Cellular context-dependent effects of H2ax and p53 deletion on the development of thymic lymphoma

H2AX and Artemis each cooperate with p53 to suppress lymphoma. Germline H2ax(-/-)p53(-/-) mice die of T-cell receptor-β(-) (TCR-β(-)) thymic lymphomas with translocations and other lesions characteristic of human T-cell acute lymphoblastic leukemia. Here, we demonstrate that mice with inactivation of H2ax and p53 in thymocytes die at later ages to TCR-β(-) or TCR-β(+) thymic lymphomas containing a similar pattern of translocations as H2ax(-/-)p53(-/-) tumors. Germline Artemis(-/-) p53(-/-) mice die of lymphomas with antigen receptor locus translocations, whereas Artemis(-/-)H2ax(-/-)p53(-/-) mice die at earlier ages from multiple malignancies. We show here that Artemis(-/-) mice with p53 deletion in thymocytes die of TCR-β(-) tumors containing Tcrα/δ translocations, other clonal translocations, or aneuploidy, as well as Notch1 mutations. Strikingly, Artemis(-/-) mice with H2ax and p53 deletion in thymocytes exhibited a lower rate of mortality from TCR-β(-) tumors, which harbored significantly elevated levels of genomic instability. Our data reveal that the cellular origin of H2ax and p53 loss impacts the rate of mortality from and developmental stage of thymic lymphomas, and suggest that conditional deletion of tumor suppressor genes may provide more physiologic models for human lymphoid malignancies than germline inactivation.     

Recombinase-activating gene (RAG) 2-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice

The majority of Atm-deficient mice die of malignant thymic lymphoma by 4-5 mo of age. Cytogenetic abnormalities in these tumors are consistently identified within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA breaks that occur during V(D)J recombination. Since V(D)J recombination is a recombinase-activating gene (RAG)-dependent process, we generated Rag2(-/-)Atm(-/-) mice to assess the requirement for RAG-dependent recombination in thymic lymphomagenesis. In contrast to expectation, the data presented here indicate that development of malignant thymic lymphoma in Atm(-/-) mice is not prevented by loss of RAG-2 and thus is not dependent on V(D)J recombination. Malignant thymic lymphomas in Rag2(-/-)Atm(-/-) mice occurred at a lower frequency and with a longer latency as compared with Atm(-/-) mice. Importantly, cytogenetic analysis of these tumors indicated that multiple chromosomal abnormalities occurred in each tumor, but that none of these involved the Tcr alpha/delta locus. Nonmalignant peripheral T cells from TCR-transgenic Rag2(-/-)Atm(-/-) mice also revealed a substantial increase in translocation frequency, suggesting that these translocations are early events in the process of tumorigenesis. These data are consistent with the hypothesis that the major mechanism of tumorigenesis in Atm(-/-) mice is via chromosomal translocations and other abnormalities that are secondary to aberrant responses to double-stranded DNA breaks. Furthermore, these data suggest that V(D)J recombination is a critical, but not essential, event during which Atm-deficient thymocytes are susceptible to developing chromosome aberrations that predispose to malignant transformation.     

Overlapping functions between XLF repair protein and 53BP1 DNA damage response factor in end joining and lymphocyte development

Nonhomologous end joining (NHEJ), a major pathway of DNA double-strand break (DSB) repair, is required during lymphocyte development to resolve the programmed DSBs generated during Variable, Diverse, and Joining [V(D)J] recombination. XRCC4-like factor (XLF) (also called Cernunnos or NHEJ1) is a unique component of the NHEJ pathway. Although germ-line mutations of other NHEJ factors abrogate lymphocyte development and lead to severe combined immunodeficiency (SCID), XLF mutations cause a progressive lymphocytopenia that is generally less severe than SCID. Accordingly, XLF-deficient murine lymphocytes show no measurable defects in V(D)J recombination. We reported earlier that ATM kinase and its substrate histone H2AX are both essential for V(D)J recombination in XLF-deficient lymphocytes, despite moderate role in V(D)J recombination in WT cells. p53-binding protein 1 (53BP1) is another substrate of ATM. 53BP1 deficiency led to small reduction of peripheral lymphocyte number by compromising both synapse and end-joining at modest level during V(D)J recombination. Here, we report that 53BP1/XLF double deficiency blocks lymphocyte development at early progenitor stages, owing to severe defects in end joining during chromosomal V(D)J recombination. The unrepaired DNA ends are rapidly degraded in 53BP1(-/-)XLF(-/-) cells, as reported for H2AX(-/-)XLF(-/-) cells, revealing an end protection role for 53BP1 reminiscent of H2AX. In contrast to the early embryonic lethality of H2AX(-/-)XLF(-/-) mice, 53BP1(-/-)XLF(-/-) mice are born alive and develop thymic lymphomas with translocations involving the T-cell receptor loci. Together, our findings identify a unique function for 53BP1 in end-joining and tumor suppression.     

