RAG Genes Research Articles

Continued expression of recombination-activating genes and TCR gene recombination in human peripheral T cells.

It has been reported that a small population of peripheral T lymphocytes are capable of expressing V(D)J recombinase and initiating secondary V(D)J rearrangements. To determine whether RAG-expression and secondary TCR gene recombination in peripheral T cells are an antigen-driven process in secondary lymphoid tissues, we examined naive CD4(+) T cells, activated/memory CD4(+) T cells, and germinal center T cells from human tonsils. Our results showed that low levels of RAG-1 and RAG-2 mRNA were present in all T cell subpopulations except CD3(+)/CD4(-) T cells. LM-PCR analyses for double-strand DNA breaks showed that all the T cell subsets expressing RAG genes contain double-strand signal break ends, indicating ongoing TCR gene recombination. Continued RAG gene expression, introducing and repairing of double-strand DNA breaks at the TCR loci in the periphery may have significant implications in the development of some T cell neoplasms.

Fusion gene transcripts and Ig/TCR gene rearrangements are complementary but infrequent targets for PCR-based detection of minimal residual disease in acute myeloid leukemia.

PCR-based monitoring of minimal residual disease (MRD) in acute leukemias can be achieved via detection of fusion gene transcripts of chromosome aberrations or detection of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements. We wished to assess whether both PCR targets are complementary in acute myeloid leukemia (AML). We investigated 105 consecutive AML cases for the presence of fusion gene transcripts by reverse transcriptase polymerase chain reaction (RT-PCR): AML1-ETO associated with t(8;21), CBFB-MYH11 with inv(16), PML-RARA with t(15;17), BCR-ABL with t(9;22), and MLL-AF4 with t(4;11). In 17 out of 105 AML cases (16%), fusion gene transcripts were found. Ninety-five of these AML patients (13 with fusion gene transcripts) were also investigated for the presence of IGH, IGK, TCRG and TCRD rearrangements by Southern blot and/or PCR heteroduplex analysis and sequencing. In nine out of 95 patients (9.5%), such rearrangements were found. Combined data revealed that only one patient with a fusion gene transcript had a coexistent Ig/TCR rearrangement. The nine AML patients with Ig/TCR rearrangements, as well as five additional AML patients from a previous study were investigated in more detail, revealing that Ig/TCR rearrangements in AML are immature and unusual. The presence of Ig/TCR rearrangements in AML did not correlate with RAG gene expression levels as determined by real-time quantitative PCR. In conclusion, fusion gene transcripts and Ig/TCR rearrangements are infrequent, but complementary MRD-PCR targets in AML.

Early death and severe lymphopenia caused by ubiquitous expression of the Rag1 and Rag2 genes in mice.

The recombination activating proteins (RAG1 and RAG2) are essential for V(D)J recombination of immunoglobulin chains. Expression of both genes is lymphocyte-specific and RAG levels are tightly regulated throughout lymphopoiesis and cell cycle. To assess the significance of this pattern of expression, we generated transgenic mice expressing the Rag genes both continuously throughout lymphocyte development and constitutively in most non-lymphoid tissues. The transgenes partially complement an endogenous Rag2 null mutation and lead to a partial block in early B and T lymphopoiesis when introduced on a Rag2 sufficient background. The defect in thymocyte number is restricted to the alpha beta lineage leaving the gamma delta T cell pool intact, while neither IgH phenotypic allelic exclusion nor the kappa/lambda light chain ratio are altered. Finally, the ectopic expression of the Rag genes associates with growth retardation and early death of the animals, a phenotype reminiscent of those reported for mice deficient in double-strand break repair molecules.

