 |
INTRODUCTION |
The mammalian G1 cyclins, which include cyclins D1,
D2, D3, E1 and E2, are important regulators of cellular proliferation (1-3). Quiescent cells express the G1 cyclins at minimal
levels; however, upon mitogenic stimulation, the D-type cyclins are
transcriptionally activated, and their levels rise as cells traverse
G1 (4). In the prevailing model (3), it is thought that the
D-type cyclins direct the limited phosphorylation of Rb via their
interactions with cyclin-dependent kinases (5-7).
Phosphorylation of Rb by the D-type cyclin-cyclin-dependent
kinases complexes permits the release of DNA-E2F-Rb-bound
histone deacetylase proteins, which impart a dominant transcriptional
repressing activity to promoter-bound Rb-E2F complexes (8). Loss
of histone deacetylase-mediated inhibition allows a modest
transcriptional activation of certain Rb-silenced genes, including
cyclin E (9), but other E2F-Rb-regulated genes remain repressed (8).
Once stimulated, cyclin E associates with cyclin-dependent
kinase 2 and adds additional phosphate modifications to Rb later in
G1 (10). Upon its hyperphosphorylation by the cooperative
efforts of the D-type cyclins and cyclin E, the Rb protein completely
releases the E2F transcription factor. Liberated E2F3B and E2F4, which
are the predominant E2Fs expressed (11-14) and promoter-bound (15, 16)
in G0 and early G1, then activate transcription
(17-20) of the more potent S phase-promoting E2Fs, E2F1, -2, and -3A
(21). The combined activities of E2F1, -2, and -3A then lead to the
potent activation of a large number of genes that are required for
nucleotide biosynthesis, the firing of origins of replication, and the
completion of replicative DNA synthesis (22-24). In this paradigm, E2F
activates transcription of cyclin E, which then further activates E2F
by stimulating the phosphorylation of Rb. This positive feedback loop
leads to an irreversible commitment to entry into S phase at the
restriction point (25).
In previous work, we showed that ectopic expression of E2F1 alone is
sufficient to stimulate quiescent cells to enter cell cycle (26) and
that stable expression of E2F1 can lead to oncogenic transformation
(27). Subsequently, we utilized microarray technology to screen for
genes that could account for the ability of E2F to induce cellular
proliferation (24) and apoptosis (28, 29). In addition to verifying
several known E2F targets, our microarray screen suggested that E2F1
potently activates cyclin D3 transcription. Cyclins D1 and D2 were not
activated by E2F1 expression in this screen. Here we verify that cyclin
D3 is indeed a direct E2F-regulated transcript, and we localize the E2F
regulatory element of the cyclin D3 promoter within a small and
extremely GC-rich region in the vicinity of the major transcriptional
start site. We conclude that in addition to cyclins E and A, E2F
family members can also activate one member of the D-type cyclins,
further contributing to the ability of E2F to drive the
G1 to S phase transition.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
Human cyclin D3 promoter fragments were generated
by PCR from genomic DNA, ligated into pGL3 basic vector, and sequenced. Initial PCR primers were designed to amplify 1023 bp (
1017/+6) of the
published cyclin D3 promoter sequence (30), which are numbered relative
to the ATG codon at +1. The forward (
1017F) and reverse (+6R)
PCR primers for the full-length promoter were 5'-GGGGACGCGTAGATCTCACAGAGTCTGTGCA-3' and
5'-GGGGCTCGAGCTCCATACTCGGGGCAGCGAA-3', respectively. The forward primer
added a MluI site, and the reverse primer added a
XhoI site to facilitate subcloning. Deletion mutants of
the cyclin D3 promoter construct (
362/+6,
202/+6,
159/+6, and
115/+6) were generated using reverse primer +6R with the following
forward primers: 5'-GGCCGGTACCACCTCCTAGAAAGTTCTCT-3' (
362F),
5'-GGCCGGTACCGAGCATTCCACGGTTGCTA-3' (
202F),
5'-GGGGGGTACCTGTCAGGGAAGCGGCGCG-3' (
159F) and
5'-GGGGGGTACCGGATCCGCCGCGCAGTGCCAG-3' (
115F), respectively. Each of these primers added a KpnI restriction site for
subcloning. Promoter deletion mutants
202/
146,
202/
134, and
202/
112 were generated using primer
202F in combination with
primers 5'-GGGGAAGCTTCCCTGACAGGCGCCCCG-3' (
146R),
5'-CCCCAAGCTTCGCGCGCGCGCCGCTTCCCTG-3' (
134R) and
5'-GGGGAAGCTTGGATCCCCAGCCCGCCCGCCG-3' (
112R), respectively. Primers
146R,
134R, and
112R each added a HindIII restriction site for subcloning. Construct
159/+6(del9) was created using primer
5'-CCCCGGTACCTGTCAGGGAAGCGAGGGCGGCGGGCGGGCTGG-3' (159del9F) and
reverse primer +6R. Construct
146/
111 was created by annealing oligonucleotides 5'-CGGCGCGCGCGCGGGCGGCGGGCGGGCTGGGGATCCA-3' and 5'-AGCTTGGATCCCCAGCCCGCCCGCCGCCCGCGCGCGCGCCGGTAC-3' and cloning directly into the KpnI and HindIII sites of pGL3.
