FOXM1c transactivates the human c-myc promoter
directly via the two TATA boxes P1 and P2
Inken Wierstra
1
and Ju
¨
rgen Alves
2
1 Institute of Molecular Biology, Medical School Hannover, Germany
2 Institute of Biophysical Chemistry, Medical School Hannover, Germany
c-Myc, a key regulator of proliferation, differentiation
and apoptosis, plays a central role in cell growth
control and can induce quiescent cells to enter into
S-phase [1–7]. Because c-Myc potently stimulates pro-
liferation and inhibits differentiation it possesses a high
transformation potential that is supplemented by its
cell growth and angiogenesis-promoting, cell-adhesion-
reducing, immortality and genomic-instability-causing
activities. c-myc expression correlates strictly with cell
proliferation. c-Myc regulates target genes either by
activation via E-boxes or by repression via initiator
(Inr)-dependent and Inr-independent mechanisms.
c-Myc acts as part of the Myc ⁄ Max ⁄ Mad network in
which Max is the heterodimerization partner for
c-Myc and Mad proteins, the c-Myc antagonists,
which repress target genes via E-boxes.
The forkhead ⁄ winged helix transcription factor
FOXM1, expression of which correlates strictly with
proliferation, stimulates proliferation by promoting
S- and M-phase entry and regulates genes that control
G

via the TATA box, but as an inhibitory domain (retinoblastoma protein-
independent transrepression domain and retinoblastoma protein-recruiting
negative regulatory domain) for transactivation via conventional FOXM1c-
binding sites. Each promoter with the P2 TATA box TATAAAAG is
postulated to be transactivated by FOXM1c. This was demonstrated for the
promoters of c-fos, hsp70 and histone H2B ⁄ a. A database search revealed
almost 300 probable FOXM1c target genes, many of which function in
proliferation and tumorigenesis. Accordingly, dominant-negative FOXM1c
proteins reduced cell growth approximately threefold, demonstrating a pro-
liferation-stimulating function for wild-type FOXM1c.
Abbreviations
BRE, TFIIB recognition element; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DPE, downstream promoter element;
EDA, essential domain for activation; EMSA, electrophoretic mobility shift assay; FKH, forkhead domain; GST, glutathione S-transferase;
GTF, general transcription factor; Inr, initiator; NE, neutrophile elastase; NLS, nuclear localization signal; NRD, negative regulatory domain;
OHT, 4-hydroxy-tamoxifen; PIC, preinitiation complex; RB, retinoblastoma protein; SV40, simian virus 40; TAD, transactivation domain; TAF,
TBP-associated factor; TBP, TATA-binding protein; TFIIB, transcription factor IIB; TK, thymidine kinase; TPA, 12-O-tetradecanoylphorbol-13-
acetate; TRD, transrepression domain.
FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4645
transcription factor the splice variant FOXM1c (MPP2)
binds to FOXM1-specific DNA sequences via its fork-
head domain and transactivates via its strong acidic
transactivation domain (TAD) [29–31]. This strong
TAD can be kept almost inactive by two different
inhibitory domains. The N-terminus functions as a
specific negative regulatory domain (NRD), named
NRD-N, which completely inhibits the TAD by directly
binding to it. The central domain functions as a retino-
blastoma protein (RB)-independent transrepression
domain (TRD) [29–31] and as RB-recruiting NRD-C
[31].

s, general trancription factors (GTFs),
transcription factors, coactivators and general cofac-
tors [38,49,50]. TBP binds to the minor groove of the
TATA box, thereby bending the DNA 80° towards the
major groove, unwinding the DNA by 120° and kink-
ing the TATA box at both ends by intercalation of
two phenylalanine residues. TFIIA interacts with the
N-terminal TBP stirrup, which is orientated towards
the 3¢-end of the TATA box, and with TBP helices H1
and H2. TFIIB interacts with the C-terminal TBP stir-
rup, which is orientated towards the 5¢-end of the
TATA box, and with TBP helix H1¢ [38,39,51].
The PIC can be assembled in a stepwise fashion in
reconstituted in vitro systems [38,39]. In vivo, PIC
assembly may vary among core promoters between
two extremes: (a) the stepwise assembly of individual
GTFs, and (b) recruitment of the complete holo-
enzyme in one step [45]. However, PIC assembly will
always require at least two separate steps, namely
TFIID ⁄ TFIIA binding and TFIIB ⁄ Pol II binding [46].
Here, we describe a new transactivation mechanism
by which FOXM1c transactivates the c- myc promoter
via its P1 and P2 TATA boxes. It does so by binding
to the TATA box and directly to TBP, TFIIB and
TFIIA. The P1 TATA box TATAATGC requires its
sequence context to be FOXM1c responsive. In con-
trast, the P2 TATA box TATAAAAG alone is
sufficient to confer FOXM1c responsiveness on any
minimal promoter so that each promoter with this
TATA box is postulated to be transactivated by

TATA box and a GC-box-type Sp1-binding site.
The Sp1-binding sites )44 (known; position )44
relative to the P1 transcription start site) and )66
(potential; position )66 relative to the P2 transcription
start site), as well as overlapping binding sites for
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4646 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
other transcription factors were not FOXM1c respon-
sive (Fig. 1D). Minimal promoters include only the
TATA box and the transcription start (+1). These
minimal c-myc P1 and P2 promoters were both
strongly transactivated by FOXM1c(189–762) (Fig. 1C,
D). By contrast, the minimal promoters of human
D
P1 P2
-44
-262
P1
+49
P2
P2
mintk
-66
-66
GCTT
GGCGGGAAA
GCGGGAAA E2F
gGGAA ETS-Core
TTGGCGGGAAA STAT3
GGAAA NFATc1-Consensus

pmyc(-262/+49)luc
TA b y
FOXM1c
(189-762)
+
-
-
+
+
+
pmyc(-2486/-259)
mintkluc
C
pTATA-WAF-luc
pTATA-jun-luc
pTATA-P2-luc
pTATA-P1-luc
pmintkluc
y
t
ivitcaesareficulev
i
taler
0
10
20
30
40
0213456
μg pFOXM1c(189-762)

