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Subclass-specific nuclear localization of a novel CD4 silencer binding factor.

Kim WW, Siu G - J. Exp. Med. (1999)

Bottom Line: This factor, referred to as silencer-associated factor (SAF), is a member of the helix-turn-helix factor family and shares sequence similarity with the homeodomain class of transcriptional regulators.Introduction of a specific mutation into the SAF binding site in the CD4 silencer abrogates silencer activity in transgenic mice, supporting the hypothesis that SAF is important in mediating silencer function.We thus hypothesize that the subclass-specific subcellular compartmentalization of SAF plays an important role in mediating the specificity of function of the CD4 silencer during T cell development.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and the Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
The control of CD4 expression is essential for proper T lymphocyte development. We have previously described a cis-acting silencer element required for repressing transcription of the CD4 gene. Here we report the cloning and characterization of a novel factor that binds to a critical functional site in the CD4 silencer. This factor, referred to as silencer-associated factor (SAF), is a member of the helix-turn-helix factor family and shares sequence similarity with the homeodomain class of transcriptional regulators. Introduction of a specific mutation into the SAF binding site in the CD4 silencer abrogates silencer activity in transgenic mice, supporting the hypothesis that SAF is important in mediating silencer function. Although SAF is expressed in all lymphocytes, immunofluorescence studies indicate that SAF is present primarily in the cytoplasm in T cells in which the endogenous silencer is nonfunctional, whereas it is present primarily in the nucleus in T cells in which the silencer is functional. We thus hypothesize that the subclass-specific subcellular compartmentalization of SAF plays an important role in mediating the specificity of function of the CD4 silencer during T cell development.

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Expression and binding of endogenous SAF. (A) Western blot analysis with the SAF antisera (top) and the control anti–β-actin antibody (bottom). Western blot analysis on whole cell extracts from 293T cells (lane 1): 293T cells transfected with 5, 10, and 20 μg of the CMV–SAF expression construct (lanes 2–4); the CD4+CD8+ DP AKR1G1 thymoma (lane 6); the CD8+ SP Tc L3 cell clone (lane 7); the CD4+ SP Th D10 cell clone (lanes 8 and 9); the P3X63 plasmacytoma (lane 10); the 103 pre-B cell lymphoma (lane 11); the WEHI-3B macrophage/monocyte (lane 12); and the 3T3 fibroblast (lane 13). Molecular weight standards were loaded in lane 5. (B) Antibody ablation EMSA analyses with the SAF antisera and (left) CD4+ SP Th D10 or (right) CD8+ SP Tc L3 extracts. Lanes containing S3 probe only, S3 probe with extract, and S3 probe with extract and antibody are indicated above each lane. ‘Pre’ indicates preimmune sera.
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Figure 5: Expression and binding of endogenous SAF. (A) Western blot analysis with the SAF antisera (top) and the control anti–β-actin antibody (bottom). Western blot analysis on whole cell extracts from 293T cells (lane 1): 293T cells transfected with 5, 10, and 20 μg of the CMV–SAF expression construct (lanes 2–4); the CD4+CD8+ DP AKR1G1 thymoma (lane 6); the CD8+ SP Tc L3 cell clone (lane 7); the CD4+ SP Th D10 cell clone (lanes 8 and 9); the P3X63 plasmacytoma (lane 10); the 103 pre-B cell lymphoma (lane 11); the WEHI-3B macrophage/monocyte (lane 12); and the 3T3 fibroblast (lane 13). Molecular weight standards were loaded in lane 5. (B) Antibody ablation EMSA analyses with the SAF antisera and (left) CD4+ SP Th D10 or (right) CD8+ SP Tc L3 extracts. Lanes containing S3 probe only, S3 probe with extract, and S3 probe with extract and antibody are indicated above each lane. ‘Pre’ indicates preimmune sera.

Mentions: To characterize SAF in greater detail, we generated a rabbit polyclonal antisera against SAF using the GST–SAF89–123 fusion protein as antigen. The specificity of the antisera was tested by Western blot analyses using whole cell extracts purified from a variety of cells of different phenotypes (Fig. 5 A). We can detect the induction of expression of a 14-kD species in 293T cells transfected with a CMV expression vector containing the full-length SAF cDNA, supporting the hypothesis that the 369-bp ORF is indeed the reading frame used in vivo (Fig. 5 A). In addition, we can detect the same 14-kD species in all T cell subclasses, B cells, macrophages, and fibroblasts (Fig. 5 A), indicating a wide tissue distribution of expression of SAF protein, consistent with the EMSA data discussed above. To prove that the endogenous S3 binding protein is indeed SAF, we tested whether the SAF antisera would affect endogenous S3 binding factor–DNA complex formation in EMSAs. As can be seen in Fig. 5 B, we can ablate S3–protein complex formation completely in both CD4 SP Th and CD8 SP Tc cell extracts using the SAF antisera. We cannot inhibit S3–protein complex formation significantly using either the preimmune sera or an antibody directed against Elf-1, indicating that this ablation is specific for the endogenous S3 binding protein. These data indicate that the endogenous S3 binding protein shares antigenic epitopes with SAF, supporting the hypothesis that SAF is the endogenous S3 binding factor.


