anti–Ser5-P RNAPII (Abcam), anti–Ser2-P RNAPII (Abcam), anti-MLL (Bethyl Laboratories, Inc.), anti-Bmi1 (Santa Cruz Biotechnology, Inc.), anti-STAT6 (Santa Cruz Biotechnology, Inc.), anti-Menin (Bethyl Labora- tories, Inc.), anti-Suz12 (Abcam), and anti-EZH2 (Diagenode). The anti- bodies used for EMSA were anti-STAT6 (Santa Cruz Biotechnology, Inc.), anti-CBP (Abcam), and anti-p300 (Millipore). The antibodies used for cyto- plasmic staining were anti–IFN-–FITC, anti–IL-4–PE, and anti-GATA3– Alexa Fluor 647 (BD). The generation of Th1 and Th2 cells. Th1/Th2 cells were generated as previously described (Yamashita et al., 2006). In brief, splenic CD4 T cells were stimulated with 3 µg/ml of immobilized anti–TCR- mAb plus 1 µg/ml anti-CD28 mAb under the Th1 or Th2 culture conditions for 5 d in vitro. Th1 conditions were as follows: 25 U/ml IL-2, 10 U/ml IL-12, and anti–IL-4 mAb. Th2 conditions were as follows: 25 U/ml IL-2 and 100 U/ml IL-4. These cells were used as either Th1 or Th2 cells. Establishment of fully developed Th2 cells. Splenic CD4 T cells from DO11.10 OVA-specific TCR transgenic mice were stimulated with an OVA peptide (1 µM Loh15) plus APC under Th2 culture conditions for 5 d in vitro. The Th2 cells were further cultured in vitro for 2 d in the absence of any exogenous cytokines. The cultured CD4 T cells were then restimu- lated with OVA peptide (1 µM Loh15) plus APC with IL-2 and anti–IL-4 mAb for 5 d. This cycle was then repeated more than three times. Quantitative RT-PCR. Total RNA was isolated using the TRIZOL re- agent (Invitrogen). cDNA was synthesized using oligo (dT) primer and Superscript II RT (Invitrogen). Quantitative RT-PCR was performed as described previously using a sequence detection system (ABI Prism 7500; Applied Biosystems; Yamashita et al., 2006). The primers and TaqMan probes for the detection of GATA3, Bmi1, EZH2, Menin, MLL, and HPRT were purchased from Applied Biosystems and Roche, respectively. The specific primers and Roche Universal probes used are described in Table S1. The expression was normalized by the HPRT signal. ChIP assay. ChIP was performed using ChIP assay kits (Millipore) as previ- ously described (Yamashita et al., 2006). Quantitative representations of the results are shown as relative band intensities measured by a densitometer (AE6905H [ATTO] and CS Analyzer version 2.08b). The specific primers used are described in Table S1. Real-time quantitative PCR analysis was performed on an ABI Prism 7500 real time PCR machine with TaqMan probes and primers (sequences available in Table S1). To calculate the en- richment of each protein to a particular target DNA, values obtained (via the standard curve method) for each target were divided by the amount of the corresponding target in the input fraction. Enrichments obtained from mock immunoprecipitations performed in parallel with normal IgG were then sub- tracted from the enrichment values obtained with specific antibodies ([spe- cific antibody ChIP  control Ig ChIP]/input DNA). All the enrichments are expressed as a function of the highest enrichment obtained on the locus (set to 10; Demers et al., 2007). EMSA. EMSAs were performed using a gel shift assay system (Promega) as described previously (Kimura et al., 2005). In brief, the nuclear extracts were incubated at room temperature with a 32P-labeled, double-stranded oligonucleotide in DNA-binding buffer. In some experiments, the nuclear extracts were preincubated at 4°C with specific antibodies. Electrophoresis was conducted on 4% native polyacrylamide gel (acrylamide/bisacrylamide ratio 29:0.8 in 0.5× Tris-borate-EDTA), and the radioactivity was visual- ized by autoradiography. The oligonucleotides used in this experiment were normal probe S4, 5-CTTGGCGTTCAAGAGAATTCTCAA-3; mutant probe S4, 5-CTTGGCGTTCGGTTTAATTCTCAA-3; nor- mal probe S7, 5-AGCCAACTTCCTAGGAAAAAGCTG-3; and mu- tant probe S7, 5-AGCCAACTTCAGTTTAAAAAGCTG-3. The STAT6 consensus motif is underlined, and the mutated nucleic acids are shown in italicized and bold characters. 