American Society of Hematology

Figure 2

$Figure 2. c-Myb activates GATA-3 expression by binding to a canonical site within the GATA-3 exon 1b promoter in naive CD4 T cells under Th2 cell-promoting conditions. (A) Top graphic: GATA-3 promoter locus. Transcription can initiate from either exon 1a or exon 1b. The translational start site begins within exon 2. (Bottom left graph) The amount of exon 1a or exon 1b mRNA, as measured by quantitative RT-PCR, in naive (CD45RO−) or effector/memory (CD45RO+) CD4+ T cells before stimulation (0 day) and 1, 3, and 5 days after Th2 cell-promoting stimulatory conditions. The copy number was calculated for each species using a standard curve for exon 1a or exon 1b. The amount of exon 1a mRNA was adjusted to 1.0 to show the relative copy number. (Bottom right graph) The relative expression of c-myb mRNA in the same cells as the graph on the left. Data are mean ± SD of triplicate determinations. (B) Purified nucleoprotein complex was obtained from CD4+CD45RO− cells cultured under Th1- or Th2-promoting conditions and then used for ChIP assays using either anti–c-Myb (c-Myb) or mouse IgG (mIgG). The precipitated DNA fractions were analyzed by quantitative RT-PCR with primers specific to the GATA-3 upstream exon 1a (right graph) or downstream exon 1b (left graph) promoter region. *P < .05 compared with the value of murine (m) IgG. (C) Reporter constructs used for promoter analysis. Numbers represent nucleotides referenced relative to the start of transcription (arrow) from exon 1b. Gray circle represents c-Myb binding site; and black circle, mutated c-Myb binding site. (D) Dual-Luciferase reporter assays were performed in human peripheral T cells 24 hours after cotransfection with each of the pGL3–GATA-3 promoter constructs, and either a c-Myb expression construct (c-myb) or an empty plasmid (control). T cells were stimulated with CD3/CD28 antibodies, IL-2, and IL-4 for 8 hours before the cells were collected. Data are representative of 5 independent determinations. (E) The siRNA against c-Myb or control siRNA were cotransfected with the pGL3–GATA-3 promoter construct into human primary T cells. Dual-Luciferase reporter assays were performed 24 hours after the transfection. Data are representative of at least 2 independent experiments. *P < .05.$

c-Myb activates GATA-3 expression by binding to a canonical site within the GATA-3 exon 1b promoter in naive CD4 T cells under Th2 cell-promoting conditions. (A) Top graphic: GATA-3 promoter locus. Transcription can initiate from either exon 1a or exon 1b. The translational start site begins within exon 2. (Bottom left graph) The amount of exon 1a or exon 1b mRNA, as measured by quantitative RT-PCR, in naive (CD45RO⁻) or effector/memory (CD45RO⁺) CD4⁺ T cells before stimulation (0 day) and 1, 3, and 5 days after Th2 cell-promoting stimulatory conditions. The copy number was calculated for each species using a standard curve for exon 1a or exon 1b. The amount of exon 1a mRNA was adjusted to 1.0 to show the relative copy number. (Bottom right graph) The relative expression of c-myb mRNA in the same cells as the graph on the left. Data are mean ± SD of triplicate determinations. (B) Purified nucleoprotein complex was obtained from CD4⁺CD45RO⁻ cells cultured under Th1- or Th2-promoting conditions and then used for ChIP assays using either anti–c-Myb (c-Myb) or mouse IgG (mIgG). The precipitated DNA fractions were analyzed by quantitative RT-PCR with primers specific to the GATA-3 upstream exon 1a (right graph) or downstream exon 1b (left graph) promoter region. *P < .05 compared with the value of murine (m) IgG. (C) Reporter constructs used for promoter analysis. Numbers represent nucleotides referenced relative to the start of transcription (arrow) from exon 1b. Gray circle represents c-Myb binding site; and black circle, mutated c-Myb binding site. (D) Dual-Luciferase reporter assays were performed in human peripheral T cells 24 hours after cotransfection with each of the pGL3–GATA-3 promoter constructs, and either a c-Myb expression construct (c-myb) or an empty plasmid (control). T cells were stimulated with CD3/CD28 antibodies, IL-2, and IL-4 for 8 hours before the cells were collected. Data are representative of 5 independent determinations. (E) The siRNA against c-Myb or control siRNA were cotransfected with the pGL3–GATA-3 promoter construct into human primary T cells. Dual-Luciferase reporter assays were performed 24 hours after the transfection. Data are representative of at least 2 independent experiments. *P < .05.

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