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CORRESPONDENCE Barry P. Sleckman: Sleckman{at}immunology.wustl.edu
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locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient ß T cells with translocations involving the T cell receptor / locus is directly related to the number of T cell receptor rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment. The exons encoding the variable region of lymphocyte antigen receptor chains are assembled from V, J, and in some cases D gene segments through the process of V(D)J recombination (1). The V(D)J recombination reaction is initiated by the RAG-1 and -2 proteins (hereafter referred to as RAG). After synaptic complex formation, RAG introduces DNA double-strand breaks (DSBs) at the border of recombining gene segments and their flanking recombination signals (RSs) (2–4). The resulting pairs of blunt phosphorylated signal ends (SEs) and hairpin-sealed coding ends (CEs) are processed and joined into a signal joint and coding joint, respectively, by proteins of the nonhomologous end-joining pathway of DNA DSB repair (5).
The ataxia-telangiectasia (A-T) mutated (ATM) protein is a serine/threonine kinase activated early in the response to DNA DSBs (6–9). Mutations in the ATM gene cause A-T, a disease with lymphoid phenotypes that include lymphopenia and a predisposition to lymphoid malignancies (10, 11). Most lymphoid malignancies in A-T patients have karyotypic abnormalities with chromosome breakpoints clustering near antigen receptor genes (11). In addition, 
10% of nontransformed T cells from A-T patients have translocations or large inversions involving chromosomes containing TCR genes (chromosomes 7 and 14) (11).
Like A-T patients, ATM-deficient mice are lymphopenic, and they almost invariably die from thymic lymphomas with translocations involving the TCR/ locus by 3–6 mo of age (12–16). Thymocytes from ATM-deficient mice have an increased frequency of biallelic loss of distal V gene segments, and 11% of nontransformed ß T cells in these mice have karyotypic abnormalities involving chromosome 14, which contains the TCR/ locus (17, 18). In ATM-deficient mice, the thymic lymphomas with TCR/ locus translocations are RAG dependent, and the T lymphopenia is partially rescued by TCR transgene expression (19–21). Collectively, these findings suggest that ATM functions during antigen receptor gene assembly.
RAG-induced DSBs are generated exclusively in developing lymphocytes at the G1 phase of the cell cycle (22, 23). ATM enforces the G1-S cell-cycle checkpoint through phosphorylation and stabilization of p53 and activation of the Chk2 kinase (7–9). Furthermore, p53 promotes apoptosis of cells with persistent unrepaired DSBs (7–9). In developing lymphocytes, ATM could therefore function primarily by activating the G1-S cell-cycle checkpoint in response to RAG-induced DSBs. However, mice deficient in either p53 or Chk2 are not lymphopenic and are not prone to lymphoid tumors with antigen receptor gene translocations, as are ATM-deficient mice (24, 25). Thus, ATM likely has distinct functions, in addition to its cell-cycle checkpoint/apoptotic activities, during V(D)J recombination.
Analyses of ATM-deficient cell lines did not reveal defects in V(D)J recombination of extrachromosomal plasmid substrates (26). However, ATM has been found to associate with RAG-induced DSBs generated at chromosomal loci (27). We recently analyzed ATM function during recombination of chromosomal substrates in v-abl–transformed mouse pre–B cell lines (AMuLV pre–B cells) that can be induced to undergo V(D)J recombination (28). Inhibition of the v-abl kinase with STI571 leads to G1 cell-cycle arrest, rapid induction of RAG gene expression, and, in wild-type AMuLV pre–B cells, robust rearrangement of chromosomally integrated retroviral recombination substrates (28). In contrast, although signal joint formation proceeds normally in ATM-deficient AMuLV pre–B cells, unrepaired CEs accumulate in these cells because of their loss from postcleavage complexes (28). Furthermore, these CEs are frequently aberrantly resolved as translocations or large chromosomal deletions or inversions (28). These results demonstrate that, in AMuLV pre–B cells, ATM performs an important, nonredundant function during coding joint formation.
