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. 2008 May;28(9):3076-87.
doi: 10.1128/MCB.01710-07. Epub 2008 Feb 19.

Activation of FoxM1 during G2 requires cyclin A/Cdk-dependent relief of autorepression by the FoxM1 N-terminal domain

Jamila Laoukili  1 , Monica Alvarez, Lars A T Meijer, Marie Stahl, Shabaz Mohammed, Livio Kleij, Albert J R Heck, René H Medema
Affiliations

Activation of FoxM1 during G2 requires cyclin A/Cdk-dependent relief of autorepression by the FoxM1 N-terminal domain

Jamila Laoukili et al. Mol Cell Biol. 2008 May.

Abstract

The Forkhead transcription factor FoxM1 is an important regulator of gene expression during the G(2) phase. Here, we show that FoxM1 transcriptional activity is kept low during G(1)/S through the action of its N-terminal autoinhibitory domain. We found that cyclin A/cdk complexes are required to phosphorylate and activate FoxM1 during G(2) phase. Deletion of the N-terminal autoinhibitory region of FoxM1 generates a mutant of FoxM1 (DeltaN-FoxM1) that is active throughout the cell cycle and no longer depends on cyclin A for its activation. Mutation of two cyclin A/cdk sites in the C-terminal transactivation domain leads to inactivation of full-length FoxM1 but does not affect the transcriptional activity of the DeltaN-FoxM1 mutant. We show that the intramolecular interaction of the N- and C-terminal domains depends on two RXL/LXL motifs in the C terminus of FoxM1. Mutation of these domains leads to a similar gain of function as deletion of the N-terminal repressor domain. Based on these observations we propose a model in which FoxM1 is kept inactive during the G(1)/S transition through the action of the N-terminal autorepressor domain, while phosphorylation by cyclin A/cdk complexes during G(2) results in relief of inhibition by the N terminus, allowing activation of FoxM1-mediated gene transcription.

