Gene expression in the Gitr locus is regulated by NF-κB and Foxp3 through an enhancer

Abstract

Glucocorticoid-induced TNFR (Gitr) and Ox40, two members of the TNFR superfamily, play important roles in regulating activities of effector and regulatory T cells (Treg). Their gene expression is induced by T cell activation and further upregulated in Foxp3+ Treg. Although the role of Foxp3 as a transcriptional repressor in Treg is well established, the mechanisms underlying Foxp3-mediated transcriptional upregulation remain poorly understood. This transcription factor seems to upregulate expression not only of Gitr and Ox40, but also other genes, including Ctla4, Il35, Cd25, all critical to Treg function. To investigate how Foxp3 achieves such upregulation, we analyzed its activity on Gitr and Ox40 genes located within a 15.1-kb region. We identified an enhancer located downstream of the Gitr gene, and both Gitr and Ox40 promoter activities were shown to be upregulated by the NF-κB-mediated enhancer activity. We also show, using the Gitr promoter, that the enhancer activity was further upregulated in conjunction with Foxp3. Foxp3 appears to stabilize NF-κB p50 binding by anchoring it to the enhancer, thereby enabling local accumulation of transcriptional complexes containing other members of the NF-κB and IκB families. These findings may explain how Foxp3 can activate expression of certain genes while suppressing others.

FIGURE 1

Regulation of Gitr gene expression in non-activated (Non) and CD3-activated (CD3) T cells. (A) Gitr RNA expression levels were analyzed by qRT-PCR using RNA from non-activated and CD3-activated (activated with anti-CD3) EL4 cells and T cells. Expression levels of Gitr RNA were normalized relative to 18S ribosomal RNA levels. (B) Gitr promoter activity was analyzed by luciferase reporter assay using Gitr promoter deletion mutants. All promoter fragments contain the same 3′-ends (+46) and positions of the 5′-ends are indicated in the right hand side of the graph. The promoter activity was analyzed using EL4 under indicated conditions. (C) DNase I hypersensitive assay was performed. Nuclei from indicated cells were treated by DNase I under different concentrations [low (left) to high (right), indicated by the triangle]. DNA isolated from the nuclei was digested with Kpn I and analyzed by Southern blot hybridization using the probe shown in (D). CD3-activated T cell specific DNA fragments are indicated by an arrow (HS3). (D) The activated T cell (including EL4) specific DNase I hypersensitive sites are indicated by solid arrows (HS1, HS2 and HS3) upper the map of the Ox40 and Gitr gene locus, and common DNase I hypersensitive sites in non-activated and CD3-activated cells are indicated by arrows with dotted lines below the map. Exons are indicated by gray boxes, and position of the probe for the Southern blot hybridization is indicated by a solid line. Data are representative of three (A) or four (B) independent experiments (error bars indicate the s.d. of triplicate samples), three to four independent experiments with different restriction enzymes (C and D).

FIGURE 2

Identification of an enhancer in CD3-activated T cells and Treg. Assays were performed with non-activated (Non) and CD3-activated (CD3) cells. (A) Structures of the promoter/enhancer reporter plasmids used in B, C, D and E are illustrated. DNA fragments containing HS1, HS2 or HS3 sites were inserted downstream of the luciferase gene in both orientations (sense; S and antisense; AS) and these plasmid were used in B. The reporter plasmid containing the enhancer in sense orientation was used in C, D and E. Luciferase assays were performed using EL4 cells under indicated conditions. (B) Luciferase assays were performed using EL4 cells under indicated conditions. Luciferase reporter plasmids were constructed using the Gitr promoter as shown in A. Luciferase activities were compared with that given by the negative control vector (Basic, no promoter and no enhancer). (C) The enhancer activity was detected with both the Ox40 and the Gitr promoter. The promoter in the Gitr promoter/enhancer with HS3 (sense) plasmid (shown in A) was swapped with the Ox40 promoter, and luciferase assay was performed using the Ox40 and Gitr promoter (Pro) and the Ox40 and Gitr promoter/enhancer (Pro+HS3) reporters. (D) and (E) Luciferase assays were performed using the wild type (Wt) enhancer and 5′-deletion mutants (D) and 3′-deletion mutants (E). These deletion mutants are illustrated with the possible NF-κB sites indicated by gray boxes (κB1, κB2 and κB3). (F) Acetylation of histone H4 molecules in the chromatin region containing the κB1+κB2 and κB3 sites in CD3-activated T cells (T cells were activated by anti-CD3 for indicated times) was analyzed by ChIP using anti-acetyl-histone H4 (AcH4) or control IgG (IgG). (G) Acetylation of histone H4 was also analyzed using CD4⁺CD25⁻ T cells (CD25⁻) and nTreg as described in (F). Data are representative of more than three (B to G) independent experiments (error bars indicate the s.d. of triplicate samples).

