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Journal of Virology, March 2000, p. 2084-2093, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Induction of the Cellular E2F-1 Promoter by the
Adenovirus E4-6/7 Protein
Joel
Schaley,
Robert J.
O'Connor,
Laura J.
Taylor,
Dafna
Bar-Sagi, and
Patrick
Hearing*
Department of Molecular Genetics and
Microbiology, School of Medicine, State University of New York,
Stony Brook, New York 11794
Received 2 August 1999/Accepted 30 November 1999
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ABSTRACT |
The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with
different members of the E2F family and mediates the cooperative and
stable binding of E2F to a unique pair of binding sites in the Ad5 E2a
promoter region. This induction of E2F DNA binding activity strongly
correlates with increased E2a transcription when analyzed using virus
infection and transient expression assays. Here we show that while
different adenovirus isolates express an E4-6/7 protein that is capable
of induction of E2F dimerization and stable DNA binding to the Ad5 E2a
promoter region, not all of these viruses carry the inverted E2F
binding site targets in their E2a promoter regions. The Ad12 and Ad40
E2a promoter regions bind E2F via a single binding site. However, these
promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3
E2a promoter region binds E2F very poorly, even via a single binding
site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with
this notion, we show that infection with different adenovirus isolates
induces the binding of E2F to an inverted configuration of binding
sites present in the cellular E2F-1 promoter. Transient expression of
the E4-6/7 protein alone in uninfected cells is sufficient to induce
transactivation of the E2F-1 promoter linked to chloramphenicol
acetyltransferase or green fluorescent protein reporter genes. Further,
expression of the E4-6/7 protein in the context of adenovirus infection
induces E2F-1 protein accumulation. Thus, the induction of E2F binding
to the E2F-1 promoter by the E4-6/7 protein observed in vitro
correlates with transactivation of E2F-1 promoter activity in vivo.
These results suggest that adenovirus has evolved two distinct
mechanisms to induce the expression of the E2F-1 gene. The E1A proteins
displace repressors of E2F activity (the Rb family members) and
thus relieve E2F-1 promoter repression; the E4-6/7 protein complements
this function by stably recruiting active E2F to the E2F-1 promoter to
transactivate expression.
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INTRODUCTION |
The transcription factor E2F plays a
pivotal role in the regulation of cellular proliferation in mammalian
cells and in Drosophila. Mammalian E2F DNA binding activity is a
heterodimer containing one of six known members of the E2F family
(E2F-1 through -6) in association with a partner from the DP family
(DP-1 or DP-2) (reviewed in reference 11). Different
E2F heterodimers are regulated by interactions with members of the
retinoblastoma gene family (pRb, p107, and p130) (reviewed in reference
37). E2F-1, -2 and -3-DP complexes bind to pRb,
E2F-4-DP heterodimers interact with pRb and p107, and E2F-5-DP is
preferentially bound by p130. The association of E2Fs with Rb family
members as well as the relative importance of different E2F complexes
varies with specific stages of the cell cycle (11, 37). In
growth-arrested cells, the predominant E2F activity is an E2F-5-p130
complex. In G1 phase of the cell cycle, E2F-Rb, E2F-p107
and E2F-p130 complexes are evident. The Rb family members are targets
of G1-phase cyclin/cyclin-dependent kinases, and their
phosphorylation results in their release from E2Fs (11, 37).
E2F-1-DP heterodimers are important for cellular proliferation with
cells entering the cell cycle from a G0 state, whereas
E2F-3-DP complexes are important for cell cycle progression with
continuously proliferating cells (29, 34, 56).