The pBSK-ER-E2F1 plasmid, which encodes a hemagglutinin-tagged estrogen
receptor E2F1 fusion protein (31), was a gift from Dr. Kristian Helin (European Institute of Oncology, Milan, Italy). An EcoRI and
NotI fragment from pBSK-ER-E2F1 was cloned into pcDNA3
to allow protein expression and to provide a selectable marker.
Cell Culture and Analysis--
H1299 cells obtained from Dr.
Jiandong Chen (Moffitt Cancer Center) were cultured in Dulbecco's
modified Eagle's medium supplemented with 5% fetal bovine serum.
H1299 cells were transfected with pcDNA3-ER-E2F1 plasmid using the
calcium phosphate method, and stable transfectants were selected with
G418 (0.5 mg/ml). G418-resistant colonies were isolated after 2 weeks
and expanded in the continued presence of 0.25 mg/ml G418.
ER-E2F1-positive lines were identified by Western blot using
anti-hemagglutinin and anti-E2F1 antibodies. H1299-pcDNA3 stable
cell lines were established by the same method and served as negative
controls. 4-Hydroxytamoxifen
(HT1; 300 nM) was
added to the medium to induce rapid ER-E2F1 nuclear accumulation of the
ER-E2F1 protein. Cycloheximide (CHX) was used at a final concentration
of 10 µg/ml to block new protein synthesis. Apoptosis and cell cycle
parameters were measured by flow cytometry using an Apo-BrdU Kit (DB
PharMingen) as previously described (32).
Biochemical Assays--
Total RNA was isolated from 5 × 106 H1299 cells using the RNAeasy mini kit (Qiagen). RNase
protection assays were carried out with the Riboquant hCYC1 (cyclin
family) multiprobe templates (BD PharMingen). Briefly, the multiprobe
templates were synthesized by in vitro transcription with
incorporation of [32P]dUTP and purified on Quick Spin RNA
columns (Roche Applied Science). Labeled probe (1× 106
cpm) was hybridized with 10 µg of total RNA through a temperature gradient of 90 to 56 °C over a 16-h period. Unprotected probe was
removed by RNase digestion at 30 °C for 1 h followed by
separation of protected RNA fragments on a 5% polyacrylamide-urea gel
and detection using autoradiography.
Transfections were performed using calcium phosphate with test DNAs
totaling 20 µg of DNA per 100-cm dish. Transfections included 300 ng
of expression plasmid (pcDNA3-based vectors), 10 µg of reporter
firefly luciferase reporter plasmid (pGL3, Promega), 2 µg of
Renilla luciferase reporter plasmid (pRL-TK, Promega), and
carrier DNA (sheared salmon sperm DNA) to equal 20 µg of total DNA in
each transfection. Cells were harvested 48 h after transfection, and luciferase assays were performed using the Dual-Luciferase Reporter
Assay System following the manufacturer's protocol (Promega). Experiments were done in triplicate, and the relative activities and
S.E. values were determined. To control for transfection efficiency, firefly luciferase values were normalized to the values for
Renilla luciferase.
