RAG Genes Research Articles

The Function of E2A in B-Cell Development.

Helix-loop-helix (HLH) transcription factors (TFs) play a key role in various cellular differentiation and function through the regulation of enhancer activity. E2A, a member of the mammalian E-protein family (class I HLH protein), is well known to play an important role in hematopoiesis, especially in adaptive lymphocyte development. E2A instructs B- and T-cell lineage development through the regulation of enhancer activity for B- or T-cell signature gene expression, including Rag1 and Rag2 (Rag1/2) genes. In this chapter, we mainly focus on the function of E2A in B-cell development and on the roles of E2A in establishing the enhancer landscape through the recruitment of EP300/KAT3B, chromatin remodeling complex, mediator, cohesion, and TET proteins. Finally, we demonstrate how E2A orchestrates the assembly of the Rag1/2 gene super-enhancer (SE) formation by changing the chromatin conformation across the Rag gene locus.

Spleen tyrosine kinase mediates the γδTCR signaling required for γδT cell commitment and γδT17 differentiation.

The γδT cells that produce IL-17 (γδT17 cells) play a key role in various pathophysiologic processes in host defense and homeostasis. The development of γδT cells in the thymus requires γδT cell receptor (γδTCR) signaling mediated by the spleen tyrosine kinase (Syk) family proteins, Syk and Zap70. Here, we show a critical role of Syk in the early phase of γδT cell development using mice deficient for Syk specifically in lymphoid lineage cells (Syk-conditional knockout (cKO) mice). The development of γδT cells in the Syk-cKO mice was arrested at the precursor stage where the expression of Rag genes and αβT-lineage-associated genes were retained, indicating that Syk is required for γδT-cell lineage commitment. Loss of Syk in γδT cells weakened TCR signal-induced phosphorylation of Erk and Akt, which is mandatory for the thymic development of γδT17 cells. Syk-cKO mice exhibited a loss of γδT17 cells in the thymus as well as throughout the body, and thereby are protected from γδT17-dependent psoriasis-like skin inflammation. Collectively, our results indicate that Syk is a key player in the lineage commitment of γδT cells and the priming of γδT17 cell differentiation.

A Rag GTPase dimer code defines the regulation of mTORC1 by amino acids

Amino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.

Interaction between Rag genes results in a unique synergistic transcriptional response that enhances soybean resistance to soybean aphids

BackgroundPyramiding different resistance genes into one plant genotype confers enhanced resistance at the phenotypic level, but the molecular mechanisms underlying this effect are not well-understood. In soybean, aphid resistance is conferred by Rag genes. We compared the transcriptional response of four soybean genotypes to aphid feeding to assess how the combination of Rag genes enhanced the soybean resistance to aphid infestation.ResultsA strong synergistic interaction between Rag1 and Rag2, defined as genes differentially expressed only in the pyramid genotype, was identified. This synergistic effect in the Rag1/2 phenotype was very evident early (6 h after infestation) and involved unique biological processes. However, the response of susceptible and resistant genotypes had a large overlap 12 h after aphid infestation. Transcription factor (TF) analyses identified a network of interacting TF that potentially integrates signaling from Rag1 and Rag2 to produce the unique Rag1/2 response. Pyramiding resulted in rapid induction of phytochemicals production and deposition of lignin to strengthen the secondary cell wall, while repressing photosynthesis. We also identified Glyma.07G063700 as a novel, strong candidate for the Rag1 gene.ConclusionsThe synergistic interaction between Rag1 and Rag2 in the Rag1/2 genotype can explain its enhanced resistance phenotype. Understanding molecular mechanisms that support enhanced resistance in pyramid genotypes could facilitate more directed approaches for crop improvement.

The Source of Rag5-Mediated Resistance to Soybean Aphids Is Located in the Stem.

The soybean aphid (Aphis glycines) continues to threaten soybean production in the United States. A suite of management strategies, such as planting aphid-resistant cultivars, has been successful in controlling soybean aphids. Several Rag genes (resistance against A. glycines) have been identified, and two are currently being deployed in commercial soybean cultivars. However, the mechanisms underlying Rag-mediated resistance are yet to be identified. In this study, we sought to determine the nature of resistance conferred by the Rag5 gene using behavioral, molecular biology, physiological, and biochemical approaches. We confirmed previous findings that plants carrying the Rag5 gene were resistant to soybean aphids in whole plant assays, and this resistance was absent in detached leaf assays. Analysis of aphid feeding behaviors using the electrical penetration graph technique on whole plants and detached leaves did not reveal differences between the Rag5 plants and Williams 82, a susceptible cultivar. In reciprocal grafting experiments, aphid populations were lower in the Rag5/rag5 (Scion/Root stock) chimera, suggesting that Rag5-mediated resistance is derived from the shoots. Further evidence for the role of stems comes from poor aphid performance in detached stem plus leaf assays. Gene expression analysis revealed that biosynthesis of the isoflavone kaempferol is upregulated in both leaves and stems in resistant Rag5 plants. Moreover, supplementing with kaempferol restored resistance in detached stems of plants carrying Rag5. This study demonstrates for the first time that Rag5-mediated resistance against soybean aphids is likely derived from stems.