Subclass-specific nuclear localization of a novel CD4 silencer binding factor.

Kim WW, Siu G - J. Exp. Med. (1999)

Expression and binding of endogenous SAF. (A) Western blot analysis with the SAF antisera (top) and the control anti–β-actin antibody (bottom). Western blot analysis on whole cell extracts from 293T cells (lane 1): 293T cells transfected with 5, 10, and 20 μg of the CMV–SAF expression construct (lanes 2–4); the CD4+CD8+ DP AKR1G1 thymoma (lane 6); the CD8+ SP Tc L3 cell clone (lane 7); the CD4+ SP Th D10 cell clone (lanes 8 and 9); the P3X63 plasmacytoma (lane 10); the 103 pre-B cell lymphoma (lane 11); the WEHI-3B macrophage/monocyte (lane 12); and the 3T3 fibroblast (lane 13). Molecular weight standards were loaded in lane 5. (B) Antibody ablation EMSA analyses with the SAF antisera and (left) CD4+ SP Th D10 or (right) CD8+ SP Tc L3 extracts. Lanes containing S3 probe only, S3 probe with extract, and S3 probe with extract and antibody are indicated above each lane. ‘Pre’ indicates preimmune sera.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195567&req=5

Figure 5: Expression and binding of endogenous SAF. (A) Western blot analysis with the SAF antisera (top) and the control anti–β-actin antibody (bottom). Western blot analysis on whole cell extracts from 293T cells (lane 1): 293T cells transfected with 5, 10, and 20 μg of the CMV–SAF expression construct (lanes 2–4); the CD4+CD8+ DP AKR1G1 thymoma (lane 6); the CD8+ SP Tc L3 cell clone (lane 7); the CD4+ SP Th D10 cell clone (lanes 8 and 9); the P3X63 plasmacytoma (lane 10); the 103 pre-B cell lymphoma (lane 11); the WEHI-3B macrophage/monocyte (lane 12); and the 3T3 fibroblast (lane 13). Molecular weight standards were loaded in lane 5. (B) Antibody ablation EMSA analyses with the SAF antisera and (left) CD4+ SP Th D10 or (right) CD8+ SP Tc L3 extracts. Lanes containing S3 probe only, S3 probe with extract, and S3 probe with extract and antibody are indicated above each lane. ‘Pre’ indicates preimmune sera.
Mentions: To characterize SAF in greater detail, we generated a rabbit polyclonal antisera against SAF using the GST–SAF89–123 fusion protein as antigen. The specificity of the antisera was tested by Western blot analyses using whole cell extracts purified from a variety of cells of different phenotypes (Fig. 5 A). We can detect the induction of expression of a 14-kD species in 293T cells transfected with a CMV expression vector containing the full-length SAF cDNA, supporting the hypothesis that the 369-bp ORF is indeed the reading frame used in vivo (Fig. 5 A). In addition, we can detect the same 14-kD species in all T cell subclasses, B cells, macrophages, and fibroblasts (Fig. 5 A), indicating a wide tissue distribution of expression of SAF protein, consistent with the EMSA data discussed above. To prove that the endogenous S3 binding protein is indeed SAF, we tested whether the SAF antisera would affect endogenous S3 binding factor–DNA complex formation in EMSAs. As can be seen in Fig. 5 B, we can ablate S3–protein complex formation completely in both CD4 SP Th and CD8 SP Tc cell extracts using the SAF antisera. We cannot inhibit S3–protein complex formation significantly using either the preimmune sera or an antibody directed against Elf-1, indicating that this ablation is specific for the endogenous S3 binding protein. These data indicate that the endogenous S3 binding protein shares antigenic epitopes with SAF, supporting the hypothesis that SAF is the endogenous S3 binding factor.

Bottom Line: This factor, referred to as silencer-associated factor (SAF), is a member of the helix-turn-helix factor family and shares sequence similarity with the homeodomain class of transcriptional regulators.Introduction of a specific mutation into the SAF binding site in the CD4 silencer abrogates silencer activity in transgenic mice, supporting the hypothesis that SAF is important in mediating silencer function.We thus hypothesize that the subclass-specific subcellular compartmentalization of SAF plays an important role in mediating the specificity of function of the CD4 silencer during T cell development.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and the Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
The control of CD4 expression is essential for proper T lymphocyte development. We have previously described a cis-acting silencer element required for repressing transcription of the CD4 gene. Here we report the cloning and characterization of a novel factor that binds to a critical functional site in the CD4 silencer. This factor, referred to as silencer-associated factor (SAF), is a member of the helix-turn-helix factor family and shares sequence similarity with the homeodomain class of transcriptional regulators. Introduction of a specific mutation into the SAF binding site in the CD4 silencer abrogates silencer activity in transgenic mice, supporting the hypothesis that SAF is important in mediating silencer function. Although SAF is expressed in all lymphocytes, immunofluorescence studies indicate that SAF is present primarily in the cytoplasm in T cells in which the endogenous silencer is nonfunctional, whereas it is present primarily in the nucleus in T cells in which the silencer is functional. We thus hypothesize that the subclass-specific subcellular compartmentalization of SAF plays an important role in mediating the specificity of function of the CD4 silencer during T cell development.

Show MeSH
Related in: MedlinePlus