Activation of FoxM1 during G2 requires cyclin A/Cdk-dependent relief of autorepression by the FoxM1 N-terminal domain.
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.
- SourceAvailable from: Arne Nedergaard Kousholt[Show abstract] [Hide abstract]
ABSTRACT: The maintenance of genome integrity is important for normal cellular functions, organism development and the prevention of diseases, such as cancer. Cellular pathways respond immediately to DNA breaks leading to the initiation of a multi-facetted DNA damage response, which leads to DNA repair and cell cycle arrest. Cell cycle checkpoints provide the cell time to complete replication and repair the DNA damage before it can continue to the next cell cycle phase. The G2/M checkpoint plays an especially important role in ensuring the propagation of error-free copies of the genome to each daughter cell. Here, we review recent progress in our understanding of DNA repair and checkpoint pathways in late S and G2 phases. This review will first describe the current understanding of normal cell cycle progression through G2 phase to mitosis. It will also discuss the DNA damage response including cell cycle checkpoint control and DNA double-strand break repair. Finally, we discuss the emerging concept that DNA repair pathways play a major role in the G2/M checkpoint pathway thereby blocking cell division as long as DNA lesions are present.Biomolecules. 12/2012; 2(4):579-607.
- [Show abstract] [Hide abstract]
ABSTRACT: The forkhead box transcription factor FoxM1, a positive regulator of the cell cycle, is required for β-cell mass expansion postnatally, during pregnancy, and after partial pancreatectomy. Upregulation of full-length FoxM1, however, is unable to stimulate increases in β-cell mass in unstressed mice or after partial pancreatectomy, likely due to lack of posttranslational activation. We hypothesized that expressing an activated form of FoxM1 could aid in recovery after β-cell injury. We therefore derived transgenic mice that inducibly express an activated version of FoxM1 in β-cells (RIP-rtTA; TetO-HA-Foxm1(Δ)(NRD) mice). This N-terminally truncated form of FoxM1 bypasses two post-translational controls: exposure of the forkhead DNA binding domain and targeted proteosomal degradation. Transgenic mice were subjected to streptozotocin (STZ)-induced β-cell ablation to test whether activated FoxM1 can promote β-cell regeneration. Mice expressing HA-FoxM1(Δ)(NRD) displayed decreased ad libitum-fed blood glucose and increased β-cell mass. β-cell proliferation was actually decreased in β-FoxM1* mice compared to RIP-rtTA mice seven days after STZ treatment. Unexpectedly, β-cell death was decreased two days following STZ treatment. RNA-Seq analysis indicated that activated FoxM1 alters the expression of extracellular matrix and immune cell gene profiles, which may protect against STZ-mediated death. These studies highlight a previously underappreciated role for FoxM1 in promoting β-cell survival.Molecular Endocrinology 07/2014; · 4.20 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made.Journal of Cell Science 01/2015; · 5.33 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, May 2008, p. 3076–3087
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 9
Activation of FoxM1 during G2Requires Cyclin A/Cdk-Dependent
Relief of Autorepression by the FoxM1 N-Terminal Domain?†
Jamila Laoukili,1,2* Monica Alvarez,1Lars A. T. Meijer,3Marie Stahl,1Shabaz Mohammed,4
Livio Kleij,1Albert J. R. Heck,4and Rene ´ H. Medema1*
Department of Medical Oncology, Laboratory of Experimental Oncology, University Medical Center Utrecht, Utrecht, The Netherlands1;
Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands2;
Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht,
Utrecht, The Netherlands3; and Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for
Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
Utrecht, The Netherlands4
Received 17 September 2007/Returned for modification 30 October 2007/Accepted 2 February 2008
The Forkhead transcription factor FoxM1 is an important regulator of gene expression during the G2phase.
Here, we show that FoxM1 transcriptional activity is kept low during G1/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 G2phase. Deletion of the N-terminal autoinhibitory region of FoxM1 generates a mutant of
FoxM1 (?N-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 ?N-FoxM1 mutant. We show that the intramo-
lecular 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 G1/S
transition through the action of the N-terminal autorepressor domain, while phosphorylation by cyclin A/cdk
complexes during G2results in relief of inhibition by the N terminus, allowing activation of FoxM1-mediated
FoxM1 is a transcription factor of the Forkhead family. It is
also known in the literature as Trident (in the mouse) (3),
HFH-11 (in humans) (18), WIN (in the rat) (17), or MPP-2
(partial human cDNA) (13). FoxM1 is expressed ubiquitously
in all embryonic tissues, while in adults its expression is only
observed in actively proliferating tissues (3, 18). Disruption of
the mouse FoxM1 gene results in various organ defects due to
the lack of progenitor cell proliferation (2–5, 12). Moreover,
FoxM1 controls expression of a subset of genes in the G2/M-
specific gene cluster, among which are essential regulators of
mitosis (5, 11). Consistently, the inhibition of FoxM1-mediated
gene expression results in pleiotropic cell cycle defects, includ-
ing severe delay in mitotic entry, chromosome missegregation,
and polyploidization (5, 11, 16). In addition, FoxM1-deficient
primary mouse embryonic fibroblasts (MEFs) display dramatic
changes in chromosome numbers and premature senescence,
suggesting that, in addition to promoting cell cycle progression,
FoxM1 is also required to maintain chromosomal stability (5).
