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INTRODUCTION |
The transcription factors encoded by the MYC genes form
part of a complex regulatory network implicated in diverse
tumorigenesis-relevant processes such as cell-cycle control,
growth-factor dependence, response to antimitogenic signals, and
apoptosis (1). Overexpression of the MYC genes as a result
of chromosomal translocation, gene amplification, or loss of negative
transcriptional control plays a prominent role in the etiology of many
types of tumors. The evidence for a contribution of the MYC
genes to tumorigenesis and the functional consequences of
MYC overexpression have been the focus of several recent
reviews (2, 3). Support for a critical role of MYC in cancer
comes from several transgenic mouse models of MYC-induced
tumorigenesis in which MYC expression can be reversibly
switched off after tumors have developed (4-6). Switching off
MYC expression in the tumors resulted in tumor regression, suggesting that a tumor cell requires continuous MYC
expression, although secondary genetic changes can obliterate this
MYC dependence.
The MYCN gene is found amplified in several types of
childhood tumors of mostly neuroendocrine origin, including about 25% of neuroblastomas (7). Amplification results in overexpression of
MYCN and distinguishes a subset of aggressive tumors with a poor prognosis (8). Together, these observations suggest that blocking
MYCN expression may be beneficial for neuroblastoma
patients. However, the development of a therapy based on this concept
is hampered by our lack of understanding of the transcriptional
regulation of the MYCN gene in neuroblastomas (9). Several
signals that trigger neuronal differentiation of neuroblastoma cells,
including pharmacological concentrations of all-trans
retinoic acid, cause a repression of MYCN (10). This
down-regulation of MYCN expression is essential for
differentiation because ectopic expression of MYCN blocks
differentiation (11). Neither the transcription factors nor the
regulatory elements mediating the response to these signals have been
identified as yet.
The E2F transcription factors are important regulators of cell-cycle
progression, and their activity is negatively controlled by the pRb
pathway, which is deregulated in the majority of human cancers (for
review, see Refs. 12 and 13). Recent DNA microarray analyses have
identified a large number of genes that are regulated by one or more of
the E2F proteins (14-19). The products of these target genes regulate
diverse cellular processes including cell-cycle progression, apoptosis,
differentiation, and DNA repair. There are six E2F family members that
can be classified into different subgroups based on their structure,
affinity for different members of the pRb family, and putative
function. Members of one of these subgroups, which consists of E2F-1,
E2F-2, and E2F-3, associate predominantly with pRb and, when
overexpressed in serum-starved immortalized cells, are sufficient to
induce progression into S-phase. E2F-4 and E2F-5, which form a second
subgroup, bind to all pRb family proteins and are impaired in their
ability to induce S-phase in quiescent cells. Whereas members of the
first subgroup function predominantly in transcriptional activation,
members of the second subgroup have been implicated in transcriptional repression. Although neither mutations in the RB gene nor
any of the other genetic alterations known to inactivate the pRb
pathway in different types of tumors have been detected in
neuroblastomas, recent evidence suggests that E2F activity in
neuroblastomas may nevertheless be deregulated (20, 21).
A region of 200 bp immediately upstream of the multiple transcription
start sites is highly conserved in the MYCN genes of man and
mouse and controls basal promoter activity (22).
DMS1 in
vivo-footprinting of this region revealed several sites that were
either hypersensitive or protected from modification by DMS in
neuroblastoma cells expressing MYCN but were absent in cells that do not express MYCN (23). One of the protected regions corresponded to two inversely oriented and overlapping E2F binding sites, indicating that members of the E2F family of transcription factors regulate MYCN expression in neuroblastomas.
We therefore tested whether the MYCN gene is indeed
regulated by E2F proteins in neuroblastomas. We show that the
transcription factors E2F-1, E2F-2, and E2F-3 bind the MYCN
promoter in human neuroblastoma cells in vivo, and that
blocking E2F acitivity in neuroblastoma cells via overexpression of
p16INK4A reduces MYCN mRNA levels in a
neuroblastoma cell line with amplification of MYCN. In
addition, we show that TGF-
represses the MYCN gene through the E2F-binding sites, and that all-trans retinoic
acid induced down-regulation of MYCN is associated with
changes in E2F binding to the MYCN promoter.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The human neuroblastoma cell lines SH-EP,
IMR-32, LA-N-5, and Kelly were a gift of Manfred Schwab and were
cultured as described (23). HaCaT were kindly provided by Norbert
Fusenig and cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Drugs and growth factors were
added to the cell culture medium at the following concentrations:
all-trans retinoic acid, 50 µM;
TGF-
(R&D Systems), 2.5 ng/ml.
