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Thomas M Aune - One of the best experts on this subject based on the ideXlab platform.

  • Reciprocal regulation of Rag expression in thymocytes by the zinc-finger proteins, Zfp608 and Zfp609
    Genes & Immunity, 2013
    Co-Authors: N P Reed, Eugene M Oltz, M A Henderson, Thomas M Aune
    Abstract:

    Recombination-Activating Gene 1 (Rag1) and Rag2 enzymes are required for T cell receptor assembly and thymocyte development. The mechanisms underlying the transcriptional activation and repression of Rag1 and Rag2 are incompletely understood. The zinc-finger protein, Zfp608, represses Rag1 and Rag2 expression when expressed in thymocytes blocking T-cell maturation. Here we show that the related zinc-finger protein, Zfp609, is necessary for Rag1 and Rag2 expression in developing thymocytes. Zfp608 represses Rag1 and Rag2 expression indirectly by repressing the expression of Zfp609 . Thus, the balance of Zfp608 and Zfp609 plays a critical role in regulating Rag1 and Rag2 expression, which may manifest itself not only during development of immature thymocytes into mature T cells but also in Generation of the T-cell arm of the adaptive immune system, which does not fully develop until after birth.

  • Control of thymocyte development and Recombination-Activating Gene expression by the zinc finger protein Zfp608
    Nature Immunology, 2006
    Co-Authors: Feng Zhang, Lance R Thomas, Eugene M Oltz, Thomas M Aune
    Abstract:

    The products of Recombination-Activating Gene 1 ( Rag1 ) and Rag2 are required for T cell receptor Gene assembly and thymocyte maturation, yet their transcriptional control mechanisms remain unclear. A congenic strain (called 'ZORI' here) with defects in Rag1 and Rag2 expression, thymocyte maturation and peripheral T cell homeostasis has been developed. Here, we mapped the mutation in this strain to a chromosome 18 locus containing a single known Gene encoding the zinc finger protein Zfp608. This Gene ( Zfp608 ) was highly expressed in neonatal thymus but was extinguished thereafter. In contrast to wild-type mice, ZORI mice had sustained thymocyte expression of Zfp608 throughout life. The ZORI mutation produced a thymocyte-intrinsic developmental defect. Overexpression of Zfp608 in BALB/c thymocytes substantially impaired Rag1 and Rag2 expression, indicating the underlying mechanism for the defect in ZORI thymocyte development. Thus, the normal function of Zfp608 may be to prevent Rag1 and Rag2 expression in utero .

Stephen L. Kaattari - One of the best experts on this subject based on the ideXlab platform.

  • THE RECOMBINATION ACTIVATING Gene 2 (RAG2) OF THE RAINBOW TROUT ONCORHYNCHUS MYKISS
    Immunogenetics, 1996
    Co-Authors: John D. Hansen, Stephen L. Kaattari
    Abstract:

    We have previously described the isolation and expression ofRAG1 in trout to provide an initial understanding regarding the tissues involved inV(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating Gene 2 (RAG2) of rainbow trout has now been cloned and characterized. The rainbow trout genomicRAG2 Gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical ofRAG2 sequences. The proximity ofRAG1 and −2 within this teleost is similar to that found in other vertebrates. The Genes are convergently transcribed and share a 3′ untranslated (UT) region [2.8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6–8 kb). The entire 3′ UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for bothRAG Genes. Northern blot analysis of one-year-old trout demonstrated strong expression ofRAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of bothRAG1 and −2 in the thymas followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both Genes are expressed in embryos begining at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based uponRAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of theRAG Genes as well as that of the immune system itself.

  • The recombination activating Gene 2 (RAG2) of the rainbow troutOncorhynchus mykiss
    Immunogenetics, 1996
    Co-Authors: John D. Hansen, Stephen L. Kaattari
    Abstract:

    We have previously described the isolation and expression of RAG1 in trout to provide an initial understanding regarding the tissues involved in V(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating Gene 2 ( RAG2 ) of rainbow trout has now been cloned and characterized. The rainbow trout genomic RAG2 Gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical of RAG2 sequences. The proximity of RAG1 and −2 within this teleost is similar to that found in other vertebrates. The Genes are convergently transcribed and share a 3′ untranslated (UT) region [2.8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6–8 kb). The entire 3′ UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for both RAG Genes. Northern blot analysis of one-year-old trout demonstrated strong expression of RAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of both RAG1 and −2 in the thymas followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both Genes are expressed in embryos begining at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based upon RAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of the RAG Genes as well as that of the immune system itself.

