Control of CD4 gene expression: connecting signals to outcomes in T cell development

The control of CD4 gene expression is essential for proper T lymphocyte development. Signals transmitted from the T-cell antigen receptor (TCR) during the thymic selection processes are believed to be linked to the regulation of CD4 gene expression during specific stages of T cell development. Thus, a study of the factors that control CD4 gene expression may lead to further insight into the molecular mechanisms that drive thymic selection. In this review, we discuss the work conducted to date to identify and characterize the cis-acting transcriptional control elements in the CD4 locus and the DNA-binding factors that mediate their function. From these studies, it is becoming clear that the molecular mechanisms controlling CD4 gene expression are very complex and differ at each stage of development. Thus, the control of CD4 expression is subject to many different influences as the thymocyte develops.

CD4; gene expression; silencer; T cell; development

Authorship

S.D. Sarafova

Columbia University College of Physicians and Surgeons, Department of Microbiology and the Integrated Program in Cellular, Molecular and Biophysical Studies, New York, NY, USAColumbia University College of Physicians and SurgeonsUSANew York, NY, USAColumbia University College of Physicians and Surgeons, Department of Microbiology and the Integrated Program in Cellular, Molecular and Biophysical Studies, New York, NY, USA

G. Siu

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (11)

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Figure 1
- Antigen recognition by helper T (T_H) and cytotoxic T (T_C) cells. Antigen-presenting cells (APC) are shown below. See text for details. TCR, T-cell antigen receptor.

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Figure 2
- T cell development in the thymus.

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Figure 3
- The CD4 gene locus. A DNAse hypersensitivity (DH) map of the CD4 gene locus is presented. The DH sites are indicated by numbered arrows. The direction of CD4 gene transcription is indicated by a horizontal arrow. The identified transcriptional control elements are represented by striped boxes. The CD4 gene exons are represented by black boxes.

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Figure 4
- A transgenic approach for studying control of gene expression. A construct with a marker gene (labeled marker) under the control of different combinations of CD4 control elements, represented by boxes, is introduced into fertilized eggs by microinjection and the eggs are implanted into pseudo-pregnant foster mothers. Transgenic progeny are identified and their peripheral lymphocytes analyzed by flow cytometry to determine expression patterns of the marker gene in different lymphocyte populations (CD4 SP populations are shown). The dashed line represents an isotype-matched control antibody staining. The solid line represents the marker gene antibody staining.

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Figure 5
- Schematic representation of the transcription control elements of CD4 and their factors. The binding sites within a control element are designated by a letter and a number: P for promoter sites, E for mature enhancer sites, and S for silencer sites. Interacting factors are indicated above their binding sites. A question mark is used when the factor is unknown. Data were from the following references: for the promoter (13,21,23), for the mature enhancer (28; Adlam M, Allen RD & Siu G, unpublished data), and for the silencer (32,34).

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Figure 6
- Representative analysis of pTG mice. Thymocytes from mice transgenic for a construct, containing the CD4 promoter and the CD4 mature enhancer, were stained with antibodies to the CD4, CD8 and TG markers, and the cell-surface markers indicated and analyzed by flow cytometry. CD8⁺ cells were eliminated from the analysis and the remaining cells were plotted in two-color flow cytometric plots with CD4 expression on the x-axis and TG marker expression on the y-axis (left panel). The marker-positive (CD4 SP/M⁺) and marker-negative (CD4 SP/M^-) CD4 SP thymocytes were gated on and analyzed for expression of CD3, heat stable antigen (HSA), CD69, and H-2K and compared to staining in the complete CD4 SP population (CD4 SP). The CD4 SP/M^- population stains HSA⁺, CD69⁺, and H-2K^lo, consistent with a CD4 SP thymocyte undergoing positive selection. In contrast, the CD4 SP/M⁺ population is HSA^lo, CD69^lo, and H-2K^hi, consistent with a CD4 SP thymocyte that has successfully survived positive selection. Data taken from Adlam et al. (24); see references therein.