FIGURE 3

Three NF-κB binding sites in the enhancer core sequence. (A) DNA sequence of the enhancer core (286-bp) is shown. NF-κB binding sites (κB1, κB2 and κB3) are indicated in bold. Transcription factor binding to κB1, κB2 and κB3 probes (probe positions are underlined) with or without mutations (indicated by italic upper the κB sites) were used for EMSA in (B) and Fig. 4., and enhancer activity with mutations of the κB sites were analyzed in (C). (B) Transcription factor binding to the wild type (Wt) and mutant κB probes (shown in A) were analyzed using the indicated probes and a nuclear extract from CD3-activated EL4 cells with anti-CD3 for 1.5 h. (C) Enhancer activity was analyzed using the enhancer fragment with or without mutations shown in (A). Luciferase assays were performed in Non-activated (Non) and CD3-activated (CD3) EL4 cells using the indicated plasmids, and activities were compared with the negative control plasmid (no promoter and no enhancer) (Basic). Positions of the potential NF-κB binding sites (κB1, κB2 and κB3) are indicated by gray boxes. The mutated sites are indicated by X. Data are representative of more than three (B and C) independent experiments (error bars indicate the s.d. of triplicate samples).

FIGURE 4

NF-κB binding to the enhancer. Binding of transcription factors to the enhancer region was analyzed by EMSA performed using ³²P-labeled indicated probes (Fig. 3A). (A) EMSA using nuclear extracts from non-stimulated (Non) and CD3-activated (CD3) (24 h activation) EL4 cells. (B) Competition EMSA using the κB3 probe with a nuclear extract prepared from CD3-activated (1.5 h) EL4 cells. Added cold competitors (100-fold excess) are indicated by +. (C) Super-shift EMSA using a nuclear extract prepared from CD3-activated (1.5 h) EL4 cells, indicated probes, and anti-NF-κB p50 (anti-p50) and anti-NF-κB p65 (anti-p65). To block Sp1 and Sp3 binding to the κB1 and κB2 probes, cold Sp1 competitor (100 fold) was added to the reaction mixture. Added antibodies are indicated by +. Data are representative of more than three independent experiments (A to C).

FIGURE 5

NF-κB regulates Gitr and Ox40 gene expression. (A) p50 binding to κB1+κB2 and κB3 regions in CD3-activated T cells (activated by anti-CD3 for indicated times) were analyzed by ChIP using anti-p50 (p50) and control IgG (IgG). (B) Gitr and Ox40 expression on CD4+ T cells from p50 deficient (p50 KO) and wild type (Wt) mice. Non-stimulated (Non) and CD3-activated (CD3) CD4+ T cells from indicated mice were analyzed by FACS using anti-Gitr and anti-Ox40. The percentages of CD4⁺ Gitr high or CD4⁺ Ox40 high population are indicated. (C, D and E) EL4 cells were activated with anti-CD3 and cultured with or without NF-κB activation inhibitor (NAI) concentrations (nM) are indicated. (C) Gitr, (D) Ox40 or (E) Gapdh expression were analyzed by qRT-PCR (expression levels normalized relative to 18S ribosomal RNA levels). (F) Gitr and Ox40 expression in non-activated (gray) and CD3-activated (solid line) T cells with or without NAI (100 nM) analyzed by FACS. Data are representative of more than three (A to F) independent experiments (error bars indicate the s.d. of triplicate samples).

