Tài liệu Báo cáo khoa học: FOXM1c transactivates the human c-mycpromoter directly via the two TATA boxes P1 and P2 docx

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

1
⁄ S and G
2
⁄ M transition [8–27]. The activity of
FOXM1 as a conventional transcription factor is
increased by proliferation signals and reduced by anti-
proliferative signals. Furthermore, FOXM1 is assumed
to be implicated in tumorigenesis [18,23–26,28].
We have previously shown that as a conventional
Keywords
c-myc; core promoter; FOXM1; TATA box;
TATA-binding protein
Correspondence
I. Wierstra, Wißmannstr. 17, D-30173
Hannover, Germany
Fax: +49 511 883 536
Tel. +49 511 883 536
E-mail:
(Received 29 June 2006, revised 9 August
2006, accepted 15 August 2006)
doi:10.1111/j.1742-4658.2006.05468.x
FOXM1c transactivates the c-myc promoter via the P1 and P2 TATA boxes
using a new mechanism. Whereas the P1 TATA box TATAATGC requires
its sequence context to be FOXM1c responsive, the P2 TATA box TATA-
AAAG alone is sufficient to confer FOXM1c responsiveness to any minimal
promoter. FOXM1c transactivates by binding to the TATA box as well as
directly to TATA-binding protein, transcription factor IIB and transcrip-
tion factor IIA. This new transactivation mechanism is clearly distinguished
from the function of FOXM1c as a conventional transcription factor. The
central domain of FOXM1c functions as an essential domain for activation

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].

Core promoters and basal transcription complexes
were initially thought to be interchangeable at will, but
are now viewed as active participants in gene regula-
tion. Their diversity makes essential contributions to
the specificity and variability in combinatorial gene
regulation [32–34]. Core promoter elements are the
TATA box, the initiator (Inr), the downstream promo-
ter element (DPE), motif ten element (MTE) and the
transcription factor IIB (TFIIB) recognition element
(BRE). None of these elements is obligatory and sev-
eral different combinations are operational. Enhancers
can target certain core promoter elements so that their
activating effect is limited to genes with these elements
[32–35]. Basal transcription complexes are not uniform
because of TATA-binding protein (TBP)-related fac-
tors and alternative TBP-associated factors (TAF
II
s)
[36,37]. It is believed that the basal transcription com-
plex can adopt different conformations on different
core promoters and that different core promoters can
determine different rate-limiting steps in preinitiation
complex (PIC) assembly and transcription initiation, as
well as different reinitiation rates [32–34,38–48].
TBP plays a central role in the recognition of TATA
box promoters. The C-terminal ⁄ core region of TBP
has a saddle-like structure: its concave underside binds
to DNA; the convex upper surface binds to a large
variety of TAF
II

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

FOXM1c as seen for c-fos, hsp70 and histone H2B ⁄ a.
In addition to these new FOXM1c target genes, a
database search revealed nearly 300 genes with such a
TATA box sequence, many of which also play a role
in proliferation and tumorigenesis. Accordingly, dom-
inant-negative FOXM1c proteins reduce cell growth by
approximately threefold demonstrating a proliferation-
stimulating function for wild-type FOXM1c.
Results
FOXM1c transactivates the c-myc promoter,
namely the minimal P1 and P2 promoters
Human c-myc promoter was transactivated by wild-
type FOXM1c and significantly more so by the mutant
FOXM1c(189–762) (Fig. 1A), which lacks the negat-
ive-regulatory N-terminus (see below). Therefore,
FOXM1c(189–762) was used in this study. In contrast
to c-myc, FOXM1c(189–762) did not transactivate the
promoters of human c-jun, waf1(p21), ink4a(p16),
murine neutrophile elastase (NE) or the simian virus
(SV)40 early promoter (Fig. 1B; data not shown).
To map the FOXM1c-responsive element, several
c-myc–promoter constructs were analyzed (Fig. 1D).
FOXM1c(189–762) strongly transactivated the P1
and P2 promoters, but not the P0 promoter. Because
all potential FOXM1c-binding sites (C ⁄ T-AAA-C ⁄ T)
of the c-myc promoter are positioned in the non-
FOXM1c-responsive segment )2486 ⁄ )259 (Fig. 1D;
data not shown), common elements of the P1 and
P2 promoters were analyzed for FOXM1c responsive-
ness. The P1 and P2 promoters both possess a

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

GGCTT Smad
GGAAAG METS-Consensus
cGT
3x
-95
+49
P2
-224
P1
-136
+49-2486
P1
P0
-259
mintk
P0
-2486
pTATA-P1-luc
p(-44)mintkluc
pmyc(-224/-136)luc
-
+
+
TA b y
FOXM1c
(189-762)
pTATA-P2-luc
p(-66)mintkluc
pmyc(-95/+49)luc
pmycluc

