The CCAAT and GC box in the ID1 promoter are required for PML-RARα–mediated transactivation. (A) ID1 promoter region showing the presence of consensus Sp1- and NF-Y–binding sites. (B) Transactivation of ID1 promoter-luciferase constructs. Several deletion constructs were generated and transfected in combination with RARα plus RXR, or with PML-RARα. Transactivation assays were performed as described in Figure 2. Mean values from 3 independent experiments (± SD) are shown. (C) To investigate the importance of the putative Sp1- and NF-Y–binding sites in the − 121-bp upstream promoter sequence of the ID1 gene for transactivation by PML-RARα, these sites were mutated in the context of the − 963-bp promoter fragment. Mutations were introduced either alone, or in combination. Transactivation by PML-RARα was performed as described in Figure 1. Sequences were mutated as follows: GC box: CCGCCC was replaced by CTATCC; NF-Y site: ATTGG was replaced by ACACG. Transactivation was significantly lower in the promoter fragments with one mutated binding site in comparison with the wt promoter fragment (P < .01).