Transfections and Reporter Assay--
Cells were transfected in
Dulbecco's modified Eagle's medium with 10% fetal calf serum using
calcium phosphate precipitation in BBS buffer (50 mM
BES, pH 6.95, 280 mM NaCl, 1.5 mM
Na2HPO4). For transient reporter assays, cells in 6-cm
dishes were transfected with 10 µg of DNA (4 µg of reporter
construct, 0.5 µg of CMV enhancer/promoter-based expression vectors
for both DP1 and E2F, 2 µg of an eGFP expression vector, and 3 µg
of Bluescript). Cells were lysed in 100 mM potassium phosphate/0.2% Triton X-100, followed by three freeze/thaw cycles. Reporter assays were performed according to standard procedures. Luciferase activity was normalized to the activity of a co-transfected lacZ reporter construct for the assays in HaCaT cells, or to total protein of the lysates as determined by Bradford assay for the experiments involving overexpression of E2F proteins. In these cases,
the percentage of cells expressing eGFP was determined microscopically
before harvesting the cells to exclude differences in the relative
transfection efficiencies. No significant differences in the number of
eGFP-positive cells were observed between different samples. The
luciferase reporter constructs have been described (23). The following
CMV-based E2F expression vectors were used: pCMV-E2F-1 (24); pCMV-E2F-2
(25); pcDNA3-HA-E2F-3 (26); pcDNA3-HA-E2F-4 (27);
pcDNA3-HA-E2F-5 (28); pCMV-DP-1 (29).
Retroviral Infections--
Human IMR-32 cells were transfected
with an expression plasmid encoding the ecotropic receptor, and
G418-resistant cells were pooled for infection with ecotropic
retroviruses based on pBabehygro-p16. Recombinant retroviruses were
generated and used as described (30). Infected cells were selected with
100 µg/ml hygromycin and analyzed immediately without further
passaging. The p16 construct was obtained by inserting the p16 cDNA
derived from pXp16ink4 into pBabehygro (31).
Western Blotting--
SDS-gel electrophoresis, transfer to
polyvinylidene difluoride membranes, and Western blotting were
performed according to standard procedures using 50 µg of the
whole-cell lysates prepared for reporter assays. Cdk2 was used to
control for equal loading. The antibodies used were:
-HA, clone
16B12 (MMS-101P, BABCO);
-E2F1, C-20 (sc-193, Santa Cruz);
-E2F-2, C-20 (sc-633, Santa Cruz);
-Cdk2, M2 (sc-163, Santa Cruz).
Chromatin Immunoprecipitation Assay--
Chromatin
immunoprecipitation was performed according to published procedures
with some modifications. Cross-linking of the cells as well as
preparation and sonication of chromatin were carried out as described
by Boyd (32). Immunoprecipitation and reversal of cross-links was done
according to Takahashi (33). In brief, twelve 15-cm dishes of
subconfluent cells were treated with 1% formaldehyde for 10 min at
room temperature. Cross-linking was stopped by the addition of glycine
to a final concentration of 125 mM. Cells were washed with
cold phosphate-buffered saline, scraped into phosphate-buffered saline
containing protease inhibitors, pelleted by centrifugation, resuspended
in lysis buffer (5 mM Pipes pH 8, 85 mM KCl,
0.5% Nonidet P-40, and protease inhibitors), and incubated on ice for
20 min. Cells were then ruptured in a Dounce homogenizer, and nuclei
were pelleted, washed once in lysis buffer, and resuspended in
sonication buffer (10 mM Tris pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, and protease inhibitors). After 10 min, on ice the solution was
sonicated with a Bandelin Sonoplus carrying a MS73 tip at 30% maximum
power for six to ten 10-s pulses on ice. The chromatin solution was
cleared for 10 min at 14,000 rpm, frozen in liquid nitrogen, and stored
at
80 °C. After thawing, the solution was microcentrifuged as
before and then pre-cleared by the addition of a mixture of protein A
and protein G-Sepharose (previously blocked with 1 mg/ml bovine serum
albumin and 1 mg/ml sheared salmon sperm DNA in sonication buffer at
4 °C overnight) for 4 h. Aliquots of the pre-cleared chromatin
were incubated with 2 µg of each antibody overnight at 4 °C. One
aliquot was incubated without antibody. The supernatant of this sample
was later processed in parallel with the immunoprecipitates starting
with the RNase A and proteinase K digest (this sample is referred to as
"Input"). Immune complexes were captured with 40 µl of blocked
protein A/protein G-Sepharose, washed seven times with cold washing
buffer (50 mM Hepes pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% Nonidet P-40, 0.7% deoxycholate and protease
inhibitors) and once with TE, and then resuspended in 200 µl of TE.
