH/c-myc translocations. To test this, we introduced 25 tandemly arrayed ISceI endonuclease target sites⁴² into the c-myc first intron to create the c-myc^25IsceI allele (Fig. 4d; Suppl. Fig. 14). The array was used to increase ISceI cut frequency. Then, αCD40/IL4-activated peripheral B cells heterozygous for the c-myc^25IsceI allele (c-myc^25ISceI/wt) or wt control B cells (c-myc^wt/wt) were infected with ISceI-expressing or control retrovirus⁴³ and assayed for c-myc breaks via metaphase FISH. Strikingly, c-myc chromosomal breaks occurred in approximately 10% of c-myc^25ISceI/wt B cells infected with the ISceI virus, but were absent in the various control B cells (Fig. 4e, Suppl. Table 16). PCR quantification demonstrated that IgH/c-myc translocations in ISceI virus-infected activated c-myc^25ISceI/wt B cells were increased by at least 100 fold over control levels (Fig. 4a, Suppl. Fig. 15 and Table 16).

a, Frequency of *IgH/c-myc* translocations from day4 αCD40/IL4-activated wt (n=4) and *CX^c/−* (n=4) splenic B cells, or B cells harboring *c-myc^25ISceI/wt* (n=3) or *c-myc^wt/wt* (n=1) infected with either control or ISceI-expressing retrovirus (details in Suppl. Fig. 13 and 15). **b, Top:** Schematic showing *c-myc* (red) probe on chromosome 15 and *3′IgH* (green) probe on chromosome 12. **Bottom:** Representative images of *IgH/c-myc* co-localization in day 0 control and *CX^c/−* B cell interphase nuclei. c, Quantification of *IgH/c-myc* association by 3D interphase FISH in control and *CX^c/−* splenic B cells (n=3), and ES cells (n=3). Cells were analyzed at the indicated time points before or after stimulation. d, Schematic showing the wt *c-myc* allele (*c-myc^wt*) and the modified *c-myc* allele containing 25 ISceI sites (*c-myc^25ISceI*). **e, Top left:** Diagram of *c-myc* FISH probes. **Bottom left:** Representative *c-myc* abnormalities in αCD40/IL-4-activated *c-myc^25ISceI/wt* B cells infected with IsceI-expressing retrovirus, appearing as green and red signals on separate chromosome fragments (white arrows). **Bottom right:** Quantification of *c-myc* breaks by metaphase FISH on day4 αCD40/IL4-activated B cells harboring either *c-myc^25ISceI/wt* (n=4) or *c-myc^wt/wt* (n=1) alleles after infection with control or ISceI-expressing retrovirus. Data are presented as mean ± std (details in Suppl. Table 16). High titer retrovirus infection appears to inhibit end joining allowing break visualization (see online methods).

Discussion

We show that some activated CX^c/- splenic B cells harbor characteristics of postulated “editing and switching” CXP peripheral B cell lymphoma progenitors³⁶, including Igκ deletions, aberrant Igλ V(D)J recombination, Igλ translocations, and aberrant IgH CSR associated with IgH translocations to c-myc or Igλ. Moreover, our studies clearly reveal V(D)J recombination-related events in CX^c/- splenic B cells; because they leave telltale RAG-dependent Igλ breaks. We note that cultured splenic B cells do not represent GC B cells⁴⁴ and CXP tumor progenitors do not appear of GC origin³⁶. Therefore, we suggest that V(D)J recombination events in activated CX^c/- splenic B cells and putative CXP lymphoma progenitors may represent peripheral “editing” mediated by low RAG expression, for example, as found in transitional B cells⁸^,⁴⁵^,⁴⁶. While potential physiological roles for such a process are unknown, it may be relevant for peripheral B cells subjected to chronic activation, such as those in gut-associated lymphoid tissues where CXP tumors arise³⁶. In this context, we find RAG-dependent Igλ breaks in CX^c/- mesenteric lymph node B cells taken directly from mice (unpublished data).

