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1
Steedman, Carolyn. "What a Rag Rug Means." Journal of Material Culture 3, no. 3 (November 1998): 259–81. http://dx.doi.org/10.1177/135918359800300301.
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Lee, Minji, Jong Hyun Kim, Ina Yoon, Chulho Lee, Mohammad Fallahi Sichani, Jong Soon Kang, Jeonghyun Kang, et al. "Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway." Proceedings of the National Academy of Sciences 115, no. 23 (May 21, 2018): E5279—E5288. http://dx.doi.org/10.1073/pnas.1801287115.
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A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.
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Shen, Kuang, and David M. Sabatini. "Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms." Proceedings of the National Academy of Sciences 115, no. 38 (September 4, 2018): 9545–50. http://dx.doi.org/10.1073/pnas.1811727115.
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The mechanistic target of rapamycin complex 1 (mTORC1) growth pathway detects nutrients through a variety of sensors and regulators that converge on the Rag GTPases, which form heterodimers consisting of RagA or RagB tightly bound to RagC or RagD and control the subcellular localization of mTORC1. The Rag heterodimer uses a unique “locking” mechanism to stabilize its active (GTPRagA–RagCGDP) or inactive (GDPRagA–RagCGTP) nucleotide states. The Ragulator complex tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that upon activation stimulates mTORC1 activity through the Rag GTPases. How Ragulator and SLC38A9 control the nucleotide loading state of the Rag GTPases remains incompletely understood. Here we find that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer. Altogether, Ragulator and SLC38A9 act on the Rag GTPases to activate the mTORC1 pathway in response to nutrient sufficiency.
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Naik, Abani Kanta, Aaron T. Byrd, Aaron C. K. Lucander, and Michael S. Krangel. "Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes." Journal of Experimental Medicine 216, no. 1 (December 13, 2018): 231–43. http://dx.doi.org/10.1084/jem.20181402.
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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.
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Anandapadamanaban, Madhanagopal, Glenn R. Masson, Olga Perisic, Alex Berndt, Jonathan Kaufman, Chris M. Johnson, Balaji Santhanam, Kacper B. Rogala, David M. Sabatini, and Roger L. Williams. "Architecture of human Rag GTPase heterodimers and their complex with mTORC1." Science 366, no. 6462 (October 10, 2019): 203–10. http://dx.doi.org/10.1126/science.aax3939.
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The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
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Eastman, Quinn M., Isabelle J. Villey, and David G. Schatz. "Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3788–97. http://dx.doi.org/10.1128/mcb.19.5.3788.
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ABSTRACT V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.
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Hall, Lucinda M. C., Stuart C. Fawell, Xiaoju Shi, Marie-Claire Faray-Kele, Joseph Aduse-Opoku, Robert A. Whiley, and Michael A. Curtis. "Sequence Diversity and Antigenic Variation at the rag Locus of Porphyromonas gingivalis." Infection and Immunity 73, no. 7 (July 2005): 4253–62. http://dx.doi.org/10.1128/iai.73.7.4253-4262.2005.
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ABSTRACT The rag locus of Porphyromonas gingivalis W50 encodes RagA, a predicted tonB-dependent receptor protein, and RagB, a lipoprotein that constitutes an immunodominant outer membrane antigen. The low G+C content of the locus, an association with mobility elements, and an apparent restricted distribution in the species suggested that the locus had arisen by horizontal gene transfer. In the present study, we have demonstrated that there are four divergent alleles of the rag locus. The original rag allele found in W50 was renamed rag-1, while three novel alleles, rag-2 to rag-4, were found in isolates lacking rag-1. The three novel alleles encoded variants of RagA with 63 to 71% amino acid identity to RagA1 and each other and variants of RagB with 43 to 56% amino acid identity. The RagA/B proteins have homology to numerous Bacteroides proteins, including SusC/D, implicated in polysaccharide uptake. Monoclonal and polyclonal antibodies raised against RagB1 of P. gingivalis W50 did not cross-react with proteins from isolates carrying different alleles. In a laboratory collection of 168 isolates, 26% carried rag-1, 36% carried rag-2, 25% carried rag-3, and 14% carried rag-4 (including the type strain, ATCC 33277). Restriction profiles of the locus in different isolates demonstrated polymorphism within each allele, some of which is accounted for by the presence or absence of insertion sequence elements. By reference to a previously published study on virulence in a mouse model (M. L. Laine and A. J. van Winkelhoff, Oral Microbiol. Immunol. 13:322-325, 1998), isolates that caused serious disease in mice were significantly more likely to carry rag-1 than other rag alleles.
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Petiniot, Lisa K., Zoë Weaver, Melanie Vacchio, Rhuna Shen, Danny Wangsa, Carrolee Barlow, Michael Eckhaus, et al. "RAG-Mediated V(D)J Recombination Is Not Essential for Tumorigenesis in Atm-Deficient Mice." Molecular and Cellular Biology 22, no. 9 (May 1, 2002): 3174–77. http://dx.doi.org/10.1128/mcb.22.9.3174-3177.2002.