Both the immunoprecipitates and the input sample were digested with 10 µg each of RNase A and proteinase K at 55 °C for 3 h. After
incubation at 65 °C overnight, the Sepharose was pelleted, and the
supernatant containing the co-precipitated DNA was applied to a PCR
purification column (Qiagen) according to the instructions of the
manufacturer. The purified DNA was eluted in 50 µl of 10 mM Tris. 1 µl of the eluted DNA was used for PCR. The
following antibodies were used: anti-Gadd-45 (sc-H165; Santa Cruz),
anti-acetylated H3 and H4 (06-599 and 06-866; Upstate Biotechnologies); the antibodies specific for E2F family members and
pocket proteins have been used for chromatin immunoprecipitation experiments before (33). The following primers were used:
MYCN: 5'-AATGACAAGCAATTGCCAGGC-3' and
5'-AGGCGCCAAGGCTTTCGCG-3'; GAP-43: 5'-GGGGCTGGGGGAAGTGATTAGTC-3' and 5'-CCCCCTCTCCACCTCTTTTCTGC-3'; p107 (33);
-prothymosin (34). The
p107 and
-prothymosin PCRs were run for 36 cycles and the GAP-43 PCR for 38 cycles. In the case of the
MYCN primers, the PCR was run for 30 cycles with DNA from
Kelly and IMR-32, and for 36 cycles for DNA from SH-EP, to compensate
for the amplification of the MYCN locus in Kelly and
IMR-32.
The PCRs for the chromatin immunoprecipitation with pocket
protein-specific antibodies were performed on an ABI 7000 using SYBR
Green. First, the mean cycle threshold (Ct) values of triplicate reactions of the input samples were analyzed. Chromatin preparations were only used when the input samples showed a Ct difference of 0.5 or
less. Then, the Ct values of PCR reactions using the immunoprecipitated chromatin as a template were subtracted from the Ct values of the PCR
reactions with the negative control sample (immunoprecipitation of
chromatin in the absence of antibody; when the Ct values of the
-Gadd-45 samples were used as negative-control samples, very similar
results were obtained). The mean values of these
Ct values of
triplicate PCR reactions were than compared between untreated and
retinoic acid-treated samples.
Reverse Transcriptase-PCR--
Total cytoplasmic RNA was
isolated with the RNeasy Kit (Qiagen), and 1 µg was
reverse-transcribed using Superscript II and random primers. An aliquot
of cDNA first strands corresponding to 50 ng of RNA was used for
the PCR amplification. Pilot experiments established that the PCR
conditions used are within the linear range of amplification. Selective
results were confirmed with real-time PCR on an ABI 7000 using SYBR
Green. Primer sequences are available upon request.
 |
RESULTS |
E2F-1, E2F-2, and E2F-3 Activate the MYCN Promoter through
E2F-binding Sites--
To test whether transcription factors of the
E2F-family are able to activate the MYCN promoter in
neuroblastoma cells, we transiently co-transfected human neuroblastoma
cells with E2F expression vectors and a reporter construct in which
luciferase expression is controlled by 230 base pairs of the human
MYCN gene, which encompass the major transcription start
sites and three closely spaced putative E2F-binding sites, two of which
overlap (Fig. 1A). In SH-EP
cells lacking amplification and expression of MYCN, this
reporter construct was activated 3- to 5-fold by E2F-1, 2- to 3-fold by
E2F-2, and 8- to 10-fold by E2F-3 (Fig. 1A). E2F-4 and E2F-5
did not influence the activity of the MYCN promoter in this
assay. Similar results were obtained with two neuroblastoma cell lines
with amplified MYCN, IMR-32, and Kelly (data not shown).
