53BP1 alters the landscape of DNA rearrangements and suppresses AID-induced B cell lymphoma

B cell lymphoma in 53BP1^−/−IgkAID mice

Both AID expression and loss of 53BP1 have been associated with development of human B cell lymphomas (Kuppers, 2005),(Shaffer et al., 2002),(Okazaki et al., 2007),(Takeyama et al., 2008). However, neither 53BP1 mutation, nor AID over-expression alone is sufficient to induce B cell malignancy in mice (Ward et al., 2003),(Ward et al., 2005),(Robbiani et al., 2009),(Morales et al., 2006).To test the idea that the combination of deregulated AID expression and loss of 53BP1 is required for B cell lymphomagenesis we bred AID transgenic mice (IgkAID) (Robbiani et al., 2009) with 53BP1^−/− mice (Ward et al., 2003). Consistent with previous work, 53BP1^−/− mice did not develop lymphomas during an observation period of 12 months (Fig.1a and (Morales et al., 2006)). In contrast, during the same period 14 of the 28 53BP1^−/−IgkAID mice died, of which 5 were analyzed and found to have lymphomas (Fig.1a and b). Of these, 4 were B cell lymphomas, all of which were surface immunoglobulin negative (sIg-) (Fig.1b and Fig.S1a). We conclude that 53BP1 suppresses the development of AID-induced B cell lymphoma.

To further analyze the 53BP1^−/−IgkAID lymphomas we performed whole genome sequencing of 3 B cell tumors. To control for genetic variation we subtracted variations found in the genomes of C57BL/6, 53BP1^−/−, 129/Sv, and 17 other inbred strains of laboratory mice (Keane et al., 2011). The tumor genomes were primarily diploid with the exception of tumor 3, which carried a duplication of chromosome 14 and a ~40Mb duplication on chromosome 11 (Fig. S2b).

As expected for B cell lymphomas, sequence and copy number variation analysis showed alterations in the Ig genes (Fig.1c and d). Two tumors appeared to be derived from B cell precursors because the V_H and V_k gene segments were in germline configuration (Fig.1c and d). The third tumor was more mature B cell lymphoma displaying a deletion of the entire V_H region 5’ to IgH-V2-6 (GM16594_201) and a deletion of the V_k locus from Igκ-V2-112 to Igκ-J1 (Fig. 1c and d). However, all three of the lymphomas were unusual in that the J_H and Eμ regions were also deleted (Fig.1e). To confirm the sequencing results, we amplified the J_H4, Eμ, and Cμ regions of the IgH locus from 53BP1^−/−IgkAID lymphomas by PCR. As a control for PCR amplification, we used B cell tumors with intact IgH loci from P53^−/−IgkAID mice, (Robbiani et al., 2009). In contrast to the control, 53BP1^−/−IgkAID lymphomas showed loss of J_H4 and Eμ regions of the IgH locus (Fig.1f and S1b). Finding that the J_H4-Eμ region is deleted in 53BP1^−/− IgkAID lymphomas is consistent with the observation that components of the DNA damage response protect DSBs from resection during V(D)J recombination and CSR (Bothmer et al., 2010),(Difilippantonio et al., 2008),(Helmink et al., 2011).

In addition to deletions in IgH genes, and similar to what has been reported for other cancer genomes, 53BP1^−/−IgkAID B cell lymphomas showed numerous rearrangements (Fig.S2a). There were 313, 221 and 248 acquired structural variations including deletions, inversions and translocations in tumors 1, 2 and 3 respectively (Table S1). Although AID target genes such as Pax5, Ebf1, Il4ra and Irf4 (Fig S3) (Hakim et al., 2012),(Klein et al., 2011),(Liu et al., 2008) were involved in only a minority of these structural variations, analysis by Monte Carlo Simulation revealed that the proximity of rearrangement breakpoints to AID target genes was significantly increased over randomly expected (p<0.01). Further, chromosomal rearrangements in 53BP1^−/−IgkAID lymphomas differed from those reported for other cancers (Stephens et al., 2009), and from AID-induced rearrangements found in wild-type B cells (Klein et al., 2011), in that the breakpoints were biased to intergenic regions (Fig.S1c). Thus, loss of 53BP1 results in a unique cancer genome landscape.

