Molecular Mechanisms Underlying FOXP3 Induction in Human T Cells

Pierre-Yves Mantel, Nadia Ouaked, Beate Rückert, Christian Karagiannidis, Roland Welz, Kurt Blaser and Carsten B. Schmidt-Weber
J Immunol March 15, 2006, 176 (6) 3593-3602; DOI: https://doi.org/10.4049/jimmunol.176.6.3593

Abstract

FOXP3 is playing an essential role for T regulatory cells and is involved in the molecular mechanisms controlling immune tolerance. Although the biological relevance of this transcription factor is well documented, the pathways responsible for its induction are still unclear. The current study reveals structure and function of the human FOXP3 promoter, revealing essential molecular mechanisms of its induction. The FOXP3 promoter was defined by RACE, cloned, and functionally analyzed using reporter-gene constructs in primary human T cells. The analysis revealed the basal, T cell-specific promoter with a TATA and CAAT box 6000 bp upstream the translation start site. The basal promoter contains six NF-AT and AP-1 binding sites, which are positively regulating the trans activation of the FOXP3 promoter after triggering of the TCR. The chromatin region containing the FOXP3 promoter was bound by NF-ATc2 under these conditions. Furthermore, FOXP3 expression was observed following TCR engagement. Promoter activity, mRNA, and protein expression of T cells were suppressed by addition of cyclosporin A. Taken together, this study reveals the structure of the human FOXP3 promoter and provides new insights in mechanisms of addressing T regulatory cell-inducing signals useful for promoting immune tolerance. Furthermore, the study identifies essential, positive regulators of the FOXP3 gene and highlights cyclosporin A as an inhibitor of FOXP3 expression contrasting other immunosuppressants such as steroids or rapamycin.

T cells play a key role in adaptive immunity and enable the immune system to develop specific immune responses. T cell activation is tightly regulated allowing responses against pathogens, while maintaining tolerance of harmless Ags. Disequilibrated immune tolerance causes autoimmune disease or allergy. Thus, immune tolerance is an important mechanism that allows to distinguish between self and nonself (1, 2). Regulatory T cells (Tregs)3 are critical regulators of immune tolerance, and their suppressive control of effector T cells was observed in experimental systems (3) and humans (4, 5). Tregs are defined by their function, and express the transcription factor FOXP3 and/or suppressive cytokines (IL-10, TGF-β) as well as CTLA-4 and/or CD25 (6, 7, 8, 9, 10). FOXP3 is a transcription factor, belonging to the forkhead family (11), and it has been shown that FOXP3, overexpressed in Jurkat cells, can act as a repressor of transcription of the IL-2 promoter by competing with the binding of NF-AT (12) or by directly interacting with NF-AT or NF-κB (13). The CD25+ Tregs express constitutively high amount of FOXP3 and represent ∼5–10% of the total CD4+ population. Despite the great relevance of these cells in immunology and clinical issues, the origin and pathways of Treg induction are still unclear. Interestingly, it could be demonstrated that ectotrophic expression of FOXP3 in T cells was sufficient to restore autoimmune symptoms of mice depleted of CD25+ T cells (14, 15). In fact, genetic defects of the human ortholog cause the immune dysregulation polyendocrinopathy enteropathy, X-linked syndrome (11). Patients with immune dysregulation polyendocrinopathy enteropathy, X-linked syndrome suffer from a neonatal onset of insulin-dependent diabetes, infections, enteropathy, thrombocytopenia and anemia, endocrinopathy, eczema, and cachexia (16), and transgenic mice lacking FOXP3 are developing a severe autoimmune disease (17, 18, 19).

These evidences indicate that FOXP3 is a gene, which is involved in the generation or maintenance of regulatory T cell phenotypes, which is essential for maintaining immune tolerance. Interestingly, it has been shown that its expression can also be induced in the CD4+CD25− population by activation (20), corticosteroids (21), estrogen (22), and TGF-β (23, 24), suggesting that FOXP3 can be induced in peripheral T cells, which may become crucial for therapeutic interventions. We therefore investigated the FOXP3 promoter to systematically reveal signals inducing FOXP3 expression.

The 5′-flanking region of the human FOXP3 gene was cloned, and the promoter activity was characterized in primary CD4+ T cells. The data demonstrate that the proximal promoter is localized in the region between −511/+176 bp upstream the 5′-noncoding region and contains several common features of basal promoter such as a GC and a TATA box. Our results demonstrate that the promoter is inducible by activation in a NF-AT-AP-1-dependent manner, which is inhibited by cyclosporin A (CsA).

Materials and Methods

Localization of the human FOXP3 promoter by RACE

The cDNA from CD4+ T cells was amplified with the anchor primer and two nested antisense primers: RACE FOXP3 + 987, RACE FOXP3 + 521 (Table I⇓) designed from the FOXP3 cDNA sequence. The PCR products were purified and cloned into pCR2.1 vector (Invitrogen Life Technologies) for sequencing of the 5′ cDNA ends.

View this table:
Table I.