Western blotting showed high levels of E2F-1, E2F-2, E2F-3, and E2F-4
in the transfected cells (Fig. 1B). E2F-5 was expressed at
about 20% of E2F-3 and E2F-4, respectively. A point-mutant reporter
construct lacking the two overlapping E2F-sites was not activated by
E2F-1 but was still responsive to E2F-3 (Fig. 1C). A
construct lacking all three E2F-sites was only weakly activated by
E2F-3 compared with the wild-type construct. Therefore, the proximal
promoter of the MYCN gene in neuroblastoma cells is
activated by E2F-1, E2F-2, and E2F-3, but not E2F-4 and E2F-5, and this activation is largely dependent on intact E2F-binding sites.

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Fig. 1.
E2F-1, E2F-2, and E2F-3 activate the
MYCN promoter through E2F-binding sites. A,
activation of the MYCN promoter by E2F-1, E2F-2, and E2F-3,
but not E2F-4 and E2F-5. SH-EP human neuroblastoma cells were
transiently transfected with expression vectors for E2F proteins and a
reporter construct, in which the luciferase gene is controlled by 230 base pairs of the human MYCN promoter. Luciferase activity
was normalized to protein concentration of the lysates. Transfection
efficiencies of different samples were compared by counting the
percentage of eGFP positive cells before lysis. No significant
differences in the number of eGFP-positive cells were observed. The
normalized luciferase data are represented as -fold activation with the
reporter activity in the absence of ectopic E2F set to 1. Error bars
represent the standard deviation of triplicate samples. B,
expression of the different E2F proteins in the transiently-transfected
cells. Detection of E2F proteins in the transfected cells was done by
Western blotting. In the experiment shown, E2F-3-E2F-5 were expressed
as HA-tagged proteins and detected with a HA-specific antibody to allow
direct comparison of protein amounts. Untagged versions of E2F-3 and
E2F-4 gave very similar results with regard to reporter activation.
C, activation of the MYCN promoter by E2F
proteins depends on intact E2F-binding sites. Transient transfection of
SH-EP human neuroblastoma cells with expression vectors for E2F
proteins and the different MYCN
promoter-dependent luciferase constructs indicated at the
top. The open boxes represent the three E2F
binding-sites present in the proximal MYCN promoter.
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E2F-1, E2F-2, and E2F-3 Bind to the MYCN Promoter in Vivo--
If
E2F proteins do play a role in the maintenance of MYCN
expression in neuroblastoma, they should bind to the MYCN
promoter in MYCN-expressing neuroblastoma cells. To test
this prediction, ChIP assays with antibodies specific for E2F-1, E2F-2,
E2F-3, and E2F-4 were performed with cross-linked chromatin from three neuroblastoma cell lines. Co-precipitated chromatin was used as template for PCR amplification using primers that amplify a 190-base pair fragment of the proximal MYCN promoter, including all
three E2F binding sites. In the two cell lines Kelly and IMR-32, which show amplification and overexpression of MYCN, E2F-1, E2F-2,
and E2F-3 but not E2F-4 bound to the proximal MYCN promoter
(Fig. 2). In contrast, in SH-EP cells
lacking detectable MYCN expression, no association of any of
the E2F proteins with the MYCN promoter could be detected.
Binding of E2F-1, E2F-2, and E2F-3 to the promoter of another
E2F-responsive gene, p107 (RBL1), was readily
detected in all three cell lines including SH-EP. No product for any of the imunoprecipitated chromatin samples was obtained with primers amplifying part of intron 1 of the PTMA gene encoding
-prothymosin, which harbors a MYC-responsive E-box motif
but no E2F-binding site. There were marked differences in the abundance
of different E2F proteins at the MYCN promoter in Kelly
versus IMR-32 cells, with E2F-1 being the predominant
E2F-binding activity in Kelly, whereas E2F-1-E2F-3 were present in
equal amounts in IMR-32. From these data, we conclude that members of
the E2F family bind the proximal MYCN promoter in
vivo specifically in neuroblastoma cells with strong
MYCN expression.

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Fig. 2.
E2F proteins bind the MYCN
promoter in neuroblastoma cells in vivo.
