Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice

Dudley, Darryll D.; Sekiguchi, JoAnn; Zhu, Chengming; Sadofsky, Moshe J.; Whitlow, Scott; DeVido, Jeffrey; Monroe, R. J.; Bassing, Craig H.; Alt, Frederick W.

doi:10.1084/jem.20030627

Cited by 70 publications

(74 citation statements)

References 66 publications

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“…We also observed numerous aberrant DNA ends within the rearranging J H locus in developing mutant B lymphocytes. This phenotype is unique to the RAG1-S723C mice as RAG1/2 cleavage products of unpredicted sizes have not been reported in NHEJ deficiencies 26,[58][59][60][61][62] nor in truncated, "core" RAG1 63 or core RAG2 knockin mice. 64 Together, these findings indicate that the RAG1-S723C mutation results in aberrant DNA DSB formation during lymphocyte development.…”

Section: Discussionmentioning

confidence: 99%

Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Giblin

Chatterji

Westfield

et al. 2009

Blood

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The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating doublestrand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence. IntroductionThe immense diversity of genes encoding the variable regions of antigen receptors is generated through rearrangement of component V, D, and J segments via a cut-and-paste mechanism known as V(D)J recombination. 1 These site-specific chromosomal rearrangements are initiated in progenitor lymphocytes by the recombination activating gene 1 (RAG1) and RAG2 proteins that comprise a lymphoid-specific endonuclease, RAG1/2. 2,3 DNA double-strand breaks (DSBs) are generated by RAG1/2 at specific recombination signal sequences (RSSs) adjacent to the numerous rearranging V, D, and J coding exons. 4 The broken ends are then processed and joined by ubiquitously expressed nonhomologous end-joining (NHEJ) DNA repair factors, including Ku70, Ku80, the DNAdependent protein kinase catalytic subunit (DNA-PKcs), Artemis, DNA ligase IV, XRCC4 and Cernunnos/XLF. 1,5,6 The RAG1/2 endonuclease initially binds to RSSs that are composed of a conserved heptamer and nonamer separated by relatively nonconserved spacer sequences of either 12 or 23 nucleotides. 4 Efficient cleavage requires recombination between one 12 RSS and one 23 RSS in the context of a synaptic, paired complex mediated by RAG1/2. RAG1/2 nicks the duplex DNA at the junction between the RSS heptamer and coding sequences then uses the resulting 3Ј OH in a direct transesterification reaction with the phosphodiester backbone of the opposing strand. The products of RAG1/2 cleavage are blunt, 5Ј phosphorylated signal ends and covalently closed, hairpin coding ends. Sequence elements within the nonconserved 12 and 23 spacers 7 and coding ...

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Section: Discussionmentioning

confidence: 99%

Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Giblin

Chatterji

Westfield

et al. 2009

Blood

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“…Cell sorting of DN2 and DN3 thymocytes was performed by using a MoFlo cell sorter (Cytomation, Fort Collins, CO) after staining of CD4-depleted thymocytes (Miltenyi Biotec, Auburn, CA) with FITC-conjugated anti-CD8␣, FITC-conjugated anti-CD4, FITC-conjugated anti-TCR␤, FITC-conjugated anti-B220, FITC-conjugated anti-TCR␥␦, phycoerythrin-conjugated anti-CD25, and CY5-conjugated anti-CD44. D␤1-J␤1.2 and V␤14-J␤1.2 primers pairs and conditions were described (24,25). Amplification conditions were 94°C for 3 min, then 94°C for 45 s, 60°C for 75 s, 72°C for 60 s (25 cycles), and 72°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Restriction of endogenous T cell antigen receptor β rearrangements to Vβ14 through selective recombination signal sequence modifications

