PML-RARα binds Sp1 and binds to the endogenous ID1 promoter. (A) To test binding of PML-RARα to Sp1 and NF-Y, GST pull-down experiments were performed. Empty beads and beads loaded with GST or GST-NF-YA (left panels) were incubated with in vitro–translated Sp1 (positive control) or in vitro–translated PML-RARα. GST-Sp1 (right panels) was incubated with in vitro–translated PML-RARα or PML-RARαΔCC. Beads were washed and subsequently resuspended in loading buffer. Protein was resolved on SDS-PAGE. Immunostaining was performed with anti-Sp1 or anti-RARα antibody. Clear interactions between NF-YA and Sp1 and between PML-RARα and Sp1 were observed. To show binding of PML-RARα to the endogenous RARβ (B) and ID1 (C) genes, ChIP assays were performed in PML-RARα–positive NB4 cells and in PML-RARα–negative U937 cells. Cells were treated with ATRA for 30 minutes. ChIP was done with anti-PML antiserum. 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. Mean values plus or minus SD of 4 independent experiments are shown.