Limits...
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.

Show MeSH
Egr-1 mRNA and DNA binding activity in the thymus is  MHC dependent. (A) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. (B) Competitive RT-PCR was used to determine the level of expression of CD4 and  Egr-1 genes in thymocytes derived from wild-type or MHC-deficient  mice. (C) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear  extracts derived from 5 × 105 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum  (NRS). For comparison, a binding reaction containing recombinant Egr-1  is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196139&req=5

Figure 5: Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. (A) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. (B) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. (C) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 105 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.

Mentions: The neonatal and adult rat thymus has been reported to express Egr-1 mRNA (37). Our results in the DPK system suggested that Egr-1 expression in normal thymus might be the result of positive selection. To address this issue, we used competitive RT-PCR to analyze Egr-1 gene expression in normal thymocytes and in thymocytes derived from double gene knockout mice that do not express class I or class II MHC molecules (39, 40). These latter animals have normal numbers of double positive thymocytes but lack single positive thymocytes due to the inability of TCR bearing immature thymocytes to undergo positive selection (Fig. 5 A). Freshly isolated thymocytes from MHC-deficient animals expressed approximately 10–20-fold less Egr-1 mRNA than normal thymocytes (Fig. 5 B). In contrast, expression of the CD4 gene was similar in both groups of animals, consistent with the expression of this coreceptor by >90% of thymocytes in both normal and MHC-deficient mice (Fig. 5 A). To analyze functional Egr-1 protein expression, gel mobility shift assays were performed using nuclear extracts derived from freshly isolated thymocytes. Consistent with the pattern of Egr-1 mRNA expression, Egr-1 DNA binding activity was detected in nuclear lysates of thymocytes derived from wild-type but not MHC-deficient animals (Fig. 5 C). This complex has an identical mobility to that obtained with recombinant Egr-1 and supershifts with an anti–Egr-1 antibody (Fig. 5 C).


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)

Egr-1 mRNA and DNA binding activity in the thymus is  MHC dependent. (A) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. (B) Competitive RT-PCR was used to determine the level of expression of CD4 and  Egr-1 genes in thymocytes derived from wild-type or MHC-deficient  mice. (C) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear  extracts derived from 5 × 105 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum  (NRS). For comparison, a binding reaction containing recombinant Egr-1  is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196139&req=5

Figure 5: Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. (A) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. (B) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. (C) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 105 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.
Mentions: The neonatal and adult rat thymus has been reported to express Egr-1 mRNA (37). Our results in the DPK system suggested that Egr-1 expression in normal thymus might be the result of positive selection. To address this issue, we used competitive RT-PCR to analyze Egr-1 gene expression in normal thymocytes and in thymocytes derived from double gene knockout mice that do not express class I or class II MHC molecules (39, 40). These latter animals have normal numbers of double positive thymocytes but lack single positive thymocytes due to the inability of TCR bearing immature thymocytes to undergo positive selection (Fig. 5 A). Freshly isolated thymocytes from MHC-deficient animals expressed approximately 10–20-fold less Egr-1 mRNA than normal thymocytes (Fig. 5 B). In contrast, expression of the CD4 gene was similar in both groups of animals, consistent with the expression of this coreceptor by >90% of thymocytes in both normal and MHC-deficient mice (Fig. 5 A). To analyze functional Egr-1 protein expression, gel mobility shift assays were performed using nuclear extracts derived from freshly isolated thymocytes. Consistent with the pattern of Egr-1 mRNA expression, Egr-1 DNA binding activity was detected in nuclear lysates of thymocytes derived from wild-type but not MHC-deficient animals (Fig. 5 C). This complex has an identical mobility to that obtained with recombinant Egr-1 and supershifts with an anti–Egr-1 antibody (Fig. 5 C).

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