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)

B
ytivitcaesareficulevitaler
0
2,5
5
7,5
10
pwaf1
(p21)luc
pmyc
luc
pjun
luc
C
FOXM1c(189-762)
C
)267-981(c1MXOF
c1MXOF
ytivitcaesareficulevi
taler
pmycluc
0
5
10
15
20
25
30
A
mintk

-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

between P1 or P2 and the c-jun or waf1 ⁄ (p21) promot-
ers, and vice versa, showed that the c-myc P1 and P2
TATA boxes are themselves the FOXM1c-responsive
elements (Fig. 2B,C). Both are necessary for FOXM1c
responsiveness because replacing them with the TATA
box of a non-FOXM1c-responsive promoter abolished
transactivation by FOXM1c(189–762) (Fig. 2B,C). The
P2–TATA box is sufficient as the FOXM1c-responsive
element because insertion of it into a nonresponsive
minimal promoter resulted in very strong transactiva-
tion by FOXM1c(189–762) (Fig. 2B). The P1 TATA
box requires its sequence context to function as the
FOXM1c-responsive element because insertion of it
into the minimal promoters of c-jun and waf1(p21)
did not result in transactivation by FOXM1c(189–762)
(Fig. 2C). Figure 2D shows the sequence differences
between the TATA boxes used. To our knowledge,
transactivation of a promoter by a transcription factor
via its TATA box has not been described previously
and thus represents a new mechanism.
FOXM1c domains required for transactivation
of the c-myc promoter
FOXM1c transactivates by two different mechanisms:
(a) the reporter construct p(MBS)
3
-mintk-luc via its
FOXM1c-binding sites as a conventional transcription
factor [29–31]; and (b) the P1 and P2 promoters of
c-myc via their TATA boxes by a new mechanism.
Several FOXM1c mutants (Fig. 3F) the expression

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

taken into account (Fig. 3A). Thus, these two
mutants with deletions in the central domain (amino
acids 349–572) showed that amino acids 349–425 are
essential for transactivation of the P1 and P2 promot-
ers. Therefore, amino acids 349–425 are referred to as
the essential domain for activation (EDA). The cen-
tral domain has opposing functions for transactiva-
tion of the c-myc promoter via its TATA boxes,
where it functions as the EDA, and for transactiva-
tion as a conventional transcription factor, where it
Fig. 2. The FOXM1c-responsive elements are the P1 and P2 TATA boxes. (A ,B) 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. (C) TATA boxes and transcription start sites (+1) are bold and underlined. Symbols below the
nucleotide sequences explain the composition of hybrid promoters. It is indicated whether the reporter constructs are transactivated by
FOXM1c(189–762) (¼ +) or not (¼ –). TA, transactivation. (D) Differences of TATA boxes of non-FOXM1c-responsive (¼ –) promoters to the
FOXM1c-responsive (¼ +) TATA boxes c-myc-P1 and c-myc-P2. Nucleotides that deviate from the c-myc TATA box are bold. Nucleotides
that are identical to the c-myc TATA box are replaced by a dash. For c-jun and TK both possible TATA box positions are shown. c-myc-P0
and ink4a(p16) are TATA-less (¼ –) non-FOXM1c responsive promoters. TA by FOXM1c, transactivation by FOXM1c(189–762); NE, murine
neutrophile elastase; TK, thymidine kinase of HSV; SV40early, early promoter of simian virus (SV)40.
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4648 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 4649
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4650 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
functions as an inhibitory domain [29–31] (Fig. 3E,F).
Consequently, FOXM1c(189–348; 573–762)NLS can
be used to discriminate between these mechanisms:
(a) if it transactivates considerably more strongly
than FOXM1c(189–762), FOXM1c functions as a

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

repressed (–) and the c-myc-promoter is transactivated very strongly (+ + + + +), strongly (+ + +) or repressed (–) or neither transactivated
nor repressed (). Note that the indicated transactivation for FOXM1(189–425; 568–762) is corrected by expression (see text).
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 4651
box is transactivated by FOXM1c. Therefore, the pro-
moters of human c-fos, hsp70 and histone H2B ⁄ a which
all possess the c-myc P2 TATA box TATAAAAG
(Fig. 4C) were tested. As postulated, these three pro-
moters were transactivated by FOXM1c(189–762), but
not transactivated or considerably less so (Fig. 4A,B)
by FOXM1c(189–348; 573–762)NLS. This also held
true for a ‘minimal’ hsp70 promoter (Fig. 4B) showing
that FOXM1c transactivates the hsp70 promoter via its
TATA box. The parental vectors used to construct
the reporter plasmids were not FOXM1c responsive
(Figs 1B,D and 4A,B; data not shown). This transacti-
vation of the c-fos, hsp70 and histone H2B ⁄ a promoters
confirmed that each promoter with the c-myc P2 TATA
box is transactivated by FOXM1c. Comparison of the
six promoters used showed that, in the sequences flank-
ing the c-myc P2 TATA box, almost every nucleotide
was found at almost every position (Fig. 4C). Thus the
c-myc P2 TATA box TATAAAAG alone is sufficient
as the FOXM1c-responsive element. A database search
for promoters with this TATA box gave a list of almost
300 potential FOXM1c target genes (Fig. S1).
FOXM1c binds directly to components of the
basal transcription complex
To characterize this new mechanism by which
FOXM1c transactivates the c-myc P1 and P2 promoters

