Forkhead Box M1 Regulates the Transcriptional Network of Genes Essential for Mitotic Progression and Genes Encoding the SCF (Skp2-Cks1) Ubiquitin Ligase

Abstract

The Forkhead box m1 (Foxm1) gene is critical for G 1 /S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1 ؊/؊ mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and p27 Kip1 . Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21Cip1 and p27 Kip1 . We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G 1 /S transition. Moreover, early-passage Foxm1 ؊/؊ MEFs display premature senescence as evidenced by high expression of the senescence-associated ␤-galactosidase, p19 ARF , and p16 INK4A proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.

Transgenic expression of the forkhead box M1 transcription factor induces formation of lung tumors

“…We transfected A549 cells with 100 nM l À1 of either siFoxm1 or mutant control siFoxm1 using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in serum-free tissue culture medium as previously described (Wang et al, 2005;Kim et al, 2006). Forty-eight hours after transfection, the A549 cells were stimulated with PMA (100 nM) and LPS (2.5 mg ml À1 ) in the presence of siRNA to induce Cox-2 expression as described (Chang et al, 2005;Tong et al, 2006).…”

“…Nuclear extracts from siRNA-transfected A549 cells were cross-linked by addition of formaldehyde, sonicated and used for the immunoprecipitation with Foxm1 rabbit antiserum as previously described (Wang et al, 2005). Reverse cross-linked ChIP DNA samples were subjected to PCR using the oligonucleotides listed in Table 1.…”

“…Foxm1 then directly stimulates the transcription of genes essential for progression into DNA replication and mitosis, including cyclin A2, cyclin B1, Cdc25B phosphatase, Aurora B kinase and Polo-like kinase 1 (Costa et al, 2005). The Foxm1 protein also stimulates transcription of Skp2 and Cks1 genes, which are specificity subunits of the Skp1-Cullin-F-Box ubiquitin ligase complex that targets the Cdk inhibitors p27 kip1 and p21 cip1 for degradation during the G 1 /S transition (Wang et al, 2005). We previously demonstrated that Foxm1 À/À mice exhibit embryonic lethality due to severe abnormalities in development of the heart, liver and lung (Krupczak-Hollis et al, 2004;Kim et al, 2005a).…”

Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box m1 transcription factor

“…Foxm1 Ϫ/Ϫ livers displayed abnormal accumulation of polyploid hepatoblasts resulting from diminished DNA replication and a failure to enter mitosis (Krupczak-Hollis et al, 2004). Mouse embryonic fibroblasts (MEF) from our Foxm1 Ϫ/Ϫ embryos were unable to grow in culture due to a block in mitotic progression and undergo cellular senescence at passage three with high expression levels of senescence-associated ␤-galactosidase and cell cycle inhibitors p21 Cip1 , p27 Kip1 , p16 Ink4A , and p19 ARF proteins (Wang et al, 2005). This mitotic block was also associated with diminished protein levels of the Polo-like kinase 1 and Aurora B kinase (Krupczak-Hollis et al, 2004;Kim et al, 2005a;Wang et al, 2005), both of which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis (Glover et al, 1998;Adams et al, 2001).…”

“…Mouse embryonic fibroblasts (MEF) from our Foxm1 Ϫ/Ϫ embryos were unable to grow in culture due to a block in mitotic progression and undergo cellular senescence at passage three with high expression levels of senescence-associated ␤-galactosidase and cell cycle inhibitors p21 Cip1 , p27 Kip1 , p16 Ink4A , and p19 ARF proteins (Wang et al, 2005). This mitotic block was also associated with diminished protein levels of the Polo-like kinase 1 and Aurora B kinase (Krupczak-Hollis et al, 2004;Kim et al, 2005a;Wang et al, 2005), both of which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis (Glover et al, 1998;Adams et al, 2001). Foxm1 is required for differentiation of hepatoblast precursor cells toward billiary epithelial cell lineage because Foxm1 Ϫ/Ϫ livers fail to develop intrahepatic bile ducts (Krupczak-Hollis et al, 2004).…”