Genome-wide rearrangements in the absence of 53BP1

The increased resection in the J_H4-Eμ region and bias towards intergenic rearrangements in 53BP1^−/−IgkAID B cell lymphomas might be intrinsic or result from selection during the transformation process. To examine the role of 53BP1 in recombinational repair of DSBs in primary B lymphocytes in the absence of selection, we produced translocation capture (TC-Seq) libraries from 53BP1 mutant B lymphocytes and compared them to wild type controls (Oliveira et al., 2012),(Klein et al., 2011).To produce the TC-Seq libraries we created a DSB at c-myc by infecting activated B cells that carry an I-SceI recognition site in c-myc (Myc^I) with a retrovirus encoding for the I-SceI meganuclease (Robbiani et al., 2008). Rearrangements between the I-SceI break in c-myc and DSBs elsewhere in the genome were amplified, and paired-end libraries were generated and analyzed by high-throughput sequencing (Oliveira et al., 2012),(Klein et al., 2011).

236,575 unique rearrangements from 100 million AID over-expressing (AID^rv) 53BP1^−/−MYC^I cells (hereafter referred to as 53BP1^−/−AID^rv) and 33,029 rearrangements from 100 million 53BP1^−/−MYC^IAID^−/− cells (hereafter referred to as 53BP1^−/−AID^−/−) were analyzed and compared to 53BP1 proficient controls (Fig.2a and (Klein et al., 2011)). Similar to the wild type controls, we found rearrangements between the I-SceI-induced DSB in c-myc (chromosome 15) and every other chromosome in the genome in both 53BP1^−/−AID^rv and 53BP1^−/−AID^−/− samples. However, the majority of rearrangements were mapped to chromosome 15 indicating that the bias for intra-chromosomal rearrangements is maintained in the absence of 53BP1 (Klein et al., 2011),(Chiarle et al., 2011),(Lieberman-Aiden et al., 2009),(Stephens et al., 2009),(Mahowald et al., 2009). This intra-chromosomal bias is most evident within 350kb around I-SceI site (Klein et al., 2011),(Chiarle et al., 2011).The profile of rearrangements in the 350kb region around the I-SceI site on chromosome 15 was indistinguishable in the presence or absence of 53BP1 irrespective of AID expression (Fig.2b). Because of the bias to proximal recombination, a region encompassing 50Kb on either side of the DSB was omitted from further analysis.

In addition to the bias for chromosomal rearrangement in cis (to chromosome 15), AID over-expressing wild-type B cells also showed a large number of rearrangements to chromosome 12 (Fig.2a). This pattern results from the presence of the IgH locus on chromosome 12, which is the primary target of AID (Klein et al.). In contrast to the wild-type cells, rearrangements to chromosome 12 are far less apparent in AID over-expressing 53BP1 deficient cells (Fig.2a). Specifically, rearrangements to IgH locus represent 10.3% of all rearrangements to chromosomes other than chromosome 15 in wild-type samples but only 0.76% in the absence of 53BP1.

Chromosome rearrangements and transcription

Transcriptionally active genes tend to segregate together in euchromatic regions of the nucleus (Kalhor et al., 2011),(Lieberman-Aiden et al., 2009),(Schoenfelder et al., 2010),(Sproul et al., 2005). TC-Seq experiments revealed that I-SceI induced DSBs in c-myc or IgH in B lymphocytes preferentially translocate to transcriptionally active genes (Klein et al., 2011),(Chiarle et al., 2011). Similarly, cancer associated rearrangements are preferentially genic (Stephens et al., 2009). On the other hand, the rearrangements in AID-induced 53BP1 deficient lymphomas are biased towards intergenic regions (Fig.S1c).

To determine whether the altered preference for intergenic rearrangements in 53BP1 deficient lymphomas is due to selection we analyzed the rearrangements in TC-Seq libraries produced from primary B cells cultured for only a short time in vitro (Fig.3a). The frequency of intergenic rearrangements was significantly increased in 53BP1^−/−AID^−/− libraries compared to wild type (54.1 % vs. 50.07% respectively; P<0.0001). This bias for intergenic rearrangements was even more pronounced in 53BP1^−/−AID^rv samples, partly due to the relative decrease in rearrangements to the IgH locus (54% vs. 45% in wild type; P<0.0001). Consistent with this observation, the enrichment of genic rearrangements at transcription start sites (TSS) found in wild-type cells was not apparent in 53BP1 deficient samples (Fig.3b) (Klein et al., 2011). Finally, the preference for transcribed genes among genic rearrangements was less significant in 53BP1 deficient than in wild-type samples (for example fe=1.2 vs. fe=1.4 for medium transcribed genes) (Fig.3c). We conclude that rearrangement bias for genic regions and actively transcribed genes is decreased in the absence of 53BP1.