Primera

Cloning of the FOXP3 promoter, and construction of deletion and mutant constructs

The human FOXP3 promoter containing −1657 bp from transcription start site (TSS) was amplified by PCR using FOXP3 promoter sequence-specific primers from position −1657 to +176. The genomic DNA extracted from CD4+ T cells of a healthy donor was used as a template. The FOXP3 promoter amplicon was cloned into the pGL3 basic vector (Promega) to generate the pGL3 FOXP3 −1611/+176. Series of deletion constructs were generated. The PCR products were subcloned in the pGL3 basic vector. Site-directed mutagenesis in the FOXP3 promoter region was introduced using the QuickChange kit (Stratagene), according to the manufacturer’s instructions. The constructs were generated by using pGL3 −511, −348, −307, or −211 as template. Primers that were used to generate the individual constructs are listed in Table I⇑.

Bioinformatics

Genomic sequences spanning the 5′ untranslated region (UTR) of the FOXP3 gene were analyzed using the alignment software m-Vista: 〈www.gsd.lbl.gov/vista/VistaInput〉 (25), allowing to identify conserved regions. Transcription factor binding sites were identified using TESS (〈www.cbil.upenn.edu/cgi-bin/tess/tess33〉) and Genomatix (〈www.genomatix.derang〉) program, which uses matrices of the Transfac database.

Isolation of CD4+ T cells

CD4+ T cells were isolated from blood of healthy volunteers using the anti-CD4 magnetic beads (Dynal Biotech), as previously described (26). The purity of CD4+ T cells was initially tested by FACS and was ≥95%.

Flow cytometry

For analysis of FOXP3 expression at the single-cell level, cells were first stained with the mAb CD25 (Beckman Coulter); after fixation and permeabilization, cells were incubated with PE-conjugated mAb PCH101 (anti-human FOXP3; eBioscience), based on the manufacturer’s recommendations, and subjected to FACS (EPICS XL-MCL; Beckman Coulter).

Transfections and reporter gene assays

T cells were rested in serum-free AIM-V medium (Invitrogen Life Technologies) overnight. An amount of 3.5 μg of the FOXP3 promoter luciferase reporter vector and 0.5 μg of phRL-TK was added to 3 × 106 CD4+ T cells resuspended in 100 μl of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega), according to the manufacturer’s instructions. Data were normalized by the activity of Renilla luciferase. HeLa, Chinese hamster ovary (CHO), and Jurkat were transfected using LipofectAMINE 2000 (Invitrogen Life Technologies), according to the manufacturer’s protocol.

Quantitative real-time PCR

The PCR primers and probes detecting FOXP3 were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems), as follows: EF-1α forward primer and reverse primer, as described (27); FOXP3 forward primer, A 5′-GAA ACAG CAC ATT CCC AGA GTT C-3′; FOXP3 reverse primer, A 5′-ATG GCC CAG CGG ATG AG-3′. The prepared cDNAs were amplified using SYBR-PCR Mastermix (Applied Biosystems), according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Relative quantification and calculation of the range of confidence were performed using the comparative ΔΔCT method, as described (28). All amplifications were conducted in triplicates.

Western blotting

For FOXP3 analysis on the protein level, 1 × 106 cells CD4+CD25− were lysed and loaded next to a protein-mass ladder (Magicmark; Invitrogen Life Technologies) on a NuPAGE 4–12% bis-Tris gel (Invitrogen Life Technologies). The proteins were electroblotted onto a polyvinylidene difluoride membrane (Amersham Biosciences). After blocking, the membranes were incubated with a 1/200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4°C. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). To confirm sample loading and transfer, membranes were incubated in stripping buffer and reblocked for 1 h, then reprobed using anti-actin (C-2; Santa Cruz Biotechnology).

Pull-down assay

CD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37°C. The cells were pelleted, resuspended in buffer C (20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors (Sigma-Aldrich), and 0.1% Nonidet P-40), and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1/3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 μg of poly(deoxyinosinic-deoxycytidylic acid) (Sigma-Aldrich) and 70 μl of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4°C. The beads were washed twice with buffer C/D (1/3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen Life Technologies), heated to 70°C for 10 min, and the eluants were loaded next to a protein-mass ladder (Magicmark; Invitrogen Life Technologies) on a NuPAGE 4–12% bis-Tris gel (Invitrogen Life Technologies). The proteins were electroblotted onto a polyvinylidene difluoride membrane (Amersham Biosciences) and detected using an anti-NF-ATc mAb (Santa Cruz Biotechnology). The blots were developed, as described above. Accumulated signals were analyzed using AIDA software (Raytest).

EMSAs

EMSAs were performed, as previously described (29). Nuclear extracts of CD4+ T cells were prepared, as described. Briefly, the cells were treated with a hypo-osmotic buffer, containing 10 mM KCl, 1 mM DTT, 0.5 mM EDTA, and 10 mM HEPES (pH 7.9) (all Sigma-Aldrich), and a mix of protease inhibitors (Complete; Boehringer Mannheim), followed by addition of Nonidet P-40 (Sigma-Aldrich) to a 1% final concentration. Nuclei were pelleted by a brief spin in a microcentrifuge and washed once with the buffer described above. Nuclei were lysed in 50 μl of a high-salt buffer containing 400 mM NaCl, 50 mM DTT, 20 mM HEPES, 0.5 mM EDTA, and a mix of protease inhibitors (Complete; Boehringer Mannheim). The nuclear debris of this lysate was removed by centrifugation at 4°C, and the supernatant was stored in a fresh tube at −70°C. Nuclear extracts were controlled for equal protein content by a protein assay, as described by the manufacturer (Bio-Rad).