Chromatin immunoprecipitation from the three human neuroblastoma cell
lines Kelly, IMR-32, and SH-EP. Kelly and IMR-32 harbor amplified
MYCN, whereas no MYCN expression is detected in
SH-EP lacking MYCN amplification. The chromatin was
precipitated with polyclonal antibodies specific for different members
of the E2F family or Gadd-45 as a negative control. Coprecipitated
chromatin was analyzed with primer pairs that are specific for the
proximal MYCN promoter including the three E2F-binding
sites, the E2F-responsive region of the p107 promoter, or
part of intron 1 of the -prothymosin gene lacking an
E2F-binding site. In the case of the MYCN PCR, 36 cycles
were used with the DNA from SH-EP cells but only 30 cycles with the DNA
from Kelly and IMR-32 to compensate for the amplification of the
MYCN locus in the latter two cell lines.
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Inhibition of E2F Activity Reduces Expression of Endogenous
MYCN--
Having demonstrated binding of E2F proteins to the
MYCN promoter in vivo, we next asked whether
blocking E2F activity would cause a reduction of endogenous
MYCN expression. Overexpression of p16INK4a, an
inhibitor of Cdk4 and Cdk6, leads to the accumulation of hypophosphorylated pRb and G1 arrest (35). IMR-32 cells do
not produce detectable amounts of p16INK4a protein (20). We
generated pools of IMR-32 cells expressing the ecotropic receptor and
subsequently infected these cells with pBABEhygro-p16INK4A, an ecotropic retrovirus
driving expression of p16INK4a, or an empty control virus.
RNA was isolated from hygromycin-selected cell populations and used for
RT-PCR analysis. Cells that had been infected with the
p16INK4A-containing retrovirus strongly
expressed p16INK4A compared with cells that had
been infected with the control virus (Fig.
3). p16INK4A caused a
down-regulation of both CCNE1 and MYCN (Fig. 3).
These results were confirmed by real-time PCR (data not shown). We
conclude that E2F activity is required for maximum expression of
MYCN in neuroblastomas.

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Fig. 3.
Inhibition of E2F activity through
overexpression of the Cdk-inhibitor p16INK4A
reduces MYCN expression. IMR-32 cells
expressing the ecotropic receptor were infected with a retrovirus
containing the CDKN2A cDNA or an empty control virus.
Pools of infected cells were selected for 2 days with hygromycin. RNA
was then isolated and analyzed by RT-PCR for the expression of
CDKN2A, the E2F target gene CCNE1,
MYCN, and S14, encoding a ribosomal protein, as a
control.
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Loss of Binding of E2F-2, E2F-3, and E2F-4, Reduction of Histone
Acetylation, and Recruitment of pRb during Retinoic Acid-induced
Differentiation--
Retinoic acid (RA) triggers neuronal
differentiation of some neuroblastoma cell lines including LA-N-5,
which carry amplified MYCN. Morphological differentiation,
which is completed after about 10 days of treatment, is preceded by a
reduction of MYCN expression as early as 6 h after
addition of RA (10). During the differentiation process, the amount of
E2F-1 protein remains constant and the amount of E2F-4 increases
sharply (36). The repression of MYCN through RA has been
mapped to the proximal promoter, which, however, lacks a sequence
resembling a retinoic acid response element (37). To test whether the
E2F-binding sites contribute to the RA-mediated down-regulation of
MYCN, LA-N-5 cells were treated with RA or the solvent
control ethanol for 12 days and subjected to chromatin
immunoprecipitation with a panel of E2F-specific antibodies. At this
time, most of the cells had extended long cellular processes indicative
of neuronal differentiation, and MYCN mRNA levels were
reduced (data not shown). ChIP assay showed that in untreated LA-N-5
cells, like in Kelly and IMR-32, E2F-1, E2F-2, and E2F-3, were
associated with the MYCN promoter (Fig.
4A). In contrast to Kelly and
IMR-32 cells, E2F-3 was the predominant E2F species in LA-N-5 (compare
Figs. 2 and 4A). Furthermore, unlike Kelly and IMR-32, E2F-4
was detected at the MYCN promoter in untreated LA-N-5 cells.