-44
3x
ATCT
CCGCCCACC
Fig. 1. FOXM1c transactivates the minimal P1 and P2 promoters of c-myc. (A, B) RK13 cells were transiently transfected with expression
plasmids for the FOXM1c proteins or as control (c) with the empty vector and with the indicated reporter constructs. The relative luciferase
activity of each reporter construct in the control (c) was set as 1. (C) RK13 cells were transiently transfected with the indicated amounts of
pFOXM1c(189–762) and with the indicated reporter constructs. The relative luciferase activity of each reporter construct in the absence of
pFOXM1c(189–762) was set as 1. (D) c-myc sequences are shown as black lines, TATA boxes as black boxes, transcription start sites (+1)
as arrows, Sp1-binding sites are shown as dark gray boxes and sequences of the thymidine kinase (TK) promoter of herpes simplex virus
(HSV) as a light gray box. Numbers give the nucleotides of c-myc relative to the transcription start (+1) of P2. p()44)mintkluc and
p()66)mintkluc contain three adjacent copies of the indicated nucleotide sequences. Sp1-binding sites are marked bold and underlined. Bind-
ing sites for other transcription factors are indicated below. It is indicated whether the reporter constructs are transactivated by
FOXM1c(189–762) (¼ +) or not (¼ –). TA, transactivation; P0, P1, P2, c-myc promoters; mintk, minimal TK promoter of HSV.
I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes
FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4647
c-jun, waf1(p21) or herpes simplex virus (HSV) thymi-
dine kinase (TK) were not transactivated by
FOXM1c(189–762) (Fig. 1C).
The P1 and P2 TATA boxes are the
FOXM1c-responsive elements
The existence of FOXM1c-responsive and -nonrespon-
sive minimal promoters offered the possibility of con-
structing hybrid minimal promoters (Fig. 2C) to map
the responsive element exactly. Hybrids exchanging the
TATA box half and the transcription start (+1) half
between c-myc P1 or c-myc P2 and c-jun promoters
showed that the TATA box halves of the P1 and P2
promoters both transfer FOXM1c responsiveness
(Fig. 2A). Hybrids exchanging only the TATA boxes

levels of which have been compared previously [30]
were analyzed for transactivation of c-myc promoter
constructs (Fig. 1D). Two mutants lacking either part
of the TAD (amino acids 721–762) or part of the
forkhead domain (amino acids 235–332), and thereby
the complete recognition helix 3 (amino acids 277–
290) [53], repressed or did not transactivate the P1
and P2 promoters (Fig. 3A,B). Therefore, both the
intact DNA-binding domain (DBD) and the intact
TAD are essential for transactivation of the P1 and
P2 promoters (Fig. 3E,F). Wild-type FOXM1c trans-
activated the P1 and P2 promoters considerably less
than FOXM1c(189–762) (Fig. 3A). The N-terminus
(amino acids 1–232) in trans repressed transactivation
of the P1 and P2 promoters by FOXM1c(189–762)
(Fig. 3D), which can be explained by the direct interac-
tion of the N-terminus (amino acids 1–194) with the
TAD (amino acids 721–762) [30]. Therefore, the N-ter-
minus as NRD represses transactivation of the P1 and
P2 promoters by directly binding to the TAD. In sum-
mary, the forkhead domain (i.e. the DBD) TAD and
N-terminus, have the same functions for transactiva-
tion of the c-myc promoter via its TATA boxes and for
transactivation as a conventional transcription factor
(Fig. 3E,F) [30].
FOXM1c(189–348; 573–762)NLS did not transacti-
vate the P1 and P2 promoters (Fig. 3C). In contrast,
FOXM1c(189–425; 568–762) transactivated the P1
and P2 promoters as strongly as FOXM1c(189–762)
if the lower expression level of the former [30] was

conventional transcription factor; and (b) if it does
not transactivate, FOXM1c functions via the TATA
box.
FOXM1c transactivates other genes involved in
cell proliferation that possess the c-myc P2 TATA
box TATAAAAG
The c-myc P2 TATA box is sufficient to transfer very
strong transactivation by FOXM1c(189–762) to a non-
responsive minimal promoter (Fig. 2). Consequently, it
was postulated that each promoter with this TATA
Fig. 4. FOXM1c transactivates other proliferation-associated genes with the c-myc P2 TATA box TATAAAAG. (A, B) RK13 cells were transiently
transfected with expression plasmids for the FOXM1c proteins or as control (c) with the empty vector and with the indicated reporter con-
structs. The relative luciferase activity of each reporter construct in the control (c) was set as 1. phsp70luc contains the hsp70 promoter
sequence from )2400 to +150. phsp70-TATA-luc contains the hsp70 promoter sequence from )32 to +150, i.e. a ‘minimal’ hsp70 promoter. (C)
Summary of the flanking nucleotides of the TATA box TATAAAAG (bold and underlined) in the six promoters that are activated (¼ +) by
FOXM1c. The transcription start site (+1) is bold and underlined. Symbols below the sequences explain the composition of hybrid promoters.
Fig. 3. FOXM1c domains required for c-myc promoter transactivation. (A–C) RK13 cells were transiently transfected with expression plas-
mids for the indicated FOXM1c proteins or as control (c) with the empty vector and with the indicated reporter constructs. The relative lucif-
erase activity of each reporter construct in the control (c) was set as 1. (D) RK13 cells were transiently transfected with the expression
plasmid for FOXM1c(189–762) or as control (c) with the empty vector and with the indicated reporter constructs. The indicated amounts of
pFOXM1c(1–232) were cotransfected. (E) Functions of FOXM1c domains for transactivation of the c-myc promoter via the P1 and P2 TATA
boxes and for transactivation of p(MBS)
3
-mintk-luc as a conventional transcription factor [29–31] and whether their functions in these two dif-
ferent transactivation mechanisms are equivalent or opposite. TA, transactivation; IA, interaction; P1, P2, P1- or P2-promoter of c-myc. (E, F)
TAD, transactivation domain; DBD, DNA-binding domain; TRD, transrepression domain; EDA, essential domain for activation; NRD, negative
regulatory domain. (F) FOXM1c(189–348; 573–762)NLS possesses the nuclear localization signal (NLS) of SV40 large T between amino acids
348 and 573. FKH, forkhead domain. p(MBS)
3
-mintk-luc is transactivated very strongly (+ + + + +), strongly (+ + +) or weakly (+) or

