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. 2008 Mar 13;27(12):1696-704.
doi: 10.1038/sj.onc.1210814. Epub 2007 Sep 24.

An N-terminal inhibitory domain modulates activity of FoxM1 during cell cycle

H J Park  1 , Z Wang, R H Costa, A Tyner, L F Lau, P Raychaudhuri
Affiliations

An N-terminal inhibitory domain modulates activity of FoxM1 during cell cycle

H J Park et al. Oncogene. .

Abstract

The FoxM1 transcription factor plays critical roles in the expression of genes that are essential for cell proliferation. FoxM1 null or depleted cells fail to progress through mitosis, as expression of several mitotic genes depends upon FoxM1. The transcriptional activity of FoxM1 is stimulated by cyclin-cdk-mediated phosphorylation at a site within the transcriptional activation domain. Here, we characterize the role of an N-terminal inhibitory domain in the transcriptional activity of FoxM1. Deletion of the N-terminal 232 amino-acid residues increases the transcriptional and transforming activities of FoxM1. Moreover, while the activity of the full-length FoxM1 is stimulated by growth factors, the activity of the N-terminal deletion mutant is constitutively high in all phases of the cell cycle. The N-terminal deletion also eliminates the requirement for cyclin-cdk to activate FoxM1. We provide evidence that the N-terminal domain interacts with the C-terminal half of the transcription factor to attenuate its transcriptional activity. Moreover, the N-terminal fragment inhibits the transcriptional activity of FoxM1 in G1/S cells, but not in G2/M cells. Our results suggest that cyclin-cdk phosphorylates FoxM1 to counteract the inhibition by the N-terminal domain to fully activate FoxM1 in G2/M phase.

