maintained (Fig. 6 F). The recruitment of MLL at the GATA3 gene locus was not detected in Menin-deficient Th2 cells after third cycle cultivation (Fig. 6 G, left). H3-K4Me3 and H3-K9Ac levels were decreased at both the upstream and the coding regions after stimulation in the presence of anti–IL-4 mAb (Fig. 6 G, left). Interestingly, no significant increase in the binding of Bmi1 and EZH2 and in the level of H3-K27Me3 was observed even after stimulation in the presence of anti–IL-4 mAb (Fig. 6 G, right). The total H3 levels were not changed in Menin-deficient Th2 cells even after the third cycle cultivation (Fig. S6 B). These results indicate that the PcG complex did not reassociate with the GATA3 gene locus in Th2 cells even in the absence of the binding of the Menin–TrxG complex. The ability to pro- duce IL-4 was assessed after restimulation with anti-TCR mAb and was found to be diminished in the Menin-deficient Th2 cells after the two-cycle stimulation in the presence of anti–IL-4 mAb (Fig. 6 H). The antigenic stimulation with IL-4 for 1 wk did not restore the production of IL-4 in the Th2 cells that had received two-cycle stimulation in the presence of anti–IL-4 mAb (unpublished data), indicating that the ability to become IL-4–producing cells was lost in the absence of Menin. Menin-deficient naive CD4 T cells expressed an equivalent amount of GATA3 transcript to WT CD4 T cells (Fig. 6 I, 0 cycle). The expression of GATA3 transcript in Menin-deficient Th2 cells was not maintained in the presence of anti–IL-4 mAb (Fig. 6 I, second cycle). Collectively, once Th2 cells have successfully developed, they maintain the GATA3 expression and Th2 function via the recruitment of the Menin–TrxG complex to the GATA3 gene locus even in the absence of IL-4–mediated STAT6 activation. 2502 Figure 7. Schematic illustration of the transcriptional regulation of the GATA3 gene in naive CD4 T cells, developing Th2 cells, and developed Th2 cells. In naive CD4 T cells, the PcG complex binds to the upstream region of the proximal promoter of the GATA3 gene, and the expression of GATA3 mRNA is moderate. After stimulation through TCR in the presence of IL-4 (Th2 culture conditions), STAT6 is activated, and STAT6 associated with the HAT complex binds to the GATA3 gene locus. HAT-dependent histone acetylation spreads to the upstream region, resulting in the dissociation of PcG complex. A high-level GATA3 mRNA expression is achieved in an IL-4/STAT6-dependent but Menin–TrxG- independent manner. The role of Menin–TrxG recruitment in GATA3 transcription appears to be minimal at this stage. Once Th2 cells are developed, the Menin–TrxG complex binds to the whole GATA3 gene region, including the upstream region of the proximal promoter. A broad range of H3-K9Ac and H3-K4Me3 is observed. The Menin–TrxG complex bound to the GATA3 locus maintains the high expres- sion levels of GATA3 in the absence of IL-4. IL-4/STA6-independent but Menin–TrxG- dependent regulation of the transcription of GATA3 is operating. DISCUSSION This study dissects two distinct pro- cesses that are criti- cal in the regulation of transcription of the GATA3 gene in Th2 cells based on the binding of PcG and TrxG complexes (Fig. 7). In naive CD4 T cells, the PcG complex binds to the upstream region of the GATA3 proximal promoter, whereas the accumulation of the Menin–TrxG complex is restricted to a part of the coding region. In developing Th2 cells, IL-4– mediated STAT6 activation induces the dissociation of the PcG complex, which is accompanied by the acetylation of H3-K9 and noncoding transcripts at the upstream region of the GATA3 gene locus. Histone hyperacetylation is suffi- cient to cause the dissociation of the PcG complex and the induction of noncoding transcripts. High-level transcription of the GATA3 gene is induced in these cells (Fig. 7, middle). The role of binding of the Menin–TrxG complex in GATA3 transcription appears to be minimal at this stage. In fully de- veloped Th2 cells, high-level expression of GATA3 can be maintained in the absence of IL-4 (Fig. 7, bottom). IL-4/ STAT6-independent but Menin–TrxG-dependent regula- tion of GATA3 transcription is operating. A broad range binding of Menin was confirmed by a ChIP-seq assay (Fig. S8). The binding of Menin–TrxG complex to the GATA3 gene is required for the long-term maintenance of GATA3 ex- pression and Th2 cytokine production. Because the supply of IL-4 is limiting in the body after the clearance of infectious organisms, this could be the mechanism by which memory Th2 cells can maintain Th2 function for a long time in vivo. STAT6 plays several distinct roles in the regulation of GATA3 transcription. Although STAT6 is well known to be Maintaining Th2-specific GATA3 gene expression | Onodera et al.