decrease but rather a slightly increased expression of Bmi1, EZH2, Menin, and MLL in the STAT6-deficient Th2 cells was seen with TSA treatment, suggesting that neither PcG complex dissociation nor TrxG complex recruitment in the TSA-treated Th2 cells is controlled by the expression levels of these molecules (Fig. S4). Thus, histone hyperacetylation itself is responsible for the dissociation of the PcG complex accompanied with down-regulation of H3-K27Me3 and noncoding transcripts at the upstream region of the proximal promoter of the GATA3 gene; however, this alone was not sufficient for the recruitment of the TrxG complex. The activation of STAT6 induced TrxG recruitment and up-regulation of mature GATA3 transcript during Th2 cell differentiation Next, we assessed whether STAT6 activation itself induced the recruitment of Menin and mature transcripts. STAT6VT, an autoactivated STAT6 molecule (Daniel et al., 2000), and a STAT6VT DNA-binding mutant (STAT6VT dDBD) mole- cule (Fig. 5 A) were introduced into STAT6-deficient CD4 T cells cultured under Th2 conditions. The STAT6VT- and STAT6VT dDBD–introduced cells were harvested 3 d after infection and then were subjected to a ChIP assay. STAT6VT was found to induce the recruitment of Menin between the distal and the proximal promoter and also the coding region (Fig. 5 B, probes (5), (4), and (+3)). STAT6VT induced the reduction of H3-K27Me3 and the up-regulation of H3- K4Me3 (Fig. 5 C). However, STAT6VT dDBD did not in- duce the recruitment of Menin, the reduction of H3-K27Me3, or the up-regulation of H3-K4Me3 (Fig. 5, B and C). The total H3 levels were not influenced by STAT6VT introduction (Fig. 5 C). In addition, the transcripts between the distal and the proximal promoter (probes (10) to (4)) were restored by the ectopic expression of STAT6VT in STAT6-deficient developing Th2 cells (Fig. 5 D, left). Furthermore, STAT6VT rescued the mature GATA3 transcript (Fig. 5 D, right), whereas STAT6VT dDBD had no effect on GATA3 transcription (Fig. 5 D). These results indicate that the activation of STAT6 itself induced the recruitment of Menin and the mature GATA3 transcript during the development from naive CD4 T cells to Th2 cells. The first alternative exons of the GATA3 transcripts (1a and 1b) are spliced to a common exon 1, which contains the translation start site (Fig. 5 E; Asnagli et al., 2002; Scheinman and Avni, 2009). First, the levels of transcripts of both exon 1a-1 and exon 1b-1 were assessed in WT and STAT6-deficient Th2 cells in the presence of TSA (Fig. 5 F). We confirmed that the exon 1b-1 transcript was much more abundant than that of exon 1a-1 (Scheinman and Avni, 2009). The levels of both transcripts were very low in STAT6-deficient Th2 cells. Interestingly, TSA treatment partially rescued the exon 1a-1 transcript but not the exon 1b-1 transcript (Fig. 5 F). The re- sult was similar to that seen with noncoding and mature tran- scripts, respectively, as shown in Fig. 4 C. Then, we assessed the effect of the ectopic expression of STAT6VT on these two transcripts and observed that rescue was partial for the 2500 exon 1a-1 transcript but was substantial in the case of the exon 1b-1 transcript (Fig. 5 G). Thus, the effect of STAT6VT on exon 1b-1 transcript was similar to that seen on GATA3 mature transcripts, as shown in Fig. 5 D. STAT6VT dDBD showed no obvious effect on exon 1a-1 and exon 1b-1 tran- scripts (Fig. 5 G). These results indicate that both exon 1a-1 and exon 1b-1 transcripts are dependent on STAT6 activa- tion and that the former is associated with noncoding tran- scripts of the upstream region of the GATA3 gene and the latter is associated with the mature transcripts. Menin was required for the maintenance of GATA3 expression in developed Th2 cells Menin is an essential component for DNA binding of the TrxG–MLL complex (Guru et al., 1998). A CD4-Cre–driven conditional knockout mouse for Menin (Menin-deficient mice) was used to assess the role of Menin in the TrxG re- cruitment in the expression of the GATA3 gene. In Menin- deficient mice, equivalent numbers of CD4 and CD8 T cells developed in the thymus, but a 50% reduction in the number of splenic CD4 cells was observed in comparison with WT control (Fig. S5 A). The cell surface phenotype of splenic CD4 T cells was found to be within the normal range (Fig. S5 B). Proliferative responses induced with immobilized anti-TCR mAb were not impaired but somewhat slightly enhanced in Menin-deficient CD4 T cells (unpublished data). The lack of Menin mRNA in the conditional Menin-deficient Th1 and Th2 cells was confirmed (Fig. S5 C). Menin-deficient CD4 T cells cultured under Th2 conditions expressed an equiva- lent amount of GATA3 transcript and protein to WT CD4 T cells (after the first cycle of stimulation; Fig. 6, A and B) and differentiated normally into effector Th2 cells (Fig. 6 D). To further probe the role of Menin, the binding of MLL and the status of H3-K4Me3 at the GATA3 gene locus were as- sessed in the Menin-deficient Th2 cells. We could not detect the recruitment of MLL at the GATA3 gene locus in Menin- deficient Th2 cells (Fig. 6 C, left). Interestingly, the levels of H3-K4Me3 were decreased at the upstream region of the GATA3 proximal promoter (probes (5) and (4)), whereas this was not obvious at the coding region (Fig. 6 C, left, probe (+3)). The binding of the PcG proteins, Bmi1 and EZH2, and those of H3-K27Me3 after the first cycle stimula- tion were reduced equivalently between the WT and Menin- deficient Th2 cells (Fig. 6 C, right). The total H3 levels were not changed in Menin-deficient Th2 cells (Fig. S6 A). These results indicate that STAT6-dependent but Menin–TrxG- independent GATA3 transcription is induced during the first cycle of Th2 cell differentiation. Interestingly, although the expression levels of GATA3 transcript in WT Th2 cells was maintained even when the cells were stimulated with anti-TCR mAb in the presence of anti–IL-4 mAb (Fig. S7, second to third cycle), the expres- sion of GATA3 transcript in Menin-deficient Th2 cells was not maintained in the presence of anti–IL-4 mAb (after the third cycle of stimulation; Fig. 6 E). The high expression of GATA3 protein in Menin-deficient Th2 cells was also not Maintaining Th2-specific GATA3 gene expression | Onodera et al.