required for the expression of the GATA3 gene, STAT6- binding sites that are critical for the induction of GATA3 transcription have not been identified. We identified two STAT6-binding sites within the intron 1 and intron 3 regions of the GATA3 gene locus at which STAT6 rapidly accumu- lated after IL-4 stimulation, resulting in the recruitment of the HAT complex (Fig. 3). Therefore, Th2-specific GATA3 transcription appears to be mediated by the direct binding of STAT6. These binding sites are located several kilobases downstream from the transcriptional start site. However, this is not unusual because there are several examples in which transcription is induced by the binding of STAT family pro- teins at the distal locus control region (LCR; Jothi et al., 2008; Liao et al., 2008). We also demonstrate that STAT6 binding recruited the HAT complex to the GATA3 gene and induced the spread- ing of histone hyperacetylation with the resultant dissociation of the PcG complex at the upstream region of the GATA3 gene. The two STAT6-binding sites are also located several kilobases downstream from the region where the displace- ment of PcG by TrxG occurs. Two likely mechanisms can be entertained. First, these STAT6-binding sites may act as cis- regulatory elements and exert enhancer activity for GATA3 mRNA expression. At the -globin gene locus, communica- tion between the LCR and the active -globin genes is gen- erally thought to occur via a looping mechanism whereby the LCR and the -globin genes come into direct physical con- tact through their interacting proteins and the intervening DNA sequences are looped out (Dean, 2006). The STAT6- binding sites may be close to the proximal or distal promoter through the high-ordered chromatin configuration, and thus, the STAT6 complex may control the dissociation of the PcG complex from the distant region of the STAT6-binding sites. Alternatively, the STAT6–HAT complex may spread from its binding sites toward the upstream region during Th2 cell differentiation and induce the dissociation of the PcG com- plex. This model is called the tracking or spreading model (Bondarenko et al., 2003; Dean, 2006). For example, ery- throid activators NF-E2 and GATA1 are important for the recruitment of the HAT complex to the LCR and for his- tone acetylation at the LCR and the distant -globin gene (Johnson et al., 2001). The accumulation of STAT6 was ob- served at only two sites at the early phase of Th2 cell differ- entiation (Fig. 3, B and C), whereas a few days after the initial stimulation, the binding of STAT6 was broadly detected throughout the GATA3 gene by a ChIP assay (unpublished data). Thus, we would favor the second model. The induction of Th2-specific up-regulation of GATA3 transcription has been suggested to result from the activation of the distal promoter of GATA3 (Amsen et al., 2007; Fang et al., 2007). However, recent studies by other investigators have indicated that the Th2-specific increase of GATA3 tran- scripts was dependent on the proximal promoter (Scheinman and Avni, 2009; Yu et al., 2009). As shown in Fig. 5 (F and G), we demonstrated that exon 1b-1 transcripts in- duced by the proximal promoter were much more abundant JEM VOL. 207, October 25, 2010 A r t i c l e in comparison with exon 1a-1 transcript, and thus, it would appear to us that the majority of the Th2-specific increase of mature GATA3 transcripts is dependent on the proximal promoter. In contrast, the transcript induced by the distal promoter may reflect the existence of noncoding transcripts, which may play an important role in the dissociation of the PcG complex at the GATA3 gene (Hekimoglu and Ringrose, 2009). In fact, the induction of noncoding transcripts and that of exon 1a-1 junction-containing transcripts occurs in similar fashion. The induction of noncoding transcripts at the upstream region of the proximal promoter including the re- gion of the distal promoter was STAT6 dependent, and the noncoding transcripts were induced by the treatment with TSA (Fig. 4 C). The transcripts from the distal promoter are spliced and form the exon 1a-1 junction (Fig. 5 E, middle). These exon 1a-1 junction-containing transcripts were also STAT6 dependent and significantly up-regulated by the treat- ment with TSA (Fig. 5 F). The polycomb response element (PRE) and the trithorax response element (TRE) have been well established in Drosophila (Schwartz and Pirrotta, 2008). Non- coding transcripts have been detected around the PRE/TRE, and they play an important role for the binding of the PcG and TrxG complexes (Schmitt et al., 2005; Hekimoglu and Ringrose, 2009). Therefore, the identification of the PRE/ TRE in the region between exon 1a and exon 1b of the GATA3 gene locus would be an interesting issue. In summary, this study provides the first evidence indi- cating that the activation of the transcription factor STAT6 induces the displacement of the PcG complex by the TrxG complex, which then leads to control of the expression of the GATA3 gene in Th2 cells. STAT6-induced histone acetyla- tion appeared to be responsible for the dissociation of the PcG complex. The recruitment of the Menin–TrxG complex was found to be crucial for the maintenance of high GATA3 expression in developed Th2 cells. Therefore, STAT6- dependent displacement of the PcG by the TrxG complex would allow for a permissive chromatin status of the GATA3 gene locus in the developed Th2 cells in which a STAT6- independent regulation of transcription operates. MATERIALS AND METHODS Mice. C57BL/6 and BALB/c mice were purchased from CLEA. STAT6- deficient mice (Takeda et al., 1996) were provided by S. Akira (Osaka Uni- versity, Suita, Osaka, Japan). Menin-deficient mice (Crabtree et al., 2003) were purchased from The Jackson Laboratory. CD4-Cre transgenic mice were purchased from Taconic. All mice used in this study were maintained under specific pathogen-free conditions and ranged from 6 to 8 wk of age. All experimental protocols using mice were approved by the Chiba Univer- sity animal committee. All animal care was performed in accordance with the guidelines of Chiba University. Reagents. Recombinant mouse IL-12 was purchased from BD, and recombi- nant mouse IL-4 was purchased from TOYOBO. The OVA peptide (residues 323–339; ISQAVHAAHAEINEAGR) was synthesized by BEX Corporation. Antibodies. The antibodies used for the ChIP assay were anti–acetyl- histone H3-K9 (Millipore), anti–trimethyl-histone H3-K4 (LP Bio), anti–trimethyl-histone H3-K27 (Millipore), anti–histone H3 (Abcam), 2503