FoxM1 protein levels vary during cell cycle progression.
Both FoxM1 mRNA and protein levels are barely detectable in
quiescent cells, whereas they increase in cells stimulated to
reenter the cell cycle (3). FoxM1 expression reaches maximum
levels in late G1or early S phase and is sustained at these levels
throughout G2and mitosis (3). While the earliest findings have
reported that phosphorylation of FoxM1 occurs mainly during
mitosis (3), others have shown that phosphorylation of FoxM1
is initiated by cyclin-cdk complexes in early G1and continues
during the G2and M phases of the cell cycle (8). FoxM1
associates with cyclin E-cdk2 complexes in the G1and S phases
of the cell cycle, whereas it preferentially binds the cyclin
B-cdk1 complex in the G2phase (8). Moreover, Raf/MEK/
mitogen-activated protein kinase signaling can stimulate
FoxM1 nuclear translocation through phosphorylation in late S
phase (7). This suggest that phosphorylation of FoxM1 in-
volves multiple regulatory kinases at different stages of the cell
cycle. In addition, FoxM1 interacts with the cell cycle-inhibi-
tory pocket protein pRb and the cdk-activating phosphatase
Cdc25B in G1and in G1/S, respectively (8). These proteins are
important cell cycle regulators and might regulate FoxM1 tran-
scriptional activity via their effects on cdk activity. Remarkably,
although FoxM1 is expressed as early as late G1, many of its
target genes are only induced in G2(5). Therefore, FoxM1
activity must somehow be inhibited during early S phase and
mitotic exit in order to prevent the unscheduled expression of
mitotic proteins in S and G1. However, the mechanism respon-
sible for this level of regulation is poorly understood. Here, we
set out to study this aspect of FoxM1 function through analysis
of the regulation of FoxM1 at the posttranscriptional level
during an ongoing cell cycle. We show that the N-terminal
* Corresponding author. Mailing address: Laboratory of Experi-
mental Oncology, Department of Medical Oncology, University Med-
ical Center, Stratenum 2.118, Universiteitsweg 100, 3584 CG Utrecht,
The Netherlands. Phone: 31-88-7568066. Fax: 31-88-7568479. E-mail
for J. Laoukili: firstname.lastname@example.org. E-mail for Rene ´ H. Medema:
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 19 February 2008.
region of FoxM1, shown to act as an autorepressor domain (9,
14), is involved in cell cycle-dependent inactivation of FoxM1
during G1and S phases. Increased FoxM1 transcriptional ac-
tivity occurs specifically during G2and is dependent on active
cyclin A/cdk complexes. We show that a truncated form of
FoxM1 protein that is lacking the N-terminal autorepressor
domain (?N-FoxM1) is constitutively active throughout the
cell cycle and no longer requires cyclin A for its activation.
Similarly, mutating two RXL/LXL motifs in the C terminus of
FoxM1 disrupts the intramolecular interaction between N- and
C-terminal domains, causing this mutant to behave like ?N-
FoxM1. Our data strongly suggest that cyclin A/cdk-mediated
phosphorylation of FoxM1 is involved in relieving the repres-
sive function that resides within the N-terminal region of
FoxM1. These findings provide a new mechanism of regulation
of transcriptional activity of FoxM1 during the cell cycle.
MATERIALS AND METHODS
Cell culture, transfections, and drugs. U2OS cells, 293-T cells, immortalized
MEFs, and FoxM1-ER-inducible cell lines were maintained in Dulbecco’s mod-
ified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics.
Thymidine, nocodazole, and MG132 were added at final concentrations of 2.5
mM, 250 ng/ml, and 5 ?M, respectively, and were all purchased from Sigma.
Cells were transfected with plasmid DNA using the standard calcium phosphate
transfection protocol. Small inhibitory RNA (siRNA) oligonucleotides were
transfected with HiPerFect (Qiagen) following the manufacturer’s protocol.
Antibodies. Rabbit anti-Cdk2 (sc-163), mouse anti-Cdk1 (sc-54), rabbit anti-
cyclin A (sc-751), mouse anti-cyclinB1 (sc-245), goat antiactin (sc-1616), and
rabbit anti-FoxM1 MPP-C20 (sc-502), MPP-K19 (sc-500), and MPP-H300 (sc-
13016) antibodies were all purchased from Santa Cruz Biotechnology. Mouse
anti-flag-M2 (f3165), anti-flag–agarose beads (A2220), and mouse anti-?-tubulin
(t5168) were from Sigma. HA-11 affinity matrix (AFC-101P) and mouse anti-
hemagglutinin (anti-HA; mms-101p) were purchased from Covance. Rabbit anti-
CENP-F (ab5) and antiphosphothreonine (9381) were from Abcam and Cell
Signaling, respectively. The following secondary antibodies were used: peroxi-
dase-conjugated goat anti-rabbit, goat anti-mouse, and rabbit anti-goat antibod-
ies were from DAKO, and chicken anti-mouse/Alexa 488 and goat anti-rabbit/
Alexa 568 were from Molecular Probes.
