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1.  Mechanisms That Can Promote Peripheral B Cell Lymphoma in ATM-deficient Mice 
Cancer immunology research  2014;2(9):857-866.
The Ataxia Telangiectasia-mutated (ATM) kinase senses DNA double-strand breaks (DSBs) and facilitates their repair. In humans, ATM deficiency predisposes to B- and T-cell lymphomas, but in mice leads only to thymic lymphomas. We tested the hypothesis that increased DSB frequency at a cellular oncogene could promote B-cell lymphoma by generating ATM-deficient mice with a V(D)J recombination target (“DJβ” cassette) within c-myc intron one (“DA” mice). We also generated ATM-deficient mice carrying an Eμ-Bcl-2 transgene (“AB” mice) to test whether enhanced cellular survival could promote B-cell lymphomas. About 30% of DA or AB mice and nearly 100% of mice harboring the combined genotypes (“DAB” mice) developed mature B-cell lymphomas. In all genotypes, B-cell tumors harbored oncogenic c-myc amplification generated by breakage-fusion-bridge (“BFB”) from dicentric chromosomes formed through fusion of IgH V(D)J recombination-associated DSBs on chromosome 12 to sequences downstream of c-myc on chromosome 15. AB tumors demonstrate that B lineage cells harboring spontaneous DSBs leading to IgH/c-myc dicentrics are blocked from progressing to B cell lymphomas by cellular apoptotic responses. DA and DAB tumor translocations were strictly linked to the cassette, but occurred downstream, frequently in a 6-kb region adjacent to c-myc that harbors multiple cryptic V(D)J recombination targets, suggesting that bona fide V(D)J target sequences may activate linked cryptic targets. Our findings indicate that ATM deficiency allows IgH V(D)J recombination DSBs in developing B cells to generate dicentric translocations that via BFB cycles lead to c-myc-activating oncogenic translocations and amplifications in mature B cells.
PMCID: PMC4156541  PMID: 24913718
ATM deficiency; B Cell Lymphoma; V(D)J recombination; c-myc Amplification; Breakage-Fusion-Bridge
2.  Mechanisms of Programmed DNA Lesions and Genomic Instability in the Immune System 
Cell  2013;152(3):417-429.
Chromosomal translocations involving antigen receptor loci are common in lymphoid malignancies. Translocations require DNA double-strand breaks (DSBs) at two chromosomal sites, their physical juxtaposition, and their fusion by end joining. Ability of lymphocytes to generate diverse repertoires of antigen receptors and effector antibodies derives from programmed genomic alterations that produce DSBs. We discuss these lymphocyte-specific processes, with a focus on mechanisms that provide requisite DSB target specificity and mechanisms that suppress DSB translocation. We also discuss recent work that provides new insights into DSB repair pathways and influences of three-dimensional genome organization on physiological processes and cancer genomes.
PMCID: PMC4382911  PMID: 23374339
3.  A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency 
The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes.
We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations.
We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology.
Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity.
Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process.
PMCID: PMC4005599  PMID: 24290284
Recombination-activating gene 1; V(D)J recombination; severe combined immune deficiency; Omenn syndrome; autoimmunity; genotype-phenotype correlation; immune repertoire
4.  Functional Overlaps Between XLF and The ATM-dependent DNA Double Strand Break Response 
DNA repair  2014;16:11-22.
Developing B and T lymphocytes generate programmed DNA Double Strand Breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the Immunoglobulin Heavy chain (IgH) Class Switch Recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via Classical Non-Homologous End-Joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.
PMCID: PMC4017585  PMID: 24674624
Cernunnos; ATM; DNA-PKcs; NHEJ; V(D)J recombination; 53BP1
5.  Microbial colonization influences early B-lineage development in the gut lamina propria 
Nature  2013;501(7465):10.1038/nature12496.
The RAG1/RAG2 endonuclease ("RAG") initiates the V(D)J recombination reaction that assembles Ig heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B cell antigen-binding receptor ("BCR"). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3–5. This “receptor editing” process, which also can lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen-encounter at the Rag-expressing immature B cell stage helps shape pre-immune BCR repertoires. As the major site of post-natal B cell development, the bone marrow is the principal location of primary Ig repertoire diversification in mice. Here, we report that early B cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP Ig repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B lineage cells that harbor intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B, and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing BM counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B cell development occurs in the intestinal mucosa, where it is regulated by extra-cellular signals from commensal microbes that influence gut Ig repertoires.