  • The recombination activating Gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phyloGenetic analysis
    Immunogenetics, 1995
    Co-Authors: John D. Hansen, Stephen L. Kaattari
    Abstract:

    The characterization of Genes involved in the Generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating Genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) Genes, leading to the Generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and-T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue-and lymphocyte-specific expression. The polymerase chain reaction, coupled with deGenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this Gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.

Eugene M Oltz - One of the best experts on this subject based on the ideXlab platform.

  • Reciprocal regulation of Rag expression in thymocytes by the zinc-finger proteins, Zfp608 and Zfp609
    Genes & Immunity, 2013
    Co-Authors: N P Reed, Eugene M Oltz, M A Henderson, Thomas M Aune
    Abstract:

    Recombination-Activating Gene 1 (Rag1) and Rag2 enzymes are required for T cell receptor assembly and thymocyte development. The mechanisms underlying the transcriptional activation and repression of Rag1 and Rag2 are incompletely understood. The zinc-finger protein, Zfp608, represses Rag1 and Rag2 expression when expressed in thymocytes blocking T-cell maturation. Here we show that the related zinc-finger protein, Zfp609, is necessary for Rag1 and Rag2 expression in developing thymocytes. Zfp608 represses Rag1 and Rag2 expression indirectly by repressing the expression of Zfp609 . Thus, the balance of Zfp608 and Zfp609 plays a critical role in regulating Rag1 and Rag2 expression, which may manifest itself not only during development of immature thymocytes into mature T cells but also in Generation of the T-cell arm of the adaptive immune system, which does not fully develop until after birth.

  • Control of thymocyte development and Recombination-Activating Gene expression by the zinc finger protein Zfp608
    Nature Immunology, 2006
    Co-Authors: Feng Zhang, Lance R Thomas, Eugene M Oltz, Thomas M Aune
    Abstract:

    The products of Recombination-Activating Gene 1 ( Rag1 ) and Rag2 are required for T cell receptor Gene assembly and thymocyte maturation, yet their transcriptional control mechanisms remain unclear. A congenic strain (called 'ZORI' here) with defects in Rag1 and Rag2 expression, thymocyte maturation and peripheral T cell homeostasis has been developed. Here, we mapped the mutation in this strain to a chromosome 18 locus containing a single known Gene encoding the zinc finger protein Zfp608. This Gene ( Zfp608 ) was highly expressed in neonatal thymus but was extinguished thereafter. In contrast to wild-type mice, ZORI mice had sustained thymocyte expression of Zfp608 throughout life. The ZORI mutation produced a thymocyte-intrinsic developmental defect. Overexpression of Zfp608 in BALB/c thymocytes substantially impaired Rag1 and Rag2 expression, indicating the underlying mechanism for the defect in ZORI thymocyte development. Thus, the normal function of Zfp608 may be to prevent Rag1 and Rag2 expression in utero .

John D. Hansen - One of the best experts on this subject based on the ideXlab platform.

  • THE RECOMBINATION ACTIVATING Gene 2 (RAG2) OF THE RAINBOW TROUT ONCORHYNCHUS MYKISS
    Immunogenetics, 1996
    Co-Authors: John D. Hansen, Stephen L. Kaattari
    Abstract:

    We have previously described the isolation and expression ofRAG1 in trout to provide an initial understanding regarding the tissues involved inV(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating Gene 2 (RAG2) of rainbow trout has now been cloned and characterized. The rainbow trout genomicRAG2 Gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical ofRAG2 sequences. The proximity ofRAG1 and −2 within this teleost is similar to that found in other vertebrates. The Genes are convergently transcribed and share a 3′ untranslated (UT) region [2.8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6–8 kb). The entire 3′ UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for bothRAG Genes. Northern blot analysis of one-year-old trout demonstrated strong expression ofRAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of bothRAG1 and −2 in the thymas followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both Genes are expressed in embryos begining at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based uponRAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of theRAG Genes as well as that of the immune system itself.