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

FOXM1c, i.e. to the EDA (amino acids 349–425)
(Fig. 3F), but not to amino acids 1–379 or 574–762.
TAF
II
250 interacted with amino acids 380–477 of
FOXM1c, but not with amino acids 1–379. TFIIAa ⁄ b
and TFIIAc both probably interacted with amino acids
359–477 of FOXM1c.
In summary, FOXM1c binds directly, via its essen-
tially required EDA (amino acids 349–425) (Fig. 3F),
to the components TBP, TFIIAa ⁄ b and TFIIB of the
basal transcription complex, which are positioned at or
near the TATA box, respectively. FOXM1c(189–762)
and FOXM1c(189–425; 568–762), which bound to
TBP and TFIIB, transactivated the c-myc P1 and P2
promoters, whereas FOXM1c(189–348; 573 762)NLS,
which did not bind to TBP or TFIIB, failed to transac-
tivate both promoters (Figs 3A,C,F, 5A, Fig. S2A,F,G;
data not shown). Consequently, these interactions
should be important for the new mechanism by which
FOXM1c transactivates via the c-myc P1 and P2
TATA boxes.
Binding of TBP and FOXM1c to the P1 and P2
TATA boxes
Because TBP binds to all TATA boxes the question
arose: what is the difference between the FOXM1c-
responsive TATA boxes of c-myc P1 and c-myc P2
versus the non-FOXM1c-responsive TATA boxes of
c-jun, waf1(p21) and HSV TK? The TBP ⁄ TFIIA com-
plex bound to the c-myc P2 TATA box (P2) with the

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)

RASMOL drawing of the cocrystal structure of the C-ter-
minal ⁄ core region of human TBP complexed with the TATA element of the adenovirus major late promoter [49]. TBP segments are colored
as indicated in the table. DNA is shown in gray. (Lower) Quantification of the pull-down assay in (B). Contribution (%) made by the TBP seg-
ments to total GST–FOXM1c(1–477) binding and which elements of the TBP saddle they included. H, a helix; S, b strand.
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 4653
(amino acids 235–332) is sufficient for this DNA bind-
ing. The order of binding affinities for the different
TATA boxes was similar for GST–FOXM1c(195–596)
as for the TBP ⁄ TFIIA complex (Fig. 7C; data not
shown). For comparison, the best conventional
FOXM1c-binding site HFH-11 [30] was bound by
GST–FOXM1c(195–596) with lower affinity than the
c-myc P1 and P2 TATA boxes (Fig. 8B).
To examine in vivo binding of FOXM1c to the endog-
enous c-myc promoter chromatin immunoprecipitation
(ChIP) assays were performed. Figure 8E shows that
the c-myc P1 ⁄ P2 TATA box region was enriched mark-
edly more with a FOXM1c-specific antibody than with
a control antibody (a-b-Gal), indicating that in vivo
FOXM1c binds to the c-myc promoter. As a negative
control, the NE promoter (TATA box region) was less
immunoprecipitated with the FOXM1c-specific anti-
body than with the control antibody (Fig. 8E), indicat-
ing that in vivo this promoter is not bound by FOXM1c.
Dominant-negative FOXM1c reduces cell growth
c-Myc, a key factor for cell-growth control, potently
stimulates cell proliferation, promotes apoptosis and
represses differentiation and entry into quiescence.
c-Fos also stimulates proliferation, HSP70 and histone

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)
++

HA-TBP
++
WB: α-HA
IP: α-FOXM1c
WB: α-FOXM1c
IP: α-HA
A
HA-TFIIAγWB: α-HA
FOXM1c
(189-762)
FOXM1c
(189-762)
WB: α-FOXM1c
HA-TFIIAγ
++
FOXM1c
(189-762)
++
IP: α-HA
WB: α-FOXM1c
D
myc-TFIIAαβWB: α-myc
myc-TFIIAαβ
FOXM1c
(189-762)
WB: α-FOXM1c
FOXM1c
(189-762)
++
myc-TFIIAαβ