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ABSTRACT Atm-deficient mice die of malignant thymic lymphomas characterized by translocations within the Tcrα/δ locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA (dsDNA) breaks that occur during RAG-dependent V(D)J recombination. We recently demonstrated that development of thymic lymphoma in Atm−/− mice was not prevented by loss of RAG-2. Thymic lymphomas that developed in Rag2−/− Atm−/− mice contained multiple chromosomal abnormalities, but none of these involved the Tcrα/δ locus. These findings indicated that tumorigenesis in Atm−/− mice is mediated by chromosomal translocations secondary to aberrant responses to dsDNA breaks and that V(D)J recombination is an important, but not essential, event in susceptibility. In contrast to these findings, it was recently reported that Rag1−/− Atm−/− mice do not develop thymic lymphomas, a finding that was interpreted as demonstrating a requirement for RAG-dependent recombination in the susceptibility to tumors in Atm-deficient mice. To test the possibility that RAG-1 and RAG-2 differ in their roles in tumorigenesis, we studied Rag1−/− Atm−/− mice in parallel to our previous Rag2−/− Atm−/− study. We found that thymic lymphomas occur at high frequency in Rag1−/− Atm−/− mice and resemble those that occur in Rag2−/− Atm−/− mice. These results indicate that both RAG-1 and RAG-2 are necessary for tumorigenesis involving translocation in the Tcrα/δ locus but that Atm deficiency leads to tumors through a broader RAG-independent predisposition to translocation, related to a generalized defect in dsDNA break repair.
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Shi, Xiaoju, Shirley A. Hanley, Marie-Claire Faray-Kele, Stuart C. Fawell, Joseph Aduse-Opoku, Robert A. Whiley, Michael A. Curtis, and Lucinda M. C. Hall. "The rag Locus of Porphyromonas gingivalis Contributes to Virulence in a Murine Model of Soft Tissue Destruction." Infection and Immunity 75, no. 4 (February 5, 2007): 2071–74. http://dx.doi.org/10.1128/iai.01785-06.
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ABSTRACT The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.
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Shetty, Keerthi, and David G. Schatz. "Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination." Molecular and Cellular Biology 35, no. 21 (August 24, 2015): 3701–13. http://dx.doi.org/10.1128/mcb.00219-15.
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V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the “12/23 rule”) is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.
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Rogala, Kacper B., Xin Gu, Jibril F. Kedir, Monther Abu-Remaileh, Laura F. Bianchi, Alexia M. S. Bottino, Rikke Dueholm, et al. "Structural basis for the docking of mTORC1 on the lysosomal surface." Science 366, no. 6464 (October 10, 2019): 468–75. http://dx.doi.org/10.1126/science.aay0166.
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The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)–Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP–RagC/D•GDP) permits mTORC1 association. We used cryo–electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor “claw” threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.
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Bories, JC, JM Cayuela, P. Loiseau, and F. Sigaux. "Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression." Blood 78, no. 8 (October 15, 1991): 2053–61. http://dx.doi.org/10.1182/blood.v78.8.2053.2053.
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Abstract Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V- (D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.
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Bories, JC, JM Cayuela, P. Loiseau, and F. Sigaux. "Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression." Blood 78, no. 8 (October 15, 1991): 2053–61. http://dx.doi.org/10.1182/blood.v78.8.2053.bloodjournal7882053.
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Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V- (D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.
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Gennery, Andrew. "Recent advances in understanding RAG deficiencies." F1000Research 8 (February 4, 2019): 148. http://dx.doi.org/10.12688/f1000research.17056.1.
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Recombination-activating genes (RAG)1 and RAG2 initiate the molecular processes that lead to lymphocyte receptor formation through VDJ recombination. Nonsense mutations in RAG1/RAG2 cause the most profound immunodeficiency syndrome, severe combined immunodeficiency (SCID). Other severe and less-severe clinical phenotypes due to mutations in RAG genes are now recognized. The degree of residual protein function may permit some lymphocyte receptor formation, which confers a less-severe clinical phenotype. Many of the non-SCID phenotypes are associated with autoimmunity. New findings into the effect of mutations in RAG1/2 on the developing T- and B-lymphocyte receptor give insight into the development of autoimmunity. This article summarizes recent findings and places the genetic and molecular findings in a clinical context.
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Nagawa, Fumikiyo, Kei-ichiro Ishiguro, Akio Tsuboi, Tomoyuki Yoshida, Akiko Ishikawa, Toshitada Takemori, Anthony J. Otsuka, and Hitoshi Sakano. "Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination." Molecular and Cellular Biology 18, no. 1 (January 1, 1998): 655–63. http://dx.doi.org/10.1128/mcb.18.1.655.
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ABSTRACT We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.
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Chatterji, Monalisa, Chia-Lun Tsai, and David G. Schatz. "Mobilization of RAG-Generated Signal Ends by Transposition and Insertion In Vivo." Molecular and Cellular Biology 26, no. 4 (February 15, 2006): 1558–68. http://dx.doi.org/10.1128/mcb.26.4.1558-1568.2006.
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ABSTRACT In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated “core” RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.
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Yoshikawa, Genki, Kazuko Miyazaki, Hiroyuki Ogata, and Masaki Miyazaki. "The Evolution of Rag Gene Enhancers and Transcription Factor E and Id Proteins in the Adaptive Immune System." International Journal of Molecular Sciences 22, no. 11 (May 31, 2021): 5888. http://dx.doi.org/10.3390/ijms22115888.
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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.
