and (4)) occurred normally in STAT6-deficient Th2 cells in the presence of TSA (Fig. 4 A, top). H3-K27Me3 at the GATA3 locus was also reduced in the presence of TSA. The total H3 levels were not influenced by TSA treatment. JEM VOL. 207, October 25, 2010 A r t i c l e Figure 5. STAT6 activation was necessary and sufficient for recruitment of Menin and up-regulation of mature GATA3 mRNA during Th2 cell differentiation. (A) Schematic representation of STAT6 WT, full-length STAT6VT, and DNA-binding mutant (dDBD) with location of the transactivation (TA) and DNA-binding domain indicated. STAT6VT and STAT6VT dDBD contain V547A and T548A mutations. (B–D) Freshly isolated WT or STAT6-deficient CD4 T cells were stimulated under Th2 condi- tions for 2 d, and then the cells were mock infected or infected with a retrovirus vector containing a STAT6VT cDNA (pMXs- STAT6VT-IRES-hNGFR) or a STAT6VT dDBD cDNA (pMXs- STAT6VT-dDBD-IRES-hNGFR). hNGFR-positive infected cells were enriched by magnetic cell sorting. (B) The levels of Menin bind- ing to the GATA3 gene locus were assessed by a ChIP assay. (C) Effect of STAT6VT introduction on the histone modifications was determined by a ChIP assay with quantitative PCR. (B and C) The relative intensity (mean of three samples) is shown with SDs. (D) Noncoding transcripts and mature GATA3 mRNA were measured by quantitative PCR. (E) Schematic illustration of the GATA3 exon 1a, exon 1b, and exon 1 and the primer sites for the amplification of the 1a (exon 1a-1)- and 1b-containing tran- script (exon 1b-1). Arrows represent the position of primer pairs for PCR detection. Squiggly lines represent the break in a long section of the gene. (F and G) The levels of GATA3 exon 1a– containing transcript and exon 1b–containing transcript were measured by exon-specific quantitative PCR analysis. (F) TSA treatment was performed as described in Fig 4. (D, F, and G) The relative expression (mean of three samples) is shown with SDs. (B–D, F, and G) Two independent experiments were performed with similar results. In contrast to the dissociation of the PcG complex, no obvious recruitment of Menin or MLL was de- tected in the TSA-treated STAT6-deficient Th2 cells (Fig. 4 B, top). Interestingly, TSA treatment induced H3-K4Me3 in addition to the H3-K9Ac at the GATA3 locus (Fig. 4 B, bottom). Next, the effect of TSA on the GATA3 transcription was examined. The transcripts between the distal and the proximal promoter (probes (10) to (4)) were not induced in STAT6-deficient Th2 cells, and these transcripts were induced by the treatment with TSA (Fig. 4 C, top left). However, the TSA treatment did not in- duce mature GATA3 transcript (Fig. 4 C, top right). Recent studies indicate that the phosphorylation status at Ser2 and Ser5 is associated with the transcrip- tional active and poised status of the RNAPII com- plex (for review see Brookes and Pombo, 2009). Consequently, we performed ChIP assays to assess the binding of Ser2- and Ser5-phosphorylated RNAPII at the GATA3 gene locus. No obvious difference in the binding pattern was observed in both WT and STAT6-deficient Th2 cells (Fig. S3). The TSA treat- ment resulted in the accumulation of RNAPII (both Ser2- and Ser5-phosphorylated RNAPII) between the distal and the proximal promoter (Fig. 4 C, bottom left, probes (10) to (4)) but not at the coding region of the proximal promoter (Fig. 4 C, bottom right, probe (+3)). No obvious 2499