In differentiated cells, there was virtually no binding of E2F-2 and
E2F-4 to the MYCN promoter, and the binding of E2F-3 was
drastically reduced. In contrast, the binding of E2F-1 to the
MYCN promoter was unaltered. The loss of binding of E2F-2,
E2F-3, and E2F-4 to the MYCN promoter was accompanied by a
reduction in the acetylation of histones H3 and H4, consistent with a
drop in transcriptional activity of the MYCN gene (Fig.
4A). To exclude the possibility that the chromatin from the
RA-treated cells in general produced less signal despite a similar
intensity of the input lanes, the proximal promoter of the
GAP-43 gene, which is up-regulated during neuronal
differentiation, was also analyzed. Acetylation of the histones H3 and
H4 at the GAP-43 promoter was unchanged upon RA treatment.
As expected, no binding of any of the E2F proteins was detected.

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Fig. 4.
Changes in the binding of E2F and pocket
proteins to the MYCN promoter during retinoic
acid-induced differentiation. LA-N-5 cells were either treated
with 5 × 10 5 M of all-trans
retinoic acid or the solvent control ethanol for 12 days. ChIP assay
was then performed using a panel of E2F-specific antibodies, antibodies
that recognize acetylated histones H3 and H4, and pocket
protein-specific antibodies. The Gadd-45 specific antibody served as a
negative control. The co-precipitated chromatin was analyzed by PCR as
in Fig. 2. A, reduced binding of E2F-2, E2F-3, and E2F-4 to
the MYCN promoter in differentiated LA-N-5 cells. The
GAP-43 gene is induced during neuronal differentiation and
served as a control. B, increased binding of pRb to the
MYCN promoter in differentiated LA-N-5. PCR was performed on
an ABI 7000 using SYBR Green. The Ct values of PCR reactions using the
immunoprecipitated chromatin as a template were subtracted from the Ct
values of the PCR reactions with the negative control sample
(immunoprecipitation of chromatin in the absence of antibody). Shown
are the mean values and standard deviations of the Ct values of
triplicate PCR reactions.
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E2F proteins can repress target genes by associating with pocket
proteins and recruiting a variety of co-repressor complexes (38). To
test whether pocket proteins are bound to the MYCN promoter
in undifferentiated or differentiated LA-N-5 cells and to exclude the
possibility that masking of the antibody epitopes on the E2F proteins
by associated pocket proteins in RA-treated cells prevented their
detection in the ChIP assay, we performed another chromatin
immunoprecipitation with a panel of pocket-protein-specific antibodies,
which have previously been shown to detect repressive E2F complexes in
ChIP assays (33). We detected low levels of pRb, p107, and p130 at the
MYCN promoter in undifferentiated cells (Fig.
4B). In differentiated cells there were no changes in
binding of p107 and p130; in contrast, binding of pRb increased upon
differentiation. Thus, down-regulation of MYCN transcription
in the course of RA-triggered differentiation of neuroblastoma cells is
accompanied by a loss of binding of E2F-2, E2F-3, and E2F-4, a
reduction in histone acetylation, and recruitment of pRb.
Repression of MYCN by TGF-
Requires the E2F-binding
Sites--
TGF-
has been shown to down-regulate MYCN
expression (39). E2F-binding sites can confer TGF-
responsiveness to
a promoter (40). In addition to several E2F-binding sites, the
MYCN promoter also contains a putative TGF-
-inhibitory
element (TIE; consensus sequence: GnnTTGGnG) (41) that partly overlaps
the third E2F-binding site (Fig.
5A). To determine whether
either the E2F-binding sites or the TIE are required for the regulation
of MYCN by TGF-
, HaCaT human keratinocytes were
transiently transfected with wild-type and mutant MYCN
promoter-dependent luciferase constructs and then either
treated with TGF-
for 26 h or left untreated. The wild-type MYCN promoter was repressed 5-fold by TGF-
in HaCaT cells
(Fig. 5B). A promoter lacking the two overlapping
E2F-binding sites showed a reduction in basal activity compared with
the wild-type construct, and this residual activity was not reduced
further by TGF-
. In contrast, a promoter with a mutation destroying
both the TIE and the third E2F-binding site was almost as active as the
wild-type promoter and was repressed by TGF-
to the same extent as
the wild-type promoter. Thus, in HaCaT cells, the two overlapping
E2F-binding sites of the MYCN promoter mediate the major
part of the promoter activity as well as its responsiveness to TGF-
,
whereas neither the putative TIE nor the third E2F-binding site
contribute to promoter activity or TGF-
responsiveness.