Our findings of RAG-initiated chromosomal breaks and translocations in Xrcc4-deficient peripheral B cells raises the possibility that translocations in some human peripheral B cell lymphomas, such as follicular lymphomas, might be initiated by V(D)J recombination in the periphery¹⁵^,³⁰. Our findings also demonstrate that AID and RAG can collaborate to generate frequent IgH/Igλ translocations in peripheral CX^c/- B cells. It is particularly notable that these IgH/Igλ translocations offer no obvious cellular selective advantage. Therefore, their appearance as clonal translocations in CXP lymphomas simply may reflect the frequent occurrence of these translocations in tumor progenitors due to mechanistic factors that include the two loci being frequently broken and spatially proximal. In the latter context, our findings demonstrate that the co-localization of two loci on different chromosomes can be quite focal, implicating aspects of particular loci themselves, beyond broader chromosomal territories⁴⁷, as important factors in determining spatial proximity and translocation frequency. Finally, analyses of oncogenic translocations in NHEJ-deficient pro-B and B cell lymphomas³⁶^,⁴⁸ suggested A-EJ may be translocation prone relative to NHEJ⁴⁹^,⁵⁰. The high frequency of specific translocations catalyzed by A-EJ in non-transformed CX^c/- B cells supports this notion.

Methods Summary

Generation of mouse strains utilized

CX^c/− mice were generated as previously described¹⁰ and crossed into AID-deficient mice³⁸ to generate CX^c/−A^-/- or mice carrying floxed RAG2 alleles⁴⁰ to generate CX^c/−RAG^c lines. We inserted a cassette containing 25 tandem ISceI target sites into the 1^st intron of c-myc by gene targeting (details in online Methods). Mice were analyzed as outlined in the text at 8–16 weeks of age. The Institutional Animal Care and Use Committee of Children's Hospital (Boston, Massachusetts) approved all animal work.

Splenic B cell Purification, Activation in Culture, Retroviral Infection and CSR Assays

CD43⁻ B cells were isolated from spleen, cultured, and assayed for CSR as previously described¹⁰^,²². Cells were sampled on various days for DNA isolation, flow cytometry analyses and metaphase preparation. Retroviral infection was performed as previously described⁴³ (details in online Methods).

Two-color FISH and telomere-FISH

Metaphase spreads from αCD40/IL4-activated B cell cultures were prepared and two-color FISH to detect IgH, Igκ, Igλ or c-myc chromosomal aberrations and telomere staining (T-FISH) to detect general aberrations were performed as previously described¹⁰. FISH probes are detailed in online Methods.

3D interphase FISH

3D FISH was performed as described previously³² (details in online Methods). Images of approximately 50 serial optical sections spaced by 0.2 microns were captured with Marianas spinning disk confocal microscope (63×) with a CCD detector (Intelligent Imaging Innovations) and analyzed with Slidebook software (Intelligent Imaging Innovations).

PCR assay to detect IgH/c-myc or IgH/Igλ translocations

IgH/c-myc translocation junctions were amplified by PCR from genomic DNA prepared from αCD40/IL4 activated splenic B cells using primers previously described²⁶. PCR products were run on agarose gels and hybridized with an internal c-myc oligo. IgH/Igλ translocations were amplified using nested primers for Sμ and Jλ. PCR products were hybridized with Jλ and IgH probes, the bands positive for both probes were cloned into the pGEM-T vector (Promega), sequenced and analyzed using Lasergene software and the NCBI database. Primer sequences and PCR conditions are detailed in online Methods.

Supplementary Material

Suppl. Figure 1. Overview of V(D)J recombination, receptor editing, and IgH CSR in B cells.

a, Diagram of Primary and Secondary V(D)J recombination during B cell development. See text for details. b, Diagram of IgH V(D)J recombination and CSR. See text for details.

Suppl. Figure 2. Distribution of different types of Igλ and Igκ abnormalities in activated CX^c/− or CXP splenic B cells.

The bar graphs show the distribution of the different types of Igκ and Igλ aberrations found in 4 day αCD40/IL4-activated CX^c/− or CXP splenic B cells categorized as the proportion of metaphases with either breaks alone, breaks plus translocations, or translocations alone.

Suppl. Figure 3. Kinetics of Igκ and Igλ breaks in activated CX^c/− splenic B cells.