Expression of the recombination activating genes (RAG1 and RAG2) is not detectable in Epstein-Barr virus-associated human lymphomas

The expression of the recombination activating genes (RAG1 and RAG2) is largely restricted to immature lymphoid cells. Previous studies have suggested that Epstein-Barr virus (EBV) infection may lead to a re-induction of RAG expression in mature B lymphocytes. To assess the significance of this mechanism for the pathogenesis of malignant lymphomas, we have examined the expression of RAG genes in 11 cases of EBV-associated endemic Burkitt's lymphoma (BL), 25 cases of Hodgkin's disease (HD, 17 EBV(+), 8 EBV(-)) and 10 cases of follicular non-Hodgkin's lymphoma (NHL). Using in situ hybridization, expression of the RAGs was detected in cortical thymocytes in normal thymus and in the tumor cells of 2 of 3 lymphoblastic NHL. By contrast, there was no detectable RAG expression in the BL, HD and follicular NHL cases. Our results indicate that re-induction of RAG expression does not occur in human lymphomas in vivo. Thus, it is unlikely to play a role in the development of translocations involving immunoglobulin gene loci which are characteristically found in BL and follicular NHL. Moreover, our study shows that in situ hybridization is a suitable method for the analysis of RAG expression in human tissue sections.

Recombinase activating gene enzymes of lymphocytes.

Expression of T-cell receptor and surface immunoglobulins on T and B lymphocytes, respectively, is strictly dependent on the variable, (diversity) joining exon (V(D)J) recombination process, which is initiated by the lymphoid-specific recombinase activating gene proteins 1 and 2 (RAG1 and RAG2). Recent advances have highlighted the functional organization of the RAG1 and RAG2 proteins and have provided important information on the regulation of RAG gene expression. Depending on the severity of their effects on the V(D)J recombination process, mutations of the RAG genes account for a spectrum of combined immune deficiencies in humans.

Antigen-independent appearance of recombination activating gene (RAG)-positive bone marrow B cells in the spleens of immunized mice.

Splenic B lineage cells expressing recombination activation genes (RAG(+)) in mice immunized with 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken gamma-globulin (NP-CGG) and the adjuvant aluminum-hydroxide (alum) have been proposed to be mature B cells that reexpress RAG after an antigen encounter in the germinal center (GC), a notion supported by findings of RAG expression in peripheral B lymphocyte populations activated in vitro. However, recent studies indicate that these cells might be immature B cells that have not yet extinguished RAG expression. Here, we employ RAG2-green fluorescent protein (GFP) fusion gene knock-in mice to show that RAG(+) B lineage cells do appear in the spleen after the administration of alum alone, and that their appearance is independent of T cell interactions via the CD40 pathway. Moreover, splenic RAG(+) B lineage cells were detectable in immunized RAG2-deficient mice adoptively transferred with bone marrow (BM) cells, but not with spleen cells from RAG(+) mice. Although splenic RAG(+) B cells express surface markers associated with GC B cells, we also find the same basic markers on progenitor/precursor BM B cells. Finally, we did not detect RAG gene expression after the in vitro stimulation of splenic RAG(-) mature B cells with mitogens (lipopolysaccharide and anti-CD40) and cytokines (interleukin [IL]-4 and IL-7). Together, our studies indicate that RAG(+) B lineage cells from BM accumulate in the spleen after immunization, and that this accumulation is not the result of an antigen-specific response.

Cutting edge: recombinase-activating gene expression and V(D)J recombination in CD4+CD3low mature T lymphocytes.

The recombinase-activating genes, RAG-1 and RAG-2, can be expressed by a subset of B cells within germinal centers, where they mediate secondary V(D)J rearrangements. This receptor revision mechanism could serve either receptor diversification or tolerance-induced functions. Alternatively, it might rescue those cells the receptors of which have been damaged by somatic mutation. Less is known about the occurrence of similar mechanisms in T cells. Here we show that mature T cells with defective TCR surface expression can express RAG genes and are capable of initiating secondary V(D)J rearrangements. The possibility that a cell rescue mechanism based on the generation of a novel Ag receptor might be active in peripheral T cells is envisaged.

Stimulation of V(D)J recombination by histone acetylation.

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.

