binding of STAT6 to the GATA3 S4 and S7 regions (Fig. 3 C). In splenic B cells, phosphorylation and nuclear translocation of STAT6 were induced by IL-4, but STAT6 binding at the GATA3 gene locus was not detected (unpublished data). The putative STAT6-binding sites are located at positions 1.5 kb (S4) and 10.2 kb (S7) relative to the translational start site (Fig. 3 A). To determine whether these motifs are re- quired for the binding of STAT6, an electrophoretic mobil- ity shift assay (EMSA) was used with nuclear extracts prepared from IL-4–stimulated T cells and incubated with radiolabeled double-stranded DNA oligonucleotide probes containing the putative STAT6-binding sites or mutated STAT6-binding sequence (probes S4, Mut-S4, S7, and Mut-S7). IL-4 treat- ment resulted in the formation of a nucleoprotein complex at these sites in a STAT6-dependent manner for probes S4 and S7 (Fig. 3 D) but no specific bands for the mutated oligo- nucleotides (Fig. 3 D, probes Mut-S4 and Mut-S7). Next, a supershift EMSA was performed using WT CD4 T cells stimulated in the presence of IL-4 (Fig. 3 E). Whereas no band shift was observed with control antibodies (Fig. 3, com- pare D [second through eighth lanes] with E [first through fifth lanes]), bands were supershifted by preincubation with antibodies against STAT6 (Fig. 3 E, fourth through eighth lanes), indicating that these complexes contain STAT6. Antibodies specific for HAT complex molecules, anti-p300, or anti–CREB-binding protein (CBP) mAb were also in- cluded in the supershift analysis. The intensity of the STAT6 complex band was reduced by the addition of anti-p300 mAb (second and sixth lanes) and anti-CBP mAb (Fig. 3 E, third and seventh lanes), indicating that p300 and CBP are compo- nents in the STAT6 complex. Thus, STAT6 appears to asso- ciate with the p300/CBP-containing HAT complex after IL-4 stimulation. Trichostatin A (TSA) treatment was sufficient for the dissociation of the PcG complex from the GATA3 locus To further understand the role for histone modification in this process, the effect of histone deacetylase inhibitor TSA in the displacement of PcG complex by TrxG complex was ex- amined. WT and STAT6-deficient CD4 T cells were cul- tured under Th2 conditions in the absence or presence of TSA for 5 d, and then the cells were subjected to a ChIP assay. The dissociation of the PcG complex (Bmi1 and EZH2) at the upstream region of the proximal promoter (probes (5) Figure 4. TSA treatment induced dissociation of PcG complex at the upstream region of the GATA3 gene locus. Freshly prepared WT or STAT6- deficient CD4 T cells were stimulated under Th2 culture conditions, and where indicated, 10 ng/ml TSA was added on day 2. 3 d later, the cells were sub- jected to ChIP assays using the indicated antibodies. (A) Effects of TSA treatment on the dissociation of Bmi1 and EZH2 and the status of H3-K27Me3. The total H3 levels were included as a control. (B) Effects of TSA treatment on the recruitment of TrxG complexes (MLL and Menin) and the status of H3- K4Me3 and H3-K9Ac. (A and B) The relative intensity (mean of three samples) is shown with SDs. (C) The levels of noncoding transcripts at the GATA3 gene locus and mature GATA3 mRNA and HPRT in cells stimulated as in A and B were determined by quantitative RT-PCR. The relative intensity compared with HPRT (mean of three samples with SDs) is shown. Effect of TSA treatment on the recruitment of Ser5-P and Ser2-P RNAPII was determined by a ChIP assay with quantitative PCR (bottom). (A–C) Positions of TaqMan probes described in Fig. 1 B are indicated in the parenthesis. Three independent experiments were performed with similar results. 2498 Maintaining Th2-specific GATA3 gene expression | Onodera et al.