Metaphases prepared from day 2, day 3 or day 4 αCD40/IL4-activated splenic B cells were analyzed via two-color FISH using Igκ or Igλ BAC probes as outlined in Figure 1. At least three mice were analyzed for each point (see Suppl. Table 6 for raw data). Data are presented as mean±s.e.m.

Suppl. Figure 4. Representative Igλ abnormalities visualized by 3D interphase FISH.

Interphase nuclei were isolated from day 4 αCD40/IL4-activated CX^c/− splenic B cells and analyzed by 3D FISH using 5′ (red) and 3′ (green) Igλ BAC probes as outlined in Figure 1d. Representative images with isolated centromeric or telomeric signals are shown. Among 14 Igλ locus breaks scored, 3 had split signals (the distance between green and red signals greater than 1.5μm), 3 had isolated red signals only, and 8 had isolated green signals only.

Suppl. Figure 5. Igκ and Igλ abnormalities in activated CX^c/− splenic B cells are not initiated by AID.

Metaphases from αCD40/IL4-activated splenic B cells (day 4) were analyzed by FISH for hybridization to Igκ or Igλ probes as outlined in Figure 1. Representative Igκ or Igλ breaks and translocations in CX^c/−AID^−/− splenic B cells are shown.

Suppl. Figure 6. Deletion of RAG conditional alleles in resting and activated splenic CX^c/−RAG^c B cells.

DNA samples were prepared from day 0, day 3, or day 4 αCD40/IL4-activated CX^c/−RAG^c/- B cells and from kidney (Kid) as a control. Genomic DNA was digested with StuI and hybridized with a 5′ RAG2 probe.

Suppl. Figure 7. Lack of Igκ and Igλ breaks or translocations in the same CX^c/− metaphases.

Metaphases from αCD40/IL4-activated CX^c/− B cells (day 4) were analyzed by sequential FISH hybridization first with 5′ and 3′ Igκ probes and then 5′ and 3′ Igλ probes as indicated. Upper: Representative metaphases with an intact Igκ locus and a broken/translocated Igλ locus in CX^c/− splenic B cells are shown. Bottom: Quantification of these sequential FISH analyses.

Suppl. Figure 8. Increased Jκ-Cκ deletion in metaphases harboring Igλ abnormalities.

Metaphases prepared from day 4 aCD40/IL-4 activated wt (n=2) or CX^c/− (n=3) splenic B cells were first analyzed via two color FISH to identify those with Igλ breaks and translocations. Subsequently the Igλ signals were stripped and the metaphases were assayed by two color FISH using a 5′ Igκ BAC probe (to identify chromosome 6 in the region of Igκ) and a Jκ-Cκ probe (indicated in the schematic map of Igκ locus) to assay for Igκ specific deletions. Upper: Bar graph showing that metaphases from wt B cells (n=207) have an intact Igκ locus in greater than 90% (open bars) while nearly 50% of metaphases from CX^c/- B cells with Igλ abnormalities (n=21) (black bars) had deletions of the Cκ region on either one or both alleles (scored as absence of Jκ-Cκ probe signal on the chromosome carrying the 5′ Igκ signal). Bottom: Map of Igκ locus showing position of Jκ-Cκ probe and indicating potential mechanisms for Igκ deletions via rearrangements to 3′RS.

Suppl. Figure 9. Frequent IgH and Igλ abnormalities in activated CX^c/− peripheral B cells.