E2F binds to the promoter regions of a number of cellular genes
involved in DNA synthesis and regulation of the cell cycle. E2F binding
sites in the b-myb, cdc2, cdc6,
c-myc, cyclin A, cyclin E, dhfr, E2F-1,
orc1, and thymidine kinase gene promoters have been shown to
be involved in the induction of transcription in resting cells
following serum stimulation (2, 3, 9, 18, 25, 28, 33, 40, 44, 45,
50, 55). Ectopic E2F-1 expression is sufficient to induce
quiescent cells to enter S phase, although unregulated E2F-1
overexpression may result in the induction of p53-mediated apoptosis
(22, 23, 29, 31, 32, 46, 47, 51, 59). Inhibition of cellular
proliferation, a hallmark of normal Rb function, is relieved when the
Rb-E2F interaction is disrupted by mutation of Rb or dissociation of Rb
from E2F by the DNA tumor virus transforming proteins large T antigen,
E1A, or E7 (reviewed in references 8 and
37). The Rb family members bind to sequences in E2Fs
that overlap the activation domains of these transcription factors
(7, 14, 20, 43), and a model whereby Rb family proteins
repress E2F activities by physically masking the transactivation
domains can be easily envisioned. Further, Rb family members act as
dominant repressors of promoter regions to which E2F-Rb complexes are
linked by virtue of recruitment of complexes containing histone
deacetylases (1, 4, 5, 12, 35, 57). Thus, E2F binding sites serve to repress as well as to activate cellular promoters, depending on the nature of the E2F complexes found in the cell. These
observations may explain why E2F-1 may act as either an oncogene or a
tumor suppressor, depending on the context in which activity is
analyzed (13, 27, 53, 60, 61).
E2F was first described as a nuclear activity that bound to an inverted
repeat in the adenovirus type 5 (Ad5) E2a promoter (30). The
binding activity of E2F to these sites is stimulated by Ad infection
dependent on the activities of two viral proteins. E2F transcriptional
activity is positively regulated by the Ad E1A gene products which
avidly bind to Rb family members and dissociate them from E2Fs
(8). Free E2F is then bound by the Ad E4-6/7 protein, which
forms a complex with E2F and induces the cooperative and stable binding
of E2F to the inverted binding sites in the Ad5 E2a promoter (Fig.
1A) (16, 17, 26, 36, 48). The induction of E2F binding to the Ad5 E2a promoter in vitro is directly correlated with transcriptional activation of the E2a promoter in vivo
(38, 39, 41, 42, 49). The dissociation of Rb family members
from E2Fs by the E1A proteins also results in activation of cellular
genes containing E2F response elements and the induction of cell cycle
progression (8). In this report, we show that while
different Ad isolates are capable of induction of E2F dimerization and
DNA binding, not all of these viruses carry the inverted binding site
targets in their E2a promoter regions. We hypothesize that these
viruses may have evolved E4-6/7 products in order to activate other
E2F-responsive genes; in confirmation of this idea, we demonstrate that
different Ad isolates induce E2F binding to an inverted binding site in
the cellular E2F-1 promoter. This induction of E2F binding results in
transactivation of the cellular E2F-1 promoter and an increase in E2F-1
protein levels. These results suggest that Ad has evolved two distinct
mechanisms to induce expression of the E2F-1 gene. The E1A proteins
displace repressors of E2F activity (the Rb family members) and thus
relieve E2F-1 promoter repression; the E4-6/7 protein complements this
function by stably recruiting active E2F to the E2F-1 promoter to
transactivate expression.
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FIG. 1.
(A) Model for Ad induction of E2F DNA binding. The left
depicts interaction of the E4-6/7 protein with free E2F-DP
heterodimers. The right depicts the induction of E2F DNA binding to the
Ad5 E2a promoter region by E4-6/7 protein-mediated dimerization. The
inverted E2F binding sites in the Ad5 E2a promoter are indicated by
inverted arrows. (B) Alignment of E4-6/7 proteins from different Ad
serotypes. E4 ORF7 of the Ad5 E4-6/7 protein is shown at the top in
alignment with homologous sequences found in Ad12, Ad40, and Ad9. Dark
letters indicate amino acid identities; conserved amino acid changes
are indicated by asterisks across the top. The two regions in Ad5
E4-6/7 required for stable interaction with a E2F-DP heterodimer are
indicated at the top of the sequence (termed E2F interaction), while
the region of E4-6/7 that directs dimerization is shown at the bottom
of the sequence (termed E2F induction).
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MATERIALS AND METHODS |
Extract preparation and gel mobility shift assays.
