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. 2005 Dec;25(24):10875–10894. doi: 10.1128/MCB.25.24.10875-10894.2005

FIG. 5.

FIG. 5.

FoxM1 regulates transcription of the mitotic regulators Aurora B kinase, survivin, CENPA, and CENPB. (A) FoxM1 is required for expression of mitotic regulatory proteins. U2OS C3 cells were transfected with 100 nM of FoxM1 siRNA #1 or #2 and induced for GFP-FoxM1b with Dox, and protein extracts were isolated 72 h after transfection. Western blot analysis was used to measure siFoxM1 suppression of induced GFP-FoxM1b protein and Aurora B kinase, CENPA, Plk1, survivin, Aurora A kinase, or INCENP. (B) FoxM1 regulates mRNA levels of CENPA, survivin, and CENPB. RNA was isolated from siFoxM1 #2-transfected or untransfected U2OS cells and used for QRT-PCR analysis with the indicated human gene primers. This analysis demonstrated that FoxM1-depleted U2OS cells exhibited a statistically significant decrease in mRNA expression of CENPA (**, P = 0.008), survivin (**, P = 0.004), and CENPB (**, P = 0.01). (C and D) Compared to control MEFs, Foxm1−/− MEFs expressed reduced levels of PLK1 protein as determined by Western blotting (C) and reduced mRNA levels of Cdc25B (**, P = 0.004) and CENPA (**, P = 0.007) as determined by quantitative real-time RT-PCR (D) analysis. (E) FoxM1b activates transcription of the Aurora B kinase promoter in cotransfection assays. U2OS cells were cotransfected with CMV-FoxM1b expression vector and the −749 bp Aurora B kinase (AurkB) promoter fused to the luciferase reporter and, 24 h following transfection, protein extracts were prepared and analyzed for dual luciferase activity as described previously (42). Triplicate plates were used to calculate the mean fold induction of transcriptional activity by CMV-FoxM1B transfection ± SD (**, P = 0.004). Promoter expression with CMV-empty vector transfection was set at 1. (F) FoxM1 regulates transcription of Cdc25B, Aurora B kinase, CENPA, survivin, and CENPB promoters as determined by quantitative ChIP assays. FoxM1-depleted or untreated U2OS cells were processed for ChIP assay as described in Materials and Methods. The cross-linked and sonicated human chromatin was IP with antibodies specific to FoxM1, CBP, RNA polymerase II, or rabbit serum (control), and the amount of promoter DNAs associated with the IP chromatin was quantitated by real-time PCR with primers specific to the indicated regions of the human Cdc25B, Aurora B kinase, CENPA, survivin, and CENPB promoter. FoxM1 ChIP promoter binding in untreated U2OS cells was set at 1 ± SD. FoxM1-depleted U2OS cells showed diminished binding of FoxM1, RNA polymerase II, or CBP coactivator protein to the endogenous human promoter regions of Cdc25b, AurkB, CENPA, survivin, and CENPB genes. The asterisks in panels B, D, E, and F indicate statistically significant changes, with P values calculated by the Student t test as follows: *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. The IgG control ChIP assays produced 0.1 of the FoxM1 binding levels observed with untreated U2OS cells and similar to the FoxM1-depleted U2OS cells.