Plasmids and oligonucleotides. The 6xDB and CENP-F luciferase reporters
have been described previously (5). HA-Nt-FoxM1, glutathione S-transferase
(GST), GST-Nt-FoxM1, and GST-Ct-FoxM1 constructs were from W. Korver,
and corresponding proteins were purified from bacterial cultures. Full-length
human FoxM1 and ?N-FoxM1 mutant were gifts from B. Luscher. FoxM1AAA
(T600A/T611A/S638A), FoxM1T600A, FoxM1T611A, FoxM1T600A/T611A,
FoxM1R716A, and FoxM1 R716A/L722A were obtained by site-directed mu-
tagenesis in both full-length FoxM1 and ?N-FoxM1. RNA interference (RNAi)-
insensitive expression constructs were also obtained by site-directed mutagenesis.
Correctly mutated plasmids were identified through direct sequence analysis.
Expression constructs for cyclinB1, empty pSuper, and pS-cyclinB1 were de-
scribed previously (5). Short hairpin RNA (shRNA)-targeting vectors for cyclin
A (pS-cycA) and for cdk2 (pS-cdk2) and cdk1 (pS-cdk1) were gifts from M. van
Vugt and R. Wolthuis, respectively. Expression plasmids for cyclin A, DNcdk2,
and DNcdk1 were kind gifts from S. van den Heuvel. Double-stranded RNA
oligonucleotides used for cyclin A2 RNAi were purchased from Ambion.
Reporter assays. Cells were transfected using the standard calcium phosphate
transfection protocol. Luciferase activity was determined 48 h after transfection,
using the dual luciferase kit (Promega) according to the manufacturer’s instruc-
tions. Relative luciferase activity was expressed as the ratio of firefly luciferase
activity to control Renilla luciferase activity.
Western blot analysis, immunoblotting, and kinase assays. Western blot anal-
ysis was performed as described elsewhere (5). For immunoblotting and in vitro
kinase assays, cells were lysed in ELB buffer (50 mM HEPES, pH 7.5, 150 mM
NaCl, 5 mM EDTA, and 0.1% NP-40) and 100 ?g of protein was used for
immunoprecipitation with the appropriate antibody coupled to protein A/G-
agarose beads. For immunoblotting, the precipitates were extensively washed,
resuspended in sample buffer, boiled, and analyzed in Western blot assays. For
kinase assays, the immunoprecipitates were extensively washed and incubated in
kinase buffer (50 mM HEPES pH 7.5, 5 mM MgCl2, 2.5 mM MnCl2, 1 mM
dithiothreitol [DTT]) with 1 ?l of substrate (histone H1, GST, GST-Nt-FoxM1,
or GST-Ct-FoxM1), 50 ?M cold ATP, and 2.5 ?Ci [?-32P]ATP for 30 min at
30°C. Samples were then denatured in sample buffer, boiled, and loaded on
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Results were
visualized by Coomassie blue staining of the gel followed by autoradiography.
For cyclin A/cdk2-dependent disruption of the N-/C-terminal complexes of
FoxM1, washed immunoprecipitates were incubated with 100 ng of active re-
combinant cyclin A/cdk2 (Cell Signaling) for 30 min prior to analysis of the
complexes in immunoblot assays.
RT-PCR and chromatin immunoprecipitation. Total RNA was isolated by
using either TRIzol reagent or the Qiagen RNeasy kit, according to the manu-
facturer’s instructions. Reverse transcription-PCR (RT-PCR) and chromatin
immunoprecipitations were performed as previously described (5).
FACS and immunofluorescence analysis. Fluorescence-activated cell sorter
(FACS) and immunofluorescence analyses were performed as described previ-
Tryptic phosphopeptide analysis. For analysis of in vitro phosphorylation by
cyclin A/cdk2, FoxM1 and respective phospho site mutants were expressed in
293T cells and immunoprecipitated using anti-FoxM1 antibodies. The resulting
immunocomplexes were incubated in kinase buffer (50 mM HEPES pH 7.5, 10
mM Mg-acetate, 1 mM DTT) in the presence of 50 ?M ATP and 5 ?Ci
[?-32P]ATP, with or without 100 ng of recombinant cyclin A/cdk2 (Cell Signal-
ing), for 30 min at 30°C. Phosphorylated FoxM1 was processed for two-dimen-
sional phosphopeptide mapping. For analysis of in vivo phosphorylation of
FoxM1, endogenous FoxM1 in control or cyclin A-depleted U2OS cells was
labeled using 2 mCi [32P]orthophosphate per 10-cm dish. Phosphorylated FoxM1
was immunoprecipitated, separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, and electroblotted to nitrocellulose. After exposing the blot
to X-ray film, bands of interest were cut out for tryptic phosphopeptide analysis.
Tryptic phosphopeptide analysis was performed as described earlier (1).
Phosphopeptide analysis by mass spectrometry. Epitope-tagged FoxM1 was
expressed in U2OS cells and 10 15-cm dishes were harvested, after which FoxM1
was isolated by immunoprecipitation. Subsequently, immunoprecipitated pro-
teins were reduced in 10 mM DTT and alkylated in 50 mM iodoacetamide,
followed by digestion using sequencing-grade trypsin (Roche Diagnostics, In-
gelheim, Germany) overnight at a protein/protease ratio of 50:1. Phosphopeptide
enrichment was performed as previously described (10). A home-built nanoflow
vented column system, comprised of an LC-Packings Ultimate quaternary sol-
vent delivery system, a thermostatted FAMOS autosampler, and a Switchos
six-port switching module (LC-Packings, Amsterdam, The Netherlands), coupled
on-line to a linear ion trap-Fourier transform ion cyclotron resonance mass
spectrometer (Thermo Electron, Bremen, Germany), was used for all analyses.