Western blots were performed as previously described (28) using
monoclonal antibodies against cyclin D3 (14781A; BD PharMingen). Western blots were stripped and reprobed with an antibody to actin (A5441; Sigma) to ensure equivalent loading. Electrophoretic mobility shift assays (EMSAs) and antibody supershift assays were performed as
previously described. Briefly, EMSA assays included 20 µg of total
protein extract from E2F1-DP1 baculovirus-infected Sf9 or uninfected Sf9 cells. EMSA probes were generated by restriction digestion of the appropriate luciferase reporter plasmids (
202/+6, KpnI/XhoI;
202/
146,
KpnI/HindIII;
202/
112,
KpnI/BamHI;
159/+6, KpnI/XhoI;
115/+6,
BamHI/XhoI;
159/
112,
KpnI/BamHI). [
-32P]dATP was
incorporated into band-purified DNA fragments using the Klenow fragment
of DNA polymerase I. E2F consensus (sc-2507; Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) and E2F mutant consensus (sc-2508; Santa Cruz
Biotechnology) competitor oligonucleotides were added at 20 ng/10-µl
reaction (100-fold excess). An E2F1 monoclonal antibody (sc-251; Santa
Cruz Biotechnology) was used to identify the putative E2F1-DP1 complex.
The 15-bp competitor oligonucleotides used in Fig. 5C were
desalted, annealed, and used in competition assays without further
purification. The sequences of the upper strands of the
competitor oligonucleotides were as follows: 1 (5'-GTACCGGCGCGCGCG-3'), 2 (5'-GGCGCGCGCGCGGGC-3'), 3 (5'-GCGCGCGGGCGGCGG-3'), 4 (5'-CGGGCGGCGGGCGGG-3'), 5 (5'-GGCGGGCGGGCTGGG-3'), 6 (5'-GCGGGCTGGGGATCC-3'), 7 (5'-CTGGGGATCCAAGCT-3'), and C15 (5'-AAGTTTCGCGCCCTT-3').
For reference, the boldface letters in each
oligonucleotide represent the key E2F1 regulatory element as determined
in Fig. 5 (although oligonucleotide 3 contains part of the sequence, it
does not bind E2F; thus, the element is apparently too close to the
5'-end of the oligonucleotide to bind E2F. Underlined bases in the
oligonucleotides listed above are not from the cyclin D3 promoter but
represent cloning sites that were present in the relevant luciferase vectors.
Chromatin immunoprecipitation (ChIP) assays were performed as
previously described (16, 28). Briefly, asynchronously growing H1299
cells were treated with formaldehyde to create protein-DNA cross-links,
and the cross-linked chromatin was then extracted, diluted with ChIP
buffer, and sheared by sonication. After preclearing with protein A
beads, blocked with 1% salmon sperm DNA and 1% bovine serum albumin,
the chromatin was divided into equal samples for immunoprecipitation
with either anti-E2F1 polyclonal antibody (sc-193; Santa Cruz
Biotechnology), anti-RhoA polyclonal antibody (sc-179; Santa Cruz
Biotechnology), or no antibody. The immunoprecipitates were pelleted by
centrifugation, and heating reversed the cross-linking. After proteins
and any contaminating RNA were removed by treatment with proteinase K
and RNase, PCR primers (
202F and +6R) that generate a 230-bp product
were used to detect the presence of specific DNA sequences. PCR primers
corresponding to the human actin promoter (negative control) were
previously described (15).
 |
RESULTS |
Generation of Cell Lines Expressing ER-E2F1--
Our previous
microarray analysis utilized an adenovirus to express E2F1; thus, it
was possible that the activation of the cyclin D3 promoter was a
secondary consequence of E2F1 expression. To verify that activation of
cyclin D3 by E2F1 was direct, we created a cell line that
constitutively expresses a hemagglutinin-tagged estrogen receptor-E2F1
fusion protein (ER-E2F1). In this construct, E2F1 is fused to a
transcriptionally inactive mutant of the murine estrogen receptor that
is unable to bind estrogen yet retains high affinity for HT. In the
absence of HT, the ER-E2F1 fusion protein is excluded from the nucleus
and thus cannot directly affect transcription. Upon the addition of HT,
however, the fusion protein rapidly enters the nucleus and induces
transcription of E2F1 target genes. The advantage of this system is
that, since the fusion protein is preexisting when HT is added,
induction can occur in the absence of new protein synthesis. Therefore, promoters that are activated upon HT addition in the presence of the
protein synthesis inhibitor CHX are most likely regulated by a direct mechanism.
ER-E2F1-expressing H1299 cell lines were derived by transfecting the
pcDNA3-ER-E2F1 plasmid followed by selection in G418. After
G418-resistant colonies were isolated and screened by Western blot with
both hemagglutinin and E2F1 antibodies (data not shown), five colonies
expressing detectable levels of the ER-E2F1 protein were chosen for
further experiments. For negative controls, G418-resistant cell lines
were also derived with the empty pcDNA3 plasmid. To identify the
ER-E2F1 cell line with the tightest HT regulation, the five
ER-E2F1-expressing lines were transfected with an E2F reporter plasmid
in which the adenovirus E2 promoter was fused to firefly luciferase.
