The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf − / − PAXX −/− E18.5 embryos is severely affected with the block of B-and T-cell maturation at the stage of IgH and TCRβ gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development. Cell Death and Differentiation (2018) 25, 444-452; doi:10.1038/cdd.2017; published online 27 October 2017All living organisms are subjected to multitude sources of DNA damage during their lifespan, either as a result of external assault or endogenous physiological processes.1 Among endogenous sources of physiological DNAdsb is the somatic rearrangement of immunoglobulin (Ig) and TCR genes in B and T lymphocytes, respectively, during the diversification of the adaptive immune system through V(D)J recombination.2 DNA double-stranded breaks (DNAdsb) are considered the most toxic lesions. DNAdsbs are repaired by two main mechanisms: the homologous recombination (HR) in cycling cells, when a sister chromatid is available as DNA repair template, and the non-homologous end joining (NHEJ) during all phases of the cell cycle.NHEJ proceeds via the simple religation of DNA ends without the need for a repair template.3 Briefly, the NHEJ is composed of seven core factors comprising the Ku70/80/ DNA-PKcs (DNA-dependent protein kinase catalytic subunit) complex, which recognizes and protects the broken DNA ends, the Artemis endo/exonuclease, which participates, when needed, in processing the DNA ends and the XRCC4/ DNA-Ligase IV/Xlf complex, which ultimately reseals the DNA break. The critical function of the NHEJ apparatus in various aspects of higher eukaryote development has been extensively perceived in several animal and human pathological conditions. As emblematic examples, loss of function of either XRCC4 or DNA ligase IV results in embryonic lethality in mice 4,5 and mutations in Artemis or DNA-PKcs result in severe combined immunodeficiency conditions in both men and mice, owing to aborted V(D)J recombination. 6 In addition, defects in NHEJ results in genetic instability and the propensity to develop various types of cancers, notably leukemia and lymphomas. Recently, a new DNA repair factor, PAXX (PAralog of XRCC4 and Xlf, also known as C9orf142 or XLS), has been identified independently by three laboratories based on bioinformatic...

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Repair of DNA double-strand breaks by the nonhomologous end joining pathway is central for proper development of the adaptive immune system. This repair pathway involves eight factors, including XRCC4-like factor (XLF)/Cernunnos and the paralog of XRCC4 and XLF, PAXX nonhomologous end joining factor (PAXX). Xlf−/− and Paxx−/− mice are viable and exhibit only a mild immunophenotype. However, mice lacking both PAXX and XLF are embryonic lethal because postmitotic neurons undergo massive apoptosis in embryos. To decipher the roles of PAXX and XLF in both variable, diversity, and joining recombination and immunoglobulin class switch recombination, here, using Cre/lox-specific deletion to prevent double-KO embryonic lethality, we developed two mouse models of a conditional Xlf KO in a Paxx−/− background. Cre expressed under control of the iVav or CD21 promoter enabled Xlf deletion in early hematopoietic progenitors and splenic mature B cells, respectively. We demonstrate the XLF and PAXX interplay during variable, diversity, and joining recombination in vivo but not during class switch recombination, for which PAXX appeared to be fully dispensable. Xlf/Paxx double KO in hematopoietic progenitors resulted in a shorter lifespan associated with onset of thymic lymphomas, revealing a genome caretaking function of XLF/PAXX.

The aim of this work was to delineate the effects of chronic ingestion of strontium 90 ((90) Sr) at low concentrations on the hematopoiesis and the bone physiology. A mouse model was used for that purpose. Parent animals ingested water containing 20 kBq l(-1) of (90) Sr two weeks before mating. Offspring were then continuously contaminated with (90) Sr through placental transfer during fetal life, through lactation after birth and through drinking water after weaning. At various ages between birth and 20 weeks, animals were tested for hematopoietic parameters such as blood cell counts, colony forming cells in spleen and bone marrow and cytokine concentrations in the plasma. However, we did not find any modification in (90) Sr ingesting animals as compared with control animals. By contrast, the analysis of bone physiology showed a modification of gene expression towards bone resorption. This was confirmed by an increase in C-telopeptide of collagen in the plasma of (90) Sr ingesting animals as compared with control animals. This modification in bone metabolism was not linked to a modification of the phosphocalcic homeostasis, as measured by calcium, phosphorus, vitamin D and parathyroid hormone in the blood. Overall these results suggest that the chronic ingestion of (90) Sr at low concentration in the long term may induce modifications in bone metabolism but not in hematopoiesis.

90Sr is one of the radionuclides released after nuclear accidents that can significantly impact human health in the long term. 90Sr accumulates mostly in the bones of exposed populations. Previous research has shown that exposure induces changes in bone physiology both in humans and in mice. We hypothesize that, due to its close location with bone marrow stromal cells (BMSCs), 90Sr could induce functional damage to stromal cells that may explain these biological effects due to chronic exposure to 90Sr. The aim of this work was to verify this hypothesis through the use of an in vitro model of MS5 stromal cell lines exposed to 1 and 10 kBq.mL−1 of 90Sr. Results indicated that a 30-minute exposure to 90Sr induced double strand breaks in DNA, followed by DNA repair, senescence and differentiation. After 7 days of exposure, MS5 cells showed a decreased ability to proliferate, changes in cytokine expression, and changes in their ability to support hematopoietic progenitor proliferation and differentiation. These results demonstrate that chronic exposure to a low concentration of 90Sr can induce functional changes in BMSCs that in turn may explain the health effects observed in following chronic 90Sr exposure.

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We developed a Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4 deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA Ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core NHEJ DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double KO settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights in the understanding of the clinical manifestations of human XRCC4 deficient condition, in particular its absence of immune deficiency.

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