FIGURE 6

NF-κB p50 regulates Gitr expression in Treg. (A) p50 binding to the κB1+κB2 and κB3 regions in nTreg and CD4⁺CD25⁻ T cells were analyzed by ChIP using anti-p50 (p50) and control IgG (IgG). (B) CD4⁺CD25⁻ T cells from Foxp3-GFP reporter mice were cultured with TGF-β+anti-CD3+anti-CD28 for 48 h, and Foxp3⁺ iTreg cells were isolated by sorting with GFP (93% were Foxp3+ T cells). p50 binding to the κB1+κB2 and κB3 regions in iTreg and CD4⁺CD25⁻ T cells was analyzed by ChIP using anti-p50 (p50) and control IgG (IgG). (C) Splenocytes and thymocytes were isolated from p50 deficient (p50 KO) and wild type (Wt) mice. These cells were stained with anti-CD4, anti-Foxp3, and anti-Gitr. Gitr expression in CD4⁺Foxp3⁺ population was analyzed. Data are representative of more than three (A to C) independent experiments (error bars indicate the s.d. of triplicate samples).

FIGURE 7

Binding of NF-κB p50 and Foxp3 to κB sites. (A) DNA sequences of the κB1, the Foxp3 and the CD40 NF-κB (from CD40 promoter) (CD40) competitors. NF-κB binding sequences are indicated by bold, and a potential Foxp3 binding sequence is indicated by bold italic. (B) DNA pull-down assays were performed using a biotinylated the enhancer probe containing the κB1+κB2 sites (enhancer) or the same length of the Gitr promoter probe and a nuclear extract prepared from stimulated CD4⁺CD25⁻ T cells with TGF-β+anti-CD3+anti-CD28 for 72 h. The co-precipitated proteins were analyzed by immunoblotting with anti-p50 and anti-Foxp3. (C) DNA pull down assays were performed using the κB probe and nuclear extracts prepared from non-stimulated (Non) and TGF-β+anti-CD3+anti-CD28 stimulated (72 h) CD4⁺CD25⁻ T cells (left panel). Competition DNA pull down assay was performed using the same probe and nuclear extract (from stimulated cells) and using non-biotinylated the κB1 or Foxp3 competitor (shown in A) (right panel). Co-precipitated proteins were analyzed by immunoblotting with anti-p50, anti-Foxp3, anti-p65, anti-c-Rel and anti-Bcl-3. (D) DNA pull down assays were performed with a biotinylated κB probe and the non-biotinylated CD40 NF-κB competitor (shown in A). Co-precipitated proteins were analyzed by immunoblotting with anti-p50, anti-Foxp3. (E) p50 was precipitated by anti-p50 or control IgG (without DNA), and co-precipitated FLAG-tagged Foxp3 was detected using an anti-FLAG. Data are representative of more than three (B to E) independent experiments.

FIGURE 8

NF-κB p50 and Foxp3 co-operate to upregulate enhancer activity. (A and B) The Gitr promoter-enhancer luciferase reporter plasmid (Pro8+Enhancer) was co-transfected with empty Vector, p50, and/or p65 (A) or c-Rel (B) expression plasmids with or without Foxp3 expression plasmid. Co-transfected expression plasmids are indicated under the graph. Total amount of DNA for transfection was adjusted by addition of DNA from the empty vector. Luciferase activity with negative control plasmid Basic (no promoter and no enhancer) and Pro8 (no enhancer) are also shown. *P<0.05, **P<0.005 (Student’s t-test). (C) Gitr promoter-enhancer luciferase reporter plasmid (Pro8+Enhancer) was co-transfected with 4 μg of p50 expression plasmid (p50) and different amounts of Foxp3 expression plasmid (0 to 8 μg). DNA amounts of the expression plasmids are shown under the graph. The total amount of DNA was adjusted with addition of DNA from the empty vector. (D) Model of NF-κB p50 and Foxp3 binding to the κB1 site. (E) Model of NF-κB p50, Foxp3 and NF-κB p65 or c-Rel binding to the κB site. Data are representative of more than three (A to C) independent experiments (error bars indicate the s.d. of triplicate samples).