++
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-

ern blots (WB) are indicated. The input control represents 1 ⁄ 30
of the volume used in the coimmunoprecipitations. a-FOXM1c,
a-FOXM1c(C-20). (B) The control antibody a-C was a-cytochrome c.
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4654 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
number of colonies about threefold, whereas the con-
trols Gal–Engr and Engr–ER–myc had no significant
effect (Fig. 9A–C). Therefore, this strong negative effect
on cell growth of the two former proteins depended spe-
cifically on their FOXM1c parts, which recruited them
to FOXM1c target genes. Thus, they exerted their
growth-inhibitory effect by repression of FOXM1c tar-
get genes that are normally activated by FOXM1c. Con-
sequently, FOXM1c should have a positive effect on cell
proliferation. This proliferation-stimulating function of
FOXM1c confirms previous results for FOXM1 [11–
21,23–27]. Because no increase in apoptosis was found
in FoxM1-deficient mice livers [18,20] or pancreas [27],
compared with control organs, and because RNAi of
FoxM1 did not induce apoptosis in breast cancer cell
lines [23] it is unlikely that the strong negative effect on
cell growth of the two dominant-negative forms of
FOXM1c is based on an increased rate of apoptosis.
C
P1 5'-ACCGGCCCTT T A T AA TGC GAGGGTCTG-3'
P2 5'-TCGCGCTGAG
T A T AAAAG CCGGTTTTCG-3'
AdML 5'-GTTCCTGAAGGGGGGC
T A T AAAAG GGGGTGGGGGCGCGTT-3'
jun 5'-GACTGGTAG

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

0 4 V S
L M d A
TBP+TFIIA
P1
TBP
TBP+
TFIIA
T
F
D M C
0 4 V S
L M d A
WAF P2 jun mintk
TBP+TFIIA
P2
TBP
T
F
TBP+
TFIIA
B
α A H -
α A H -
α P B T -
α P B T -
0 4 V S
TBP+TFIIA c
P2
P1 P2 AdML
F

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

= >
CATATTAA
TATTAAGG
TK
CAGATAAG
GATAAGTG
c-jun
TATATCAG
waf1(p21)
TATAATGC
c-myc
-P1
TATAAAAG
c-myc
-P2
>=
GST-FOXM1c(195-596)
≈> ≈ >
DNA binding affinity
Fig. 7. TBP binds to the P1 and P2 TATA boxes. (A, B) EMSAs were performed with radioactively labeled oligonucleotides P1 or P2 and with
purified TBP and TFIIA or as control (c) without TBP and TFIIA. For supershifts (A), the antibodies a-HA and a-TBP were used. For competi-
tions (A, B), unlabeled oligonucleotides were used in excess. (A) P2, P1 and AdML, 5-, 20- or 100-fold; SV40, 100-fold. (B) mintk, WAF, P1,
P2, and jun, 5-, 20- or 100-fold; CMD, SV40 and AdML, 100-fold, S, supershift; F, free probe; T, gel slot. (C) Binding affinities of the
TBP ⁄ TFIIA complex and GST–FOXM1c(195–596) for the different TATA boxes. For c-jun and TK both possible TATA box positions are
shown. The TATA box definitions of Patikoglou et al. [50] and Bucher [90] and the general TATA box consensus sequence are indicated. (D)
In the oligonucleotides TATA boxes (bold and underlined), E-boxes (CMD) and binding sites for Sp1 (SV40, )66, WAF), FOXM1c (HFH-11),
E2F, STAT3, ETS, NFATc1, Smad and METS ()66) (underlined) are marked. For transcription factor binding sites in the oligonucleotide )66
see p()66)mintkluc in Fig. 1D.
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 4655

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

250
c
cym-
RE
-
r
gnE
5 μg
fo rebmun evitaler
% ni e
talp r
ep seinoloc
0
c
rgnE-laG
r
gnE
-
)3
4
7-
9
81
(c
1MX
O
F
)
26
7

-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

25
75
D
FOXM1c
HL-60
0 h 29 h
FOXM1c
(189-743)-
Engr
FOXM1c
(189-762)
FOXM1c
(189-587;
744-762)
FOXM1c
(189-566)-
Engr
10 μg
C
OHTconstruct
average
fold reduction
of number of
colonies
number of
colonies
in %
+
Engr-ER-myc 0,93107
Engr-ER-myc

