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Induction of the early growth response (Egr) family of transcription factors during thymic selection.

Shao H, Kono DH, Chen LY, Rubin EM, Kaye J - J. Exp. Med. (1997)

Bottom Line: A similar pattern of expression is found for family members Egr-2 and Egr-3.In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1.The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+ thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

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The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. (A) Total RNA isolated from  DPK cells cultured with immobilized anti-CD3ε mAb for the indicated  times (shown in hours), was subjected to RT-PCR analysis using Egr-1  or CD4 primers. (B) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site (left) or overlapping Egr-1 and Sp1 sites (right). (C) DPK cells were cultured with  DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and  subjected to a competitive RT-PCR assay (see Materials and Methods).  Note the different scales for Egr-1 and CD4 mRNA expression.
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Figure 1: The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. (A) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. (B) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site (left) or overlapping Egr-1 and Sp1 sites (right). (C) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.

Mentions: The DPK cell line was derived from a CD4+8+ thymic lymphoma of an AND TCR transgenic mouse (38, 45). This cell line possess the phenotype of immature thymocytes, including expression of RAG-1,2 and TdT (41), and can be triggered to differentiate into CD4+8− cells upon activation by a pigeon cytochrome c peptide and Ek bearing antigen-presenting cells, or alternatively, by Ek bearing thymic epithelial cells in vivo or in vitro in the absence of antigen (38, 46). Although anti-CD3 mAb alone is a relatively poor inducer of DPK cell differentiation, it is active at high concentration or in conjunction with accessory molecule stimulation (41, 47). To identify genes involved in the early phase of TCR mediated double positive thymocyte differentiation, we used representational difference analysis to compare mRNA derived from DPK cells that were cultured in the presence or absence of anti-CD3ε mAb (see Materials and Methods). This approach allowed us to identify the Egr-1 immediate early gene, encoding a zinc finger transcription factor, as one such candidate gene. Subsequent RT-PCR analysis confirmed that DPK cells express little Egr-1 mRNA before activation, but express high levels as early as 1 h after anti-CD3ε mAb stimulation (Fig. 1 A). Levels of Egr-1 mRNA remain high for at least 5 h but decline by approximately fivefold 1 d after initial stimulation (Fig. 1 A). To ascertain whether the induction of Egr-1 mRNA resulted in DNA binding activity, we performed an electrophoretic mobility shift assay using nuclear extracts derived from DPK cells activated by antiCD3ε mAb. Nuclear extracts from DPK cells in the absence of activation had no detectable complexes binding to an oligonucleotide probe containing an Egr-1 consensus binding sequence (Fig. 1 B), consistent with the low level of Egr-1 mRNA. In contrast, a single binding complex was observed in activated DPK cells (Fig. 1 B). This complex has an identical mobility to that obtained using recombinant Egr-1, and supershifts with an anti-Egr-1 antibody (not shown). The inducibility of Egr-1 DNA binding activity is in contrast to Sp1 DNA binding activity which is constitutively expressed in DPK cells and is not affected by stimulation (Fig. 1 B).


Induction of the early growth response (Egr) family of transcription factors during thymic selection.

Shao H, Kono DH, Chen LY, Rubin EM, Kaye J - J. Exp. Med. (1997)

The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. (A) Total RNA isolated from  DPK cells cultured with immobilized anti-CD3ε mAb for the indicated  times (shown in hours), was subjected to RT-PCR analysis using Egr-1  or CD4 primers. (B) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site (left) or overlapping Egr-1 and Sp1 sites (right). (C) DPK cells were cultured with  DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and  subjected to a competitive RT-PCR assay (see Materials and Methods).  Note the different scales for Egr-1 and CD4 mRNA expression.
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Related In: Results  -  Collection

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Figure 1: The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. (A) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. (B) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site (left) or overlapping Egr-1 and Sp1 sites (right). (C) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.
Mentions: The DPK cell line was derived from a CD4+8+ thymic lymphoma of an AND TCR transgenic mouse (38, 45). This cell line possess the phenotype of immature thymocytes, including expression of RAG-1,2 and TdT (41), and can be triggered to differentiate into CD4+8− cells upon activation by a pigeon cytochrome c peptide and Ek bearing antigen-presenting cells, or alternatively, by Ek bearing thymic epithelial cells in vivo or in vitro in the absence of antigen (38, 46). Although anti-CD3 mAb alone is a relatively poor inducer of DPK cell differentiation, it is active at high concentration or in conjunction with accessory molecule stimulation (41, 47). To identify genes involved in the early phase of TCR mediated double positive thymocyte differentiation, we used representational difference analysis to compare mRNA derived from DPK cells that were cultured in the presence or absence of anti-CD3ε mAb (see Materials and Methods). This approach allowed us to identify the Egr-1 immediate early gene, encoding a zinc finger transcription factor, as one such candidate gene. Subsequent RT-PCR analysis confirmed that DPK cells express little Egr-1 mRNA before activation, but express high levels as early as 1 h after anti-CD3ε mAb stimulation (Fig. 1 A). Levels of Egr-1 mRNA remain high for at least 5 h but decline by approximately fivefold 1 d after initial stimulation (Fig. 1 A). To ascertain whether the induction of Egr-1 mRNA resulted in DNA binding activity, we performed an electrophoretic mobility shift assay using nuclear extracts derived from DPK cells activated by antiCD3ε mAb. Nuclear extracts from DPK cells in the absence of activation had no detectable complexes binding to an oligonucleotide probe containing an Egr-1 consensus binding sequence (Fig. 1 B), consistent with the low level of Egr-1 mRNA. In contrast, a single binding complex was observed in activated DPK cells (Fig. 1 B). This complex has an identical mobility to that obtained using recombinant Egr-1, and supershifts with an anti-Egr-1 antibody (not shown). The inducibility of Egr-1 DNA binding activity is in contrast to Sp1 DNA binding activity which is constitutively expressed in DPK cells and is not affected by stimulation (Fig. 1 B).

Bottom Line: A similar pattern of expression is found for family members Egr-2 and Egr-3.In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1.The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+ thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

Show MeSH