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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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EMSAs using the S3 probe and CD8 SP Tc L3 nuclear extracts (A), whole cell extracts from the CD4 SP Th clone D10, the DP thymoma AKR1G1, and the DN thymoma S49 (B), or whole cell extracts from D10 and L3 (C). Arrows indicate putative SAF-containing complex; addition of increasing amounts of different nonradioactive competitor oligonucleotides are indicated above each lane.
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Figure 2: EMSAs using the S3 probe and CD8 SP Tc L3 nuclear extracts (A), whole cell extracts from the CD4 SP Th clone D10, the DP thymoma AKR1G1, and the DN thymoma S49 (B), or whole cell extracts from D10 and L3 (C). Arrows indicate putative SAF-containing complex; addition of increasing amounts of different nonradioactive competitor oligonucleotides are indicated above each lane.

Mentions: In previous studies, we identified three factor binding sites in the CD4 silencer using DNAse footprinting. Additional EMSA analyses identified one major and several minor factor–DNA complexes when the footprinted S3 region was used as a radioactive probe; we have concentrated our further analyses on the major S3 binding complex 11. Using o-phenanthroline copper footprinting, we narrowed the recognition site of the major S3 binding factor to a 16-bp region (reference 11 and Fig. 1). This region contains a 5-bp direct repeat (CTGTG) separated by 6 bp. A comparison of this 16-bp region with known binding site motifs revealed consensus LEF-1 26272829 and ETS 30 recognition sites; however, we were unable to demonstrate that either LEF-1 or an ETS family protein binds to S3 using biochemical approaches (data not shown). To identify a more precise recognition site for the major endogenous S3 binding factor, we designed a series of mutant S3 oligonucleotides to be used as competitors in EMSAs (Fig. 1 A). As we have reported previously 11, we can detect a major DNA–protein complex with the 16-bp S3 probe using nuclear extracts isolated from CD4−CD8+ Tc cells; complex formation can be completely inhibited by the addition of nonradioactive S3 probe but not linker, indicating that the S3 binding factor binds specifically to the S3 probe (Fig. 2 A). Oligonucleotides that contain mutations in both CTGTG repeats failed to compete away the major S3 binding complex (Fig. 1 and Fig. 2, M1 and M4). However, those containing mutations in either of the CTGTG repeats are still capable of competing for S3 complex formation (Fig. 1 and Fig. 2, M5 and M6). Oligonucleotides that contain mutations in sequences between the two CTGTG repeats also compete for complex formation efficiently (Fig. 1 and Fig. 2, M3; Fig. 1, M2; and data not shown); oligonucleotides that contain mutations in the spacer sequence directly adjacent to the CTGTG repeats also compete for complex formation, albeit somewhat less efficiently than do oligonucleotides with central mutations (Fig. 1 and Fig. 2; compare M7 to M2 and M3). We can detect three complexes with the S3 probe; the two slower mobility complexes appear to have similar sequence specificities and thus may represent modified versions of the same factor, whereas the fastest mobility complex does not appear to be reproducible from experiment to experiment (Fig. 2 A and data not shown). We can also detect S3 binding complexes in whole cell extracts purified from the D10 (CD4+CD8− Th), S49 (DN thymoma), AKR1G1 (DP thymoma), and the L3 and B18 (CD4−CD8+ SP Tc) T cell clones (Fig. 2b and Fig. c, and data not shown). Our data thus indicate that S3 binding proteins are present in T cells of all developmental phenotypes and bind to one of the two CTGTG direct repeats in the S3 probe.


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

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

EMSAs using the S3 probe and CD8 SP Tc L3 nuclear extracts (A), whole cell extracts from the CD4 SP Th clone D10, the DP thymoma AKR1G1, and the DN thymoma S49 (B), or whole cell extracts from D10 and L3 (C). Arrows indicate putative SAF-containing complex; addition of increasing amounts of different nonradioactive competitor oligonucleotides are indicated above each lane.
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Related In: Results  -  Collection

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Figure 2: EMSAs using the S3 probe and CD8 SP Tc L3 nuclear extracts (A), whole cell extracts from the CD4 SP Th clone D10, the DP thymoma AKR1G1, and the DN thymoma S49 (B), or whole cell extracts from D10 and L3 (C). Arrows indicate putative SAF-containing complex; addition of increasing amounts of different nonradioactive competitor oligonucleotides are indicated above each lane.
Mentions: In previous studies, we identified three factor binding sites in the CD4 silencer using DNAse footprinting. Additional EMSA analyses identified one major and several minor factor–DNA complexes when the footprinted S3 region was used as a radioactive probe; we have concentrated our further analyses on the major S3 binding complex 11. Using o-phenanthroline copper footprinting, we narrowed the recognition site of the major S3 binding factor to a 16-bp region (reference 11 and Fig. 1). This region contains a 5-bp direct repeat (CTGTG) separated by 6 bp. A comparison of this 16-bp region with known binding site motifs revealed consensus LEF-1 26272829 and ETS 30 recognition sites; however, we were unable to demonstrate that either LEF-1 or an ETS family protein binds to S3 using biochemical approaches (data not shown). To identify a more precise recognition site for the major endogenous S3 binding factor, we designed a series of mutant S3 oligonucleotides to be used as competitors in EMSAs (Fig. 1 A). As we have reported previously 11, we can detect a major DNA–protein complex with the 16-bp S3 probe using nuclear extracts isolated from CD4−CD8+ Tc cells; complex formation can be completely inhibited by the addition of nonradioactive S3 probe but not linker, indicating that the S3 binding factor binds specifically to the S3 probe (Fig. 2 A). Oligonucleotides that contain mutations in both CTGTG repeats failed to compete away the major S3 binding complex (Fig. 1 and Fig. 2, M1 and M4). However, those containing mutations in either of the CTGTG repeats are still capable of competing for S3 complex formation (Fig. 1 and Fig. 2, M5 and M6). Oligonucleotides that contain mutations in sequences between the two CTGTG repeats also compete for complex formation efficiently (Fig. 1 and Fig. 2, M3; Fig. 1, M2; and data not shown); oligonucleotides that contain mutations in the spacer sequence directly adjacent to the CTGTG repeats also compete for complex formation, albeit somewhat less efficiently than do oligonucleotides with central mutations (Fig. 1 and Fig. 2; compare M7 to M2 and M3). We can detect three complexes with the S3 probe; the two slower mobility complexes appear to have similar sequence specificities and thus may represent modified versions of the same factor, whereas the fastest mobility complex does not appear to be reproducible from experiment to experiment (Fig. 2 A and data not shown). We can also detect S3 binding complexes in whole cell extracts purified from the D10 (CD4+CD8− Th), S49 (DN thymoma), AKR1G1 (DP thymoma), and the L3 and B18 (CD4−CD8+ SP Tc) T cell clones (Fig. 2b and Fig. c, and data not shown). Our data thus indicate that S3 binding proteins are present in T cells of all developmental phenotypes and bind to one of the two CTGTG direct repeats in the S3 probe.

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