2504 TSA treatment. STAT6-deficient splenic CD4 cells were cultured under Th2 conditions, and 10 nM TSA (Sigma-Aldrich) was added in the culture on day 2. After another 3-d culture, CD4 T cells were collected for the ChIP assay and RT-PCR. Retroviral vectors and infection. The pMX-IRES-hNGFR plasmid was generated as previously described (Shinnakasu et al., 2006). Retrovirus vec- tors containing STAT6VT cDNA (pMXs-STAT6VT-IRES-hNGFR; Daniel et al., 2000) or STAT6VT dDBD cDNA (pMXs-STAT6VT-dDBD-IRES- hNGFR) were used. The infected cells were enriched by magnetic cell sorting with anti-hNGFR (clone C40-1457; BD) and were subjected to a ChIP assay. A resting and restimulation culture system. Naive CD4 T cells from Menin-deficient mice were cultured under Th2 conditions for 5 d. The Th2 cells were further cultured for 2 d in the absence of cytokines added and then re- stimulated with anti-TCR mAb in the presence of IL-2 and anti–IL-4 mAb for an additional 5 d. This cycle was then repeated. PcG/TrxG binding and histone modifications were detected by ChIP assays, and the GATA3 expression was determined using quantitative RT-PCR at the end of the each cycle. Immunoblot analysis. Cytoplasmic extracts and nuclear extracts were pre- pared using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific). The antibodies used for the immunoblot analysis were anti–histone H3 (Abcam), anti-Erk1 (Santa Cruz Biotechnology, Inc.), anti- MLL (Bethyl Laboratories, Inc.), anti-Bmi1 (Santa Cruz Biotechnology, Inc.), anti-Menin (Bethyl Laboratories, Inc.), and anti-EZH2 (Diagenode). ChIP-seq and Illumina sequencing. For ChIP-seq analysis, immuno- precipitate and input samples were prepared using ChIP-Seq Sample Prep kit (Illumina, Inc.). Adaptor-ligated DNA fragments were size-fractionated by 12% acrylamide gel, and the 150–250-bp fraction was recovered. DNA thus obtained was amplified by 18 cycles of PCR. 1 ng of the DNA was used for the sequencing reaction of the GAIIx (Illumina, Inc.) according to the man- ufacturer’s instructions. 150,000–250,000 clusters were generated per tile, and 36 cycles of the sequencing reactions were performed. Short-read se- quences were aligned to the mouse genome sequences (mm9 as from the University of California, Santa Cruz Genome Browser) using the Eland pro- gram. Sequences allowing no more than two mismatches per sequence were used for the analysis. Online supplemental material. Fig. S1 shows the real PCR product bands of the results shown in Fig. 1 C. Fig. S2 shows expression of Bmi1, EZH2, Menin, and MLL in naive CD4 T cells, Th2 cells, and fully devel- oped Th2 cells. Fig. S3 shows the binding pattern of phosphorylated RNA- PII at the GATA3 gene locus. Fig. S4 shows that expression of Bmi1, EZH2, Menin, and MLL was not affected by TSA treatment in the STAT6- deficient Th2 cells. Fig. S5 shows phenotypic characterization of peripheral CD4 T cells in Menin-deficient mice. Fig. S6 shows the levels of total H3 at the GATA3 gene locus in Menin-deficient Th2 cells. Fig. S7 shows IL-4/ STAT6-independent maintenance of GATA3 expression in developed Th2 cells. Fig. S8 shows binding of Menin protein to specific regions around the GATA3 gene locus. Table S1 shows primers and probes used for ChIP and RT-PCR. Online supplemental material is available at http://www.jem .org/cgi/content/full/jem.20100760/DC1. We are grateful to Drs. Ralph Kubo and Anjana Rao for their helpful comments and constructive criticisms in the preparation of the manuscript. We thank Ms. Kaoru Sugaya, Hikari K. Asou, Satoko S. Norikane, and Mr. Toshihiro Ito for their excellent technical assistance. This work was supported by the Global Centers of Excellence Program (Global Center for Education and Research in Immune System Regulation and Treatment), Ministry of Education, Culture, Sports, Science and Technology (MEXT; Japan), and by grants from MEXT (Grants-in-Aid for Scientific Research on Priority Areas #17016010 and #20060003, Scientific Research [B] #21390147, Scientific Research [C] #21591808 and #20590485, and Young Scientists [B] # 20790367, [Start-up] #19890041, Maintaining Th2-specific GATA3 gene expression | Onodera et al.