Cell, Volume 126

Supplemental Data

FOXP3 Controls Regulatory T Cell

Function through Cooperation with NFAT Yongqing Wu, Madhuri Borde, Vigo Heissmeyer, Markus Feuerer, Ariya D. Lapan, James C. Stroud, Darren L. Bates, Liang Guo, Aidong Han, Steven F. Ziegler, Diane Mathis, Christophe Benoist, Lin Chen, Anjana Rao

Supplemental Experimental Procedures

Crystallization of the NFAT1:FOXP2:DNA Ternary Complex

The forkhead domain of human FOXP2 (residues 502-584) and the Rel homology region (RHR) of human NFAT1 (residues 392-678) were prepared as previously described (Chen et al., 1998)(Chen et al., 1998; Stroud et al., 2006). Various DNA fragments containing the murine or human ARRE2 site were synthesized by IDT and purified as described previously (Chen et al., 1998; Stroud et al., 2006). The forkhead domain of FOXP2 exists in solution as slow-exchanging monomer and dimer (Stroud et al., 2006). Using gel filtration, we separated the monomer and dimer for biochemical analysis and crystallization. The forkhead domain of FOXP2 is identical between human and mouse. The human (5’-GGAAAAACTGTTTCA-3’) and murine (5’-GGAAAATTTGTTTCA-3’) ARRE2 sites are also nearly identical and show no detectable difference in DNA binding assays (see below, Y. Wu, unpublished data). We therefore pursued parallel crystallization of complexes formed by different lengths of NFAT1 RHR, FOXP2 and DNA to enhance our chance of obtaining crystals. So far we have only been able to obtain diffracting crystals of the FOXP2:DNA binary complex and the NFAT1/FOXP2/DNA ternary complexes with the murine ARRE2 DNA. For the NFAT1:FOXP2:DNA ternary complex, NFAT1 RHR, the FOXP2 forkhead domain, and DNA were mixed at 1:1:1 molar ratio at approximately 10 mg/mL in 5 mM HEPES, pH 7.63, 2 mM dithiothreitol (DTT), 0.5 mM EDTA and 150 mM NaCl (S75 buffer). Crystals were grown by hanging drop at 18 °C using a reservoir buffer of 50 mM cacodylic acid (pH 6.30), 12% (w/v) PEG 4000, 100 mM NaCl, 10 mM CaCl2, 5 mM MgCl2, 4% (v/v) glycerol. Crystals belong to the space group P21 with cell dimensions a=65.455 Å, b=157.447 Å, c=67.666 Å, and •=118.67°.

Data Collection, Structure Determination, and Analysis

The NFAT1:FOXP2:DNA complex crystals were stabilized in the harvest/cryoprotectant buffer: 50 mM cacodylic acid (pH 6.30), 24% (w/v) PEG 4000, 100 mM NaCl, and flash frozen with liquid nitrogen for cryocrystallography. The Data were collected at the ALS BL8.2.1 beam line at the Lawrence Berkeley National Laboratory. Data were reduced using DENZO and SCALEPACK (Otwinowski, 1997). Initial phases were determined by molecular replacement using the coordinates of NFAT RHR-N (Chen et al., 1998; Stroud et al., 2006). Molecular replacement, refinement and final analysis were done with CNS (Brunger, 1998). The statistics of crystallographic analysis are presented in Suppl. Table 1. Figures of structure illustration were prepared using MOLSCRIPT (Kraulis, 1991), Pymol (DeLano Scientific). Model building and structural comparisons were carried out in O (Jones, 1991).

Reporter Assays

Jurkat cells (10 x 106) were transfected by electroporation with HA-tagged NFAT1 and/or myc-tagged human FOXP3 expression plasmids and luciferase reporter plasmids as described below, together with a renilla luciferase reporter plasmid (Promega) for normalization (Macian et al., 2000). Twenty-four hours later, cells were stimulated for 6 hours with 1 µM ionomycin +/- 10 nM PMA in the presence of 2 mM CaCl2 and luciferase activity was measured. When CA-NFAT1 was used, endogenous NFAT activity was inhibited 24 h after transfection by pretreating cells with 1 µM cyclosporin A for 30 minutes before addition of stimuli. Results are plotted as relative luciferase units after normalisation to renilla, except in the case of Suppl. Figure 1C where luciferase values are normalised to the values obtained with control cells transfected with reporter plasmid alone. Reporter plasmids: 3x NFAT:AP-1, 3 tandem copies of the ARRE2 site from the murine IL-2 promoter (Hedin et al., 1997; Macian et al., 2000); GAL4-NFAT1 (1-144), GAL4-NFAT-TAD (Luo et al., 1996; Okamura et al., 2000); 2x-κ3 (long) (McCaffrey et al., 1994), tandem copies of sequences spanning the κ3 site of the TNF promoter (a gift from A.E. Goldfeld laboratory); 6x-NFκΒ (Krappmann et al., 2001), 3 tandem copies of the sequence 5’-AGCTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA-3’ comprised of two copies of an NFκΒ •binding site (a gift from C. Scheidereit).