SEVERE COMBINED IMMUNODEFICIENCY CAUSED BY DEFECTS IN COMMON CYTOKINE RECEPTOR γc SIGNALING PATHWAYS

Severe combined immunodeficiency (SCID) syndromes are a group of congenital disorders characterized by severe impairment of cellular and humoral immunity, leading to death at an early age. Human SCID comprises genotypically and phenotypically heterogeneous conditions of which the genetic basis for many of the underlying immunologic defects have been recently elucidated. All SCID phenotypes are defined by the absence of mature T cells. Other characteristics, including the presence or absence of B cells and natural killer (NK) cells and the particular genes involved, help to distinguish the various subtypes of these disorders (Fig. 1). One of the principal difficulties inherent in deciphering the pathogenesis of SCID syndromes is that different mutations in a single gene may give rise to distinct clinical conditions and that a similar clinical phenotype can result from mutations in different genes (Figs. 1 and 2). Complete deficiencies in the recombinase activating genes RAG1 and RAG2 block the generation of mature T and B lymphocytes by preventing antigen receptor gene rearrangements, 70 whereas NK cell development proceeds normally (T − NK + B − SCID). Deficiencies in RAG may not be the only cause of this SCID subtype: defects in other genes involved in the recombination process, such as Ku 70 and 86 proteins and DNA-dependent protein kinases, 4 could result in T − NK + B − SCID. Not all defects in the RAG genes generate typical T − NK + B − SCID. Mutations associated with partial RAG function recently were described in Omenn's syndrome, an immunodeficiency characterized by oligoclonal T- and B-cell development. 86 Defining the genetic basis of SCID syndromes remains a challenge. One prominent clinical immunologist described SCID syndromes as experiments of nature: a fitting characterization in that through these rare conditions, scientists and clinicians often can extract information regarding the essential roles of various gene products in the differentiation of the human lymphoid system. One of the most common subtypes of SCID is characterized by the selective blockade of T- and usually NK-cell differentiation, with the presence of poorly functioning B cells (T − NK − B + SCID). This immunologic phenotype is caused by deficiencies in signaling through a group of cytokine receptors that share the common cytokine receptor γ chain (γ c ). Like all SCIDs, disorders characterized by perturbations in the function of γ c -dependent cytokines can give rise to considerable heterogeneity with respect to the immunologic presentation and the molecular basis for the underlying disease pathology. This article summarizes the clinical considerations for T − NK − B + SCID and its atypical variants, the role of the γ c cytokine network in normal lymphopoiesis and in the pathophysiology of these diverse human SCID syndromes, and analyzes γ c mutations in typical and atypical SCID syndromes.

RAG MUTATIONS IN SEVERE COMBINED IMMUNODEFICIENCY AND OMENN'S SYNDROME

RAG gene mutations determine different forms of primary immunodeficiencies. A complete lack of RAG function leads to a block of B- and T-lymphocyte development resulting in a B~T~ SCID. On the other hand, hypomorphic RAG alleles allow T-cell but not B-cell differentiation, which is observed in patients with Omenn’s syndrome. These observations confirm that an undisturbed generation of T- and B-cell repertoires is required for effective immune responses, and that B-cell development tends to rely more strictly on an intact V(D)J recombination machinery. The observation that several of the OS clinical features resemble autoimmune manifestations raises the question whether RAG mutations may contribute to human autoimmune diseases. Presently, RAG defects can be confirmed reliably at the genetic level. The knowledge of RAG mutations in diseased children helps to facilitate prenatal and family diagnoses. RAG deficiencies are treated by provision of allogeneic hematopoietic stem cells. At present, gene therapy is not a therapeutic option, but it might become an attractive alternative in the future.

Recombination-activating genes, transposition, and the lymphoid-specific combinatorial immune system: a common evolutionary connection.