Antigen receptor gene assembly at all loci in developing B and T cells relies on efficient coding joint formation, yet the T lymphopenia of ATM deficiency is much more profound than the B lymphopenia (12, 15). In addition, in developing ATM-deficient T cells, the most dramatic block in development is at the stage where TCR chain genes are assembled and expressed (12). Finally, the antigen receptor gene translocations found in malignant and nonmalignant T cells from ATM-deficient mice primarily involve the TCR/ locus (18). Thus, whether ATM deficiency leads to broad defects in coding joint formation at all antigen receptor loci in vivo, as well as how these defects contribute to the lymphoid phenotypes of A-T, is not clear.
In this paper, using ligation-mediated PCR (LMPCR) and Southern blot approaches, we show that unrepaired CEs, but not SEs, accumulate at higher levels during the assembly of TCR and Ig genes in developing ATM-deficient T and B lymphocytes, respectively. Furthermore, we show that in ATM-deficient ß T cells, the frequency of translocations and unrepaired DSBs involving the chromosome containing the TCR/ locus is related to the number of TCR rearrangements that can be made in developing ATM-deficient thymocytes. These findings unequivocally demonstrate that ATM deficiency leads to broad defects in V(D)J recombination during both TCR and Ig gene assembly in developing B and T cells. Moreover, these results have important implications for the role of V(D)J recombination defects in the lymphoid phenotypes of A-T.
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RESULTS |
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chain genes are assembled and expressed (12). To determine whether ATM functions during TCR chain gene coding and/or signal joint formation, wild-type (Atm+/+) and Atm–/– DP thymocytes were purified by flow cytometric cell sorting, and genomic DNA from these cells was assayed for J56 CEs and SEs by LMPCR (Fig. 1, A and B).
To optimize for CE detection, genomic DNA was treated with a DNA polymerase to blunt DNA ends before linker ligation. Whereas the analyses of Atm+/+ and Atm–/– DP thymocytes revealed nearly equivalent levels of J56 SEs, considerably higher levels of J56 CEs were detected in Atm–/– DP thymocytes (Fig. 1 B). Thus, in agreement with our findings in AMuLV pre–B cells, ATM deficiency leads to coding joint, but not signal joint, defects during TCR chain gene assembly, as indicated by the increased accumulation of unrepaired J56 CEs in ATM-deficient DP thymocytes.
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SEs and CEs in Atm+/+ and Atm–/– thymocytes. TCRß and chain genes are assembled in CD4–:CD8– (double negative [DN]) thymocytes that express CD25 (29). CD25+ DN thymocytes were purified from Atm+/+ and Atm–/– mice by flow cytometric cell sorting, and genomic DNA from these cells was assayed for Jß1.1, Jß1.2, and J1 CEs and SEs by LMPCR (Fig. 1, C–E). As both the TCRß and loci have D gene segments, these analyses will also detect CEs generated by cleavage at DJß1.1, DJß1.2, and DJ1 rearrangements (Fig. 1, C, D, F, and G).
Atm+/+ and Atm–/– CD25+ DN thymocytes had nearly equivalent levels of J1 SEs (Fig. 1 E). These cells also had near equivalent levels of Jß1.1, Jß1.2, and 3'Dß1 SEs (detected by the LMPCR for Jß1 CEs; Fig. 1, C–E). However, as was observed for the TCR locus, there were higher levels of unrepaired Jß1.1, Jß1.2, and J1 CEs in Atm–/– DN thymocytes as compared with Atm+/+ DN thymocytes (Fig. 1 E). Sequence analyses revealed that the Jß1.1 and J1 LMPCR products were heterogeneous, and several included Dß and D gene segment nucleotides, respectively (Fig. 1, F and G). Collectively, these data demonstrate that ATM deficiency leads to broad defects in coding joint formation during different steps (V to J, D to J, and V to DJ) of TCR gene assembly at multiple loci (TCR, ß, and ) in developing thymocytes.
