Li et al. 10.1073/pnas.0700298104. |
SI Methods
Human CD4+CD25+ T Cells.
Human FOXP3+CD4+CD25+ T cells were obtained by in vitro expansion as follows: 200 million 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, anti-CD28 coated beads using a 3 bead to 1 cell ratio or using a cell based aAPC expressing CD64 and CD86 loading with anti-CD3 Ab (1) in the presence of high levels of IL-2 (300 U/ml) and cultured in RPMI medium 1640 with 10% FCS for the next 20-25 days.Cloning of Human FOXP3 cDNA.
Total RNA was isolated from 5 million expanded human FOXP3+CD4+CD25+ T cells. Based on the nucleotide sequence of FOXP3 in the human genome (www.ensembl.org/Homo_sapiens/), we designed the following two primers, 5'-CAAGGATCCGTATGCCCAACCCCAGG-3' and 5'-ACAGTCTAGATCAGGGGCCAGGTGTAGGG-3', to amplify FOXP3 cDNA with KOD Hot Start DNA Polymerase (Cat. no. 71086; Novagen, Inc). Two PCR amplicons were separately digested with BamHI and XbaI and cloned into the mammalian expressor vectors pIRESpuromycin-myc2 with two myc epitope tags at the N terminus or pIRESpuromycin-HA2 with two HA eptitope tags, or pIRESpuromycin-FLAG2 with two FLAG eptitope tags. DNA sequencing results showed that we cloned two FOXP3 cDNAs, one corresponding to the full length, which was used in all of the following experiments as well as a splice variant lacking exon 2.Preparation of Nuclear Extracts.
Nuclear extracts were prepared according to Nishiya et al. with some modification (2). In brief, 20 million Human FOXP3+CD4+CD25+ T cells were washed with ice-cold DPBS and were incubated in hypotonic buffer containing 10 mM Hepes, pH7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1´ complete protease inhibitor cocktails (Cat. No. 1-697-498; Roche Biochem.), 1 mM Na3VO4 on ice for 15 min. Cells were then lysed in the presence of 0.6% Nonidet P-40 on ice for 15 min with vortexing. The nuclei were collected by centrifugation for 30 sec, and were resuspended in 100 ml of 20 mM Hepes pH7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1´ protease inhibitor cocktails, 1 mM Na3VO4, followed by rotation for 30 min at 4°C. The nuclear suspension was centrifuged at 16,060 ´ g , 4°C for 15 min. The nuclear protein containing supernatant concentration was measured with BCA Protein Assay Kit (Pierce) using BSA as a standard.Plasmids, Reagents, and Antibodies.
The following antibodies were used: Affinity Purified anti-human FOXP3 monoclonal antibody hFOXY from eBioscience; anti-myc (9E10); HRP conjugated 9E10 and anti-HA-probe (F-7), anti-HDAC9 (H-45), anti-HDAC7 (C-18) from Santa Cruz Biotechnology; anti-FLAG M2; rabbit anti-HDAC7 (KG-17), anti-beta actin (AC-15), affinity-purified nonspecific IgG from Sigma; anti-TIP60 (07-038) from Upstate; anti-FOXP3 monoclonal Antibody 221D (3); HRP conjugated anti-HA 3F10 from Roche; HRP conjugated Rat anti-mouse kappa chain (04-6620) from Zymed; Cy3-conjugated anti-rabbit and FITC-conjugated anti-mouse IgG from Jackson ImmunoResearch Laboratory. Histone deacetylation inhibitor Trichostatin A (T 8552) was from Sigma. We are grateful to Dr. Eric Verdin for pcDNA-FLAG-HDAC7 constructs (4), Dr. He-Jin Lee for pCMV2-FLAG-wild type TIP60 (5), and Dr. Craig N Robson for pcDNA-FLAG-HAT-deficient TIP60 (Q377E/G380E, MutTip60) constructs (6). pcDNA-FLAG-HDAC-deficient HDAC7(H669F/H670F/H709F/H710F) construct was made by site-mutant kit. N-terminal Myc-tagged, HA-tagged, and FLAG-tagged series FOXP3 constructs 3a, N1, C1, C2, C3, C4 were made by PCR using full-length FOXP3a as template except 3b using FOXP3b as template, then subcloned to the BamHI-XhoI sites of pIRESpuromycin-myc2, -HA2, -FLAG2 tagged empty vectors. The primers are the following:3a (1-431aa) and 3b (with 71-105aa deletion): 5'-aag gat cca tgc cca acc cca ggc ctg-3' and 5'-aat ctc gag tca ggg gcc agg tgt agg g-3'; N1 (1-336aa): 5'-aag gat cca tgc cca acc cca ggc ctg-3' and 5'-tac tcg agc atg ttg tgg aac ttg aag-3'; C1 (221-431aa): 5'-gcg gat ccg acc atc ttc tgg atg ag-3' and 5'-aat ctc gag cat gtt gtg gaa c-3'; C3 (191aa-431aa): 5'-agg gat cct acc cac tgc tgg caa atg g-3' and 5'-aat ctc gag tca ggg gcc agg tgt agg g-3'; C4 (106aa-431aa): 5'tgg gat ccc tct caa cgg tgg-3' and 5'-aat ctc gag tca ggg gcc agg tgt agg g-3'.
