Mycoplasma superantigen is a CDR3-dependent ligand for the T cell antigen receptor.

Superantigens are defined as proteins that activate a large number of T cells through interaction with the Vbeta region of the T cell antigen receptor (TCR). Here we demonstrate that the superantigen produced by Mycoplasma arthritidis (MAM), unlike six bacterial superantigens tested, interacts not only with the Vbeta region but also with the CDR3 (third complementarity-determining region) of TCR-beta. Although MAM shares typical features with other superantigens, direct interaction with CDR3-beta is a feature of nominal peptide antigens situated in the antigen groove of major histocompatibility complex (MHC) molecules rather than superantigens. During peptide recognition, Vbeta and Valpha domains of the TCR form contacts with MHC and the complex is stabilized by CDR3-peptide interactions. Similarly, recognition of MAM is Vbeta-dependent and is apparently stabilized by direct contacts with the CDR3-beta region. Thus, MAM represents a new type of ligand for TCR, distinct from both conventional peptide antigens and other known superantigens.

Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells.

The products of the Rag1 and Rag2 genes drive genomic V(D)J rearrangements that assemble functional immunoglobulin and T cell antigen receptor genes. Expression of the Rag genes has been thought to be limited to developmentally immature lymphocyte populations that in normal adult animals are primarily restricted to the bone marrow and thymus. Abundant RAG1 and RAG2 protein and messenger RNA was detected in the activated B cells that populate murine splenic and Peyer's patch germinal centers. Germinal center B cells thus share fundamental characteristics of immature lymphocytes, raising the possibility that antigen-dependent secondary V(D)J rearrangements modify the peripheral antibody repertoire.

The functional domains of the murine Thy-1 gene promoter.

The Thy-1 gene promoter resembles a "housekeeping" promoter in that it is located within a methylation-free island, lacks a canonical TATA box, and displays heterogeneity in the 5'-end termini of the mRNA. Using transgenic mice, we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner. It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences.

Transcriptional unit of the murine Thy-1 gene: different distribution of transcription initiation sites in brain.

Structural analysis of the mouse Thy-1.2 gene has shown that the major promoter of the gene is characterized by a tissue-specific DNase I-hypersensitive site and is located within a methylation-free island. The gene is regulated at the transcriptional level, and steady-state mRNA analysis reveals that the previously reported exon Ib contributes at most 5% of the total mRNA. The major promoter uses several transcription initiation sites within a region of 100 base pairs. The frequency of usage of these sites in brain is markedly different from that in other tissues.

A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.

The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered.

The RAG1 homeodomain recruits HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1-RAG2.

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies.

The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed beta-propeller structure, with the second beta-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and DeltaI273 within the predicted second beta-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second beta-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.

Biochemistry of V(D)J recombination.

The genes that encode immunoglobulin and T cell receptor proteins are assembled from component gene segments in a reaction known as V(D)J recombination. The reaction, and its crucial mediators RAG1 and RAG2, are essential for lymphocyte development and hence for adaptive immunity. Here we consider the biochemistry of this reaction, focusing on the DNA transactions and the proteins involved. We discuss how the RAG proteins interact with DNA and how coordinate cleavage of the DNA at two sites might be achieved. Finally, we consider the RAG proteins and V(D)J recombination from an evolutionary point of view.

Cellular and molecular analysis of lymphoid development using Rag-deficient mice.

The establishment of a functional immune system with diverse antigen receptors is dependent on the V(D)J recombination activating gene products Rag-1 and Rag-2. These two proteins constitute the key lymphoid components required for the activation of antigen receptor rearrangement. Both Rag-1 and Rag-2 are required for the catalysis of the initial stages of V(D)J recombination. Thus, functional disruption of either the Rag-1 or Rag-2 genes by homologous recombination, leads to immunodeficiency due to lymphoid arrest at a stage prior to the recombination of the antigen receptor loci. In Rag-deficient mice, both B- and T-cell differentiation is eliminated due to the absence of antigen receptors. Lymphoid development can be restored by the introduction of rearranged antigen receptor transgenes that give rise to monoclonal populations of fully mature B- or T-cells. The absence of the major conventional populations of B- and T-cells from the Rag-deficient mice provided an excellent background for studying the molecular and cellular mechanisms of lymphoid differentiation. The Rag-deficient background has been used as a system for: the functional analysis of Rag-1 and Rag-2; studying the developmental functions of antigen receptors and other molecules of the immune system; the molecular analysis of the early stages of the B- and T-cell lineages; the co-development of lymphocytes with stroma cells; the identification of minor subpopulations of the developing immune system; the involvement of lymphoid populations in the onset of pathogenesis. In addition, the development of the "blastocyst complementation assay" methodology, based on the phenotype of the Rag-/- mice, allowed the functional analysis of numerous lymphoid specific components.