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Hao, Bingtao, Abani Kanta Naik, Akiko Watanabe, Hirokazu Tanaka, Liang Chen, Hunter W. Richards, Motonari Kondo, et al. "An anti-silencer– and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development." Journal of Experimental Medicine 212, no. 5 (April 6, 2015): 809–24. http://dx.doi.org/10.1084/jem.20142207.
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Rag1 and Rag2 gene expression in CD4+CD8+ double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.
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Meng, Jin, and Shawn M. Ferguson. "GATOR1-dependent recruitment of FLCN–FNIP to lysosomes coordinates Rag GTPase heterodimer nucleotide status in response to amino acids." Journal of Cell Biology 217, no. 8 (May 30, 2018): 2765–76. http://dx.doi.org/10.1083/jcb.201712177.
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Folliculin (FLCN) is a tumor suppressor that coordinates cellular responses to changes in amino acid availability via regulation of the Rag guanosine triphosphatases. FLCN is recruited to lysosomes during amino acid starvation, where it interacts with RagA/B as a heterodimeric complex with FLCN-interacting proteins (FNIPs). The FLCN–FNIP heterodimer also has GTPase-activating protein (GAP) activity toward RagC/D. These properties raised two important questions. First, how is amino acid availability sensed to regulate lysosomal abundance of FLCN? Second, what is the relationship between FLCN lysosome localization, RagA/B interactions, and RagC/D GAP activity? In this study, we show that RagA/B nucleotide status determines the FLCN–FNIP1 recruitment to lysosomes. Starvation-induced FLCN–FNIP lysosome localization requires GAP activity toward Rags 1 (GATOR1), the GAP that converts RagA/B to the guanosine diphosphate (GDP)-bound state. This places FLCN–FNIP recruitment to lysosomes under the control of amino acid sensors that act upstream of GATOR1. By binding to RagA/BGDP and acting on RagC/D, FLCN–FNIP can coordinate nucleotide status between Rag heterodimer subunits in response to changes in amino acid availability.
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Schulz, Danae, Lothar Vassen, Kwan T. Chow, Sarah M. McWhirter, Rupesh H. Amin, Tarik Möröy, and Mark S. Schlissel. "Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1." Journal of Experimental Medicine 209, no. 1 (December 26, 2011): 187–99. http://dx.doi.org/10.1084/jem.20110645.
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Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5′ of the B cell–specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.
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Shaw, Albert C., Wojciech Swat, Roger Ferrini, Laurie Davidson, and Frederick W. Alt. "Activated Ras Signals Developmental Progression of Recombinase-activating Gene (RAG)-deficient Pro-B Lymphocytes." Journal of Experimental Medicine 189, no. 1 (January 4, 1999): 123–29. http://dx.doi.org/10.1084/jem.189.1.123.
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To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras–RAG). Similar to the effects of an immunoglobulin (Ig) μ heavy chain (HC) transgene, activated Ras caused progression of RAG1–deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of λ5, RAG2, and germline κ locus transcripts. However, these Ras–RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig μ HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.
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Thwaites, Daniel T., Clive Carter, Dylan Lawless, Sinisa Savic, and Joan M. Boyes. "A novel RAG1 mutation reveals a critical in vivo role for HMGB1/2 during V(D)J recombination." Blood 133, no. 8 (February 21, 2019): 820–29. http://dx.doi.org/10.1182/blood-2018-07-866939.
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Abstract The Recombination Activating Genes, RAG1 and RAG2, are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo.
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Kondratenko, I. V., O. E. Pashchenko, Y. A. Rodina, M. V. Belevtcev, Den M. Van, and A. A. Bologov. "Clinical and laboratory phenotypes of severe combined immunodeficiencies with mutations in RAG1/RAG2 genes." Russian Journal of Allergy 9, no. 4 (December 15, 2012): 26–32. http://dx.doi.org/10.36691/rja689.
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Background. The RAG1 and RAG2 proteins are key players in the V(D)J recombination process leading to the assembly of antigen receptor genes. Defects in RAG1/RAG2 genes are caused to different phenotypes of severe combined immunodeficiencies (SCID). Objective — to determine the clinical and laboratory manifestations in patients with RAG1 / RAG2 mutations from one single center, to identify the phenotype-genotype correlations. Materials and methods. We described 4 children with RAG1 mutations. Diagnosis of SCID was confirmed by criteria’s of European society of immunodeficiencies (ESID). Results. In two patients we observed Omenn syndrome, in 1 — classic T -B -NK + SCID, in 1 — «sof» T +B -NK + SCID. One patient with Omenn syndrome and patient with «soft» SCID had same RAG1 mutations. Conclusions. RAG 1 / RAG2 mutations are caused to severe life-threatening combined immunodeficiency, requiring radical therapy. We found no genotype-phenotype correlations in patients with RAG1 defects.
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Yannoutsos, Nikos, Patrick Wilson, Wong Yu, Hua Tang Chen, Andre Nussenzweig, Howard Petrie, and Michel C. Nussenzweig. "The Role of Recombination Activating Gene (RAG) Reinduction in Thymocyte Development in Vivo." Journal of Experimental Medicine 194, no. 4 (August 20, 2001): 471–80. http://dx.doi.org/10.1084/jem.194.4.471.