Following transfection, HT (or solvent control) was added to induce
nuclear accumulation of ER-E2F1. Fig.
1A shows that all five ER-E2F1
positive cell lines induced the reporter gene upon the addition of HT.
Cell line H1299-ER-E2F1-15, which produced the most dramatic response
(a 9-fold induction), was selected for subsequent experiments. As
expected, H1299-pcDNA3 control cells did not activate the Ad E2
promoter in response to HT.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
HT treatment activates transcription of an
AdE2 promoter in ER-E2F1-expressing H1299 cell lines.
A, five H1299 cell lines stably expressing ER-E2F1 and a
control line were transfected with 20 µg of pGL3-AdE2 by the calcium
phosphate method. After 12 h of incubation, cells were washed with
phosphate-buffered saline, and fresh medium containing HT
(HT) or vehicle only (EtOH) was added. Cells were
collected, and luciferase levels were determined 48 h after
transfection. B, H1299-ER-E2F1 line 15 was treated with HT
for 48 h, and cell cycle parameters and apoptosis levels were
determined by propidium iodide and Apo-BrdU staining,
respectively.
|
|
To determine whether the ER-E2F1 fusion protein in ER-E2F1-15 was
expressed at sufficient levels to alter cell growth properties, cell
cycle parameters were determined before and after the addition of HT.
As demonstrated in Fig. 1B, a 48-h treatment with HT
resulted in a 15% increase in the fraction of cells in S phase and a
corresponding decrease in cells in G0/G1. In
addition to its ability to induce S phase, E2F1 is also a potent
inducer of apoptosis. Not surprisingly, Fig. 1B reveals that
nearly 24% of H1299-ER-E2F1-15 cells underwent apoptosis after
48 h of HT treatment, whereas less than 1% of the cells were
apoptotic in the absence of HT (Fig. 1B). Thus, the ER-E2F1
fusion protein expressed in H1299-ER-E2F1-15 cells functioned as
expected in the presence of HT.
Cyclin D3 Expression Is Directly Activated by E2F1--
To verify
direct activation of the cyclin D3 promoter by E2F1, the cell line
ER-E2F1-15 and a control line (pcDNA3) were treated with solvent
only, CHX alone, HT alone, or a combination of both CHX and HT.
Following 16 h of treatment, cyclin D3 message levels were
measured by an RNase protection assay. Fig.
2A reveals that the cyclin D3
mRNA increased dramatically in the presence of HT alone and even
more dramatically in the presence of both HT and cycloheximide. Since
immediate early mRNAs such as cyclin D3 are stabilized in the
presence of cycloheximide, the observed effects of cycloheximide alone
or in combination with HT are expected (33). These results
suggest that the induction of the cyclin D3 mRNA is a direct effect
of the ER-E2F1 fusion protein and does not involve synthesis of another
protein that is activated or repressed by E2F1. Cyclin D3 protein
expression was also measured by Western blot. In Fig. 2B,
48 h after drug treatment, the cyclin D3 protein was also induced
in the presence of HT, reflecting the increase in mRNA. Levels of
cyclin D3 mRNA and protein did not change in response to HT in the
control cell line, as expected. These results demonstrate that cyclin
D3 is a direct target of E2F1. In contrast to cyclin D3, cyclin D1 and
D2 were repressed following HT treatment (data not shown).

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 2.
Cyclin D3 expression is directly activated by
E2F1. A, H1299-ER-E2F1 and H1299-pcDNA3 cells were
treated with solvent only, with CHX alone, HT alone, or a combination
of both CHX and HT for 16 h. Expression of cyclin D3 mRNA was
measured by an RNase protection assay. B, H1299-ER-E2F1 and
H1299-pcDNA3 cells were treated as indicated, and the expression of
cyclin D3 was by Western blot. The actin Western blot served as a
loading control.
|
|
E2F1, -2, and -3A Trans-activate the Cyclin D3 Promoter--
To
determine whether the activation of cyclin D3 expression by E2F1 was
mediated via the promoter, we used luciferase reporter constructs to
examine the effect of E2F1 expression on the level of transcription
from the cyclin D3 promoter. Previous analysis (30) has mapped the
cyclin D3 promoter to a region from nucleotide position
1017 to +6
relative to the ATG start codon. To identify E2F1-responsive elements,
we tested luciferase reporter activity of three cyclin D3 promoter
constructs (
1017/+6,
362/+6, and
202/+6). As demonstrated in Fig.