-
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

ER-HBD
566
189
Engr-RPD
Fig. 9. Reduction of cell growth by dominant-negative FOXM1c. (A) Summary of colony formation assays. (Right) U2OS cells were transfected
with expression plasmids for the indicated proteins or as control with the empty vector. The absence (–) or presence (+) of 4-hydroxy-tamoxi-
fen (OHT) is indicated. For each protein the average of colony-formation assays is indicated. The colony number in the control was set to
100%. The fold reduction of the number of colonies is indicated. (Left) FOXM1c segments are shown as black lines, the repressor domain of
Drosophila Engrailed (Engr-RPD) as a light gray box, the GAL4-DBD (GAL) as a white box, the modified hormone-binding domain of the murine
estrogen-receptor (ER-HBD) as a white box, the 9E10-epitope of c-Myc as a dark gray box and the remainder of c-Myb as a black box. (B, C)
U2OS cells were transfected with 5 lg (B) or 10 lg (C) of expression plasmids for the indicated proteins or as control (c) with 5 or 10 lgof
the empty vector and with a neomycin resistance plasmid (pCMVneoBam). After splitting the cells 1 : 20 on three plates they were grown in
selection medium with neomycine (G418). Where indicated the selection medium contained 100 n
M 4-hydroxy-tamoxifen (¼ +OHT) to activate
Engr–ER-myc [91]. The average for the number of colonies per plate was calculated for three identical plates (number of colonies per plate). In
the control (c) the average for the number of colonies per plate was set as 100% (relative number of colonies per plate in %). (D) Total cell
lysates of HL-60 cells standardized for equal cell number were analyzed in a western blot using a-FOXM1c(C-20) as the primary antibody.
0 h, exponentially growing HL-60 cells; 29 h, HL-60 cells treated with TPA for 29 h.
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 4657
differentiated HL-60 cells (Fig. 9D). This significant
decline in FOXM1c expression during HL-60 cell dif-
ferentiation is in accordance with the decline in expres-
sion of the splice variants HFH-11 and Trident during
differentiation of Caco-2 cells or T lymphocytes,
respectively [9,10].
E7 enhances transactivation of c-myc by FOXM1c
DNA tumor viruses exploit the DNA replication
machinery of their host cells to replicate viral DNA.
Therefore, such viruses must interfere with the differ-

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

in strong transactivation by FOXM1c (Fig. 2). There-
fore, we postulated that each promoter with the TATA
box TATAAAAG is transactivated by FOXM1c. This
was confirmed for the human promoters of c-fos,
hsp70 and histone H2B ⁄ a, which each possess this
TATA box (Fig. 4). A database search revealed almost
A
B
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
c FOXM1c
pmycluc
1,3 2,8
12,5
17,5
22,5
0
2,5
5
7,5
10
15
20
11
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(-224/-136)luc
cFOXM1c
1,1 1,8
11
0
2

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

i c u l e v i t a l e r
pmyc(-95/+49)luc
cFOXM1c
(189-762)
11
0
2,5
5
7,5
10
12,5
15
17,5
20
1,8 2,3
A B 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(-224/-136)luc
c FOXM1c
(189-762)
11
0
2
4
6
8
10
12
14
16

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-

mid for HPV16 E7 or as control (K) the
empty vector were cotransfected. The relat-
ive luciferase activity of each reporter con-
struct in the combination of control (c) and
control (K) was set to 1. Numbers above
the columns indicate the factor by which
HPV16 E7 enhances the transactivation by
FOXM1c, i.e. the relative luciferase activity
of each reporter construct in the control (K)
was always set to 1.
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4658 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
300 potential cellular FOXM1c target genes with this
TATA box (supplementary Fig. S1). By contrast, the
c-myc P1 TATA box requires its sequence context to
function as the FOXM1c-responsive element (Fig. 2)
so it is uncertain whether other promoters with this
TATA box are also transactivated by FOXM1c.
Transactivation via the TATA box requires three
essential components of FOXM1c (Figs 3,5,6,8,
supplementary Fig. S2): the forkhead domain, which
binds to the TATA box; the EDA, i.e. the central
domain, which directly binds to components of the
basal transcription complex, namely to TBP and to
TFIIB as well as probably to TFIIA; and the TAD for
transactivation. The N-terminus, which serves as a
NRD to control the high transactivation potential of
FOXM1c, the forkhead domain and the TAD have
equal functions for FOXM1c’s two transactivation
mechanisms. By contrast, the central domain has oppo-