Defects in coding joint formation in developing ATM-deficient B cells
To determine whether ATM functions during coding and/or signal joint formation at Ig loci in developing B cells, IgH and IgL
chain gene CEs and SEs were assayed by LMPCR of genomic DNA from Atm+/+ and Atm–/– bone marrow (Fig. 2).
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1 SEs at the IgL locus (Fig. 2 C). However, considerably higher levels of JH1 and J1 CEs were detected in Atm–/– bone marrow (Fig. 2 C). Sequence analyses demonstrated that the LMPCR products were heterogeneous and were generated by linker ligation to DJH1, JH1, and J1 CEs (unpublished data). Collectively, these data demonstrate that developing ATM-deficient B cells also have defects in coding joint formation, as indicated by an increased accumulation of unrepaired CEs during DH to JH and VH to DJH rearrangements at the IgH locus and V to J rearrangements at the IgL locus.
Quantitative assessment of CE accumulation in ATM-deficient thymocytes
The LMPCR analyses demonstrate that unrepaired CEs exist at higher levels in developing Atm–/– lymphocytes. However, this type of analysis cannot be used to determine the fraction of alleles that have unrepaired CEs, which is needed to assess the potential developmental impact of coding joint defects in ATM-deficient mice. As most antigen receptor loci contain many gene segments, it would be difficult to quantitatively assay the total accumulation of unrepaired CEs at a specific locus. Accordingly, we generated Atm+/+ and Atm–/– mice that were homozygous for the TCRsJ allele (TCRsJ/sJ:Atm+/+ and TCRsJ/sJ:Atm–/– mice, respectively; Fig. 3 A) (30).
The wild-type TCR locus (TCR+) has 100 V and 61 J gene segments, and all V to J rearrangements occur by deletion (31). The TCRsJ allele is identical to the wild-type TCR locus except that the 61 J gene segments have been replaced with 2 J gene segments (J61 and J56) through a multistep gene-targeting approach (Fig. 3 A) (30). Thus, rearrangement of any of the 100 V gene segments on the TCRsJ allele must occur to 1 of the 2 closely linked (0.4 kb) J gene segments.
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signal and coding joints in TCRsJ/sJ thymocytes should reveal heterogeneously sized fragments, as any 1 of the 100 V gene segments can rearrange to either of the 2 J gene segments in each individual cell. However, as all of these rearrangements will involve the generation of a SE and CE at one of the two closely linked J gene segments, we reasoned that these DNA ends may be detected as discrete fragments by Southern blotting.
Southern blot analyses of TCRsJ/sJ:Atm+/+ and TCRsJ/sJ:Atm–/– thymocyte DNA digested with two restriction enzymes and hybridized to probe 8 (P8; Fig. 3 B) upstream of J61 revealed fragments of the expected size for germline TCRsJ alleles and many different-sized fragments generated by heterogeneous signal joint formation (Fig. 3 C). Importantly, prominent fragments of the expected size for J61 SEs were also observed at equal intensities in TCRsJ/sJ:Atm+/+ and TCRsJ/sJ:Atm–/– thymocyte DNA (Fig. 3 C). These fragments were sensitive to exonuclease V digestion, further demonstrating that they likely represent unrepaired SEs (Fig. 3, D and E). Notably, prominent fragments of the expected size for J56 SEs were not detected (Fig. 3 C). This would be expected if, like the wild-type TCR locus, rearrangement of the TCRsJ allele is ordered, with the more 5' J gene segment (J61) being used before the 3' J gene segment (J56) (32–34). In this regard, P8 would not detect J56 SEs on TCRsJ alleles that had undergone a V to J61 rearrangement, which deletes the P8-hybridizing region from the chromosome (Fig. 3 B). LMPCR analyses, however, confirmed that J56 SEs are present at nearly equivalent levels in TCRsJ/sJ:Atm+/+ and TCRsJ/sJ:Atm–/– thymocytes (Fig. 3 F).