Nonradioactive Electrophoretic Mobility Shift Assays (EMSAs)

Recombinant wild-type NFAT1-DBD was expressed as His6-tagged proteins and purified as previously described (Macian et al., 2000). Recombinant GST-tagged murine Foxp1 forkhead domain (Li and Tucker, 1993) (aa 461-544) was kindly provided by Phil Tucker (University of Texas, Austin), and the GST tag was removed by precision protease (GE Healthcare) prior to binding reactions. Recombinant His6-tagged human FOXP3 forkhead domain (aa 335-419) was generated by PCR subcloning into the pET28 bacterial expression vector (Novagen). Binding reactions for Foxp1 and FOXP3 were performed in a buffer containing 10 mM Hepes, 5 mM DTT, 100 mM NaCl, 0.2 mM EDTA and 5% glycerol in a total volume of 10 µl. The sequence of the NFAT:AP-1 (ARRE2) (Jain et al., 1993) probe is 5’-CAAAGAGGAAAATTTGTTTCATACAGAAGG-3’. After 20 minutes of incubation at room temperature, DNA-protein complexes were separated from free probe by electrophoresis in a 6% polyacrylamide gel. The electrophoresis mobility shift assay (EMSA) for NFAT1 and FOXP2 was performed in a buffer of 20 mM Hepes (pH 7.7), 100 mM NaCl, 1 mM DTT, and 10% glycerol with various concentrations of the NFAT1 RHR and the forkhead domain of FOXP2. The binding reactions were incubated at room temperature for 30 min, run on 5% native polyacrylamide gel in 0.5x TBE buffer and stained by ethidium bromide.

For Foxp1 (Suppl. Fig. 1D), EMSAs were performed with the Il2 promoter ARRE2 element (10 µM), recombinant NFAT1 DNA-binding domain (5 and 10 µM) and the forkhead domain of Foxp1 (5 and 10 µM). For FOXP2 (Suppl. Fig. 1E), EMSAs were performed with the Il2 promoter ARRE2 element (3.75 µM), recombinant NFAT1 DNA-binding domain (1 and 4 µM in lanes 2 and 4; 2 µM in lanes 3 and 9-12) and the forkhead domain of FOXP2 (1, 2, 4 and 8 µM as indicated in lanes 5-12). For FOXP3 (Fig. 1C), EMSAs were performed with the Il2 promoter ARRE2 element (5 µM), recombinant NFAT1 DNA-binding domain (1 and 2 µM in lanes 2, 3; 2 µM in lanes 6, 7) and the forkhead domains of FOXP3 (5 and 10 µM). Because of the weak affinity of the FOXP2 forkhead domain for ARRE2, we could not quantitatively measure the degree to which the presence of NFAT enhanced FOXP2’s affinity for DNA. However, under conditions where the FOXP forkhead domains alone barely bind the ARRE2 DNA, they can be readily recruited to DNA by NFAT.

Chemical Crosslinking of NFAT and FOXP2 on DNA

For the chemical cross-linking (Suppl. Fig. 1F), various combinations of the NFAT1 RHR (~ 12 µM), the forkhead domain of FOXP2 (~30 µM) and DNA (ARRE2 or a non-specific DNA control, ~ 12 µM) were incubated with 2.7 mM disuccinimidyl suberate (DSS) for one hour at room temperature in 20 µl buffer: 25 mM HEPES, pH 7.61, 100 mM NaCl, in 20 µl volumes. At the end of reaction time, 1 µl Tris, pH 7.5, was added to quench the reaction. Samples were then dissolved in denaturing loading dye, analyzed via SDS-PAGE with Coomassie blue staining. We used both the human and murine ARRE2 DNA in the EMSA and chemical cross-linking assays and obtained essentially the same results.

