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Summary Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1−/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity., Graphical abstract, Highlights • XRCC1 prevents endogenous PARP1 trapping during DNA base excision repair • PARP1 trapping impedes base excision repair and increases sensitivity to base damage • In the absence of PARP1, XRCC1 is dispensable for DNA base excision repair, Demin et al. show that the essential role of the scaffold protein XRCC1 during DNA base excision repair is to prevent toxic “trapping” of PARP1 on SSB intermediates, which otherwise block this essential repair process and lead to increased cellular sensitivity to DNA base damage.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

SummaryDNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

The DNA damage response protein ATM has long been known to influence class switch recombination in ex vivo–cultured B cells. However, an assessment of B cell–intrinsic requirement of ATM in humoral responses in vivo was confounded by the fact that its germline deletion affects T cell function, and B:T cell interactions are critical for in vivo immune responses. In this study, we demonstrate that B cell–specific deletion of ATM in mice leads to reduction in germinal center (GC) frequency and size in response to immunization. We find that loss of ATM induces apoptosis of GC B cells, likely due to unresolved DNA lesions in cells attempting to undergo class-switch recombination. Accordingly, suboptimal GC responses in ATM-deficient animals are characterized by decreased titers of class-switched Abs and decreased rates of somatic hypermutation. These results unmask the critical B cell–intrinsic role of ATM in maintaining an optimal GC response following immunization.

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4(−/−)Tp53(−/−) mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs(−/−)Tp53(−/−) mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.

DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKIN) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here we report the cryoEM structures of DNA-PKcs (catalytic subunit) bound to a DNA end, or complexed with Ku70/80 and DNA, in both inactive and activated forms at resolutions of 3.7 Å overall, and 3.2 Å for FATKIN. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as ‘trapping’. To understand the molecular nature of ‘trapping’ in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

Revealing the seasonal and interannual variations in leaf-level photosynthesis is a critical issue in understanding the ecological mechanisms underlying the dynamics of carbon dioxide exchange between the atmosphere and shrub ecosystem. Artemisia ordosica is a dominant shrub species in semi-arid area of northwest China. Photosynthetic gas exchange, leaf nitrogen content(LN), specific leaf area (SLA) and some environmental factors were measured simultaneously on clear days (rotated every 10 days) of the growing season from 2011 to 2018, to quantify the temporal variations and environmental controls of photosynthetic parameters. Our results demonstrated that mean value of light-response curve parameters, the maximum photosynthetic capacity (Pmax), appear quality efficiency (AQE), respiration in dark (Rd), light saturated point (LSP) and light compensated point (LCP) had a gradual decline with the growth (spring> summer>autumn). Structural equation modeling (SEM) was used to elucidate the direct and indirect effects of biophysical factors on Pmax. The driven factors of Pmax in growing season changed, but stomatal conductance (gs) was the dominant factor in all stages. The gs was influenced by SLA and LN，and the soil water content at a depth of 10cm (SWC10) affected the Pmax in spring. In summer, Pmax was significantly positively related with gs and transpiration rate (Tr), and gs was influenced by SLA, LN and soil water content at a depth of 30cm (SWC30). In autumn, Pmax was significantly positively correlated with gs, while was significantly negatively correlated with air temperature (Ta). This simulation based on situ ecophysiological research suggest that Pmax of A. ordosica responded to the environment factors of seasonal and interannual variations, which is not the inherent genetic characteristics. Soil water content is the major environmental factor influencing Pmax in spring and summer, while Ta is the major one in autumn. Knowledge of how environmental change will affect the photosynthesis of A. ordosica in the future is essential for their protection, adaptation strategies and carbon fixation prediction in shrub ecosystems.

Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM gene. Typically, it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM (c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T.

Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.

Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocyte-specific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution., T-cell prolymphocytic leukemia (T-PLL) is a rare malignancy with a poor prognosis. Here, the authors investigate the genomic landscape, gene expression profiles and functional mechanisms in 111 patients, highlighting TCL1 overexpression and ATM aberrations as core lesions which co-operate to impair DNA damage processing.