Nuclear
and cytoplasmic extracts were prepared according to the method of
Dignam and Roeder (10). The cytoplasmic supernatant obtained
after isolation of the nuclei was adjusted to 100 mM KCl, spun at
100,000 × g for 1 h, and saved as the cytoplasmic fraction. Cytoplasmic and nuclear fractions were dialyzed against DB
(20 mM HEPES [pH 7.5], 100 mM KCl, 10% glycerol, 5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride [PMSF]), and the dialysate was cleared
by centrifugation at 25,000 × g. In vitro DNA binding
assays were performed as described previously (43). Briefly,
binding reaction mixtures (20 µl) contained 5 to 10 µg of nuclear
or cytoplasmic extract, 2 µg of sonicated salmon sperm DNA, and
20,000 cpm (double-site probe) or 40,000 cpm (single-site probe) of
32P-labeled E2F recognition sites (1 to 2 fmol of DNA) in
DB supplemented with Nonidet P-40 (final concentration, 0.1%). Binding
reactions were incubated for 1 to 2 h at room temperature,
followed by electrophoresis on a 4% 30:1 polyacrylamide gel run in
0.5× Tris-borate-EDTA at 4°C. The Ad5 E2a E2F double-site probe
contains nucleotides 
30 to 73 from the Ad5 E2a promoter plus
additional vector sequences; the sequence of one strand is
5'-AATTCGTAGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTAGTCCCGG-3'. The E2F sites are underlined, and vector sequences are in
italics. The E2F single-site probe contains Ad nucleotides 270 to 293 from the E1A enhancer and flanking vector sequences; the sequence of one strand is
5'-AATTCCCCCATTTTCGCGGGAAAACTGAATCCTCGA-3'.
The cellular E2F-1 promoter probe corresponds to nucleotides 44
to 12 (25, 28); the sequence of one strand is
5'-AATTCCGGGCTCTTTCGCGGCAAAAAGGATTTGGCGCGTAAAAGG-3'. The sequences of one strand of the probes for the E2a promoter regions of different Ad serotypes are as follows: Ad3,
5'-AATTCGTGATTCCGCCGTTTTCAAAATGAGCGCGGGCAAGGGCTACTC-3'; Ad12,
5'-AATTCGTCACTTTTCCCGCCTGTTGAAAGTCCGCGCGCGGGCTTTTTTACTC-3'; and Ad40,
5'-AATTCGTAATTTGGCGCCTAAAAAAAGCGCGGGTGTTTAGTC-3'.
Probe fragments were labeled with [
-32P]dATP and
Klenow DNA polymerase; specific activities were 5,000 to 10,000 cpm/fmol.
For bacterial expression of E4-open reading frame 7 (ORF7), a 50-ml
overnight culture of
Escherichia coli DH5 transformed
with
pGEX-ORF7 (Ad5 or Ad40) was grown overnight to saturation.
A 500-ml
culture was then inoculated and allowed to grow for 1
h.
Isopropyl-
-
D-thiogalactopyranoside was added to 0.1 mM,
and
incubation was continued for 4 h. Bacteria were harvested,
washed
with phosphate-buffered saline-(PBS) 1 mM PMSF, and resuspended
in 25 ml TNE (10 mM Tris [pH 7.5], 100 mM NaCl, 1 mM EDTA) containing
10 mM dithiothreitol, 1 mM PMSF, and 1% Triton X-100. Aliquots
were
sonicated, and soluble material was recovered following centrifugation
at 25,000 ×
g for 20 min. The soluble pool was loaded
on a column
of glutathione-agarose (Sigma), the bound material was
washed
with DB-100, and glutathione
S-transferase
(GST)-ORF7 protein
was eluted in DB-100-20 mM reduced
glutathione.
Virus infections, transfection and microinjection assays, and
immunofluorescence assay.
A549 cells were maintained in
Dulbecco's modified minimal essential medium (DMEM) containing 10%
calf serum. REF52 and ATCC HeLa cells were maintained in DMEM
containing 10% fetal bovine serum (FBS). HepG2 cells were maintained
in minimal essential medium containing 10% FBS. Different Ad serotypes
were purchased from the American Type Culture Collection and propagated
on A549 cells. Purified virus particles were purified by CsCl
equilibrium gradient centrifugation following standard approaches
(54). A549 cells were infected at a multiplicity of 500 particles/cell for 1 h. Nuclear and cytoplasmic extracts were
prepared 6 to 8 h after infection.