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Assembly of T cell receptor (TCR)α/β genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRβ recombination in CD4−CD8−CD25+ thymocytes. In these cells, TCRβ expression leads to clonal expansion, RAG downregulation, and TCRβ allelic exclusion. At the subsequent CD4+CD8+ stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCRα locus. This second wave of RAG expression is terminated upon expression of a positively selected α/β TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4+CD8+ T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4+CD8+ cells but a 50–70% reduction in the number of mature CD4+CD8− and CD4−CD8+ thymocytes. TCRα rearrangement is restricted to the 5′ end of the Jα cluster and there is little apparent secondary TCRα recombination. Comparison of the TCRα genes expressed in wild-type or mutant mice shows that 65% of all α/β T cells carry receptors that are normally assembled by secondary TCRα rearrangement. We conclude that RAG reinduction in CD4+CD8+ thymocytes is not required for initial TCRα recombination but is essential for secondary TCRα recombination and that the majority of TCRα chains expressed in mature T cells are products of secondary recombination.
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Nilavar, Namrata M., Mayilaadumveettil Nishana, Amita M. Paranjape, Raghunandan Mahadeva, Rupa Kumari, Bibha Choudhary, and Sathees C. Raghavan. "Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs." Biochemical Journal 477, no. 18 (September 24, 2020): 3567–82. http://dx.doi.org/10.1042/bcj20200361.
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Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.
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Nagaoka, Hitoshi, Gloria Gonzalez-Aseguinolaza, Moriya Tsuji, and Michel C. Nussenzweig. "Immunization and Infection Change the Number of Recombination Activating Gene (Rag)-Expressing B Cells in the Periphery by Altering Immature Lymphocyte Production." Journal of Experimental Medicine 191, no. 12 (June 19, 2000): 2113–20. http://dx.doi.org/10.1084/jem.191.12.2113.
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Recombination activating gene (RAG) expression in peripheral B cells increases after immunization with (4-hydroxy-3-nitrophenyl) acetyl coupled to chicken gamma globulin (NP-CGG) in alum. This increase could result from reinduction of RAG expression or, alternatively, from accumulation of RAG-expressing immature B cells in the periphery. We have used mice that carry a green fluorescent protein (GFP) RAG indicator transgene (RAG2-GFP) to characterize the RAG-expressing B cells in immunized spleens. Most of the RAG2-GFP–expressing B cells in unimmunized spleen are immature B cells. Injection with NP-CGG in alum initially suppresses lymphopoiesis in the bone marrow and decreases the number of immature RAG2-GFP–expressing B cells in the spleen. Recovery of lymphopoiesis in the bone marrow coincides with accumulation of RAG-expressing immature B cells in the spleen. Most of the RAG-expressing cells that accumulate in the spleen after immunization do not proliferate and they are not germinal center cells. Neither the initial suppression of lymphopoiesis nor the subsequent accumulation of RAG-expressing cells in the spleen is antigen dependent, since similar changes are seen with alum alone. Furthermore, such changes in the numbers of developing and circulating immature lymphoid cells are seen after injection with complete Freund's adjuvant or malaria infection. Our experiments suggest that adjuvants and infectious agents cause previously unappreciated alterations in lymphopoiesis resulting in the accumulation of RAG-expressing immature B cells in the spleen.
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Meru, Nadine, Andreas Jung, Irith Baumann, and Gerald Niedobitek. "Expression of the recombination-activating genes in extrafollicular lymphocytes but no apparent reinduction in germinal center reactions in human tonsils." Blood 99, no. 2 (January 15, 2002): 531–37. http://dx.doi.org/10.1182/blood.v99.2.531.
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Abstract V(D)J recombination in lymphocytes is mediated by 2 recombination-activating genes, RAG1 and RAG2,which are expressed during lymphocyte development in bone marrow and thymus. Prompted by studies reporting re-expression of the RAGs in germinal center B cells, the expression of RAGs and terminal deoxynucleotidyl transferase (TdT) in human lymphoid tissues was examined using in situ hybridization and immunohistochemistry, respectively. Here it is shown that RAGs and TdT are not reinduced in germinal center reactions. However, RAG+/TdT+ cells are frequently present in extrafollicular areas of tonsils mainly at the boundary between lymphoid tissue and fibrous scaffold. Phenotypic analyses suggest that these cells are B cells. Finally, it is shown that RAG+/TdT+ cells are found more frequently in tonsils than in other peripheral lymphoid tissues. This may reflect an increased influx of RAG+/TdT+ cells as a result of higher antigenic stimulation at this site. Alternatively, this observation may indicate that the tonsils are an additional site of lymphocyte ontogeny.
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Anbazhagan, Kolandaswamy, Vincent Fuentes, Eliane Bissac, Remy Nyga, Naomi Taylor, Jacques Rochette, and Kaiss Lassoued. "The Human Pre-B Cell Receptor Signaling Cascade Is Regulated Via PI-3Kinase and MAPK Pathway." Blood 118, no. 21 (November 18, 2011): 1314. http://dx.doi.org/10.1182/blood.v118.21.1314.1314.