The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1(S563F) and Bard1(K607A)) or Brca1 (Brca1(S1598F)). Brca1(S1598F) abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1-mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1(S563F/S563F) and Bard1(K607A/K607A) mice, unlike Brca1(S1598F/S1598F) mice, are not tumor-prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, since SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1-mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.

Ataxia telangiectasia mutated (ATM) is a protein kinase and a master regulator of DNA-damage responses. Germline ATM inactivation causes ataxia-telangiectasia (A-T) syndrome with severe lymphocytopenia and greatly increased risk for T-cell lymphomas/leukemia. Both A-T and T-cell prolymphoblastic leukemia patients with somatic mutations of ATM frequently carry inv(14;14) between the T-cell receptor α/δ (TCRα/δ) and immunoglobulin H loci, but the molecular origin of this translocation remains elusive. ATM(-/-) mice recapitulate lymphocytopenia of A-T patients and routinely succumb to thymic lymphomas with t(12;14) translocation, syntenic to inv(14;14) in humans. Here we report that deletion of the TCRδ enhancer (Eδ), which initiates TCRδ rearrangement, significantly improves αβ T cell output and effectively prevents t(12;14) translocations in ATM(-/-) mice. These findings identify the genomic instability associated with V(D)J recombination at the TCRδ locus as the molecular origin of both lymphocytopenia and the signature t(12;14) translocations associated with ATM deficiency.

Non-homologous end-joining (NHEJ) is a major DNA double strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate specific NHEJ factor that can be auto-phosphorylated or trans-phosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated trans-phosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strictly auto-phosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcsKD/KD mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations.

The genome instability syndrome, ataxia-telangiectasia (A-T) is caused by null mutations in the ATM gene, that lead to complete loss or inactivation of the gene’s product, the ATM protein kinase. ATM is the primary mobilizer of the cellular response to DNA double-strand breaks (DSBs) – a broad signaling network in which many components are ATM targets. The major clinical feature of A-T is cerebellar atrophy, characterized by relentless loss of Purkinje and granule cells. In Atm-knockout (Atm-KO) mice, complete loss of Atm leads to a very mild neurological phenotype, suggesting that Atm loss is not sufficient to markedly abrogate cerebellar structure and function in this organism. Expression of inactive (“kinase-dead”) Atm (Atm(KD)) in mice leads to embryonic lethality, raising the question of whether conditional expression of Atm(KD) in the murine nervous system would lead to a more pronounced neurological phenotype than Atm loss. We generated two mouse strains in which Atm(KD) was conditionally expressed as the sole Atm species: one in the CNS and one specifically in Purkinje cells. Focusing our analysis on Purkinje cells, the dynamics of DSB readouts indicated that DSB repair was delayed longer in the presence of Atm(KD) compared to Atm loss. However, both strains exhibited normal life span and displayed no gross cerebellar histological abnormalities or significant neurological phenotype. We conclude that the presence of Atm(KD) is indeed more harmful to DSB repair than Atm loss, but the murine central nervous system can reasonably tolerate the extent of this DSB repair impairment. Greater pressure needs to be exerted on genome stability to obtain a mouse model that recapitulates the severe A-T neurological phenotype.

XRCC4-like factor (XLF/Cernunnos) is a component of the nonhomologous end-joining (NHEJ) pathway of double-strand DNA break repair. XLF-deficient patients develop a severe progressive lymphocytopenia. Although NHEJ is required for V(D)J recombination and lymphocyte development, XLF-deficient mice have normal V(D)J recombination, highlighting the need for an alternative mechanism for the lymphocytopenia. Here, we report that XLF-deficient mice recapitulate the age-dependent lymphocytopenia of patients. We show that XLF deficiency leads to premature aging of hematopoietic stem cells (HSCs), measured by decreased functional capacity in transplantation assays, preferential myeloid reconstitution, and reduced self-renewal at a young age. We propose that premature aging of HSCs, together with previously reported defects in class-switch recombination and memory immune response, underlies the progressive and severe lymphocytopenia in XLF-deficient patients in the absence of measurable V(D)J recombination defects.

Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency. Background: ATM acts as a master controller of the DSB response by phosphorylating numerous DSB response proteins, some of which, including histone H2AX, function in homologous recombination (HR). Results: HR is not impaired in mouse ATM-null ES cells. Conclusion: ATM is dispensable for HR, including H2AX-dependent HR. Significance: This study helps clarify the perception that ATM has a general role in HR, including that controlled by H2AX.

Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway that is required for assembly of antigen receptor variable region gene segments by V(D)J recombination. Recombination activating gene endonuclease initiates V(D)J recombination by generating DSBs between two V(D)J coding gene segments and flanking recombination signal sequences (RS), with the two coding ends and two RS ends joined by C-NHEJ to form coding joins and signal joins, respectively. During C-NHEJ, recombination activating gene factor generates two coding ends as covalently sealed hairpins and RS ends as blunt 5′-phosphorylated DSBs. Opening and processing of coding end hairpins before joining by C-NHEJ requires the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). However, C-NHEJ of RS ends, which do not require processing, occurs relatively normally in the absence of DNA-PKcs. The XRCC4-like factor (XLF) is a C-NHEJ component that is not required for C-NHEJ of chromosomal signal joins or coding joins because of functional redundancy with ataxia telangiectasia mutated kinase, a protein that also has some functional overlap with DNA-PKcs in this process. Here, we show that XLF has dramatic functional redundancy with DNA-PKcs in the V(D)J SJ joining process, which is nearly abrogated in their combined absence. Moreover, we show that XLF functionally overlaps with DNA-PKcs in normal mouse development, promotion of genomic stability in mouse fibroblasts, and in IgH class switch recombination in mature B cells. Our findings suggest that DNA-PKcs has fundamental roles in C-NHEJ processes beyond end processing that have been masked by functional overlaps with XLF.

Expression of a kinase-deficient ATM protein leads to severe genomic instability and embryonic lethality., Ataxia telangiectasia (A-T) mutated (ATM) kinase orchestrates deoxyribonucleic acid (DNA) damage responses by phosphorylating numerous substrates implicated in DNA repair and cell cycle checkpoint activation. A-T patients and mouse models that express no ATM protein undergo normal embryonic development but exhibit pleiotropic DNA repair defects. In this paper, we report that mice carrying homozygous kinase-dead mutations in Atm (AtmKD/KD) died during early embryonic development. AtmKD/− cells exhibited proliferation defects and genomic instability, especially chromatid breaks, at levels higher than Atm−/− cells. Despite this increased genomic instability, AtmKD/− lymphocytes progressed through variable, diversity, and joining recombination and immunoglobulin class switch recombination, two events requiring nonhomologous end joining, at levels comparable to Atm−/− lymphocytes. Together, these results reveal an essential function of ATM during embryogenesis and an important function of catalytically inactive ATM protein in DNA repair.

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI–induced DSBs in XRCC4-deficient pro–B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.

The loss of a classical non-homologous end-joining (NHEJ) repair factor, XLF, shows strong effects in non-lymphoid cells, but in lymphoid cells its absence surprisingly has only modest effects on V(D)J recombination. Frederick Alt and colleagues show that in lymphoid cells, two other repair factors — ATM kinase and histone protein H2AX — have functional redundancy with XLF. Thus, mice that are deficient in both ATM and XLF have compromised conventional NHEJ, although alternative end-joining is retained. The results hint that the redundant function in end-joining that XLF has with both ATM and H2AX may be related to a role for ATM in chromatin accessibility. Although loss of XLF, a classical non-homologous DNA end-joining (NHEJ) repair factor, shows strong effects in non-lymphoid cells, in lymphoid cells its absence has only modest effects on V(D)J recombination. This study now shows that in lymphoid cells, two other repair factors — ATM kinase and histone protein H2AX — have functional redundancy with XLF. Thus, mice deficient in both ATM and XLF have compromised conventional NHEJ, although alternative end-joining is retained. The results hint that the redundant function in end-joining that XLF has with both ATM and H2AX may have to do with an ATM role in chromatin accessibility. Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ3,4,5,6; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes7,8,9. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.