Spectra were processed using the Bioworks 3.3 software (Thermo, Bremen,
Germany), and the subsequent data analysis was conducted using the Mascot
(version 2.2) software platform (Matrix Science, London, United Kingdom). The
IPI human database (version 3.36; containing 69,012 sequences and 29,002,682
residues) was searched with trypsin, allowing two missed cleavages, carbamido-
methyl (C) as fixed modification and oxidation (M), N-acetylation (protein N
terminus), deamidation (NQ), phosphorylation (ST), and phosphorylation (Y) as
variable modifications. The peptide tolerance was set to 10 ppm, and tandem
mass spectrometry tolerances to 0.9-Da phosphorylated peptides with MASCOT
scores of 50 or higher were considered to be reliable, whereas the remaining
phosphorylated peptides were confirmed manually.
FoxM1 transcriptional activity is restricted to the G2/M
phases of the cell cycle. Our recent data have shown that most
known FoxM1 target genes are not induced until the cell
reaches G2phase (5), although FoxM1 itself is expressed from
S phase onwards. Thus, additional levels of regulation must
exist to control proper timing of FoxM1 activation during G2.
In order to examine regulation of FoxM1 transcriptional ac-
tivity throughout the cell cycle, we analyzed the transactivation
of luciferase reporters containing the 6XDB Forkhead-binding
motif or CENP-F promoter, a specific FoxM1-responsive gene
(5), at different phases of the cell cycle. For this purpose,
FoxM1 was transiently expressed in human U2OS osteosar-
coma cells from a cytomegalovirus promoter-driven expression
VOL. 28, 2008RELIEF OF FoxM1 AUTOREPRESSION IN G2
vector to ensure cell cycle-independent FoxM1 expression
throughout the cell cycle (Fig. 1A). We found that FoxM1
activity was low in cells synchronized at the G1/S transition and
increased only as the cells entered the G2phase after release
from the G1/S block (10 to 12 h after release) (Fig. 1A),
whereas expression of FoxM1 was relatively constant (Fig. 1B).
Induction of FoxM1 activity in G2was not due to enhanced
DNA binding, as chromatin immunoprecipitation assays
showed that FoxM1 is already bound to its target promoters in
G1/S (see Fig. S1A in the supplemental material). Interest-
ingly, while the majority of cells appeared to have completed S
phase at 10 h in these experiments, luciferase activity peaked
some 2 h later (Fig. 1A). However, analysis of luciferase
mRNA showed that mRNA levels are readily induced at 10 h
after the release (see Fig. S1B in the supplemental material),
suggesting that this delay is a consequence of slow accumula-
tion of the luciferase enzyme. These data suggest that FoxM1
activation occurs as cells progress to G2phase, even if it is
constitutively expressed throughout the cell cycle. Transcrip-
tional activation of FoxM1 correlates well with the appearance
of a slower-migrating band 10 h after release (Fig. 1B, upper
panel). This shifted band was also present in extracts prepared
from cells trapped in G2/M by nocodazole treatment (Fig. 1B,
upper panel), whereas it disappeared in cells that reentered the
G1phase after 3 h of release from the nocodazole block (Fig.
1B, lower panel). We further showed that this shifted band
corresponds to a phosphorylated form of FoxM1, since phos-
phatase treatment prevented the shift (Fig. 1C).
FoxM1 transcriptional activity is enhanced by cyclin A/cdk
function. Previous data from our lab showed that FoxM1 ac-
tivity is highest in G2/M-arrested cells (5). Treatment of cells
with nocodazole, which blocks cell cycle progression in G2/M,
enhanced FoxM1 activity, whereas removal of serum arrests
cells in G0with very low FoxM1 transcriptional activity (5). In
addition, expression of the cdk inhibitors p16 and p21, or a
dominant negative form of cdk2 (DNcdk2), caused a dramatic
reduction in FoxM1 activity (5). Similarly, expression of a dom-
inant negative form of cdk1 (DNcdk1) also abolished FoxM1
transcriptional activity (Fig. 2A). Interestingly, the effect of
DNcdk1 was reverted when cyclin A was coexpressed (Fig.
2A), suggesting that the inhibitory effect of DNcdk1 on FoxM1
activity is due to titration of the endogenous cyclin A. Indeed,
we found that maximum activity of FoxM1 coincided with
maximum cyclin A-associated kinase activity 12 h after release
from the G1/S thymidine block (see Fig. S1C in the supple-
mental material). Consistently, exogenous expression of cyclin
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) G2cell 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
(?PPase) treatment in the absence or presence of phosphatase inhibitors (PPase Inh). Endogenous and ectopically expressed FoxM1 proteins were
viewed by Western blotting.