Site-Directed Mutagenesis.
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'. And the following primers were used to make HDAC deficient HDAC7 (H669F/H670F/H709F/H710F, MutHDAC7): 1) H669F/H670F with 5'- gtg gtg cgg ccc cca gga ttc ttt gca gat cat tca aca gc-3' and 5'- gct gtt gaa tga tct gca aag aat cct ggg ggc cgc acc ac; 2) then use the H669F/H670F mutated construct as template to further mutate H709F/H710F with 5'- gta gac tgg gac gtg ttc ttt ggc aac ggc acc cag c -3' and 5'- gct ggg tgc cgt tgc caa aga aca cgt ccc agt cta c-3'. All of the mutants were made with QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's standard procedure, and confirmed by DNA sequencing.shRNA Vectors and Reagent.
TRC shRNAs (Lenti) targeting human TIP60, TRCN0000020315 (sh15), 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).Cells and Transfections.
Human HEK 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, penicillin/streptomycin and glutamine. The cells were transfected with 2 mg of each plasmid using Fugene 6 transfect reagent (Roche) according to the manufacturer's instruction manual. Forty-eight hours posttransfection cells were used in the indicated assay.Cell Lysis, Immunoprecipitation, and Immunoblotting.
Cell lysates were obtained by cell lysis in RIPA buffer (50 mM Tris•HCl, pH 7.4, 0.5% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, with 1 mM PMSF, 1 mg/ml each of Aprotinin, leupeptin and pepstatin, 1 mM Na3VO4, and 1 mM NaF), followed by immunoprecipitation with indicated antibodies, SDS/PAGE, and analyzed by Western blotting with standard procedures. ECL or ECLplus Western blotting detection reagents were used (Amersham Pharmacia Biosciences).Dual Luciferase Assay.
Jurkat transfections and all luciferase assays were performed as previously described (7). Briefly, Jurkat E6.1 T cells were harvested at ≈0.4 ´ 106 cells per ml growing in antibiotic-free RPMI medium 1640 plus 10% FCS medium, and resuspended at 20 ´ 106 cells per ml. A total of 0.5 ml of cells and 20 mg of FOXP3 expression vectors or the control empty vector, 15 mg of IL-2-Luciferase and 2 mg of TK-Renilla luciferase vectors, as indicated, were electroporated in a 0.4-cm gap cuvette (Invitrogen) at LV (low voltage), 310 V, 10 ms, using a BTX ECM 2001 electroporation system (Harvard Apparatus, MA). Twenty to 24 h after the electroporation, cells were stimulated with 50 ng/ml PMA and 1 mM 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 presented are the mean of three separate experiments, and the error bars indicate standard deviations.IL-2 ELISA.
Twenty-four hours after transiently transfecting FLAG-tagged FOXP3a or empty control vector, Jurkat T cells were treated with 400 nM TSA for 4 h followed by three washes in RPMI medium 1640, then stimulated with plate-bound anti-TCR V beta 8.1 monoclonal antibody plus 1 mg/ ml soluble anti-CD28 antibody (PharMingen) for 16 h. IL-2 secretion was determined by the Human IL-2 (Interleukin-2) ELISA Ready-SET-Go! Kit according to the standard protocol provided by the manufacturer (eBioscience).1. Maus MV, Thomas AK, Leonard DG, Allman D, Addya K, Schlienger K, Riley JL, June CH (2002) Nat Biotechnol 20:143-148.
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4. Dequiedt F, Kasler H, Fischle W, Kiermer V, Weinstein M, Herndier BG, Verdin E (2003) Immunity 18:687-698.
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6. Gaughan L, Logan IR, Cook S, Neal DE, Robson CN (2002) J Biol Chem 277:25904-25913.
7. Shapiro VS, Mollenauer MN, Weiss A (1998) J Immunol 161:6455-6548.