STAT6-deficient mice expressed equivalent levels of GATA3 mRNA, they failed to up-regulate GATA3 mRNA during in vitro differentiation into Th2 cells (Fig. 2, A and B). The re- duced expression of GATA3 protein in STAT6-deficient Th2 cells was confirmed by the intracellular staining of GATA3 (Fig. 2 C). WT and STAT6-deficient CD4 T cells were cul- tured under Th2 conditions for 5 d, and then the cells were subjected to a ChIP assay with quantitative PCR. In the case of WT and STAT6-deficient naive CD4 T cells, equivalent binding of Bmi1, Menin, and MLL and equivalent histone modifications at the upstream region and coding region of the GATA3 gene (probes (5) and (4)) were observed (Fig. 2 D). In contrast, the binding of Bmi1 and the level of H3-K27Me3 at the upstream region of the proximal promoter (probes (5) and (4)) were reduced in WT but not in STAT6–deficient effector Th2 cells (Fig. 2 D, left). In addition, the recruitment of Menin and MLL and the induction of H3-K4Me3 and H3-K9Ac were significantly impaired in STAT6-deficient Th2 cells (Fig. 2 D, right). Although less characteristic, a simi- lar pattern was observed at the coding region (Fig. 2 D, probe (+3)). The total H3 levels were not affected in STAT6- deficient naive and Th2 cells. The displacement of the PcG complex by the TrxG complex and corresponding histone modifications observed in Th2 cells were not observed in Th1 cells (Fig. 2 E). These results indicate that the activation of the STAT6 is crucial for the displacement of the PcG complex by the TrxG complex and that this is accompanied by the altera- tion in histone modification of the GATA3 gene locus during Th2 cell differentiation. Identification of the STAT6-binding sites within the GATA3 gene locus A sequence analysis identified 11 putative STAT6 consensus sites within the GATA3 locus (from 20 kb upstream to 20 kb Figure 2. The displacement of the PcG by the TrxG complex at the GATA3 gene locus was induced in a STAT6-dependent manner. (A and B) GATA3 expression in STAT6-deficient naive CD4 T cells and Th2 cells was determined by a quantitative PCR. The relative intensity compared with HPRT (mean of three samples with SD) is shown. (C) The levels of GATA3 protein expression were determined by intracellular staining. (D) PcG and TrxG binding to the GATA3 locus and histone modifications in STAT6-deficient naive CD4 T cells and Th2 cells were determined by a ChIP assay with a quantita- tive PCR analysis as described in Fig. 1 D. Positions of TaqMan probes described in Fig. 1 B are indicated in the parenthesis. The total H3 levels were in- cluded as a control. (E) PcG and TrxG binding to the GATA3 gene locus and histone modifications in Th1, Th2, and STAT6-deficient Th2 cells were determined by a ChIP assay. Positions of TaqMan probes described in Fig. 1 B are indicated. (D and E) The relative intensity (mean of three samples) is shown with SDs. (A–E) Three independent experiments were performed with similar results. 2496 Maintaining Th2-specific GATA3 gene expression | Onodera et al.