To determine whether the differences in translocation partner choice were due to differences in transcription, we compared the transcriptome of 53BP1 deficient and wild-type B cells by RNA-Seq and Polymerase II chromatin immunoprecipitation sequencing (ChIP-Seq). We found that neither the RNA content nor polymerase loading was significantly different between 53BP1 deficient and wild-type B cells (S=3.2536e+10 for Pol II ChIP-Seq and S=3932969 for RNA-Seq)(Fig.S4). To attempt to evaluate global nuclear architecture in 53BP1^−/− cells we performed chromosome conformation capture sequencing (4C-Seq) using IgH as bait (Fig.S5). We found no differences that could explain the skewing of the 53BP1 deficient samples toward intergenic region rearrangement, instead there was significant correlation between contacts in wild-type and 53BP1^−/− cells (Spearman’s r=0.67)(Fig.S5). However, the resolution of the technique is only to 200Kbs and therefore it is insufficiently sensitive to make definitive conclusions about alterations in genic regions, which average only 16.3Kb. We conclude that transcription and large-scale chromosome contacts are not significantly altered in the absence of 53BP1.

Translocation hotspots

To determine whether rearrangement hotspots differ in the absence of 53BP1, we searched the 53BP1^−/− libraries for local accumulations of reads (Klein et al., 2011) and compared them to 53BP1 sufficient controls (Table S2). The 27 hotspots in 53BP1^−/−AID^−/− resembled those found in AID^−/− samples in that 9 were in the Pvt1 gene, which is adjacent to c-myc, and 7 in the vicinity of cryptic I-SceI sites (Klein et al., 2011),(Chiarle et al., 2011) (Fig.4a, bottom left). In addition, there was a highly significant overlap between the 79 rearrangement hotspots found in 53BP1^−/−AID^rv and 53BP1^+/+AID^rv libraries that involved AID target gene sites (Klein et al., 2011),(Chiarle et al., 2011) (Fig.4a). In agreement with the reduced rearrangements to genic regions in the absence of 53BP1 (Fig.3), hotspots were less frequent in genic regions for 53BP1^−/− cells compared to 53BP1^+/+ irrespective of AID expression (Fig.4a). We conclude that 53BP1 deficiency changes the landscape of rearrangement hotspots resulting in increased probability of rearrangement to intergenic regions.

DNA end resection

53BP1 is required for protection of broken DNA ends from resection during class switch recombination(Bothmer et al., 2010), and it facilitates homologous recombination in the absence of BRCA1 by allowing formation of ssDNA (Bunting et al., 2010). To determine whether there is increased resection of DNA ends during chromosome rearrangements in the absence of 53BP1 we compared the shape and size of the hotspots obtained from 53BP1^−/−AID^rv and 53BP1^+/+AID^rv libraries. Visual inspection of translocations involving the IgH locus revealed that the hotspots were broader in the absence of 53BP1, with events extending both 5′ and 3′ when compared to wild-type (Fig.4b). Furthermore, while 53BP1^+/+AID^rv sample had a defined peak of translocations in switch regions, the 53BP1 deficient sample had two peaks located 5′ and 3′ to the wild-type peak, consistent with DNA resection (Fig.4b). Visual inspection of non-Ig hotspots also showed broader profiles in the absence of 53BP1 suggestive of resection (Fig.4d and Fig.S6). To quantify this effect we analyzed the distribution of translocations in the 16 prominent hotspots present 53BP1^+/+AID^rv and compared them to 53BP1^−/−AID^rv samples (Fig. 4c). This analysis shows that in the wild-type translocations spread ~10kb on the either side of the hotspot peak, while in the absence of 53BP1 they reach up to 20kb upstream and downstream of the hotspot peak. Our results suggest that in the absence of 53BP1 DSBs are resected more extensively compared to wild-type before joining.

Mice

Mice bearing I-SceI recognition sites in the first intron of c-myc (Myc^I/I) (Robbiani et al., 2008) were crossed to 53BP1^−/− (Ward et al., 2003) and to AID^−/−(Muramatsu et al., 2000) and then intercrossed to obtain 53BP1^−/−MYC^I/I and 53BP1^−/−MYC^IAID^−/− mice for experiments. 53BP1^−/−IgkAID mice were generated by intercrossing of 53BP1^−/− (Ward et al., 2003) and IgkAID (Robbiani et al., 2009) mice. All experiments were performed in accordance with protocols approved by the Rockefeller University and National Institutes of Health (NIH) Institutional Animal Care and Use Committee.

