FOXM1 regulates the gene transcription of CDC25A by direct DNA binding as well as the E2F pathway.

<p>(A) Schematic diagram of CDC25A promoter showing the 3′ distal end containing two E2F binding sites, the 5′ proximal end including three putative FOXM1 binding sites and excluding E2F binding sites, and a 224 bp fragment containing the three putative FOXM1 binding sites. (B) FOXM1 increased the transactivation of full-length and truncated CDC25A promoter constructs, indicating both direct and indirect FOXM1 induction. U2OS cells were co-transfected with pACT-FOXM1 or pACT empty plasmid and the reporter plasmids containing full length and truncated CDC25A promoters. pRL-SV40, a <i>Renilla</i> luciferase report vector, was co-transfected as the control. Data were normalized to <i>Renilla</i> luciferase activities and are presented as the mean ± SD (N = 5). ***, p<0.001. (C) FOXM1 recruits p300, but not PCAF or CBP, to activate CDC25A promoter. U2OS cells were co-transfected with pACT-FOXM1 (or pACT control) and pCMV-p300, or pCMV-pCAF, or pCMV-CBP (or empty vector control), along with pGL3-CDC25A promoter for 48 h. pRL-SV40 was co-transfected as the normalizing control. Data are normalized to <i>Renilla</i> activity, and represented as the mean ± SD. The difference was analyzed by paired t test. ***p<0.001, Δ>0.05. (D) Representation of the wild-type and mutated E2F consensus binding sequences at CDC25A promoter. (E) Two E2F binding sites are required for FOXM1-activated CDC25A promoter transactivation. U2OS cells were co-transfected with pACT-FOXM1 or empty vector control and the reporter plasmids containing the indicated CDC25A promoters (wild-type or mutated E2F binding sites). Data are presented as the mean fold induction of FOXM1 activity over control ± SD. *** p<0.001.