Ataxia-telangiectasia mutated (ATM) is required for resistance to radiation-induced DNA breaks. Here we use chromatin immunoprecipitation to show that ATM also localizes to breaks associated with V(D)J recombination. ATM recruitment to the recombining locus correlates approximately with recruitment of the break-initiating factor RAG1 and precedes efficient break repair, consistent with localization of ATM to normal recombination intermediates. A product of ATM kinase activity, Ser 18-phosphorylated p53, was detected similarly at these breaks, arguing that ATM phosphorylates target proteins in situ. We suggest routine surveillance of intermediates in V(D)J recombination by ATM helps suppress potentially oncogenic translocations when repair fails. Ataxia-telangiectasia is characterized by cerebellar degeneration, immunodeficiency, and a high frequency of malignancy, usually lymphoid in origin (Morrell et al. 1986). The gene mutated in this disease (ataxia-telangiectasia mutated, or ATM) is required for resistance to DNA double-strand break (DSB) inducing agents such as ionizing radiation, and is an important trigger for the cellular response to DSBs (for review, see Rotman and Shiloh 1999). ATM may act as a primary sensor of DSBs, first binding directly to DSBs and consequently activating through phosphorylation several downstream effectors of the DNA damage response, including Nbs1, BRCA1, Chk2, and p53 (for review, see Durocher and Jackson 2001). For example, after treatment with ionizing radiation, phosphorylation of p53 at Ser 15 (Ser 18 in mice) in cells is largely ATM dependent (for review, see Giaccia and Kastan 1998) and is required for effective p53-dependent responses to this stimulus (Chao et al. 2000).In contrast to its important role in mediating resistance to exogenous DSB-inducing agents, genetic evidence for a role for ATM in repair of DSB intermediates in V(D)J recombination is less clear. V(D)J recombination is an essential step in the generation of a diverse repertoire of immunoglobulins and T-cell receptors (for review, see Gellert 1997). RAG1 and RAG2 proteins initiate V(D)J recombination by introducing DSBs precisely adjacent to recombination-targeting signals that flank segments of immunoglobulin and T-cell receptor coding sequence. Efficient resolution of broken species requires factors implicated in end-joining DSB repair, including Ku, XRCC4, ligase IV, and the ATM-related kinase DNA-PKcs, but not ATM. Cells from patients with AT support normal levels of V(D)J recombination using an extra-chromosomal substrate assay (Hsieh et al. 1993), and mature antigen receptor-bearing lymphocytes are readily observed in ATM-deficient mice (Barlow et al. 1996;Elson et al. 1996;Xu et al. 1996).Are V(D)J recombination intermediates recognized by ATM as DNA damage? The lack of requirement for ATM in V(D)J recombination might suggest that ATM is excluded from breaks associated with this pathway, possibly through masking of ends by RAG1 and RAG2, to avoid counter-productive apoptotic responses during this n...

The first step in assembling immunoglobulin and T-cell receptors by V(D)J recombination has similarities to transposon excision. The excised transposon-like element then integrates into DNA targets at random in vitro, but whether this activity significantly threatens the genomic integrity of its host has been unclear. Here, we recover examples where the putative transposon associated with V(D)J recombination integrated into the genome of a pre-B-cell line. Transposition accounted for a surprisingly high proportion (one-third) of integrations, while most of the remaining events had parallels to other aberrant V(D)J recombination pathways linked to oncogenic translocation. In total, transposition occurred approximately once every 50,000 V(D)J recombinations. Transposition may thus contribute significantly to genomic instability.

The relationship between chromatin modification and lymphocyte development is still poorly understood. Here we show a correlation between methylation of lysine 4 on histone 3 (H3-K4) and activation of several loci required for the pre-B cell to immature B-cell developmental transition. A critical step in this transition is the induction of V(D)J recombination at the Igkappa locus. Upon activation of Igkappa recombination, a >10-fold enrichment of both di- and trimethylated H3-K4 is observed at Jkappa targeting signals, but not at an analogous targeting signal in the T-cell receptor alpha locus or, surprisingly, at several Vkappa signals. However, H3-K4 methylation is restricted to the actively recombining fraction of Jkappa recombination targeting signals, consistent with a direct relationship between H3-K4 methylation and signal activity. Correlations between increased H3-K4 methylation and induction of transcription are also observed at some, but not all, loci where transcription is induced. H3-K4 methylation may therefore be a widely used but not universal means for controlling chromatin activity in this developmental transition.

Reproducibility is a cornerstone of the scientific method and sets apart science from pseudoscience. Unfortunately, a majority of scientists have experienced difficulties in reproducing their own or someone else’s results. This inability to confirm scientific findings negatively impacts individual scientists, funding bodies, academic journals, pharmaceutical drug development and the public’s perception of science. Factors causing irreproducible results can arise from nearly every aspect of the scientific process, and typically reflect a lack of in-depth training in reproducible research practices. Here, we present the Reproducibility for Everyone (R4E) initiative, a collaboration between researchers from diverse scientific disciplines and industry partners who aspire to promote open and reproducible research practices. We have developed a customizable workshop series targeting researchers at all levels and across disciplines. Our workshop series covers the conceptual framework of reproducible research practices followed by an overview of actionable research practices. To date, we have reached more than 2000 researchers through over 25 workshops held at international conferences and local meetings. By incorporating further contributions from the scientific community, we hope to expand this valuable resource for teaching transparent and reproducible research practices. Our initiative demonstrates how a shared set of materials may form the basis for a global initiative to improve reproducibility in science. The workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at www.repro4everyone.org.

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