Primary T Cell Cultures and Retroviral Transductions

CD4+ cells were isolated from spleens and lymph nodes of mice with positive selection using anti-CD4 magnetic beads (Dynal) and stimulated with anti-CD3 (mAb 145-2C11) and anti-CD28 (mAb 37.5, BD Pharmingen) for 48 hours as described (Ansel et al., 2004). For retroviral transductions, Phoenix Ecotropic packaging cells were transfected with retroviral expression plasmids (pRV IRES-GFP or MSCV IRES-Thy1.1, either empty or encoding wild-type myc-tagged FOXP3 and PCR-generated mutant derivatives that were fully-sequenced to confirm the mutations). Retrovirus-containing supernatants were collected 48 and 72 hours post-transfection. The MSCV-Thy1.1 vector was a generous gift from Dr. A. Abbas. CD4+ isolated T cells were infected by spin-infection with retrovirus-containing supernatants and 8 µg/ml polybrene during the last 6-8 hours of stimulation.

CD4+CD25+ regulatory T cells were isolated from the spleens and lymph nodes of C57BL/6J mice with positive selection using anti-CD4 magnetic beads (Dynal) followed by two rounds of CD25 positive selection (Miltenyi Biotec). T cells were stimulated for 48 hours with 1 µg/ml anti-CD3 (mAb 145-2C11) and 1µg/ml anti-CD28 (mAb 37.5, BD Pharmingen) in the presence of 2000 units/ml of IL-2 for 48 hours, and kept in culture in the presence of 2000 units/ml IL-2, as described previously (Masteller et al., 2005).

Flow Cytometry and Intracellular Staining

T cells were stained intracellularly for IL-2, CTLA-4 and FOXP3 at day 3 or 4 after retroviral infection, and cell-surface staining for CD25 and GITR was performed at day 2 and 5 after infection respectively. Intracellular staining for CTLA4 and FOXP3 was performed on unstimulated T cells. Staining for IL-2 expression was performed after stimulation for 6 hours with 0.1-1 µg /ml anti-CD3 and 0.2-1 µg/ml CD28 in 12-well plates coated with goat-anti-hamster antibody, with Brefeldin A (10 µg/ml; Sigma) added during the last 2-3 hours of stimulation. Cells were washed in PBS-1%BSA, fixed with 4% paraformaldehyde in PBS for 15 minutes at 25 °C, washed in PBS, and permeabilized in saponin buffer (PBS, 0.5% saponin (Sigma), 1% BSA and 0.1% sodium azide). Nonspecific antibody binding was blocked with Fc block (Pharmingen) before intracellular staining with allophycocyanin-conjugated anti-IL-2 (Ebiosciences), phycoerythrin-CTLA-4 (BD Pharmingen), or phycoerythrin-conjugated anti-FOXP3 (E-biosciences). In some experiments, cell surface staining was completed before fixation. Cells were washed in saponin buffer and in PBS and were analyzed with a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo software. Flow cytometry data are presented both as contour plots and as plots of mean fluorescence intensity (MFI) of IL-2, CTLA-4 and CD25, normalised to that of the GFP negative population within each sample, against the mean fluorescence intensity of GFP. FOXP3 expression was also evaluated by western blotting and immunocytochemistry using an IgG-purified rabbit antibody raised against the mouse Foxp3 (Cell-tech Chiroscience).