53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis

p53-binding protein 1 (53BP1) participates in the cellular response to DNA double-stranded breaks where it associates with various DNA repair/cell cycle factors including the H2AX histone variant. Mice deficient for 53BP1 (53BP1(-/-)) are sensitive to ionizing radiation and immunodeficient because of impaired Ig heavy chain class switch recombination. Here we show that, as compared with p53(-/-) mice, 53BP1(-/-)/p53(-/-) animals more rapidly develop tumors, including T cell lymphomas and, at lower frequency, B lineage lymphomas, sarcomas, and teratomas. In addition, T cells from animals deficient for both 53BP1 and p53 (53BP1(-/-)/p53(-/-)) display elevated levels of genomic instability relative to T cells deficient for either 53BP1 or p53 alone. In contrast to p53(-/-) T cell lymphomas, which routinely display aneuploidy but not translocations, 53BP1(-/-)/p53(-/-) thymic lymphomas fall into two distinct cytogenetic categories, with many harboring clonal translocations (40%) and the remainder showing aneuploidy (60%). We propose that 53BP1, in the context of p53 deficiency, suppresses T cell lymphomagenesis through its roles in both cell-cycle checkpoints and double-stranded break repair.     

Functional redundancy between repair factor XLF and damage response mediator 53BP1 in V(D)J recombination and DNA repair

The classical nonhomologous DNA end-joining (C-NHEJ) double-strand break (DSB) repair pathway in mammalian cells maintains genome stability and is required for V(D)J recombination and lymphocyte development. Mutations in the XLF C-NHEJ factor or ataxia telangiectasia-mutated (ATM) DSB response protein cause radiosensitivity and immunodeficiency in humans. Although potential roles for XLF in C-NHEJ are unknown, ATM activates a general DSB response by phosphorylating substrates, including histone H2AX and 53BP1, which are assembled into chromatin complexes around DSBs. In mice, C-NHEJ, V(D)J recombination, and lymphocyte development are, at most, modestly impaired in the absence of XLF or ATM, but are severely impaired in the absence of both. Redundant functions of XLF and ATM depend on ATM kinase activity; correspondingly, combined XLF and H2AX deficiency severely impairs V(D)J recombination, even though H2AX deficiency alone has little impact on this process. These and other findings suggest that XLF may provide functions that overlap more broadly with assembled DSB response factors on chromatin. As one test of this notion, we generated mice and cells with a combined deficiency for XLF and 53BP1. In this context, 53BP1 deficiency, although leading to genome instability, has only modest effects on V(D)J recombination or lymphocyte development. Strikingly, we find that combined XLF/53BP1 deficiency in mice severely impairs C-NHEJ, V(D)J recombination, and lymphocyte development while also leading to general genomic instability and growth defects. We conclude that XLF is functionally redundant with multiple members of the ATM-dependent DNA damage response in facilitating C-NHEJ and discuss implications of our findings for potential functions of these factors.     