a, Shown is an example of a metaphase with an Igκ break joined to an IgH break. Left: Igκ FISH shows a chromosomal translocation involving Igκ Sequential hybridizations of the same metaphase with chromosome 12 and chromosome 6 paints (center) followed by IgH FISH (right) revealed a translocation involving chromosome 12 (red) and chromosome 6 (green) with 3′IgH and 3′Igκ sequences at the breakpoint. b, Upper: Diagram of Igλ and IgH probes. Lower: Examples of metaphases with simultaneous Igλ and IgH breaks, as revealed by sequential Igλ FISH followed by IgH FISH. Metaphase 1: The broken Igλ locus (3′Igλ probe, red signal, left panel) is joined to a broken IgH locus (3′IgH probe, green signal, right panel) to form a dicentric chromosome, while the telomeric portion of chromosome 16 (5′Igλ probe, green signal, left panel) is fused with the telomeric portion of chromosome 12 (5′IgH probe, red signal, right panel) in an acentric fragment. The other chromosome 12 in the same metaphase is also involved in formation of a dicentric chromosome, which is only positive for the 3′IgH probe (green signal, right panel). Metaphase 2: Both the IgH and the Igλ loci are broken; and the chromosomal fragments containing the 5′Igλ probe (green, left panel) and the 5′IgH probe (red, right panel) are involved in translocations with different chromosomes. Metaphase 3: The Igλ locus is broken, with the 5′Igλ probe (green) joined to another chromosome to form a translocation (left panel), whereas the IgH locus is intact (right panel). c, Example is shown of a metaphase with an Igλ break joined to IgH break. Sequential hybridization of the same metaphase with Igλ probes, chromosome 12 and chromosome 16 paints and IgH probes revealed the telomeric portion of chromosome 16, containing the 5′Igλ probe (green, left panel) joined to the centromeric portion of chromosome 12, containing the 3′IgH probe (red, right panel).

Suppl. Figure 10. PCR assay to detect IgH/Igλ translocations in day 4 activated control and CX^c/− splenic B cells.

a, Schematic representation of the PCR assay used for IgH/Igλ translocations. Primers used to detect chromosome 12/16 translocations are represented as horizontal black arrows. The internal oligonucleotide probe used in Southern blot experiments is shown as a horizontal black bar. b, Representative Southern blots with the IgH or Jλ1 probe are shown for activated control, CX^c/− and CX^c/−A^−/− peripheral B cells (day 4 of αCD40/IL4 activation). No IgH/Igλ translocation was detected in 1.5×10⁶ cells from CX^c/−A^−/− mice. In each reaction DNA from ∼20000 cells was used.

Suppl. Figure 11. Sequence analyses of junctions of IgH/Igλ translocations.

IgH/Igλ translocations were cloned from 4 day αCD40/IL4-activated CX^c/⁻ (n=3) B cells by PCR. Sequences are aligned with genomic IgH (AJ851868) or Igλ (NG_004051) locus sequences. Mutations, insertions, or deletion were found in the Sμ regions (underlined). Microhomology at the junctions is underlined.

Suppl. Figure 12. Spatial proximity of IgH and Igκ.

The 3′ IgH probe and one of the three indicated Igκ region probes (F6, Igκ, I9) (diagrammed in lower panel) were used for 3D interphase FISH on control or CX^c/− peripheral B cell nuclei. The bar graph in the upper panel shows the percentage of nuclei in which the 3′IgH was found co-localized (closer than 0.5μm) with each of the Igκ regions as indicated. At least three mice were analyzed for each point (see Suppl. Table 12). The percentage of co-localization is presented as mean ± s.e.m based on results of at least 3 different experiments per sample.

Suppl. Figure 13. PCR assay to detect IgH/c-myc translocations in day 4 activated control and CX^c/− splenic B cells.

a, Schematic representation of the PCR assay used for IgH/c-myc translocations. Primers used to detect derivative chromosome 12 (der12) translocations are represented as horizontal black arrows. The internal oligonucleotide probe used in Southern blot experiments is shown as a horizontal black bar (T13). b, Representative Southern blots with the T13 probe are shown for activated control and CX^c/− peripheral B cells (day 4 of αCD40/IL4). In each reaction DNA from 100000 or 50000 cells was used (see Suppl. Table 15). c, Representative Southern blots with T13 probe are shown for activated control and CX^c/−RAG2^c splenic B cells at day 2 and day 3 of activation with αCD40/IL4. Each reaction used DNA from 50000 cells.

Suppl. Figure 14. Gene targeting strategy and Southern blot analyses of c-myc^25ISceI allele.

Top Diagram: Schematic maps of the targeting strategy for insertion of 25 ISceI sites into the 1^st intron of c-myc locus. The position of the 5′ and 3′ probes used for ES cell screening are shown in red. Bottom Panels: Southern blot analysis of a targeted clone before and after Neo cassette deletion.