  • The recombination activating Gene 2 (RAG2) of the rainbow troutOncorhynchus mykiss
    Immunogenetics, 1996
    Co-Authors: John D. Hansen, Stephen L. Kaattari
    Abstract:

    We have previously described the isolation and expression of RAG1 in trout to provide an initial understanding regarding the tissues involved in V(D)J recombination of antigen receptors in this teleost. Here we report that the recombination activating Gene 2 ( RAG2 ) of rainbow trout has now been cloned and characterized. The rainbow trout genomic RAG2 Gene (1602 base pairs) displays an average of 60% and 75% similarity at the nucleotide and amino acid level when compared with clones from other species and was found to contain an acidic region in the carboxyl terminal end, which is typical of RAG2 sequences. The proximity of RAG1 and −2 within this teleost is similar to that found in other vertebrates. The Genes are convergently transcribed and share a 3′ untranslated (UT) region [2.8 kilobases (kb)] which is much shorter than that found in higher vertebrates (6–8 kb). The entire 3′ UT region was also sequenced and used in conjunction with cDNA clones to identify the polyadenylation sites for both RAG Genes. Northern blot analysis of one-year-old trout demonstrated strong expression of RAG2 in the thymus, with a much weaker signal being detected in the pronephros. Using reverse transcriptase-polymerase chain reaction, we detected the highest expression of both RAG1 and −2 in the thymas followed by the pronephros, with much fainter signals being observed in the spleen, mesonephros, and liver. Finally, both Genes are expressed in embryos begining at approximately day 10 post-fertilization. Taken together, these findings indicate that the thymus and pronephros most likely serve as the primary lymphoid tissues in trout, based upon RAG expression. In addition, the trout sequences may provide further insight into the evolution and origins of the RAG Genes as well as that of the immune system itself.

  • The recombination activating Gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phyloGenetic analysis
    Immunogenetics, 1995
    Co-Authors: John D. Hansen, Stephen L. Kaattari
    Abstract:

    The characterization of Genes involved in the Generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating Genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) Genes, leading to the Generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and-T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue-and lymphocyte-specific expression. The polymerase chain reaction, coupled with deGenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this Gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.

Michel C. Nussenzweig - One of the best experts on this subject based on the ideXlab platform.

  • A cis element in the recombination activating Gene locus regulates Gene expression by counteracting a distant silencer
    Nature immunology, 2004
    Co-Authors: Nikos Yannoutsos, Vasco M. Barreto, Ziva Misulovin, Anna Gazumyan, Nikolaus Rajewsky, B. R. Peixoto, Thomas Eisenreich, Michel C. Nussenzweig
    Abstract:

    We have identified a silencer and an antisilencing element that interact at a distance of 85 kilobases to regulate expression of the recombination activating Genes Rag1 and Rag2 in thymocytes. Transgenic experiments showed that Rag promoter-proximal cis elements directed tissue-specific expression and that a Runx-dependent intergenic silencer suppressed expression in developing T cells. Deletion of the antisilencing element from the genomic Rag locus unmasked the intergenic silencer and abrogated Rag expression in developing CD4(+)CD8(+) T cells. We speculate that the Rag antisilencing element belongs to a class of cis elements that might be useful for genome diversification by activating Genes encoded by otherwise silent transposable elements.

  • The Role of Recombination Activating Gene (RAG) Reinduction in Thymocyte Development in Vivo
    The Journal of experimental medicine, 2001
    Co-Authors: Nikos Yannoutsos, Patrick C. Wilson, Hua Tang Chen, André Nussenzweig, Howard T. Petrie, Michel C. Nussenzweig
    Abstract:

    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.

  • Immunization and Infection Change the Number of Recombination Activating Gene (Rag)-Expressing B Cells in the Periphery by Altering Immature Lymphocyte Production
    The Journal of experimental medicine, 2000
    Co-Authors: Hitoshi Nagaoka, Gloria Gonzalez-aseguinolaza, Moriya Tsuji, Michel C. Nussenzweig
    Abstract:

    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.