Upon DNA damage, histone H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other phosphoinositide 3-kinase-related protein kinases. To elucidate further the potential overlapping and unique functions of ATM and H2AX, we asked whether they have synergistic functions in the development and maintenance of genomic stability by inactivating both genes in mouse germ line. Combined ATM/H2AX deficiency caused embryonic lethality and dramatic cellular genomic instability. Mechanistically, severe genomic instability in the double-deficient cells is associated with a requirement for H2AX to repair oxidative DNA damage resulting from ATM deficiency. We discuss these findings in the context of synergies between ATM and other repair factors.

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy. DOI: http://dx.doi.org/10.7554/eLife.14709.001, eLife digest Cancer is a genetic disease. To remain healthy, therefore, it is essential that cells do not accrue too many dangerous mutations in their DNA that allow cancers to grow and develop. An enzyme called ATM helps to do just that. DNA damage activates ATM, which, in turn, adds phosphate groups to other proteins. These newly tagged proteins then stop cells dividing until the DNA has been repaired. Human cancers often switch off ATM, either by completely deleting the enzyme or mutating it. This renders ATM unable to add phosphate groups to proteins, and so allows the cancer cells to continue proliferating even in the face of DNA damage. Yamamoto et al. wanted to know whether cancers that completely lack ATM behave differently from cancers that contain an inactive version of the enzyme. Studying mice that were engineered to have an inactive version of ATM in their blood cells showed that such mice developed blood cancers faster than mice have no ATM in their blood cells. In particular, cancer cells with the inactive form of the ATM enzyme accumulated more DNA damage than cells that lacked the enzyme completely. Using biochemical techniques, Yamamoto et al. then showed that the inactive form of ATM can prevent other enzymes from repairing DNA. Drugs that inhibit one of these repair enzymes – called topo-isomerase I – are already used in cancer treatments. These drugs were particularly effective on tumors with the inactive version of the ATM enzyme. As ATM is commonly mutated in human cancers, the next steps that follow on from this research are to develop methods to test which cancers contain the inactive form of the ATM enzyme. Clinical trials could then investigate how effectively topo-isomerase I inhibitors treat these specific types of cancer. DOI: http://dx.doi.org/10.7554/eLife.14709.002

Bcl11b is a transcription factor important for T cell development and also a tumor-suppressor gene that is hemizygously inactivated in ~10 % human T cell acute lymphoblastic leukemia (T-ALL) and several murine T-ALL models, including ATM−/− thymic lymphomas. Here we report that heterozygous loss of Bcl11b (Bcl11b+/−) unexpectedly reduced lethal thymic lymphoma in ATM−/− mice by suppressing lymphoma progression, but not initiation. The suppression was associated with a T cell-mediated immune response in ATM−/−Bcl11b+/− mice, revealing a haploid insufficient function of Bcl11b in immune modulation against lymphoma and offering an explanation for the complex relationship between Bcl11b status with T-ALL prognosis. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0191-8) contains supplementary material, which is available to authorized users.

Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.