Suppl. Figure 15. PCR assay to detect IgH/c-myc translocations in day 4-activated c-myc^25IsceI/wt splenic B cells infected with control or ISceI virus.

a, Schematic representation of the PCR assay used for IgH/c-myc translocations. Primers used to detect derivative chromosome 12 (der12) translocations are represented as horizontal black arrows. The internal oligonucleotide probe used in Southern blot experiments is shown as a horizontal black bar (T15). b, Representative Southern blots with the T15 probe are shown for activated c-myc^25IsceI/wt and c-myc^wt/wt peripheral B cells (day 4 of αCD40/IL4) infected with either control or ISceI virus. In each reaction, DNA from 50000 (control-infected samples) or 5000 (ISceI-infected samples) cells was used (see Suppl. Table 16).

Supplementary Table 1. Frequency of IgH breaks in Xrcc4-deficient peripheral B cells activated with αCD40/IL4 for 4 days

Supplementary Table 2. General Genomic Instability Measured by Telomere-FISH

Supplementary Table 3. Frequency of Igλ breaks in Xrcc4-deficient peripheral B cells activated with αCD40/IL4 for 4 days

Supplementary Table 4. Frequency of Igκ breaks in Xrcc4-deficient peripheral B cells activated with αCD40/IL4 for 4 days

Supplementary Table 5. Absence of Igκ and Igλ breaks in Xrcc4-deficient ES cells

Supplementary Table 6. Kinetics of Igλ and Igκ abnormalities in activated Xrcc4-deficient peripheral B cells

Supplemental Table 7. Igλ Abnormalities in CXc/- B cells measured by 3D Interphase FISH

Supplemental Table 8. IgH-Igλ, translocations measured by two color IgH-Igλ, FISH on control or CX^c/− B cells activated with αCD40/IL4 for 4 days

Supplementary Table 9. Igλ-IgH co-localization in X^c/c and CX^c/- B cells and other cell types measured by 3D interphase FISH

Supplementary Table 10. Igλ-IgH co-localization in activated control and CX^c/- B cells measured by 3D interphase FISH

Supplementary Table 11. IgH-chr16 control loci co-localization in X^c/c and CX^c/- B cells and other cell types by Interphase FISH

Supplementary Table 12. Igκ-IgH co-localization in control and CX^c/- B cells and other cell types by 3D Interphase FISH

Supplementary Table 13. IgH-cmyc co-localization in B cells and other cell types by 3D Interphase FISH

Supplementary Table 14. No c-myc abnormalities detected in activated Xrcc4-deficient B cells by two color FISH

Supplementary Table 15. Kinetics of IgH-cmyc translocations in Xrcc4-deficient peripheral B cells

Supplementary Table 16. c-myc breaks and IgH/c-myc translocation in ISceI targeted B cells

NIHMS212258-supplement-supplement_1.pdf^{(4.9MB, pdf)}

Acknowledgments

We thank Alt lab members for discussions, and Y.L. Chen, J. M. Bianco and M. Moghimi for technical assistance. This work was supported by NIH grant 5P01CA92625 and a Leukemia and Lymphoma Society of America (LLS) SCORE grant (to F.W.A. and K.R.). M.G. is and J.H.W. was a Special Fellow of LLS. J.H.W. and D.R.W. are supported by an NIH training grant and C.T.Y. was supported by an NCI training grant. A.N. is supported by the Intramural Research program of the NIH, NCI, Center for Cancer Research. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

Footnotes

Supplementary Information is linked to the online version of the paper at www.nature.com/nature

Author contributions F.W.A., J.H.W., M.G. and C.T.Y. planned studies and interpreted data. J.H.W. performed the majority of experiments, including mouse breeding, B cell studies, FISH, and IgH/Igλ PCR studies. C.T.Y. bred mice and performed B cell analyses. M.G. generated and analyzed c-myc^25IsceI/wt mice and performed FISH and IgH/c-myc translocation studies. P.G., T.H., and E.H. provided technical assistance. S.D. and A.N. provided expertise in 3D interphase FISH. A.A.Z. generated the 25 IsceI array. D.R.W. performed RAG expression studies and mesenteric lymph node B cell analyses. K.R. provided RAG conditional knock-out mice and helped interpret data. F.W.A., J.H.W. and M.G. wrote the paper.

Author information Reprints and permissions information is available at www.nature.com/reprints.

The authors declare no competing financial interests.

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