The hallmarks that distinguish the gnathostome (jawed vertebrates) adaptive immune system from the immune system of agnathans (jawless fish), which both diverged from a common ancestor over 500 million years ago (MYA), include the presence of somatically rearranging antigen receptors (immunoglobulins, Ig; T cell receptors, TCR; new antigen receptor, NAR) via recombination activating molecules (Ragl and 2) and specific antigen-presentation molecules (MHC class I and II: Du Pasquier and Flajnik 1999). The process of somatic V(D)J recombination of antigen receptor gene segments, mediated by the Rag gene products, is the most significant characteristic of adaptive immunity in the jawed vertebrates. During this process a complex of Rag1 and Rag2 proteins initiates recombination by introducing DNA double strand breaks (DSBs) at evolutionarily conserved recombination signal sequences (RSS). These RSS flank the Ig and TCR (and NAR) germline variable region (VDJ) segments which are found in all gnathostomes (Du Pasquier and Flajnik 1999; Schatz et al. 1992). Expression of the Rag proteins is regulated at both the transcriptional and post-transcriptional levels, and co-expression of Rag1 and Rag2 is limited to lymphoid committed cells. In order to guarantee that the V(D)J recombinase acts only within the B and T lymphocyte lineage, the reaction is tightly regulated at multiple stages, including expression of the Rag genes themselves as well as controlled access of the recombination machinery to its substrates within chromatin.

Two genes encoding a putative multidrug efflux pump of the RND/MFP family are cotranscribed with an rpoH gene in Bradyrhizobium japonicum.

The rpoH3 gene of Bradyrhizobium japonicum codes for one of three sigma32-type transcription factors in this organism and is flanked by rag (rpoH3-associated) genes comprising the chromosomal arrangement ragABrpoH3ragCD. The first genes in this cluster code for a classical two-component regulatory system with an unknown function (Narberhaus et al., 1997. Mol. Microbiol. 24, 93-104). The deduced proteins of the last two genes display a high sequence similarity to heavy metal or multidrug efflux pumps of the RND (Resistance/Nodulation/cell Division)-MFP (Membrane Fusion Protein) family. Reverse transcription and polymerase chain reaction (RT-PCR) analysis demonstrated that ragC is cotranscribed with rpoH3. Mutant strains carrying disrupted rag genes or an extented deletion of the rag locus exhibited neither an apparent growth defect nor a deficiency in the symbiotic interaction with soybean roots. The minimal inhibitory concentrations (MICs) of various metals and organic compounds were identical for the wild-type and mutant strains. Moreover, translational lacZ fusions to each of the first four genes of the rag cluster showed a very low expression under all conditions tested; hence, the substrate for the putative efflux pump has not been uncovered.

Intrathymic Restriction and Peripheral Expansion of the T-Cell Repertoire in Omenn Syndrome

Mutations in the human RAG genes that impair, but do not abolish, recombination activity lead to Omenn syndrome, a severe primary immune deficiency that is associated with clinical and pathological features of graft-versus-host disease and oligoclonal expansion of activated, autologous T cells. We have analyzed the mechanisms accounting for peripheral oligoclonality of the T-cell repertoire. Predominance of few T-cell receptor clonotypes (both within TCRAB- and within TCRGD-expressing lymphocytes) is already detectable in the thymus and is further selected for in the periphery, with a different distribution of clonotypes in different tissues. These data indicate that oligoclonality of the T-cell repertoire in Omenn syndrome is due both to intrathymic restriction and to peripheral expansion. Moreover, the RAG genes defect that causes Omenn syndrome directly affects early stages of V(D)J recombination, but does not alter the process of double-strand-break DNA repair, including N and P nucleotide insertion.

The rag locus of Porphyromonas gingivalis: a novel pathogenicity island.

Previous studies in our laboratories of the serum IgG antibody response of periodontal patients have demonstrated the presence of an immunodominant surface antigen (Mr 55 kDa) in the outer membrane of Porphyromonas gingivalis W50. Genetic analysis of this antigen revealed that the corresponding gene forms part of a small operon which may have arisen via horizontal gene transfer into the genome of this strain. On the basis of sequence homology, the 55 kDa antigen (RagB) and the product of a cotranscribed gene (RagA) may act in concert at the surface of the bacterium to facilitate active transport, mediated through the periplasmic spanning protein, TonB, or form part of a signal transduction system in this organism. The rag locus is present in only a proportion of P. gingivalis laboratory strains and clinical isolates. Analysis of the distribution of ragB in subgingival samples by PCR demonstrated that rag+ P. gingivalis are more frequently detected in deep periodontal pockets than shallow sites in periodontal patients. These findings indicate that the rag genes may influence the virulence potential of P. gingivalis strains which harbour this locus and may thus be considered a novel pathogenicity island. Furthermore, horizontal gene transfer between organisms in subgingival plaque may represent a significant force in the evolution of these bacteria with ramifications for both diagnosis and targeted treatment of periodontal disease.