The effect of Me2+ cofactors at the initial stages of V(D)J recombination.

V(D)J site-specific recombination mediates the somatic assembly of the antigen receptor gene segments. This process is initiated by the recombination activating proteins RAG1 and RAG2, which recognize the recombination signal sequences (RSS) and cleave the DNA at the coding/RSS junction. In this study, we show that RAG1 and RAG2 have the ability to directly interact in solution before binding to the DNA. RAG1 forms a homodimer, which leads to the appearance of two distinct RAG1.RAG2 complexes bound to DNA. To investigate the properties of the two RAG1.RAG2 complexes in the presence of different Me2+ cofactors, we established an in vitro Mg2+-based cleavage reaction on a single RSS. Using this system, we found that Mg2+ confers a specific pattern of DNA binding and cleavage. In contrast, Mn2+ allows aberrant binding of RAG1.RAG2 to single-stranded RSS and permits cleavage independent of binding to the nonamer. To determine the contribution of Me2+ ions at the early stages of V(D)J recombination, we analyzed specific DNA recognition and cleavage by RAG1.RAG2 on phosphorothioated substrates. These experiments revealed that Me2+ ions directly coordinate the binding of RAG1.RAG2 to the RSS DNA.

Tissue-specific control elements of the Thy-1 gene.

We have exploited the structural homology, but different patterns of expression of the murine and human Thy-1 genes to map a number of tissue-specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally-regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent.

Immunoglobulin gene hypermutation in germinal centers is independent of the RAG-1 V(D)J recombinase.

Antigen-driven somatic hypermutation in immunoglobulin genes coupled with stringent selection leads to affinity maturation in the B-lymphocyte populations present in germinal centers. To date, no gene(s) has been identified that drives the hypermutation process. The site-specific recombination of antigen-receptor gene segments in T and B lymphocytes is dependent on the expression of two recombination activating genes, RAG-1 and RAG-2. The RAG-1 and RAG-2 proteins are essential for the cleavage of DNA at highly conserved recombination signals to make double-strand breaks and their expression is sufficient to confer V(D)J recombination activity to non-lymphoid cells. Until very recently, expression of the V(D)J recombinase in adults was believed to be restricted to sites of primary lymphogenesis. However, several laboratories have now demonstrated expression of RAG-1 and RAG-2 and active V-to-(D)J recombination in germinal center B cells. This observation of active recombinase in germinal centers raises the issue of RAG-mediated nuclease activity as a component of V(D)J hypermutation. Here, we show that a transgenic kappa-light chain gene in a RAG-1-/- genetic background can acquire high frequencies of mutations. Thus, the RAG-1 protein is not essential for the machinery of immunoglobulin hypermutation. The genetic approaches to identifying the genes necessary for somatic hypermutation will require further studies on DNA-repair and immunodeficient models.

Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex.

During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.

RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination.

Recent studies have demonstrated that DNA cleavage during V(D)J recombination is mediated by the RAG1 and RAG2 proteins. These proteins must therefore bind to the recombination signals, but the specific binding interaction has been difficult to study in vitro. Here, we use an in vivo one-hybrid DNA binding assay to demonstrate that RAG1, in the absence of RAG2, can mediate signal recognition via the nonamer, with the heptamer acting to enhance its binding. A region of RAG1 with sequence similarity to bacterial invertases is essential for DNA binding. Localization of RAG2 to the signal is dependent upon the presence of RAG1 and is substantially more efficient with a 12 bp spacer signal than with a 23 bp spacer signal.