we analyzed whether FOXM1c binds to their TATA
boxes (Fig. 8) and whether it interacts with components
of the basal transcription complex (Figs 5 and 6).
In pull-down experiments (Fig. 5, Fig. S2), FOXM1c
bound to TBP, TFIIB, TFIIAa ⁄ b, TFIIAc and
TAF
II
250 (TAF1) [52], but not to TFIIEa. These inter-
actions are direct for TBP, TFIIB and TFIIAa ⁄ b
because they could be verified using in vitro-translated
proteins (Fig. 5). The respective interaction domains of
FOXM1c were each mapped to its central domain (see
below; Fig. 5, Fig. S2). Therefore, the interactions of
TAF
II
250 and ⁄ or TFIIAc with FOXM1c may be indi-
rect via TBP or TFIIAa ⁄ b, respectively. The inter-
actions of FOXM1c with TBP, TFIIAa ⁄ b, TFIIAc and
TAF
II
250 are also found in vivo because these proteins
could be coimmunoprecipitated with FOXM1c (Fig. 6).
TBP bound strongly to FOXM1c ( 28% of the
input TBP was pulled down) (Fig. 5B). Deletion
mutants of TBP showed that FOXM1c binds predom-
inantly to the C-terminal half of the conserved TBP
saddle (Fig. 5B,C), which is orientated towards the
5¢-end of the TATA box [38,49,50].
More detailed mapping (Fig. 5, Fig. S2) showed that
TBP and TFIIB both bound to amino acids 380–425 of

same very high affinity as to the identical TATA box
of the adenovirus 2 major late promoter (AdML)
(Fig. 7A), which is bound very strongly by TBP [50].
Its binding affinity for the c-myc P1 TATA box (P1)
was lower, although still high (Fig. 7A). Its binding
affinity for the FOXM1c-responsive TATA boxes of
c-myc P1 and c-myc P2 was higher than for the non-
responsive TATA boxes of c-jun (jun), waf1(p21)
(WAF) and HSV TK (mintk) (Fig. 7B,C).
GST–FOXM1c(233–334), which comprised the
forkhead domain (amino acids 235–332), and GST–
FOXM1c(195–596) bound to the c-myc P1 and
c-myc P2 TATA boxes (Fig. 8C,D). These protein–DNA
complexes were supershifted with an antibody [a-GST,
a-FOXM1c(1B1)] that recognized the two GST–
FOXM1c fusion proteins, but not with a control anti-
body [a-FOXM1c(7E4)] (Fig. 8C,D; data not shown).
These protein–DNA complexes were competed by an
excess of unlabeled c-myc P1 TATA box or c-myc P2
TATA box, respectively, but not by an excess of
unlabeled control oligonucleotides (Fig. 8A,B,D). Thus
FOXM1c binds in a sequence-specific manner and with
high affinity to the c-myc P1 TATA box and the
c-myc P2 TATA box, and the forkhead domain
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4652 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
Fig. 5. Direct binding of FOXM1c to TBP, TFIIA and TFIIB. (A, B) Pull-down assays were performed in the presence of ethidium bromide
[87] with purified GST or the indicated GST–fusion proteins and the indicated in vitro-translated proteins. Bound in vitro-translated proteins
were detected following SDS ⁄ PAGE by autoradiography. The input control represents 1 ⁄ 10 of the volume used in the pull-down assays. (B)
Amount (%) of the input bound to GST–FOXM1c(1–477). wt, wild-type. (C) (Upper)

H2B are required for its execution. Consequently,
transactivation of the four respective genes by FOXM1c
should increase proliferation. By contrast, repression of
these genes by dominant-negative FOXM1c should
reduce proliferation. FOXM1c(189–743)–Engr and
FOXM1c(189–566)–Engr were constructed by replacing
the TAD (amino acids 721–762) or its C-terminal half
with the repressor domain of Drosophila Engrailed
(Figs 9A and S3C). These two dominant-negative forms
of FOXM1c repressed p(MBS)
3
-mintk-luc, the c-myc
P1 promoter and the c-myc P2 promoter (Fig.
S3A,B; data not shown). Thus they functioned as
repressors for all FOXM1c target genes regardless whe-
ther activation is via TATA box binding or binding to
the conventional target sequences.
In colony-formation assays, both FOXM1c(189–
743)–Engr and FOXM1c(189–566)–Engr reduced the
HA-TBP
FOXM1c
(189-762)
FOXM1c
(189-762)
WB: α-HA
WB: α-FOXM1c
HA-TBP
FOXM1c
(189-762)
++

++
IP: α-FOXM1c
WB: α-myc
C
HA-
TAF
II
250
WB: α-HA
Co-IP
input
C
++
HA-TAF
II
250
++++
IP:
α-FOXM1c
++
IP: α-C
+
B
Fig. 6. In vivo binding of FOXM1c to TBP, TAF
II
250 and TFIIA. Co-
immunoprecipitations (Co-IP) were performed with total cell lysates
of COS-7 cells transiently transfected with expression plasmids for
the indicated proteins. The antibodies used in the coimmunoprecipi-
tations (IP) and the primary antibodies used in the (following) west-

CAGA T AAGTG TTGAGCTCGGG-3'
WAF 5'-G
GGGCGGTTG T A T A TCAG GGCCGCGCTGAG-3'
mintk 5'-GATCCTTCG
CA T A TT AAGG TGACGCGTGTG-3'
-66 5'-TCAGA
GGCTTGGCGGGAAA AAGAACG-3'
SV40 5'-GGAACT
GGGCGGAGTTAGGGG-3'
CMD 5'-TCAGAC
CACGTGGTCGGG-3'
HFH-11 5’-TCGACGAAAAAA
ACAAA T AACAACGTACTCGA-3’
D
α A H -
α A H -
α P B T -
α P B T -
TBP+TFIIA
P1 P2
c
P1
TBP+
TFIIA
TBP
T
F
A
D M C
mintk WAF P1 jun