Nuclear extracts were incubated with annealed oligonucleotides (Table I⇑), which correspond to the FOXP3 promoter sequences, as indicated in the figures. The two strands of the oligonucleotides were first labeled with [32P-γ]ATP using the T4 kinase (Invitrogen Life Technologies). Subsequently, the oligonucleotides were separated from free [32P-γ]ATP by running the labeling mix over a chromaspin-10 column (BD Clontech). Following annealing, single-stranded oligonucleotides were eliminated by gel purification of the column eluate on a 20% polyacrylamide gel. The eluted probe was precipitated, and the binding reactions for the TATA site were conducted for 30 min at room temperature with 2 μg of nuclear extract in 10 mM HEPES (pH 7.9), 10% glycerol, 1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5 μg of poly(deoxyinosinic-deoxycytidylic acid), 1 mM PMSF, and 30,000 cpm of probe. For the GC box, 3 μg of nuclear extract was incubated, as previously described (30). The reaction was incubated for 10 min at room temperature and loaded on a 5% nondenaturating polyacrylamide gel. Following electrophoresis, the gel was dried and subjected to autoradiography and phosphor imaging.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed using ChIP assay kit following the recommendations of the supplier (Upstate Biotechnology). For precipitation, a polyclonal Ab against acetylated histone H4 was used along with an isotype-matched rabbit IgG control. The PCR addressed for the FOXP3 promoter region −246 to −511 and was performed using the following primers: 5′-GTG CCC TTT ACG AGT CAT CTG-3′ and 5′-GTG CCC TTT ACG AGT CAT CTG-3′. The PCR products were visualized using an ethidium bromide gel. For ChIP assay addressing the NF-AT binding to the chromatin, an anti-NF-ATc2 (4G6-G5; Santa Cruz Biotechnology) was used, and primer addressing the FOXP3 promoter region −1540 to −1470 to the following primer was used: 5′-TTT GCA GGG TGC TGG GA-3′ and 5′-GTA GAC CAG CCC CCA GGG-3′, and quantitative RT-PCR was performed.

FACS sorting of CD4+CD25+

PBMC were isolated from buffy coat by density gradient centrifugation over Ficoll/Hypaque. Cells were stained with PE anti-CD25 and anti-PE magnetic beads (Miltenyi Biotec), and CD25+ cells were enriched using the Midi-MACS system (Miltenyi Biotec). CD25-enriched or -depleted cell populations were stained with FITC anti-CD4 and sorted into CD4+CD25− and CD4+CD25high on a FACStarPlus (BD Biosciences).

Suppression assay

Samples in triplicate, containing 5 × 104 CD4+CD25− and 1 × 104 of preactivated or resting CD4+CD25+T cells/well, were incubated in 96 round-bottom plates, which were coated previously with 1 μg/ml anti-CD3 mAb or a matched isotype control. Cells were cultured for 4 days, pulsed for the last 10 h with 1 μCi of [3H]thymidine (Hartmann), and harvested on glass fiber filters using an automated multisample harvester (LKB; Pharmacia-Wallac). Filters were transferred in sample bags with liquid scintillation fluid and analyzed using a beta scintillation counter (Pharmacia-Wallac). Round-bottom 96-well plates were coated with 1 μg/μl anti-CD3 for 1 h at 37°C and subsequently washed with PBS.

Results

Localization of the FOXP3 promoter in human CD4+ T cells

To map the 5′ end of the human FOXP3 gene, 5′-RACE was performed, using nested PCR. The first primer was located in exon 11, and the second in exon 6 of the FOXP3 gene. The mRNA was isolated from CD4+ T cells of a healthy donor, reverse transcribed, and used as template for 5′-RACE. Sequence analysis of 11 clones revealed that the TSS is located 6211 bp upstream of the translation start site. The UTR is interrupted by an intron 0 of 6011 bp. An alignment of the sequences of human, mouse, and rat FOXP3 gene was performed, and several conserved regions (Fig. 1⇓A) were identified including 11 exons (Fig. 1⇓A, dark blue) and some conserved noncoding sequences (Fig. 1⇓A, red, CNS). Interestingly, the region preceding the UTR is highly conserved (Fig. 1⇓, A and B) and contains several transcription factor binding sites. On the basis of these sites, a putative promoter scheme was generated and tested in the following experiments (Fig. 1⇓C).

FIGURE 1.
FIGURE 1.

Human, mouse, and rat alignment of the FOXP3 core promoter. A, m-Vista alignment of human/mouse genomic sequences (human accession AF235097; mouse accession AF277994). m-Vista criteria that were applied require 75% identity for at least 100-bp length. The conserved regions are in red, the exons in dark blue, and the UTR in light blue. B, Sequence conservation of the human (top: GenBank accession no. AF235097), mouse (middle: GenBank accession no. AF277994), and rat (bottom: GenBank accession no. NW_048035). The TSS is indicated by a broken arrow. Transcription factor binding to the regions of interest is indicated (factor name above and position below). C, Scheme of the 5′ UTR region of the human FOXP3 gene, indicating the sites analyzed in this study.