J56 and J61 CEs were assayed by Southern blot analysis using two different restriction enzymes and a probe (CI; Fig. 4 A) downstream of the J56 gene segment (Fig. 4 A).
In addition to fragments expected for heterogeneous VJ coding joints and germline TCRsJ alleles, these analyses revealed a novel fragment of expected size for unrepaired J56 CEs in TCRsJ/sJ:Atm–/– thymocyte DNA (Fig. 4, A and B). This fragment was not present in TCRsJ/sJ:Atm+/+ thymocyte DNA (Fig. 4 B). Furthermore, hybridization with SJA probe 6 (PS6; Fig. 4 A) to a region between the J56 and J61 gene segments revealed a fragment of expected size for J61 CEs only in TCRsJ/sJ:Atm–/– thymocytes (Fig. 4, A and B). Quantification of these novel fragments, as described in Materials and methods, revealed that 17% (8% J56 and 9% J61) of TCRsJ alleles in TCRsJ/sJ:Atm–/– thymocytes have unrepaired CEs. LMPCR analyses confirmed the increased accumulation of J56 and J61 CEs in TCRsJ/sJ:Atm–/– thymocytes (Fig. 4 C).
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56 CE fragment (Fig. 4, D and E). Treatment of genomic DNA with mung bean nuclease, which opens hairpin-sealed CEs, before exonuclease V treatment led to a nearly complete loss of hybridization (Fig. 4, F and G). Thus, approximately half of the unrepaired CEs in ATM-deficient thymocytes are hairpin sealed, raising the possibility that ATM may have some function in the hairpin opening process. Collectively, these data demonstrate that a substantial fraction of TCRsJ/sJ:Atm–/– thymocytes have unrepaired J CEs that exist in either hairpin-sealed or opened configurations.
Reduced chromosome 14 breaks and translocations in TCRsJ/sJ:Atm–/– ß T cells
ATM-deficient mice have increased numbers of nonmalignant ß T cells with karyotypic abnormalities involving chromosome 14, which contains the TCR/ locus (11, 18). Our analyses of V(D)J recombination in Atm–/– AMuLV pre–B cells demonstrated that unrepaired CEs are frequently resolved aberrantly as translocations or large chromosomal deletions or inversions (28). In DP thymocytes, each TCR allele usually undergoes several V to J rearrangements (see Discussion). Thus, the increased frequency of chromosome 14 aberrations observed in Atm–/– ß T cells could be due, in part, to the cumulative risk that any one of these multiple rearrangements would be aberrantly resolved.
To investigate this possibility, cytogenetic analyses were performed on metaphases from proliferating TCR+/+:Atm–/– and TCRsJ/sJ:Atm–/– ß T cells, as described in Materials and methods. As discussed above, the TCRsJ allele can undergo only two V to J rearrangements, whereas the wild-type TCR allele has the potential to undergo many more V to J rearrangements. Metaphases were hybridized with red whole chromosome paint (WCP) for chromosome 14 and green WCP for chromosome 15, which serves as a control as it does not contain antigen receptor loci. Chromosome 14 or 15 aberrations were not observed in TCR+/+:Atm+/+ or TCRsJ/sJ:Atm+/+ ß T cells (Fig. 5).
In close agreement with published studies, 8% of TCR+/+:Atm–/– ß T cells had chromosome 14 aberrations (Fig. 5) (18). In contrast, only 4% of TCRsJ/sJ:Atm–/– ß T cells had chromosome 14 aberrations (Fig. 5). Notably, the chromosome 14 aberrations in TCR+/+:Atm–/– and TCRsJ/sJ:Atm–/– ß T cells were equally divided between translocations and replicated chromosomal breaks (Fig. 5 C). From these analyses, it is not possible to determine whether the observed chromosome 14 aberrations are derived from TCR or TCR chain gene rearrangements. However, the TCR locus is unaltered on the TCRsJ allele, making it unlikely that the change in the frequency of chromosome 14 aberrations is caused by differences in the TCR locus–derived translocations. Collectively, these findings demonstrate that ATM-deficient mice with TCR alleles that undergo fewer V to J rearrangements have fewer peripheral ß T cells with chromosome 14 aberrations. In addition, they suggest that RAG-induced DSBs generated in ATM-deficient thymocytes can persist unrepaired, as indicated by the chromosome 14 breaks in mature ATM-deficient ß T cells.