The Evolution of Rag Gene Enhancers and Transcription Factor E and Id Proteins in the Adaptive Immune System.

Adaptive immunity relies on the V(D)J DNA recombination of immunoglobulin (Ig) and T cell receptor (TCR) genes, which enables the recognition of highly diverse antigens and the elicitation of antigen-specific immune responses. This process is mediated by recombination-activating gene (Rag) 1 and Rag2 (Rag1/2), whose expression is strictly controlled in a cell type-specific manner; the expression of Rag1/2 genes represents a hallmark of lymphoid lineage commitment. Although Rag genes are known to be evolutionally conserved among jawed vertebrates, how Rag genes are regulated by lineage-specific transcription factors (TFs) and how their regulatory system evolved among vertebrates have not been fully elucidated. Here, we reviewed the current body of knowledge concerning the cis-regulatory elements (CREs) of Rag genes and the evolution of the basic helix-loop-helix TF E protein regulating Rag gene CREs, as well as the evolution of the antagonist of this protein, the Id protein. This may help to understand how the adaptive immune system develops along with the evolution of responsible TFs and enhancers.

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.

The Interplay Between Chromatin Architecture and Lineage-Specific Transcription Factors and the Regulation of Rag Gene Expression.

Cell type-specific gene expression is driven through the interplay between lineage-specific transcription factors (TFs) and the chromatin architecture, such as topologically associating domains (TADs), and enhancer-promoter interactions. To elucidate the molecular mechanisms of the cell fate decisions and cell type-specific functions, it is important to understand the interplay between chromatin architectures and TFs. Among enhancers, super-enhancers (SEs) play key roles in establishing cell identity. Adaptive immunity depends on the RAG-mediated assembly of antigen recognition receptors. Hence, regulation of the Rag1 and Rag2 (Rag1/2) genes is a hallmark of adaptive lymphoid lineage commitment. Here, we review the current knowledge of 3D genome organization, SE formation, and Rag1/2 gene regulation during B cell and T cell differentiation.

Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System.

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

Illuminating the core of adaptive immunity-how the regulatory genome controls Rag chromatin dynamics.

E2A specifies adaptive immunity by instructing large-scale topological changes for Rag gene super-enhancer formation (see the related Research Article by Miyazaki et al.).

The Resistant Soybean-Aphis glycines Interaction: Current Knowledge and Prospects

Soybean aphids (Aphis glycines Matsumura) are invasive insect pests of soybean, and they cause significant yield losses. Resistance to soybean aphids is conferred by Resistance to Aphis glycines (Rag) genes. Since the first discovery of aphid-resistant soybean genotypes in 2004, several studies have attempted to characterize Rag genes from aphid-resistant soybean genotypes. To date, 12 Rag genes and four quantitative trait loci for aphid resistance have been reported on soybean chromosomes 07, 08, 13, 16, and 17. Although candidate genes have been proposed for several discovered Rag loci, additional studies are needed to pinpoint, validate, and further explain the potential mechanisms of Rag gene action. A major challenge to utilizing host plant resistance is the discovery of virulent aphid biotypes that can colonize aphid-resistant soybean. This occurrence suggests the need for additional studies to devise strategies to enhance the effectiveness of aphid-resistant soybean. In this mini review, we discuss current knowledge on the resistant soybean-Aphis glycines interaction, potential mechanisms of Rag gene action, opportunities to discover new Rag genes, and prospects for utilization of host plant resistance to manage soybean aphids. A clearer understanding of host plant resistance to soybean aphids will guide researchers on strategies for developing soybean varieties with more durable aphid resistance, reducing the present challenge of virulent aphid biotypes.

