Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient ...

Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient lymphocytes target chromosomal breakpoints in cis Grace K. Mahowalda, Jason M. Barona, Michael A. Mahowalda, Shashikant Kulkarnia, Andrea L. Bredemeyera, Craig H. Bassingb,c, and Barry P. Sleckmana,1 aDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; bDepartment of Pathology and Laboratory Medicine, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA19104; and cAbramson Family Cancer Research Institute, Philadelphia, PA 19104

Canonical chromosomal translocations juxtaposing antigen receptor genes and oncogenes are a hallmark of many lymphoid malignancies. These translocations frequently form through the joining of DNA ends from double-strand breaks (DSBs) generated by the recombinase activating gene (RAG)-1 and -2 proteins at lymphocyte antigen receptor loci and breakpoint targets near oncogenes. Our understanding of chromosomal breakpoint target selection comes primarily from the analyses of these lesions, which are selected based on their transforming properties. RAG DSBs are rarely resolved aberrantly in wild-type developing lymphocytes. However, in ataxia telangiectasia mutated (ATM)-deficient lymphocytes, RAG breaks are frequently joined aberrantly, forming chromosomal lesions such as translocations that predispose (ATM)deficient mice and humans to the development of lymphoid malignancies. Here, an approach that minimizes selection biases is used to isolate a large cohort of breakpoint targets of aberrantly resolved RAG DSBs in Atm-deficient lymphocytes. Analyses of this cohort revealed that frequently, the breakpoint targets for aberrantly resolved RAG breaks are other DSBs. Moreover, these nonselected lesions exhibit a bias for using breakpoints in cis, forming small chromosomal deletions, rather than breakpoints in trans, forming chromosomal translocations. ataxia telangiectasia mutated 兩 chromosomal translocation 兩 DNA double-strand break repair 兩 V(D)J recombination

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ouble-strand breaks (DSBs) in DNA are generated by genotoxic agents and cellular endonucleases as intermediates in several important physiologic processes including V(D)J recombination, Ig class switch recombination (CSR), DNA replication, gene transcription, and meiosis. DNA DSBs activate a highly conserved cellular response that prevents cell cycle progression, initiates repair of the broken DNA ends, and promotes apoptosis of cells with persistent un-repaired DSBs (1, 2). Broken DNA ends from a single DSB are usually rejoined; however, in some processes, such as V(D)J recombination and CSR, DNA ends arising from two DSBs are joined in a regulated fashion, generating a new gene product (1–4). Broken DNA ends from distinct DSBs can also be joined aberrantly, leading to the formation of potentially dangerous chromosomal lesions such as translocations, deletions, and inversions. All developing lymphocytes generate programmed DNA DSBs during V(D)J recombination, a process that joins variable (V), joining (J), and in some cases, diversity (D) gene segments to generate the second exon of antigen receptor genes (4). The V(D)J recombination reaction is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which together form the RAG endonuclease (5). RAG introduces DNA DSBs at the border of two recombining gene segments and their flanking RAG recognition sequences, termed recombination signals (RSs) (5). DNA cleavage by RAG occurs after an appropriate RS pair (12/23 compatible) forms a synaptic complex, generating www.pnas.org兾cgi兾doi兾10.1073兾pnas.0902545106