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-

able. Therefore, FOXM1c can distinguish different
TATA boxes, in contrast to TBP, by their nucleotide
sequences and, like TBP, by indirect readout [57,58].
The c-myc P2 TATA box is bound by the
TBP ⁄ TFIIA complex with the highest affinity (Fig. 7).
How could FOXM1c cooperate with TBP at such a
good TATA box? Because the A-tract is very rigid [59]
and bent towards the minor groove [60] it is more dif-
ficult for TBP to bend the c-myc P2 TATA box TAT-
AAAAG towards the major groove than it is to bend
other more flexible TATA boxes [58]. However, once
TBP is bound to this TATA box the resulting complex
is more stable than at other TATA boxes and accord-
ingly this TATA box leads to higher reinitiation rates
[41,42,50,57,61] so that, in vivo, TATAAAAG is the
optimal TATA box. Upon DNA binding, the forkhead
domain of HNF-3c bends the DNA towards the major
groove [53] so that FOXM1c is expected to also bend
the c-myc P2 TATA box towards the major groove.
TBP binds to DNA, which is slightly pre-bent towards
the major groove, with higher affinity and more rap-
idly than to straight DNA (or DNA pre-bent towards
the minor groove) and dissociates more slowly [62].
Consequently, in the presence of FOXM1c, TBP may
bind faster and the resulting TBP ⁄ TATA complex may
be more stable. Thus, the high rigidity and the bending
towards the minor groove of the A-tract in the c-myc
P2 TATA box TATAAAAG make this TATA box a
good target for FOXM1c, which may help TBP to
bind this TATA box by pre-bending it towards the

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-

dimerized with c-Jun) [70–72] both stimulate G
1
⁄ S
transition via their target genes which are cell-cycle
regulators. The chaperone HSP70, which prevents
apoptosis, is required during protein synthesis [73,74],
and so is histone H2B to package replicated DNA into
chromatin.
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 4659
Identification of c-myc as new direct FOXM1c target
gene is confirmed by several findings: (a) c-myc expres-
sion was upregulated in Foxm1b-transgenic mice com-
pared with wild-type mice post partial hepatectomy
and post CCl
4
liver injury [11,12]; (b) many FOXM1-
regulated genes are c-Myc target or regulated genes
(Fig. S3D); (c) expression of both c-Myc [1–4,6] and
FOXM1 [8–13,17,19] correlates strictly with prolifer-
ation; and (d) the liver regeneration phenotype of
Foxm1b-transgenic mice was most similar to that of
c-myc transgenic mice [11].
Among the genes described as being regulated by
FOXM1 [11,12,21] five have the FOXM1c-responsive
TATA box TATAAAAG, namely matrix metalloprote-
inase 10, histone 1 H2Bj, histone 2 H2Be, transthyretin
and c-myc (Fig. S1). Other FOXM1-regulated genes
include many c-Myc and several c-Fos( ⁄ c-Jun) target
or regulated genes such that FOXM1c may regulate

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-

genesis. In contrast, some other genes fulfill antiprolif-
erative functions in growth arrest and differentiation
(e.g. C ⁄ EBPa, GADD45b, GADD45c, GADD34, JunB ,
HES6, DP-2, ferritin light chain, PAG, TFF3, myosta-
tin, ODC antizyme). In proliferating FOXM1-expres-
sing cells, these genes might be inaccessible to
FOXM1c due to repressive chromatin structures.
In summary, we identified c-myc,c-fos, hsp70 and
histone H2B ⁄ a as new FOXM1c target genes which
are transactivated by FOXM1c via their TATA boxes
via a new mechanism. Consistently dominant-negative
FOXM1c proteins reduced cell growth approximately
threefold, demonstrating a proliferation-stimulating
function for FOXM1c. In accordance, FOXM1c
expression declined significantly during HL-60 differen-
tiation. The c-myc P2 TATA box TATAAAAG alone
is sufficient as the FOXM1c-responsive element so that
each gene with this TATA box is postulated to be
transactivated by FOXM1c as evidenced for c-fos,
hsp70 and histone
H2B ⁄ a. Many of the almost 300
postulated FOXM1c target genes play a role in prolif-
eration and tumorigenesis. FOXM1c transactivates by
binding to the TATA box as well as directly to TBP,
TFIIB and TFIIA. The central domain of FOXM1c
distinguishes between this new transactivation mechan-
ism, where it acts as EDA, and the function as conven-
tional transcription factor, where it acts as inhibitory
domain [29–31]. Identification of c-myc as new direct
FOXM1c target gene strongly supports the role of

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

¨
scher) was cloned by insertion of the
human c-myc promoter ()2486 to +49) of pMC41P2CAT
as a HindIII ⁄ XbaI (blunt) fragment into HindIII ⁄ BglII
(blunt)-opened pXP-2 [84]. pjunluc (from M. Austen,
Develo Gen AG, Go
¨
ttingen, Germany) was cloned by inser-
tion of the human c-jun promoter of p-1600 ⁄ +170JUN-
CAT5 as a BglII fragment into BglII-opened pXP-2.
pwaf1(p21)luc (from S. Gehring, DLR Projekttra
¨
ger des
BMBF, Bonn, Germany) was cloned by insertion of the
human p21(waf1, cip1) promoter of pWWP-CAT as a Hin-
dIII fragment into HindIII-opened pXP-2. pfosluc [original
name pfL711; W. Ernst (AppliChem GmbH, Darmstadt,
FOXM1c activates c-myc via its two TATA boxes I. Wierstra and J. Alves
4660 FEBS Journal 273 (2006) 4645–4667 ª 2006 The Authors Journal compilation ª 2006 FEBS
Germany) and A. Nordheim (Interfukulta
¨
res Institut fu
¨
r
Zellbiologie, Universita
¨
tTu
¨
bingen, Germany)] was cloned
by insertion of the human c-fos promoter ()711 to +39) of

