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Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

Maruyama T, Li J, Vaque JP, Konkel JE, Wang W, Zhang B, Zhang P, Zamarron BF, Yu D, Wu Y, Zhuang Y, Gutkind JS, Chen W - Nat. Immunol. (2010)

Bottom Line: We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells).Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter.Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

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Enrichment of E2A binding to the Foxp3 promotera, Schematic analysis of E protein binding sites (E-Boxes) at the Foxp3 promoter. b, Relative E2A binding ability (E47/control IgG, ChIP-qPCR assay) to the indicated E boxes in TCR and TGF-β treated CD4+CD25− T cells. E2A shows strongest binding to the E-boxes located at +327/+513. c, Analysis of relative E2A binding (E47/control IgG, ChIP-qPCR assay at +327/+513) to Foxp3 promoter in WT CD4+CD25− T cells 12-24 h after TCR stimulation with or without TGF-β. Data represent mean ± s.d. of E2A binding of four independent experiments. ** P < 0.01. d, A positive correlation between E2A binding and TBP binding to the Foxp3 promoter in WT CD4+CD25− T cells at 12-24 h after TCR and TGF-β treatment. White bar indicates the cells treated with CD3- and CD28-antibodies alone; black bar indicates plus TGF-β. Data are displayed as normalized ratios of E47/control IgG or TBP/Control IgG (mean ± s.d. of duplicate wells in one representative experiment of two, ChIP-qPCR assay at +327/513). e, Relative E2A binding ability (E47/control IgG, at +327/+513) in purified CD4+CD25+ (CD25+) Treg cells and CD4+CD25− (CD25−) T cells. (mean ± s.d. of triplicate wells in one representative experiment of two).
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Figure 3: Enrichment of E2A binding to the Foxp3 promotera, Schematic analysis of E protein binding sites (E-Boxes) at the Foxp3 promoter. b, Relative E2A binding ability (E47/control IgG, ChIP-qPCR assay) to the indicated E boxes in TCR and TGF-β treated CD4+CD25− T cells. E2A shows strongest binding to the E-boxes located at +327/+513. c, Analysis of relative E2A binding (E47/control IgG, ChIP-qPCR assay at +327/+513) to Foxp3 promoter in WT CD4+CD25− T cells 12-24 h after TCR stimulation with or without TGF-β. Data represent mean ± s.d. of E2A binding of four independent experiments. ** P < 0.01. d, A positive correlation between E2A binding and TBP binding to the Foxp3 promoter in WT CD4+CD25− T cells at 12-24 h after TCR and TGF-β treatment. White bar indicates the cells treated with CD3- and CD28-antibodies alone; black bar indicates plus TGF-β. Data are displayed as normalized ratios of E47/control IgG or TBP/Control IgG (mean ± s.d. of duplicate wells in one representative experiment of two, ChIP-qPCR assay at +327/513). e, Relative E2A binding ability (E47/control IgG, at +327/+513) in purified CD4+CD25+ (CD25+) Treg cells and CD4+CD25− (CD25−) T cells. (mean ± s.d. of triplicate wells in one representative experiment of two).