3078 LAOUKILI ET AL.MOL. CELL. BIOL.
A, but not cyclin B, enhanced FoxM1 transcriptional activity
(Fig. 2B, left panel), suggesting that FoxM1-dependent trans-
activation might be specifically mediated via cyclin A-associ-
To further confirm the requirement for cyclin A in transac-
tivation by FoxM1, expression of endogenous cyclin A was
repressed by an shRNA-targeting vector. Specific removal of
cyclin A, but not cyclin B, through RNAi-mediated depletion
was able to cause a strong reduction in FoxM1 activity, similar
to what is observed after expression of a dominant negative
version of cdk1 (Fig. 2C). In addition, cyclin A-associated
kinases appear to be required for in vivo phosphorylation of
endogenous FoxM1, as depletion of cyclin A with two different
shRNA-targeting vectors resulted in a strong reduction of
phosphorylated forms of FoxM1 in G2phase (Fig. 3A; see also
Fig. S2A in the supplemental material). In addition, removal of
cyclin A caused cells to accumulate in G2and resulted in a
strong reduction in the mitotic population after nocodazole
treatment (Fig. 3B), similar to what we have observed follow-
ing depletion of FoxM1 (5). Down-regulation of cyclin A also
led to a reduction in the expression of two well-known target
genes of FoxM1 during G2/M progression, CENP-F and cyclin
B, at both protein and mRNA levels (Fig. 3C). This was ob-
served both in G2cells released from a thymidine block and in
nocodazole-arrested cells (Fig. 3C). Codepletion of cyclin A
and FoxM1 does not lead to a further reduction of FoxM1
target genes (Fig. 3D), indicating that cyclin A and FoxM1 act
together, rather than independently, to regulate these genes.
Also, while depletion of cyclin A caused a clear reduction in
CENP-F and cyclin B mRNA levels in wild-type MEFs, this
effect was much less pronounced in FoxM1-deficient MEFs
(see Fig. S2B in the supplemental material). These data sug-
gest that cyclin A acts through FoxM1 to activate transcription
of genes required for proper G2/M progression.
Deletion of the N-terminal region of FoxM1 generates a
constitutively active form of FoxM1. Removal of the N-termi-
nal domain, right up to the start of the DNA-binding domain
(?N-FoxM1), results in a protein with increased activity (Fig.
4A). To define the contribution of the N terminus of FoxM1 to
the cell cycle-dependent regulation of its transcriptional activ-
ity, we examined the activity of this truncated mutant during
different stages of the cell cycle. In contrast to the full-length
protein, transactivation by the ?N-FoxM1 variant was hardly
affected upon synchronization of cells by serum starvation or
by the addition of thymidine (Fig. 4B). Likewise, the activity of
this mutant remained constantly high throughout the cell cycle,
whereas full-length FoxM1 activity only increased during the
G2phase (10 h to 12 h after release from the G1/S block) (Fig.
4C). This is not due to differences in the subcellular localiza-
tion or protein expression, since both full-length FoxM1 and
?N-FoxM1 localize to the nucleus and show similar expression
levels throughout the cell cycle (Fig. 4C and D; see also Fig. S3
in the supplemental material). These data show that ?N-
FoxM1 behaves as a constitutively active form of FoxM1 whose
activity is independent of cell cycle progression. As cyclin A-
containing complexes directly phosphorylate FoxM1 and en-
hance its transcriptional activity, we next examined the effect of
cyclin A on the transcriptional activity of the ?N-FoxM1 mu-
tant. As shown in Fig. 2A, the activity of full-length FoxM1 was
dramatically reduced in cells expressing DNcdk1 (by 90%) and
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.
VOL. 28, 2008RELIEF OF FoxM1 AUTOREPRESSION IN G2
fully restored by overexpression of cyclin A. In contrast, the
activity of the ?N-FoxM1 mutant was only slightly affected by
DNcdk1 (less than ?20%) (Fig. 5A). Furthermore, depletion
of cyclin A by shRNA had no apparent effect on the activity of
?N-FoxM1, while it strongly decreased the activity of full-
length FoxM1 (Fig. 5B). The difference in cyclin A dependence
between the wild type and ?N-FoxM1 was observed over a
range of FoxM1 concentrations, indicating that the observed
difference was not an artifact of saturation of our assay system
(see Fig. S4A in the supplemental material). Our observations
strongly suggest that cyclin A/cdk complexes activate FoxM1
during the G2phase primarily through relief of repression
mediated by its autoinhibitory N-terminal domain. However,
the fact that ?NFoxM1 transcriptional activity was slightly
inhibited by DNcdk1 points to the possibility that an additional
mechanism(s) may contribute to cyclin A/cdk-mediated activa-
tion of FoxM1.
The N terminus of FoxM1 contains two conserved cdk phos-
phorylation (S/T)P sites at amino acid serine 4 (S4P) and
serine 35 (S35P), and at least the latter appears to be phos-
phorylated in vivo (see Fig. S5E in the supplemental material).
To determine the contribution of these sites in G2-dependent
activation of FoxM1, we mutated both sites to alanine and
examined the transcriptional activity of this mutant throughout
the cell cycle. Both wild-type FoxM1 and the FoxM1S4A/S35A
mutant show a similar pattern of activation as cells progress
from G1/S to the G2phase of the cell cycle (data not shown),
indicating that additional residues in FoxM1 serve as sub-
strates for cyclin A/cdk complexes to mediate release of the
N-terminal inhibitory domain. Additional cdk phosphorylation
sites (T600/T611/S638; amino acid numbering in the FoxM1C
variant) in the C-terminal transactivation domain (TAD) may
contribute to FoxM1 activation by cyclin/cdk complexes (6).