Plasmid DNA transfections were performed by the calcium phosphate
precipitation procedure (
58). HepG2 cells were split and
plated the day before transfection. The following day, the cells
were
transfected with 1 µg of the E2a-CAT reporter vector or 2
µg of the
E2F-1-CAT reporter plasmid DNA, 18 µg of salmon sperm
DNA, and
various amounts of effector plasmid per 100-mm-diameter
dish, as
indicated in the text. The cells were incubated for 4
h with the
calcium phosphate precipitate, the medium was removed,
and
Tris-buffered saline solution containing 20% glycerol was
added for 1 min. The cells were then washed three times with Tris-buffered
saline,
and fresh medium containing 10% serum or 0.5% serum was
added, as
indicated in the text. Total cell extracts were prepared
24 h
later, and chloramphenicol acetyltransferase (CAT) enzymatic
activity
in cellular extracts was assayed using a fluorescent
chloramphenicol
substrate (FAST-CAT; Molecular Probes, Eugene,
Oreg.). CAT activity was
quantified with a phosphorimager. The
results presented represent the
average of five experiments. The
E2a-CAT vector was previously
described (
42). The E2F-1-CAT
vector was constructed from a
plasmid provided by Peggy Farnham
(University of Wisconsin) and
contains positions
176 to +36 of
the murine E2F-1 promoter region
(
25) linked to the CAT gene.
The E2F-1-GFP (green
fluorescent protein) plasmid contains the
same segment of the murine
E2F-1 promoter linked to the GFP coding
region (
62).
For microinjection experiments, REF52 cells plated on gridded glass
coverslips were grown to subconfluency and subsequently
serum starved
for 36 h in DMEM containing 0.5% FBS. DNA was prepared
by
dilution of microinjection buffer (50 mM HEPES [pH 7.2], 100
mM KCl,
5 mM NaH
2PO
4). E2F-1-GFP (25 ng/µl, along
with empty cytomegalovirus
[CMV] vector [25 ng/µl]) was injected
into the nucleus followed
by maintenance of the cells in DMEM
containing 0.5% FBS or with
serum stimulation in DMEM containing 20%
FBS. Cells coinjected
with E2F-1-GFP and CMV-E4-6/7 (
42)
(each at 25 ng/µl) were maintained
in DMEM containing 0.5% FBS.
Cells were fixed 16 h after microinjection
in 3.7% formaldehyde
for 1 h and washed three times in PBS prior
to slide mounting. GFP
fluorescence was observed using a fluorescein
filter on an Axiovert 135 microscope (Zeiss). For immunofluorescence
assays, ATCC HeLa cells were
seeded on coverslips and infected
with 200 to 250 Ad particles/cell for
1 h. Coverslips were harvested
at 8 and 16 h after infection,
washed with PBS, fixed with 3%
formaldehyde, and permeabilized using
0.2% Triton X-100. Blocking
as well as primary and secondary antibody
application were performed
in PBS containing 2% bovine serum albumin.
Primary antibodies
included E4-6/7 monoclonal antibody M45
(
41) and a rabbit polyclonal
antibody specific to the C
terminus of E2F-1 (sc-193; Santa Cruz
Biotechnology). Secondary
antibodies were tetramethyl rhodamine
isothiocyanate-labeled anti-mouse
and fluorescein isothiocyanate-labeled
anti-rabbit conjugates. All
coverslips were processed with Fluoromount-G
(Southern Biotechnologies,
Inc.), and immunofluorescence was detected
as described above for
GFP.
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RESULTS |
Functional conservation of E4-6/7 proteins.
The importance of
the function of a viral protein in natural infections can be inferred
if that protein is encoded by the genomes of related, but
evolutionarily diverse, viruses. The E4 regions of Ad serotypes 2, 5, 9, 12, and 40 have been sequenced. We examined these regions for the
capacity to encode proteins homologous to the Ad5 E4 ORF7 coding
region, the segment of the Ad5 E4-6/7 protein sufficient to bind to E2F
and induce E2F dimerization on an inverted binding site
(42). Each of the viruses, representing five of the six Ad
subgroups, encoded hypothetical proteins with significant homology to
Ad5 ORF7 (Fig. 1B), including the two segments required for stable
binding to E2F (E2F interaction) and the region that mediates E2F
dimerization (E2F induction) (41). To determine if these
viruses exhibited the ability to induce stable E2F binding
characteristic of the Ad5 E4-6/7 product, nuclear extracts were
prepared from A549 cells infected with Ad3, Ad5, Ad9, and Ad12 and
analyzed by mobility shift assay using a probe corresponding to the Ad5
E2a inverted E2F binding sites. A549 cells were used for this
experiment since this cell line may be productively infected by all of
the Ad serotypes under study. These results are shown in Fig.