a pair of coding ends and a pair of signal ends (5). These DNA ends are then processed and joined by the nonhomologous end-joining (NHEJ) pathway of DNA DSB repair to form a coding joint and signal joint, respectively (4). The generation and repair of RAG-mediated DSBs occurs in developing lymphocytes only during the G1-phase of the cell cycle (6). Several lymphoid malignancies are associated with canonical translocations involving antigen receptor loci and potential oncogenes such as c-Myc and Bcl-2 (7, 8). Breakpoint analyses demonstrate that many of these translocations form through the aberrant joining of RAG-mediated DNA breaks at antigen receptor loci to specific chromosomal regions, hereafter referred to as breakpoint targets, near potential oncogenes. The formation of these lesions likely also requires the generation of a DSB at the breakpoint target, which could potentially occur through off-target RAG activity (9–12). Because these lesions are selected based on their transforming ability, it is not possible to know whether there is a significant mechanistic bias for the use of these breakpoint targets. However, analyses of mice in which the c-Myc-coding region was replaced with that of N-Myc demonstrated that the bias for the targeting of c-Myc over N-Myc by IgH translocations in pro-B cell tumors is not due to selection for the specific oncogenic properties of c-Myc (13). Rather, the formation of pro-B cell tumors in these mice suggests that there are mechanistic constraints that favor breakpoint targeting of the c-Myc locus in these cells. Although the aberrant resolution of RAG DSBs occurs extremely rarely in wild-type lymphocytes, these lesions are generated at higher frequencies in lymphocytes with DNA repair defects (14–16). In this regard, deficiency in the ataxia telangiectasia mutated (ATM) serine threonine kinase in both mice and humans leads to an increased incidence of lymphoid tumors with RAG-dependent translocations involving antigen receptor loci (17–21). In addition, mature, nontransformed Atm-deficient lymphocytes have an increased frequency of translocations and other chromosomal aberrations involving antigen receptor loci (18, 19, 22–24). Murine pre-B cells transformed through the expression of the viral abl kinase, hereafter referred to as abl pre-B cells, have been used to study the generation and repair of chromosomal RAG-generated DSBs (25). Inhibition of the abl kinase with STI571 leads to G1 cell cycle arrest, induction of RAG expression, and robust rearrangement of the endogeAuthor contributions: G.K.M., J.M.B., C.H.B., and B.P.S. designed research; G.K.M., J.M.B., and S.K. performed research; A.L.B. contributed new reagents/analytic tools; G.K.M., J.M.B., M.A.M., and B.P.S. analyzed data; and G.K.M. and B.P.S. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1To

whom correspondence should be addressed. E-mail: [email protected]..

This article contains supporting information online at www.pnas.org/cgi/content/full/ 0902545106/DCSupplemental.

PNAS 兩 October 27, 2009 兩 vol. 106 兩 no. 43 兩 18339 –18344

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Edited by Frederick W. Alt, Harvard Medical School, Boston, MA, and approved August 31, 2009 (received for review March 7, 2009)

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Results have previously analyzed chromosomally integrated retroviral V(D)J recombination substrates that undergo rearrangement by deletion (pMX-DELCJ) or inversion (pMX-INV) in wild-type and Atm-deficient abl pre-B cells (Fig. S1) (25). After induction of V(D)J recombination with STI571, these retroviral substrates undergo robust rearrangement with efficient coding joint formation in both wild-type and Atm⫺/⫺ abl pre-B cells (Fig. S2) (25). However, in Atm⫺/⫺ abl pre-B cells. unrepaired coding ends accumulate in 10–20% of cells, and approximately 10% of coding ends are resolved aberrantly as translocations or chromosomal deletions or inversions (Fig. S2) (25). In contrast, ⬍1% of substrates exhibit aberrant joining in wild-type abl pre-B cells (25). One of the challenges in elucidating the parameters governing breakpoint target selection is the difficulty in obtaining large numbers of breakpoint sequences. Because RAG breaks are aberrantly resolved at such a high frequency in Atm⫺/⫺ abl pre-B cells, we sought to isolate a large cohort of aberrantly resolved coding ends from these cells. To this end, we generated seven Atm⫺/⫺ abl pre-B cell lines, each with a single copy of the pMX-DELCJ or pMX-INV recombination substrate integrated at a unique chromosomal location. Cleavage of the substrate by RAG results in a chromosomal coding end at the IRES that is either centromeric (Atm⫺/⫺:DELCJ-4, -21, -50, -84 and Atm⫺/⫺:INV-26) or telomeric (Atm⫺/⫺:DELCJ-46 and -70) (Fig. 1A and Fig. S1). In all of the cells analyzed, the retroviral substrate recombines robustly, leading to the formation of normal coding joints and the accumulation of un-repaired coding ends (Fig. S2). We previously identified breakpoints in abl pre-B cell subclones isolated after the induction of V(D)J recombination (25). However, the breakpoint targets isolated by this approach could be biased by the impact of specific aberrant joints on cell division. In addition, aberrant joints that result in formation of dicentric or acentric chromosomes are unlikely to propagate efficiently in dividing cells, hindering our ability to recover these lesions. To circumvent these limitations, we developed a high-throughput inverse PCR (I-PCR) approach, described in detail in Materials and Methods, to isolate the chromosomal breakpoint targets of aberrantly resolved coding ends from retroviral substrates in G1-phase abl pre-B cells (Fig. 1B). Briefly, genomic DNA is digested with MseI, which cuts at TTAA, and ligated at dilute concentrations favoring circularization, followed by digestion with KpnI to linearize the circularized products and PCR amplification of the chromosomal breakpoint targets (Fig. 1B). Most locations (86%) in the mouse genome are within 0.5 kb of an MseI site; thus, the I-PCR was optimized to amplify products of ⬍1 kb in size. I-PCR amplification of genomic DNA from Atm⫺/⫺:DELCJ and Atm⫺/⫺:INV abl pre-B cells that had been treated with STI571 to induce V(D)J recombination yielded a diverse spectrum of products, primarily between 0.5 and 1.0 kb in size (Fig. 1C). As expected, I-PCR of genomic DNA from abl pre-B