Mentions: Id3 lacks the basic region needed for DNA binding but retains the functional dimerization domain, and therefore is regarded as an inhibitor for basic HLH protein E2A binding to its target genes by forming inactive heterodimers24. We therefore hypothesized that TGF-β1 might influence E2A binding to the Foxp3 promoter and thence regulate gene transcription. E2A proteins (E47 and E12) possess a DNA-binding domain and bind the CANN TG (E-box) DNA motif as protein dimmers to regulate gene transcription during the development of both T and B cells24. We first analyzed Foxp3 with the help of Genomatix® matinspector software and found that the sequence between −1.1kb and +1.1kb with respect to the transcription initiation site (+1), contained multiple E-box elements with a canonical nucleotide sequence (CANNTG), which are expected to bind members of the E protein family of transcription factors31-34 (Fig. 3a). Using naïve CD4+CD25− T cells isolated from spleens of WT mice, we analyzed whether TGF-β1 influenced E2A binding to the Foxp3 promoter. We treated CD4+ CD25− T cells with CD3- and CD28-specific antibodies and TGF-β1 overnight and determined E2A binding to the Foxp3 promoter with chromatin immunoprecipitation (ChIP)-coupled quantitative PCR (qPCR) assay. ChIP-qPCR was performed with an antibody against E47, a major E2A protein component24, to precipitate chromatin from CD4+CD25− T cells, followed by qPCR analysis of the precipitated DNA. To determine where E2A binds in the Foxp3 gene, we analyzed three different regions of the Foxp3 gene; 1) the 5′ promoter region (amplicon 1; −995/−798), 2) the proximal promoter plus a region comprising the untranslated 5′mRNA (amplicon 2; +327/+513), and 3) the region located next to the enhancer containing the Smad-binding domain (amplicon 3; +2335/+2532)15 (Fig. 3a). We observed increased binding of E2A to a regulatory region of Foxp3 comprising the proximal promoter and its untranslated 5′ mRNA region (+327/+513, Fig. 3b). We then quantitatively compared the relative E2A binding to this region in the Foxp3 gene in T cells treated with TCR alone and TCR plus TGF-β1 and found that TGF-β1 significantly enhanced E2A binding to the promoter compared to the control (Fig. 3c). TGF-β1-driven enhancement of E2A binding to the Foxp3 promoter occurred 12-24 h following TGF-β1 stimulation, which correlated with the detected expression of Foxp3 mRNA (>12h) in TGF-β1 treated T cells (data not shown). This enrichment of E2A binding is probably not due to the changes in protein levels, as neither treatment significantly affected whole E2A protein expression levels (Supplementary Fig. 6a). Increased E2A binding was associated with TBP (TATA binding protein) binding, a marker of active transcription, within the Foxp3 promoter, strongly suggesting that both factors were recruited to this Foxp3 gene regulatory region (Fig. 3d). We validated the essential role of TGF-β signaling in promoting E2A binding to the Foxp3 promoter by utilizing Tgfbr1f/fCd4-cre+ CD4+CD25− T cells that lack TGF-β signaling. There was no upregulation of E2A binding to the Foxp3 promoter in response to TGF-β1 treatment (Supplementary Fig. 6b). In addition to TGF-β1-treated naïve CD4+ T cells, freshly isolated CD4+CD25+ Treg cells from WT spleen also showed enrichment of E2A binding at the Foxp3 promoter compared to CD4+CD25− T cells (Fig. 3e). These data collectively support a positive role of E2A in the activation of Foxp3 transcription in response to TGF-β1 treatment, which can be explained by the direct binding of E2A to the E-box rich region within the regulatory region of the Foxp3.


Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

Maruyama T, Li J, Vaque JP, Konkel JE, Wang W, Zhang B, Zhang P, Zamarron BF, Yu D, Wu Y, Zhuang Y, Gutkind JS, Chen W - Nat. Immunol. (2010)