Chromatin Immunoprecipitation

Control and FOXP3-transduced T cells were stimulated with PMA (5 nM) and ionomycin (0.5 µM) for 30 minutes. Stimulation was terminated by addition of fixation buffer (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA). Fixation was stopped by addition of glycine (to final concentration of 125 mM). Cells were washed twice with cold PBS, Solution I (100 mM Hepes pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) followed by 10’ incubation at 4 degrees, and Solution II (100 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) followed by 10’ incubation at 4 degrees. Cells were resuspended in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% Sodium deoxycholate, PMSF, leupeptin, and aprotinin) and sonicated 8 x 20”, with 1’ incubation on ice between sonications. After centrifugation, lysates were precleared with protein A beads (previously incubated with 100 µg/ml salmon sperm DNA to block nonspecific DNA binding) and incubated overnight with the following antibodies: anti-67.1 (antibody to a peptide near the N-terminus of NFAT1), anti-T2B1 (antibody to a peptide at the C-terminus of NFAT1 isoform C), anti-FOXP3 (Cell-tech Chiroscience), anti-myc (9E10) or control rabbit pre-immune serum. The next day, following incubation with protein A beads, the immunoprecipitates were washed with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA), high salt buffer (50 mM Tris pH 8.0, 500 mM NaCl, 0.1% SDS, 1% NP-40, 1 mM EDTA), and LiCl buffer (50 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 0.5% sodium deoxycholate), and twice with TE (10 mM Tris pH 8, 1 mM EDTA). Beads were incubated with RNase A for 30’ at 37 degrees C, followed by proteinase K digestion for 4 hours at 50 degrees C, and incubated at 65 degrees C overnight for removal of crosslinks. Genomic DNA was precipitated and used as template for real-time PCR reactions (Sybr Green, Applied Biosystems) using a BioRad icycler. The results in Fig. 6 show the average and standard deviation of triplicates, with the exception of the CD25 promoter in Fig. 6A and 6B, which shows the average of duplicates. Experiments on expanded CD4+CD25+ T regulatory cells were performed similarly, except that cells were stimulated for 30 minutes with ionomycin (1 µM) with or without a 30 min preincubation with CsA (2 µM). The following primer pairs were used:

distal Il2 promoter forward 5’-CAGGATGGTTTTGACAAAGAGAACA-3’,

distal Il2 promoter reverse 5’- TTCATACAATGAACGCCTTCTGTATG-3’,

Ctla4 promoter forward 5’-TATACTCTCCAAGACTCCACGT-3’,

Ctla4 promoter reverse 5’-GGTTTAGCTGTTACGTCGTAAAGA-3’,

Cd25 promoter forward 5’-GAGATCACAGAACAGAGTAGGC-3’,

Cd25 promoter reverse 5’-CTCTCAGTCTGTCATCTTGGC-3’.

Mice

Mice were maintained in specific pathogen-free barrier facilities and used in accordance with protocols approved by animal care and use committees at the respective institutions. All mice were used at 4-10 weeks of age. C57BL/6J and BALB/cJ mice were purchased from Jackson Laboratories. Tcrα -/- DO11.10 TCR-transgenic mice were bred in the Harvard Medical School animal facility. NOD/Lt and BDC2.5/NOD mice were maintained in the Joslin Diabetes Center barrier facility and bred under specific-pathogen-free conditions. Diabetes was monitored by measuring glucose in the urine (Diastix, Bayer, Elkhart, IN) and confirmed by measuring blood glucose levels (Glucometer Elite,

Bayer). Animals were considered diabetic if measured glucose levels were above 300 mg/dl in two consecutive measurements.

Induction of Diabetes in NOD Mouse Model

CD4+ T cells were isolated from BDC2.5/NOD mice, activated under Th1 conditions (Ansel et al., 2004), and infected as described above with MSCV IRES-Thy1.1 retrovirus, empty or encoding wild-type or mutant FOXP3. Two days after infection, Thy1.1+ cells were sorted and transferred into neonatal NOD mice at a 1:2 ratio (0.5 x105 : 1 x 105) with untransduced BDC2.5/NOD Th1 cells (“effector” cells). Recipient mice were monitored for six weeks for presentation of diabetes. For histological scoring of insulitis, paraffin sections of formalin-fixed pancreata were examined after hematoxylin–eosin staining. Multiple sections were taken from at least three different levels and 110–140 islets were examined for each group. Insulitis refers to islet lesions with a clear islet infiltrate exhibiting direct lymphocyte beta-cell contact. ‘‘Aggressive insulitis’’ refers to an extensive infiltrate, where lymphoid cells invade the entire islet, with extensive signs of beta cell damage (Andre et al., 1996).

Figure S1. FOXP3 Represses Transcription Driven by the NFAT:AP-1 Complex and FOXP Proteins Form a Cooperative Complex with NFAT on DNA

(A-C) Jurkat cells were transfected with reporter and expression plasmids, and luciferase activity was measured as described in Experimental Procedures. Results are representative of 2-3 independent experiments.

(A) FOXP3 inhibits the activity of wildtype NFAT1 on the ARRE2 composite NFAT:AP-1 site (left) , but does not repress the activity of a GAL4 fusion protein in which the GAL4 DNA binding domain is fused to the NFAT1 transactivation domain (right)(Okamura et al., 2000). Results are representative of 2-3 independent experiments.