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Abstract Abstract 1314 Background: Pre-B cell receptor (pre-BCR) constitutes a major check point in the early steps of mouse and human B cell development. Several functions have been attributed to this receptor which include a delivery of proliferation and survival signals, increased sensitivity to interleukin-7 (IL-7) and down modulation of recombinase activating genes (RAG) and surrogate light chain (SLC) encoding genes. Pre-BCR is also involved in shaping the VH repertoire and preventing autoimmunity. Finally, there is increasing evidence that pre-BCR might be implicated in leukemogenesis. Most of the functions of pre-BCR have been predicted based on studies in knockout mice and leukemic cell lines. In a previous study we have shown that pre-BCR aggregation resulted in the activation of src and Syk kinases which in turn activated the PI-3K/Akt, Btk, PLCγ-2 and Ras/MAPK. In this study, we examined the pre-BCR signalling cascade using human normal primary pre-B cells with a particular focus on transcription factors activation and Rag modulation and their regulatory aspects. Methods: Pre-B cells were sorted from adult human bone marrow samples, treated or not with inhibitors of Syk (BAY61–3606), Akt (LY294002) and MEKK1 (UO126) prior to crosslink the pre-BCR by means of F(ab')2 anti-μHC. The effect of Pre-BCR signaling was examined by quantifying the transcript levels of Rag1, Rag2, E2A, EBF1, Pax5, FoxO1 and FoxO3, IRF4/8. Activation of transcription factors such as NF-κB p50, c-Fos, IRF4 and FoxO3A, was assessed by analyzing their nuclear translocation by immunofluorescence microscopy. Results: We show that NF-κB p50 is translocated into nucleus within 3h after pre-BCR stimulation. Crosslinking of pre-BCR also resulted in an enhancement of nuclear c-Fos translocation. BAY61-3606 (Syk inhibitor) treatment resulted in complete apoptosis (100 % cell death within 48h). Although treatment of normal pre-B cells with LY294002 or U0126 did not alter cell survival, nuclear translocation of pre-BCR-induced p50 NF-κB was prevented by former and enhanced by later. Conversely, c-Fos nuclear expression was inhibited by U0126 and slightly but consistently enhanced by LY294002 in association with a decrease in its cytoplasmic location. Pre-BCR stimulation also induced IRF4 translocation to the nucleus. Pre-BCR stimulation also resulted in the down regulation of Rag1 (− 48 %, P<0.01), Pax5 (− 40%, P<0.01) and E2A (− 35 %, P< 0.01) transcripts, whereas EBF1 and FoxO1 and 3 expression remained unchanged. In LY294002-treated cells, Rag1/Rag2 expression was up regulated (+130%, P< 0.01 and +251%, P< 0.01, respectively) following pre-BCR crosslinking, whereas in the presence of U0126 the pre-BCR induced Rag1/Rag2 down modulation remained unchanged. Conclusion: Our results indicate that the pre-BCR has the potential to promote pre-B cell proliferation, survival and differentiation by activating NF-kB, c-Fos and IRF4. It also has the ability to protect pre-B cells from genome instability by down-regulating Rag1/2, probably through down modulation of Pax5 and E2A. We bring evidence that PI-3 K/Akt pathway plays a crucial role in the regulation of the pre-BCR signaling cascade and that Akt-mediated NF-kB and c-Fos activation is antagonized by MAPK. Up-regulation of Rag transcripts upon Akt inhibition suggests either a feed-back negative loop or a dual effect of pre-BCR on Rag expression with an Akt-dependent Rag down regulation and an accessory pathway that enhances Rag expression. Disclosures: No relevant conflicts of interest to declare.
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Miyazaki, Kazuko, Hitomi Watanabe, Genki Yoshikawa, Kenian Chen, Reiko Hidaka, Yuki Aitani, Kai Osawa, et al. "The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells." Science Immunology 5, no. 51 (September 4, 2020): eabb1455. http://dx.doi.org/10.1126/sciimmunol.abb1455.
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Cell type–specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage–specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage–specific enhancer (R-TEn) and the two B cell–specific elements, R1B and R2B. By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.
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Thomson, Daniel, Hezrin Shahrin, Paul Wang, Carol Wadham, Timothy P. Hughes, Andreas Schreiber, and Susan Branford. "High Recombination Activating Gene (RAG) Expression and RAG Mediated Recombination Is Associated with Oncogenic Rearrangement Observed with Tyrosine Kinase Inhibitor Resistant CML." Blood 132, Supplement 1 (November 29, 2018): 3001. http://dx.doi.org/10.1182/blood-2018-99-111212.
