H3-K4 (Milne et al., 2002; Nakamura et al., 2002), whereas the PcG complex maintains a repressive chromatin structure via the methylation of histone H3-K27 (Cao et al., 2002). The mammalian TrxG complexes contain RbBP5, Ash2L, and WDR5, which are also related to the components in the yeast Set1 complex, and a catalytic subunit that harbors the SET domain (Yokoyama et al., 2004). In contrast, PcG molecules form multimeric and heterogeneous complexes and maintain the early-determined gene expression patterns of key develop- mental regulators such as homeobox genes (Satijn and Otte, 1999; van Lohuizen, 1999). There are at least two types of PcG complexes, PRC1 (polycomb repressive complex 1) and PRC2 (Ringrose and Paro, 2004). Ring1B, Ring1A, Bmi1, Mel-18, M33, Pc2, Rae-28/Mph1, and Mph2 are members of a multimeric protein complex that show similarity to the PRC1 identified in Drosophila melanogaster. PRC2 is another PcG complex that contains Eed, Suz12, Ezh1, and Ezh2, and it possesses intrinsic methyltransferase activity for histone H3- K27 (Cao et al., 2002). In mature lymphocytes, PcG gene products appear to play several roles in the differentiation pro- cess and cell fate. Bmi1 controls the stability of GATA3 pro- tein in developing Th2 cells (Hosokawa et al., 2006) and also memory CD4 T cell survival through the direct repression of the Noxa gene (Yamashita et al., 2008). Mixed-lineage leuke- mia (MLL) is a member of TrxG molecules and controls the maintenance of Th2 cytokine gene expression in memory Th2 cells (Yamashita et al., 2006). Menin was initially identi- fied as a product of the MEN1 tumor suppressor gene and is known to be an essential component for DNA binding of the TrxG–MLL complex (Guru et al., 1998). This study investigates the molecular mechanisms under- lying the PcG complex– and TrxG complex–mediated regu- lation of GATA3 transcription. In naive CD4 T cells, the PcG complex bound to the upstream region of the GATA3 proxi- mal promoter, whereas the accumulation of the Menin–TrxG complex was restricted to a part of the coding region. IL-4– mediated STAT6 activation induced the displacement of the PcG complex by the TrxG complex at the upstream region of the GATA3 gene locus. After Th2 cell differentiation, the binding of Menin–TrxG complex was required for the main- tenance of GATA3 expression and Th2 cytokine production. This study revealed two distinct molecular processes that are critical in the regulation of GATA3 transcription in Th2 cells: (1) IL-4/STAT6-mediated displacement of the PcG complex by the TrxG complex leading to the induction of GATA3 transcription during Th2 cell differentiation and (2) STAT6- independent maintenance of GATA3 expression and Th2 function via recruitment of the Menin–TrxG complex. RESULTS Dissociation of PcG complex and recruitment of TrxG complex to the GATA3 gene locus during Th2 cell differentiation The expression of GATA3 mRNA is regulated in a tissue- specific manner. Naive CD4 T cells express a moderate level of GATA3, and in vitro differentiated Th2 cells and fully 2494 developed Th2 cells express higher levels of GATA3, whereas splenic B cells express little GATA3 mRNA (Fig. 1 A, left). A similar tissue-specific profile in the protein expression of GATA3 mRNA was observed (Fig. 1 A, right). Fully devel- oped Th2 cells were established as described in Materials and methods. A schematic representation of the GATA3 gene locus, with the location of specific primer pairs and probes for quantitative PCR used in this study, is shown in Fig. 1 B. First, the histone modification and the binding of the PcG and TrxG complexes at the GATA3 gene locus were deter- mined by chromatin immunoprecipitation (ChIP) assays. In B cells, Bmi1 bound to the upstream region of the proximal promoter and the region around exon 1 and exon 2 (primers #1 to #11; Fig. 1 C, dark blue line). The histone H3-K27 trimethylation (H3-K27Me3) signals were enriched at a broader but similar region (primers #1 to #15). Only weak signals for Menin and MLL binding and H3-K4Me3 were detected in B cells. However, in naive CD4 T cells, the Bmi1 signal was enriched only in the upstream region of the proxi- mal promoter (primers #1 to #7; Fig. 1 C, turquoise line) but was low at the proximal promoter and the coding regions (primers #7 to #20). The signal pattern for H3-K27Me3 was almost exactly the same as that of Bmi1. In sharp contrast, Menin and MLL binding and H3-K4Me3 signals were en- riched from the downstream region of the proximal promoter to exon 2. Fully developed Th2 cells showed strong enrich- ment of Menin and MLL signals from the beginning of the distal promoter to exon 5 and H3-K4Me3 signals to exon 3 (Fig. 1 C, red line). No significant Bmi1 accumulation and H3-K27Me3 were detected in fully developed Th2 cells. The real PCR product bands are shown in Fig. S1 (A and B). We detected comparable levels of histone H3 at all regions in these three types of cells (Fig. S1, C and D). The signals for H3-K4Me3 showed a similar pattern to that of Menin and MLL in all cells, whereas the H3-K27Me3 profile was similar to that of Bmi1. These results indicate that the increase in the expression of GATA3 mRNA correlates with the dissocia- tion of Bmi1 and the association of Menin and MLL to the GATA3 gene locus, particularly in the upstream region of the proximal promoter (primers #3 to #6). Next, the change of chromatin status and the PcG/TrxG binding at the GATA3 gene locus during Th2 cell differentia- tion were assessed using a quantitative PCR assay system (Fig. 1 D). PcG proteins, Bmi1, EZH2, and Suz12 bound to the upstream region of GATA3 proximal promoter in naive CD4 T cells (Fig. 1 D, left, probes (15), (5), and (4)). The sig- nal for H3-K27Me3 was also enriched in the upstream region of the proximal promoter. A progressive dissociation of Bmi1, EZH2, and Suz12 and decreased H3-K27Me3 were observed during Th2 cell differentiation. Essentially no PcG association was observed in fully developed Th2 cells (Fig. 1 D, left, probes (15), (5), and (4)). In contrast, the binding of Menin, MLL, and RNA polymerase II (RNAPII) was restricted to the coding region in naive CD4 T cells (Fig. 1 D, right). H3-K4Me3 and H3-K9Ac signals were enriched around the proximal promoter (probe (0)). This accumulation of Menin, Maintaining Th2-specific GATA3 gene expression | Onodera et al.