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DISCUSSION |
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The structure of most antigen receptor loci prohibits the simple quantitative assessment of the fraction of loci with unrepaired CEs or SEs. However, through Southern blot analysis of ATM-deficient thymocytes with a modified TCR locus (TCRsJ), we show that 17% of alleles have unrepaired J CEs (Fig. 4 B). That 2% of peripheral TCRsJ/sJ:Atm–/– ß T cells have chromosome 14 breaks suggests that at least some J CEs generated in ATM-deficient DP thymocytes can persist unrepaired for the period of time it takes these cells to be positively selected and released into the periphery (Fig. 5). These findings are in remarkable agreement with analyses of ATM-deficient AMuLV pre–B cells, which revealed that 10–20% of retroviral recombination substrates develop persistent unrepaired CEs after induction of V(D)J recombination (28). Several lines of evidence, including interphase fluorescent in situ hybridization, revealed that unrepaired CEs accumulate in these cells due, at least in part, to their loss from postcleavage complexes (28). Although it is not possible to perform similar types of analyses on developing lymphocytes, we suspect that the loss of CEs from postcleavage complexes also contributes to the coding joint defect observed in developing ATM-deficient lymphocytes in vivo.
Cytogenetic analyses revealed that 2% of proliferating TCRsJ/sJ:Atm–/– ß T cells have chromosome 14 translocations, suggesting that some J CEs are aberrantly resolved (Fig. 5). In agreement, cytogenetic analyses of ATM-deficient AMuLV pre–B cells that had undergone recombination revealed that 1.5% had chromosomal translocations involving the retroviral recombination substrate CEs (28). However, Southern blot analysis of clones revealed that 14% of the retroviral substrates had aberrantly resolved CEs (28). This difference (1.5% by cytogenetic and 14% by clonal analysis) is caused by the inability of cytogenetic approaches to detect many chromosomal deletions and inversions involving the retroviral substrate (28). By analogy, we would expect that the true fraction of TCRsJ/sJ:Atm–/– ß T cells with aberrant TCRsJ allele rearrangements is likely greater than the 2% with translocations detected using cytogenetic approaches (Fig. 5).
Defects in coding joint formation were found at both Ig and TCR loci and during D to J and V to DJ rearrangements in developing B and T cells, yet the lymphopenia of ATM deficiency is more profound in the T cell than the B cell compartment (15). It is possible that distinct features of TCR gene assembly contribute to the more pronounced T lymphopenia of ATM deficiency. The generation of translocations or large chromosomal deletions or inversions involving an antigen receptor locus would likely prevent subsequent rearrangements. Inactivation of a single allele in this manner would require the cell to generate an in-frame rearrangement on the alternate allele if it is to continue development, whereas inactivation of both alleles would preclude any further development. The developmental impact of such a defect should be greatest for loci that must undergo multiple rearrangements, as each additional rearrangement increases the possibility of inactivating the allele. In this regard, it is notable that in ATM-deficient mice the most profound block in T cell development occurs at the DP thymocyte stage, where TCR chain genes are being assembled and expressed (12).
Developing T cells must assemble TCR chain genes that are in frame and encode TCR chains that form nonautoreactive ß TCRs capable of positive selection. Most randomly generated ß TCRs are not capable of mediating positive selection (35–37). The large V and J gene segment clusters are positioned such that all V to J rearrangements occur by deletion (31). Thus, VJ rearrangements that are not in frame, or that are in frame but do not encode a TCR chain that forms a selectable ß TCR, can be replaced through rearrangement of an upstream V to a downstream J gene segment on the same allele. In addition, TCR chains that form autoreactive ß TCRs can, in some instances, be similarly replaced, rescuing the cell from negative selection (38–40). These revision rearrangements proceed in an orderly fashion using progressively more 3' J gene segments in the locus (32, 33). That most developing DP thymocytes must undergo multiple V to J rearrangements on each allele, as they attempt to generate a selectable ß TCR, is supported by the defect in positive selection observed in TCRsJ/sJ mice (30).