Enrichment of E2A binding to the Foxp3 promotera, Schematic analysis of E protein binding sites (E-Boxes) at the Foxp3 promoter. b, Relative E2A binding ability (E47/control IgG, ChIP-qPCR assay) to the indicated E boxes in TCR and TGF-β treated CD4+CD25− T cells. E2A shows strongest binding to the E-boxes located at +327/+513. c, Analysis of relative E2A binding (E47/control IgG, ChIP-qPCR assay at +327/+513) to Foxp3 promoter in WT CD4+CD25− T cells 12-24 h after TCR stimulation with or without TGF-β. Data represent mean ± s.d. of E2A binding of four independent experiments. ** P < 0.01. d, A positive correlation between E2A binding and TBP binding to the Foxp3 promoter in WT CD4+CD25− T cells at 12-24 h after TCR and TGF-β treatment. White bar indicates the cells treated with CD3- and CD28-antibodies alone; black bar indicates plus TGF-β. Data are displayed as normalized ratios of E47/control IgG or TBP/Control IgG (mean ± s.d. of duplicate wells in one representative experiment of two, ChIP-qPCR assay at +327/513). e, Relative E2A binding ability (E47/control IgG, at +327/+513) in purified CD4+CD25+ (CD25+) Treg cells and CD4+CD25− (CD25−) T cells. (mean ± s.d. of triplicate wells in one representative experiment of two).
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Figure 3: Enrichment of E2A binding to the Foxp3 promotera, Schematic analysis of E protein binding sites (E-Boxes) at the Foxp3 promoter. b, Relative E2A binding ability (E47/control IgG, ChIP-qPCR assay) to the indicated E boxes in TCR and TGF-β treated CD4+CD25− T cells. E2A shows strongest binding to the E-boxes located at +327/+513. c, Analysis of relative E2A binding (E47/control IgG, ChIP-qPCR assay at +327/+513) to Foxp3 promoter in WT CD4+CD25− T cells 12-24 h after TCR stimulation with or without TGF-β. Data represent mean ± s.d. of E2A binding of four independent experiments. ** P < 0.01. d, A positive correlation between E2A binding and TBP binding to the Foxp3 promoter in WT CD4+CD25− T cells at 12-24 h after TCR and TGF-β treatment. White bar indicates the cells treated with CD3- and CD28-antibodies alone; black bar indicates plus TGF-β. Data are displayed as normalized ratios of E47/control IgG or TBP/Control IgG (mean ± s.d. of duplicate wells in one representative experiment of two, ChIP-qPCR assay at +327/513). e, Relative E2A binding ability (E47/control IgG, at +327/+513) in purified CD4+CD25+ (CD25+) Treg cells and CD4+CD25− (CD25−) T cells. (mean ± s.d. of triplicate wells in one representative experiment of two).
Mentions: Id3 lacks the basic region needed for DNA binding but retains the functional dimerization domain, and therefore is regarded as an inhibitor for basic HLH protein E2A binding to its target genes by forming inactive heterodimers24. We therefore hypothesized that TGF-β1 might influence E2A binding to the Foxp3 promoter and thence regulate gene transcription. E2A proteins (E47 and E12) possess a DNA-binding domain and bind the CANN TG (E-box) DNA motif as protein dimmers to regulate gene transcription during the development of both T and B cells24. We first analyzed Foxp3 with the help of Genomatix® matinspector software and found that the sequence between −1.1kb and +1.1kb with respect to the transcription initiation site (+1), contained multiple E-box elements with a canonical nucleotide sequence (CANNTG), which are expected to bind members of the E protein family of transcription factors31-34 (Fig. 3a). Using naïve CD4+CD25− T cells isolated from spleens of WT mice, we analyzed whether TGF-β1 influenced E2A binding to the Foxp3 promoter. We treated CD4+ CD25− T cells with CD3- and CD28-specific antibodies and TGF-β1 overnight and determined E2A binding to the Foxp3 promoter with chromatin immunoprecipitation (ChIP)-coupled quantitative PCR (qPCR) assay. ChIP-qPCR was performed with an antibody against E47, a major E2A protein component24, to precipitate chromatin from CD4+CD25− T cells, followed by qPCR analysis of the precipitated DNA. To determine where E2A binds in the Foxp3 gene, we analyzed three different regions of the Foxp3 gene; 1) the 5′ promoter region (amplicon 1; −995/−798), 2) the proximal promoter plus a region comprising the untranslated 5′mRNA (amplicon 2; +327/+513), and 3) the region located next to the enhancer containing the Smad-binding domain (amplicon 3; +2335/+2532)15 (Fig. 3a). We observed increased binding of E2A to a regulatory region of Foxp3 comprising the proximal promoter and its untranslated 5′ mRNA region (+327/+513, Fig. 3b). We then quantitatively compared the relative E2A binding to this region in the Foxp3 gene in T cells treated with TCR alone and TCR plus TGF-β1 and found that TGF-β1 significantly enhanced E2A binding to the promoter compared to the control (Fig. 3c). TGF-β1-driven enhancement of E2A binding to the Foxp3 promoter occurred 12-24 h following TGF-β1 stimulation, which correlated with the detected expression of Foxp3 mRNA (>12h) in TGF-β1 treated T cells (data not shown). This enrichment of E2A binding is probably not due to the changes in protein levels, as neither treatment significantly affected whole E2A protein expression levels (Supplementary Fig. 6a). Increased E2A binding was associated with TBP (TATA binding protein) binding, a marker of active transcription, within the Foxp3 promoter, strongly suggesting that both factors were recruited to this Foxp3 gene regulatory region (Fig. 3d). We validated the essential role of TGF-β signaling in promoting E2A binding to the Foxp3 promoter by utilizing Tgfbr1f/fCd4-cre+ CD4+CD25− T cells that lack TGF-β signaling. There was no upregulation of E2A binding to the Foxp3 promoter in response to TGF-β1 treatment (Supplementary Fig. 6b). In addition to TGF-β1-treated naïve CD4+ T cells, freshly isolated CD4+CD25+ Treg cells from WT spleen also showed enrichment of E2A binding at the Foxp3 promoter compared to CD4+CD25− T cells (Fig. 3e). These data collectively support a positive role of E2A in the activation of Foxp3 transcription in response to TGF-β1 treatment, which can be explained by the direct binding of E2A to the E-box rich region within the regulatory region of the Foxp3.

Bottom Line: We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells).Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter.Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

Show MeSH
Related in: MedlinePlus