TBP
T
TBP+
TFIIA
T >> c > a ~ g
A >> t
T >> a ~ c
A >> t
T >> a
A >> g > c ~ t
A ~ T > g > c
G ~ A > c ~ t
T >> c > g ~ a
A >> T
T >> c
A >> t
A > t
A >> g
A > t >> g
G ~ A >> c ~ t
T
A
T
A
A / T
A
A / T
A / G / C / T
Patikoglou et al. (1999) Bucher (1990) general
TBP/TFIIA

Fig. 8. FOXM1c binds to the P1 and P2 TATA boxes. (A–D) EMSAs were performed with radioactive labeled oligonucleotides P1 or P2 and
with purified GST–FOXM1c(233–334), GST–FOXM1c(195–596) or, as a control, GST. For supershifts (C, D) the antibodies a-FOXM1c(1B1),
a-FOXM1c(7E4) and a-GST were used. 7E4 recognizes an epitope within amino acids 1–188 of FOXM1c, and 1B1 recognizes an epitope
within amino acids 297–334 of FOXM1c [30]. For competitions (A, B, D), unlabeled oligonucleotides were used in excess. (A) P1 and CMD,
10-, 20- or 50-fold; )66 and P2, 5-, 10- or 20-fold; SV40, 10- or 20-fold. (B) Left gel: P1, P2 and HFH-11, two- or fivefold; CMD, fivefold; right
gel: P1, P2, jun, WAF, mintk, CMD and HFH-11, fivefold. (D) P1 and P2, 20-fold. S, supershift; F, free probe; T, gel slot; NS, nonspecific
complex. (E) ChIP assays in exponentially growing human HL-60 cells were conducted with the indicated antibodies and precipitates were
analyzed using primers specific for the c-myc P1 ⁄ P2 TATA boxes region. Primers specific for the NE promoter (TATA box region) were used
as control.
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4656 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
Human promyelocytic HL-60 leukemia cells differ-
entiate toward macrophages in response to 12-O-tetra-
decanoylphorbol-13-acetate (TPA). Like c-Myc [54],
FOXM1c was well expressed in exponentially growing
(0 h), undifferentiated HL-60 cells, but was almost
not expressed in TPA-treated (29 h) differentiating or
A
B
+ OHT
5 μg
etalp rep sein
o
loc fo rebmu
n
0
50
100
150
200

-9
81
(c1M
X
O
F
)
267-44
7
;78
5
-9
8
1
(
c
1MX
OF
r
g
nE-)66
5
-9
8
1(c
1MX
OF
125
100
50

-
1,0991
FOXM1c(189-566)-Engr
-
2,8635,5
FOXM1c(189-587; 744-762)
-
1,0694
FOXM1c(189-762)
-
1,0694
-
1100control
+
1100control
Gal-Engr
-
1,1686
FOXM1c(189-743)-Engr
-
2,9234,8
762189
762189
744587
189
743
Engr-RPD
GAL
Engr-RPD
Engr-RPD

entiation program of their host cells and drive
these differentiated cells from G
0
⁄ G
1
- into S-phase,
resulting in the transformation of the infected cells.
Oncoprotein E7 of the transforming human papilloma-
virus 16 (HPV16) binds directly to FOXM1c [28].
Figure 10A shows that the transactivation of the c-myc
promoter by FOXM1c and FOXM1c(189–762) was
significantly enhanced by HPV16 E7. HPV16 E7
increased the transactivation of both the c-myc P1 and
P2 promoters by these two FOXM1c proteins
(Fig. 10B,C). This activation of c-myc by HPV16 E7
contributes to transformation by HPV16 because
c-Myc induces S-phase entry and inhibits differenti-
ation [1–7].
Discussion
FOXM1c transactivates the human c-myc promoter
via both its P1 TATA box TATAATGC and its
P2 TATA box TATAAAAG (Figs 1,2). Thus
FOXM1c can transactivate via two different mecha-
nisms: (a) as a conventional transcription factor by
binding to a conventional FOXM1c-binding site [29–
31]; and (b) using a new mechanism by binding to
the TATA boxes of the c-myc P1 and P2 promoters.
The c-myc P2 TATA box alone is sufficient as the
FOXM1c-responsive element, so that its insertion into
a non-FOXM1c-responsive minimal promoter resulted

4
6
8
7
5
1
3
A - C:
K
HPV16 E7
C
y t i v i t c a e s a r e f i c u l e v i t a l e r
pmyc(-95/+49)luc
c FOXM1c
1,8 2,5
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
1 1
y t i
v
i t c a e s a
r e f

1,1 1,6
y t i
v
i t
c a e
s a
r e f
i
c u
l e v i t a l e r
pmycluc
c FOXM1c
(189-762)
11
0
10
20
30
40
50
60
70
1,3 2,2
Fig. 10. Transactivation of c-myc by
FOXM1c is enhanced by HPV16 E7. (A–C)
RK13 cells were transiently transfected with
expression plasmids for the indicated
FOXM1c proteins or as control (c) with the
empty vector and with the indicated repor-
ter constructs. Either the expression plas-

sing functions: (a) for transactivation via TATA boxes
it is essentially required as EDA (Fig. 3); (b) for trans-
activation via conventional FOXM1c-binding sites it
plays a dual inhibitory role as RB-independent TRD
[30] and RB-recruiting NRD-C [31]. These opposing
functions imply two different conformations of the cen-
tral domain depending on the DNA sequence bound,
such that it either makes different protein–protein inter-
actions or the same protein–protein interactions have
different effects. Such allosteric effects of DNA-binding
sites on the conformation and function of transcription
factors are well known [55,56]. Direct interaction of the
central domain with itself (data not shown) may be
involved in these conformational changes.
The characteristics of this new mechanism by which
FOXM1c transactivates via TATA boxes distinguish
FOXM1c from all known groups of transcriptional
regulators, such that it cannot be classified as a con-
ventional transcription factor, GTF, TAF
II
, coactiva-
tor or general positive cofactor. Our results confirm
the role of core promoters, and in particular the
TATA box, as active participants in gene regulation,
which make important contributions to specificity and
variability in combinatorial gene regulation [32–34].
Because the forkhead domain proteins bind DNA as
monomers in the major groove [53] FOXM1c should
also bind to the c-myc P1 and P2 TATA boxes in the
major groove, where all four base pairs are distinguish-