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Abstract Background Patients with chronic myeloid leukemia (CML) who develop blast crisis are resistant to TKI therapy. A key focus in CML research is the identification of genetic factors that promote blast crisis and TKI resistance. By using an integrative genomic approach we recently reported frequent structural variation in CML patients, particularly at lymphoid blast crisis (LBC) (Blood, 2018). Developing lymphocytes are uniquely equipped with a molecular toolkit capable of programmed DNA damage and structural variant formation; the V(D)J recombination pathway. Recombination activating genes (RAG1 and RAG2) are involved in cleavage and recombination of immunoglobulin genes to provide diversity in antibodies and T cell receptors. Off target RAG activity is reported to occur in lymphoid malignancies and cause oncogenic structural rearrangements. However, RAG expression and the extent of RAG mediated structural variation in CML are largely uncharacterized. Aim To elucidate the role of RAG mediated recombination as a source of oncogenic structural rearrangement in CML. Methods In a study of samples of 49 patients at chronic phase (CP) diagnosis (Dx), 20 at LBC and 19 at myeloid blast crisis (MBC), we performed whole exome sequencing and/or RNAseq. Bioinformatic analyses included fusion detection (Manta & STAR), gene expression analysis (EdgeR), and copy number variation analysis (in house developed tool). Unsupervised motif detection of sequences surrounding breakpoints was performed with MEME, and fusions were visualized with Jcircos. To identify off target RAG mediated mutation we interrogated the breakpoints of structural variants, excluding those associated with the Philadelphia translocation and those solely involving antigen receptor gene rearrangement. Results 33 structural variants were identified in 22 patients with samples at Dx and/or blast crisis involving genes regularly mutated in hematologic malignancy such as MLL, MECOM, RUNX1 and IKZF1. Differential expression analysis between patients at Dx, MBC and LBC revealed >1000 genes that were differentially expressed, P<0.001. Amongst the most upregulated genes at LBC were RAG1, RAG2 and DNTT, reflected in the most enriched gene ontology: V(D)J recombination. V(D)J recombination genes were sufficient alone in stratifying LBC samples from CP Dx and MBC [Figure 1A]. Furthermore, RAG expression was detectably elevated at Dx in most patients who subsequently developed LBC [Figure 1B]. To identify off target RAG mediated structural variation, we interrogated the sequence surrounding breakpoints of the 33 identified structural variants (66 breakpoints) that were separated into groups of high (n=40) and low (n=26) RAG expression. The canonical heptamer CACAGTG recombination signal sequence (RSS) was enriched only at breakpoints in the high RAG group with sequences from 31/40 breakpoints making up the motif [Figure 1C]. The comparable group of low RAG breakpoints were not enriched for the motif. Another indicator of RAG involvement is the addition of non-template nucleotides at the breakpoint, consistent with DNTT nucleotidyl-transferase activity. This was detected almost exclusively at breakpoints in patients with high RAG, 16/20 structural variants, compared to 1/13 with low RAG. There was an association between high RAG expression and structural variants bearing the RAG RSS motif at breakpoints with nucleotide additions. The most frequently observed of these were intragenic deletions of IKZF1 in 7 patients, including 3 at Dx with subsequent LBC, 3 at LBC and 1 at MBC. A number of novel structural variants also had the RAG recombination signature: IKZF1-IGK fusion, RUNX1 deletion and recurrent HBS1L-MYB intergenic locus deletion. RAG mediated structural variation was further investigated on a genomic level using putative low level fusions identified from RNAseq. By assessing the immunoglobulin/T cell receptor genes, as known sites of RAG activity, we found enrichment of interchromosomal fusions involving these genes in samples with high RAG, compared to Dx and MBC with low RAG [Figure 1D]. An agnostic motif enrichment search at sequences surrounding putative fusions showed enrichment of a motif resembling the recombination signal sequence. Conclusion Our results implicate RAG as a driver of structural variation and the formation of novel oncogenic rearrangements in CML. Figure 1. Figure 1. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cepheid: Honoraria.
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Notarangelo, Luigi D., Anna Villa, and Klaus Schwarz. "RAG and RAG defects." Current Opinion in Immunology 11, no. 4 (August 1999): 435–42. http://dx.doi.org/10.1016/s0952-7915(99)80073-9.
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Lee, Baeck-seung, Joseph D. Dekker, Bum-kyu Lee, Vishwanath R. Iyer, Barry P. Sleckman, Arthur L. Shaffer, Gregory C. Ippolito, and Philip W. Tucker. "The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination." Molecular and Cellular Biology 33, no. 9 (February 25, 2013): 1768–81. http://dx.doi.org/10.1128/mcb.00987-12.
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Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a lox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
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Gärtner, Frank, Frederick W. Alt, Robert J. Monroe, and Katherine J. Seidl. "Antigen-Independent Appearance of Recombination Activating Gene (Rag)-Positive Bone Marrow B Cells in the Spleens of Immunized Mice." Journal of Experimental Medicine 192, no. 12 (December 11, 2000): 1745–54. http://dx.doi.org/10.1084/jem.192.12.1745.
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Splenic B lineage cells expressing recombination activation genes (RAG+) in mice immunized with 4-hydroxy-3-nitrophenyl-acetyl coupled to chicken γ-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.
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Wu, Zeguang, Narmadha Subramanian, Eva-Maria Jacobsen, Kerstin Laib Sampaio, Johannes van der Merwe, Manfred Hönig, and Thomas Mertens. "NK Cells from RAG- or DCLRE1C-Deficient Patients Inhibit HCMV." Microorganisms 7, no. 11 (November 10, 2019): 546. http://dx.doi.org/10.3390/microorganisms7110546.
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The recombination-activating genes (RAGs) and the DNA cross-link repair 1C gene (DCLRE1C) encode the enzymes RAG1, RAG2 and Artemis. They are critical components of the V(D)J recombination machinery. V(D)J recombination is well known as a prerequisite for the development and antigen diversity of T and B cells. New findings suggested that RAG deficiency impacts the cellular fitness and function of murine NK cells. It is not known whether NK cells from severe combined immunodeficiency (SCID) patients with defective RAGs or DCLRE1C (RAGs−/DCLRE1C−-NK) are active against virus infections. Here, we evaluated the anti-HCMV activity of RAGs−/DCLRE1C−-NK cells. NK cells from six SCID patients were functional in inhibiting HCMV transmission between cells in vitro. We also investigated the expansion of HCMV-induced NK cell subset in the RAG- or DCLRE1C-deficient patients. A dynamic expansion of NKG2C+ NK cells in one RAG-2-deficient patient was observed post HCMV acute infection. Our study firstly reveals the antiviral activity of human RAGs−/ DCLRE1C−-NK cells.