A recent modeling study predicted that, on average, each TCR allele undergoes five V to J rearrangements in developing thymocytes (41). In contrast, the TCRsJ allele can undergo only two V to J rearrangements. In this regard, it is notable that TCR+/+:Atm–/– ß T cells have twice as many chromosome 14 breaks and translocations as compared with TCRsJ/sJ:Atm–/– ß T cells (Fig. 5). Thus, there is a close correlation between the frequency of chromosome 14 breaks and translocations in ATM-deficient ß T cells and the potential number of V to J rearrangements that these cells can make during thymocyte development. Collectively, these findings suggest that, in ATM-deficient cells, loci that undergo multiple rearrangements have an increased probability of generating a persistent unrepaired DSB or of sustaining an aberrant rearrangement, both of which should inactivate the allele and prevent further rearrangements.
What is the developmental impact of TCR locus inactivation in ATM-deficient thymocytes? Although it is not possible to answer this question with certainty, an estimate of the potential developmental impact can be made. In ATM-deficient AMuLV pre–B cells, 14% of rearrangements lead to CEs that are aberrantly resolved in ways (translocations, deletions, and inversions) that would inactivate an antigen receptor locus (28). If 14% of TCR gene rearrangements are also resolved aberrantly and an average of five V to J rearrangements occur on each TCR allele, then on average there would be a 53% (1 – 0.865) possibility that a single TCR allele would be inactivated in developing ATM-deficient DP thymocytes. Furthermore, approximately one third (0.53 x 0.53 = 0.28) of ATM-deficient DP thymocytes would be expected to inactivate both TCR alleles, precluding further development of these cells.
In addition to unrepaired TCR CEs, unrepaired TCRß chain gene CEs are also found in ATM-deficient thymocytes (Fig. 1 E). Thus, defects in TCRß chain gene assembly may also contribute to the T lymphopenia of ATM deficiency. However, the only constraint on TCRß chain gene assembly is that it must be in frame and encode a TCRß chain that can form a pre-TCR (42). As a result, it is possible that developing thymocytes may undergo fewer TCRß chain gene rearrangements than TCR chain gene rearrangements. Moreover, the developmental impact of defects in TCRß chain gene assembly may be blunted by the cellular expansion that occurs during the DN to DP transition (42). In contrast, positive selection is not accompanied by a substantial cellular expansion that could compensate for defects in TCR chain gene assembly in ATM-deficient thymocytes.
Defects in coding joint formation during IgH and IgL chain gene assembly were also found in developing ATM-deficient B cells (Fig. 2 C), yet B cell development is minimally compromised in ATM-deficient mice (15). Like TCRß chain gene assembly, IgH chain genes need only to be in frame and encode an IgH chain that can form a pre–B cell receptor (BCR) (43, 44). Furthermore, the pro– to pre–B cell transition is accompanied by a cellular expansion that could compensate for defects in IgH chain gene assembly (43, 44). IgL chain genes are assembled in pre–B cells, and they must encode an IgL chain that forms a nonautoreactive BCR (43, 44). As is the case for TCR chain gene assembly, IgL chain gene assembly is not followed by a substantial cellular expansion that could compensate for defects in this process (43, 44). Like the TCR locus, the structure of the IgL locus permits VJ rearrangements to be replaced with new VJ rearrangements, on the same allele, as pre–B cells attempt to generate a nonautoreactive BCR (45). However, unlike the ß TCR, there is no requirement for positive selection of the BCR. Thus, the additional constraint of positive selection may mandate more TCR chain gene rearrangements in DP thymocytes than IgL chain gene rearrangements in pre–B cells and, as such, contribute to the more profound T cell lymphopenia of ATM deficiency.