major groove.
Effects that depend specifically on the TATA box
TATAAAAG have also been described for other tran-
scriptional regulators and other genes demonstrating
the special role of this TATA box in gene regulation
[35,43,63–68]. The existence of a protein with the prop-
erties of FOXM1c is suggested by the results of Lee
et al. [69] who showed that modifications of only the
major groove, but not the minor groove, of the adeno-
virus major late TATA box TATAAAAG decreased
markedly (six- to eightfold) the levels of both basal
and activator-mediated transcription in nonfractionat-
ed nuclear extracts, whereas they had no effect in a
cell-free system reconstituted with purified factors.
FOXM1 promotes G
1
⁄ S and G
2
⁄ M transition [11–
21,23–27]. Consistent with this, dominant-negative
FOXM1c proteins reduced cell growth approximately
threefold (Fig. 9A–C) indicating that wild-type
FOXM1c should stimulate cell proliferation. Accord-
ingly, we identified c-myc,c-fos, hsp70 and histone
H2B ⁄ a as new FOXM1c target genes (Figs 1 and 4),
which either stimulate proliferation or are required for
its execution and all play a role in G
1
⁄ S transition.
Transcription factors c-Myc [3–7] and c-Fos (hetero-

them indirectly (Fig. S3D). Thus activation of the
c-myc promoter may represent a major part of
FOXM1’s function, in particular for its role in stimu-
lating G
1
⁄ S transition. Nevertheless, the c-myc promo-
ter is regulated by not only FOXM1c, but also a large
variety of other transcription factors [75–80].
Among the postulated FOXM1c target genes with
the TATA box TATAAAAG (Fig. S1) are transcrip-
tion factors, cell-cycle regulators, proto-oncogenes,
genes involved in proliferation and tumorigenesis,
apoptosis-associated genes, subunits of the translation
and the basal Pol III transcription machinery, cyto-
skeletal and extracellular matrix proteins, factors of
the immune and endocrine systems, components of
signaling pathways and of the ubiquitin–proteasome
pathway, and genes of tumor viruses. Activation of
cell-cycle regulators, proto-oncogenes and genes
involved in proliferation and tumorigenesis [e.g. c-Myc,
c-Fos, ATF2, STAT5, DP-2, Evi3, ID1, Skp1, Vav1,
aurora kinase C, 70 kDa heatshock proteins, DnaJ,
SnoN, histones, ribosomal proteins, a translation elon-
gation factor, RNA helicases, a topoisomerase, sub-
units of the basal Pol III transcription complex
(POLR3C, TFIIIA ), matrix metalloproteinases, growth
factors (KIT ligand, REG1, amphiregulin, resistin-like
a), integrin av, carbonic anhydrase, CXCR4 and
CYR61] matches perfectly the proliferation-stimulating
role of FOXM1 and its assumed implication in tumori-

FOXM1 as a typical proliferation gene.
Experimental procedures
Plasmids and antibodies
pmintkluc [81], p(MBS)
3
-mintk-luc, expression plasmids
for FOXM1c, GST–FOXM1c(233–334), HPV16 E7
[28], FOXM1c(189–762), FOXM1c(189–587; 744–762),
FOXM1c(1–347; 574–762) [82] and GST–TBP [83], as well
as the plasmids pBS-FOXM1c(189–762) (from J. M.
Lu
¨
scher-Firzlaff and B. Lu
¨
scher, Abteilung Biochemie
und Molekularbiologie, Institut fu
¨
r Biochemie, Universita
¨
ts-
linikum der RWTH, Aachen, Germany), pFOXM1c(189–
263; 297–762), pFOXM1c(189–347; 574–762), pFOXM1c
(189–348; 573–762)NLS, pFOXM1c(189–425; 568–762),
pFOXM1c(1–232), pGST–FOXM1c(195–596), pGST–
FOXM1c(359–762), pGST–FOXM1c(195–477), pGST–
FOXM1c(1–477) and pGST–FOXM1c(1–379) have been
described previously [30]. pmycluc (from J. M. Lu
¨
scher-
Firzlaff and B. Lu

pF711 as a EcoRI (blunt) ⁄ NaeI fragment into SmaI-opened
pXP-2. pH2B ⁄ aluc (from W. Albig, Institut fu
¨
r Biochemie
und Molekulare Zellbiologie, Abteilung Molekularbiologie,
Universita
¨
tGo
¨
ttingen, Germany; original name pRTL1)
contains the cDNA for the luciferase from Photinus pyralis
under the control of the human histone h2b ⁄ a promoter.
We also used pNEluc (from A. Friedman, Division of
Pediatric Oncology, The Johns Hopkins Oncology Center,
Baltimore, MD, USA), phsp70luc (original name pHB-
Luc), phsp70-TATA-luc (original name pHS-TATA-Luc)
(both from H. Ariga, Hokkaido University, Sapporo,
Japan), pGal0 (from C. Dang, The Johns Hopkins Univer-
sity School of Medicine, Baltimore, MD, USA), pGEX-
ERT (from K. Weston, CRUK Centre for Cell and Mole-
cular Biology, Institute of Cancer Research, London, UK),
pCMVneoBam (from B. Vogelstein, Howard Hughes Medi-
cal Institute and The Sidney Kimmel Comprehensive Can-
cer Center, The Johns Hopkins Medical Institutions,
Baltimore, MD, USA), pCMVHAXhTAFII250 (from
R. Tjian, Department of Molecular and Cell Biology and
Howard Hughes Medical Institute, University of California,
Berkeley, CA, USA), pSG5-3’HA-hTBP, pSG5-myc-
hTFIIAa ⁄ b, pSG5-HA-hTFIIAc, pSG5-hTFIIB (all from
H. G. Stunnenberg, Department of Molecular Biology,