PMCID: PMC3807868  PMID: 23965619
6.  Genome-Wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells 
Cell  2011;147(1):107-119.
While chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for Activation Induced-cytidine Deaminase (AID)-dependent IgH class-switching. DSBs translocated very widely across the genome, but were preferentially targeted to transcribed chromosomal regions and also to numerous AID-dependent and AID-independent hotspots, with the latter being comprised mainly of cryptic genomic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short micro-homologies. We discuss implications of our findings for diverse fields including gene therapy and cancer genomics.
PMCID: PMC3186939  PMID: 21962511
7.  Chromosomal translocations are guided by the spatial organization of the genome 
Cell  2012;148(5):908-921.
The extent to which the three dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We now have generated a high resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and mapped translocations from target DNA double strand breaks (DSBs) within it via high throughput genome-wide translocation sequencing. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high frequency DSBs at these loci and their co-localization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing-radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and sub-chromosomal domains in a manner directly related to pre-existing spatial proximity. Our studies reveal the power of combining two high-throughput genomic methods to address long-standing questions in cancer biology.
PMCID: PMC3320767  PMID: 22341456
Translocations; 3D nuclear organization; DNA double-strand breaks; genome stability
8.  Immature B cells preferentially switch to IgE with increased direct Sμ to Sε recombination 
The Journal of Experimental Medicine  2011;208(13):2733-2746.
To be added.
Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cμ (IgM) constant regions (CH) exons with downstream CH exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cμ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cμ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cμ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cμ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.
PMCID: PMC3244039  PMID: 22143888
9.  Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases 
Nature biotechnology  2014;33(2):179-186.
Although great progress has been made in the characterization of off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN-nucleases revealed off-target hotspots for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases by more than 10-fold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.
PMCID: PMC4320661  PMID: 25503383
10.  Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination 
Microbiology spectrum  2015;3(1):10.1128/microbiolspec.MDNA3-0037-2014.
The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination.
PMCID: PMC4481323  PMID: 26104555
11.  ATM Damage Response and XLF Repair Factor are Functionally Redundant In Joining DNA Breaks 
Nature  2010;469(7329):250-254.
Classical non-homologous DNA end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Deficiencies for C-NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for C-NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF) is mutated in certain immunodeficient human patients and has been implicated in C-NHEJ3,4,5,6. Yet, 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 measurably impact these processes7,8,9. Here, we show that XLF, ATM, and H2AX all have fundamental roles in processing and joining ends during V(D)J recombination; but that these roles have been masked by unanticipated functional redundancies. Thus, combined ATM/XLF-deficiency nearly blocks mouse lymphocyte development due inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs C-NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in C-NHEJ are mediated via ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, suggesting 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 indeed has a role in this process.
PMCID: PMC3058373  PMID: 21160472
12.  Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency 
The Journal of Experimental Medicine  2010;207(7):1541-1554.
The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.
PMCID: PMC2901061  PMID: 20547827
13.  ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification 
The Journal of Experimental Medicine  2010;207(7):1369-1380.
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.
PMCID: PMC2901073  PMID: 20566716
14.  Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4 
The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; “MH-mediated” joins) or no homologies (“direct” joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via “alternative” end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.
PMCID: PMC2822597  PMID: 20142431
15.  Mechanisms Promoting Translocations in Editing and Switching Peripheral B Cells 
Nature  2009;460(7252):231-236.
V(D)J recombination assembles immunoglobulin (Ig) heavy or light chain (IgH or IgL) variable region exons in developing bone marrow B cells, while class switch recombination (CSR) exchanges IgH constant region exons in peripheral B cells. Both processes employ DNA double strand breaks (DSBs) repaired by non-homologous end-joining (NHEJ). Errors in either V(D)J recombination or CSR can initiate chromosomal translocations, including oncogenic IgH/c-myc translocations of peripheral B cell lymphomas. Collaboration between these processes also has been proposed to initiate translocations. However, occurrence of V(D)J recombination in peripheral B cells is controversial. Here, we report that activated NHEJ-deficient splenic B cells accumulate V(D)J recombination-associated IgL chromosomal breaks, as well as CSR-associated IgH breaks, often in the same cell. Moreover, IgL breaks frequently are joined to IgH breaks to form translocations, a phenomenon associated with specific IgH/IgL co-localization. IgH and c-myc also co-localize in these cells; correspondingly, introduction of frequent c-myc DSBs robustly promotes IgH/c-myc translocations. Our studies reveal peripheral B cells that attempt secondary V(D)J recombination and elucidate a role for mechanistic factors in promoting recurrent translocations in tumors.