It is also possible that developing T cells may have unique requirements for ATM function in addition to its role in antigen receptor gene assembly. For example, recent studies in macrophages have demonstrated that ATM deficiency leads to constitutive c-Jun N-terminal kinase (JNK) activation (46). JNK signals are involved in negative selection; therefore, if JNK activity is also perturbed in ATM-deficient thymocytes, this may contribute to the T lymphopenia of ATM deficiency (47). In addition, like ATM-deficient mice, mice that are deficient in both ATM and RAG eventually succumb to thymic lymphomas (21). However, these lymphomas do not have translocations involving the TCR/ locus (21). This demonstrates that ATM deficiency can promote thymocyte transformation through defects in processes other than the assembly of antigen receptor genes. Therefore, although we demonstrate unequivocally that ATM-deficient lymphocytes have defects in V(D)J recombination, the relative contribution of these and other defects to the lymphoid phenotypes of A-T remains to be determined.
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MATERIALS AND METHODS |
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Southern blot analyses.
Southern blot analyses of genomic DNA and PCR products were performed as previously described (30, 48). P8, the CI probe, and the RAG-2 probe (PR2) have been previously described (49). PS6 is a 0.4-kb PCR fragment amplified from TCRsJ/sJ genomic DNA using the PS6-1 and PS6-2 oligonucleotides (Table S1, available at http://www.jem.org/cgi/content/full/jem.20061460/DC1). For exonuclease V digestion, 15 µg of thymocyte DNA was treated with increasing concentrations of exonuclease V, using the manufacturer's buffer conditions (USB Corporation), for 1 h at 37°C before restriction enzyme digestion and Southern blot analyses. For mung bean nuclease assays, 15 µg of thymocyte DNA was digested with 12.5 U of mung bean nuclease (New England Biolabs, Inc.) in 200 µl using the manufacturer's buffer supplemented with 0.4 mM ZnSO4 at 25°C for 1 h before treatment with exonuclease V, as described in this section.
Quantification of Southern blot fragments.
Quantifications of Southern blot fragments were performed on a PhosphorImager (Storm 840; GE Healthcare) using ImageQuant software (GE Healthcare). The percentage of TCRsJ alleles with unrepaired J56 CEs was calculated from the Southern blot of StuI-digested DNA probed with the CI probe (Fig. 4 B) or PR2 as a DNA loading control (Fig. 4 B).
The following formula was used to determine the percentage of TCRsJ allele J56 CEs:
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The hybridization intensity of the CI probe hybridizing the J56 CE fragment (J56CEThy) and PR2 hybridizing the RAG-2 gene fragment (R2Thy) from TCRsJ/sJ:Atm–/– thymus DNA is used. In addition, the hybridization intensity of the CI probe hybridizing the germline TCRsJ allele fragment (CIKid) and PR2 hybridizing the RAG-2 gene fragment (R2Kid) from TCRsJ/sJ:Atm–/– kidney DNA is used.
The percentage of TCRsJ alleles with unrepaired J61 CEs was similarly calculated from Southern blotting of StuI-digested DNA probed with PS6 (Fig. 4 B) using the following formula:
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The hybridization intensity of PS6 hybridizing the J61 CE fragment (J61CEThy) from TCRsJ/sJ:Atm–/– thymus DNA and the hybridization intensity of PS6 hybridizing the germline TCRsJ allele fragment (PS6Kid) from TCRsJ/sJ:Atm–/– kidney DNA are used. R2Thy and R2Kid are as described in this section.
The flowing formulas were used for quantifying the retention of J61 SEs and J56 CEs, respectively, after exonuclease V or mung bean nuclease and exonuclease V treatment:
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The intensities of the J56 CE (J56CE), J61 SE (J61SE), or TCRsJ germline fragments in lanes that were either not treated (–ExoV) or treated with different concentrations of exonuclease V (+ ExoV) are used (Fig. 3 D; and Fig. 4, D and F).
LMPCR.