¨
scher)
and pXHmintkluc [28] were cloned by insertion of the
polylinker of pXP-2 as a BamHI ⁄ BglII fragment into
BamHI-cut pmintkluc in both possible directions. To clone
pmyc()2486 ⁄ )92)mintkluc part of the c-myc promoter
insert of pmycluc was transferred as a HindIII ⁄ XhoI frag-
ment into HindIII ⁄ SalI-opened pmintkluc. pmyc()262 ⁄
)92)mintkluc was made from pmyc()2486 ⁄ )92)mintkluc by
SmaI ⁄ HindIII digestion, Klenow fill-in reaction and religa-
tion. pmyc()262 ⁄ )9 2)luc was made from p myc()262 ⁄ )92)mi-
ntkluc by BamHI ⁄ BglII digestion, Klenow fill-in reaction
and religation. pmyc()224 ⁄ )92)mintkluc was made from
pmyc()262 ⁄ )92)mintkluc by BamHI (partial)
⁄ BstYI diges-
tion and religation. pmyc()224 ⁄ )136)luc was made from
pmyc()224 ⁄ )92)mintkluc by NotI ⁄ BglII digestion, Klenow
fill-in reaction and religation. pmyc()224 ⁄ )92)luc was made
from pmyc()224 ⁄ )92)mintkluc by BamHI ⁄ BglII digestion,
Klenow fill-in reaction and religation. pTATA-P1-luc was
created by ligating into XhoI ⁄ HindIII-digested pXP-2 the
annealed product of the oligonucleotides indicated in
Table S1. pTATA-P2-luc, pTATA-jun-luc, pTATA-WAF-
luc, pP1-jun-luc, pP2-jun-luc, pjun-P1-luc, pjun-P2-luc,
pP1(junTATA)luc, pP2(junTATA)luc, pP1(WAFTATA)
luc, pP2(WAFTATA)luc, pjun(P1TATA)luc, pjun(P2TA-
TA)luc, pWAF(P1TATA)luc and pWAF(P2TATA)luc
were created by ligating into XhoI ⁄ HindIII-opened pXP-1
[84] the annealed product of the oligonucleotides indica-
ted in Table S1. pXP-1 and pXP-2 are identical vectors

EcoRI-digested pBluescript KS
+
(Stratagene, La Jolla, CA),
transfer of the EcoRI fragment of pFOXM1c(189–762) into
this EcoRI-cut new construct; (b) insertion of the BamHI ⁄
I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes
FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4661
NotI fragment of pGEX-ERT into BamHI ⁄ NotI-digested
pBluescript KS
+
, transfer of the EcoRI ⁄ KpnI fragment of
pFOXM1c(189–762) into this EcoRI ⁄ KpnI-cut new con-
struct, (i) EcoNI ⁄ BspMI digestion [for pFOXM1c(189–
743)–Engr] of the resulting construct followed by Klenow
fill-in reaction and religation, (ii) ScaI ⁄ EcoRI digestion [for
pFOXM1c(189–566)–Engr] of the resulting construct fol-
lowed by Klenow fill-in reaction and religation, (i) + (ii)
transfer of the inserts of these new constructs as NheI ⁄ NotI
fragments into the NheI ⁄ NotI-opened construct which
was cloned in (a); (c) transfer of the inserts of the two
constructs which were cloned in (b) as HindIII ⁄ BamHI
fragments into HindIII ⁄ BglII-digested pEQ176P2; (d) inser-
tion of the HindIII fragment of the construct with deletion
(i) which was cloned in (c) into HindIII-opened pEQ176P2
resulting in pFOXM1c(189–743)–Engr, which possesses
the cDNA for the repressor domain of Drosophila Eng-
railed (amino acids 3–289) 3’ of the FOXM1c cDNA; (e)
insertion of the NheI ⁄ NotI fragment of the construct
with deletion (ii), which was cloned in (c) into NheI ⁄ NotI-
opened pFOXM1c(189–743)–Engr, resulting in pFOXM1c

were seeded into 6-cm dishes at a density of 1.5 · 10
5
cells
per plate. Five or 10 lg of the indicated expression plas-
mids or the empty control vector were transfected into the
cells together with a neomycin-resistance plasmid (pCMV-
neoBam). For each individual experiment, cell cultures were
split into triplicate dishes 1 : 20 after 24 h. After 48 h,
0.5 lgÆmL
)1
G418 (neomycin) was added. After 14–21 days,
the selected colonies were stained with Giemsa (Riedel-de
Haen, Seelze, Germany) and counted. 4-Hydroxy-tamoxifen
(100 nm) was added 24 h after transfection, where appro-
priate.
HL-60 cells were grown in RPMI-1640 medium with
10% fetal bovine serum and 1% penicillin ⁄ streptomycin.
For differentiation, logarithmically growing cultures
(10
5
cellsÆmL
)1
) of HL-60 cells, which were grown in flasks
coated with 2% agarose M (Pharmacia) to prevent adher-
ence, were tretaed with 1.6 · 10
)8
m TPA (Sigma-Aldrich,
Munich, Germany).
Electrophoretic mobility shift assays
Oligonucleotides were end-labeled with [