α-Methylacyl-CoA racemase (AMACR), an enzyme involved in branched-chain fatty acid β-oxidation that is normally expressed at high levels in human liver, is specifically and consistently overexpressed at both mRNA and protein levels in human prostate cancer and potentially other cancer types. To characterize the mechanisms underlying transcriptional regulation of AMACR at the genetic and epigenetic levels, we performed a series of methylation and reporter assays in prostate cancer tissues and cell lines. The results ruled out altered methylation patterns as the cause of overexpression in prostate cancer cells. However, promoter deletion analysis identified an 8-bp nonclassic CCAAT enhancer element located ∼80 bp upstream of the transcriptional initiation site that is responsible for AMACR expression in both prostate cancer cell lines and cell lines of liver origin. Deletion or mutation of this element completely abolished AMACR promoter activity. Ectopic expression of CCAAT/enhancer binding protein β increased luciferase activity driven by a wild-type AMACR promoter sequence but not by the sequence in which the putative CCAAT/enhancer binding protein binding element had been mutated. These results implicate a nonclassic CCAAT enhancer element in the AMACR gene promoter as playing a critical role in the regulation of AMACR gene expression.

Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends., DNA double-strand breaks (DSBs), a highly deleterious form of DNA damage, are associated with multiple types of broken ends. Here, the authors identify a XRCC4-like factor that functions in the non-homologous end-joining DNA repair pathway to repair DSBs with complex broken ends.

The Mre11 protein has well-documented functions in the repair of DNA double-strand breaks via homologous recombination. Now, several new studies reveal that Mre11 also has a role in mammalian DNA double-strand break repair by nonhomologous end joining.

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.

The BCL11B transcription factor is required for normal T-cell development, and has recently been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) induced by TLX overexpression or Atm deficiency. To comprehensively assess the contribution of BCL11B inactivation to human T-ALL, we performed DNA copy number and sequencing analyses of T-ALL diagnostic specimens, revealing monoallelic BCL11B deletions or missense mutations in 9% (n = 10 of 117) of cases. Structural homology modeling revealed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for this transcription factor to bind DNA. BCL11B haploinsufficiency occurred across each of the major molecular subtypes of T-ALL, including early T-cell precursor, HOXA-positive, LEF1-inactivated, and TAL1-positive subtypes, which have differentiation arrest at diverse stages of thymocyte development. Our findings provide compelling evidence that BCL11B is a haploinsufficient tumor suppressor that collaborates with all major T-ALL oncogenic lesions in human thymocyte transformation.

Antigen receptor variable region exons are assembled during lymphocyte development from variable (V), diversity (D), and joining (J) gene segments. Each germ-line gene segment is flanked by recombination signal sequences (RSs). Recombination-activating gene endonuclease initiates V(D)J recombination by cleaving a pair of gene segments at their junction with flanking RSs to generate covalently sealed (hairpinned) coding ends (CEs) and blunt 5′-phosphorylated RS ends (SEs). Subsequently, nonhomologous end joining (NHEJ) opens, processes, and fuses CEs to form coding joins (CJs) and precisely joins SEs to form signal joins (SJs). DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activates Artemis endonuclease to open and process hairpinned CEs before their fusion into CJs by other NHEJ factors. Although DNA-PKcs is absolutely required for CJs, SJs are formed to variable degrees and with variable fidelity in different DNA-PKcs–deficient cell types. Thus, other factors may compensate for DNA-PKcs function in SJ formation. DNA-PKcs and the ataxia telangiectasia-mutated (ATM) kinase are members of the same family, and they share common substrates in the DNA damage response. Although ATM deficiency compromises chromosomal V(D)J CJ formation, it has no reported role in SJ formation in normal cells. Here, we report that DNA-PKcs and ATM have redundant functions in SJ formation. Thus, combined DNA-PKcs and ATM deficiency during V(D)J recombination leads to accumulation of unjoined SEs and lack of SJ fidelity. Moreover, treatment of DNA-PKcs– or ATM-deficient cells, respectively, with specific kinase inhibitors for ATM or DNA-PKcs recapitulates SJ defects, indicating that the overlapping V(D)J recombination functions of ATM and DNA-PKcs are mediated through their kinase activities.