Therefore, we mutated all three sites into alanine in both
full-length FoxM1 and ?N-FoxM1 and assessed their tran-
scriptional activities throughout the cell cycle. We found that
mutation of T600/T611/S638 sites into alanine strongly re-
duced transcriptional activation of full-length FoxM1 (FL3A)
during the G2phase (Fig. 5C). However, mutation of these
sites in ?N-FoxM1 (?N3A) had no effect on its activity (Fig.
5C), although both FL3A and ?N3A showed strongly de-
creased phosphorylation (see Fig. S4B and C in the supple-
mental material). Similarly, the 3A FoxM1 mutant was less
phosphorylated by active recombinant cyclin A/cdk2 in vitro
(Fig. 5D). Finally, we assessed the functionality of these mu-
tants by testing their ability to promote G2/M progression in
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.
3080LAOUKILI ET AL.MOL. CELL. BIOL.
the absence of endogenous FoxM1. U2OS cells were cotrans-
fected with the FoxM1 shRNA-targeting vector and RNAi-
insensitive expression plasmids encoding the various proteins.
Cells lacking FoxM1 fail to enter mitosis and show a significant
delay in G2, resulting in a low mitotic index in the presence of
nocodazole (5) (Fig. 5E). As expected, full-length FoxM1, ?N-
FoxM1, and ?N3A mutants were able to reverse the mitotic
entry defect in FoxM1-depleted cells to a large extent, while
the FL3A mutant failed to do so significantly (Fig. 5E, left
panel). Collectively, these data suggest that cyclin A/cdk-de-
pendent phosphorylation of the C-terminal TAD is required
for relief of inhibition by the N-terminal autorepressor domain
to promote proper G2/M progression.
Phosphorylation of FoxM1 by cyclin A/cdk complexes. Be-
cause exogenous expression of cyclin A induces a strong acti-
vation of FoxM1, we examined whether FoxM1 protein was a
direct substrate for cyclin A-containing cdk complexes. We
performed in vitro kinase assays using GST-FoxM1 variants as
a substrate. We found that cyclin A/cdk complexes can strongly
phosphorylate both N-terminal and C-terminal domains of
FoxM1 in vitro (see Fig. S5A in the supplemental material). To
verify if cyclin A/cdk complexes can phosphorylate the T600/
T611/S638 residues, we first analyzed if all sites were relevant
to cell cycle-dependent regulation of FoxM1. Mutating single
residues within this triad did not result in loss of FoxM1 acti-
vation in G2(not shown), but a double T600/T611 mutant fully
reproduced the functional effects seen with the triple mutant
(see Fig. S5B in the supplemental material), indicating that the
S638 site is not relevant for this aspect of FoxM1 regulation.
To test if T600 and T611 can be phosphorylated by cyclin
A/cdk2, we immunoprecipitated ectopically expressed single
and double T600/T611 mutants of FoxM1 and incubated these
immunocomplexes with active recombinant cyclin A/cdk2 com-
plexes. In vitro-phosphorylated proteins were subsequently an-
alyzed by tryptic phosphopeptide mapping. As shown in Fig.
S5C of the supplemental material, several peptides in FoxM1
are phosphorylated by cyclin A/cdk2, two of which appear to
correspond to a T600- and a T611-containing peptide (see Fig.
S5C in the supplemental material), as they are missing in the
peptide maps of the corresponding T600/T611 single or double
alanine mutants. Of these, the T611 peptide runs at the ex-
pected mobility as predicted with Mobility software, while the
T600 peptide displays a different running behavior, possibly as
a result of incomplete cleavage of the protein.
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 ?N-FoxM1. (B) Transactivation
of 6xDB by FoxM1 or ?N-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 ?N-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 ?N-FoxM1 protein levels were
viewed by Western blotting. (D) Nuclear localization of full-length FoxM1 and ?N-FoxM1 introduced in U2OS cells, as visualized by anti-FoxM1
and 4?,6?-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy.
VOL. 28, 2008 RELIEF OF FoxM1 AUTOREPRESSION IN G2
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 ?N-FoxM1 in U2OS cells expressing DNcdk1 alone or together with cyclin A. (B) Transactivation of 6xDB
by FoxM1 or ?N-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 ?N-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 of32P 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.
3082LAOUKILI ET AL.MOL. CELL. BIOL.
In addition, we analyzed the phosphopeptide map of in
vivo-phosphorylated endogenous FoxM1 obtained from
[32P]orthophosphate-labeled control or cyclin A-depleted
U2OS cells. This showed that T611, and possibly also T600 to
a lesser extent, is also phosphorylated in vivo in a cyclin A-de-
pendent manner (see Fig. S5D in the supplemental material).
Phosphorylation of T611 in vivo was further confirmed by mass
spectrometry using exogenously expressed FoxM1 isolated
from U2OS cells (see Fig. S5E in the supplemental material).
These data show that FoxM1 is phosphorylated on T611 and
possibly T600, in a cyclin A-dependent manner, both in vitro
and in vivo.