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Figure 7
- The transgenic constructs used to identify the thymocyte enhancer. A, Transgenic constructs. Gray boxes - marker gene; open boxes - transcriptional control elements; arrows - additional DNAse hypersensitivity (DH) sites present, as identified originally in Figure 3. B, Histograms depicting marker expression in CD4 SP and double-positive (DP) thymocytes, using the constructs from A. Solid line - marker expression; dashed line - isotype-matched control. The constructs which contain DH sites 11-17 (cosC and lB) have clear marker expression in the DP thymocytes, while the constructs that do not contain DH sites 11-17 (pA5UA, pF and pTG) have no expression in the DP thymocytes and only a portion of the CD4 SP thymocytes express the marker gene. Data are from Adlam et al. (24).

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Figure 8
- Functional redundancy of the silencer binding sites. FACS analysis of marker expression in peripheral lymphocytes is shown for CD4 SP and CD8 SP cells. The constructs contain the marker gene driven by the CD4 promoter and enhancer and either the full-length silencer (pTGSil), no silencer (pTG), or silencers with different sites deleted as indicated above each plot. The solid line represents the marker stain; the dashed line represents the isotype-matched control stain. A marker-positive population in the CD8 SP cells indicates a break in silencing. No break of silencing is observed in the CD8 SP population in the single-site deletion silencer mutants (D1, D2 and D3). The lower panels show the double and triple deletions of the binding sites; all break silencing in the CD8 SP population with the exception of D1/3. Data are from Duncan et al. (31).

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Figure 9
- Possible models for subclass specific regulation using ubiquitous or nonsubclass specific factors. For simplicity, only one of the numerous possibilities is shown in each case. A, Phosphorylation determines the presence or absence of activity of the factor. B, Sequestration of the factor away from the nucleus (top) or away from its binding site (bottom) prevents it from participation in the element regulation. C, Association with yet unidentified subclass-specific cofactor(s) determine(s) the functionality of the element. D, Different combinations of cofactors occur at different stages of T cell development to positively or negatively regulate an element's function. E, The same factor binds to different configurations of its binding site and adopts different conformations, which allow interactions with different cofactors. Arrow: transcription; crossed-out arrow: lack of transcription; ovals and boxes: different transcription factors except in top part of B, where ovals represent a cell and its nucleus.

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Figure 10
- Activity of the transcriptional control elements throughout T cell development. A timeline of T cell development is presented with the phenotypes of the cells indicated as in Figure 2. For each stage, the activity of each transcriptional element is indicated under it. Plus represents an active state and minus represents an inactive state, except for the promoter, where a minus represents a basal transcription activity. See text for details.

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Figure 11
- Comparison between the organization of CD4 and bithorax gene loci. Exons are represented by vertical lines and control elements are represented by boxes. The genes are indicated above together with the direction of their transcription (horizontal arrows). The regulatory elements are indicated below with arrows pointing to the boxes for clarity.

Figure 1 - Antigen recognition by helper T (T_H) and cytotoxic T (T_C) cells. Antigen-presenting cells (APC) are shown below. See text for details. TCR, T-cell antigen receptor.

Figure 2 - T cell development in the thymus.

Figure 3 - The CD4 gene locus. A DNAse hypersensitivity (DH) map of the CD4 gene locus is presented. The DH sites are indicated by numbered arrows. The direction of CD4 gene transcription is indicated by a horizontal arrow. The identified transcriptional control elements are represented by striped boxes. The CD4 gene exons are represented by black boxes.

Figure 4 - A transgenic approach for studying control of gene expression. A construct with a marker gene (labeled marker) under the control of different combinations of CD4 control elements, represented by boxes, is introduced into fertilized eggs by microinjection and the eggs are implanted into pseudo-pregnant foster mothers. Transgenic progeny are identified and their peripheral lymphocytes analyzed by flow cytometry to determine expression patterns of the marker gene in different lymphocyte populations (CD4 SP populations are shown). The dashed line represents an isotype-matched control antibody staining. The solid line represents the marker gene antibody staining.

Figure 5 - Schematic representation of the transcription control elements of CD4 and their factors. The binding sites within a control element are designated by a letter and a number: P for promoter sites, E for mature enhancer sites, and S for silencer sites. Interacting factors are indicated above their binding sites. A question mark is used when the factor is unknown. Data were from the following references: for the promoter (13,21,23), for the mature enhancer (28; Adlam M, Allen RD & Siu G, unpublished data), and for the silencer (32,34).