Chromatin structure

Because FOXP3 is specifically expressed in T cells, we analyzed whether chromatin in the area of the putative promoter is accessible to the transcriptional machinery in T cells by ChIP. Histone H4 hyperacetylation is a typical feature of active transcription (31); we therefore analyzed chromatin hyperacetylation of the FOXP3 promoter by comparing cells of lymphoid and nonlymphoid origin as well as T cells characterized by low or high FOXP3 expression. T cells were depleted of CD25+, and intracellular FACS staining revealed that FOXP3 expression was virtually absent in the remaining cells (0.4%; Fig. 2⇓A). The frequency of FOXP3+ T cells increased following T cell activation predominantly in the CD25+ subset (11.1% after 72 h; Fig. 2⇓A). It occurred as a possibility that activation-induced FOXP3+ T cells expand from the 0.4% of CD25−FOXP3+ T cells; however, on the basis of known T cell division kinetics (doubling maximally in 48 h), the FOXP3 expression must predominantly arise from the FOXP3− T cells. We demonstrated that histone hyperacetylation is detectable in CD4+ T cells, particularly in activated or FACS-sorted CD25+high Treg cells, but absent in HeLa and Jurkat cells. Lower levels were observed in resting CD4+CD25− and CD4+CD45RA+ T cells (Fig. 2⇓B), showing that the FOXP3 promoter region is in an open conformation and accessible to the transcription machinery in the CD4+CD25− cells, and that activation might play an important role in mobilizing the chromatin structure. The acetylation levels correspond to the FOXP3 mRNA expression levels of these cells (Fig. 2⇓C).

FIGURE 2.
FIGURE 2.

Chromatin configuration of FOXP3. A, Kinetic FACS staining indicating intracellular FOXP3 expression in CD25-depleted CD4+ T cells upon activation. The dot blots are representative of three independent experiments. B, The acetylation status of histone H4 in the nucleosomes associated with the FOXP3 core promoter region was assessed by ChIP assay in naive (CD45RA+), resting (CD25−), activated or regulatory (CD25+) T cells as well as in Jurkat or HeLa cells. Cells were lysed, and proteins were cross-linked with formaldehyde and immunoprecipitated with Ab to acetyled histone H4 (anti-acetyl H4) or control Ab (rabbit IgG). Shown is the PCR for the FOXP3 gene after reversing the cross-linking. The input represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments. C, Expression level of FOXP3 mRNA measured by RT-PCR in cells, as described under B. Bars show the mean ± SD of three independent experiments.

The FOXP3 promoter region contains cell-specific activity

The chromatin accessible region described above was functionally investigated for trans activational activity. To identify potential regulatory elements in the 5′-flanking region of the human FOXP3 gene, a series of promoter-luciferase 5′-deletion constructs were generated to test whether the FOXP3 promoter fragment also reflects cell specificity. We transfected the identical constructs into cells of lymphoid and nonlymphoid origin that do not express FOXP3. High trans activation was observed in primary CD4+ T cells, whereas no activity was detected in HeLa, CHO (data not shown), nor Jurkat cells independently of the promoter fragment size (Fig. 3⇓). The longest construct was designed from position −1657 to +176 and displayed a promoter activity in CD4+ T cells 3-fold higher than that of the control plasmid, pGL3 basic (Fig. 3⇓). We designed 5′ deletions (−1210, −511, −465, −423, −348, −307, −211, and −90) to identify the proximal promoter, which we could localize in a fragment of −511 bp from transcription start site. The −511/+176 region is highly conserved between humans, mice, and rats (Fig. 1⇑, A and B). A 6.8-fold increase in luciferase activity was measurable with the fragment of −511/+176 compared with pGL3 basic vector. In contrast, the smaller constructs (–307, −211, or −90/+176) show lower luciferase activity. Although the construct −307/+176 shows low activity, it is essential for the activity of the −511/+176, because a deletion of −245 to +176 region out of −511 (−511/−245 construct) shows no activity in CD4+ T cells (Fig. 3⇓). Thus, the construct −511/+176 showed the most prominent reporter activities, whereas larger fragments didn’t show any significant increase in activity over the −511/+176 construct. These results together with the open chromatin configuration suggest that the first 500 bp of basal FOXP3 promoter confer cell specificity and trans activation. Having demonstrated that the promoter is active in CD4+ T cells, we performed site-directed mutagenesis to further characterize the promoter.

FIGURE 3.
FIGURE 3.

The putative FOXP3 promoter is tissue specific. T cells, Jurkat, and HeLa cells were transfected with empty vector or vector containing the putative FOXP3 promoter region. Bars show the mean ± SD of arbitrary light units normalized for Renilla luciferase of experiments performed with six independent donors (CD4+ T cells) or six independent experiments (in the case of Jurkat and HeLa cells; samples were measured as triplets).

