Figure 2

PML/RARα represses the promoter activity of CDKN2D through the ER8 site. (a) Schematic representation of the CDKN2D promoter region shows the different truncations/mutations used in this study. Numbering is indicated with respect to the transcriptional start site (TSS) of CDKN2D (bent arrow). □ represents the wild-type ER8 site and ⊠ represents the mutated ER8 site. (b) PML/RARα repressed the promoter activity of CDKN2D in a dose-dependent manner. The CDKN2D promoter reporter construct (-900 bp) and increasing amounts of pSG5-PML/RARα expression plasmid (PML/RARα) were cotransfected into 293T cells. Luciferase values were normalized to those of control. Values were the mean±S.E.M. obtained from at least three independent experiments. (c–d) The ER8 site located between −273 bp and −488 bp of the CDKN2D promoter is critical for PML/RARα-mediated repression. The 293T cells were transfected with different truncations/mutations of the CDKN2D promoter reporter construct and the pSG5-PML/RARα expression plasmid or the empty vector. Results in 293T cells (c) are represented as fold repression. The NB4 cells were transfected with different truncations/mutations of the CDKN2D promoter reporter construct. Results in NB4 cells (d) are relative to the empty vector (pGL3-basic). * indicates P<0.05