¨
ger and were dissolved in BC-100
buffer (20 mm Tris ⁄ HCl pH 7.2–7.3, 100 mm KCl, 20%
glycerol, 0.2 mm EDTA pH 8.0, 0.5 mm Pefabloc SC,
10 mm b-mercaptoethanol, 100 ngÆlL
)1
BSA). For super-
shift experiments an antibody, and for competition experi-
ments a 2–100-fold molar excess of cold oligonucleotide
was added to the mixture prior to oligonucleotide addi-
tion. The DNA–protein complexes with GST–FOXM1c
fusion proteins were separated by electrophoresis on a
nondenaturing 5% polyacrylamide gel in running buffer
(25 mm Tris base, 25 mm boric acid, 0.5 mm EDTA
pH 8.0) at 4 °C and 5 VÆcm
)1
. The DNA–protein com-
plexes with TBP + TFIIA were separated by electrophor-
esis on a nondenaturing 5% polyacrylamide gel in
0.5 · TBE (89 mm Tris base, 89 mm boric acid, 2 mm
EDTA pH 8.0) at 4 °C and 5 VÆcm
)1
. Radioactive bands
were visualized by autoradiography. The oligonucleotides
used are indicated in Fig. 7D.
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4662 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
GST fusion proteins, in vitro transcription
⁄
translation, GST pull-downs, coimmuno-

ously providing antibodies and H. Burckhardt for
technical assistance. We are grateful to T. Oelgeschla
¨
-
ger for the generous gift of purified proteins, plasmids,
antibodies and oligonucleotides as well as for helpful
suggestions.
References
1 Henriksson M & Lu
¨
scher B (1996) Proteins of the Myc
network: essential regulators of cell growth and differ-
entiation. Adv Cancer Res 68, 111–182.
2 Facchini LM & Penn LZ (1998) The molecular role of
myc in growth and transformation: recent discoveries
lead to new insights. FASEB J 12, 633–651.
3 Grandori C, Cowley SM, James LP & Eisenman RN
(2000) The Myc ⁄ Mad ⁄ Max network and the transcrip-
tional control of cell behaviour. Annu Rev Cell Dev Biol
16, 653–699.
4 Oster SK, Ho CSW, Soucle EL & Penn LZ (2002) The
myc oncogene: Marvelousl yComplex. Adv Cancer Res
84, 81–154.
5 Pelengaris S, Khan M & Evan G (2002) c-Myc: more
than just a matter of life and death. Nat Rev Cancer 2,
764–776.
6 Hurlin PJ & Dezfouli S (2004) Functions of Myc:Max
in the control of cell proliferation and tumorigenesis.
Int Rev Cytol 238, 183–226.
7 Adhikary S & Eilers M (2005) Transcriptional regula-

RH (2002b) Increased levels of Forkhead Box M1B
transcription factor in transgenic mouse hepatocytes
prevent age-related proliferation defects in regenerating
liver. Proc Natl Acad Sci USA 98, 11468–11473.
14 Wang X, Krupczak-Hollis K, Tan Y, Dennewitz MB,
Adami GR & Costa RH (2002a) Increased hepatic
Forkhead Box M1B (FoxM1B) levels in old-aged mice
stimulated liver regeneration through diminished p27
Kip1 protein levels and increased Cdc25B expression.
J Biol Chem 277, 44310–44316.
15 Wang X, Kiyokawa H, Dennewitz MB & Costa RH
(2002b) The Forkhead Box m1b transcription factor is
essential for hepatocyte DNA replication and mitosis
during mouse liver regeneration. Proc Natl Acad Sci
USA 99, 16881–16886.
16 Wang IC, Chen YJ, Hughes D, Petrovic V, Major ML,
Park HJ, Tan Y, Ackerson T & Costa RH (2005)
Forkhead box M1 regulates the transcriptional network
of genes essential for mitotic progression and genes
I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes
FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4663
encoding the SCF (Skp2-Cks1) ubiquitin ligase. Mol
Cell Biol 25, 10875–10894.
17 Kalinichenko VV, Gusariva GA, Tan Y, Wang IC,
Major ML, Wang X, Yoder HM & Costa RH (2003)
Ubiquitous expression of the Forkhead Box M1B
transgene accelerates proliferation of distinct pulmon-
ary cell types following lung injury. J Biol Chem 39,
37888–37894.
18 Kalinichenko VV, Major ML, Wang X, Petrovic V,

amplification and mitotic catastrophe. Cancer Res 65,
5181–5189.
24 Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac
CJ, Kalinichenko VV, Lyubimov A & Costa RH
(2006) Increased levels of the FoxM1 transcription fac-
tor accelerate development and progression of prostate
carcinomas in both TRAMP and LADY transgenic
mice. Cancer Res 66, 1712–1720.
25 Kim IM, Ackerson T, Ramakrishna S, Tretiakova M,
Wang IC, Kalin TV, Major ML, Gusarova GA, Yoder
HM, Costa RH et al. (2006) The Forkhead Box m1
transcription factor stimulates the proliferation of
tumor cells during development of lung cancer. Cancer
Res 66, 2153–2161.
26 Liu M, Dai B, Kang SH, Ban K, Huang FJ, Lang FF,
Aldape KD, Xie TX, Pelloski CE, Xie K et al. (2006)
FoxM1B is overexpressed in human glioblastomas and
critically regulates the tumorigenicity of glioma cells.
Cancer Res 66, 3593–3602.
27 Zhang H, Ackermann AM, Gusarova GA, Lowe D,
Feng X, Kopsombut UG, Costa RH & Gannon M
(2006) The Foxm1 transcription factor is required to
maintain pancreatic beta cell mass. Mol Endocrinol 20,
1853–1866.
28 Lu
¨
scher-Firzlaff JM, Westendorf JM, Zwicker J,
Burkhardt H, Henriksson M, Mu
¨
ller R, Pirollet F &

enhancer. Nature 344 , 260–262.
36 Hochheimer A & Tjian R (2003) Diversified transcrip-
tion initiation complexes expand promoter selectivity
and tissue-specific gene expression. Genes Dev 17,
1309–1320.
37 Mu
¨
ller F & Tora L (2004) The multicoloured world
of promoter recognition complexes. EMBO J 23,
2–8.
38 Burley SK & Roeder RG (1996) Biochemistry and
structural biology of transcription factor IID (TFIID).
Annu Rev Biochem 65, 769–799.
39 Roeder RG (1996) The role of general initiation factors
in transcription by RNA polymerase II. Trends Bio-
chem Sci 21, 327–335.
40 Verrijzer CP & Tjian R (1996) TAFs mediate
transcriptional activation and promoter selectivity.
Trends Biochem Sci 21, 338–342.
41 Yean D & Gralla J (1997) Transcription reinitiation: a
special role for the TATA-box. Mol Cell Biol 17, 3809–
3816.
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4664 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
42 Yean D & Gralla JD (1999) Transcription reinitiation
rate: a potential role for TATA-box stabilization of the
TFIID:TFIIA:DNA complex. Nucleic Acids Res 27,
831–838.
43 Damania B, Liberman P & Alwine JC (1998) Simian
virus 40 large T antigen stabilizes the TATA-binding