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Fig. 5.
Two overlapping E2F-binding sites are
essential for TGF- -mediated repression of
MYCN. A, location of regulatory
elements that may mediate down-regulation by TGF- in the promoters
of MYCN and MYC. The putative TGF- -inhibitory
element, TIE, in the MYCN promoter overlaps one of the
E2F-binding sites. There is only one E2F-binding site in the
MYC promoter. This E2F site overlaps with the TIE.
B, mutation of the overlapping E2F-binding sites, but not
the TIE, abolishes the TGF- responsiveness of the MYCN
promoter. Various wild-type and mutant
MYCN-promoter-dependent luciferase constructs
were transiently transfected into HaCaT cells together with a
SV40-lacZ expression vector. Immediately after transfection,
cells were split equally into two new dishes, and 2.5 ng/ml TGF- was
added to one of the dishes. After 26 h lysates were prepared and
reporter activity measured. Luciferase activity was normalized to the
activity of a co-transfected lacZ reporter construct. The
error bars represent the standard deviation observed with
triplicate samples.
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|
 |
DISCUSSION |
We have shown here that E2F proteins regulate MYCN
transcription and are required for full activity of the MYCN
promoter in neuroblastomas. This conclusion is based on the following
observations: first, the activating members of the E2F family, E2F-1,
E2F-2, and E2F-3, activate the MYCN promoter in transient
transfections in an E2F-site-dependent manner; second,
E2F-1, E2F-2, and E2F-3 bind to the MYCN promoter in
vivo specifically in neuroblastoma cells exhibiting
MYCN expression; third, inhibition of E2F activity through
overexpression of p16INK4A reduces MYCN mRNA
levels; fourth, several signals known to down-regulate MYCN
expression either require intact E2F-binding sites for their regulation
of the MYCN gene or are associated with changes in the
binding of E2F proteins to the MYCN promoter. These data
support the existence of a positive feedback loop linking E2F and
N-Myc, which may at least in part explain the maintenance of
MYCN expression in aggressive neuroblastomas (for
discussion, see Ref. 9).
The status of the pRb pathway in neuroblastomas and its role in
neuroblastoma tumorigenesis is far from clear. Mutations in RB itself or other genes implicated in the pathway are rare
in neuroblastomas. There is, however, evidence that the pRb pathway may
become inactivated in neuroblastomas by other means. One is the
inactivation of CDKN2A (p16ink4A) by
promoter methylation in some neuroblastomas (20). However, other
neuroblastomas contain large amounts of p16INK4A (42, 43).
This observation may be explained by the overexpression in
neuroblastomas of Id2, which has been suggested to sequester and
inactivate pRb and thus bypass p16INK4A (21). IMR-32 is one
of the cell lines that, because of the direct transcriptional
activation of ID2 by N-Myc, produce large amounts of Id2
(21). However, IMR-32 also lacks detectable p16INK4A
protein (20). If overexpression of Id2 were indeed a way of blocking
pRb function in neuroblastomas, one would expect that overexpression of
p16INK4A, which controls pRb upstream of Id2, would not be
able to reestablish negative control over E2F activity. In U2OS
osteosarcoma cells, Id2 can indeed reverse the cell-cycle arrest
induced by p16INK4A (44). We tested whether this is also
true for neuroblastoma cells by introducing
p16INK4A into IMR-32 by retroviral infection and found that
p16INK4A repressed both CCNE1 and
MYCN, suggesting that overexpressed Id2 is not able to
relieve negative control of E2F activity in neuroblastoma cells. This
is consistent with the observation that the majority of primary
neuroblastomas do not express detectable amounts of
p16INK4A protein and that the lack of p16INK4A
in primary neuroblastomas is associated with a poor prognosis (20).
The MYC promoter is the target of multiple mitogenic and
antimitogenic signals, some of which regulate MYC expression
via an E2F site in the MYC promoter (45, 46). Our results
provide evidence that the E2F-binding sites in the MYCN
promoter are also involved in the response to different signals. The
conservation of E2F-binding sites in the proximal promoter regions of
both MYC and MYCN and the shared role of these
E2F-binding sites in repressing promoter activity in response to
external signals points to similarities in the regulatory logic of the
two genes despite their very different spatiotemporal expression
profiles. We hypothesize that after duplication of the ancient
MYC gene into MYC and MYCN, the
preservation in both genes of the regulatory elements required for
tight negative control was essential, given their oncogenic potential.