LMPCR was performed as previously described (48). In brief, 4 µg of thymus DNA that was treated with the Klenow DNA polymerase (New England Biolabs, Inc.) in the presence of dNTP before ligation to the BW or ANC linker (48, 50). Heminested PCR was performed with Taq polymerase (Roche Molecular Systems, Inc.), according to the manufacturer's recommendation, using 1 mM MgCl2. All primary PCRs were performed at 94°C for 5 min, followed by 17 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 60 s. This was followed by a final incubation at 72°C for 7 min. All secondary PCRs were performed at 94°C for 5 min, followed by 23–36 cycles (Table S1) at 94°C for 30 s, 58°C for 30 s, and 72°C for 60 s. This was followed by a final incubation at 72°C for 7 min. Primer sequences are provided in Table S1. Oligonucleotides used as probes for PCR products are also listed in Table S1. IL-2 control PCR was performed as previously described (32).
Flow cytometric cell sorting.
To purify CD25+ DN thymocytes, total thymocytes were depleted with CD4- or CD8-expressing cells using magnetic beads, according to the manufacturer's instruction (Dynal), followed by flow cytometric purification of CD25+ DN thymocytes (FACSDiva; Beckton Dickinson) using PE-Cy7–conjugated anti-CD25 (BD Biosciences). The resulting cells were >95% CD25+ DN thymocytes. DP thymocytes were purified to >95% purity by flow cytometric cell sorting using FITC-conjugated anti-CD8 and PE-Cy7–conjugated anti-CD4 (BD Biosciences).
Metaphase WCP analysis.
Metaphases were prepared from ConA-stimulated peripheral T cells, as previously described (28). Fluorescent in situ hybridization of metaphase chromosomes using the mouse WCPs (Applied Spectral Imaging) for chromosomes 14 (red) and 15 (green) were performed using the manufacturer's recommended procedure. In brief, denatured probes were allowed to reanneal at 37°C for 20 min and were hybridized to denatured chromosomes for 16 h in a humidified chamber at 37°C. Stringency washing was done in 0.4x SSC at 70°C for 4 min, followed by 4x SSC/0.1% Tween 20. DAPI-counterstained slides were mounted in antiquench mount (Vectashield; Vector Laboratories) and analyzed on an epifluorescence microscope (Axioplan 2ie; Carl Zeiss MicroImaging, Inc.). Image acquisition and processing was done using ISIS software (Metasystems).
Online supplemental material.
Table S1 presents a list of oligonucleotides used for LMPCR and probes. For each SE or CE LMPCR, the allele-specific oligonucleotides used as PCR primers or Southern probes are grouped with their names and sequences indicated. For TCR locus LMPCR, the primers used for detecting SEs and CEs in the wild-type TCR locus (TCR+) or TCRsJ locus (TCRsJ) are grouped, respectively. The cycle numbers for the primary and secondary reactions of the heminested PCR are also indicated alongside the allele-specific primers that were used. Primers A, B, and C are linker-specific primers. PS6-1 and PS6-2 are used to amplify PS6 for thymocyte DNA Southern analyses. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20061460/DC1.
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Acknowledgments |
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B.P. Sleckman is supported by National Institutes of Health (NIH) grants AI47829 and AI49934 and American Cancer Society grant RSG-05-070-01-LIB. T.K. Pandita is supported by NIH grants NS34746 and CA10445. C.H. Bassing is a Pew Scholar. C.-Y. Huang is supported by a postdoctoral training grant from the NIH. Mice were housed in a facility supported by National Center for Research Resources grant RR012466.
The authors have no conflicting financial interests.
Note added in proof. Since the acceptance of this manuscript, another paper has appeared that also reports an increase in TCR CEs in ATM-deficient mice (Vacchio, M.S., A. Olaru, F. Livak, and R.J. Hodes. 2007. Proc. Natl. Acad. Sci. USA. 104:6323–6328).
Submitted: 10 July 2006
Accepted: 19 January 2007
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REFERENCES |
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