Binding of PML-RARα to the endogenous p21, RAR-β, ID1, and ID2 genes. (A) To show binding of PML-RARα and NF-Y to the endogenous p21, RAR-β, ID1, and ID2 genes, ChIP assays were performed. As anti-PML antibodies do not discriminate between the unrearranged PML protein and the PML-RARα fusion protein, FLAG-tagged PML-RARα was used. Cells were transfected with or without FLAG-PML-RARα and cultured in the presence or absence of 10⁻⁶ M ATRA. ChIP was performed using anti-FLAG antibodies, anti–NF-YA antibodies, and control IgGs. The Y-axis shows the recovery (%) of ID1/ID2/p21 or RARβ sequences relative to the input. Mean values of 3 independent experiments (± SD) are shown. (B) Dominant-negative and gain-of-function model for PML-RARα. Genes that are regulated through a retinoic acid–responsive element (RARE) may be bound by PML-RARα (1). Competition with normal, unrearranged retinoid receptors results in a dominant-negative silencing of the gene by PML-RARα in the absence of ligand. Addition of high-dose retinoic acid may reverse the silencing, allowing transcription. Sp1- and NF-Y–regulated genes may be targeted by PML-RARα through interaction with Sp1 (2). Tethering of PML-RARα to these promoters renders them responsive to retinoic acid, representing a gain-of-function for the PML-RARα fusion protein.