B cell cultures

Resting B lymphocytes were isolated from mouse spleens by immunomagnetic depletion with anti-CD43 MicroBeads (Miltenyi Biotech) and cultured at 0.5 × 10⁶ cells/ml in RPMI medium. B cells were stimulated by addition of 500ng/ml RP105 (BD Pharmingen), 25 μg/ml lipopolysaccharide (LPS) (Sigma) and 5 ng/ml mouse recombinant IL-4 (Sigma). Retroviral supernatants were harvested 72 hours after co-transfection of BOSC23 cells with pCL-Eco and pMX-IRES-GFP-derived plasmids encoding for I-SceI-mCherry or AID-GFP. B cells were spinoculated at 1150 × g for 1.5 hr in the presence of 10 μg/ml polybrene at 20 and 44 hours after initiation of culture. After 4 hr at 37°C, viral supernatants were replaced with LPS and IL-4 in supplemented RPMI. For dual infection, retroviral supernatants were added simultaneously. Dually infected B cells were sorted for double positive cells after a 96 hour culture period using a FACSAria (Becton Dickson).

TC-Seq libraries and computational analysis

Translocation libraries were made as described (Oliveira et al., 2012). Briefly, genomic DNA was extracted with phenol/chloroform and then fragmented by sonication at low power for 7 cycles (30″ on/30″ off) in a Bioruptor (Diagenode) to yield a 500–1350bp distribution of DNA fragments. DNA was blunted by End-It DNA Repair Kit (Epicentre) and A-tailed. DNA fragments were then ligated to linkers. Native loci were eliminated by restriction digestion with I-SceI. Pooled linker-ligated DNA was divided into 2 equal parts for semi-nested ligation-mediated PCR using biotinylated forward or reverse primers. Forward and reverse enrichment streams were kept separate for the entire remainder of the protocol. Computational analysis of TC-Seq libraries was done as described (Klein et al., 2011).

Chromatin Immunoprecipitation and sequencing (ChIP-Seq)

ChIP-Seq was performed as described. In brief, cells were fixed with 1% paraformaldehyde at 37°C for 10 minutes followed by sonication. Chromatin fragments were then immunoprecipitated with antibodies specific for serine 5-phosphorylated RNA Polymerase II (Abcam). Immunoprecipitates were processed following Illumina’s protocol and sequenced on a Genome Analyzer.

Flow cytometry

Moribund mice were analyzed for tumor development by necropsy. Single cell suspensions were made from tumors and analyzed by flow cytometry as previously described (Robbiani et al., 2009). All antibodies used were from BD.

PCR

Genomic DNA (gDNA) from B cell tumors of 53BP1^−/−IgkAID and P53^−/− IgkAID mice (Robbiani et al., 2009) was analyzed by qPCR as described (Robbiani et al., 2009) using the following primers: 5′-AGAATGGCCTCTCCAGGTCT-3′ and 5′-TGCAATGTTCAGAAAACTCCATA-3′ for amplification of J_H4, 5′-ACCTGGGAATGTATGGTTGTGGCT -3′ and 5′-TTACCATTTGCGGTGCCTGGTTTC-3′ for amplification of Eμ, 5′-ACCTGGCAACCTATGAAACCCTGA-3′ and 5′-AACACTAGCCACACCCTTAGCACT-3′for amplification of Cμ, 5′-TGACCAACGCGACCTCATCTCTAA-3′ and 5′-AAGGGCTCTCTGCTGGTATTCACA-3′ for amplification of Cd74. Amplification products were analyzed additionally by gel electrophoresis.

Whole-genome library generation and analysis

Whole-genome libraries were produced according to the Paired-end Sample Preparation kit (Illumina) protocol with minor modifications. In brief, 5 μg gDNA from three 53BP1^−/−IgkAID sIg-B cell tumors, C57/BL6 tails, 53BP1^−/− spleenocytes and 129/Sv embryonic stem cells was sonicated to an average fragment size of 500bp, respectively. For converting DNA ends into blunt ends the End-it DNA end repair kit (Epicenter Biotechnologies) was used. Purification steps, addition of dATP to 3′ DNA ends and adapter ligation was performed according to the Illumina protocol. Post adapter ligation DNA fragments were separated on a 2% agarose gel and separated by electrophoresis. 538-575bp fragments were extracted from gels, purified and used for pre-amplification by PCR according to the Illumina protocol. Purified PCR products were analyzed for their quality by Bioanalyzer (Agilent) and subjected to paired-end sequencing.