(B) FOXP3 inhibited CA-NFAT1 activity on the ARRE2 site in resting cells, where CA-NFAT1 cooperates with basally-active AP-1 (Fos-Jun) proteins in the nucleus; in cells stimulated with PMA, where AP-1 is activated and reporter activity increases; and in cells stimulated with both PMA and ionomycin, where both AP-1 and calcineurin-independent Ca2+ signalling are activated. FOXP3 did not inhibit the activity of CA-NFAT1 activity on palindromic NFκB-like site at which NFAT forms dimers (McCaffrey et al., 1994). Identical dose-response curves for inhibition were observed in all cases (see normalized data in Fig. 1A, B).

(C) FOXP3 does not repress the activity of endogenous NFκB. The results are representative of 2 independent experiments. A previous report (Bettelli et al., 2005) suggested that FOXP3 repressed NFκB reporter activity driven by overexpressed RelA (NFκB p65); however the authors did not rule out that FOXP3 indirectly suppressed the reporter by decreasing RelA expression from the expression plasmid used.

(D, E) Cooperative binding of the recombinant NFAT1 DNA-binding domain with the forkhead domains of Foxp1 (C) and FOXP2 (D) on the Il2 promoter ARRE2 element. Numbers in (D) give concentrations in µM. Consistent with previously published observations (Li et al., 2004), FOXP forkhead domains bind the ARRE2 site weakly. Addition of the NFAT1 DBD did not result in competition for DNA binding, but rather produced an upper or supershifted NFAT1:FOXP:DNA complex.

(F) Sequence-dependent crosslinking of NFAT1 and FOXP2 on the ARRE2 site. All reactions contain disuccinimidyl suberate (DSS) except for lanes 1 and 9. NS, nonspecific DNA. FOXP2 crosslinked efficiently to NFAT1 on ARRE2 DNA (lane 6) but much less efficiently on non-specific DNA (lane 5) or in the absence of DNA (lane 4). Thus the ternary NFAT:FOXP2 complex assembles specifically on the ARRE2 site.

Figure S2. Structural Comparison of NFAT1:FOXP2:DNA Complexes within the Asymmetric Unit and with the NFAT:AP-1:DNA Complex

(A) Superposition of the complex of NFAT1 (RHR-N: light yellow; RHR-C: light green), Fos (light red), Jun (light blue), and DNA (orange) with the complex of NFAT1 (RHR-N: yellow; RHR-C: green), FOXP2 (red) and DNA (magenta). This is a side view to show that Fos-Jun and FOXP2 occupy the same space on the ARRE2 and that the DNA (orange) bends upward in the NFAT1:Fos-Jun:DNA complex.

(B) Top view to show the DNA (magenta) bends to the side in the NFAT1/FOXP2/DNA complex. In this view only the DNA (orange) of the NFAT:Fos-Jun:DNA complex is shown for comparison.

(C) Superposition of the two ternary NFAT1:FOXP2:DNA complexes (green and black) in the asymmetric unit reveals similar overall structure except for different orientations of RHR-C. The folded-up conformation of NFAT in several complexes is explained by protein-protein interactions between RHR-N and RHR-C, but the interface is sufficiently malleable to allow flexibility in the relative orientations of RHR-N and RHR-C (Stroud and Chen, 2003). The fg loops of RHR-C from the two complexes superimpose very well. This loop is involved in direct contacts with Wing1 of FOXP2. Thus, in spite of the variable orientation of RHR-C, the NFAT:FOXP interface is nearly identical in the two complexes of the asymmetric unit.

A B

C

Figure S3. Sequence Alignment of the RHR of NFAT1-4 (Abbreviated NF1-4)

The secondary structure is shown above the alignment (Chen et al., 1998). The numbering for NFAT1 is used. Residues interacting with DNA in the NFAT1:FOXP2:ARRE2 structure are shaded magenta, residues interacting with FOXP2 are shaded orange-red, and residues interacting with both FOXP2 and DNA are shaded cyan. Residues interacting with Fos and Jun in the NFAT1:AP-1:ARRE2 structure (Chen et al., 1998) are outlined in red and blue respectively.