3A, transient co-transfection of E2F1 resulted in strong activation of all three of the cyclin D3
promoter constructs. The smallest of these constructs (
202/+6) was
fully responsive to exogenous E2F, suggesting that this small region
contains the E2F1-responsive element.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
E2F1 trans-activates cyclin D3
promoter/reporter constructs dependent upon its DNA binding and
trans-activation domains. H1299 cells were transfected with 10 µg of each reporter construct and 300 ng of E2F expression plasmid or
mock vector (pcDNA3). Carrier DNA was used to bring total
DNA/transfection to 20 µg. A, the luciferase activity of
each reporter in absence of E2F1 expression is normalized to 1. B, the indicated E2F1 mutants were compared for the
ability to induce the 202/+6 promoter/reporter.
E2F11-284 lacks the trans-activation domain, and
E2F1E132 lacks the ability to bind DNA. C, the
indicated E2F family members (human cDNAs) were compared for the
ability to induce the 202/+6 promoter/reporter. Each of these
pcDNA3 vectors has been previously described and expresses
comparably high levels of the E2F proteins (55).
|
|
Previous studies using various E2F1 mutants have demonstrated that both
the DNA-binding domain and the transcriptional activation/Rb-binding domains of E2F1 are required for trans-activation to occur. To determine whether both of these functional domains are required for
cyclin D3 promoter activation, two previously described E2F1 mutant
proteins were tested for the ability to activate the cyclin D3
promoter. The first was E2F1E132, which lacks the ability
to bind DNA (34), and the second was E2F11-284, which
lacks the trans-activation domain (35, 36). As expected, both E2F1 mutants had diminished ability to trans-activate the cyclin D3 promoter
(Fig. 3B), demonstrating that activation of the cyclin D3
promoter is dependent on the ability of E2F1 to bind DNA and to
activate transcription.
Based upon structural and functional relatedness and on cell cycle
expression pattern, the E2F family members are classified into two
major subfamilies (37): the growth-promoting E2Fs (including E2F1, -2, and -3A) and the growth-restraining E2Fs (including E2F3B, -4, -5, and
-6). Growth-promoting E2Fs are primarily expressed at the
G1/S boundary and are capable of driving quiescent
fibroblasts into S phase upon overexpression. In contrast, the
growth-restraining E2Fs are expressed constitutively during the cell
cycle and induce S phase less efficiently or not at all when
overexpressed. To determine which of the E2Fs most potently modulate
the cyclin D3 promoter, several E2F family members were tested in
luciferase reporter assays. Fig. 3C shows that the cyclin D3
promoter is most potently activated by the growth-promoting E2Fs
(especially E2F1 and E2F2), whereas growth-restraining E2Fs have much
smaller effects, if any. These results suggest that cyclin D3 is a
target of multiple growth-promoting E2F1s and is not an E2F1-specific target.
E2F1 Binds to Cyclin D3 Promoter in Vivo and in Vitro--
The
experiments described above utilized ectopic E2F expression, which is
subject to the concern that the observed regulation is not
physiological. Thus, ChIP assays were performed on native H1299
cells to ascertain whether endogenous E2F1 binds to the cyclin D3
promoter in vivo. As shown in Fig.
4A, PCR primers that span the
region of
202 to +6 of the cyclin D3 promoter clearly detect cyclin
D3 promoter DNA in ChIP samples generated using an E2F1 antibody.
However, chromatin immunoprecipitations with either an irrelevant
antibody (anti-RhoA) or no added antibody resulted in the absence of
detectable cyclin D3 promoter DNA. PCR primers specific for the human
actin promoter (negative control) did not detect actin promoter DNA in
E2F1 ChIP samples, as expected from previous work demonstrating that
E2F1 does not bind the actin promoter (15). These data demonstrate that
E2F1 associates with the cyclin D3 promoter under physiological
conditions.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4.