Chromosome conformation capture on chip (4C) followed by deep sequencing

The 4C assay was performed as previously described (Hakim et al., 2012). In brief, ten million mouse B cells were crosslinked in 2% formaldehyde at 37 °C for 10 min and the reaction was quenched by the addition of glycine. Cells were then washed with cold PBS and lysed and nuclei were incubated at 37 °C for 1 h in 500 μl of restriction buffer (New England DNA digestion was performed with 400 U of HindIII or BglII (New England Biolabs) at 37 °C overnight. After heat inactivation (65 °C for 30 min), the reaction was diluted to a final volume of 7 ml with ligation buffer containing 100 U T4 DNA Ligase (Roche) and incubated at 16 °C overnight. Samples were then treated with 500 μg Proteinase K (Ambion) and incubated overnight at 65 °C to reverse formaldehyde crosslinking. DNA was then purified by phenol extraction and ethanol precipitation. For circularization, the ligation junctions were digested with Csp6I (Fermentas) or DpnII (New England Biolabs) at 37 °C overnight. After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche). Three micrograms of 4C library DNA was amplified with Expand Long template PCR System (Roche). Thermal cycle conditions were DNA denaturing for 2 min at 94 °C, followed by 30 cycles of 15 s at 94 °C, 1 min at 60 °C, 3 min at 68 °C, and a final step of 7 min at 68 °C. Baits were amplified with inverse PCR primers as follows: IgH with HindIII: IgH_R_4C 5′-CCAGACATGTGGGCTGAGAT-3′, Igh_Hind_Read 5′-CTACCCACCTAACTCCAAGC-3′; 4C amplified DNA was micro-sequenced with the Illumina platform.