The homeodomain region of Rag-1 reveals the parallel mechanisms of bacterial and V(D)J recombination.

The V(D)J recombinase subunits Rag-1 and Rag-2 mediate assembly of antigen receptor gene segments. We studied the mechanisms of DNA recognition by Rag-1/Rag-2 using surface plasmon resonance. The critical step for signal recognition is binding of Rag-1 to the nonamer. This is achieved by a region of Rag-1 homologous to the DNA-binding domain of the Hin family of bacterial invertases and to homeodomain proteins. Strikingly, the Hin homeodomain can functionally substitute for the Rag-1 homologous region. Rag-1 also interacts with the heptamer but with low affinity. Rag-2 shows no direct binding to DNA. Once the Rag-1/Rag-2 complex is engaged on the DNA, subsequent cleavage is directed by the heptamer sequence. This order of events remarkably parallels mechanisms that mediate transposition in bacteria and nematodes.

TBP binding and the rate of transcription initiation from the human beta-globin gene.

DNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the site of initiation (-4 to +7). Point mutants which specifically abolish the binding of each of these proteins were tested in a beta-globin locus control region (LCR) construct which allows quantitative comparisons at physiological levels of transcription. Only mutants which drastically affect the binding of TBP resulted in decreased levels of transcription. A threshold value of TBP binding of 15-30% of wild type was sufficient to give normal levels of transcription. This indicates that the association of TF IID with the TATA box is not limiting in the rate of initiation of transcription.

A novel adhesion molecule in the murine thymic microenvironment: functional and biochemical analysis.

The rat monoclonal antibody (mAb) 4F1, raised against mouse thymic stromal cells, recognizes cortical epithelium in tissue sections of mouse thymus; however, in flow cytometry, activated leucocytes (T cells, B cells, and macrophages) and transformed thymocytes are also positive for the 4F1-antigen (4F1-Ag). Western blotting, under both reducing and nonreducing conditions, demonstrates that the molecule to which 4F1 binds is expressed in four forms, 29, 32, 40, and 43 kD, all of which carry N-linked carbohydrate; and that the structure is identical on epithelium and lymphocytes. The 4F1-Ag on cortical epithelium is partially sensitive to PI-PLC treatment, whereas on transformed epithelial and lymphoid cell lines, it was resistant to this enzyme. The molecule, therefore, may exist in both transmembrane and phosphoinositol-linked forms. In functional blocking experiments, mAb 4F1 gave inhibition of both T-cell proliferation in MLR and of cytotoxic T-cell killing of alloantigenic targets; it also blocked adhesion of transformed thymocytes to thymic epithelial cells in vitro. These molecular and functional characteristics suggest that the 4F1-Ag is a novel adhesion molecule that may be involved both in intrathymic T lymphocyte differentiation and in peripheral T-cell function.

Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination.

As a probe of whether RAG-1 and RAG-2 gene products activate other genes or form part of the recombinase itself, certain mutants of the RAG genes were assayed for their ability to activate variable-diversity-joining region [V(D)J] recombination in a plasmid substrate in fibroblasts. The results indicate that the N-terminal one-third of RAG-1, including a zinc-finger-like domain, and an acidic domain of RAG-2 are dispensable for activating V(D)J recombination in a fibroblast, although they contribute quantitatively. In contrast, deletion of the C-terminal segment of RAG-1, which has homology to a topoisomerase-like protein from yeast, abolished recombination activation. These results do not support the hypothesis that the RAG gene products are transcription factors and suggest the possibility that they are parts of the recombination machinery.

Differential regulation of a Thy-1 gene in transgenic mice.

We have generated Thy-1.1-transgenic Thy-1.2 mice to study the developmental expression of the Thy-1 gene in detail by transcriptional and immunological methods. In brain, the expression of the injected gene was identical to that of the endogenous gene in a tissue- and development-specific manner. In lymphoid tissue, the transferred gene was also expressed correctly in the early phases of T-cell lineage development; however, as the T cells matured, the transcription of the transferred gene, but not the endogenous gene, was suppressed. This result shows that different regulatory elements are used to express the Thy-1 gene in early and late lymphoid development.