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Figures

FIG. 1.
FIG. 1.
Cell cycle-dependent transcriptional activation of FoxM1. (A) U2OS osteosarcoma cells were cotransfected with 6xDB or CENP-F luciferase reporter, synchronized at the G1/S transition by 24-h administration of thymidine, and released from the block for the indicated times. DNA profiles of cell cultures after release from the G1/S block (top panels) were determined by FACS using propidium iodide staining. Transactivation of the 6xDB construct and of the CENP-F promoter (CENP-Fp) by endogenous FoxM1 (Mock) or by ectopic FoxM1 was measured in a dual luciferase assay. In all experiments relative luciferase reporter activity was expressed as the ratio of firefly luciferase activity to control Renilla luciferase activity. (B) U2OS cells were blocked at the G1/S transition by thymidine treatment and released from the thymidine block at the indicated times. Endogenous FoxM1 protein levels in these cells were viewed by Western blotting (upper panel). Nocodazole-blocked cells (mitotic shake-off) were released from the nocodazole block after 3 h in the absence or presence of the proteasome inhibitor MG132. Endogenous FoxM1 protein levels in these cells were viewed by Western blotting (lower panel). (C) G2 cell lysates (12 h after release from the thymidine block) were prepared from U2OS cells transfected with either mock or FoxM1 expression vectors and subjected to lambda phosphatase (λPPase) treatment in the absence or presence of phosphatase inhibitors (PPase Inh). Endogenous and ectopically expressed FoxM1 proteins were viewed by Western blotting.
FIG. 2.
FIG. 2.
Regulation of FoxM1 transcriptional activity by cyclin A/cdk's. (A) Luciferase assay in U2OS cells transiently cotransfected with control, or FoxM1 expression vectors and 6xDB luciferase reporter in combination with DNcdk1, alone or in combination with ectopically expressed cyclin A (left panel). The expression of all constructs was confirmed by Western blotting (right panel). (B and C) Transactivation of 6xDB by FoxM1 was measured in U2OS cells that were either transfected with empty vector (mock), cyclin A- or cyclin B-expressing vectors (B), or with shRNA-targeting vectors against cyclin A (pS-cycA) or cyclin B (pS-cyB) (C). Presented data are the averages of three independent experiments performed in duplicate. Expression levels of all constructs were viewed by Western blotting.
FIG. 3.
FIG. 3.
Cyclin A is required for FoxM1 target gene expression and for proper G2/M progression. (A) Western blot analysis of endogenous FoxM1 protein in human U2OS cells transfected with empty pSuper or two different pS-cycA-targeting constructs. Cells were blocked at the G1/S transition by 24-h administration of thymidine and released from the thymidine block for 12 h or 14 h in the presence of nocodazole. (B) U2OS cells cotransfected with spectrin-green fluorescent protein (GFP) plasmid and empty pSuper or pS-cycA targeting constructs were synchronized at G1/S transition by thymidine treatment and released from the block for 14 h in the absence (left graphs) or presence of nocodazole (right graph). The left graphs show FACS analysis of DNA profiles of spectrin-GFP-positive cells using propidium iodide staining. The right panel shows the quantification of the mitotic cell population in nocodazole-treated cells using phospho-histone H3 staining (pH 3). (C) Western blot analysis of FoxM1 target gene (CENP-F and cyclin B1) expression in U2OS cells expressing pS or pS-cycA targeting vectors blocked at the G1/S transition by 24-h administration of thymidine and released from the thymidine block for 14 h in the presence or absence of nocodazole (left panel). RT-PCR analysis of CENP-F and cyclin B1 mRNA levels in U2OS cells transfected with control siRNA or siRNA oligonucleotides targeting cyclin A and released from a thymidine block for 12 h (right panel). (D) Western blot analysis of FoxM1 target gene expression in human U2OS cells that were either transfected with empty pSuper RNAi vector (pS), with an RNAi-expressing construct targeting FoxM1 (pS-FoxM1), or with an RNAi-expressing construct targeting cyclin A (pS-cycA) or were cotransfected with both pS-FoxM1 and pS-cyclin A targeting constructs.
FIG. 4.
FIG. 4.
Deletion of the N-terminal region of FoxM1 leads to a hyperactive mutant that is constitutively active throughout the cell cycle. (A) Transactivation of the 6xDB luciferase reporter was measured in U2OS cells expressing full-length FoxM1 or ΔN-FoxM1. (B) Transactivation of 6xDB by FoxM1 or ΔN-FoxM1 in U2OS cells blocked in G1/S by thymidine treatment or by serum starvation (serum free). (C) 6xDB luciferase reporter transactivation by full-length FoxM1 or ΔN-FoxM1 in U2OS cells that were blocked at the G1/S transition by thymidine treatment and released from the thymidine block for the indicated times. Endogenous as well as ectopically expressed FoxM1 or ΔN-FoxM1 protein levels were viewed by Western blotting. (D) Nuclear localization of full-length FoxM1 and ΔN-FoxM1 introduced in U2OS cells, as visualized by anti-FoxM1 and 4′,6′-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy.
FIG. 5.
FIG. 5.