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Teng, Wendi, Wenjing Yin, Liang Zhao, Changwei Ma, Jiaqiang Huang, and Fazheng Ren. "Resveratrol metabolites ameliorate insulin resistance in HepG2 hepatocytes by modulating IRS-1/AMPK." RSC Advances 8, no. 63 (2018): 36034–42. http://dx.doi.org/10.1039/c8ra05092a.
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RSV metabolites R3G and R4G protected HepG2 cell from insulin resistance by improving glucose uptake and glycogen synthesis, along with inhibiting ROS generation and modulating the RS-1/AMPK signaling pathway.
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Dudley, Darryll D., JoAnn Sekiguchi, Chengming Zhu, Moshe J. Sadofsky, Scott Whitlow, Jeffrey DeVido, Robert J. Monroe, Craig H. Bassing, and Frederick W. Alt. "Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice." Journal of Experimental Medicine 198, no. 9 (October 27, 2003): 1439–50. http://dx.doi.org/10.1084/jem.20030627.
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RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.
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Steinel, Natalie C., Baeck-Seung Lee, Anthony T. Tubbs, Jeffrey J. Bednarski, Emily Schulte, Katherine S. Yang-Iott, David G. Schatz, Barry P. Sleckman, and Craig H. Bassing. "The Ataxia Telangiectasia mutated kinase controls Igκ allelic exclusion by inhibiting secondary Vκ-to-Jκ rearrangements." Journal of Experimental Medicine 210, no. 2 (February 4, 2013): 233–39. http://dx.doi.org/10.1084/jem.20121605.
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Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre–B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.
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Weinstock, David M., and Maria Jasin. "Alternative Pathways for the Repair of RAG-Induced DNA Breaks." Molecular and Cellular Biology 26, no. 1 (January 1, 2006): 131–39. http://dx.doi.org/10.1128/mcb.26.1.131-139.2006.
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ABSTRACT RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be ≥40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70 − / − cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.
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Lovely, Geoffrey A., Robert C. Brewster, David G. Schatz, David Baltimore, and Rob Phillips. "Single-molecule analysis of RAG-mediated V(D)J DNA cleavage." Proceedings of the National Academy of Sciences 112, no. 14 (March 23, 2015): E1715—E1723. http://dx.doi.org/10.1073/pnas.1503477112.
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The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites.
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Mundy, Cynthia L., Nadja Patenge, Adam G. W. Matthews, and Marjorie A. Oettinger. "Assembly of the RAG1/RAG2 Synaptic Complex." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 69–77. http://dx.doi.org/10.1128/mcb.22.1.69-77.2002.
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ABSTRACT Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.
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Yoon, Mee-Sup, and Jie Chen. "Distinct amino acid–sensing mTOR pathways regulate skeletal myogenesis." Molecular Biology of the Cell 24, no. 23 (December 2013): 3754–63. http://dx.doi.org/10.1091/mbc.e13-06-0353.
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Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase–independent manner. Our results uncover amino acid–sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.
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Zhang, Zhuo-Qian, Jing Wang, Zachary Hoy, Achsah Keegan, Samir Bhagwat, Francis Gigliotti, and Terry W. Wright. "Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome." Infection and Immunity 83, no. 12 (September 14, 2015): 4594–603. http://dx.doi.org/10.1128/iai.00763-15.
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Pneumocystisis a respiratory fungal pathogen that causes pneumonia (Pneumocystispneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4+T cell-dependent clearance ofPneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages forPneumocystisclearance has not been determined. Therefore, RAG2−/−mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected withPneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2−/−mice developed severe disease but effectively clearedPneumocystisand resolved IRIS. Neither RAG/IFN-γR−/−nor RAG/IL-4Rα−/−mice displayed impairedPneumocystisclearance. However, RAG/IFN-γR−/−mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα−/−mice. RAG/IFN-γR−/−mice had elevated numbers of lung CD4+T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8+T suppressor cells. Impaired lung CD8+T cell responses in RAG/IFN-γR−/−mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impairPneumocystisclearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8+T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.
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Nagawa, Fumikiyo, Masami Kodama, Tadashi Nishihara, Kei-ichiro Ishiguro, and Hitoshi Sakano. "Footprint Analysis of Recombination Signal Sequences in the 12/23 Synaptic Complex of V(D)J Recombination." Molecular and Cellular Biology 22, no. 20 (October 15, 2002): 7217–25. http://dx.doi.org/10.1128/mcb.22.20.7217-7225.2002.
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ABSTRACT In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre- and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.
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Kuijpers, Taco W., Hanna IJspeert, Ester M. M. van Leeuwen, Machiel H. Jansen, Mette D. Hazenberg, Kees C. Weijer, Rene A. W. van Lier, and Mirjam van der Burg. "Idiopathic CD4+ T lymphopenia without autoimmunity or granulomatous disease in the slipstream of RAG mutations." Blood 117, no. 22 (June 2, 2011): 5892–96. http://dx.doi.org/10.1182/blood-2011-01-329052.