Figure S4. DNA Binding Assay of FOXP3 and Its NFAT-Interaction Mutants

The forkhead domains of FOXP3 mutants (EARR, Insert and WRR) were purified similarly to the wild type protein, and used in EMSA assays as described (see Experimental Procedures). Top panels: Binding of FOXP3 and mutants to the V1P probe containing the consensus FOXP site (Wang et al., 2003). Binding reactions contained V1P DNA (2.5 µM) with increasing amounts of wild type and mutant FOXP3 (3.8 µM, 7.6 µM, 15.2 µM, and 30.4 µM). For the WRR mutant, the binding and running buffers also contain 0.1% Triton X-100. Under these conditions, the DNA complex of the WRR mutant has a significantly slower mobility than that of the wild type FOXP3, whereas that of the EARR mutant has a slightly slower mobility. These mobility differences are likely due to the charge differences between the wild type protein and various mutants. Bottom panels: Binding of FOXP3 and mutants to ARRE2 DNA in the presence of NFAT1 DNA-binding domain. Binding reactions contained ARRE2 DNA (2.5 µM) with 3.8 µM NFAT1 and increasing amounts of wild type and mutant FOXP3 (1.9 µM, 3.8 µM, 7.6 µM and 15.2 µM).

Figure S5. The Insert and RR Mutants Show 2- to 3-Fold Impairment Relative to Wild-Type FOXP3, in Their Ability to Repress NFAT:AP-1 Reporter Activity and IL2 Expression

(A) Jurkat T cells were transfected with CA-NFAT1, NFAT:AP-1 reporter, renilla luciferase reporter plasmid, and increasing concentrations of either wild-type or mutant FOXP3. The next day, cells were treated with CsA for 30 minutes to inhibit endogenous NFAT activity, then stimulated with PMA and ionomycin for 6 hours. The values from each stimulation are normalized to the activity of constitutively-active NFAT1 alone. Results are representative of at least three independent experiments.

(B) Nuclear localization of FOXP3 proteins was determined by immunocytochemistry using anti-FOXP3 antibodies. Nuclei were identified by DAPI stain (not shown).

(C) T cells from DO11+/- Cα -/- mice were infected with wild-type and mutant FOXP3-IRES-GFP retroviruses. Three days later, cells were restimulated for 6 h with anti-CD3 and anti-CD28 and IL-2 expression was evaluated by intracellular cytokine staining.

(D) The mean fluorescence intensities (MFI) of intracellular IL-2 and GFP were determined for each increment of log GFP expression as described in Experimental Procedures. The data in C and D are plotted with the GFP axis on a linear (left) or logarithmic (right) scale. Relative to wild-type FOXP3, the two mutant proteins show a comparable 2- to 3-fold shift in dose-response curves in both the reporter and IL2 expression assays (compare D, right graph with the reporter assay in A).

Figure S6. Analysis of Th1 Cells Used in the Cotransfer Experiments

(A) Th1 cells from BDC2.5/NOD mice, used for the experiment shown in Figure 8, were stained for Thy1.1 expression, intracellular FOXP3 levels, and surface expression of CD103 and GITR.

(B) Similar expression levels of wild-type and mutant FOXP3 proteins, shown by overlay of fluorescence histograms of Thy1.1 positive cells stained for intracellular FOXP3.

(C) Expression levels of wildtype and mutant FOXP3 proteins in retrovirally-transduced NIH 3T3 cells were monitored by immunoblotting using an anti-FOXP3 antibody.

(D) Subcellular localization of FOXP3 proteins in retrovirally-transduced T cells was determined by immunocytochemistry using anti-FOXP3 antibodies. Nuclei were identified by DAPI stain.

(E) FOXP3 expression and nuclear localization in retrovirally-infected 3T3 cells was assessed as in C. Nuclei were identified by DAPI stain.

Figure S7. Histological Analysis of Pancreata from Cotransfer Experiments

(A) The average score of insulitis from three independent quantifications in the histology sections from mice day 6 after transfer is shown for the indicated groups. Histology was performed as shown in B.

(B) A representative histological section of pancreas from a mouse that received effector T cells plus FOXP3-transduced Th1 BDC2.5 cells, stained with hematoxylin–eosin and examined on day 42 after transfer. The insulitis remains non-aggressive, indicative of the presence of fully-functional regulatory T cells.

