Figure 6
Figure 6. ETO2 directly represses the Pf4 promoter in transactivation assays via GATA, GAT, and E-box sites. (A) Comparison of the nucleotide sequence of the 5′ upstream region of the Pf4 gene from 6 species. Sequences highlighted in bold or by a box indicate a GAT, E-box, ETS, and GATA motifs. *Conserved nucleotides. Coordinates in nucleotides are with respect to the transcriptional start site. (B) Schematic representation of the luciferase gene reporter construct (pGL4.10) under the control of the enhancer/promoter regions of the rat Pf4 gene. Coordinates in nucleotides are with respect to the Pf4 gene transcriptional start site. Sequence motifs for transcription factors are marked. ↱shows the position of the transcriptional start site. The luciferase gene is drawn as an open box. (C) 293T fibroblasts were transiently transfected with either a promoterless luciferase gene (pGL4.10) or a luciferase gene regulated by 1200 nucleotides 5′ of the transcriptional start site of the rat Pf4 gene (rPF4 1200). Cells were also transfected with expression vectors expressing the indicated transcription factors. The triangle represents transfection with increasing amounts of ETO2 expression vector (50, 150, and 300 ng). Expression of luciferase in cells transfected with rPF4 1200 with expression vectors for all 6 transcriptional regulators (C6: GATA1, SCL, E2A, LDB1, LMO2, and FLI1) was set to 100%. (D) Western blot analysis confirms expression of transfected proteins in 293T cells in the absence (+ C6) or presence (+ C6 + E) of the ETO2 expression plasmid. Expression of mSIN3A protein serves as a loading control. (E) Expression of luciferase protein in 293T cells transfected with either full-length rPF4 1200 or truncated forms of the rat Pf4 promoter (rPF4 151 and rPF4 97) with C6 factors in the presence or absence of 150 ng ETO2 expressing plasmid. (F) Transcriptional effect of point mutations of the GAT motif at −130 and GATA site at −30 and E-box motif at −102 (relative to the transcriptional start site) in the construct rPF4 151 on luciferase gene expression were tested in 293T cells in the presence of the transcription factors indicated at the bottom of the diagram. In panels C, E, and F, the results are the mean plus or minus 2 SD of 3 to 5 independent experiments.

ETO2 directly represses the Pf4 promoter in transactivation assays via GATA, GAT, and E-box sites. (A) Comparison of the nucleotide sequence of the 5′ upstream region of the Pf4 gene from 6 species. Sequences highlighted in bold or by a box indicate a GAT, E-box, ETS, and GATA motifs. *Conserved nucleotides. Coordinates in nucleotides are with respect to the transcriptional start site. (B) Schematic representation of the luciferase gene reporter construct (pGL4.10) under the control of the enhancer/promoter regions of the rat Pf4 gene. Coordinates in nucleotides are with respect to the Pf4 gene transcriptional start site. Sequence motifs for transcription factors are marked. ↱shows the position of the transcriptional start site. The luciferase gene is drawn as an open box. (C) 293T fibroblasts were transiently transfected with either a promoterless luciferase gene (pGL4.10) or a luciferase gene regulated by 1200 nucleotides 5′ of the transcriptional start site of the rat Pf4 gene (rPF4 1200). Cells were also transfected with expression vectors expressing the indicated transcription factors. The triangle represents transfection with increasing amounts of ETO2 expression vector (50, 150, and 300 ng). Expression of luciferase in cells transfected with rPF4 1200 with expression vectors for all 6 transcriptional regulators (C6: GATA1, SCL, E2A, LDB1, LMO2, and FLI1) was set to 100%. (D) Western blot analysis confirms expression of transfected proteins in 293T cells in the absence (+ C6) or presence (+ C6 + E) of the ETO2 expression plasmid. Expression of mSIN3A protein serves as a loading control. (E) Expression of luciferase protein in 293T cells transfected with either full-length rPF4 1200 or truncated forms of the rat Pf4 promoter (rPF4 151 and rPF4 97) with C6 factors in the presence or absence of 150 ng ETO2 expressing plasmid. (F) Transcriptional effect of point mutations of the GAT motif at −130 and GATA site at −30 and E-box motif at −102 (relative to the transcriptional start site) in the construct rPF4 151 on luciferase gene expression were tested in 293T cells in the presence of the transcription factors indicated at the bottom of the diagram. In panels C, E, and F, the results are the mean plus or minus 2 SD of 3 to 5 independent experiments.

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