Analysis of mutations and recombination activity in RAG-deficient patients

Mutations in the recombination activating genes (RAG1 or RAG2) can lead to a variety of immunodeficiencies. Herein, we report 5 cases of RAG deficiency from 5 families: 3 of Omenn syndrome, 1 of severe combined immunodeficiency, and 1 of combined immunodeficiency with oligoclonal TCRγδ(+) T cells, autoimmunity and cytomegalovirus infection. The genetic defects were heterogeneous and included 6 novel RAG mutations. All missense mutations except for Met443Ile in RAG2 were located in active core regions of RAG1 or RAG2. V(D)J recombination activity of each mutant was variable, ranging from half of the wild type activity to none, however, a significant decrease in average recombination activity was demonstrated in each patient. The reduced recombination activity of Met443Ile in RAG2 may suggest a crucial role of the non-core region of RAG2 in V(D)J recombination. These findings suggest that functional evaluation together with molecular analysis contributes to our broader understanding of RAG deficiency.     

The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

The enormous repertoire of the vertebrate specific immune system relies on the rearrangement of discrete gene segments into intact antigen receptor genes during the early stages of B-and T-cell development. This V(D)J recombination is initiated by a lymphoid-specific recombinase comprising the RAG1 and RAG2 proteins, which introduces double-strand breaks in the DNA adjacent to the coding segments. Much of the biochemical research into V(D)J recombination has focused on truncated or “core” fragments of RAG1 and RAG2, which lack approximately one third of the amino acids from each. However, genetic analyses of SCID and Omenn syndrome patients indicate that residues outside the cores are essential to normal immune development. This is in agreement with the striking degree of conservation across all vertebrate classes in certain non-core domains. Work from multiple laboratories has shed light on activities resident within these domains, including ubiquitin ligase activity and KPNA1 binding by the RING finger domain of RAG1 and the recognition of specific chromatin modifications as well as phosphoinositide binding by the PHD module of RAG2. In addition, elements outside of the cores are necessary for regulated protein expression and turnover. Here the current state of knowledge is reviewed regarding the non-core regions of RAG1 and RAG2 and how these findings contribute to our broader understanding of recombination.     

Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1

Summary: V(D)J recombination generates functional immunoglobulin and T‐cell receptor genes in developing lymphocytes. The recombination‐activating gene 1 (RAG1) and RAG2 proteins catalyze site‐specific DNA cleavage in this recombination process. Biochemical studies have identified catalytically active regions of each protein, referred to as the core regions. Here, we review our progress in the identification and characterization, in biophysical and biochemical terms, of topologically independent domains within both the non‐core and core regions of RAG1. Previous characterizations of a structural domain identified in the non‐core region of RAG1 from residues 265–380, referred to as the zinc‐binding dimerization domain, are discussed. This domain contains two zinc‐binding motifs, a RING finger and a C₂H₂ zinc finger. Core RAG1 also consists of multiple domains, each of which functions individually in one or more of the essential macromolecular interactions formed by the intact core protein. Two structural domains referred to as the central and the C‐terminal domains that include residues 528–760 and 761–979 of RAG1, respectively, have been identified. The interactions of the central and C‐terminal domains in core RAG1 with the recombination signal sequence (RSS) have contributed additional insight to a developing model for the RAG1–RSS complex.     

The "dispensable" portion of RAG2 is necessary for efficient V-to-DJ rearrangement during B and T cell development

Previous in vitro studies defined the minimal regions of RAG1 and RAG2 essential for V(D)J recombination. In order to characterize the role of the C-terminal "dispensable" portion of RAG2, we generated core-RAG2 knock-in mice. We found that the core-RAG2-containing recombinase complex is selectively defective in catalyzing V-to-DJ rearrangement at the IgH and TCRbeta loci, resulting in partial developmental blocks in B and T lymphopoiesis. Analysis of recombination intermediates showed defects at the cleavage phase of the reaction. We also observed a reduction in overall recombinase activity in core-RAG2-expressing thymocytes, leading us to suggest that the interaction of a defective recombinase with RSS sequences unique to VH and Vbeta gene segments may underlie the specific V-to-DJ rearrangement defect in core-RAG2 mice.     

V(D)J recombination catalyzed by mutant RAG proteins lacking consensus DNA-PK phosphorylation sites

The process of antigen receptor gene rearrangement, V(D)J recombination, involves DNA cleavage by the RAG-1 and RAG-2 proteins. Cleavage generates covalently sealed (hairpin) DNA ends, termed coding ends, which must be opened by an endonuclease prior to joining. Resolution of these hairpin ends requires the activity of the DNA-dependent protein kinase (DNA-PK), a protein kinase whose specific role is yet undetermined. It has been suggested that phosphorylation of one or both RAG proteins by DNA-PK is required to activate or recruit the hairpin-opening nuclease. Furthermore, very recent work has shown that RAG proteins themselves can open hairpins. These data raise the possibility that DNA-PK-mediated phosphorylation of the RAG proteins could regulate the hairpin opening reaction. To test this hypothesis, we constructed mutant versions of RAG-1 and RAG-2 in which all four DNA-PK consensus phosphorylation sites were removed by site-directed mutagenesis. Our data provide conclusive evidence that phosphorylation of these conserved serine/threonine residues is not required for hairpin opening or joining of V(D)J recombination intermediates.     