2A. Different E2F complexes were observed
using nuclear extract from uninfected A549 cells (lane 1) that
correspond to free E2F activity as well as E2F in association with pRb
and p107. The identification of the different E2F complexes was
confirmed using antibodies to E2F-1, E2F-4, and E2F binding partners
pRb and p107, which specifically supershift individual complexes
evident in lane 1 (data not shown). Infection of A549 cells with the
different Ad serotypes induced a new and abundant E2F complex in
comparison to nuclear extract from uninfected cells. The presence of
the Ad5 E4-6/7 protein in the Ad-E2F complex previously was confirmed
using monoclonal antibodies against this viral protein (26, 36,
48). These antibodies recognize the amino-terminal region of the
E4-6/7 protein which is variable among Ad serotypes; we found that the
anti-E4-6/7 monoclonal antibodies M45 and M41 (41) did not
recognize the Ad-E2F complexes formed with Ad serotypes other than 2 or
5 (data not shown). The Ad-induced complexes were efficiently competed
when a 500-fold molar excess of a specific E2F binding site was
included in the binding reaction when added at the same time as the
probe DNA. Each of the Ad-E2F complexes was stable to competitor
challenge when the competitor DNA was added after a 1-h preincubation
with probe DNA. Thus, each of the Ad serotypes tested induced a stable
E2F complex bound to an inverted E2F binding site characteristic of the
Ad5-induced E2F/E4-6/7 dimer. We note that the Ad12 E4-6/7 protein is
predicted to be 125 amino acids in length, in contrast to the
150-amino-acid Ad2 and Ad5 protein; this is consistent with the faster
mobility of the Ad12-induced E2F complex observed in lane 13.
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FIG. 2.
Induction of E2F DNA binding to the Ad5 E2a promoter by
different Ad serotypes. (A) Nuclear extracts from uninfected A549 cells
(Uninf.; lanes 1 to 3) or A549 cells infected with Ad5 (lanes 4 to 6),
Ad3 (lanes 7 to 9), Ad9 (lanes 10 to 12), or Ad12 (lanes 13 to 15) were
used in gel mobility shift assays with a probe corresponding to the Ad5
E2a inverted E2F binding sites. The first lane of each set (lanes 1, 4, 7, 10, and 13) represents a binding reaction with no specific
competitor DNA added. E2F complexes found in uninfected cells are
indicated on the left, and the Ad-induced E2F complex is indicated on
the right (Ad-E2F). In binding reactions shown in lanes 2, 5, 8, 11, and 14, a 500-fold molar excess of unlabeled DNA corresponding to an
E2F single binding site (see Materials and Methods) was added to the
binding reaction coincident with the addition of probe DNA. In binding
reactions shown in lanes 3, 6, 9, 12, and 15, a 500-fold molar excess
of unlabeled E2F single binding site DNA was added after a 60-min
preincubation of the binding reaction with probe DNA. The binding
reactions were incubated an additional 15 min before loading on the
gel. (B) Bacterially expressed E4 ORF7 proteins were used in binding
reactions with HeLa cell-free E2F activity. Lane 1 shows a binding
reaction with HeLa cell cytoplasmic extract plus the Ad5 E2a probe.
Free E2F binding activity is indicated on the left. In lanes 2 to 4, an
Ad5 GST-E4-ORF7 fusion protein was added to the HeLa cell extract prior
to the addition of probe DNA. In lanes 5 to 7, an Ad40 GST-E4-ORF7
fusion protein was added. Lanes 2 and 5 represent binding reactions
without the addition of specific competitor DNA. In lanes 3 and 6, a
500-fold molar excess of unlabeled E2F binding site was added
coincident with the probe as described for panel A. In lanes 4 and 7, a
500-fold molar excess of unlabeled E2F binding site was added after a
60-min binding reaction as described in for panel A.
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To confirm that the E4-6/7 protein from an Ad other than Ad2 and Ad5
was capable of E2F induction, the region homologous to
ORF7 of Ad40 was
isolated by PCR and the protein was expressed
as a GST fusion product
in bacteria. When incubated with uninfected
HeLa cell cytoplasmic
extract (an abundant source of free E2F
activity), bacterially
expressed Ad40 ORF7 stabilized E2F binding
on the Ad5 E2a inverted
binding site in the same manner as the
Ad5-encoded protein (Fig.
