Mice We purchased Thy1.2+ C57BL/6 (B6) and B6/Thy1.1+ mice (Jackson Laboratory), and B6/Rag12/2 mice (Taconic). Mice were housed under pathogen-free conditions and used for experiments at 6–12 weeks of age. Antibodies and flow cytometry Conjugated mAbs were purchased from eBioscience (eBioscience), flow cytometry was performed using a Cyan ADP Color flow cytometer (Beckman Coulter), and data were analyzed by FlowJo 8 software (TreeStar). Plasmids and mutagenesis The indicated mouse Foxp3 cDNA lysines (K16, K17, K18, and K19) were substituted by arginine (R) or glutamine (Q) with the Quik-Change mutagenesis kit (Stratagene). After sequences were confirmed, fragments were recloned into MinR1 vector for retroviral expression. All plasmids used were sequenced (Napcore, The Children’s Hospital of Philadelphia) to verify the correct configurations; sequences were analyzed using Lasergene software (DNASTAR). Retroviral transduction of primary T cells Retroviruses were generated by cotransfection of WT MinR1- Foxp3 vector (WT-Foxp3), MinR1-Foxp3 vectors containing the indicated mutants, or parental MinR1 vector (EV), plus pCLeco (Invitrogen, CA) helper plasmid into the 293T-based Phoenix ecotropic packaging cell line using Lipfectamine 2000 (11668-019, Invitrogen). Virus containing supernatant was used to infect purified CD4+ T cells that were isolated with CD4+ T cell isolation kits (130-049-201, Miltenyi Biotec). Isolated CD4+ T cells were stimulated for 20 h using phorbol 12-myristate 13-acetate (PMA, 3 ng/mL), ionomycin (1 mM) and mouse IL-2 (10 U/mL, Roche). Activated T cells were infected with 48 h and 72 h viral supernatants harvested from transfected Phoenix cells, plus 10 U/ mL mouse IL-2 and 4 mg/mL Polybrene (Sigma), followed by centrifugation for 90 min at 3,200 ramp. Cells cultured at 37u C with 5% CO2 for 2–3 d were used in the suppression assays or for RNA isolation. Treg suppression assays Purified CD4+CD252 T cells and CD4+CD25+ Treg cells from wild-type C57BL/6 mice were isolated by CD4+CD25+ regulatory T cell isolation kit (130-091-041, Miltenyi Biotec). CD4+CD252 T cells were labeled with CFSE as effector cells and activated CD4+ T cells transduced with different Foxp3 constructs were utilized as Treg cells. Accordingly, 56105 CFSE-labeled effector T cells were stimulated with CD3 (5 mg/mL) in the presence of an equal number of irradiated syngeneic APC, isolated using kits from Miltenyi Biotec (130-049-101), and varying numbers of Treg cells [16]. Suppression of effector T cell proliferation was determined by flow cytometric analysis of CFSE dilution after 72 h. Homeostatic proliferation Congenic Thy1.1+ CD4+CD252 T cells (16106), purified using a Treg isolation kit (130-091-041, Miltenyi Biotec), were mixed with 16106 Thy1.2+ CD4+ cells transduced with different Foxp3 constructs, and adoptively transferred to Rag12/2 mice [16]. Spleen and lymph nodes were isolated after 7 days and the total number of Thy1.1+ CD4+ T cells determined by flow cytometry. Lysine Acetylation and Foxp3 Function Quantitative PCR (qPCR) Total RNA was extracted using an RNeasy Kit (Qiagen), cDNA was synthesized with TaqMan reverse transcription reagents (N808-0234, Applied Biosystems), and real-time PCR was performed using TaqMan Universal PCR Master Mix (4304437) and specific primers from Applied Biosystems. Transfection 293T cells were maintained (37 uC, 5% CO2) in RPMI-1640 plus 10% heat-inactivated FBS, penicillin and streptomycin. Cells were grown to 80–90% confluence, transfected with Foxp3 (12 mg), HA-p300 (12 mg) or empty vector using Lipofectamine 2000 (Invitrogen), harvested 48 h later, and cell lysates were prepared with using RIPA lysis and extraction buffer (Pierce). Nucleotide pull-down assay Cell lysates (100 mg) from 293T cells transfected with empty vector, WT-Foxp3 or Foxp3 mutants in the absence or presence of p300 expression vectors were diluted in buffer (20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.1% NP-40), and incubated overnight at 4 uC with 10 mg poly (deoxyinosinic- deoxycytidylic acid) (Roche) and 50 ml of streptavidin-agarose beads (Sigma) coated with biotinylated oligonucleotide containing putative Foxp3 binding sites: 59-CAAGGTAAACAAGAGTAAA- CAAAGTC-39 [30]. Beads were washed 5 times in dilution buffer, resuspended in 60 ml SDS sample loading buffer (BioRad), heated at 95 uC for 10 min, separated by SDS-PAGE, transferred to PVDF membranes and proteins detected using rat anti-mouse Foxp3 mAb or anti-acetyl-lysine Ab. Microarrays RNA was prepared from CD4+ T cells transduced with EV, WT-Fopx3, Foxp3-K17R or Foxp3-K18R using an RNeasy Kit (Qiagen) and hybridized to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays at the Nucleic Acid and Protein Core of The Children’s Hospital of Philadelphia. Microarray data were analyzed by R/Bioconductor software (CBMi, The Children’s Hospital of Philadelphia) and MAYDAY 2.12 software [45], assembled and processed in compliance with MIAME standards. Data were deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible through GEO Series accession number GSE27082 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE27082). Statistical analysis Data were analyzed by GraphPad Prism software and are presented as mean 6 SD; using a standard t-test; p,0.05 was considered significant and p,0.01 was considered highly significant. Supporting Information Figure S1 K17R and K18R did not inhibit the activity of NFAT1 on NFAT:AP-1 site. 293T cells were transfected with NFAP1-luciferase vector, NFAT1 and WT Foxp3 or K17R or K18R. Forty hours later, cells were stimulated with 1 mM ionomycin, 10 nM PMA, and 2 mM CaCl2 for 6 hours, dual luciferase activity was measured and results normalized to renilla. (PPT) Author Contributions Conceived and designed the experiments: YL WWH. Performed the experiments: YL LW RH UHB. Analyzed the data: YL UHB WWH. Wrote the paper: YL WWH. PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e29035