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,

NCMLS, Radboud University Nijmegen, the Netherlands),
pSG-hTFIID-B1 and the expression plasmid for GST–
TFIIB (both from F. Holstege and M. Timmers, Depart-
ment of Physiological Chemistry, University Medical Center
Utrecht, the Netherlands) as well as plasmids for in vitro
transcription ⁄ translation of TBPwt(1–339), TBP(1–267),
TBP(1–236), TBP(1–180), TFIIB, TFIIAa ⁄ b, and TFIIEa
(all from P. Carlsson, Department of Cell and Molecular
Biology, Go
¨
teborg, Sweden), TFIIEa (from T. Oelgeschla
¨
-
ger, Transcription Laboratory, Marie Curie Research Insti-
tute, Oxted, UK), TFIIAa ⁄ b, TFIIAc and TFIIB (all
from R. G. Roeder, Laboratory of Biochemistry and
Molecular Biology, The Rockefeller University, New York,
NY, USA). pGL3-Control was from Promega (Madison,
WI) and pGEX-3X from Pharmacia (Pfizer, Karlsruhe,
Germany).
pmyc()262 ⁄ +49)luc was made from pmycluc by SmaI ⁄
HindIII digestion, Klenow fill-in reaction and religation.
pmyc()95 ⁄ +49)luc was made from pmycluc by BamHI ⁄
XhoI digestion, Klenow fill-in reaction and religation. To
clone pmyc()2486 ⁄ )259)mintkluc part of the c-myc promo-
ter insert of pmycluc was transferred as a HindIII ⁄ SmaI
fragment into HindIII ⁄ SmaI-opened pHXmintkluc. pHX-
mintkluc (from J. M. Lu
¨
scher-Firzlaff and B. Lu

¨
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

except for opposing orientation of the identical polylinker
[84]. p(-44)mintkluc was created by ligating into XhoI ⁄ Hin-
dIII-digested pHXmintkluc the annealed product of the
oligonucleotides indicated in Table S1. p(-66)mintkluc was
created by ligating into XhoI ⁄ HindIII-digested pXHmintk-
luc the annealed product of the oligonucleotides indicated
in Table S1. To clone pGST–FOXM1c(359–565) the
FOXM1c insert of pGal–FOXM1c(359–565) [30] was trans-
ferred as a XhoI ⁄ EcoRI fragment into XhoI ⁄ EcoRI-cleaved
pGEX-4T-1 (Pharmacia). To clone pEngr–ER-myc the
insert of pGEX-ERT was transferred as a BamHI fragment,
which encompasses the cDNA for the repressor domain of
Drosophila Engrailed (amino acids 2–289), the cDNA for
the modified hormone-binding domain of the murine
estrogen receptor (amino acids 281–599), the cDNA for the
9E10-epitope of c-Myc (amino acids 410–419) and the
cDNA for a remainder of c-Myb (amino acids 188–199)
into BglII-opened pEQ176P2 [85]. pGal–Engr was
cloned by the following steps: transfer of the insert of
pGEX-ERT as a BamHI fragment into BamHI-opened
pGal0, transfer of part of the insert of the resulting con-
struct as a Hin dIII fragment, which encompasses the cDNA
for the DBD of GAL4 (amino acids 1–147), the cDNA
for the repressor domain of Drosophila Engrailed (amino
acids 2–289) and the cDNA for a remainder of c-Myb
(amino acids 186–199) into HindIII-cleaved pEQ176P2.
pFOXM1c(189-566)–Engr and pFOXM1c(189-743)–Engr
were cloned by the following steps: (a) insertion of the
Bam
HI ⁄ EcoRI fragment of pGEX-ERT into BamHI ⁄