Recombination-activating gene proteins: more regulation, please

Summary: Developing B and T cells assemble gene segments in order to create the variable regions of immunoglobulin and T‐cell receptors required by our adaptive immune response. The chemistry of this recombination pathway requires a specific nuclease and a more general repair pathway for double‐strand breaks. A complex of the recombination‐activating gene 1 (RAG1) and RAG2 proteins provides the nuclease activity. In fact, RAG1 and RAG2 probably coordinate many steps involving the coding and signaling DNA sequences. Studies using deletion and truncation mutants of the RAG proteins demonstrate that each of these contain a functional core region, representing about two‐thirds of the polypeptides. While the core regions are sufficient to catalyze recombination in test systems, the full‐length proteins seem to show more complicated behaviors in vivo. A plausible explanation is that regions outside the core help in the proper regulation of recombination. The non‐core region of RAG1 has been found to contain a ubiquitin ligase. Regulatory functions may contribute to autoregulation of the proteins involved, fidelity of the reaction, protection of the cell from translocations, coordination of recombination with the cell cycle, and possibly modification of the chromatin structure of target DNA.     

RAG deficiencies: Recent advances in disease pathogenesis and novel therapeutic approaches

The RAG1 and RAG2 proteins initiate the process of V(D)J recombination and therefore play an essential role in adaptive immunity. While null mutations in the RAG genes cause severe combined immune deficiency with lack of T and B cells (T⁻B⁻ SCID) and susceptibility to life-threatening, early-onset infections, studies in humans and mice have demonstrated that hypomorphic RAG mutations are associated with defects of central and peripheral tolerance resulting in immune dysregulation. In this review, we provide an overview of the extended spectrum of RAG deficiencies and their associated clinical and immunological phenotypes in humans. We discuss recent advances in the mechanisms that control RAG expression and function, the effects of perturbed RAG activity on lymphoid development and immune homeostasis, and propose novel approaches to correct this group of disorders.     

A novel missense RAG-1 mutation results in T-B-NK+ SCID in Athabascan-speaking Dine Indians from the Canadian Northwest Territories

DNA double-strand repair factors in the non-homologous end joining (NHEJ) pathway resolve DNA double-strand breaks introduced by the recombination-activating gene (RAG) proteins during V(D)J recombination of T and B lymphocyte receptor genes. Defective NHEJ and subsequent failure of V(D)J recombination leads to severe combined immunodeficiency disease (SCID). We originally linked T(-)B(-)NK(+) SCID in Athabascan-speaking Native Americans in the Southwestern US and Northwest Territories of Canada to chromosome 10. However, despite a common ancestry, the null mutation in the Artemis gene that we found to be causal in the SCID among the Navajo and Apache Indians was not present in the Dine Indians in the Northwest Territories. We now report a novel homozygous missense mutation (R776W) in RAG-1 in three children with T(-)B(-)NK(+) SCID from two related families of Athabascan-speaking Dine Indians in the Canadian Northwest Territories. As expected, we found no increased sensitivity to ionizing radiation in patient fibroblasts. The impaired activity of this RAG-1 mutant in V(D)J recombination was confirmed by the EGFP-based V(D)J recombination assays. Overexpression of wild type RAG-1 in patient fibroblasts complemented V(D)J recombination, with recovery of both coding and signal joint formation. Our results indicate that the novel R776W missense mutation in RAG-1 is causal in the T(-)B(-)NK(+) SCID phenotype in Athabascan-speaking Dine Indians from the Canadian Northwest Territories.     

A direct interaction between the RAG2 C terminus and the core histones is required for efficient V(D)J recombination

V(D)J recombination is a tightly controlled process of somatic recombination whose regulation is mediated in part by chromatin structure. Here, we report that RAG2 binds directly to the core histone proteins. The interaction with histones is observed in developing lymphocytes and within the RAG1/RAG2 recombinase complex in a manner that is dependent on the RAG2 C terminus. Amino acids within the plant homeo domain (PHD)-like domain as well as a conserved acidic stretch of the RAG2 C terminus that is considered to be a linker region are important for this interaction. Point mutations that disrupt the RAG2-histone association inhibit the efficiency of the V(D)J recombination reaction at the endogenous immunoglobulin locus, with the most dramatic effect in the V to DJ(H) rearrangement.     

Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJ recombination in nonlymphoid cells

V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation "beyond the 12/23 rule" (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences.     

The RAG1 N-terminal domain is an E3 ubiquitin ligase

RAG1 and RAG2 initiate V(D)J recombination, which is the assembly of immunoglobulin and T cell receptor genes. The N-terminal region of RAG1 can be deleted, leaving an enzymatic "core" able to catalyze the complete reaction. Here we report that the N-terminal portion of RAG1 has a distinct enzymatic role separate from the rest of the protein. It acts as an E3 ligase in the ubiquitylation of a test substrate and formation of polyubiquitin chains in vitro. This finding suggests a new way in which V(D)J recombination can be regulated and coupled to other aspects of cell physiology.     

Disruption of the RAG2 zinc finger motif impairs protein stability and causes immunodeficiency

Although the RAG2 core domain is the minimal region required for V(D)J recombination, the noncore region also plays important roles in the regulation of recombination, and mutations in this region are often related to severe combined immunodeficiency. A complete understanding of the functions of the RAG2 noncore region and the potential contributions of its individual residues has not yet been achieved. Here, we show that the zinc finger motif within the noncore region of RAG2 is indispensable for maintaining the stability of the RAG2 protein. The zinc finger motif in the noncore region of RAG2 is highly conserved from zebrafish to humans. Knock‐in mice carrying a zinc finger mutation (C478Y) exhibit decreased V(D)J recombination efficiency and serious impairment in T/B‐cell development due to RAG2 instability. Further studies also reveal the importance of the zinc finger motif for RAG2 stability. Moreover, mice harboring a RAG2 noncore region mutation (N474S), which is located near C478 but is not zinc‐binding, exhibit no impairment in either RAG2 stability or T/B‐cell development. Taken together, our findings contribute to defining critical functions of the RAG2 zinc finger motif and provide insights into the relationships between the mutations within this motif and immunodeficiency diseases.     

Evidence of a critical architectural function for the RAG proteins in end processing,protection, and joining in V(D)J recombination

In addition to creating the DNA double strand breaks that initiate V(D)J recombination, the RAG proteins are thought to play a critical role in the joining phase of the reaction. One such role, suggested by in vitro studies, might be to ensure the structural integrity of postcleavage complexes, but the significance of such a function in vivo is unknown. We have identified RAG1 mutants that are proficient in DNA cleavage but defective in their ability to interact with coding ends after cleavage and in the capture of target DNA for transposition. As a result, these mutants exhibit severe defects in hybrid joint formation, hairpin coding end opening, and transposition in vitro, and in V(D)J recombination in vivo. Our results suggest that the RAG proteins have an architectural function in facilitating proper and efficient V(D)J joining, and a protective function in preventing degradation of broken ends prior to joining.     

Detection of RAG mutations and prenatal diagnosis in families presenting with either T-B- severe combined immunodeficiency or Omenn's syndrome

It has been recently shown that mutations in both of the recombination activating genes RAG1 and RAG2 are involved in each of the two different types of severe combined immunodeficiency (SCID) syndromes: T-B- SCID and Omenn's syndrome (OS). The objective of the study was to search for novel mutations in the RAG genes and to offer prenatal diagnosis for families that have been identified as at risk of T-B- SCID or OS. Mutation analyses of polymerase chain reaction products of RAG1/RAG2 genes were performed in 14 cases (T-B- SCID = 6 and OS = 8). Consanguinity was reported in seven (50%) families. Four missense mutations in the RAG2 gene in six of eight OS patients and in four of six T-B- SCID patients were detected. The C1845T transition leading to a Tre215Ile substitution is a novel mutation. All but one of the patients were homozygous for the detected mutations, possibly reflecting the consanguinity in these families and the relative rarity of the disease-causing mutations. In addition, three putative polymorphic sites were found. Prenatal diagnosis was offered to seven families, but three of them declined genetic counseling for religious reasons. In the remaining families, four pregnancies were successfully completed, and in one case, the family chose to have an abortion because of a homozygous mutation. Mutations in RAG1/RAG2 genes were detected in only some of the T-B- SCID or OS patients, and the molecular basis for the remaining cases has yet to be elucidated. Important factors such as religious beliefs need to be considered when offering prenatal diagnosis to certain families.     