by the TATA-box-binding protein has been con-
served throughout evolution. Genes Dev 13, 3217–
3230.
51 Hampsey M (1998) Molecular genetics of the RNA
polymerase II general transcriptional machinery.
Microbiol Mol Biol Rev 62, 465–503.
52 Tora L (2002) A unified nomenclature for TATA box
binding protein (TBP)-associated factors (TAFs)
involved in RNA polymerase II transcription. Genes
Dev 16, 673–675.
53 Clark KL, Halay ED, Lai E & Burley SK (1993)
Co-crystal structure of the HNF-3 ⁄ fork head-DNA-
recognition motif resembles histone H5. Nature 364,
412–420.
54 Ayer DE & Eisenman RN (1993) A switch from Myc:
Max to Mad:Max heterocomplexes accompanies
monocyte ⁄ macrophage differentiation. Genes Dev 7,
2110–2119.
55 Latchman DS (2001) Transcription factors: bound to
activate or repress. Trend Biochem Sci 26, 211–213.
56 Lefstin JA & Yamamoto KR (1998) Allosteric effects of
DNA on transcriptional regulators. Nature 392, 885–
888.
57 Starr DB, Hoopes BC & Hawley DK (1995) DNA
bending is an important component of site-specific
recognition by the TATA binding protein.
J Mol Biol
250, 434–446.
58 Bareket-Samish A, Cohen I & Haran TE (2000) Signals
for TBP ⁄ TATA-box recognition. J Mol Biol 299, 965–

. J Biol Chem 268, 24460–24466.
66 Takeda S, North DL, Diagana T, Mayagoe Y, Lakich
MM & Whalen RG (1995) Myogenic regulatory factors
can activate TATA-containing promoter elements via
an E-box independent mechanism. J Biol Chem 270,
15664–15670.
67 Diagana TT, North DL, Jabet C, Fiszman MY, Ta-
keda S & Whalen RG (1997) The transcriptional activ-
ity of a muscle-specific promoter depends critically on
the structure of the TATA element and its binding pro-
tein. J Mol Biol 265, 480–493.
68 Johannessen M, Olsen PA, So
¨
rensen R, Johansen B,
Seternes OM & Moens U (2003) A role of the TATA-
box and the general co-activator hTAF
II
130 ⁄ 135 in
promoter-specific trans-activation by simian virus 40
small t antigen. J Gen Virol 84, 1887–1897.
69 Lee DK, Wang KC & Roeder RG (1997) Functional
significance of the TATA element major groove in
I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes
FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4665
transcription initiation by RNA polymerase II. Nucleic
Acids Res 25, 4338–4345.
70 Shaulian E & Karin M (2002) AP-1 as a regulator of
cell life and death. Nat Cell Biol 4, E131–E136.
71 Eferl R & Wagner EF (2003) AP-1: a double edged
sword in tumorigenesis. Nat Rev Cancer 3, 859–868.

81 Bousset K, Oelgeschla
¨
ger M, Henriksson M, Schreek S,
Burkhardt H, Litchfield DW, Lu
¨
scher-Firzlaff JM &
Lu
¨
scher B (1994) Regulation of transcription factors
c-Myc, Max and c-Myb by casein kinase II. Cell Mol
Biol Res 40, 501–511.
82 Lu
¨
scher-Firzlaff JM, Lilischkis R & Lu
¨
scher B (2006)
Regulation of transcription factor FOXM1c by cyclin
E ⁄ CDK2. FEBS Lett 580, 1716–1722.
83 Austen M, Lu
¨
scher B & Lu
¨
scher-Firzlaff JM (1997)
Characterization of the transcriptional regulator YY1.
J Biol Chem 272, 1709–1717.
84 Nordeen SK (1988) Luciferase reporter gene vectors for
analysis of promoters and enhancers. Biotechniques 6,
454–457.
85 Firzlaff JM, Lu
¨

acetylation at the cyclin D2 promoter. Genes Dev 15,
2042–2047.
90 Bucher P (1990) Weight matrix descriptions of four
eukaryotic RNA polymerase II promoter elements
derived from 502 unrelated promoter sequences. J Mol
Biol 212, 563–568.
91 Schmidt M, Nazarov V, Stevens L, Watson R & Wolff
L (2000) Regulation of the resident chromosomal copy
of c-myc by c-Myb is involved in myeloid leukemogen-
esis. Mol Cell Biol 20, 1970–1981.
92 Wang X, Bhattacharyya D, Dennewitz MB, Kalini-
chenko VV, Zhou Y, Lepe R & Costa RH (2003)
Rapid hepatocyte nuclear translocation of the Fork-
head Box M1B (FoxM1B) transcription factor caused a
transient increase in size of regenerating transgenic
hepatocytes. Gene Expr 11, 149–162.
93 Kim IM, Ramakrishna S, Gusarova GA, Yoder HM,
Costa RH & Kalinichenko VV (2005) The Forkhead
Box M1 transcription factor is essential for embryonic
development of pulmonary vasculature. J Biol Chem
280, 22278–22286.
94 Nasi S, Ciarapica R, Jucker R, Rosati J & Soucek L
(2001) Making decisions through Myc. FEBS Lett 490,
153–162.
95 Patel JH, Loboda AP, Showe MK, Showe LC &
McMahon SB (2004) Analysis of genomic targets
reveals complex functions of MYC. Nat Rev Cancer 4,
562–568.
96 Lee LA & Dang CV (2006) Myc target transcriptomes.
Curr Topics Microbiol Immunol 302, 145–167.

I. Wierstra and J. Alves FOXM1c activates c-myc via its two TATA boxes
FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS 4667