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.     

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.     

Making V(D)J rearrangement visible: quantification of recombination efficiency in real time at the single cell level

V(D)J recombination is of fundamental importance for the diversity of immunoglobulin and T cell receptor genes. An enhanced green fluorescent protein (EGFP) based assay was successfully developed to monitor V(D)J recombination efficiency. This assay makes V(D)J recombination visible at the single cell level in real time. Surprisingly, despite a high (60% to 90%) transfection efficiency, the EGFP based V(D)J recombination efficiency was found to be low ( approximately 1%) in 293 cells. The EGFP based V(D)J recombination efficiency correlated well with that achieved by the classical V(D)J recombination assay. The EGFP based V(D)J recombination efficiency depended on the relative RAG (recombination activating gene)-1 and RAG-2 but not Artemis expression vector concentrations used for co-transfection. A rise of RAG-1 dosage increased recombination efficiency. In contrast, a surplus of RAG-2 inhibited V(D)J recombination efficiency. The test differentiates RAG null mutants as seen in human severe combined immunodeficiency (SCID).     

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 bounty of RAGs: recombination signal complexes and reaction outcomes

Summary: V(D)J recombination is a form of site‐specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell‐specific proteins, recombination activating genes RAG‐1 and RAG‐2, and ubiquitously expressed architectural DNA‐binding proteins and DNA‐repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein‐DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence‐specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein‐DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post‐cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein‐DNA complexes that support them, and factors and forces that control them.     

A plant homeodomain in RAG-2 that binds Hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement

V(D)J recombination is initiated by the recombination activating gene (RAG) proteins RAG-1 and RAG-2. The ability of antigen-receptor-gene segments to undergo V(D)J recombination is correlated with spatially- and temporally-restricted chromatin modifications. We have found that RAG-2 bound specifically to histone H3 and that this binding was absolutely dependent on dimethylation or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction required a noncanonical plant homeodomain (PHD) that had previously been described within the noncore region of RAG-2. Binding of the RAG-2 PHD finger to chromatin across the IgH D-J(H)-C locus showed a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the RAG-2 PHD finger abolished binding to H3K4me3 and greatly impaired recombination of extrachromosomal and endogenous immunoglobulin gene segments. Together, these findings are consistent with the interpretation that recognition of hypermethylated histone H3 K4 promotes efficient V(D)J recombination in vivo.     

A short peptide at the C terminus is responsible for the nuclear localization of RAG2

The RAG1 and RAG2 proteins are the lymphoid-specific factors essential for V(D)J recombination, the process that leads to the diversification of antigen receptors on B and T lymphocytes. Nucleolar/nuclear localization of RAG1 is mediated by four basic domains, which are the binding sites for the nuclear transport proteins SRP1 and RCH1, and by a nuclear localization signal (NLS) in the fifth basic domain. The C-terminal region of RAG2 from amino acids (aa) 417 to 484 shows a homology with the PHD domain of other proteins involved in chromatin-mediated gene regulation by protein-protein interactions. Mutations in this domain were shown to be responsible for several diseases and in some case lead to altered subcellular localization of proteins. We found that the C-terminal PHD domain of RAG2 is not responsible for the nuclear localization of the protein. We report here the characterization of a region (aa 491-527) in the C-terminal domain of RAG2, downstream of the putative PHD domain, which directs the nuclear localization of the protein.     

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.     

Comparative analysis of invertebrate Tc6 sequences that resemble the vertebrate V(D)J recombination signal sequences (RSS).

Invertebrate cells lack the p53 recombination checkpoint but contain mobile DNA sequences that transpose by a mechanism in part shared with excision of the V(D)J recombination signal sequences (RSS). In this work, inversion, deletion, and duplication of sequences associated with an invertebrate C. elegans Tc6 element is described. The structure of this C. elegans sequence and other dispersed Tc6 elements suggests that covalently closed 'hairpin' structures are not unique to excision of the V(D)J RSS by the RAG proteins, but rather can be generated by transposases at transposon termini leading to characteristic inversion and duplication events. Comparative analysis of recombination events at invertebrate sequences resembling the vertebrate V(D)J RSS may be useful in understanding V(D)J recombination-mediated recombination events in malignant vertebrate cells or genetic diseases such as ataxia telangectasia, in which the p53 recombination checkpoint is defective.     

RAG1 and RAG2 in V(D)J recombination and transposition

RAG1 and RAG2 are the key components of the V(D)J recombinase machinery that catalyses the somatic gene rearrangements of antigen receptor genes during lymphocyte development. In the first step of V(D)J recombination--DNA cleavage--the RAG proteins act together as an endonuclease to excise the DNA between two individual gene segments. They are also thought to be involved in the subsequent DNA joining step. In vitro, the RAG proteins catalyze the integration of the excised DNA element into target DNA completing a process similar to bacterial transposition. In vivo, this reaction is suppressed by an unknown mechanism. The individual roles of RAG1 and RAG2 in V(D)J recombination and transposition reactions are discussed based on mutation analyses and structure predictions.     

Regions of RAG1 protein critical for V(D)J recombination

The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutations for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conserved sequence. We show here that this “dispensable” region of RAG1 is not necessary for coding joint formation or for recombination of an integrated substrate, and that this portion is not functionally redundant with the “dispensable” region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.     

New concepts in the regulation of an ancient reaction: transposition by RAG1/RAG2

Summary: The lymphoid‐specific factors, recombination‐activating gene 1 (RAG1) and RAG2, initiate V(D)J recombination by introducing DNA double‐stand breaks at specific sites in the genome. In addition to this critical endonuclease activity, the RAG proteins catalyze other chemical reactions that can affect the outcome of V(D)J recombination, one of which is transposition. While the transposition activity of the RAG proteins is thought to have been critical for the evolution of modern antigen‐receptor loci, it has also been proposed to contribute to chromosomal translocations and lymphoid malignancy. A major challenge has been to determine how the transposition activity of the RAG proteins is regulated in vivo. Although a variety of mechanisms have been suggested by recent studies, a clear resolution of this issue remains elusive.     

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.     

Role of recombination activating genes in the generation of antigen receptor diversity and beyond

V(D)J recombination is the process by which antibody and T‐cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non‐homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence‐specific nuclease and its pathological role as a structure‐specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence‐specific and structure‐specific nuclease. It also deals with the off‐target cleavage of RAGs and its implications in genomic instability.     

Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo

To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.     

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.