E2F1 binds to the cyclin D3 promoter in
vivo and in vitro. A,
chromatin immunoprecipitation (ChIP) assays were performed using no
antibody, an irrelevant antibody (anti-RhoA, sc-179; Santa Cruz
Biotechnology), or an antibody against E2F1 (sc-193; Santa Cruz
Biotechnology). PCR primers for the cyclin D3 promoter (top)
or the actin promoter (bottom) were used to detect promoter
fragments in immunoprecipitates. The input lane
represents 0.02% of total chromatin used in ChIP assays, consistent
with previously published ChIP experiments. B, EMSA assays
were performed using the 202/+6 cyclin D3 promoter region as probe.
The first lane represents an extract of
uninfected Sf9 cells. The second lane ( )
represents Sf9 cells co-infected with baculoviruses that express
recombinant human E2F1 and DP1. The protein-DNA complex formed by the
recombinant E2F1-DP1 is indicated by an arrow. The
third (WT) and fourth (MT)
lanes represent the addition of a 100-fold excess of cold
competitor oligonucleotides. The WT oligonucleotide (sc-2507; Santa
Cruz Biotechnology) is a 25-bp oligonucleotide that contains the E2F1
consensus (TTTCGCGC), whereas the MT oligonucleotide (sc-2508; Santa
Cruz Biotechnology) possesses an inactivating dinucleotide substitution
(TTTCGATC). In the final lane
(Anti-E2F1), 100 ng of E2F1 antibody was included in the
binding reaction, and an arrow indicates the supershifted
E2F1-DP1 complex.
|
|
As a complementary approach to ChIP assays, an EMSA was performed to
determine whether recombinant E2F1-DP1 would bind the cyclin D3
202/+6 promoter region in vitro. Fig. 4B
demonstrates that recombinant E2F1-DP1 present in extracts of
Sf9 cells co-infected with E2F1- and DP1-expressing
baculoviruses (38) binds to the cyclin D3
202/+6 promoter fragment,
whereas no such binding activity is detectable in uninfected Sf9
cell extracts. The addition of an excess of double-stranded
oligonucleotides containing a consensus E2F1 binding site (WT)
abolished formation of the E2F1-DP1 complex on the cyclin D3 promoter,
whereas the addition of a mutated version (MT) of the consensus
oligonucleotide had no effect. The addition of an E2F1 antibody (100 ng) resulted in the retarded mobility of the putative E2F1-DP1-cyclin
D3 promoter complex. Together, these data support the conclusion that
E2F1 activates cyclin D3 via direct binding to the promoter in a manner
dependent upon its transcriptional activation domain.
Identification of the E2F1 Regulatory Region of the Cyclin D3
Promoter--
To further define the E2F1-responsive region of the
cyclin D3 promoter, we generated several additional promoter
constructs. Constructs
202/
146,
202/
134, and
202/
112
possessed deletions from the 3'-end of the promoter. Fig.
5A reveals that deletion of
sequences downstream of
134 did not abolish induction by E2F1; in
fact, constructs
202/
134 and
202/
112 were both activated nearly
4-fold by E2F1. However further deletion to
146 (
202/
146) abolished the E2F1 response. Constructs
159/+6 and
115/+6 had additional nucleotides deleted from the 5'-end of the promoter. Fig.
5A reveals that deletion of sequences upstream of
159
(
159/+6) had little effect on the E2F1 response. In contrast, further
deletion of sequences upstream of
115 (
115/+6) abolished E2F1
responsiveness. Taken together, these results demonstrate that the
critical E2F1-responsive element resides within a highly GC-rich region
between
159 and
134.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 5.
The E2F1 regulatory element is located
between 143 and 135 of the cyclin D3 promoter. A,
luciferase reporter assays were used to compare the response of the
indicated cyclin D3 promoter constructs with the expression of E2F1.
B, EMSAs were used to compare the binding affinity of
recombinant E2F1-DP1 to the indicated fragments of the cyclin D3
promoter. Probes were labeled and normalized, and equal counts of
radioactivity were loaded in each panel (10,000 cpm/lane).