Basal transcriptional elements are located in the core promoter

To further investigate the functionality of the basal FOXP3 promoter located within the first 500 bp, we investigated binding sites characteristic for eukaryotic promoters. Putative transcription factor binding sites were identified using TESS and GENOMATIX programs. In fact, several common features of eukaryotic core promoters such as the TATA, GC, and CAAT boxes were identified. The TATA box (TATAAAA) is located −44 bp upstream of the transcription start site. This sequence is conserved between humans, mice, and rats (Fig. 1⇑A). Because the TATA box is an important feature of eukaryotic promoters and is generally located −30 to −25 bp upstream the TSS (32), we investigated the element using site-directed mutagenesis of the fragment −211/+176 (TATAAAAG was mutated to TcTcgAAGC) and could demonstrate that the mutations, which eliminate TATA binding sites, dramatically reduce by 47.64% (Fig. 4⇓A) reporter activity. EMSA of the TATA box sequence of the FOXP3 promoter (TTA GAA GAG ACT CGG TAT AAA AGC AAA GTT GTT TT) bound by nuclear extracts from CD4+ T cells confirmed that nuclear proteins are binding to this promoter element. Only one complex could be detected, which could be competed by preincubation with unlabeled oligonucleotides specific for TATA box consensus sequence (GCA GAG CAT ATA AAA TGA GGT AGG A), which abolished in a dose-dependent manner the formation of the complex (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

Basal elements of the human FOXP3 promoter. A, The human FOXP3 contains functional TATA box and GC box. The region −211/+176, which contains the TATA box, w as mutated in the pGL3 FOXP3 −211/+176. The mutated TATA box transfected into CD4+ T cells, and the luciferase activity was measured. The effect of mutagenesis is shown as percentage relative to wild-type pGL3 FOXP3 −211/+176. Results are given as the mean ± SEM of three independent experiments in triplicate. B, Binding of specific nuclear factors to the TATA box. EMSA of the region −60 to −14 region of the FOXP3 gene promoter is shown. The competition experiments were performed by preincubating nuclear extracts with 10- and 100-fold excess of TATA oligonucleotides (lanes 3 and 4). The region −307/+176, which contains the GC box, was mutated in the pGL3 FOXP3 −307/+176. C, The mutated GC box was transfected into CD4+ T cells, and the luciferase activity was measured. Effect of mutagenesis is shown as percentage relative to wild-type pGL3 FOXP3 −307/+176. Results are shown as the mean ± SEM of three independent experiments performed in triplicate. D, Binding of specific nuclear factors to the GC box is demonstrated by EMSA of the −124 to −173 region of the FOXP3 gene promoter. The competition experiments were performed by preincubating nuclear extracts with 10- and 100-fold excess of Sp1 oligonucleotides (lanes 3 and 4) or mutated Sp1 oligonucleotides (lanes 5 and 6). The supershift assays were performed with antiserum against Sp1 protein (Sp1 Ab; lane 7), Sp3 protein (Sp3 Ab; lane 8), and Sp1 and Sp3 proteins (Sp1 + Sp3 Abs; lane 9). Supershifted bands are observed in lanes 7–9 along with an increased mobility of the remaining bands carrying unidentified factors, because the GC box is bound by multiple factors.

The GC box is another basic element of eukaryotic promoters, which is located 138 bp upstream the TSS. A site-specific mutation (GC Sp1–142) was introduced to destroy transcription factor binding site into the −307/+176 fragment, and luciferase assays were conducted. The mutation in the GC box decreased trans activational activity by 42.84% (Fig. 4⇑C). The GC box is known to be bound by Sp transcription factor family members. Sp1 acts as a potent activator, and Sp3 can act as an activator or a suppressor, possibly by competing with Sp1 for the binding. Nuclear extracts from CD4+ T cell formed two specific complexes (Fig. 4⇑D), which were dose dependently competed by the addition of specific Sp1-binding oligonucleotides at a 10× and 100× molar excess (ATT CGA TCG GGG CGG GGC GAG C), but not by mutated Sp1 oligonucleotides (ATT CGA TCG GTT CGG GGC GAG C). The addition of an antiserum against Sp1 shifted a band on the EMSA, and the remaining complex I or II migrates slightly faster, indicating that the complex becomes smaller. Similar observations were made using an anti-Sp3 antiserum, demonstrating that Sp1 and Sp3 are binding to this sequence. Of note, the GC box sequence can be bound also by other factors, which explains the binding of slightly faster migrating complexes upon addition of anti-SP-1 and 3 Abs (lanes 7–9 (33)). Furthermore, the CAAT box was analyzed and mutated, as described for the TATA and GC box. The mutation in the CAAT also reduced the luciferase activity of the −307/+176 fragment (data not shown).

Regulation of FOXP3 expression in the CD4+CD25− cells by activation of the TCR

The experiments showed that the promoter construct was active in lymphocytes and contains basic elements such as a TATA and a GC box. Because T cell activation is important for regulation of immune-relevant genes, we investigated whether FOXP3 mRNA and FOXP3 promoter fragments respond to T cell activation. FOXP3 mRNA can be induced (18.8-fold at 24 h, 30.4-fold at 48 h, and 11.7-fold at 72 h; Fig. 5⇓A) in the CD4+CD25− following T cell activation. Resting CD4+CD25+ were used as a control. Of note, FOXP3 expression in resting CD4+CD25+ T cells was 80-fold higher than in CD4+CD25− cells and could just slightly be increased by activation (1.9-fold; see Fig. 9⇓B). In analogy to up-regulated FOXP3 mRNA, T cell activation also induced FOXP3 reporter activity in the CD4+CD25− cell fraction. The smaller fragments and empty vector were only slightly responsive to activation in contrast to the fragments starting from −348/+176, which were strongly induced ∼80-fold compared with the empty vector or 10-fold compared with the corresponding unstimulated cells (Fig. 5⇓B).

FIGURE 5.
FIGURE 5.

FOXP3 is up-regulated by TCR cross-linking. A, CD4+CD25− T cells were stimulated with anti-CD3 and anti-CD28, and the FOXP3 mRNA level was measured by real-time PCR. Bars show the mean ± SD of three independent experiments. B, The FOXP3 promoter can be activated by TCR cross-linking. CD4+CD25− T cells were cotransfected with a Renilla luciferase vector plus the luciferase vector containing the putative promoter region and were cultured in medium or in medium containing PMA and ionomycin. Results given are the mean ± SD of luciferase light units normalized for Renilla luciferase of the same sample. Results are representative of three independent experiments.

CsA inhibits FOXP3 expression in human CD4+CD25− T cells

We identified NF-AT and AP-1 transcription factor binding sites located in the region between −348 and −511, which are known to be involved in T cell activation (Fig. 1⇑B). NF-AT is activated by the Ca2+-calcineurin pathway and blocked by the immunosuppressive drug CsA. Therefore, we analyzed the effect of CsA on the induction of FOXP3 mRNA and promoter activity. CD4+CD25− T cells were activated in the presence or absence of CsA, and the mRNA was quantified after 24, 48, and 72 h. The FOXP3 mRNA was potently inhibited by CsA, but not by MAPK inhibitors (Fig. 6⇓A). CsA inhibition of FOXP3 mRNA induction was maintained throughout the 72-h time course (Fig. 6⇓B), while cell viability was maintained (data not shown). The CsA sensitivity of FOXP3 was confirmed at the protein level (Fig. 6⇓C). FOXP3 promoter fragments, which responded to activation, were potently inhibited by CsA. This indicates that the calcineurin-dependent NF-AT mobilization plays a crucial role in the trans activation of the FOXP3 promoter (Fig. 6⇓D).

FIGURE 8.
FIGURE 8.

NF-ATc2 regulates human FOXP3 promoter activity in CD4+CD25− T cells. A, Mutation of NF-AT sites decrease promoter activity as well as its induction by activation by PMA and ionomycin. Bars show the mean ± SD of three independent experiments. B and C, Nuclear extracts were prepared from CD4+ T cells activated 2 h with PMA and ionomycin. Biotinylated NF-AT-383 (B) and NF-AT-328 (C) oligonucleotides were absorbed by streptavidin-agarose beads and then incubated with the nuclear extracts. Then the amounts of NF-ATC2 protein in the precipitates were assessed by immunoblotting with anti-NF-ATC2 mAb. Total nuclear extracts were also run as controls. Two independent experiments were done with similar results. D, CD4+CD25− T cells were activated using anti-CD3 and anti-CD28 and analyzed by ChIP for NF-AT binding to the FOXP3 promoter. Quantitative fluorogenic PCR was performed. Data are expressed as the ratio of immunoprecipitated to input sequence and are mean ± SD of two separate experiments. E, Overexpression of CD4+CD25− cells with NF-ATc2 with the 511 FOXP3 promoter construct increases the luciferase activity of the FOXP3 promoter constructs. NF-ATc2 could not further increase the −90 luciferase activity. Results shown are the mean ± SD of one experiment performed in triplicate. Two independent experiments were done with similar results.

FIGURE 6.
FIGURE 6.

CsA inhibits FOXP3 induction in the CD4+CD25− T cells. A, CD4+CD25− T cells were activated with anti-CD3 and anti-CD28 with CsA and different MAPK inhibitors. CsA potently inhibits FOXP3 induction. Bars show the mean ± SD of three independent experiments. B, CD4+CD25− T cells were activated with anti-CD3 and anti-CD28 with CsA; cells were harvested at different time points, as indicated in the figure. Bars show the mean ± SD of three independent experiments. C, Western blot analysis of FOXP3 in CD4+CD25− T cells after activation with anti-CD3 and anti-CD28 and with treatment with CsA (1 μM). Two independent experiments were done with similar results. D, CD4+CD25− T cells were transfected with the FOXP3 promoter constructs and activated with PMA and ionomycin and treated or not with CsA (1 μM). Bars show the mean ± SD of three independent experiments.

NF-AT and AP-1 are positive trans activators of FOXP3

Because the construct −348 was the shortest construct showing TCR responsiveness, we mutated the AP-1 binding sites in the construct −348. Mutation of the AP-1 site at position −306, which is closest to the TSS, has only a weak effect on promoter activity (Fig. 7⇓). In contrast, mutation of the AP-1 site located −324 strongly reduced the trans activational activity of the promoter (3-fold; Fig. 7⇓). The background was also reduced, suggesting that those factors play an important role in the constitutive promoter activity. Mutations of the NF-AT and AP-1 sites in the constructs −511 had a dramatic effect on the basal activity and inducibility by T cell activation (Fig. 8⇑A). Loss of the NF-AT-490 and −328 in the construct −511 decreased the activity by 38%, and activation induces only three times instead of four times in the wild type. Loss of the NF-AT binding site −383 and AP-1 −476 decreases the activity by 55%, and the induction following activation was only of 2.2-fold (Fig. 8⇑A). The binding of NF-ATc2 to the NF-AT sites on −490 and −328 was proven by pull-down assay, using cell lysates of activated CD4+ T cells. NF-ATc2 was bound to the FOXP3 promoter oligonucleotides used for precipitation, but it was not precipitated by the mutated version (Fig. 8⇑B) or by the wild-type oligonucleotides competed by the excess of NF-AT consensus oligonucleotides (data not shown). To verify whether NF-AT binds the FOXP3 promoter area on the chromatin under natural conditions, we performed ChIP analysis. Starting from 2 h after activation of CD4+ T cells with anti-CD3 and anti-CD28, the binding of NF-ATc2 could be shown and was maximal after 5 h and decreases thereafter (Fig. 8⇑D). Overexpression of NF-ATc2 dramatically increased promoter activity of the −511 construct (3-fold, relative to the empty pcDNA3 vector), which could be further increased by activation (6.5-fold; Fig. 8⇑E). The overexpression of NF-ATc2 had only a minor influence on the activity of the −90/+176 construct used as a control.