Mutations in XLF/Cernunnos (XLF) cause lymphocytopenia in humans, and various studies suggest an XLF role in classical nonhomologous end joining (C-NHEJ). We now find that XLF-deficient mouse embryonic fibroblasts are ionizing radiation (IR) sensitive and severely impaired for ability to support V(D)J recombination. Yet mature lymphocyte numbers in XLF-deficient mice are only modestly decreased. Moreover, XLF-deficient pro-B lines, while IR-sensitive, perform V(D)J recombination at nearly wild-type levels. Correspondingly, XLF/p53-double-deficient mice are not markedly prone to the pro-B lymphomas that occur in previously characterized C-NHEJ/p53-deficient mice; however, like other C-NHEJ/p53-deficient mice, they still develop medulloblastomas. Despite nearly normal V(D)J recombination in developing B cells, XLF-deficient mature B cells are moderately defective for immunoglobulin heavy-chain class switch recombination. Together, our results implicate XLF as a C-NHEJ factor but also indicate that developing mouse lymphocytes harbor cell-type-specific factors/pathways that compensate for the absence of XLF function during V(D)J recombination.

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 Δ/Δ ES cells). Analyses of Cer Δ/Δ ES cells showed that they produce no readily detectable Cernunnos-XLF protein. Based on transient V(D)J recombination assays, we find that Cer Δ/Δ 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 Δ/Δ 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.

Overexpression of α-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fatty acid β-oxidation, in prostate cancer has been reported. Here, we report that an enzyme downstream from AMACR in the peroxisomal branched chain fatty acid β-oxidation pathway—D-bifunctional protein (DBP)—is also upregulated in prostate cancer at both mRNA and protein levels, accompanied by increased enzymatic activity. Furthermore, our data suggest that pristanoyl-CoA oxidase (ACOX3), which is expressed at extremely low level in other human organs studied including the liver, might contribute significantly to peroxisomal branched chain fatty acid β-oxidation in human prostate tissue and some prostate cancer cell lines. In contrast to these results for peroxisomal enzymes, no significant expression changes of mitochondrial fatty acid β-oxidation enzymes were observed in prostate cancer tissues through comprehensive quantitative RT-PCR screening. These data for the first time provide evidence for the selective over-activation of peroxisomal branched chain fatty acid β-oxidation in prostate cancer, emphasizing a new metabolic change during prostate oncogenesis. © 2004 Wiley-Liss, Inc.

Abstract 4177 The BCL11B transcription factor, previously identified as a central player in normal α/β T-cell development, has recently been found to play critical roles in the maintenance of T-cell identity, with biallelic Bcl11b inactivation in T-cell precursors severely impairing their continued development into the T-cell lineage. Previous work has demonstrated that Bcl11b haploinsufficiency accelerates the onset of thymic lymphomas in p53-mutant mice, as well as T-lymphoid blast crisis in a mouse model of chronic myeloid leukemia induced by BCR-ABL. Furthermore, we have recently identified recurrent monoallelic Bcl11b deletions in 91% of T-cell acute lymphoblastic leukemias (T-ALL) arising in Atm-deficient mice, further supporting a role for Bcl11b haploinsufficiency in T-cell leukemogenesis. In order to determine whether BCL11B inactivation plays a role in the molecular pathogenesis of human T-ALL, we analyzed BCL11B status in primary T-ALL patient samples by array CGH and sequencing analysis. Monoallelic BCL11B deletions were identified in 6% of cases (n = 3 of 47) analyzed by array CGH, including one microdeletion within the BCL11B locus, one small deletion involving BCL11B and 6 additional genes, and one large 26 Mbp deletion of the distal arm of chromosome 14. BCL11B sequencing revealed heterozygous missense mutations in an additional 9% (n = 4 of 43) of primary T-ALL patient samples and in 19% (n = 3 of 16) of T-ALL cell lines. Structural homology modeling revealed that many of the mutations identified, including 3 of the 4 in the primary patient samples, disrupted key amino acids within BCL11B zinc finger domains that are involved in DNA recognition or structural stabilization required for zinc finger domain-mediated transcriptional activity. Analysis of TCRγ rearrangement status and gene expression data revealed that most cases with BCL11B inactivation were characterized by biallelic TCRγ rearrangements together with an early thymocyte precursor (ETP) gene expression signature, indicating developmental arrest at a prethymocyte stage of T-cell development, which we and others have shown confers an increased risk of treatment failure (Coustan-Smith et al. Lancet Oncol 2009; Gutierrez et al. J Clin Oncol 2010). Given that BCL11B expression during normal T-cell development increases markedly at the prethymocyte stage, our findings suggest that BCL11B inactivation may be directly responsible for developmental arrest at a prethymocyte stage during thymocyte transformation. Our findings provide compelling evidence that BCL11B is a tumor suppressor in a subset of human T-ALLs with a high risk of treatment failure. Disclosures: No relevant conflicts of interest to declare.

