Figure 6.

c-Myb, GATA-3, and Menin bind to GATA-3's minimal promoter in CD4⁺ naive cells under Th2 cell-promoting conditions, whereas MLL binds the same promoter region in a complex with c-Myb, GATA-3, and Menin in CD4⁺ effector/memory cells.(A) Purified nucleoprotein complex was obtained from primary human whole CD4⁺ cells after the stimulation with IL-4, IL-2, and antibodies against CD3/CD28 for 3 days. ChIP assays were carried out with GATA-3, CBP, p300, MLL1, and Menin antibodies and mouse IgG (mIgG) or rabbit IgG (rIgG). The precipitated DNA fractions were analyzed by quantitative RT-PCR with primers specific to the GATA-3 downstream minimal promoter region. (B) ChIP assays were performed with primary human CD4⁺ CD45RO⁻ (naive) cells after stimulation with IL-4, IL-2, and antibodies against CD3/CD28. (C) ChIP assays were carried out with primary human CD4⁺ CD45RO⁺ (effector/memory) cells after a 3-day stimulation with IL-4, IL-2, and antibodies against CD3/CD28. (D-E) Re-ChIP assays were carried out with human CD4⁺ CD45RO⁺ (D) or CD4⁺ CD45RO⁻ (E); cells stimulated as described in panel C. The first ChIP assays were performed with antibodies against MLL or Menin. The second ChIP (Re-ChIP) assays were done with antibodies against c-Myb or GATA-3. Controls for the Re-ChIP assay were performed with antirabbit IgG (rIgG) and antimouse IgG (mIgG) antibodies. Data are representative of at least 2 independent experiments. *P < .05. **P < .01.