The N-terminal-truncated FoxM1 mutant is insensitive to inactivation of cyclin A/cdk complexes. (A) 6xDB luciferase reporter transactivation by full-length FoxM1 or ΔN-FoxM1 in U2OS cells expressing DNcdk1 alone or together with cyclin A. (B) Transactivation of 6xDB by FoxM1 or ΔN-FoxM1 was measured in U2OS cells at 12 h after release from the G1/S block. U2OS cells were either transfected with empty pSuper RNAi vector (1) or with an RNAi-expressing construct targeting cyclin A (2) or cyclin B (3). Endogenous cyclin A and cyclin B as well as ectopically expressed FoxM1 or ΔN-FoxM1 protein levels were viewed by Western blotting. *, nonspecific band. (C) Transactivation of 6xDB by the indicated constructs was measured in U2OS cells synchronized at the G1/S transition by 24-h administration of thymidine (0 h) and at 12 h after release from the G1/S block. Expression of all constructs was determined by Western blotting. (D) In vitro phosphorylation of the Flag-tagged wild type and 3A FoxM1 mutant. The flag-tagged wild-type and 3A proteins were transfected in 293T cells and immunoprecipitated using anti-flag antibody. The immunoprecipitates were then used in kinase assays with a radioactive label in the absence or presence of active purified cycA/cdk2 complex (Cell Signaling). Kinase activity was viewed by autoradiography (upper panel). Quantification of 32P incorporation is shown (lower panel). (E) Quantification of the mitotic cell fraction in U2OS cells transfected with pS or pS-FoxM1 targeting vector in combination with RNAi-insensitive forms of the indicated proteins. The percentage of the mitotic cell population was measured using pH 3 staining 16 h after release from the G1/S transition in the presence of nocodazole. Expression of all constructs was determined by Western blotting.
FIG. 6.
FIG. 6.
The N-terminal domain represses FoxM1 transcriptional activity during G1/S through interaction with the C-terminal transactivation domain. (A) Transactivation of the 6xDB luciferase reporter was measured in U2OS cells transfected with empty vector or full-length FoxM1- or ΔN-FoxM1-expressing vectors in the absence or presence of an exogenously expressed HA-tagged N-terminal fragment of FoxM1 (HA-Nt-FoxM1) at the G1/S transition. Expression of endogenous and ectopically expressed FoxM1 proteins was viewed by Western blotting with anti-FoxM1 antibody. (B) Subcellular localizations of full-length FoxM1 and ΔN-FoxM1 were visualized in U2OS cells transiently expressing the HA-Nt-FoxM1 fragment by using an anti-FoxM1 antibody that recognizes the C-terminal domain of the protein in combination with HA and 4′,6′-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. (C) Transactivation of the 6xDB luciferase reporter was measured in U2OS cells transfected with empty vector or vectors expressing full-length FoxM1 or ΔN-FoxM1 in the absence or presence of increasing amounts of HA-Nt-FoxM1 fragment (0, 0.5, and 1 μg, respectively) at the indicated times after release from the thymidine block. Presented data are the averages of four independent experiments performed in duplicate. (D) Immunoprecipitation of the HA-Nt-FoxM1 fragment transiently expressed in U2OS cells in combination with full-length FoxM1 or ΔN-FoxM1. U2OS cells were either transfected with empty pSuper RNAi vector (pS) or with an RNAi-expressing construct targeting cyclin A. Cells were synchronized at the G1/S transition by 24-h administration of thymidine (0 h) and released from the G1/S block for 12 h. Expression of all constructs was detected by Western blotting.
FIG. 7.
FIG. 7.
Autoinhibition of FoxM1 transcriptional activity requires binding of the N-terminal domain to RXL/LXL motifs in the C-terminal transactivation domain. (A) Transactivation of 6xDB luciferase reporter was measured in U2OS cells transfected with empty, wild-type full-length FoxM1, or ΔN-FoxM1 vector or mutants carrying the R716A single mutation or R716A/L722A double mutation at the G1/S transition (right graph). Expression of all constructs was detected by Western blotting (lower panel). (B) Transactivation of the 6xDB luciferase reporter was measured in U2OS cells transfected with empty vector, wild-type full-length FoxM1, full-length FoxM1R716A single, or FoxM1R716A/L722A double mutants in U2OS cells cotransfected with empty pSuper or pS-cycA targeting constructs (left panel) or cotransfected with empty or HA-Nt-FoxM1-expressing vector (lower panel). (C) Immunoprecipitation of the HA-Nt-FoxM1 fragment in 293T cells transiently coexpressed with wild-type FoxM1 or FoxM1R716A single or FoxM1R716A/L722A double mutants. Protein levels of all constructs were viewed by Western blotting.
FIG. 8.
FIG. 8.
Autoinhibition of the FoxM1 transcriptional activity is released upon phosphorylation by active cyclin A/cdk2 complexes during G2. (A) The immunoprecipitates containing HA-Nt-FoxM1 and wild-type Flag-FoxM1 (Flag-WT) or HA-Nt-FoxM1 and Flag-FoxM13A mutant (Flag-FL3A) were incubated with commercial active cyclin A/cdk2 in the presence or absence of ATP. FoxM1 and cdk2 protein levels were viewed by Western blotting. (B) Model illustrating the cell cycle-dependent regulation of FoxM1 transcriptional activity. During G1/S, FoxM1 activity is repressed by its own N-terminal domain, most likely via direct interaction with the C-terminal TAD. As cells progress to the G2 phase, phosphorylation by cyclin A/cdk complexes promotes the inactivation of the N-terminal autorepressor domain by disrupting the interaction between the N terminus and the C-terminal TAD, allowing activation of the FoxM1 protein.
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