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Figures

Figure 1
Figure 1
Deletion of the N-terminal 232 residues increases the transcriptional activity of forkhead box m1 (FoxM1). (a) Schematic diagram of FoxM1. (b) U2OS, Saos2 or NIH3T3 cells were transiently transfected with the 6X-FoxM1-TATA luciferase reporter and a cytomegalovirus (CMV)-Renilla luciferase construct in conjunction with the control (CMV), the wild-type FoxM1 (WT) or the N-terminal deletion mutant (N-del)vector. Extracts of the transfected cells were analysed for luciferase activity. Luciferase activity is plotted as a percentage of WT FoxM1B transcriptional activity following normalization to Renilla luciferase activity. (c) U2OS cells were electroporated with the wild-type FoxM1 or the N-del-expressing plasmids. The relative expression of Aurora B was determined by western blot analysis. The numbers above the lanes indicate fold increase. (d) RNA was isolated from NIH3T3 cells electroporated with the wild-type FoxM1 or N-del and was used for QRT–PCR with Skp2 primers. Data are shown as mean ± s.e.m. (**P<0.01; Student’s t-test).
Figure 2
Figure 2
Cells expressing the N-terminal deletion mutant (N-del) of forkhead box m1 (FoxM1) exhibit increased transforming abilities. NIH3T3 cell lines expressing green fluorescent protein (GFP) (MOCK), GFP-FoxM1 wild type (WT) or GFP-N-del (N-del) were established. (a) For focus formation assay, cells were allowed to grow for 10–14 days and then stained with crystal violet. The number of foci in the plot represents the mean number from three plates. (b) Cells were suspended in 0.35% agarose and plated on 0.7% bottom agar layer. The appearance of colonies was analysed after 21 days. (c) Cell invasion was assayed using Matrigel-coated invasion chamber. After 24 h incubation, cells on the lower surface of the membrane were stained and counted. Data are shown as mean ± s.e.m. (P<0.01; Student’s t-test). (d) Cells were plated on 100 mm dishes in complete medium and incubated overnight. The next day, media were substituted with Dulbecco’s modified of Eagle’s medium containing 2% fetal bovine serum. Then cells were trypsinized and counted at the indicated time point. Data shown are the average of three plates.
Figure 3
Figure 3
The N-terminal deletion (N-del) of forkhead box m1 (FoxM1) is active independently of growth factor pathways. U2OS cells were transfected with the 6X-FoxM1-TATA luciferase reporter plasmid in conjunction with the empty cytomegalovirus (CMV), the wild-type FoxM1 (WT) or the N-del-expressing plasmid. Sixteen hours after transfection, cells were either serum starved for 72 h (a) or treated with 50 μM phosphatidylinositol 3–kinase (PI3) (LY294002) or 50 μM mitogen-activated protein (MAP) kinase (U0126) inhibitors for 16 h (b). The relative luciferase activity is shown.
Figure 4
Figure 4
The N-terminal deletion (N-del) of forkhead box m1 (FoxM1) is constitutively active during cell cycle. Transactivation of the 6X-FoxM1-TATA luciferase reporter was analysed in U2OS cells transfected with either the empty cytomegalovirus (CMV), the wild-type FoxM1 (WT)or the N-del-expressing plasmids (a). Twelve hours after transfection, cells were treated with 5 μg ml−1 aphidicolin (G1/S) for 24 h or 50 ng ml−1 nocodazole (G2/M) for 16 h. (b) U2OS cells were transfected with plasmids expressing the WT FoxM1, FoxM1/L641A, the N-del or N-del/L641A along with the reporter 6X-FoxM1-TATA luciferase. The relative luciferase activities are shown. (c) U2OS cells transfected with WT (left) or N-del (right) with increasing levels of dominant-negative Cdk1 or 10 μM Cdk inhibitor (roscovitine). The relative luciferase activities are shown.
Figure 5
Figure 5
The N-terminal domain of forkhead box m1 (FoxM1) interacts with its transactivation domain and inhibits its transcriptional activity. (a) Glutathione-S-transferase (GST)-fusion proteins containing various fragments of FoxM1 (GST1–138 or GST 232–335)were used in pull-down assay along with lysates from NIH3T3 cells transfected with a plasmid expressing T7-epitope-tagged transactivation domain of FoxM1 (residues between 365 and 748). The binding of T7-365–748 proteins was determined by western blot with T7 antibody. The lower panel shows a Coomassie staining of the GST-fusion proteins (arrows). (b) U2OS cells were transfected with plasmids expressing green fluorescent protein (GFP1–232) and T7-epitope-tagged FoxM1. The extracts were subjected to immunoprecipitation using GFP or a control antibody. The immunoprecipitates were analysed for T7-FoxM1 by western blot assays. (c and d) U2OS cells were transfected with 6XFoxM1 reporter plasmid and an increasing amount (c) or a constant amount (d) of the plasmid expressing the N-terminal fragment of FoxM1 (GFP1–232) in conjunction with a constant amount of T7-FoxM1. (d) Sixteen hours after transfection, cells were treated with nocodazole (noco) for another 16 h. (e) U2OS cells transfected with 6 × FoxM1-TATA luciferase reporter and plasmids expressing T7-FoxM1B, N-terminal fragment of FoxM1 (GFP1-232) and Cdc25B. Relative luciferase activity is shown.
Figure 6
Figure 6
The N-terminal fragment inhibits forkhead box m1 (FoxM1) target genes. Exponentially growing NIH3T3 cells were electroporated with either 5 μg cytomegalovirus (CMV) empty vector or vector expressing the N-terminal fragment of FoxM1 green fluorescent protein (GFP-1-232). RNA was isolated and used for QRT–PCR with primers specific to Skp2, Cdc25B, Aurora B or 4EBP1. Data are shown as mean ± s.e.m. (**P<0.01; Student’s t-test).
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References

    1. Gusarova GA, Wang IC, Major ML, Kalinichenko VV, Ackerson T, Petrovic V, et al. A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment. J Clin Invest. 2007;117:99–111. - PMC - PubMed
    1. Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV, et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res. 2006;66:1712–1720. - PMC - PubMed
    1. Kalinichenko VV, Major ML, Wang X, Petrovic V, Kuechle J, Yoder HM, et al. Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor. Genes Dev. 2004;18:830–850. - PMC - PubMed
    1. Kim IM, Ackerson T, Ramakrishna S, Tretiakova M, Wang IC, Kalin TV, et al. The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer Res. 2006;66:2153–2161. - PubMed
    1. Korver W, Roose J, Clevers H. The winged-helix transcription factor Trident is expressed in cycling cells. Nucleic Acids Res. 1997;25:1715–1719. - PMC - PubMed

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