Increase in double-stranded DNA break-related foci in early-stage thymocytes of aged mice

Cellular and molecular mechanisms involved in aging are notoriously complex. Aging-related immune decline of T lymphocyte function is partly caused by attrition of thymic T cell development, which involves programmed creation and repair of DNA breaks for generating T cell receptors. Aging also leads to significant alterations in the cellular DNA repair ability. We show that higher levels of gamma-phosphorylated H2AX (pH2AX), which marks DNA double-stranded breaks (DSBs), were detectable in early thymocyte subsets of aged as compared to young mice. Also, while only 1–2 foci of nuclear accumulation of pH2AX were detectable in these early thymocytes from young mice, cells from aged mice showed higher numbers of pH2AX foci. In CD4−CD8− double-negative (DN) thymocytes of aged mice, which showed the highest levels of DSBs, there was a modest increase in levels of the DNA repair protein MRE11, but not of either Ku70, another DNA repair protein, or the cell cycle checkpoint protein p53. Thus, immature thymocytes in aged mice show a marked increase in DNA DSBs with only a modest enhancement of repair processes, and the resultant cell cycle block could contribute to aging-related defects of T cell development.     

Abnormal rearrangement within the alpha/delta T-cell receptor locus in lymphomas from Atm-deficient mice

Atm-deficient mice (Atm(-/-)) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm(-/-) mice. All Atm(-/-) tumors are of thymic lymphoblastoid origin, display an immature CD3(-) and CD4(+)/CD8(+) phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptoralpha/delta (Tcralpha/delta) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% of Atm(-/-) peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at the Tcralpha/delta locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcralpha/delta locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks. (Blood. 2000;96:1940-1946)     

Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX

In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form gamma-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX(Delta)/Delta) mouse embryonic stem (ES) cells. H2AX(Delta)/Delta ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ per se because H2AX(Delta)/Delta ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX(Delta)/Delta ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability.     

Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells

Nonhomologous DNA end-joining (NHEJ) is a major pathway of DNA double-strand break (DSB) repair in mammalian cells, and it functions to join both specifically programmed DSBs that occur in the context of V(D)J recombination during early lymphocyte development as well as general DSBs that occur in all cells. Thus, defects in NHEJ impair V(D)J recombination and lead to general genomic instability. In human patients, mutations of Cernunnos-XLF (also called NHEJ1), a recently identified NHEJ factor, underlie certain severe combined immune deficiencies associated with defective V(D)J recombination and radiosensitivity. To characterize Cernunnos-XLF function in mouse cells, we used gene-targeted mutation to delete exons 4 and 5 from both copies of the Cernunnos-XLF gene in ES cell (referred to as Cer(Delta/Delta) ES cells). Analyses of Cer(Delta/Delta) ES cells showed that they produce no readily detectable Cernunnos-XLF protein. Based on transient V(D)J recombination assays, we find that Cer(Delta/Delta) ES cells have dramatic impairments in ability to form both V(D)J coding joins and joins of their flanking recombination signal sequences (RS joins). Cer(Delta/Delta) ES cells are highly sensitive to ionizing radiation and have intrinsic DNA DSB repair defects as measured by pulse field gel electrophoresis. Finally, the Cernunnos-XLF mutations led to increased spontaneous genomic instability, including translocations. We conclude that, in mice, Cernunnos-XLF is essential for normal NHEJ-mediated repair of DNA DSBs and that Cernunnos-XLF acts as a genomic caretaker to prevent genomic instability.     

ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes

Mutations in ATM (ataxia-telangiectasia mutated) cause ataxia-telangiectasia (AT), a disease characterized by neurodegeneration, sterility, immunodeficiency, and T-cell leukemia. Defective ATM-mediated DNA damage responses underlie many aspects of the AT syndrome, but the basis for the immune deficiency has not been defined. ATM associates with DNA double-strand breaks (DSBs), and some evidence suggests that ATM may regulate V(D)J recombination. However, it remains unclear how ATM loss compromises lymphocyte development in vivo. Here, we show that T-cell receptor beta (TCRbeta)-dependent proliferation and production of TCRbeta(low) CD4+CD8+ (DP) thymocytes occurred normally in Atm-/- mice. In striking contrast, the postmitotic maturation of TCRbeta(low) DP precursors into TCRbeta(int) DP cells and TCRbeta(hi) mature thymocytes was profoundly impaired. Furthermore, Atm-/- thymocytes expressed abnormally low amounts of TCRalpha mRNA and protein. These defects were not attributable to the induction of a BCL-2-sensitive apoptotic pathway. Rather, they were associated with frequent biallelic loss of distal Va gene segments in DP thymocytes, revealing that ATM maintains Tcra locus integrity as it undergoes V(D)J recombination. Collectively, our data demonstrate that ATM loss increases the frequency of aberrant Tcra deletion events, which compromise DP thymocyte maturation and likely promote the generation of oncogenic TCR translocations.     