Characterization of immune function and analysis of RAG gene mutations in Omenn syndrome and related disorders

Omenn syndrome was recently found to be caused by missense mutations in RAG1 or RAG2 gene that result in partial V(D)J recombination activity. Although the clinical hallmarks of the disease are well defined, there have been several cases with clinical findings similar to, but distinct from Omenn syndrome. The data on immune functions and RAG gene mutations of such cases are limited. We described five Japanese infants from four unrelated families, including two cases of Omenn syndrome and three cases of related disorders. Sibling cases with typical Omenn phenotype were found to be compound heterozygotes of R396C and L885R mutations in RAG1. The former has been reported in European cases and may constitute a hot spot. The latter is a novel missense mutation. Infants with related disorders exhibited erythroderma, eosinophilia, hypogammaglobulinaemia, decreased number of B cells and skewing to Th2, and their lymph node specimens showed architectural effacement, lymphocyte depletion and histiocytic hyperplasia, each of which is seen characteristically in Omenn syndrome. However, in these cases serum IgE levels were low or undetectable. We found no mutation in RAG genes except for a K820R substitution in RAG1, which was regarded to be a functional polymorphism, in two of these cases. Our study suggests that RAG missense mutation may be a genetic abnormality unique to Omenn syndrome with characteristic clinical and laboratory findings. Variations of Omenn syndrome, or related disorders, may represent a different type of immunodeficiency, distinct from abnormalities in lymphoid-specific recombinase activity.     

Homozygous deletion of RAG1, RAG2 and 5′ region TRAF6 causes severe immune suppression and atypical osteopetrosis

Mutations of several genes have been implicated in autosomal recessive osteopetrosis (OP), a disease caused by impaired function and differentiation of osteoclasts. Severe combined immune deficiencies (SCID) can likewise result from different genetic mutations. We report two siblings with SCID and an atypical phenotype of OP. A biallelic microdeletion encompassing the 5′ region of TRAF6, RAG1 and RAG2 genes was identified. TRAF6, a tumor necrosis factor receptor‐associated family member, plays an important role in T cell signaling and in RANKL‐dependent osteoclast differentiation and activation but its role in human OP has not been previously reported. The RAG proteins are essential for recombination of B and T cell receptors, and for the survival and differentiation of these cells. This is the first study to report a homozygous deletion of TRAF6 as a cause of human disease.     

Coding Joint Diversity in Mature and Immature B-Cell Lines

Antigen receptor gene rearrangement is regulated by many factors in B and T lymphocytes. The sequences of the gene segments themselves, their associated recombination signal sequences (RSS), expression of the RAG genes and the chromatin accessibility of the particular gene segments to be rearranged all influence the outcome of recombination and thus antigen receptor diversity. In the present study, we have evaluated the effect of variations in RAG activity level on the junctional diversity of coding joint sequences. Using the pre-B-like 204-1-8 and the mature B DR3 cell lines under different transfection conditions, we were able to investigate recombination activity levels that varied 100-fold. We evaluated the sequences of the coding joints for junctional diversity resulting from nucleotide addition or deletion. Surprisingly, we found that the sequence of coding joints of these recombinants did not exhibit significant variation despite the large difference in recombination frequency. Our results indicate that the fidelity of the joining phase of V(D)J recombination is not jeopardized by varying RAG activity.     

Synergistic requirement of orphan nonamer-like elements and DNA bending enhanced by HMGB1 for RAG-mediated nicking at cryptic 12-RSS but not authentic 12-RSS

V(D)J recombination is initiated by the specific binding of the recombination activating gene (RAG) complex to the heptamer and nonamer elements within recombination signal sequence (RSS). The break points associated with some chromosomal translocations contain cryptic RSSs, and mistargeting of RAG proteins to these less conserved elements could contribute to an aberrant V(D)J recombination. Recently, we found RAG-dependent recombination in the hotspots of TEL-AML1 t(12;21)(p13;q22) chromosomal translocation by an extrachromosomal recombination assay. Here, we describe using in vitro cleavage assays that RAG proteins directly bind to and introduce nicks into TEL and AML1 translocation regions, which contain several heptamer-like sequences. The cryptic nicking site within the TEL fragment was cleaved by RAG proteins essentially depending on a 12-RSS framework, and the nicking activity was enhanced synergistically by both HMGB1 and orphan nonamer-like (NL) sequences, which do not possess counterpart heptamers. In addition, we found that DNA bending stimulated by HMGB1 is indispensable for the HMGB1- and orphan NL element-dependent enhancement of RAG-mediated nicking at the cryptic 12-RSS. Collectively, we would propose the mechanism of HMGB1-dependent enhancement of RAG-mediated nicking at a cryptic RSS through enhanced DNA bending.     

Transposition mediated by RAG1 and RAG2 and the evolution of the adaptive immune system

The RAG1 and RAG2 proteins together initiate V(D)J recombination by performing cleavage of chromosomal DNA adjacent to antigen receptor gene segments. Like the adaptive immune system itself,RAG1 andRAG2 are found only in jawed vertebrates. The hypothesis thatRAG1 andRAG2 arose in evolution as components of a transposable element has received dramatic support from our recent finding that the RAG proteins are a fully functional transposase in vitro. This result strongly suggests that antigen receptor genes acquired their unusual structure as a consequence of the insertion of a transposable element into an ancestral receptor gene by RAG1 and RAG2 approx 450 million years ago.