PMCID: PMC2907259  PMID: 19587764
16.  Long-range Oncogenic Activation of IgH/c-myc Translocations by the IgH 3’ Regulatory Region 
Nature  2009;462(7274):803-807.
B cell malignancies, such as human Burkitt’s lymphoma (BL), often harbor translocations that link c-myc or other proto-oncogenes to the immunoglobulin heavy chain locus (IgH)1. The nature of elements that activate oncogenes within such translocations has been a longstanding question. Translocations within IgH involve DNA double strand breaks (DSBs) initiated either by the RAG1/2 endonuclease during V(D)J recombination or by activation induced cytidine deaminase (AID) during class switch recombination (CSR)2-4. V(D)J recombination in progenitor B (pro-B) cells assembles IgH variable region exons upstream of μ constant region (Cμ) exons, which are the first of several sets of CH exons (“CH genes”) within a CH locus that spans several hundred kilobases5,6. In mature B cells, CSR deletes Cμ and replaces it with a downstream CH gene6. An enhancer (iEμ) between the variable region exons and Cμ promotes V(D)J recombination in developing B cells7. In addition, the IgH 3’ regulatory region (IgH3’RR) lies downstream of the CH locus and modulates CSR by long-range transcriptional enhancement of CH genes8-10. Transgenic mice bearing iEμ or IgH3’RR sequences fused to c-myc are predisposed to B lymphomas, demonstrating such elements can confer oncogenic c-myc expression11-16. However, in many B cell lymphomas, IgH/c-myc translocations delete iEμ and place c-myc up to 200kb upstream of the IgH3’RR1. We now address the oncogenic role of the IgH3’RR by inactivating it in two distinct mouse models for B cell lymphoma with IgH/c-myc translocations. The IgH3’RR is dispensable for pro-B lymphomas with V(D)J recombination-initiated translocations, but required for peripheral B cell lymphomas with CSR-associated translocations. As the IgH3’RR is not required for CSR-associated IgH breaks or IgH/c-myc translocations in peripheral B cell lymphoma progenitors, we conclude this regulatory region confers oncogenic activity via long-range and developmental stage-specific activation of translocated c-myc genes.
PMCID: PMC2802177  PMID: 20010689
17.  Vβ Cluster Sequences Reduce the Frequency of Primary Vβ2 and Vβ14 Rearrangements 
European journal of immunology  2008;38(9):2564-2572.
T cell receptor (TCR) β variable region exons are assembled from numerous gene segments in a highly ordered and regulated manner. To elucidate mechanisms and identify cis-acting elements that control Vβ rearrangement, we generated an endogenous TCRβ allele with only the Vβ2, Vβ4, and Vβ14 segments. We found that αβ T lineage cells containing this Vβ2-4-14 allele and a wild-type TCRβ allele developed normally, but exhibited a significant increase in Vβ2+ and Vβ14+ cells. To quantify Vβ rearrangements on the Vβ2-4-14 allele, we generated αβ T cell hybridomas and analyzed TCRβ rearrangements. Despite the deletion of almost all Vβ segments and 234 kb of Vβ cluster sequences, the Vβ2-4-14 allele exhibited only a slight decrease in Vβ rearrangement as compared to the wild-type TCRβ allele. Thus, cis-acting control elements essential for directing Vβ rearrangement across large chromosomal distances are not located within the Vβ cluster. We also found a significant increase in the frequency of Vβ rearrangements involving Vβ2 and Vβ14, but not Vβ4, on the Vβ2-4-14 allele. Collectively, our data suggest that Vβ cluster sequences reduce the frequency of Vβ2 and Vβ14 rearrangements by competing with the productive coupling of accessible Vβ2 and Vβ14 segments with DJβ1 complexes.
PMCID: PMC2736105  PMID: 18792409
V(D)J recombination; T cell receptor beta; gene-targeted mutation
18.  Oncogenic transformation in the absence of Xrcc4 targets peripheral B cells that have undergone editing and switching 
The Journal of Experimental Medicine  2008;205(13):3079-3090.
Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks (DSBs) during V(D)J recombination in developing lymphocytes and during immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in peripheral B lymphocytes. We now show that CD21-cre–mediated deletion of the Xrcc4 NHEJ gene in p53-deficient peripheral B cells leads to recurrent surface Ig-negative B lymphomas (“CXP lymphomas”). Remarkably, CXP lymphomas arise from peripheral B cells that had attempted both receptor editing (secondary V[D]J recombination of Igκ and Igλ light chain genes) and IgH CSR subsequent to Xrcc4 deletion. Correspondingly, CXP tumors frequently harbored a CSR-based reciprocal chromosomal translocation that fused IgH to c-myc, as well as large chromosomal deletions or translocations involving Igκ or Igλ, with the latter fusing Igλ to oncogenes or to IgH. Our findings reveal peripheral B cells that have undergone both editing and CSR and show them to be common progenitors of CXP tumors. Our studies also reveal developmental stage-specific mechanisms of c-myc activation via IgH locus translocations. Thus, Xrcc4/p53-deficient pro–B lymphomas routinely activate c-myc by gene amplification, whereas Xrcc4/p53-deficient peripheral B cell lymphomas routinely ectopically activate a single c-myc copy.
PMCID: PMC2605230  PMID: 19064702
19.  Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching 
The Journal of Experimental Medicine  2008;205(7):1567-1572.
Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.
PMCID: PMC2442634  PMID: 18541713
20.  DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination 
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining CH exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs–deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs−/− B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.
PMCID: PMC2275379  PMID: 18316419
Journal of internal medicine  2008;263(2):128-141.
Ageing, or increased mortality with time, coupled with physiologic decline, is a nearly universal yet poorly understood biological phenomenon. Studies in model organisms suggest that two conserved pathways modulate longevity: DNA damage repair and insulin/Igf1-like signaling. In addition, homologs of yeast Sir2 – the sirtuins – regulate lifespan in diverse organisms. Here, we focus on one particular sirtuin, SIRT6. Mice lacking SIRT6 develop a degenerative disorder that in some respects mimics models of accelerated ageing [1]. We discuss how sirtuins in general and SIRT6 specifically relate to other evolutionarily conserved pathways affecting ageing, and how SIRT6 might function to ensure organismal homeostasis and normal lifespan.
PMCID: PMC2486832  PMID: 18226091
Ageing; DNA Damage; Metabolism
22.  Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation▿ †  
Molecular and Cellular Biology  2007;27(24):8807-8814.
Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.
PMCID: PMC2169418  PMID: 17923681
23.  Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice 
The Journal of Experimental Medicine  2003;198(9):1439-1450.
RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.
PMCID: PMC2194253  PMID: 14581608
antigen receptor; DNA cleavage; RS; hybrid joint; immune deficiency
24.  Defective DNA Repair and Increased Genomic Instability in Artemis-deficient Murine Cells 
In developing lymphocytes, the recombination activating gene endonuclease cleaves DNA between V, D, or J coding and recombination signal (RS) sequences to form hairpin coding and blunt RS ends, which are fused to form coding and RS joins. Nonhomologous end joining (NHEJ) factors repair DNA double strand breaks including those induced during VDJ recombination. Human radiosensitive severe combined immunodeficiency results from lack of Artemis function, an NHEJ factor with in vitro endonuclease/exonuclease activities. We inactivated Artemis in murine embryonic stem (ES) cells by targeted mutation. Artemis deficiency results in impaired VDJ coding, but not RS, end joining. In addition, Artemis-deficient ES cells are sensitive to a radiomimetic drug, but less sensitive to ionizing radiation. VDJ coding joins from Artemis-deficient ES cells, which surprisingly are distinct from the highly deleted joins consistently obtained from DNA-dependent protein kinase catalytic subunit–deficient ES cells, frequently lack deletions and often display large junctional palindromes, consistent with a hairpin coding end opening defect. Strikingly, Artemis-deficient ES cells have increased chromosomal instability including telomeric fusions. Thus, Artemis appears to be required for a subset of NHEJ reactions that require end processing. Moreover, Artemis functions as a genomic caretaker, most notably in prevention of translocations and telomeric fusions. As Artemis deficiency is compatible with human life, Artemis may also suppress genomic instability in humans.
PMCID: PMC2193825  PMID: 12615897
Artemis; DNA repair; genomic instablility; telomere fusions; VDJ recombination
25.  Activated Ras Signals Developmental Progression of Recombinase-activating Gene (RAG)-deficient Pro-B Lymphocytes  
To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras–RAG). Similar to the effects of an immunoglobulin (Ig) μ heavy chain (HC) transgene, activated Ras caused progression of RAG1–deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of λ5, RAG2, and germline κ locus transcripts. However, these Ras–RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig μ HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.
PMCID: PMC1887686  PMID: 9874569
B cell development; pre-B cell receptor; signal transduction; Ras; recombinase-activating gene 2–deficient blastocyst complementation

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