Unique and redundant functions of ATM and DNA-PKcs during V(D)J recombination

Lymphocyte antigen receptor genes are assembled through the process of V(D)J recombination, during which pairwise DNA cleavage of gene segments results in the formation of four DNA ends that are resolved into a coding joint and a signal joint. The joining of these DNA ends occurs in G(1)-phase lymphocytes and is mediated by the non-homologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. The ataxia telangiectasia mutated (ATM) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), two related kinases, both function in the repair of DNA breaks generated during antigen receptor gene assembly. Although these proteins have unique functions during coding joint formation, their activities in signal joint formation, if any, have been less clear. However, two recent studies demonstrated that ATM and DNA-PKcs have overlapping activities important for signal joint formation. Here, we discuss the unique and shared activities of the ATM and DNA-PKcs kinases during V(D)J recombination, a process that is essential for lymphocyte development and the diversification of antigen receptors

Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells require T cell help

In K/BxN mice, arthritis is induced by autoantibodies against glucose-6-phosphate-isomerase (GPI). To investigate B cell tolerance to GPI in nonautoimmune mice, we increased the GPI-reactive B cell frequency using a low affinity anti-GPI H chain transgene. Surprisingly, anti-GPI B cells were not tolerant to this ubiquitously expressed and circulating autoantigen. Instead, they were found in two functionally distinct compartments: an activated population in the splenic marginal zone (MZ) and an antigenically ignorant one in the recirculating follicular/lymph node (LN) pool. This difference in activation was due to increased autoantigen availability in the MZ. Importantly, the LN anti-GPI B cells remained functionally competent and could be induced to secrete autoantibodies in response to cognate T cell help in vitro and in vivo. Therefore, our study of low affinity autoreactive B cells reveals two distinct but potentially concurrent mechanisms for their activation, of which one is T cell dependent and the other is T cell independent

Regional gene repression by DNA double-strand breaks in G1 phase cells

DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate th

Out-of-frame T cell receptor beta transcripts are eliminated by multiple pathways In Vivo

Non-productive antigen receptor genes with frame shifts generated during the assembly of these genes are found in many mature lymphocytes. Transcripts from these genes have premature termination codons (PTCs) and could encode truncated proteins if they are not either inactivated or destroyed by nonsense-mediated decay (NMD). In mammalian cells, NMD can be activated by pathways that rely on the presence of an intron downstream of the PTC; however, NMD can also be activated by pathways that do not rely on these downstream introns, and pathways independent of NMD can inactivate PTC-containing transcripts. Here, through the generation and analysis of mice with gene-targeted modifications of the endogenous T cell receptor beta (Tcrb) locus, we demonstrate that in T cells in vivo, optimal clearance of PTC-containing Tcrb transcripts depends on the presence of an intron downstream of the PTC

Cell circuits between B cell progenitors and IL-7+ mesenchymal progenitor cells control B cell development

B cell progenitors require paracrine signals such as interleukin-7 (IL-7) provided by bone marrow stromal cells for proliferation and survival. Yet, how B cells regulate access to these signals in vivo remains unclear. Here we show that proB and IL-

Integrated signaling in developing lymphocytes: The role of DNA damage responses

Lymphocyte development occurs in a stepwise progression through distinct developmental stages. This ordered maturation ensures that cells express a single, non-autoreactive antigen receptor, which is the cornerstone of a diverse adaptive immune response. Expression of a mature antigen receptor requires assembly of the antigen receptor genes by the process of V(D)J recombination, a reaction that joins distant gene segments through DNA double-strand break (DSB) intermediates. These physiologic DSBs are generated by the recombinase-activating gene (RAG) -1 and -2 proteins, and their generation is regulated by lymphocyte and developmental stage-specific signals from cytokine receptors and antigen receptor chains. Collectively, these signals ensure that V(D)J recombination of specific antigen receptor genes occurs at discrete developmental stages. Once generated, RAG-induced DSBs activate the ataxia-telangiectasia mutated (ATM) kinase to orchestrate a multifaceted DNA damage response that ensures proper DSB repair. In response to RAG DSBs, ATM also regulates a cell type-specific transcriptional response, and here we discuss how this genetic program integrates with other cellular cues to regulate lymphocyte development

Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination

Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes

Ataxia-telangiectasia mutated (ATM)–deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor α/δ locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient αβ T cells with translocations involving the T cell receptor α/δ locus is directly related to the number of T cell receptor α rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment