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International Immunology Advance Access originally published online on June 22, 2007
International Immunology 2007 19(7):825-835; doi:10.1093/intimm/dxm043
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

FOXP3 is a homo-oligomer and a component of a supramolecular regulatory complex disabled in the human XLAAD/IPEX autoimmune disease

Bin Li1, Arabinda Samanta1, Xiaomin Song1, Kathryn T. Iacono1, Patrick Brennan1, Talal A. Chatila2, Giovanna Roncador3, Alison H. Banham4, James L. Riley1, Qiang Wang1, Yuan Shen1, Sandra J. Saouaf1 and Mark I. Greene1

1 Department of Pathology and Laboratory Medicine, University of Pennsylvania, 252 John Morgan Building, 36th and Hamilton Walk, Philadelphia, PA 19104-6082, USA
2 Department of Pediatrics, The David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA 90095, USA
3 Monoclonal Antibodies Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain
4 Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford OX3 9DU, UK

Correspondence to: M. I. Greene; E-mail: greene{at}reo.med.upenn.edu


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We have found that FOXP3 is an oligomeric component of a large supramolecular complex. Certain FOXP3 mutants with single amino acid deletions in the leucine zipper domain of FOXP3 are associated with the X-linked autoimmunity-allergic dysregulation (XLAAD) and immunodysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome in humans. We report that the single amino acid deletion found in human XLAAD/IPEX patients within the leucine zipper domain of FOXP3 does not disrupt its ability to join the larger protein complex, but eliminates FOXP3 homo-oligomerization as well as heteromerization with FOXP1. We found that the zinc finger–leucine zipper domain region of FOXP3 is sufficient to mediate both homodimerization and homotetramerization. However, the same domain region from XLAAD/IPEX FOXP3 containing an E251 deletion prevents oligomerizaton and the protein remains monomeric. We also found that wild-type FOXP3 directly binds to the human IL-2 promoter, but the E251 deletion in FOXP3 in XLAAD/IPEX patient's T cells disrupts its association with the IL-2 promoter in vivo and in vitro, and limits repression of IL-2 transcription after T-cell activation. Our results suggest that compromising FOXP3 homo-oligomerization and hetero-oligomerization with the FOXP1 protein impairs DNA-binding properties leading to distinct biochemical phenotypes in humans with the XLAAD/IPEX autoimmune syndrome. This study explains some features of the pathogenesis of a disease syndrome that arises as a consequence of specific assembly failure of a transcriptional repressor due to certain mutations within the FOXP3 leucine zipper.

Keywords: FOXP3, IPEX, oligomerization, regulatory T cell, XLAAD


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Maintenance of tolerance to self-antigens is essential for the prevention of autoimmunity but is an incompletely understood process. CD4+CD25+ regulatory T cells have been reported to act as dominant regulators of immune activation and immune tolerance (1–3). In humans or in the murine species, one consistent feature of CD4+CD25+ regulatory T cells is the expression of the forkhead family transcription factor FOXP3 (4, 5). FOXP3 acts as a ‘sufficient’ regulator of the development and function of peripheral CD4+CD25+ regulatory T cells (4, 6), but the molecular mechanisms underlying FOXP3-mediated immunological regulation are still poorly understood (7–9).

‘X-linked autoimmunity and allergic dysregulation syndrome’ (XLAAD) or ‘Immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome’ (IPEX) (10–13) is a fatal recessive disorder of humans that develops in early childhood. These individuals fail to develop CD4+CD25+ T cells and experience varied symptoms that include diarrhea, dermatitis, insulin-dependent diabetes, thyroiditis and anemia. Massive T-cell infiltration into the skin and gastrointestinal tract is also observed (14). Several XLAAD/IPEX mutations are found in the forkhead domain of FOXP3, indicating the potential disruption of DNA binding. In addition, two independent studies identified single amino acid deletions at E251 or K250 within the leucine zipper domain (10, 15). Mutations in this region could potentially result in aberrant FOXP3 function by affecting its homoassociation or association with the highly conserved leucine zipper domain of other subfamily members, such as FOXP1, FOXP2 and FOXP4 (10, 16, 17).

Here, we report that the FOXP3 delE251 mutation found in human patients that suffer from the XLAAD/IPEX syndrome does not disrupts its association with the large supramolecular complex in vivo, but the mutant FOXP3 species can neither homo-oligomerize with itself nor heteroassociate with FOXP1. Surprisingly, we found that the purified zinc finger and leucine zipper domain of FOXP3 forms a homotetramer, rather than a homodimer, as suggested by previous studies that only studied association without use of purified proteins (18, 19). We also found that endogenous wild-type FOXP3 associated with the IL-2 promoter in vivo, whereas FOXP3 from XLAAD/IPEX patient T cells containing the E251 deletion lacks this association. Moreover, wild-type FOXP3 is more efficient in its ability to repress IL-2 transcription than the delE251 mutant. Our results reveal that oligomerization induced by the zinc finger and leucine zipper domains of FOXP3 is essential for FOXP3-mediated immunological regulation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Human IPEX patient T cells
Human IPEX patient T-cell lines containing the E251 deletion mutation of FOXP3 (delE251) and control lines expressing wild-type FOXP3 have been described (10). These are primary PHA plus IL-2 driven T-cell lines. The cells were mixed with irradiated (2500 rad) PBMC feeders (1:2 ratio) derived from normal hosts. PHA and IL-2 (1–10 µg PHA-P and 100 units ml–1 IL-2) were added to the culture, cells were then split after 3 days and expanded with IL-2 alone. The expansion phase lasted for 10–14 days with fresh IL-2 supplemented media added every 2–4 days. The feeder cycle was repeated until necessary cell numbers were achieved.

RNA isolation and reverse transcription-PCR
Total RNA was isolated from two million FACStar sorted mouse CD4+CD25+ or CD4+CD25– cells with Agilent Total RNA Isolation Mini Kit (Pat No. 5185-6000, Agilent Technologies, Palo Alto, CA, USA) and reverse transcribed with StrataScriptTM First-strand synthesis kit (Cat. # 200420, Stratagene, La Jolla, CA, USA). Transcription of Foxp1, Foxp2, FOXP3 and Foxp4 was measured by non-saturating PCR using the following primers: Foxp1 5'-GACCCACCTGCATGTGAAGTC-3' and 5'-TGGGCACGTTGTATTTGTCTG-3'; Foxp2 5'-GAGTCATCATGGCCACCGAC-3' and 5'-GGGATGGGAGATCAAGTGGTG-3'; FOXP3 5'-TCTTGCCAAGCTGGAAGACT-3' and 5'-ATCTGATGCATGAAGTGTGG-3'; Foxp4 5'-GACAGCAATGGCGAGATGAG-3' and 5'-CGCAGAGGCCGACTGTTTAC-3'. The housekeeping gene hprt primers 5'-CGTCGTGATTAGCGATGATG-3' and 5'-ACAGAGGGCCACAATGTGAT-3' were used as an endogenous reference.

Human CD4+CD25+ T cells
Human FOXP3+CD4+CD25+ T cells were obtained by in vitro expansion as follows: 200 million peripheral blood lymphoctyes (PBLs) were stained for CD4 and CD25, and using a Mo Flo high-speed sorter, the brightest (top 1%) CD4+CD25+ cells were purified. These cells were stimulated with anti-CD3 and anti-CD28-coated beads using a 3 bead to 1 cell ratio in the presence of high levels of IL-2 (300 U ml–1) and cultured in RPMI-1640 with 10% FCS for the next 20–25 days. These in vitro expanded regulatory T cells from CD25highCD4+ sub-population remain functional as indicated by their ability to mediate suppressive activity in in vitro assays (not shown). Additionally, they maintain a high level of FOXP3 expression compared with in vitro expanded CD25–CD4+ T cells (20).

Protein purification and molecular weight determination
The Zinc–LeuZip (190–263) encoding region of both wild-type FOXP3 and the E251 deletion mutant of FOXP3 were amplified by PCR, digested and sub-cloned into pET-21a-maltose-binding protein (MBP) to obtain MBP–Zinc–LeuZip (MBP-WT) and MBP–delE251–Zinc–LeuZip (MBP-delE251) expressing constructs. MBP fusion proteins or MBP alone were highly expressed in BL21 and purified by amylose affinity chromatography. The determination of protein molecular weights (MWs) in solution was based on the elution volume from a Superdex 200 gel filtration column. Gel Filtration HMW and LMW Calibration Kits (Amersham Biosciences) were used for the calibration of Superdex 200 gel filtration column (Protein KW-803).

The nucleic acid sequence encoding the c-terminal 106–431aa of FOXP3 (FOXP3-c106-431) was sub-cloned to pET28-a (Novagen). Protein was expressed in Escherichia coli BL21 (DE3) carrying one chaperone plasmid pG-Tf2 (Takara), bound to Ni-NTA resin (Qiagen) equilibrated with buffer A (50 mM NaH2PO4, pH 7.5, 300 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% Tween 20), eluted with 100–200 mM imidazole in buffer A and then further purified by Superdex-200 column (Amersham Biosciences) equilibrated in buffer B (50 mM NaH2PO4, pH 7.5, 200 mM NaCl, 1 mM DTT).

Nuclear extract size fractionation
Nuclear extracts of human CD4+CD25+ T cells were quantified with the BCATM Protein Assay Kit (Pierce) and applied to a calibrated GFC column Protein KW-803 (Shodex®, Japan) run by a HPLC apparatus (Waters Corporation, Milford, MA, USA) with the nuclear extraction buffer as the HPLC running buffer. One column volume (18.84 ml) was collected in 1.0 ml aliquots. Equal volumes of collected fractions were subjected to 8% SDS-PAGE and immunoblotting with indicated antibodies.

Plasmids and antibodies
The following antibodies were used: anti-FOXP3 mAb hFOXY and PCH101 from eBioscience; anti-myc (9E10), HA (F-7), BRG-1 (H-88) and NFATc2 (4G6-G5) from Santa Cruz Biotechnology; anti-FLAG-M2 from Sigma; anti-MEF2D from BD Biosciences PharMingen; anti-FOXP3 221D/D3 (21); anti-FOXP1 JC12 (22) and FOXP1 expression constructs were a kind gift of Edward Morrisey, University of Pennsylvania. FOXP3a (the large isoform) and FOXP3b (the small isoform-lacking exon 2) have been described previously (20).

Site-directed mutagenesis of FOXP3 delE251 and delK250
The following primers were used to make FOXP3 delK250 and delE251 mutants, respectively: 5'-GCT GGT GCT GGA GGA GAA GCT GAG TGC C-3' and 5'-GGC ACT CAG CTT CTC CTC CAG CAC CAG C-3'; 5'-CTG GTG CTG GAG AAG AAG CTG AGT GCC ATG-3'and 5'-CAT GGC ACT CAG CTT CTT CTC CAG CAC CAG-3'. All the mutants were made with QuickChangeTM site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing.

ShRNA vectors and reagent
TRC small hairpin RNA (shRNA) (Lenti) targeting human FOXP1 construct TRCN0000015664 (sh64) and the Arrest-In transfection reagent (cat no. ATR1741) were purchased from Open Biosystem. The non-target shRNA control vector was purchased from Sigma (cat no. SHC002).

Chromatin cross-linking and immunoprecipitation assay
A human IPEX patient T cell line expressing the E251 FOXP3 mutation (10) and the normal control line expressing a similar amount of wild-type FOXP3 were expanded in vitro. Chromatin cross-linking and immunoprecipitation (ChIP) analyses were performed with the EZ ChipTM Chromatin Immunoprecipitation Kit (Cat# 17-371, Upstate). ChIP antibodies included mouse IgG (#I5381, Sigma), anti-FOXP3 (hFOXY #14-5779, eBioscience) and anti-acetylated histone H4 (23-866,Upstate). PCR primers for human IL-2 primers are as follows: hIL-2F-374: 5'-CCACAATATGCTATTCACATGTTCAG-3' and hIL-2R-45: 5'-TGGCAGGAGTTGAGGTTACTG-3'.

Electrophoretic mobility shift assay
Nuclear extracts were prepared and electrophoretic mobility shift assay (EMSA) assay were performed as previously described (23). The oligonucleotide probe used corresponded to the Nuclear factor of activated T-cells (NFAT)-binding site with consensus sequence: 5'-GAGGAAAATTTGTTTCATACAGAAG-3'. In each binding reaction, 10 µg of protein from nuclear extracts and 100 000 cpm-labeled probe were used per 20 µl of binding reaction. The binding buffer composition was 20 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2, 3 mM DTT, 0.1 mg BSA, 0.25 mg ml–1 poly dI/dC and 10% glycerol. For cold competition, a 100-fold molar excess of unlabeled double-stranded probe was mixed prior to the addition of labeled probe.

Dual luciferase assay
Jurkat transient transfections and all luciferase assays were performed as previously described (24). The transfected Jurkat T cells were stimulated with 50 ng ml–1 of phorbol myristate acetate and 1 µM ionomycin for 6–7 h before lysing cells and analyzed by means of dual luciferase assay normalized with Renilla luciferase activity according to the manufacturer's protocol (Promega).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
FOXP3 normally resides within a supramolecular complex and the FOXP3 DelE251 mutant in XLADD/IPEX patient T cells is able to participate in complex formation
We studied FOXP3 derived from cells of patients with the rare genetic disease, XLAAD, also known as IPEX (10–13) in which FOXP3 is dysfunctional. Using this model system, we were able to identify certain subdomains of FOXP3 that are functionally important. Eukaryotic transcription factors usually form multiple protein complexes that affect the promoter regions of various genes. To examine the biochemical features of endogenous FOXP3, we isolated nuclear extracts from two human primary T-cell lines, A and B, expressing wild-type FOXP3 and two XLAAD/IPEX patient T-cell lines, C and D, which express a mutated FOXP3 gene with a single amino acid deletion at E251. Under native conditions, both the endogenous wild-type FOXP3 and its delE251 mutant assembled as part of a large protein complex with a MW higher than 500 kDa, although it appears that there are slightly reduced amounts of endogenous delE251-mutant protein associated within the large complex (Fig. 1A). Under reducing conditions, both wild-type FOXP3 and its delE251 mutant migrate as a monomer with a MW ~47 kDa (Fig. 1B).


Figure 1
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  		Fig. 1. Endogenous FOXP3 in T cells exists as part of a large protein complex. (A and B) Nuclear extracts (20 µg) were used to run either (A) 6% native gel electrophoresis or (B) 3–8% gradient SDS-PAGE, followed by immunoblotting with anti-human FOXP3 mAb 221D. Samples A and B are from human T-cell lines with wild-type FOXP3 and C and D are from XLAAD/IPEX patient T cells with FOXP3 delE251 mutations. A protein molecular weight (MW) marker kit for non-denaturing polyacrylamide gel electrophoresis (Sigma) was used to determine the protein MW on a native gel. (C) Nuclear extracts of FLAG-delE251 FOXP3a-expressing Jurkat T cells were size fractionated by gel filtration followed by fraction concentration, SDS-PAGE and immunoblotting with anti-FLAG, anti-BRG-1 or anti-MEF2D.

 
To further test whether delE251 mutant disrupts FOXP3 complex formation, we fractionated the nuclear extracts from FLAG-tagged delE251 FOXP3a (the large isoform) ectopically transfected Jurkat T cells by size with an HPLC gel filtration column, followed by SDS-PAGE and western blotting analysis with anti-FLAG M2 mAb. Using this approach, we found that the endogenous ~47 kDa delE251 FOXP3a protein still resided principally in two separated high MW complexes, one with MW >696 kDa (Fig. 1C, lane 4) and the other with MW between 696 and 354 kDa (Fig. 1C, lanes 7 and 8). Interestingly, BRG-1, a catalytic subunit of mammalian chromatin-remodeling complexes, which found in association with FOXP3 by MS/Qstar spectrometric analysis after co-immunoprecipitation (25), co-fractionates with both FOXP3 ensemble complexes (Fig. 1C, lanes 4, 7 and 8). We also observed that one previously described FOXP3-associated transcriptional factor NFATc2 (26, 27); as well as another transcription factor, MEF2D, known to associate with HDAC7 (28), co-fractionates with the lower MW FOXP3 complex, with MW between 354 and 696 kDa (Fig. 1C, lanes 7 and 8). Both are absent from the higher MW fractions of FOXP3-associated proteins (Fig. 1C, lane 4).

Together, these data clearly indicate that FOXP3 exists in a dynamic supramolecular complex. Of note is the presence of HDAC7, TIP60 (20) and the NFATc2 (26, 27) and MEF2D transcription factors observed in certain fractions. Clearly, the chromatin-remodeling proteins and others to be described elsewhere are likely to play defining roles in these ensembles. Most surprising, though, is the fact that the delE251 mutant of FOXP3 still associates with other proteins to form large MW complexes.

Zinc finger and leucine zipper domains are sufficient to mediate FOXP3 homotetramerization, and this assembly is disrupted in XLAAD/IPEX mutants
Because FOXP3 expressed in T cells associates with other endogenous proteins that lead to large complex formation, we also studied the behavior of FOXP3 proteins and fragments derived from it in solution. We expressed full-length FOXP3a (the large isoform) with a 6xHis tag in sf9 insect cells and purified by Ni-NTA column. The purified His-tagged full-length FOXP3a was size fractionated, followed by SDS-PAGE and immunoblotting of the individual fractions with anti-FOXP3 mAb 221D. Unexpectedly, purified full-length FOXP3a eluted from the gel filtration column as a dominant mixture of monomers, dimers, tetramers and a detectable amount of oligomeric species (Fig. 2A).


Figure 2
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  		Fig. 2. Single amino acid deletion in the leucine zipper domain of FOXP3 found in human immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome (IPEX) syndrome eliminates its homoassociation. (A) The purified His-tagged FOXP3a was size fractionated by gel filtration, then analyzed by immunoblotting of the individual fractions with anti-FOXP3. For quantification, western blot film was scanned and processed using ImageJ software (http://rsb.info.nih.gov/ij/) (B) Schematic representation of FOXP3 subdomains and K250 and E251 deletion mutants (delK250, delE251) within the leucine zipper domain found in human X-linked autoimmunity-allergic dysregulation syndrome/IPEX syndrome. (C) FOXP3 delE251 mutant disrupts its homoassociation. (D) FOXP3 delK250 mutant disrupts its homoassociation.

 
Analysis of the FOXP3 protein revealed a conserved C2H2 zinc finger and one leucine zipper motif (Fig. 2B), which could mediate DNA binding as well as function in homo- and heteroassociation, a process that has already been demonstrated for other FOXP subfamily members (16, 17, 29, 30). The forkhead domain alone has been argued to form a domain-swapped dimer (27, 31). Our data indicate that FOXP3 may exist as monomeric, dimeric, tetrameric and even higher oligomeric species with the dimeric or tetrameric associations determined by the combined zinc finger and leucine zipper domains.

We next examined whether FOXP3 could homoassociate and, more importantly, whether the E251 or K250 deletion mutants within the leucine zipper region of FOXP3 found in human XLAAD/IPEX patients affect its homoassociation properties. Full-length FOXP3 E251 and K250 deletion mutants were created by site-directed mutagenesis and sub-cloned into mammalian expression vectors containing either myc or HA epitope tags at the N-terminus. HEK293T cells were transfected with the myc-tagged wild-type FOXP3, myc-tagged FOXP3-delE251, HA-tagged wild-type FOXP3 or HA-tagged FOXP3-delE251 vectors as indicated (Fig. 2C). The HA-tagged wild-type FOXP3 species was able to homoassociate with the myc-tagged wild-type FOXP3, but not with the myc-tagged delE251 mutant and vice versa (Fig. 2C). Similar experiments were performed with FOXP3 K250 deletion mutant constructs and we found that the FOXP3 delK250 mutant also could not homoassociate (Fig. 2D). Our data suggest that the integrity of the leucine zipper domain is essential for FOXP3 homoassociation.

To further investigate homoassociation of FOXP3 at the purified protein level, the critical N-terminal 196–264 amino acids of wild-type FOXP3 containing the intact zinc finger and leucine zipper domains (Zinc–LeuZip), as well as the same fragment of the delE251-mutated FOXP3 were expressed in E. coli (Fig. 3A). The Zinc–LeuZip fragments of wild-type FOXP3 and delE251 FOXP3 were highly expressed and were purified as MBP fusion proteins. The MBP tag alone was also expressed and purified. The MBP-fused Zinc–LeuZip fragment contains 497 amino acids with a predicted MW of 54.9 kDa. The determination of protein MWs in solution was based on the elution volume from a Superdex 200 gel filtration column. Gel Filtration HMW and LMW Calibration Kits (Amersham Biosciences) were used for the calibration of Superdex 200 gel filtration column (Protein KW-803).


Figure 3
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  		Fig. 3. The zinc finger and leucine zipper domains mediate FOXP3 homotetramerization. (A) Schematic representation of FOXP3 subdomains and the wild type and delE251-mutated maltose-binding protein (MBP)–Zinc–LeuZip constructs. (B) Superdex 200 chromatography analysis of the purified MBP fusion proteins. Wild type and delE251 MBP–Zinc–LeuZip were both eluted as a single peak on Superdex 200 with a calculated molecular weight (MW) of 225 and 58.8 kDa, respectively. MBP alone eluted as a single peak with a MW of 43.5 kDa. (C) SDS-PAGE analysis of purified FOXP3 MBP-WT and MBP-delE251 Zinc–LeuZip proteins under denaturing and reducing conditions (100 mM dithiothreitol) reveals migration as a monomer.

 
Although the purified wild-type FOXP3 MBP–Zinc–LeuZip (MBP-WT) and FOXP3 delE251 MBP–Zinc–LeuZip (MBP-delE251) eluted as single peaks on gel filtration, the wild-type MBP–Zinc–LeuZip eluted with an apparent MW of 225.0 kDa, while the delE251 MBP–Zinc–LeuZip eluted as a single peak at 58.8 kDa (Fig. 3B). The peak protein fractions were concentrated and further examined by SDS-PAGE (Fig. 3C). Given the apparent MW of both the wild type and delE251 proteins of ~50 kDa on SDS-PAGE, our chromatography results indicate that the purified MBP-WT forms a homotetramer in solution (Fig. 3B, upper panel), while MBP-delE251 is monomeric (Fig. 3B, middle panel). We also developed a TEV protease cleavable MBP-tag expression vector and purified wild-type FOXP3 Zinc–LeuZip alone as a tetramer (data not shown). In summary, we have found that the zinc finger and leucine zipper domains of FOXP3 are sufficient to mediate homotetramerization, and that one amino acid deletion (E251) within the leucine zipper domain, as found in human XLAAD/IPEX patients, disrupts this homotetramerization. Just as FOXP3 associates with different supramolecular ensembles in an apparent dynamic manner, FOXP3 and fragments derived from it can be found in dimeric or tetrameric associations.

Heteroassociation of FOXP3 with FOXP1 is disrupted by the XLAAD/IPEX mutation
Expression patterns of FOXP subfamily members Foxp1, Foxp2, Foxp3 and Foxp4 in sorted murine CD4+CD25+ and CD4+CD25– T cells were analyzed. We found that while Foxp1 was expressed equally in both CD4+CD25+ and CD4+CD25– T cells, Foxp3 was expressed mainly in CD4+CD25+ T cells as expected, and both Foxp2 and Foxp4 were undetectable in these primary murine CD4+ T cells (Fig. 4A). To evaluate endogenous FOXP1 protein level in human CD4+ T cells, nuclear extracts from in vitro-expanded human CD4+CD25+ T cells or CD4+CD25– T cells were immunoprecipitated with anti-FOXP1 mAb JC12, then immunoblotted with the same antibody. We found that endogenous FOXP1 was abundantly expressed as multiple isoforms in both human CD4+CD25+ T cells and CD4+CD25– T cells (Fig. 4B). Next, we tested whether the single amino acid deletion of FOXP3 at K250 or E251 as found in human XLAAD/IPEX patients affects its ability to heteroassociate with FOXP1. We found that ectopically expressed wild-type human FOXP3, but neither K250 nor E251 deletion mutants, heteroassociated with the subfamily member FOXP1 (Fig. 4C). We also observed that endogenous FOXP1 co-precipitated with endogenous FOXP3 in human CD4+CD25+ T cells as well as in FOXP3-transfected Jurkat E6.1 T cells (Fig. 4D). Furthermore, endogenous FOXP3 was observed to co-localize with FOXP1 at many sites within the nucleus of human CD4+CD25+ T cells (Fig. 4F).


Figure 4
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  		Fig. 4. Heteroassociation of FOXP3 with FOXP1 is disrupted in human X-linked autoimmunity-allergic dysregulation syndrome (XLAAD)/immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome (IPEX) mutations. (A) FOXP1, FOXP2, FOXP3 and FOXP4 transcription levels in murine CD4+CD25+ and CD4+CD25– T cells. The control lanes contain no template. (B) FOXP1 expression level in human CD4+CD25+ and CD4+CD25– T cells. (C) FOXP3 heteroassociates with FOXP1. HEK293T cells were co-transfected with expression plasmids for FLAG-FOXP1, myc-FOXP3a, myc-delE251 FOXP3a or myc-delK250 FOXP3a as indicated. Cell lysates were immunoprecipitated with anti-FLAG, then immunoblotted with anti-myc 4E10 or anti-FLAG. (D) FOXP3 associates with FOXP1 in human CD4+CD25+ T cells. (E) Impaired endogenous FOXP3–FOXP1 association in XLAAD/IPEX patient T cells. (F) Nuclear co-localization of endogenous FOXP3 with FOXP1 in CD4+CD25+ T cells. Human CD4+CD25+ T cells were stimulated for 2 h with PMA/ionomycin, fixed, permeabilized and stained with either hFOXY anti-FOXP3 (red) in conjunction with Rabbit anti-FOXP1 (green) (left panel) or PCH101 rat anti-FOXP3 (green) plus Rabbit anti-FOXP1 (red) (right panel). (G) Endogenous knockdown of FOXP1 in FOXP3-expressing Jurkat T cells relieved FOXP3-mediated repression on IL-2 production. The result is the average ± SEM of three independent experiments.

 
To evaluate the functional consequences of FOXP1-FOXP3 heteroassociation, nuclear extracts from a human T-cell line derived from XLAAD/IPEX patient PBLs expressing delE251-mutant FOXP3 or from a normal control human T-cell line expressing wild-type FOXP3 were immunoprecipitated with the JC12 FOXP1 monoclonal antibody, then immunoblotted with anti-FOXP3 mAb 221D (Fig. 4F). These endogenous co-immunoprecipitation experiments revealed that wild-type FOXP3 heteroassociated with FOXP1, but FOXP3 from XLAAD/IPEX patient T cells containing the E251 deletion was severely impaired in its ability to heteroassociate with FOXP1 (Fig. 4F). Moreover, we found that knockdown of endogenous FOXP1 expression by lentiviral vector-mediated shRNA specific to FOXP1 TRCN0000015664 (sh64) in FOXP3-expressing Jurkat T cells partially relieved FOXP3-mediated repression of IL-2 production (Fig. 4G). These findings suggest that FOXP1–FOXP3 heteroassociation may play an important but presently undefined role in human regulatory T cells.

IPEX mutations disrupt oligomerization-dependent recruitment of FOXP3 to the IL-2 promoter in vivo
IL-2 is a critical cytokine for the regulation of peripheral T-cell tolerance and regulatory T-cell function (32). CD4+CD25+ regulatory T cells suppress polyclonal T cell activation in vitro by inhibiting IL-2 production (33). Ectopic expression of FOXP3 was sufficient to divert naive T cells toward a regulatory T-cell phenotype capable of suppressing proliferation of other T cells, through inhibition of IL-2 production (4). Putative forkhead-binding sites have been identified adjacent to the NFAT sites in the human IL-2 promoter (14). Moreover, under some circumstances, FOXP3 physically associates with NFAT and NF-{kappa}B transcription factors and has been found to block their ability to induce IL-2 (26, 27).

To elucidate the molecular basis of the functional difference between wild-type FOXP3 and its E251 deletion mutation in human XLAAD/IPEX patients, we employed a chromatin immunoprecipitation assay to test endogenous FOXP3 association with the human IL-2 promoter in vivo. A primary human T-cell line from an XLAAD/IPEX patient expressing FOXP3 with the E251 deletion (delE251) (10) and a normal primary human T-cell line expressing wild-type FOXP3 were shown to express FOXP3 equivalently (Fig. 5A). After determining that mAb hFOXY could immunoprecipitate wild-type FOXP3 and delE251 FOXP3 equally (Fig. 5B), we utilized a pair of primers to detect the recruitment of endogenous FOXP3 to the human IL-2 promoter in vivo by chromatin immunoprecipitation (Fig. 5C). Our results indicated that endogenous wild-type FOXP3 from normal human T cells associated with the human IL-2 promoter, whereas FOXP3 from human XLAAD/IPEX patient T cells containing the E251 deletion did not discernibly associate with the human IL-2 promoter (Fig. 5D). Although FOXP3 is part of a supramolecular complex, it is the oligomerized structure of wild-type FOXP3 that occurs via the zinc finger and leucine zipper domains that is required for the efficient recruitment of FOXP3 to the IL-2 promoter in vivo.


Figure 5
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  		Fig. 5. Endogenous wild-type FOXP3, but not the FOXP3 E251 deletion mutant, associates with the human IL-2 promoter in T cells. (A) Nuclear extracts from primary normal and X-linked autoimmunity-allergic dysregulation syndrome (XLAAD)/immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome (IPEX) patient (delE251) T-cell lines were immunoblotted with 221D. ß-Actin protein level was determined to show equivalence of nuclear extracts. (B) Equivalent levels of FOXP3 are immunoprecipitated by anti-FOXP3 mAb hFOXY (eBioscience) from WT and XLAAD/IPEX nuclear extracts. (C) Schematic representation of the primers used for detection of human IL-2 promoter region. (D) Chromatin cross-linking and immunoprecipitation PCR results showing that wild-type FOXP3 from primary normal T cells but not the mutant FOXP3 from delE251 T cells associates with IL-2 promoter. DNA from 1% of input for IP as positive control (input); normal mouse IgG (IgG); anti-acetyl-histone 4 antibody as the positive control for transcriptionally active chromatin (AcH4); mouse anti-human FOXP3 mAb hFOXY (FOXP3).

 
Impaired IPEX patient DelE251 FOXP3 binding to the NFAT/forkhead site of the IL2 promoter in vitro
To examine the direct binding of FOXP3 to the IL-2 promoter in vitro, we performed EMSAs using a probe from the IL-2 promoter that contains a consensus NFAT-binding site adjacent to a potential forkhead-binding site (we term this NFAT probe). Purified FOXP3-c106-431 (Fig. 6C, lane 6) and nuclear extracts from wild-type FOXP3 expressing Jurkat cells (Fig. 6C lane 3), but not the vector control extracts (Fig. 6C, lane 2), bound to the probe. This binding was specific since it was competed with cold probe or with the 400-bp human IL-2 promoter region used in the chromatin immunoprecipitation assay (Fig. 5C). Binding of the NFAT probe was drastically reduced when we used the FOXP3 E251 deletion mutant expressing nuclear extracts (Fig. 6C, lane 4), indicating that deletion of a single amino acid within the leucine zipper domain, as in XLAAD/IPEX patients, significantly impairs FOXP3 binding to the forkhead-binding site adjacent to the NFAT site in the IL-2 promoter. FOXP3 levels were equivalent in wild type and E251 deletion mutant transfectants (data not shown).


Figure 6
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  		Fig. 6. Wild-type FOXP3 is more efficient than FOXP3 E251 deletion mutant in binding to the NFAT-binding sites of the IL-2 promoter in vitro and repressing IL-2 transcription. (A) The His6-FOXP3 c-terminal 106–431aa expression construct is depicted for use in bacterial expression and purification. (B) SDS-PAGE analysis of the purified His6-FOXP3-c106-431 protein. (C) Electrophoretic mobility shift assay of FOXP3 wild type (WT) and FOXP3 E251 deletion mutant (delE251) measuring binding to the NFAT sites on the human IL-2 promoter. Nuclear extracts from Jurkat E6.1 T cells transfected with the control empty vector, wild-type FOXP3 (WT) or delE251 FOXP3 expression vectors were used in each binding reaction. Purified His6-FOXP3-C107-431 protein was used as positive control (lane 6). For cold competition, unlabeled double-stranded NFAT probe (lane 5) or unlabeled double-stranded human IL-2 promoter (–374 to +45) (lane 7) in 100-fold molar excess were mixed prior to the addition of labeled probe. For quantification, the autoradiography film was scanned and processed using ImageJ software (http://rsb.info.nih.gov/ij/) (D) IL-2 promoter luciferase assay of the repression mediated by FOXP3 wild type and FOXP3 delE251 mutant in transfected Jurkat T cells. Results presented are means of three separate experiments with SD. FOXP3 expression levels in cell lysates were analyzed with mAb 221D.

 
Reduced repression of IL-2 transcription by FOXP3 with the XLAAD/IPEX mutation
Having shown that endogenous FOXP3 (but not the FOXP3 E251 deletion mutant) associates with the IL-2 promoter in human T cells, we next examined the functional consequences of wild-type FOXP3 and the FOXP3 E251 mutant on IL-2 gene expression. IL-2 promoter activity was measured in Jurkat T cells ectopically expressing graded amounts of the wild-type FOXP3 or the FOXP3 E251 deletion mutant and co-transfected with the full-length IL-2-Luciferase reporter (24) and control TK-Renilla luciferase vectors. We found that wild-type FOXP3 repressed expression of the luciferase reporter driven by the IL-2 promoter in a dose-dependent manner (Fig. 6D, lanes 2–4). In contrast, the FOXP3 E251 mutant was less efficient but not absolutely defective in repressing IL-2 transcription (Fig. 6D, lanes 5–7). Taken together, these data provide evidence that the molecular basis for disease in human XLAAD/IPEX patients carrying the FOXP3 E251 or K250 mutation is a dysfunctional leucine zipper motif disrupting FOXP3 oligomerization and modifying its repressive function by preventing efficient interactions with sequence-specific DNA.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We found that both wild-type FOXP3 and delE251 FOXP3 exist as part of a large protein complex in primary human T cells. This indicates that formation of the multiprotein complex containing FOXP3 is not compromised in XLAAD/IPEX leucine zipper mutants of FOXP3, but rather that the mutant FOXP3 functional defect lies in another aspect of FOXP3 function such as oligomerization and DNA binding. Our studies have shown that while FOXP3 can form dimers and tetrameric forms, the mutant forms we have studied remain monomeric. It is this feature of FOXP3 oligomers embedded within a large dynamic ensemble that characterizes the functional repressive activity of FOXP3.

We determined that the leucine zipper domain in FOXP subfamily members follows a potential dimerization domain, namely the C2H2 zinc finger. To study the homoassociation of FOXP3, we expressed and purified MBP fusion proteins containing amino acids 196–264 which includes the intact zinc finger and leucine zipper domains of wild-type FOXP3 (MBP-WT) or delE251 FOXP3 (MBP-delE251) to monitor its self-association. We found that this purified wild-type FOXP3 fragment forms a tetramer exclusively. The leucine zipper domain is essential for FOXP3 tetramer formation since the delE251 Zinc–LeuZip fusion protein, having the single amino acid deletion within the leucine zipper domain as found in XLAAD/IPEX patients, remains monomeric. Our studies of purified full-length FOXP3 protein confirm formation of FOXP3 tetramers as well as the existence of purified FOXP3 monomers, dimers and higher order species in vitro in the absence of DNA. Our recent studies have shown that both wild-type FOXP3 and delE251 FOXP3 associate with histone acetyltransferase and histone deacetylases via the N-terminal 106aa–190aa proline-rich region (20), which explains the similar MWs of the supramolecular complexes of the delE251 mutant and wild-type FOXP3 under native conditions (Fig. 1A). This recently published finding (20) further explains why the purified FOXP3-C106-431 truncated protein, which also contains the 106aa–190aa proline-rich region, binds to the forkhead-binding site with a similar mobility shift compared with full-length FOXP3 (Fig. 6C).

Moreover, we found that endogenous FOXP1 heteroassociated with endogenous FOXP3 in normal human T cells but not in IPEX patient T cells, suggesting a physiological contribution of FOXP1 in the human XLAAD/IPEX autoimmune syndrome. In the immune system, FOXP1 has been shown to play an important role in macrophage differentiation (34) and is differentially expressed in B-cell populations (22). Foxp1 mRNA expression is up-regulated upon B-cell activation (35), and recently Foxp1 was found to be an essential transcriptional regulator of B-cell development (36). If FOXP3 controls regulatory T-cell function through cooperation with NFAT via its forkhead domain in a similar manner to the forkhead domain of FOXP2 (27), it is possible that FOXP1 could also compete with AP1 at the same site in vivo. We found that knockdown of endogenous FOXP1 in FOXP3-transfected Jurkat T cells by lentiviral vector-encoding shRNA partially relieved FOXP3-mediated repression of IL-2 production after TCR plus CD28 co-stimulation. Moreover, we noted that although endogenous FOXP3 was observed to co-localize with FOXP1 at many sites within the nucleus of human CD4+CD25+ T cells, the staining pattern of FOXP3 was not always coincident with FOXP1 (Fig. 4F), indicating that FOXP3 and FOXP1 might also have independent roles in regulatory T-cell function.

What is it about FOXP3 homo- and hetero-oligomerization that is so important for its physiologic function? While preparing our manuscript, another study suggested that the mutant leucine zipper domain impaired both dimerization and suppressive function of ectopically expressed FOXP3 in T cells (18, 19). Here, we have used purified FOXP3 proteins and fragments to study association patterns. We demonstrated that, rather than dimerization, the mutant leucine zipper domain impaired FOXP3 homotetramerization in vitro or oligomerization in vivo. And the oligomerization status of FOXP3 clearly plays an essential role for its efficient binding to the IL-2 promoter in wild-type FOXP3 expressing normal T cells. The idea that dimers and tetramers may mediate discrete functions was first proposed for other repressive/activating transcription factors such as p53 (37). Indeed, the deduced three-dimensional structure exhibits symmetry, such that a p53 tetramer can be considered a dimer of dimers (38).

Although the carboxyl terminal forkhead domain of FOXP3 may mediate monomeric FOXP3 DNA associations, it is clear that the leucine zipper E251 mutation disrupts FOXP3 tetramerization and dramatically impairs FOXP3 function, thus resulting in the pathogenesis of the XLAAD/IPEX syndromes. We found that the FOXP3 E251 deletion mutant poorly associated with the IL-2 promoter in human T cells, and that the FOXP3 E251 mutant was less efficient at repressing IL-2 transcription. It is possible that FOXP3, through its zinc finger and leucine zipper domain undergoes oligomerization and is in a sufficiently favorable conformation for binding with NFAT at the IL-2 promoter in T cells. This interaction may be similar but not necessarily identical to the manner in which the AP-1 transcriptional complex, a leucine zipper mediated dimer, interacts with NFAT. However, it is clear that only oligomerized forms of FOXP3 are disposed to bind to DNA efficiently.

Oligomerization is a common process necessary for optimal activity of some receptors and transcription factors. In addition, we and others have previously proposed that suppressor/regulatory T cells limit recognition of transformation-associated antigens, thus limiting immune tumor reactivity (39). Identification of small molecule inhibitors able to disrupt FOXP3 oligomerization might lead to a novel and rational approach for tumor immunotherapy.


   Acknowledgements
 
We thank G. R. Crabtree for providing human IL-2 promoter reporter constructs; E. E. Morrisey and colleagues for FOXP1 constructs and rabbit anti-FOXP1 antibody; M. Tone, V. S. Shapiro and H. Zhang for discussion and advice; S. Stoltzfus and K. Bembas for technical support. Research was supported by National Institutes of Health grants to M.I.G. who is the John Eckman Professor of Medical Science at the University of Pennsylvania. A.H.B. is supported by the Leukaemia Research Fund. The authors have no conflicting financial interests.


   Abbreviations
 
ChIP, chromatin cross-linking and immunoprecipitation
DTT, dithiothreitol
EMSA, electrophoretic mobility shift assay
HA, hemagglutinin
IPEX, immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome
MBP, maltose-binding protein
MW, molecular weight
XLAAD, X-linked autoimmunity-allergic dysregulation syndrome

   Notes
 
Transmitting editor: T. Hamaoka

Received 15 February 2007, accepted 19 March 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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