01

02

03

1. Anderson L, Henderson C, Adachi Y. Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol Cell Biol. 2001;21:1719–1729. doi: 10.1128/MCB.21.5.1719-1729.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Basu U, Meng FL, Keim C, Grinstein V, Pefanis E, Eccleston J, Zhang T, Myers D, Wasserman CR, Wesemann DR, et al. The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates. Cell. 2011;144:353–363. doi: 10.1016/j.cell.2011.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Bignell GR, Greenman CD, Davies H, Butler AP, Edkins S, Andrews JM, Buck G, Chen L, Beare D, Latimer C, et al. Signatures of mutation and selection in the cancer genome. Nature. 2010;463:893–898. doi: 10.1038/nature08768. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Boeva V, Zinovyev A, Bleakley K, Vert J-P, Janoueix-Lerosey I, Delattre O, Barillot E. Control-free calling of copy number alterations in deep-sequencing data using GC-content normalization. Bioinformatics (Oxford, England) 2011;27:268–269. doi: 10.1093/bioinformatics/btq635. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Bothmer A, Robbiani DF, Feldhahn N, Gazumyan A, Nussenzweig A, Nussenzweig MC. 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination. J Exp Med. 2010;207:855–865. doi: 10.1084/jem.20100244. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Bouwman P, Aly A, Escandell JM, Pieterse M, Bartkova J, van der Gulden H, Hiddingh S, Thanasoula M, Kulkarni A, Yang Q, et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat Struct Mol Biol. 2010;17:688–695. doi: 10.1038/nsmb.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Bunting SF, Callen E, Wong N, Chen HT, Polato F, Gunn A, Bothmer A, Feldhahn N, Fernandez-Capetillo O, Cao L, et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell. 2010;141:243–254. doi: 10.1016/j.cell.2010.03.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Chaudhuri J, Khuong C, Alt FW. Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature. 2004;430:992–998. doi: 10.1038/nature02821. [DOI] [PubMed] [Google Scholar]
9. Chiarle R, Zhang Y, Frock RL, Lewis SM, Molinie B, Ho YJ, Myers DR, Choi VW, Compagno M, Malkin DJ, et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell. 2011;147:107–119. doi: 10.1016/j.cell.2011.07.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Di Noia JM, Williams GT, Chan DT, Buerstedde JM, Baldwin GS, Neuberger MS. Dependence of antibody gene diversification on uracil excision. J Exp Med. 2007;204:3209–3219. doi: 10.1084/jem.20071768. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Difilippantonio S, Gapud E, Wong N, Huang CY, Mahowald G, Chen HT, Kruhlak MJ, Callen E, Livak F, Nussenzweig MC, et al. 53BP1 facilitates long-range DNA end-joining during V(D)J recombination. Nature. 2008;456:529–533. doi: 10.1038/nature07476. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. DiTullio RA, Jr., Mochan TA, Venere M, Bartkova J, Sehested M, Bartek J, Halazonetis TD. 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat Cell Biol. 2002;4:998–1002. doi: 10.1038/ncb892. [DOI] [PubMed] [Google Scholar]
13. Fernandez-Capetillo O, Chen HT, Celeste A, Ward I, Romanienko PJ, Morales JC, Naka K, Xia Z, Camerini-Otero RD, Motoyama N, et al. DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat Cell Biol. 2002;4:993–997. doi: 10.1038/ncb884. [DOI] [PubMed] [Google Scholar]
14. Franco S, Gostissa M, Zha S, Lombard DB, Murphy MM, Zarrin AA, Yan C, Tepsuporn S, Morales JC, Adams MM, et al. H2AX prevents DNA breaks from progressing to chromosome breaks and translocations. Mol Cell. 2006;21:201–214. doi: 10.1016/j.molcel.2006.01.005. [DOI] [PubMed] [Google Scholar]
15. Gostissa M, Alt FW, Chiarle R. Mechanisms that promote and suppress chromosomal translocations in lymphocytes. Annu Rev Immunol. 2011;29:319–350. doi: 10.1146/annurev-immunol-031210-101329. [DOI] [PubMed] [Google Scholar]
16. Hakim O, Resch W, Yamane A, Klein I, Kieffer-Kwon KR, Jankovic M, Oliveira T, Bothmer A, Voss TC, Ansarah-Sobrinho C, et al. DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature. 2012;484:69–74. doi: 10.1038/nature10909. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Helmink BA, Tubbs AT, Dorsett Y, Bednarski JJ, Walker LM, Feng Z, Sharma GG, McKinnon PJ, Zhang J, Bassing CH, Sleckman BP. H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes. Nature. 2011;469:245–249. doi: 10.1038/nature09585. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Jankovic M, Nussenzweig A, Nussenzweig MC. Antigen receptor diversification and chromosome translocations. Nat Immunol. 2007;8:801–808. doi: 10.1038/ni1498. [DOI] [PubMed] [Google Scholar]