Oligonucleotide competition and antibody supershift assays were
performed as in Fig. 4. C, a series of 15-bp partially
overlapping oligonucleotides spanning the E2F1 regulatory region of the
cyclin D3 promoter were used in competition assays. WT and MT are the
25-bp oligonucleotides used in Fig. 4. C15 corresponds to a 15-bp
double-stranded oligonucleotide containing a consensus E2F site, which
serves as a positive control. Although C15 has the same consensus
sequence as WT, it is 10 bp shorter, accounting for the reduced affinity of E2F1 to C15 relative to
WT. Oligonucleotides 1-7 are the individual sequences used as
competitors at 80, 160, and 320 ng/µl reaction, respectively, with
increasing amounts indicated by the ramp above
the lanes. D, data from Figs. 4 and 5 are
summarized. In D, BA represents binding activity,
LA represents luciferase reporter activity, and the
stars indicate the major transcription start sites. In the
bottom element, the boxed
sequence represents the essential E2F1 regulatory element of
the cyclin D3 promoter, and arrows indicate major
transcription start sites (30). Comparison of the consensus E2F binding
sequence and the essential E2F site of the cyclin D3 promoter reveal an
identical CGCGC core but lack of TTT trinucleotide in the cyclin D3
promoter.
|
|
Despite the fact that the
159 to
135 region clearly accounts for
the E2F1 responsiveness of the cyclin D3 promoter, this region does not
contain a consensus E2F binding site (TTTCGCGC). To demonstrate that
this region nonetheless accounts for E2F binding to the D3 promoter,
additional EMSAs were performed using various cyclin D3 promoter
fragments as probes. Fig. 5B demonstrates that all fragments
containing the
159/
135 region (
202/+6, 202/
134,
202/
112,
and
159/+6) bind recombinant E2F1. On the other hand, fragment
202/
146 that lacks the
159/
135 sequence does not bind
recombinant E2F1-DP1. Although fragment
115/+6 binds E2F1 in EMSA, it
is not responsive to E2F1 in luciferase reporter assays. This suggests
that whereas there is at least one functional E2F1 binding site with
the region encompassed by the
115/+6 construct, this region is
insufficient to mediate a transcriptional response in the absence of
the
159/
135 region. Thus, the
159/
135 region of the cyclin D3
promoter contains the critical E2F regulatory element.
Since the GC-rich
159/
135 region has no consensus E2F binding site,
a series of oligonucleotides spanning this short region were examined
in competition assays to empirically determine the best E2F binding
site within it. Each oligonucleotide in this series was 15 bp in length
and overlapped its 5' and 3' neighbors by 10 bp and spanned from
146
to
111. These oligonucleotides were used as competitors in E2F
electrophoretic mobility shift assays that also utilized recombinant
E2F-DP1 as a source of DNA binding activity and a fragment of the
dihydrofolate reductase promoter as a labeled probe. The data in Fig.
5C reveal that of the seven oligonucleotides tested,
oligonucleotide 2 (
145 to
131) was by far the best competitor for
E2F binding to the dihydrofolate reductase probe. In fact,
oligonucleotide 2 was nearly as good of a competitor as the C15
oligonucleotide that contains a consensus E2F1 binding site (TTTCGCGC).
Oligonucleotide 1, which overlaps oligonucleotide 2 by 10 bp, also
competed for E2F binding but was clearly less active than
oligonucleotide 2. Oligonucleotides 3-7 appeared to lack competitor
activity even at the highest doses used. The sequence common to
oligonucleotides 1 and 2 was 5'-GGCGCGCGCG-3'.
To determine that the DNA sequence common to oligonucleotides 1 and 2 was indeed the central E2F1 regulatory element of the cyclin D3
promoter, this putative binding site was removed, and the resulting
construct (
159/+6(del9)) that lacks
143 to
135 was tested for
promoter activity. As expected, Fig. 5A reveals that this
construct retained only a modest 2.3-fold induction by E2F1 as compared
with a 6-fold induction for an identical construct
159/+6 which
contains the putative E2F element. To determine whether this region is
sufficient in and of itself to support an E2F1 response, a final
construct (
146/
111) that contains the E2F regulatory element and
three transcriptional start sites was examined. Fig. 5A
reveals that this region is not sufficient to mediate transcriptional
activation by E2F1 although it is sufficient to bind recombinant E2F1
(see Fig. 5B). Thus, it appears that this element binds E2F
and is essential for a majority of the E2F1 responsiveness of the
cyclin D3 promoter; however, it is not sufficient to mediate the E2F
response and probably cooperates with other promoter elements including
other nonessential E2F binding sites.
 |
DISCUSSION |
The central role of E2F in cell growth control was firmly
established 10 years ago by the demonstration that ectopic expression of E2F1 could drive quiescent fibroblasts into S phase (26). Subsequent
work has shown that E2F2 and E2F3A are also potent inducers of S phase
(21, 31). Ironically, studies have found that certain members of the
E2F family are also potent inducers of apoptosis (21, 31, 39, 40),
demonstrating that there is a delicate, although poorly understood,
balance between cellular proliferation and survival. In an attempt to
understand how members of the E2F family control cell fate, a number of
recent studies have measured global gene expression following induction
of various members of the E2F family (22-24, 41, 42). Not
surprisingly, these studies implicate members of the E2F family in the
regulation of many genes involved in the G1 to S phase
transition, including replication activities such as replication
protein C, DNA primase, DNA topoisomerase, flap endonuclease, and DNA ligase.