M LTR

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nous Ig (Ig) ␬ light (L) chain genes and chromosomally integrated retroviral recombination substrates (25–27). Induction of V(D)J recombination by STI571 treatment of Atm⫺/⫺ abl pre-B cells leads to an accumulation of un-repaired coding ends and to the aberrant resolution of these coding ends, analogous to what is observed in Atm-deficient lymphocytes in vivo (19, 22–25). Here, we describe an experimental approach that permits high-throughput cloning of breakpoint targets from aberrantly resolved RAG DSBs generated during V(D)J recombination in Atm⫺/⫺ abl pre-B cells. Our approach minimizes selection biases, allowing for the elucidation of parameters governing breakpoint targeting of chromosomal RAGmediated DSBs that were not previously revealed through the analysis of lymphoid tumors.

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Fig. 1. Strategy for breakpoint target analysis. (A) Chromosomal integration sites (arrow) of pMX-DELCJ and pMX-INV in Atm⫺/⫺ abl pre-B cells. (B) Inverse PCR (I-PCR) strategy. The pMX-DELCJ retroviral substrate is described in Fig. S1. The MseI (M) and KpnI (K) sites are shown, as is the breakpoint target sequence (gray rectangle) and the position of primers 1–3 used for PCR. (C) I-PCR and Rag1 PCR of Atm⫺/⫺:DELCJ-50 and Atm⫹/⫹:DELCJ-119 abl pre-B cells treated with STI571 for the indicated times in hours. (Upper) I-PCR was performed on separate aliquots of DNA from individual ligations (lanes 1–5, 6 –10) or unligated (⫺) DNA. (Lower) Rag1 was amplified from 3-fold dilutions of ligated (⫹) or unligated (⫺) DNA as a loading control.

cells that were not treated with STI571, and STI571-treated wild-type abl pre-B cells (Atm⫹/⫹:DELCJ-119), yielded significantly fewer products (Fig. 1C). These findings suggest that the I-PCR products from Atm⫺/⫺ abl pre-B cells represent a diverse cohort of aberrantly joined coding ends. Importantly, because the RAG DSBs were generated and aberrantly joined in cells arrested at the G1-phase of the cell cycle, we reason that the diversity of the recovered products will be minimally affected by selection. Breakpoint Targets Are Concentrated in Antigen Receptor Loci. IPCR products were cloned and sequenced from all seven Atm⫺/⫺ abl pre-B cell lines and from one wild-type abl pre-B cell line. Mahowald et al.

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Whereas approximately 65% (1903) of the sequences obtained from Atm⫺/⫺ cells were unique, only 14% (13) of those obtained from wild-type cells were unique, consistent with our previous analyses demonstrating that coding ends are rarely resolved aberrantly in wild-type abl pre-B cells (25). Of the 1903 unique breakpoint targets isolated from Atm⫺/⫺ abl pre-B cell clones, 1,542 (81%) are joined to a breakpoint at an antigen receptor locus (Fig. 2A and Table S1). Most of these breakpoints (95%) are at the IgL␬ locus in close proximity (⬎90% within 100 bp) of a V␬ RS, a J␬ RS, or cryptic RSs in the J␬-C␬ region (Fig. 2 B and C). Although only a small cohort of aberrant joints was recovered from the wild-type abl pre-B cells, approximately 40% of these joints also involved breakpoints at the IgL␬ locus in close proximity to an RS (Table S2). Together, these findings suggest that RAG DSBs at the IgL␬ locus are frequent breakpoint targets for aberrantly resolved retroviral substrate coding ends. Genotoxic DSBs Diversify Breakpoint Target Selection. The accumulation of un-repaired coding ends at the retroviral substrate may be due, in part, to a paucity of DSBs that can serve as efficient breakpoint targets in a subset of Atm⫺/⫺ abl pre-B cells. If this is the case, we reasoned that the introduction of DSBs through exposure to ionizing radiation (IR) might provide additional breakpoint targets for these coding ends. Indeed, treatment of Atm⫺/⫺:DELCJ-50 abl pre-B cells with 4 Gy of IR (⬇100 DSBs per cell) led to a significant decrease in the accumulation of un-repaired pMX-DELCJ coding ends (Fig. 3A). Moreover, analysis of 1,622 breakpoints isolated from all seven Atm⫺/⫺ abl pre-B cell lines exposed to 4 Gy IR revealed that on average, 81% (range 64% to 93%) target nonantigen receptor loci, as compared with 19% (range 10% to 29%) observed in un-irradiated cells (Fig. 3B). As expected, there is an intermediate frequency of breakpoints targeting nonantigen receptor loci in cells that received 0.4 Gy IR (⬇10 DSBs per cell) (Fig. 3C). Thus, the introduction of genotoxic DSBs leads to a decrease in the accumulation of un-repaired substrate coding ends and to significant breakpoint target diversification for aberrantly resolved RAG breaks in Atm⫺/⫺ abl pre-B cells. Preferential Use of Breakpoint Targets in cis. In un-irradiated abl