DNA ligase IV (LIG4) is an essential component of the nonhomologous end-joining (NHEJ) repair pathway and plays a key role in V(D)J recombination. Hypomorphic LIG4 mutations in humans are associated with increased cellular radiosensitivity, microcephaly, facial dysmorphisms, growth retardation, developmental delay, and a variable degree of immunodeficiency. We have generated a knock-in mouse model with a homozygous Lig4 R278H mutation that corresponds to the first LIG4 mutation reported in humans. The phenotype of homozygous mutant mice Lig4 R278H/R278H ( Lig4 R/R ) includes growth retardation, a decreased life span, a severe cellular sensitivity to ionizing radiation, and a very severe, but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID. Genomic instability is associated with a high rate of thymic tumor development. Finally, Lig4 R/R mice spontaneously produce low-affinity antibodies that include autoreactive specificities, but are unable to mount high-affinity antibody responses. These findings highlight the importance of LIG4 in lymphocyte development and function, and in genomic stability maintenance, and provide a model for the complex phenotype of LIG4 syndrome in humans.

Summary Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMINΔB/ΔB) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMINΔB/ΔB cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells., Graphical Abstract Highlights ► ATMIN is required for ATM signaling and function in B cells ► ATMIN is required for repair of DNA breaks generated during somatic recombination ► Mice lacking ATMIN in B cells develop B cell lymphomas

Background Liver cancr is a heterogeneous disease in terms of etiology, biologic and clinical behavior. Very little is known about how many genes concur at the molecular level of tumor development, progression and aggressiveness. To explore the key genes involved in the development of liver cancer, we established a rat model induced by diethylnitrosamine to investigate the gene expression profiles of liver tissues during the transition to cirrhosis and carcinoma. Methods A rat model of liver cancer induced by diethylnitrosamine was established. The cirrhotic tissue, the dysplasia nodules, the early cancerous nodules and the cancerous nodules from the rats with lung metastasis were chosen to compare with liver tissue of normal rats to investigate the differential expression genes between them. Affymetrix GeneChip Rat 230 2.0 arrays were used throughout. The real-time quantity PCR was used to verify the expression of some differential expression genes in tissues. Results The pathological changes that occurred in the livers of diethylnitrosamine-treated rats included non-specific injury, fibrosis and cirrhosis, dysplastic nodules, early cancerous nodules and metastasis. There are 349 upregulated and 345 downregulated genes sharing among the above chosen tissues when compared with liver tissue of normal rats. The deregulated genes play various roles in diverse processes such as metabolism, transport, cell proliferation, apoptosis, cell adhesion, angiogenesis and so on. Among which, 41 upregulated and 27 downregulated genes are associated with inflammatory response, immune response and oxidative stress. Twenty-four genes associated with glutathione metabolism majorly participating oxidative stress were deregulated in the development of liver cancer. There were 19 members belong to CYP450 family downregulated, except CYP2C40 upregulated. Conclusion In this study, we provide the global gene expression profiles during the development and progression of liver cancer in rats. The data obtained from the gene expression profiles will allow us to acquire insights into the molecular mechanisms of hepatocarcinogenesis and identify specific genes (or gene products) that can be used for early molecular diagnosis, risk analysis, prognosis prediction, and development of new therapies.