2B).
This experiment confirms that E4
ORF7 from different Ad serotypes is
sufficient to direct E2F dimerization.
To substantiate the relevance of
induction of E2F binding in vitro,
we tested promoter transactivation
by the E4 ORF7 segments from
Ad5 and Ad40 in vivo. A reporter vector
containing the Ad5 E2a
promoter linked to the CAT gene was transfected
into HepG2 cells
alone or with the E4 ORF7 expression vectors. HepG2
cells were
used for this experiment since the E2a promoter displays
very
low basal activity in this cell line. Significant transactivation
of the E2a promoter (12.8 ± 2.9- and 5.8 ± 0.8-fold above
the
basal level) was observed with both Ad5 and Ad40 E4 ORF7 products.
We conclude that Ad isolates from different subgroups have conserved
the ability to induce E2F binding to an inverted E2F binding site
and
induce expression from a promoter region containing such binding
sites.
E2a promoter structure does not correlate with E4-6/7
function.
Induction of E2F DNA binding by E4-6/7 in vitro strongly
correlates with increased E2a gene expression in vivo with Ad5
(38, 39, 41, 42, 49). Based on the results described above, we anticipated that the E2a promoter regions of different Ad serotypes would retain the inverted configuration of E2F binding sites found in
Ad5. Surprisingly, when we compared the E2a promoters of Ad serogroups
where the E2a sequence has been determined, this was not found to be
the case (Fig. 3A). While the binding
site for transcription factor ATF, the TATA box, and the start site
regions were highly conserved between Ad serotypes, the interval
containing the putative E2F sites was not. Changes in both the upstream
and downstream E2F consensus binding sites (5'-TTTCGCGC-3')
as well as alterations in the spacing between these two sites,
were evident. It is well established that the correct spacing between
the inverted E2F sites in the Ad5 E2a promoter is critical for
E4-6/7-mediated induction of E2F DNA binding (17, 48).
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FIG. 3.
E2a promoter regions of different Ad serotypes do not
contain inverted E2F binding sites. (A) Alignment of the E2a promoter
regions from different Ad serotypes. The ATF binding site, TATA box,
and potential E2F sites are indicated at the top. Alignment of E2a
promoters shows strong conservation of the ATF site, the TATA box, and
start site region. The ATF-proximal and TATA-proximal potential E2F
sites are shown in a 5'-to-3' orientation in the insets above and below
the sequence. (B) Binding reactions contained HeLa cell E2F and
Ad5-induced E2F with probes corresponding to the E2a regions of
different Ad serotypes. Lanes 1 to 5 show binding reactions with HeLa
cell-free E2F activity and DNA probes corresponding to an E2F
single-site (E2F ss) and E2a regions of Ad5, Ad3, Ad12 and Ad40, as
indicated above the lanes. Lanes 6 to 10 show binding reactions with
Ad5-infected HeLa cell nuclear extract with the same set of probes as
described for lanes 1 to 5. The Ad-induced E2F complex is indicated in
the right (Ad-E2F). The faster-migrating complex is not specifically
competed by an E2F binding site and is an unknown binding activity that
serves as an internal control for the integrity of each probe DNA. (C)
The binding of HeLa cell free E2F to the E2a regions of different Ad
serotypes was analyzed using competition binding assays. Lane 1 shows a
binding reaction using HeLa cell-free E2F activity with an E2F
single-site probe. Free E2F binding activity is indicated on the left.
Lanes 2 to 16 show binding reactions where increasing molar
concentrations (5-, 25-, and 125-fold molar excess to the probe DNA) of
specific competitor DNAs were added coincident with the probe DNA. The
cold competitors corresponded to an E2F single site identical to the
probe DNA (lanes 2 to 4) and to E2a DNA of Ad5 (lanes 5 to 7), Ad3
(lanes 8 to 10), Ad12 (lanes 11 to 13), and Ad40 (lanes 14 to 16).
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To test if the different Ad E2a promoter regions were capable of
binding E2F and Ad-induced E2F activity, two approaches were
taken.
First, synthetic oligonucleotides corresponding to the
putative E2F
binding sites of the different E2a promoter regions
were used as probes
in mobility shift assays with nuclear extract
prepared from
Ad5-infected Hela cells (Fig.
3B, lanes 6 to 10).