pre-B cells, 60% (range 33–76%) of breakpoint targets at nonantigen receptor loci map to regions on the same chromosome as the retroviral recombination substrate, which we refer to as breakpoint targets in cis (Fig. 4A). Approximately 87% Mahowald et al.

(range 70–98%) of these breakpoint targets are located within a short distance (⬍200 kb) of the recombination substrate (Fig. 4 B and C). The orientation of these breakpoints suggests that the majority of aberrant joints in cis form by deletion of the intervening sequence rather than inversion (Fig. 4C). Of the small cohort of aberrant joints isolated from wild-type abl pre-B cells, those joints that did not use IgL␬ breakpoint targets also exhibited a bias for using breakpoints in cis (7 of 8), with all seven breakpoints in close proximity (⬍30 kb) to the retroviral substrate (Table S2). It is conceivable that the bias for forming aberrant joints in cis could be due, in part, to synapsis between an RS in the retroviral substrate and a cryptic RS at the breakpoint target. In this regard, 32% of the 217 breakpoint targets in cis have a potential cryptic RS within 100 bp. However, most of these cryptic RSs are predicted to be poor substrates for RAG cleavage (mean RIC score ⫺35.70 for potential 12 RSs and ⫺56.56 for potential 23 RSs) (28). The bias for the aberrant resolution of RAG DSBs using breakpoint targets in cis is particularly striking in Atm⫺/⫺ abl pre-B cells treated with IR. In these cells, the fraction of breakpoint targets recovered in cis with the retroviral substrate is 2–6-fold higher than in un-irradiated cells (Fig. 4D). Moreover, the frequency of breakpoint targets in cis among cells receiving IR is 6- to 10-fold higher than would be predicted based on the relative contribution of the chromosome harboring the substrate to the entire genome (Fig. 4E). Together, these findings demonstrate that the aberrant joining of coding ends is significantly biased toward the use of breakpoint targets at independently generated DSBs lying in close proximity on the same chromosome. Discussion We isolated and characterized a large cohort of breakpoint targets for aberrantly resolved coding ends generated during V(D)J recombination in Atm⫺/⫺ pre-B cells. Importantly, these chromosomal lesions were generated in and isolated from G1phase pre-B cells undergoing V(D)J recombination, thus minimizing potential selection biases. Our analyses reveal that in Atm⫺/⫺ pre-B cells, the most common breakpoint targets for aberrantly resolved RAG DSBs are other DSBs. Moreover, there is a significant bias for using breakpoint targets in cis with the RAG DSB. In un-irradiated cells, breakpoint targets are most frequently found at the IgL␬ locus near RSs, consistent with the notion that they are generated by RAG cleavage at the IgL␬ locus. Although this could reflect a bias for the aberrant joining of two RAG PNAS 兩 October 27, 2009 兩 vol. 106 兩 no. 43 兩 18341

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Fig. 2. Breakpoint targets at antigen receptor loci. (A) Breakpoint targets from all Atm⫺/⫺ pre-B cell lines. (B) Distribution of targeted antigen receptor loci. (C) Breakpoint targets within the IgL␬ locus, with an expanded view of four V␬ gene segments and the J␬-C␬ region. V␬ and J␬ RSs are indicated by arrows, as are known cryptic RSs such as the intron recombining sequences (IRS) and recombining sequence (RS) 3⬘ of C␬.