Loss of p19Arf in a Rag1(-/-) B-cell precursor population initiates acute B-lymphoblastic leukemia

In human B-acute lymphoblastic leukemia (B-ALL), RAG1-induced genomic alterations are important for disease progression. However, given that biallelic loss of the RAG1 locus is observed in a subset of cases, RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf(-/-)Rag1(-/-) mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model, we identified a new, Rag1-independent leukemia-initiating mechanism originating from a Sca1(+)CD19(+) precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM, a similar CD34(+)CD19(+) population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans.     

Activating Notch1 mutations in mouse models of T-ALL

Recent studies have demonstrated that most patients with T-cell acute lymphocytic leukemia (T-ALL) have activating mutations in NOTCH1. We sought to determine whether these mutations are also acquired in mouse models of T-ALL. We sequenced the heterodimerization domain and the PEST domain of Notch1 in our mouse model of TAL1-induced leukemia and found that 74% of the tumors harbor activating mutations in Notch1. Cell lines derived from these tumors undergo G(0)/G(1) arrest and apoptosis when treated with a gamma-secretase inhibitor. In addition, we found activating Notch1 mutations in 31% of thymic lymphomas that occur in mice deficient for various combinations of the H2AX, Tp53, and Rag2 genes. Thus, Notch1 mutations are often acquired as a part of the molecular pathogenesis of T-ALLs that develop in mice with known predisposing genetic alterations.     

Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient lymphocytes target chromosomal breakpoints in cis

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.     

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice

The recombination-activating gene (RAG)1 and RAG2 proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 protein ("core" RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.     

Histone methylation and V(D)J recombination

V(D)J recombination is the process by which the diversity of antigen receptor genes is generated and is also indispensable for lymphocyte development. This recombination event occurs in a cell lineage- and stage-specific manner, and is carefully controlled by chromatin structure and ordered histone modifications. The recombinationally active V(D)J loci are associated with hypermethylation at lysine4 of histone H3 and hyperacetylation of histones H3/H4. The recombination activating gene 1 (RAG1) and RAG2 complex initiates recombination by introducing double-strand DNA breaks at recombination signal sequences (RSS) adjacent to each coding sequence. To be recognized by the RAG complex, RSS sites must be within an open chromatin context. In addition, the RAG complex specifically recognizes hypermethylated H3K4 through its plant homeodomain (PHD) finger in the RAG2 C terminus, which stimulates RAG catalytic activity via that interaction. In this review, we describe how histone methylation controls V(D)J recombination and discuss its potential role in lymphoid malignancy by mistargeting the RAG complex.     

Characterization of immature thymocyte lines derived from T-cell receptor or recombination activating gene 1 and p53 double mutant mice.

The T-cell receptor (TCR) beta chain is instrumental in the progression of thymocyte differentiation from the CD4-CD8- to the CD4+CD8+ stage. This differentiation step may involve cell surface expression of novel CD3-TCR complexes. To facilitate biochemical characterization of these complexes, we established cell lines from thymic lymphomas originating from mice carrying a mutation in the p53 gene on the one hand and a mutation in TCR-alpha, TCR-beta, or the recombination activating gene 1 (RAG-1) on the other hand. The cell lines were CD4+CD8+ and appeared to be monoclonal. A cell line derived from a RAG-1 x p53 double mutant thymic lymphoma expressed low levels of CD3-epsilon, -gamma, and -delta on the surface. TCR-alpha x p53 double mutant cell lines were found to express complexes consisting of TCR-beta chains associated with CD3-epsilon, -gamma, and -delta chains and CD3-zeta zeta dimers. These lines will be useful tools to study the molecular structure and signal transducing properties of partial CD3-TCR complexes expressed on the surface of immature thymocytes.     

Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation

Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.