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

(189–566)–Engr which possesses the cDNA for the repres-
sor domain of Drosophila Engrailed (amino acids 2–289)
and the cDNA for a remainder of c-Myb (amino acids
186–199) 3’ of the FOXM1c cDNA. The protein expres-
sion of all constructs was verified (Fig. S3C; data not
shown).
a-FOXM1c(C-20) (sc-502), a-cytochrome c (H-104) (sc-
7159) and a-GAL4(DBD) (RK5C1) (sc-510) were pur-
chased from Santa Cruz (Santa Cruz, CA), a-HA(3F10)
was from Boehringer (Ingelheim, Germany) and a-TFIIB
(T41520) was from BD Transduction Laboratories (Lexing-
ton, KY). a-GST is a rat mAb. The a-FOXM1c rat mAbs
1B1 and 7E4 were as described previously [30]. a-TBP
(from T. Oelgeschla
¨
ger) and a-myc(9E10) (from G. Evan)
were gifts.
Cell culture, transient transfections, luciferase
reporter gene assays, whole-cell lysates,
colony-formation assays and differentiation of
HL-60 cells
Cell culture, transient transfections [86], luciferase reporter
gene assays [81] and whole-cell lysates were performed as
described previously [31].
For colony-formation assays, U2OS cells were cultured
in Dulbecco’s modified Eagle’s medium, supplemented with
10% fetal calf serum and 1% penicillin ⁄ streptomycin (Gib-
co ⁄ Invitrogen, Carlsbad, CA), at 37 °Cin5%CO
2
. They

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 [

32
P]ATP[cP] and
T4-polynucleotide kinase and hybridized to generate the
duplex oligonucleotide probe. For EMSAs 0.1–3 lg GST–
FOXM1c fusion protein was incubated with  0.5 ng
labeled oligonucleotide in 1· GS buffer (20 mm Hepes
pH 7.9, 2 mm MgCl
2
,40mm KCl, 5% Ficoll, 1 mm
dithiothrietol, 0.5 mm Pefabloc SC) in the presence of
0.5 lg poly(dG–dC):poly(dG–dC) and 2.4 nmol ATP in a
total volume of 18–30 lLat30°C for 30 min. For
EMSAs with TBP + TFIIA 5 ng highly purified, bacteri-
ally expressed, human 6His-TBP and 1 lL partially puri-
fied (MonoS) human TFIIA from HeLa cell nuclear
extracts were incubated with  0.5 ng labeled oligonucleo-
tide in 1· GS buffer (20 mm Hepes ⁄ KOH pH 8.4, 12 mm
Tris ⁄ HCl pH 7.2–7.3, 60 mm KCl, 5 mm MgCl
2
, 12%
glycerol, 0.12 mm EDTA pH 8.0, 100 lgÆmL
)1
BSA,
5mm dithiothrietol, 0.3 mm Pefabloc SC, 6 mm b-merca-
ptoethanol) in the presence of 0.5 lg poly(dG–dC):
poly(dG–dC) in a total volume of 20 lLat30°C for
60 min. The highly purified, bacterially expressed, human
6His-TBP and the partially purified (MonoS) human
TFIIA from HeLa cell nuclear extracts were generous
gifts of T. Oelgeschla

¨
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-

precipitations and western blotting
Preparation of GST fusion proteins, in vitro transcrip-
tion ⁄ translation, GST pull-downs [87], coimmunoprecipita-
tions and western blotting [88] were performed as described
previously [31].
ChIP assays
ChIP assays were performed as described previously [89]
using the specific antibody a-FOXM1c(K-19) (sc-500)
(Santa Cruz) and as control an a-b-galactosidase (a-b-Gal)
antibody. For each experiment, PCRs were performed in
the linear range of the amplification determined before. The
following primers were used to amplify the human gene
fragments: c-myc,5¢-TCCTCTCTCGCTAATCTCCGC-3¢
and 5¢-CCCTCCGTTCTTTTTCCCG-3¢ ; neutrophile ela-
stase (NE), 5¢-TGAATGCGATTGTGCATCCTG-3¢ and
5¢-AGGACACAGGCGAGGAAAAGAC-3¢. Thirty-four
(c-myc) or 33 (NE) PCR cycles (94 °C, 60 °C and 72 °C,
25 s each) were carried out using Hotstar Taq Polymerase
(Qiagen, Hilden, Germany).
Acknowledgements
We thank W. Albig, H. Ariga, M. Austen, P. Carls-
son, C. Dang, W. Ernst, A. Friedman, S. Gehring,
F. Holstege, B. Lu
¨
scher, J. M. Lu
¨
scher-Firzlaff,
A. Nordheim, R. G. Roeder, H. G. Stunnenberg,
M. Timmers, R. Tjian, B. Vogelstein and K. Weston
for generously providing plasmids, G. Evan for gener-

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.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Potential FOXM1c target genes.
Fig. S2. Interaction of FOXM1c with TBP, TAF
II
250,
TFIIA and TFIIB.
Fig. S3. Dominant-negative FOXM1c reduces cell
growth.
Table S1. Oligonucleotides used to clone reporter con-
structs.
This material is available as part of the online article
from

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