Virulence of Soybean Aphid, Aphis glycines (Hemiptera: Aphididae) Clones on Detached Leaves and Whole Plants

Since the discovery of the invasive soybean aphid, Aphis glycines Matsumura, in the USA in 2000, populations have expanded throughout the Midwestern USA and Canada. Its negative economic impact on soybean [Glycine max (L.) Merr.] yield provides the impetus to continue investigating the effectiveness of host plant resistance which has been affected by soybean aphid biotypes that overcome soybean plants containing Rag (resistance to A. glycines) genes. In this study, 14 aphid clones collected on soybean and buckthorn (Rhamnus cathartica L.) plants along with four known aphid biotypes (from our stock collection) were evaluated in no-choice assays by quantifying the number of aphids on soybean genotypes with Rag genes and the susceptible cultivar Williams 82. No-choice assays were conducted using detached leaves and whole plants. Inconsistent responses of the biotypes from the stock collection by using detached leaves confirm the efficacy of whole plant assays to accurately differentiate biotypes. None of the biotypes or field clones from Illinois, Indiana and South Dakota overcame the resistance of soybean genotypes LD14-8004 (Rag1) and PI437696. A soybean aphid clone from Wooster, Ohio readily colonized LD14-8002 (Rag2), but did not overcome any of the other resistance genes indicating that it is different from biotypes 3 and 4, which also overcome LD14-8002 (Rag2) and, respectively, LD14-8004 (Rag1) and LD14-8006 (Rag3).

Genome-wide association study identifies genetic factors that modify age at onset in Machado-Joseph disease

Machado-Joseph disease (MJD/SCA3) is the most common form of dominantly inherited ataxia worldwide. The disorder is caused by an expanded CAG repeat in the ATXN3 gene. Past studies have revealed that the length of the expansion partly explains the disease age at onset (AO) variability of MJD, which is confirmed in this study (Pearson’s correlation coefficient R2 = 0.62). Using a total of 786 MJD patients from five different geographical origins, a genome-wide association study (GWAS) was conducted to identify additional AO modifying factors that could explain some of the residual AO variability. We identified nine suggestively associated loci (P < 1 × 10−5). These loci were enriched for genes involved in vesicle transport, olfactory signaling, and synaptic pathways. Furthermore, associations between AO and the TRIM29 and RAG genes suggests that DNA repair mechanisms might be implicated in MJD pathogenesis. Our study demonstrates the existence of several additional genetic factors, along with CAG expansion, that may lead to a better understanding of the genotype-phenotype correlation in MJD.

Effects of aphid-resistant soybean on nontarget herbivores in Minnesota

The use of aphid-resistant soybean varieties containing Rag genes can provide an effective pest management strategy against soybean aphid (Aphis glycines Matsumura). However, the compatibility between soybean containing Rag genes and the management of other potential soybean pests (i.e., nontarget herbivores) is poorly understood. Therefore, our goal was to (1) assess the efficacy of pyramided (Rag1+Rag2) aphid-resistant soybean for reduction of soybean aphid populations, and (2) determine if aphid-resistant soybean affects populations of nontarget herbivores under field conditions. To accomplish these goals, field experiments were conducted in southeast Minnesota from 2015 to 2017 for a total of five site-years using whole-plant counts to assess soybean aphid densities and sweep-net sampling to estimate nontarget herbivore densities in plots of near-isoline soybean with Rag1+Rag2 (i.e., resistant) or no Rag genes (i.e., susceptible). Plant genotype (i.e., resistant or susceptible) affected seasonal abundance of soybean aphid, with fewer soybean aphids on aphid-resistant than susceptible soybean. Furthermore, plant genotype affected seasonal abundance of bean leaf beetle (Cerotoma trifurcata Forster), green cloverworm (Hypena scabra Fabricius), and potato leafhopper (Empoasca fabae Harris). Aphid-resistant soybean was associated with increased seasonal abundance of bean leaf beetle and reduced seasonal abundance of potato leafhopper and green cloverworm. The abundance of these herbivores was also associated with seasonal abundance of soybean aphid as a covariate. Results presented here confirm the efficacy of aphid-resistant soybean with Rag1+Rag2 against soybean aphid and show that aphid-resistant soybean can influence the seasonal abundance of nontarget herbivores in soybean. However, the magnitude of impacts of aphid-resistant soybean on nontarget herbivores was relatively small and likely of little-to-no agronomic significance, which suggests compatibility of aphid-resistant soybean with management of other potential pests.

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Identification and Genetic Characterization of Soybean Accessions Exhibiting Antibiosis and Antixenosis Resistance to Aphis glycines (Hemiptera: Aphididae).

Cultivation of aphid-resistant soybean varieties can reduce yield losses caused by soybean aphids. However, discovery of aphid biotypes that are virulent on resistant soybean greatly threatens sustained utilization of host plant resistance to control soybean aphids. The objective of this study was to identify and genetically characterize aphid resistant soybean accessions in a diverse collection of 308 plant introductions in maturity groups (MG) I and II. In large-scale screening experiments conducted in the greenhouse, we identified 12 soybean accessions (10 aphid-resistant and 2 moderately resistant), including nine previously not reported for resistance against soybean aphids. Three accessions (PI 578374, PI 612759C, and PI 603546A) and the Rag3 resistant check (PI 567543C) were susceptible when infested with a high initial aphid level but resistant when infested with a low initial aphid level, a phenomenon termed as density-dependent aphid resistance. Six accessions (PI 054854, PI 378663, PI 578374, PI 612759C, PI 540739, and PI 603546A) conferred antibiosis, five (PI 438031, PI 603337A, PI 612711B, PI 437950, and PI 096162) conferred both antibiosis and antixenosis, while one (PI 417513B) had neither when tested in no-choice and pairwise choice experiments. Molecular genotyping of the 12 accessions using single-nucleotide polymorphism (SNP) markers linked to known aphid resistance (Rag) genes revealed that PI 578374 and PI 540739 did not have any tested marker variants and could potentially carry unreported Rag genes. Genome-wide association analyses for MG I accessions identified genomic regions associated with aphid resistance on chromosomes 10 and 12 for each level of initial aphid colonization.

Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes.

Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE-Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.

RAG Deficiency: Two Genes, Many Diseases.

To review the clinical and laboratory spectrum of RAG gene defects in humans, and discuss the mechanisms underlying phenotypic heterogeneity, the basis of immune dysregulation, and the current and perspective treatment modalities. Literature review and analysis of medical records RESULTS: RAG gene defects in humans are associated with a surprisingly broad spectrum of clinical and immunological phenotypes. Correlation between in vitro recombination activity of the mutant RAG proteins and the clinical phenotype has been observed. Altered T and B cell development in this disease is associated with defects of immune tolerance. Hematopoietic cell transplantation is the treatment of choice for the most severe forms of the disease, but a high rate of graft failure has been observed. Phenotypic heterogeneity of RAG gene defects in humans may represent a diagnostic challenge. There is a need to improve treatment for severe, early-onset forms of the disease. Optimal treatment modalities for patients with delayed-onset disease presenting with autoimmunity and/or inflammation remain to be defined.

Pyramiding different aphid-resistance genes in elite soybean germplasm to combat dynamic aphid populations

The soybean aphid (Aphis glycines Matsumura), an invasive species, has posed a significant threat to soybean [Glycine max (L.) Merr.] production in North America since 2001. Use of resistant cultivars is an effective tactic to protect soybean yield. However, the variability and dynamics of aphid populations could limit the effectiveness of host-resistance gene(s). Gene pyramiding is a promising way to sustain host-plant resistance. The objectives of this study were to determine the prevalent aphid biotypes in Michigan and to assess the effectiveness of different combinations of aphid-resistance genes. A total of 11 soybean genotypes with known resistance gene(s) were used as indicator lines. Based on their responses, Biotype 3 was a major component of Michigan aphid populations during 2015–2016. The different performance of Rag-“Jackson” and Rag1-“Dowling” along with the breakdown of resistance in plant introductions (PIs) 567301B and 567324 may be explained by Biotype 3 or an unknown virulent biotype establishing in Michigan. With the assistance of flanking markers, 12 advanced breeding lines carrying different aphid-resistance gene(s) were developed and evaluated for effectiveness in five trials across 2015 to 2017. Lines with rag1c, Rag3d, Rag6, Rag3c + Rag6, rag1b + rag3, rag1c + rag4, rag1c + rag3 + rag4, rag1c + Rag2 + rag3 + rag4, and rag1b + rag1c + rag3 + rag4 demonstrated strong and consistent resistance. Due to the variability of virulent aphid populations, different combinations of Rag genes may perform differently across geographies. However, advanced breeding lines pyramided with three or four Rag genes likely will provide broader and more durable resistance to diverse and dynamic aphid populations.

The evolutionary conservation of the bidirectional activity of the NWC gene promoter in jawed vertebrates and the domestication of the RAG transposon

The RAG-1 and RAG-2 genes form a recombinase complex that is indispensable for V(D)J recombination, which generates the diversity of immunoglobulins and T-cell receptors. It is widely accepted that the presence of RAGs in the genomes of jawed vertebrates and other lineages is a result of the horizontal transfer of a mobile genetic element. While a substantial amount of evidence has been gathered that clarifies the nature of the RAG transposon, far less attention has been paid to the genomic site of its integration in various host organisms. In all genomes of the jawed vertebrates that have been studied to date, the RAG genes are located in close proximity to the NWC gene. We have previously shown that the promoter of the murine NWC genes exhibits a bidirectional activity, which may have facilitated the integration and survival of the RAG transposon in the host genome. In this study, we characterise the promoters of the NWC homologues that are present in the representatives of other jawed vertebrates (H. sapiens, X. tropicalis and D. rerio). We show that the features that are characteristic for promoters as the hosts of a successful transposon integration (in terms of the arrangement, bidirectional and constitutive activity and the involvement of the Zfp143 transcription factor in the promoter regulation) are evolutionarily conserved, which indicates that the presence of RAG genes in jawed vertebrates is a direct result of a successful transposon integration into the NWC locus.