Ranganath

Gleason

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In CD4Ϫ /CD8 Ϫ (double-negative, DN) thymocytes, TCR␤ gene segments are assembled in an ordered manner with D␤ to J␤ rearrangements occurring on both alleles before joining of V␤ segments to a preexisting DJ␤ complex (7). In-frame (productive) V␤DJ␤ rearrangements generate TCR␤ chains. At this stage, pairing of TCR␤ chains with the preexisting pT␣ chain to form preTCRs generates signals that lead to expansion of DN cells and their further development into CD4 ϩ / CD8 ϩ (double-positive, DP) thymocytes and the rearrangement of TCR␣ genes (8,9). If the first TCR␤ allele that undergoes V␤ to DJ␤ rearrangement generates a productive TCR␤ chain that pairs with pT␣, V␤ to DJ␤ rearrangement on the second allele is thought to be inhibited by a feedback mechanism that may serve to help enforce TCR␤ allelic exclusion (8, 9). However, out-of-frame V␤DJ␤ rearrangements, or rearrangements that do not lead to a TCR␤ chain that pairs with the preT␣ chain, on the first allele permit V␤ to DJ␤ rearrangements on the second allele that, if productive, generate a TCR␤ chain that pairs with pT␣ to signal differentiation. In DP thymocytes, productive assembly of TCR␣ variable region exons can lead to the generation of TCR␣ chains that, if they pair with TCR␤ chains, form ␣␤ TCRs and signal differentiation to the CD4 ϩ /CD8 Ϫ or CD4 Ϫ /CD8 ϩ (single-positive, SP) thymocyte stage (8, 9). Developmental stage-specific rearrangement of TCR␤ and TCR␣ genes and TCR␤ allelic exclusion are thought to be mediated, at least in part, through modulation of V(D)J recombinational accessibility of participating gene segments to the RAG endonuclease (8, 9). In the TCR␤ locus, V␤s have a 23-RS, J␤s a 12-RS, and D␤s a 5Ј 12-RS and a 3Ј 23-RS. Despite apparent 12/23 compatibility of V␤ 23-RSs and J␤1 12-RSs, the 5ЈD␤1 12-RS, but not the J␤1 12-RSs, can target endogenous V␤ rearrangements (10). This ''beyond 12/23 (B12/23) restriction'' can be recapitulated in transfected plasmid substrates and occurs at the level of RAG cutting of DNA substrates in vitro (11,12). The latter result demonstrates that functional synapses can occur between the RAG proteins and V␤/D␤ RSs but not V␤/J␤ RSs. In contrast,

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“…The core is sufficient to complement RAG2 in recombination of extrachromosomal substrates, although it supports markedly reduced levels of recombination on the IgH locus in cultured cells (18 -20). The mRAG1 core can also support development of both B and T cells in the mouse, but total V(D)J recombination is reduced and aberrant recombination is increased (21,22). Although the majority of the missense mutations in the human RAG1 (hRAG1) gene associated with OS result in amino acid substitutions in the core, at least three have been found in the N-TR (7,23).…”

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Biochemical and Folding Defects in a RAG1 Variant Associated with Omenn Syndrome

Simkus

Anand

Bhattacharyya

et al. 2007

The Journal of Immunology

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The RAG1 and RAG2 proteins are required to assemble mature Ag receptor genes in developing lymphocytes. Hypomorphic mutations in the gene encoding RAG1 are associated with Omenn syndrome, a primary immunodeficiency. We explored the biochemical defects resulting from a mutation identified in an Omenn syndrome patient which generates an amino acid substitution in the RAG1 RING finger/ubiquitin ligase domain (C325Y in murine RAG1) as well as an adjacent substitution (P326G). RAG1 C325Y demonstrated a 50-fold reduction in recombination activity in cultured pro-B cells despite the fact that its expression and localization to the nucleus were similar to the wild-type protein. The C325Y substitution severely abrogated ubiquitin ligase activity of the purified RAG1 RING finger domain, and the tertiary structure of the domain was altered. The P326G substitution also abrogated ubiquitin ligase activity but had a less severe effect on protein folding. RAG1 P326G also demonstrated a recombination impairment that was most pronounced when RAG1 levels were limiting. Thus, a correctly folded RAG1 RING finger domain is required for normal V(D)J recombination, and RAG1 ubiquitin ligase activity can contribute when the protein is present at relatively low levels.

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Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice

Cited by 70 publications

References 66 publications

Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Restriction of endogenous T cell antigen receptor β rearrangements to Vβ14 through selective recombination signal sequence modifications

Biochemical and Folding Defects in a RAG1 Variant Associated with Omenn Syndrome

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