The N-terminal domain can repress FoxM1 activity in trans
and is inactivated through cyclin A/Cdk2-mediated phosphor-
ylation. To demonstrate that the N terminus of FoxM1 carries
repressor activity, we next assessed FoxM1 transcriptional ac-
tivity in cells expressing the FoxM1 N-terminal fragment. We
found that transactivation by the ?N-FoxM1 mutant was dra-
matically reduced in the presence of the N-terminal fragment
during G1/S (Fig. 6A). The N-terminal fragment of FoxM1
appears to localize predominantly in the nucleus and did not
interfere with the subcellular localization of full-length FoxM1
and ?N-FoxM1 at either the G1/S block or during G2(Fig. 6B;
see also Fig. S3 in the supplemental material). Since the N-
terminal repressive action was cell cycle controlled, we specu-
lated that expression of the N-terminal fragment would not
affect FoxM1 activity during the G2phase. To test this, we
examined the effect of the N-terminal fragment on FoxM1
activity in G1/S and G2, respectively. At doses that repress
?N-FoxM1 activity in G1/S phase (0 h), the N-terminal frag-
ment had no significant effect on transcriptional activity of the
full-length FoxM1 or ?N-FoxM1 mutant in G2cells (12 h)
(Fig. 6C). Yet, at higher doses, the N-terminal fragment was
able to prevent transcriptional activation of both full-length
FoxM1 and ?N-FoxM1 during the G2phase (data not shown).
These data indicate that the N-terminal region of FoxM1 is a
potent autorepressor domain that acts to restrict FoxM1 activ-
ity in the G1and S phases. Cyclin A/cdk-dependent phosphor-
ylation of the C-terminal TAD appears to be required for relief
of autoinhibition by the N-terminal domain. Therefore, the N
terminus may exert its inhibitory action through direct binding
to the C-terminal TAD of FoxM1. To examine the possible
interaction between the N-terminal domain and FoxM1, we
performed immunoprecipitation experiments in U2OS cells
expressing full-length FoxM1 or ?N-FoxM1. We found that
the N-terminal fragment of FoxM1 strongly interacts with both
full-length FoxM1 and ?N-FoxM1 at G1/S (Fig. 6D). Interest-
ingly, the interaction was strongly reduced in G2cells, but this
reduction was prevented upon depletion of cyclin A (Fig. 6D).
These data indicate that cyclin A-dependent kinase activity
relieves FoxM1 autorepression by disrupting the interaction
between the N-terminal and C-terminal domains.
Interestingly, mutation of two RXL/LXL motifs in the C-
terminal domain of FoxM1 (R716A-X-L718A and L722A-X-
L724A) caused hyperactivation of FoxM1 in G1/S, similar to
deletion of the N-terminal domain, while the same mutations
did not further affect the activity of ?N-FoxM1 (Fig. 7A). Also,
the activity of the RXL/LXL double mutant was not affected by
depletion of cyclin A (Fig. 7B, left panel) or by expression of
the N-terminal fragment (Fig. 7B, right panel). This suggests
that the RXL/LXL motifs within the C-terminal domain are
required for interaction with the N-terminal repressor domain.
To test this directly, we coexpressed both wild-type and RXL/
LXL single or double mutants and tested their ability to inter-
act with the N-terminal domain in transfected cells. Indeed,
the double RXL/LXL mutant failed to interact with the N-
terminal mutant (Fig. 7C), consistent with a model in which
binding of the N-terminal domain to the C-terminal domain
inhibits FoxM1 activity.
Because expression of the N-terminal fragment of FoxM1
reduced transcriptional activation of full-length FoxM1 as ef-
ficiently as depletion of cyclin A, we postulated that phosphor-
ylation of FoxM1 by cyclin A/cdk complexes might cause re-
lease of inhibition by the N terminus. To test this hypothesis,
we investigated whether active cyclin A/cdk2 complexes could
disrupt the interaction between the N and C terminus of wild-
type FoxM1, but not of the 3A mutant. To this end, immuno-
precipitated complexes containing the N-terminal fragment
and FoxM1 protein were incubated in vitro with active cyclin
A/cdk2 in the presence or absence of ATP. Incubation of the
wild-type FoxM1-Nt intermolecular complexes with active cy-
clin A/cdk2 resulted in efficient disruption of the interaction
(Fig. 8A). This disruption requires ATP, as mere addition of
cyclin A/cdk2 was insufficient to release the N terminus. More-
over, incubation of cyclin A/cdk2 with the 3A FoxM1 mutant
lacking the cdk phosphorylation sites had no effect on its in-
teraction with the N terminus (Fig. 8A). Taken together, these
data suggest that cyclin A/cdk2 phosphorylates the C-terminal
TAD of FoxM1 to relieve the autoinhibition by the N-terminal
repressor domain, allowing transcriptional activation of FoxM1 in
In this study, we aimed to gain more insight into how FoxM1
activity is regulated during an ongoing cell cycle. Our data
point to the existence of a cyclin A-dependent mechanism that
controls transactivation by FoxM1, allowing a tight restriction
of FoxM1 transactivation to the G2phase of the cell cycle.