19. Kalhor R, Tjong H, Jayathilaka N, Alber F, Chen L. Genome architectures revealed by tethered chromosome conformation capture and population-based modeling. Nat Biotechnol. 2011;30:90–98. doi: 10.1038/nbt.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Keane TM, Goodstadt L, Danecek P, White MA, Wong K, Yalcin B, Heger A, Agam A, Slater G, Goodson M, et al. Mouse genomic variation and its effect on phenotypes and gene regulation. Nature. 2011;477:289–294. doi: 10.1038/nature10413. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Klein IA, Resch W, Jankovic M, Oliveira T, Yamane A, Nakahashi H, Di Virgilio M, Bothmer A, Nussenzweig A, Robbiani DF, et al. Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell. 2011;147:95–106. doi: 10.1016/j.cell.2011.07.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome research. 2009;19:1639–1645. doi: 10.1101/gr.092759.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Kumar-Sinha C, Tomlins SA, Chinnaiyan AM. Recurrent gene fusions in prostate cancer. Nat Rev Cancer. 2008;8:497–511. doi: 10.1038/nrc2402. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Kuppers R. Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer. 2005;5:251–262. doi: 10.1038/nrc1589. [DOI] [PubMed] [Google Scholar]
25. Lee AC, Fernandez-Capetillo O, Pisupati V, Jackson SP, Nussenzweig A. Specific association of mouse MDC1/NFBD1 with NBS1 at sites of DNA-damage. Cell Cycle. 2005;4:177–182. doi: 10.4161/cc.4.1.1354. [DOI] [PubMed] [Google Scholar]
26. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics (Oxford, England) 2009;25:1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009;326:289–293. doi: 10.1126/science.1181369. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Liu M, Duke JL, Richter DJ, Vinuesa CG, Goodnow CC, Kleinstein SH, Schatz DG. Two levels of protection for the B cell genome during somatic hypermutation. Nature. 2008;451:841–845. doi: 10.1038/nature06547. [DOI] [PubMed] [Google Scholar]
29. Mahowald GK, Baron JM, Mahowald MA, Kulkarni S, Bredemeyer AL, Bassing CH, Sleckman BP. Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient lymphocytes target chromosomal breakpoints in cis. Proc Natl Acad Sci U S A. 2009;106:18339–18344. doi: 10.1073/pnas.0902545106. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Morales JC, Franco S, Murphy MM, Bassing CH, Mills KD, Adams MM, Walsh NC, Manis JP, Rassidakis GZ, Alt FW, Carpenter PB. 53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis. Proc Natl Acad Sci U S A. 2006;103:3310–3315. doi: 10.1073/pnas.0511259103. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 2000;102:553–563. doi: 10.1016/s0092-8674(00)00078-7. [DOI] [PubMed] [Google Scholar]
32. Muto T, Okazaki IM, Yamada S, Tanaka Y, Kinoshita K, Muramatsu M, Nagaoka H, Honjo T. Negative regulation of activation-induced cytidine deaminase in B cells. Proc Natl Acad Sci U S A. 2006;103:2752–2757. doi: 10.1073/pnas.0510970103. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Noon AT, Shibata A, Rief N, Lobrich M, Stewart GS, Jeggo PA, Goodarzi AA. 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair. Nat Cell Biol. 2010;12:177–184. doi: 10.1038/ncb2017. [DOI] [PubMed] [Google Scholar]
34. Nussenzweig A, Nussenzweig MC. Origin of chromosomal translocations in lymphoid cancer. Cell. 2010;141:27–38. doi: 10.1016/j.cell.2010.03.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Okazaki IM, Kotani A, Honjo T. Role of AID in tumorigenesis. Adv Immunol. 2007;94:245–273. doi: 10.1016/S0065-2776(06)94008-5. [DOI] [PubMed] [Google Scholar]
36. Oliveira TY, Resch W, Jankovic M, Casellas R, Nussenzweig MC, Klein IA. Translocation capture sequencing: a method for high throughput mapping of chromosomal rearrangements. J Immunol Methods. 2012;375:176–181. doi: 10.1016/j.jim.2011.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Pasqualucci L, Neumeister P, Goossens T, Nanjangud G, Chaganti RS, Kuppers R, Dalla-Favera R. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature. 2001;412:341–346. doi: 10.1038/35085588. [DOI] [PubMed] [Google Scholar]
38. Pavri R, Gazumyan A, Jankovic M, Di Virgilio M, Klein I, Ansarah-Sobrinho C, Resch W, Yamane A, Reina San-Martin B, Barreto V, et al. Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5. Cell. 2010;143:122–133. doi: 10.1016/j.cell.2010.09.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Pavri R, Nussenzweig MC. AID targeting in antibody diversity. Adv Immunol. 2011;110:1–26. doi: 10.1016/B978-0-12-387663-8.00005-3. [DOI] [PubMed] [Google Scholar]
40. Potter M. Neoplastic development in plasma cells. Immunol Rev. 2003;194:177–195. doi: 10.1034/j.1600-065x.2003.00061.x. [DOI] [PubMed] [Google Scholar]