If true, the E2F-binding sites in the MYC genes could be
regarded as central negative control elements to restrict
MYC/MYCN expression in a dominant fashion.
Down-regulation of MYCN by TGF-
may be mediated by
E2F-4-containing repressor complexes, which have been shown to form at E2F target promoters in response to TGF-
in keratinocytes (47). Yet,
the finding that TGF-
represses MYCN via the E2F binding sites is unexpected in light of several previous reports mapping the
repressive effect of TGF-
on the MYC promoter to a
TGF-
inhibitory element (TIE), because a putative TIE is also
present in the MYCN promoter (48, 49). Very recently, the
TIE in the MYC promoter was redefined as a noncanonical
Smad-binding site, 5'-GGCT-3', adjacent to the E2F-binding site (50).
It was shown that both the E2F and Smad-binding sites are required for
repression of MYC transcription by TGF-
and that they
form a composite regulatory element that binds a repressor complex
containing Smad3, Smad4, E2F-4/5, DP1, and p107. Notably, the
overlapping E2F sites in the MYCN promoter are flanked by
putative Smad-binding sites. Recently, advanced-stage primary
neuroblastomas were reported to express strongly reduced levels of the
TGF-
type III receptor (51). In addition, whereas
ganglioneuroblastomas, which show a more differentiated phenotype than
neuroblastomas, express TGF-
, a majority of primary neuroblastomas
do not (52). Together, these observations indicate that neuroblastomas
may escape negative growth control by TGF-
.
E2F-binding sites, for example in the cdc25A gene, have
previously been implicated in mediating regulation by TGF-
(47, 53).
Neither this site nor the E2F-binding sites in other E2F target genes
such as B-myb and E2F1 contribute to the activity of the promoter in the context of transient assays in HaCaT cells, indicating that these E2F-binding sites function exclusively in transcriptional repression (54). In sharp contrast, the E2F-binding sites of the MYCN promoter are important for promoter
activation, accounting for roughly 80% of the promoter activity in
HaCaT cells in the absence of TGF-
. TGF-
blocks only the E2F
site-dependent activity but not the residual E2F
site-independent activity of the promoter.
In contrast to the down-regulation by TGF-
, the negative regulation
of MYCN by retinoic acid cannot involve an E2F-4/p107 complex because we observed a reduction rather than an increase of
binding of E2F-4 to the MYCN promoter during
differentiation. The reduction in binding of E2F-4 to the
MYCN promoter could simply be a result of reduced protein
levels in the course of differentiation. However, the amount of E2F-4
in LA-N-5 cells has previously been shown to increase during RA-induced
differentiation (36). The observation that E2F-1, unlike E2F-2 and
E2F3, remained associated with the MYCN promoter during
differentiation together with the increased binding of pRb suggests
that in the course of differentiation an E2F-1/Rb complex rather than
an E2F4/p107 complex contributes to the repression of the
MYCN gene. This would indicate that in response to different
signals, distinct E2F/pocket protein complexes mediate down-regulation
of MYCN.
The ChIP results show differences in the relative binding of different
E2F proteins to the MYCN promoter in various neuroblastoma cell lines, with E2F-1 being predominant in Kelly cells, whereas E2F-3
predominated in LA-N-5. Because MYCN is strongly expressed in all of these cell lines, E2F-1, E2F-2, and E2F-3 appear to be
redundant with regard to the activation of the MYCN
promoter. We have no explanation for the association of E2F-4 with the
MYCN promoter in undifferentiated, MYCN amplified
LA-N-5 cells. The results with the other MYCN-amplified
neuroblastoma cell lines Kelly and IMR-32, however, show that E2F-4,
although it can be found associated with the MYCN promoter,
is not required for the expression of MYCN in these cells.
In summary, we have identified E2F-1, E2F-2, and E2F-3 as the first
transcription factors known to regulate MYCN expression in
neuroblastomas. An understanding of the events that accompany activation and repression of the MYCN gene at these
E2F-binding sites may eventually suggest means of blocking expression
of MYCN in the tumor cells.