We provide evidence that although FoxM1 can already be
expressed in late G1and in S phase and can bind to its endog-
enous promoters, its transcriptional activity is kept low until
entry into G2. Our data indicate that cyclin A, but not cyclin B,
plays an important role in the regulation of FoxM1 transcrip-
tional activity during G2. Ectopic expression of cyclin A greatly
increases FoxM1 transcriptional activity, while removal of cy-
clin A through RNAi-mediated depletion leads to a strong
reduction in FoxM1 activity, similar to what is seen after ex-
pression of dominant negative versions of cdk1 and cdk2. More
importantly, cyclin A depletion results in a defect in G2/M
progression and a reduction in FoxM1 target gene expression,
similar to what is seen in FoxM1-deficient cells. Previously,
cyclin B/cdk2 was shown to phosphorylate FoxM1 (8), but the
exact contribution of this phosphorylation to FoxM1 activation
remains to be determined. Cyclin E/cdk2 was also shown to
phosphorylate and activate FoxM1 at G1/S (6, 15). A similar
observation was reported by Major and coworkers, who
showed that the cyclin E/cdk2 complex binds and phosphoryl-
ates the C-terminal region of FoxM1 in order to recruit the
transcriptional coactivator p300 (8). Accordingly, a minor
VOL. 28, 2008RELIEF OF FoxM1 AUTOREPRESSION IN G2
shifted form of FoxM1 is seen in thymidine-blocked cells (Fig.
1), suggesting that phosphorylation of FoxM1 is a multistep
process that starts as early as late G1. However, because cyclin
E/cdk2 activity is limited to the G1/S transition of the cell cycle,
it may not be sufficient for full activation of FoxM1 but could
prime FoxM1 for eventual activation by cyclin A/cdk com-
plexes in G2. Indeed, the major cyclin A-dependent shift in
FoxM1 mobility that is observed as cells progress from S to G2
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
?N-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 ?N-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 ?N-FoxM1 in the absence or presence of increasing amounts of
HA-Nt-FoxM1 fragment (0, 0.5, and 1 ?g, 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 ?N-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.
3084LAOUKILI ET AL.MOL. CELL. BIOL.
suggests that cyclin A plays a major role in promoting FoxM1
phosphorylation. Accordingly, we also found that FoxM1 is in
vitro phosphorylated by cyclin A/cdk2 complexes and that the
endogenous protein is in vivo phosphorylated during G2in a
cyclin A-dependent manner. On the basis of our observations,
it is difficult to discriminate which cyclin A/cdk complex is most
crucial for FoxM1 activation during G2phase, as both cdk1 and
cdk2 complexes could act redundantly.
We found that a truncated mutant of FoxM1 that lacks the
N-terminal domain is a hyperactive transcriptional regulator,
consistent with recent results from others (9, 14). Here, we
show that transcriptional activation of this mutant no longer
depends on cyclin A/cdk activity, as neither overexpression of
dominant negative forms of cdk’s nor depletion of cyclin A
could inhibit the activity of this mutant. These data imply that
the N-terminal repressor domain is regulated in a cell cycle-
dependent fashion, requiring cyclin A/cdk-dependent inactiva-
tion in G2. We found that phosphorylation of the C-terminal
TAD (T600 and T611 residues) is required for G2-specific
activation of FoxM1. Substitution of these residues to alanine
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 ?N-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.
VOL. 28, 2008 RELIEF OF FoxM1 AUTOREPRESSION IN G2
clearly reduced the level of FoxM1 phosphorylation by cyclin
A/cdk complexes and prevented activation of full-length
FoxM1 in G2, while it did not affect transcriptional activity of
?N-FoxM1. These data strongly suggest that cyclin A/cdk-
mediated phosphorylation of the C-terminal TAD is required
for relief of the inhibitory function of the N terminus.
Recently, it was shown that the N- and C-terminal domains
of FoxM1 can form a direct complex (14), suggesting that
inhibition by the N terminus occurs through direct interaction
of this domain with the C-terminal transactivation domain,
thereby preventing transcriptional activation of FoxM1. Im-
portantly, our data indicate that this molecular interaction
within FoxM1 can be disrupted by active cyclin A/cdk com-
plexes, allowing for full activation of FoxM1. Moreover, point
mutations in two conserved RXL/LXL sites in the TAD of
FoxM1 (R716/L718 and L722/724) strongly reduced the inter-
action with the N-terminal repressor domain of FoxM1 and
behaved as hyperactive mutants. Similar to the N-terminal
deletion mutant, mutation of the RXL/LXL motifs eliminates
the requirement of cyclin A/cdk for FoxM1 activation, indicat-
ing that these RXL/LXL motifs mediate the binding between
the N terminus and the TAD of FoxM1. In contrast, mutation
of another LXL motif present in the TAD of FoxM1 (L656A),
which has been recently shown to be required for FoxM1B
activation (9), did not show any significant effect on FoxM1C
transactivation (data not shown), suggesting that the different
FoxM1 isoforms require different modes of regulation.
Taken together, our findings uncover a crucial role for cyclin
A/cdk complexes in optimal activation of FoxM1 during the G2
phase of the cell cycle. These data are most consistent with a
model in which FoxM1 activity is kept low due to repression by
its own N-terminal domain through direct binding to the C-
terminal TAD. This binding might also allow docking of cel-
lular components, yet unknown, that further suppress FoxM1
activity. Through constitutive cyclin A/cdk action, and once
sufficient levels of phosphorylated FoxM1 protein are achieved,
repression by the N terminus is relieved, allowing full activa-
tion of FoxM1.
We thank O. Kranenburg for critically reviewing the manuscript. We
thank Lisa Caballero, Jill Meisenhelder, and Tony Hunter for help
with the Mobility software. We also thank all members of the Medema
laboratory for valuable discussions.
This work was supported by the Dutch Cancer Society (NKI200-
2192 and UU2007-3826), The Netherlands Organization of Health
Research and Development (918.46.616), and The Netherlands Pro-
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