A r t i c l e MLL, and RNAPII was observed in developing Th2 cells ac- companied by increased H3-K4Me3 and H3-K9Ac signals across the GATA3 locus (Fig. 1 D, right, probes (5) to (+3)). The accumulation was more prominent in fully developed Th2 cells. The total H3 levels were similar in these cells (Fig. 1 D, bottom left). Thus, the displacement of the PcG complex by the TrxG complex occurred during Th2 cell differentiation. Inter- estingly, the displacement was most characteristic in the region between the GATA3 proximal and distal promoter (Fig. 1 D, probes (5) and (4)). A previous study showed that the reduced expression of the PcG gene caused the dissociation of PcG molecules from the target genes in the epidermal progenitors (Ezhkova et al., 2009). However, in this study, substantial levels of PcG and TrxG mRNA and protein were expressed in naive CD4 T cells, Th2 cells, and fully developed Th2 cells (Fig. S2), suggesting that the displacement is unlikely to be explained by the expression levels of these molecules. The displacement of the PcG complex by the TrxG complex was dependent on STAT6 We assessed the role of the IL-4–STAT6 signaling pathway to help elucidate the molecular mechanisms underlying the dis- placement of the PcG complex by the TrxG complex at the GATA3 gene locus. Although naive CD4 T cells from Figure 1. Changes in the histone modifications and PcG and TrxG binding to the GATA3 locus during Th2 cell differentiation. (A) GATA3 mRNA was measured by quantitative RT-PCR (left panel shows the relative intensity, compared with HPRT; the mean ± SD of three samples). GATA3 protein expres- sion was determined by intracellular staining (right). Splenic B cells, naive CD4 T cells, effector Th2 cells (Th2), and fully developed Th2 cells (Full dev Th2) were used. Two independent experiments were performed with similar results. (B) Schematic representation of the mouse GATA3 gene locus. Open rectangles indi- cate a noncoding exon, and closed rectangles indicate a coding exon. Closed triangles show the locations of PCR primer pairs used in the ChIP assay. Positions of TaqMan probes used for real-time quantitative PCR ChIP are indicated in relative kilobases to the GATA3 translational start site. (C) A representative result of a ChIP assay using antibodies specific for the indicated proteins, with a series of primer pairs covering the GATA3 locus. PCR product band intensities relative to input in each primer pair are shown. PCR product bands are shown in Fig. S1. Five independent experiments were performed with similar results. (D) Binding of the PcG complex molecule, Bmi1, EZH2, and Suz12, modifications of histone H3-K27, and total histone H3 levels at several regions around the GATA3 gene locus were determined by ChIP assays with quantitative PCR analysis (left). Binding of the Menin–TrxG complex and RNAPII and the status of H3-K4Me3 and H3-K9Ac were also determined by quantitative ChIP assays (right). The relative intensity ([specific antibody ChIP  control Ig ChIP]/input DNA; highest signal intensity = 10; mean of three samples) is shown with SDs. Three independent experiments were performed with similar results. JEM VOL. 207, October 25, 2010 2495