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Atm-/-: Atm-/-: Atm-/-: Atm-/-: Atm-/-: Atm-/-: Atm-/-: DELCJ-4 DELCJ-21 DELCJ-46 DELCJ-50 DELCJ-70 DELCJ-84 INV-26

Fig. 4. Bias for breakpoint targets in cis. (A) Percentage of breakpoint targets in cis among breakpoints not involving antigen receptor loci (red). (B) Percentage of breakpoints in cis within 200 kb of the retroviral substrate (blue). (C) The distribution of breakpoint targets in cis within 200 kb of the retroviral substrate integration site. The IRES coding end generated by RAG cleavage at the substrate is indicated by the arrow tip. (D) Percentage of breakpoint targets in cis in cells receiving 0 or 4 Gy IR. (E) Fold overrepresentation of breakpoint targets in cis. Calculated as (# of breakpoints in cis/# total breakpoints)/(size of chromosome/size of genome).

24 h after the addition of STI571. DNA was recovered 96 h after the addition of STI571. Southern Blot Analysis. Analysis of retroviral substrate rearrangement was carried out on EcoRV-digested or EcoRV/NcoI double-digested genomic DNA using probe C4 as described (25). Inverse PCR. MseI-digested genomic DNA from abl pre-B cells was purified using the QIAquick PCR purification kit (Qiagen) and ligated at 5 ng/␮L, followed by digestion with KpnI. Five nanograms of this DNA was amplified with 5 pmol each of primer 1 (CAT AGC GAA TTC CAT TGT ATG GGA TCT GAT CTG G) and primer 2 (CCC TTG TTG AAT ACG CTT G) in a 25-␮L reaction with 1 mM MgCl2 and 100 ␮M dNTPs. PCR conditions were 17 cycles of 92 °C for 60 s, 55 °C for 60 s, and 72 °C for 60 s. One microliter of this reaction was then amplified with primer 1 and primer 3 (AGT TGC GGA TCC CTC TTT CCA CAA CTA TCC) using identical conditions as above except for an increase in the cycle number to 30. The combined products of 22 independent PCRs were digested with BamHI and EcoRI and divided into

Mahowald et al.

two fractions (0.5–1 kb, 1–1.5 kb) by fractionation on a 1% agarose gel. Each fraction was gel extracted using the QIAquick gel extraction kit (Qiagen) and cloned separately into pCR2.1-TOPO (Invitrogen). Sequence Analysis. Ligations were submitted to the Genome Sequencing Center at Washington University and sequence reads were obtained with the M13 and/or M13 REV primers using standard techniques. Sequences were aligned at the 5⬘ and 3⬘ ends with the known pCR2.1 vector and 3⬘ retroviral coding end (IRES) sequences. Those with an alignment length of ⱕ45 nt on either end, or with alignments that failed to extend beyond the primer 1 and primer 3 sequences in the IRES, were eliminated. The sequence between the 5⬘ end of the IRES and the first upstream MseI site was aligned to the mouse genome by BLASTN search against the reference assembly (July, 2007). BLASTN results were considered true alignments only if the alignment began at query position 1 and ended within 20 nt of the query end. Loading Control PCR. A 452-bp region of the Rag1 gene was amplified using serial 3-fold dilutions of the KpnI-digested ligation from I-PCR as template. PNAS 兩 October 27, 2009 兩 vol. 106 兩 no. 43 兩 18343

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67%

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PCR was performed with the R15P primer (AGA AGG AGA AGG ATT CCT CAG AGG GG) and R1IP primer (TTG GGA AGT AGA CCT GAC TGT GGG). PCR conditions were 92 °C for 3:00, followed by 30 cycles of 92 °C for 45 s, 60 °C for 60 s, and 72 °C for 90 s.

with the hCD4 –2 primer (CCA GTT TCA AGC TCA GCA TCA) and LTR-f, under the conditions described above for I-PCR. The secondary PCR product was gel purified and sequenced directly.

Positional Cloning of Retroviral Integration. Inverse PCR was performed on Tsp509I-digested genomic DNA ligated at 10 ng/␮L. The primary PCR was performed with the hCD4 –1 primer (GGG CAG AAC CTT GAT GTT GGA) and LTR-f primer (ATC AGA TGT TTC CAG GGT GC), followed by secondary PCR

ACKNOWLEDGMENTS. We thank Beth Helmink for critical review of the manuscript. B.P.S is supported by National Institutes of Health Grant AI074953 and AI047829. J.M.B. was supported by Howard Hughes Medical Institute. G.K.M. is supported by a predoctoral training grant (NIH/NIAID T32 AI007163).

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