American Society of Hematology

Figure 4

$Figure 4. NF-Y binds to the CCAAT box element from the ID1 promoter and is required for PML-RARα–mediated transactivation. (A) EMSA showing binding of NF-Y to the putative NF-Y–binding site from the ID1 promoter. Hep3B nuclear extracts were incubated with a labeled DNA probe containing the NF-Y box from the ID1 promoter region. A clearly shifted protein-DNA complex was seen (lane 1). Competition experiments were done with 100 × cold ID1 probe (lane 2) and with an unlabeled probe containing the confirmed NF-Y–binding site from the CD10 gene promoter (lane 5). In addition, a sequence without any recognizable NF-Y–binding site was used for competition (lane 3). Addition of anti–NF-YB antibody shifted the complex to a higher molecular weight complex (lane 4). Arrows indicate free probe (1), shifted DNA-protein complex (2), and the supershifted DNA-protein-antibody complex (3). (B) To detect DNA-binding of NF-Y in intact cells, ChIP assays were performed using anti–NF-YA antibodies. As a control, the nonspecific IgG fraction from human serum was used. The y-axis shows the recovery (%) of ID1 or RARβ sequences relative to the input. In U937 cells, NF-Y clearly bound to the ID1 promoter (right bars), but not to the RARβ gene (left bars). Mean values from 3 independent experiments plus or minus SD are shown. (C) To further show the importance of NF-Y for the PML-RARα–mediated transcriptional activation of ID1, transfections (as described in Figure 2) were performed using the dominant-negative NF-YA subunit (Yam29) and the 963 ID1 promoter construct. In the presence of YAm29, PML-RARα–mediated transcription was severely diminished (right bars). (D) To investigate the importance of different domains of PML-RARα, vectors encoding the DNA-binding–defective PML-RARαΔR mutant, and a mutant lacking the coiled-coil protein-protein interaction domain of PML (PML-RARαΔCC), were used. In addition, a PLZF-RARα expression construct was used. Transfections were performed as in Figure 2 using the 963 ID1 promoter construct; mean values of 3 independent experiments plus or minus SD are shown.$

NF-Y binds to the CCAAT box element from the ID1 promoter and is required for PML-RARα–mediated transactivation. (A) EMSA showing binding of NF-Y to the putative NF-Y–binding site from the ID1 promoter. Hep3B nuclear extracts were incubated with a labeled DNA probe containing the NF-Y box from the ID1 promoter region. A clearly shifted protein-DNA complex was seen (lane 1). Competition experiments were done with 100 × cold ID1 probe (lane 2) and with an unlabeled probe containing the confirmed NF-Y–binding site from the CD10 gene promoter (lane 5). In addition, a sequence without any recognizable NF-Y–binding site was used for competition (lane 3). Addition of anti–NF-YB antibody shifted the complex to a higher molecular weight complex (lane 4). Arrows indicate free probe (1), shifted DNA-protein complex (2), and the supershifted DNA-protein-antibody complex (3). (B) To detect DNA-binding of NF-Y in intact cells, ChIP assays were performed using anti–NF-YA antibodies. As a control, the nonspecific IgG fraction from human serum was used. The y-axis shows the recovery (%) of ID1 or RARβ sequences relative to the input. In U937 cells, NF-Y clearly bound to the ID1 promoter (right bars), but not to the RARβ gene (left bars). Mean values from 3 independent experiments plus or minus SD are shown. (C) To further show the importance of NF-Y for the PML-RARα–mediated transcriptional activation of ID1, transfections (as described in Figure 2) were performed using the dominant-negative NF-YA subunit (Yam29) and the 963 ID1 promoter construct. In the presence of YAm29, PML-RARα–mediated transcription was severely diminished (right bars). (D) To investigate the importance of different domains of PML-RARα, vectors encoding the DNA-binding–defective PML-RARαΔR mutant, and a mutant lacking the coiled-coil protein-protein interaction domain of PML (PML-RARαΔCC), were used. In addition, a PLZF-RARα expression construct was used. Transfections were performed as in Figure 2 using the 963 ID1 promoter construct; mean values of 3 independent experiments plus or minus SD are shown.

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