Figure S8. Functional Diversification of the TCR Signal through Assembly of Distinct NFAT Complexes

Left: Schematic representation of the T cell activation program. Integration of TCR and costimulatory signals, such as those initiated from CD28, is a key event leading to T cell activation. This signal integration is mediated in part by the cooperative assembly of the NFAT:Fos:Jun complex on composite response elements found in the promoters of many genes associated with T cell activation (Rao et al., 1997).

Right: Schematic representation of the T cell tolerance program. In the absence of co-stimulation, the TCR signal induces and maintains T cell anergy (Macian et al., 2002). In this case NFAT binds DNA without cooperation with AP-1 to different types of NFAT response elements such as κB-like sites which bind NFAT dimers, thereby activating a distinct set of anergy-associated genes (Heissmeyer et al., 2004; Heissmeyer and Rao, 2004). In regulatory T cells, the TCR signal may be rewired by yet another mechanism through cooperative assembly of the NFAT:FOXP3:DNA complex, on composite DNA elements very similar to those that support cooperative interactions between NFAT and AP-1.

Our results suggest that the NFAT:FOXP3:DNA complex regulates the expression of genes essential for the development and function of regulatory T cells, thus specifying a broad genetic program underlying immunological tolerance. However, our array data comparing gene expression in T cells transduced with wild-type and WWRR mutant FOXP3 indicate that FOXP3 has both NFAT-dependent and -independent target genes (VH, MR, and A. Rao, unpublished results).

Table S1. Crystallographic Analysis of the NFAT1:FOXP2;DNA Complex

Table 1 X-ray refinement statistics

Resolution (Å) 30.0-2.70 Å

Rwork/Rfree 24.5%/28.6%

Number of atoms

Protein 5992

DNA 1712

Water 119

B-factors

Protein 58.3 Å2

DNA 53.7 Å2

Water 27.3 Å2

R.m.s deviations

Bond lengths (Å) 0.0075

Bond angles (°) 1.31

R-factor = Σ ||Fo|-|Fc||/•Σ |Fo| where |Fo| and |Fc| are observed and calculated structure factor amplitudes, respectively. Rfree is calculated for a randomly chosen 9.6% of reflections.

Coordinates have been deposited in the RCSB Protein database under accession code 2AS5.

Table S2. Potential Target Genes of the NFAT:FOXP3 Complex Known NFAT Sites Match Human IL-2 -280 5’-GGAAAAACTGTTTCA-3’ +++++Murine IL-2 -280 5’-GGAAAATTTGTTTCA-3’ +++++Human IL-2 -135 5’-GGAAAAATGAAGGTA-3’ + Murine IL-2 -135 5’-GGAAAAACAAAGGTA-3’ + m.h IL-2 -90 5’-TGAAAATATGTGTAA-3’ +++++Human IL-2 -45 5’-GGAAAAATATTATGG-3’ ++ Murine IL-2 -45 5’-GGAAAAATAATATGG-3’ ++ Human IL-5 P site 5’-GGAAACATTTAGTTT-3’ ++ Murine IL-5 P site 5’-GGAAACCCTGAGTTT-3’ +++ hGM-CSF 330 5’-GGAGCCCCTGAGTCA-3’ +++ hGM-CSF 420 5’-GGAAAGATGACATCA-3’ + hGM-CSF 550 5’-GGAAAGCAAGAGTCA-3’ ++ CD40L (h,m) 5’-GGAAAATGTGCTTCG-3’ ++++ hINF-gamma 5’-GGAAATTTTTTGTCA-3’ ++++ hIL-13 5’-GGAAAATCCAGTGTC-3’ ++ hCTLA-4 -195 5’-GGAAAATGTACTCAA-3’ ++++ mCTLA-4 -195 5’-GGAAAATGTATTCAA-3’ ++++ Human IL-4 -63 5’-GGAAAATGTATTCAA-3’ ++++ Consensus 5’-GGAANNNNTGTTT -3’

We derived a consensus sequence for the NFAT1:FOXP3 complex based on the crystal structure: 5’-GGAANNNNTGTTT-3’, where N stands for any nucleotide with some preference for A or T. We then searched known NFAT sites (Rao et al., 1997) for adjacent forkhead binding sites that closely matched the consensus sequence. The number of + signs in the right column provide an arbitrary estimate of the strength of the match. Promoter/ enhancer elements with strong potential composite NFAT:FOXP binding sites are shown in bold in the left column. The middle column shows the sequences of the elements, with the NFAT site in bold and the forkhead binding site underlined.

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