In our own microarray work (24), we identified cyclin D3 as an
activated target of E2F1. This observation was something of a surprise
for three reasons. First, in most cell cycle models, the stimulation of
cyclin D3 in early and mid-G1 temporally precedes activation of the S phase-promoting E2Fs at the G1 to S
phase boundary. Second, previous work has carefully documented
repression of the cyclin D1 promoter by E2F1 (43) and by Myc (44, 45). Repression of cyclin D3 by E2F, and Myc has been understood to represent a negative feedback loop. Third, consensus E2F binding sites
are not conserved in the cyclin D3 promoters of mice (46), rats (47),
and humans (30), although the promoters show over 70% sequence
identity within the 700-bp region upstream of their translational start sites.
Due to the essential role played by the D-type cyclins in cell growth
control, we considered it of interest to validate our microarray data
and to determine the mechanism by E2F1 activates cyclin D3 expression.
Thus, we constructed an H1299 cell line that expresses a HT-inducible
ER-E2F1 fusion protein to verify that activation of cyclin D3 by E2F1
is direct and not an indirect consequence of E2F1 expression (Fig. 1).
Experiments in which this ER-E2F1 fusion protein is induced in the
presence and absence of cycloheximide (Fig. 2) strongly support the
hypothesis that E2F1 directly activates the endogenous cyclin D3
promoter. Further experimentation determined that E2F1, as well as
several other members of the E2F family, activates transcription of the
cyclin D3 promoter (Fig. 3). Binding of endogenous E2F1 to the cyclin D3 promoter in vivo was confirmed by CHIP assay, and direct
binding of recombinant E2F1-DP1 to the cyclin D3 promoter was confirmed by EMSA (Fig. 4). The E2F-responsive region of the cyclin D3 promoter by E2F1 was mapped to positions
143 to
135 within a GC-rich region
of the promoter that contains several of the cyclin D3 transcription
start sites (Fig. 5). The E2F1 binding sequence of the cyclin D3
promoter matches the GC core of the E2F consensus sequence perfectly,
but it completely lacks the TTT of the TTTCGCGC consensus sequence (see
Fig. 5D). Although this site lacks the TTT of the consensus
E2F binding site, it nonetheless binds with comparable affinity as a
consensus sequence (C15 in Fig. 5C) to recombinant E2F1-DP1. It may be noteworthy that the E2F regulatory elements of a growing number of well characterized promoters (48-50), including the cyclin A promoter (51), also lack consensus E2F binding
sites and are GC-rich. Thus, our finding that the cyclin D3 promoter is
activated via a nonconsensus E2F binding site is not novel but does
further support the premise that sequence analysis alone is not
sufficient to identify E2F-responsive elements (52).
We have found no evidence that E2F1 increases the expression of either
cyclin D1 or D2. In fact, in H1299-ER-E2F1-15 cells we find that HT
treatment represses expression of both cyclins D1 and D2 (data not
shown). This observation is not unprecedented, since Myc is known to
repress the cyclin D1 promoter (44, 45) but to activate the cyclin D2
promoter (53, 54). The natural question that arises is "Why would
E2F1 or Myc up-regulate one G1 cyclin and repress
another?" We suggest that these observations reflect the fact that
different D-type cyclins respond to different transcription factors and
signaling pathways with the net outcome begin determined by the
relative contributions of various signals. In this model, cyclin D3 is
merely the D-type cyclin specifically wired to respond to positive
feedback from E2F during the course of a cell cycle.
In conclusion, we have confirmed that cyclin D3 is indeed a direct
target of E2F, thus validating the power of genome-wide technologies to
identify individual E2F targets. These findings add cyclin D3 to the
list of well characterized E2F-activated cyclins that includes cyclins
A1 (51) and E1 (9). By analogy to the ability of E2F1 to activate
cyclin E1, the most straightforward interpretation of this work is that
E2F-mediated activation of cyclin D3 contributes to the phosphorylation
of Rb, thereby contributing to the potent ability of E2F to drive the
G1 to S phase transition.