FIGURE 7.
FIGURE 7.

The AP-1 sites of the FOXP3 promoter have trans activatory activity. CD4+CD25− T cells were transfected with construct containing AP-1 mutations and activated with PMA and ionomycin. AP-1 mutations decrease promoter activity, as well as its induction by activation. Bars show the mean ± SD of three independent experiments.

Regulation of FOXP3 in CD4+CD25+ Tregs

It is known that overexpression of FOXP3 is sufficient to induce a Treg phenotype; however, the significance of FOXP3 regulation in already existing T cells is not clear. We therefore investigated whether activation has any effect on FOXP3 expression in Tregs. We activated FACS-sorted CD4+CD25+ Tregs or CD4+CD25− effector T cells (Fig. 9⇓A) with plate-bound anti-CD3 and anti-CD28. After 3 days, the cells were harvested and FOXP3 mRNA was measured by real-time PCR. Resting CD4+CD25+ expressed ∼90-fold more FOXP3 than CD4+CD25− cells. Activation induced expression of FOXP3 mRNA by only 1.9-fold (Fig. 9⇓B), in contrast to 20-fold CD4+CD25− cells (Fig. 5⇑A). To test whether this increase has a functional effect on Tregs function, we compared the suppressive capacity of unstimulated to preactivated Tregs. Although FOXP3 expression did only marginally increase (1.9-fold), the activation dramatically increased the suppressive capacity of Tregs (Fig. 9⇓C). However, when Tregs were preactivated during 2 days in the presence of CsA, which was washed away before the cells were used in the suppression assay, the suppressive capacity was only marginally reduced (Fig. 9⇓C). Thus, NF-AT is important for FOXP3 induction, mediating regulatory differentiation, but does not affect the suppression of already existing Tregs, although activation potentiates suppressive capacity.

FIGURE 9.
FIGURE 9.

Activation does not induce FOXP3 expression in pre-existing Tregs. A, CD4+CD25high were FACS sorted using the shown gates. B, FACS-sorted CD4+CD25+ Tregs were activated with anti-CD3 and anti-CD28 during 3 days and treated or not with CsA (1 μM). The cells were harvested for mRNA extraction. The percentage was calculated on the basis of the ΔΔCt method. Bars, 95% confidence interval calculated on the basis of deviation of EF-1 and FOXP3 expression. The results shown are the mean ± SD of three independent experiments. C, Activation dramatically increases CD4+CD25+ Treg suppressive capacity, CsA couldn’t avoid this increase in the suppressive capacity. CD4+CD25+ Tregs were preactivated during 2 days in presence or absence of CsA. After washing the cells three times, their suppressive capacity on responder CD4+CD25− was tested. A total of 10 × 104 CD4+CD25+ Tregs was added to 5 × 104 CD4+CD25−. The results shown are the mean ± SD of three independent experiments.

Discussion

In the present study, we describe the localization and structure of the human FOXP3 promoter, as well as elements, that are essential for its induction in T cells.

The FOXP3 promoter is located −6221 bp upstream of the translation start site and the 5′ UTR is interrupted by a 6000-bp intron, which contains a splice donor site at the 5′ end and a splice acceptor site at the 3′ end, 22 bp upstream of the translation start site. The promoter is highly conserved between humans, mice, and rats. The mRNA sequence published in the present study confirms the transcription start site and the location of the intron “0” of the reference sequence (NM_014009).

The chromatin accessibility is a key mechanism of gene regulation and has been shown to be essential for many genes during T cell differentiation, like IL-4 and IFN-γ (34, 35, 36). FOXP3 has been proposed to be a lineage-specific factor for Tregs, and, therefore, the chromatin structure may be an important aspect of FOXP3 regulation (37, 38). FOXP3 was accessible in resting and activated CD4+CD25− T cells, CD4+CD45RA and CD4+CD25+, but not in Jurkat and HeLa cells, corresponding to their FOXP3 mRNA expression (4). Thus, chromatin remodeling may contribute to the cell-specific expression of FOXP3, controlling the access of the transcriptional machinery to the promoter. The CD4+CD25− population showed an open chromatin conformation of FOXP3 gene, which was further increased by activation. The nonrepressive chromatin configuration may therefore allow CD4+CD25− T cells to acquire a regulatory phenotype upon activation with the appropriate key of transcription factors.