41. Quinlan AR, Clark RA, Sokolova S, Leibowitz ML, Zhang Y, Hurles ME, Mell JC, Hall IM. Genome-wide mapping and assembly of structural variant breakpoints in the mouse genome. Genome research. 2010;20:623–635. doi: 10.1101/gr.102970.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics (Oxford, England) 2010;26:841–842. doi: 10.1093/bioinformatics/btq033. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Ramiro AR, Jankovic M, Callen E, Difilippantonio S, Chen HT, McBride KM, Eisenreich TR, Chen J, Dickins RA, Lowe SW, et al. Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. Nature. 2006;440:105–109. doi: 10.1038/nature04495. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Rappold I, Iwabuchi K, Date T, Chen J. Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. J Cell Biol. 2001;153:613–620. doi: 10.1083/jcb.153.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A, et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2) Cell. 2000;102:565–575. doi: 10.1016/s0092-8674(00)00079-9. [DOI] [PubMed] [Google Scholar]
46. Robbiani DF, Bothmer A, Callen E, Reina-San-Martin B, Dorsett Y, Difilippantonio S, Bolland DJ, Chen HT, Corcoran AE, Nussenzweig A, Nussenzweig MC. AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations. Cell. 2008;135:1028–1038. doi: 10.1016/j.cell.2008.09.062. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Robbiani DF, Bunting S, Feldhahn N, Bothmer A, Camps J, Deroubaix S, McBride KM, Klein IA, Stone G, Eisenreich TR, et al. AID produces DNA double-strand breaks in non-Ig genes and mature B cell lymphomas with reciprocal chromosome translocations. Mol Cell. 2009;36:631–641. doi: 10.1016/j.molcel.2009.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A, Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS, et al. Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat Genet. 2010;42:53–61. doi: 10.1038/ng.496. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Schultz LB, Chehab NH, Malikzay A, Halazonetis TD. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol. 2000;151:1381–1390. doi: 10.1083/jcb.151.7.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Shaffer AL, Rosenwald A, Staudt LM. Lymphoid malignancies: the dark side of B-cell differentiation. Nat Rev Immunol. 2002;2:920–932. doi: 10.1038/nri953. [DOI] [PubMed] [Google Scholar]
51. Shen HM, Bozek G, Pinkert CA, McBride K, Wang L, Kenter A, Storb U. Expression of AID transgene is regulated in activated B cells but not in resting B cells and kidney. Mol Immunol. 2008;45:1883–1892. doi: 10.1016/j.molimm.2007.10.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
52. Sproul D, Gilbert N, Bickmore WA. The role of chromatin structure in regulating the expression of clustered genes. Nat Rev Genet. 2005;6:775–781. doi: 10.1038/nrg1688. [DOI] [PubMed] [Google Scholar]
53. Stavnezer J, Guikema JE, Schrader CE. Mechanism and regulation of class switch recombination. Annu Rev Immunol. 2008;26:261–292. doi: 10.1146/annurev.immunol.26.021607.090248. [DOI] [PMC free article] [PubMed] [Google Scholar]
54. Stephens PJ, McBride DJ, Lin ML, Varela I, Pleasance ED, Simpson JT, Stebbings LA, Leroy C, Edkins S, Mudie LJ, et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature. 2009;462:1005–1010. doi: 10.1038/nature08645. [DOI] [PMC free article] [PubMed] [Google Scholar]
55. Storb U, Shen HM, Longerich S, Ratnam S, Tanaka A, Bozek G, Pylawka S. Targeting of AID to immunoglobulin genes. Adv Exp Med Biol. 2007;596:83–91. doi: 10.1007/0-387-46530-8_8. [DOI] [PubMed] [Google Scholar]
56. Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu Rev Genet. 2011;45:247–271. doi: 10.1146/annurev-genet-110410-132435. [DOI] [PubMed] [Google Scholar]
57. Takeyama K, Monti S, Manis JP, Dal Cin P, Getz G, Beroukhim R, Dutt S, Aster JC, Alt FW, Golub TR, Shipp MA. Integrative analysis reveals 53BP1 copy loss and decreased expression in a subset of human diffuse large B-cell lymphomas. Oncogene. 2008;27:318–322. doi: 10.1038/sj.onc.1210650. [DOI] [PubMed] [Google Scholar]
58. Ward IM, Difilippantonio S, Minn K, Mueller MD, Molina JR, Yu X, Frisk CS, Ried T, Nussenzweig A, Chen J. 53BP1 cooperates with p53 and functions as a haploinsufficient tumor suppressor in mice. Mol Cell Biol. 2005;25:10079–10086. doi: 10.1128/MCB.25.22.10079-10086.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
59. Ward IM, Minn K, van Deursen J, Chen J. p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice. Mol Cell Biol. 2003;23:2556–2563. doi: 10.1128/MCB.23.7.2556-2563.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
60. Yamane A, Resch W, Kuo N, Kuchen S, Li Z, Sun HW, Robbiani DF, McBride K, Nussenzweig MC, Casellas R. Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes. Nat Immunol. 2011;12:62–69. doi: 10.1038/ni.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
61. Zhang Y, Gostissa M, Hildebrand DG, Becker MS, Boboila C, Chiarle R, Lewis S, Alt FW. The role of mechanistic factors in promoting chromosomal translocations found in lymphoid and other cancers. Adv Immunol. 2010;106:93–133. doi: 10.1016/S0065-2776(10)06004-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
62. Ziv Y, Bielopolski D, Galanty Y, Lukas C, Taya Y, Schultz DC, Lukas J, Bekker-Jensen S, Bartek J, Shiloh Y. Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway. Nat Cell Biol. 2006;8:870–876. doi: 10.1038/ncb1446. [DOI] [PubMed] [Google Scholar]

Supplementary Materials