Figure 6 - Representative analysis of pTG mice. Thymocytes from mice transgenic for a construct, containing the CD4 promoter and the CD4 mature enhancer, were stained with antibodies to the CD4, CD8 and TG markers, and the cell-surface markers indicated and analyzed by flow cytometry. CD8⁺ cells were eliminated from the analysis and the remaining cells were plotted in two-color flow cytometric plots with CD4 expression on the x-axis and TG marker expression on the y-axis (left panel). The marker-positive (CD4 SP/M⁺) and marker-negative (CD4 SP/M^-) CD4 SP thymocytes were gated on and analyzed for expression of CD3, heat stable antigen (HSA), CD69, and H-2K and compared to staining in the complete CD4 SP population (CD4 SP). The CD4 SP/M^- population stains HSA⁺, CD69⁺, and H-2K^lo, consistent with a CD4 SP thymocyte undergoing positive selection. In contrast, the CD4 SP/M⁺ population is HSA^lo, CD69^lo, and H-2K^hi, consistent with a CD4 SP thymocyte that has successfully survived positive selection. Data taken from Adlam et al. (24); see references therein.

Figure 7 - The transgenic constructs used to identify the thymocyte enhancer. A, Transgenic constructs. Gray boxes - marker gene; open boxes - transcriptional control elements; arrows - additional DNAse hypersensitivity (DH) sites present, as identified originally in Figure 3. B, Histograms depicting marker expression in CD4 SP and double-positive (DP) thymocytes, using the constructs from A. Solid line - marker expression; dashed line - isotype-matched control. The constructs which contain DH sites 11-17 (cosC and lB) have clear marker expression in the DP thymocytes, while the constructs that do not contain DH sites 11-17 (pA5UA, pF and pTG) have no expression in the DP thymocytes and only a portion of the CD4 SP thymocytes express the marker gene. Data are from Adlam et al. (24).

Figure 8 - Functional redundancy of the silencer binding sites. FACS analysis of marker expression in peripheral lymphocytes is shown for CD4 SP and CD8 SP cells. The constructs contain the marker gene driven by the CD4 promoter and enhancer and either the full-length silencer (pTGSil), no silencer (pTG), or silencers with different sites deleted as indicated above each plot. The solid line represents the marker stain; the dashed line represents the isotype-matched control stain. A marker-positive population in the CD8 SP cells indicates a break in silencing. No break of silencing is observed in the CD8 SP population in the single-site deletion silencer mutants (D1, D2 and D3). The lower panels show the double and triple deletions of the binding sites; all break silencing in the CD8 SP population with the exception of D1/3. Data are from Duncan et al. (31).

Figure 9 - Possible models for subclass specific regulation using ubiquitous or nonsubclass specific factors. For simplicity, only one of the numerous possibilities is shown in each case. A, Phosphorylation determines the presence or absence of activity of the factor. B, Sequestration of the factor away from the nucleus (top) or away from its binding site (bottom) prevents it from participation in the element regulation. C, Association with yet unidentified subclass-specific cofactor(s) determine(s) the functionality of the element. D, Different combinations of cofactors occur at different stages of T cell development to positively or negatively regulate an element's function. E, The same factor binds to different configurations of its binding site and adopts different conformations, which allow interactions with different cofactors. Arrow: transcription; crossed-out arrow: lack of transcription; ovals and boxes: different transcription factors except in top part of B, where ovals represent a cell and its nucleus.

Figure 10 - Activity of the transcriptional control elements throughout T cell development. A timeline of T cell development is presented with the phenotypes of the cells indicated as in Figure 2. For each stage, the activity of each transcriptional element is indicated under it. Plus represents an active state and minus represents an inactive state, except for the promoter, where a minus represents a basal transcription activity. See text for details.

Figure 11 - Comparison between the organization of CD4 and bithorax gene loci. Exons are represented by vertical lines and control elements are represented by boxes. The genes are indicated above together with the direction of their transcription (horizontal arrows). The regulatory elements are indicated below with arrows pointing to the boxes for clarity.

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Control of CD4 gene expression: connecting signals to outcomes in T cell development

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