To identify this set of transcription factors, we analyzed the 1.6-kbp region upstream of the TSS. This region showed promoter activity, when cloned in front of a luciferase reporter gene and transfected into primary CD4+ T cells. In contrast, HeLa and CHO cells did not show any promoter activity. Thus, FOXP3 cell specificity is regulated not only at the chromatin, but also on transcription level. The serial deletion constructs revealed that a fragment of 348 bp contained the minimal promoter necessary for the induction of the gene. The deletion of 245 bp upstream of the TSS totally abrogated the promoter activity, indicating that this area contains the core promoter. The current data show that the specific mutation of the TATA (−34), the GC (−138), and CAAT boxes (−218) reduces activity of the core promoter. Furthermore, we demonstrate that the GC box is in fact bound by Sp1 and Sp3. Because these factors are characteristic for eukaryotic promoters (39), these data confirm the location of the FOXP3 promoter.

On the basis of these results, we analyzed inducible elements upstream of this area. We demonstrate that FOXP3 expression is induced following TCR engagement in CD4+CD25− T cells. Activation of CD4+CD25− T cells with anti-CD3 or PMA and ionomycin induced FOXP3 promoter activity in the −511 reporter gene. This result shows that TCR engagement acts directly on the FOXP3 promoter and confirms previous studies (20) showing that in vitro activation of CD4+CD25− cells was sufficient to generate cells expressing FOXP3, which have suppressive capacity. In fact, exposure to an Ag (40), TGF-β (24, 41, 42), estrogen (43), or glucocorticoids (21) along with T cell activation can induce FOXP3 in CD4+CD25− T cells. Therefore, activation seems to be a key event in the generation of Tregs, as it was shown previously to be essential in the differentiation process of Th1 and Th2 cells (44, 45, 46).

We narrowed down the activation dependence to the minimal FOXP3 promoter (−348), whereas the fragment that is just 41 bp shorter does not show any induction. Therefore, the activation-responsive element of the FOXP3 promoter is located between −511 and −307. NF-AT and AP-1 are well-known mediators of T cell activation and are clustered in this region. Mutations disrupting the NF-AT and AP-1 binding sites decreased the luciferase activity, revealing their role in the trans activation of the FOXP3 promoter. The activation of the FOXP3 gene is mediated by at least three NF-AT sites, which we demonstrated to be bound by NF-ATc2 and three AP-1 sites, in proximity of NF-AT sites. Those transcription factors often cooperate to induce cytokine gene expression and are forming complexes as in the promoter of IL-2 (47), IL-4 (48), IFN-γ (49), and CTLA-4 (50).

The MAPK inhibitor (PD98059) only partially inhibited activation-induced FOXP3 mRNA expression, suggesting that the AP-1 factors can be mobilized by other pathways. In contrast, CsA completely inhibited the mRNA induction of FOXP3 as well as the promoter activity. CsA is a well-known immunosuppressive drug that blocks NF-AT translocation into the nucleus by inhibition calcineurin phosphatase activity (51). We have shown previously that immunosuppressant glucocorticoids promote FOXP3 expression (21), whereas rapamycin neither enhances nor decreases FOXP3 (data not shown) (52, 53). Therefore, immunosuppressive drugs may have different mechanisms to promote tolerance induction.

Alternatively, immunosuppressive drugs may also act on pre-existing Tregs that are only marginally affected by TCR engagement in terms of FOXP3 mRNA expression, which is already high in resting Tregs. This marginal enhancement of FOXP3 expression in already existing, anergic Tregs is resistant to CsA expression, confirming previous studies showing that anergic cells are impaired in Ca2+/NF-AT mobilization (54, 55). However, preactivation shows a dramatic increase on the suppressive capacity of the CD4+CD25+ Treg cells. Pretreatment of the CD4+CD25+ cells with CsA had just a minor effect on the suppressive capacity and suggests that NF-AT is not essential in the process of suppression. In fact, CD4+CD25+ Tregs have been shown to be anergic and hyporesponsive to TCR stimulation and unable to induce Ca2+ signaling, which may explain that those cells are unable to further induce FOXP3 expression upon activation (56).

Taken together, our results indicate that the FOXP3 promoter is cell specific and is active only in primary T cells. The identified basal promoter has similarities to immunological genes carrying elements including NF-AT and AP-1, which are induced following TCR engagement. The reporter constructs provide new tools to identify mechanisms underlying tolerance induction and potential therapeutic interventions.

Acknowledgments

We thank Prof. G. Suske (University of Marburg, Marburg, Germany) for providing us Sp1 and Sp3 antiserum. We thank Prof. A. Rao (Harvard Medical School, Boston, MA) for providing the NF-ATc2 construct.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by Swiss National Foundation Grants 31-65436 and 3100A0-100164, the Ehmann Foundation Chur, the Ernst Goehner Foundation Zug, the Saurer Foundation Zurich, and the Swiss Life Zurich.

  • ↵2 Address correspondence and reprint requests to Dr. Carsten B. Schmidt-Weber, Swiss Institute of Allergy and Asthma Research, Obere Str. 22, CH-7270 Davos, Switzerland. E-mail address: Carsten.schmidt-weber{at}siaf.unizh.ch

  • ↵3 Abbreviations used in this paper: Treg, regulatory T cell; ChIP, chromatin immunoprecipitation; CHO, Chinese hamster ovary; CsA, cyclosporin A; TSS, transcription start site; UTR, untranslated region.

  • Received September 13, 2005.
  • Accepted December 23, 2005.

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