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Abstract A girl presented during childhood with a single course of extensive chickenpox and moderate albeit recurrent pneumonia in the presence of idiopathic CD4+ T lymphocytopenia (ICL). Her clinical condition remained stable over the past 10 years without infections, any granulomatous disease, or autoimmunity. Immunophenotyping demonstrated strongly reduced naive T and B cells with intact proliferative capacity. Antibody reactivity on in vivo immunizations was normal. T-cell receptor-Vβ repertoire was polyclonal with a very low content of T-cell receptor excision circles (TRECs). Kappa-deleting recombination excision circles (KRECs) were also abnormal in the B cells. Both reflect extensive in vivo proliferation. Patient-derived CD34+ hematopoietic stem cells could not repopulate RAG2−/−IL2Rγc−/− mice, indicating the lymphoid origin of the defect. We identified 2 novel missense mutations in RAG1 (p.Arg474Cys and p.Leu506Phe) resulting in reduced RAG activity. This report gives the first genetic clue for ICL and extends the clinical spectrum of RAG mutations from severe immune defects to an almost normal condition.
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Chandrasena, Desmi, Yang Wang, Carmille Bales, Jiazheng Yuan, Cuihua Gu, and Dechun Wang. "Pyramiding rag 3, rag 1b, rag 4, and rag 1c Aphid-Resistant Genes in Soybean Germplasm." Crop Science 55, no. 5 (September 2015): 2108–15. http://dx.doi.org/10.2135/cropsci2015.02.0089.
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Sekiguchi, Takeshi, Eiji Hirose, Nobutaka Nakashima, Miki Ii, and Takeharu Nishimoto. "Novel G Proteins, Rag C and Rag D, Interact with GTP-binding Proteins, Rag A and Rag B." Journal of Biological Chemistry 276, no. 10 (November 9, 2000): 7246–57. http://dx.doi.org/10.1074/jbc.m004389200.
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Ji, Yanhong, Alicia J. Little, Joydeep K. Banerjee, Bingtao Hao, Eugene M. Oltz, Michael S. Krangel, and David G. Schatz. "Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination." Journal of Experimental Medicine 207, no. 13 (November 29, 2010): 2809–16. http://dx.doi.org/10.1084/jem.20101136.
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V(D)J recombination assembles antigen receptor genes in a well-defined order during lymphocyte development. This sequential process has long been understood in the context of the accessibility model, which states that V(D)J recombination is regulated by controlling the ability of the recombination machinery to gain access to its chromosomal substrates. Indeed, many features of “open” chromatin correlate with V(D)J recombination, and promoters and enhancers have been strongly implicated in creating a recombinase-accessible configuration in neighboring chromatin. An important prediction of the accessibility model is that cis-elements and transcription control binding of the recombination-activating gene 1 (RAG1) and RAG2 proteins to their DNA targets. However, this prediction has not been tested directly. In this study, we use mutant Tcra and Tcrb alleles to demonstrate that enhancers control RAG1 binding globally at Jα or Dβ/Jβ gene segments, that promoters and transcription direct RAG1 binding locally, and that RAG1 binding can be targeted in the absence of RAG2. These findings reveal important features of the genetic mechanisms that regulate RAG binding and provide a direct confirmation of the accessibility model.
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De Ravin, Suk See, Edward W. Cowen, Kol A. Zarember, Narda L. Whiting-Theobald, Douglas B. Kuhns, Netanya G. Sandler, Daniel C. Douek, et al. "Hypomorphic Rag mutations can cause destructive midline granulomatous disease." Blood 116, no. 8 (August 26, 2010): 1263–71. http://dx.doi.org/10.1182/blood-2010-02-267583.
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Abstract Destructive midline granulomatous disease characterized by necrotizing granulomas of the head and neck is most commonly caused by Wegener granulomatosis, natural killer/T-cell lymphomas, cocaine abuse, or infections. An adolescent patient with myasthenia gravis treated with thymectomy subsequently developed extensive granulomatous destruction of midface structures, palate, nasal septum, airways, and epiglottis. His lymphocyte numbers, total immunoglobulin G level, and T-cell receptor (TCR) repertoire appeared normal. Sequencing of Recombination activating gene-1 (Rag1) showed compound heterozygous Rag1 mutations; a novel deletion with no recombinase activity and a missense mutation resulting in 50% Rag activity. His thymus was dysplastic and, although not depleted of T cells, showed a notable absence of autoimmune regulator (AIRE) and Foxp3+ regulatory T cells. This distinct Rag-deficient phenotype characterized by immune dysregulation with granulomatous hyperinflammation and autoimmunity, with relatively normal T and B lymphocyte numbers and a diverse TCR repertoire expands the spectrum of presentation in Rag deficiency. This study was registered at www.clinicaltrials.gov as #NCT00128973.
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Laufenberg, Lacee J., Kristen T. Crowell, and Charles H. Lang. "Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle." Nutrients 13, no. 4 (April 9, 2021): 1236. http://dx.doi.org/10.3390/nu13041236.
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The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol’s effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein–protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min (“refed”) or remained food-deprived (“fasted”). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V1 domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein–protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.
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Naish, John. "Tory Rag." Nursing Standard 4, no. 45 (August 1990): 51. http://dx.doi.org/10.7748/ns.4.45.51.s60.
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