Unraveling the Regulation of RAG Genes

Unraveling the Regulation of RAG Genes

IL-7 Reconstitutes Multiple Aspects of v-Abl-Mediated Signaling

The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells.

Maturation of B Cell Precursors Is Impaired in Thymic-Deprived Nude and Old Mice

We have previously reported that bone marrow B cell precursors from thymic-deprived nude and old mice express less recombination-activating gene-1 (RAG-1) mRNA than they do in young euthymic mice. We now report that both nude and old mice have decreased bone marrow pre-B cells and that fewer pre-B cells express RAG protein. This combination of events appears to be the basis for the lower level of bone marrow RAG mRNA in thymic-deprived mice. A link between thymic function and B cell development was suggested by the similar kinetics of thymic involution and of declining bone marrow RAG-1 gene expression during aging. Support for this hypothesis was obtained by demonstrating that injection of supernatant medium from activated CD8+ but not CD4+ young T cells from mice increases RAG mRNA, RAG protein, and the number of bone marrow pre-B cells in nude and old mice. Furthermore, in vivo CD8+ T cells also regulate bone marrow RAG gene expression. Thus, mice deficient in CD8+ T cells expressed levels of RAG-1 mRNA in their bone marrow that were only 10% of those observed in normal or CD4+ T cell-deficient mice. IL-16 was detected in the supernatant medium from activated T cell cultures, and injection of nanogram quantities of recombinant IL-16 (rIL-16) into nude or old mice increased the levels of RAG mRNA in bone marrow B cell precursors and the number of bone marrow pre-B cells. We conclude that the impaired development of B cells within the bone marrow of thymic-deprived nude and old mice can be reversed, at least in part, by the administration of rIL-16.

Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions.

Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.

Lack of expression of the recombination activating genes RAG-1 and RAG-2 in cutaneous T-cell lymphoma: pathogenic implications

Analyses of the karyotype and genome of cutaneous T-cell lymphoma (CTCL) cells have shown that they contain chromosome breaks and translocations. The sequence analyses of such DNA breakpoints found in various kinds of leukaemias have suggested that some of the observed translocations have been caused by illegitimate V(D)J (v = Variability; D = diversity; J = joining) recombination. To study whether illegitimate V(D)J recombination is responsible for the continuously increasing number of DNA breaks in CTCL, we used reverse transcriptase polymerase chain reaction (RT-PCR) to analyse three CTCL cell lines and biopsies from 14 CTCL patients for the expression of the RAG-1 and RAG-2 genes which are essential for V(D)J recombination. We found no RAG gene expression in any of the 17 samples analysed, indicating that illegitimate V(D)J recombination may not be the reason for the increased number of chromosomal aberrations and translocations in CTCL cells.

Rabbit RAG-2 UTRs: characterization of an unusually large 3' untranslated region.

We previously characterized the rabbit recombination activating gene-2 (RAG-2) coding region and a portion of the cDNA. Rabbit RAG-2 mRNA, however, was shown to be approximately twice as large as the predominant form expressed in other vertebrate species, suggesting that it contained additional coding and/or untranslated regions (UTR). In this report, we map and sequence the complete 5' and 3' UTRs of the rabbit RAG-2 transcript and identify and sequence the genomic regions from which they are transcribed. The data show that, with the exception of a 300 nucleotide 5' UTR, almost all of the additional sequence belongs to the 3' UTR and that the 3' UTR sequence is transcribed from a single large exon that encodes most of the coding region and all of the 3' UTR. The 3' UTR contains four poly A signal sites, the last of which is closely followed by a GU-rich region. The rabbit 3' UTR has a high level of identity with the homologous region downstream of the human RAG-2 gene but not with the mouse RAG-2 gene. The region of identity extends several hundred nucleotides beyond the transcribed region and terminates in a series of dinucleotide (TG) repeats. The data are discussed in terms of RAG gene and 3' UTR function, regulation, and evolution.