The boundaries of the
sequences chosen for probes in this analysis
overlapped with the 3'
part of the highly conserved ATF binding
site and the 5' part of the
highly conserved TATA box. Thus, the
entire intervening region was
represented in the probe DNA with
each Ad serotype. This assay showed
that whereas the Ad5 E2a sites
bound Ad-E2F in a significant manner,
the Ad12 and Ad40 sites
only weakly bound Ad-induced E2F activity and
no binding was evident
with the Ad3 E2a probe. When free E2F activity
from uninfected
HeLa cells was analyzed with the different probes, the
same pattern
of binding was found (Fig.
3B, lanes 1 to 5). In a second
approach,
the oligonucleotides corresponding to the different E2a
promoter
regions were used as competitor DNAs to test for E2F binding
(Fig.
3C). HeLa cell-free E2F was incubated with a probe corresponding
to a single E2F binding site with no specific competitor DNA in
the
binding reaction or increasing amounts of oligonucleotide
DNAs
corresponding to the different E2a promoters (Fig.
3C). These
results
demonstrated that Ad5, Ad12, and Ad40 regions were capable
of binding
E2F comparably when only a single E2F site is required
to compete for
binding, but that the Ad3 segment did not bind
E2F to a significant
level. We conclude from these experiments
that while the different Ad
serotypes evolved E4-6/7 proteins
that are capable of dimerizing E2F at
the Ad5 E2a inverted E2F
binding site, and the Ad40 E4 ORF7 product
effectively transactivates
the Ad5 E2a promoter (see above), the same
viruses did not evolve
promoter regions to take advantage of this
induction.
Ad induction of E2F binding to the cellular E2F-1 promoter.
An
intriguing explanation of these apparently contradictory observations
is that a physiologically relevant target of the E4-6/7 protein is of
cellular origin. This possibility stipulates that the putative cellular
target gene(s) contains an inverted configuration of E2F binding sites
that forms a stable DNA bound E2F complex with E4-6/7. The promoter
regions of the human and murine E2F-1 genes contain two pairs of
overlapping E2F sites with a configuration similar to that found with
the Ad5 E2a promoter region (Fig. 4A)
(25, 28). The E2F-1 promoter sequence shown in Fig. 4A is
identical in human and mouse cells. The spacing between the E2F binding
sites is 3 bp closer than that found with the Ad5 E2a promoter. To
determine if the cellular E2F-1 promoter could form a stable E2F
complex with E4-6/7, an E2F-1 promoter probe corresponding to this
region was used. Ad5-infected HeLa cell extract was incubated with the
E2F-1 promoter probe in comparison to an Ad5 E2a probe and analyzed by
mobility shift assay (Fig. 4B). A series of complexes were detected
with the E2F-1 promoter probe, but only the slowest-migrating complex
was stable to competitor challenge in an off-rate analysis (10-, 20-, and 40-min challenges with a 500-fold excess of specific E2F site
competitor DNA) in a manner identical to the Ad5 E2a probe fragment.
That this slowly migrating complex contained the Ad5 E4-6/7 protein was
verified using a specific monoclonal antibody (41) to this
viral gene product (data not shown). When extracts from Ad3-, Ad9-, and
Ad12-infected A549 cells were analyzed with the E2F-1 promoter probe, a
similar complex that was stable to competitor challenge was found (Fig. 4C). We conclude that different Ad serotypes induce stable E2F binding
to the cellular E2F-1 promoter.
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FIG. 4.
Ad induction of E2F binding to the cellular E2F-1
promoter. (A) Nucleotide sequence comparison of the Ad5 E2a promoter
E2F binding sites and similar sites found in the cellular E2F-1
promoter. Inverted E2F binding sites are indicated by arrows below the
sequences. (B) Binding of Ad-induced E2F to the E2a and E2F-1 binding
sites. Lanes 1 and 5 show binding reactions using Ad5-infected HeLa
cell extract with probes corresponding to Ad5 E2a (lane 1) or the
cellular E2F-1 promoter E2F binding sites (lane 5). The positions of
free E2F and the Ad-induced E2F (Ad-E2F) complexes are indicated on the
left. Following a 60-min binding reaction, a 500-fold molar excess of
unlabeled E2F single binding site was added, and aliquots of the
binding reaction were removed and loaded on the gel at the times
indicated above the lanes (10, 20, and 40 min after the addition of
specific competitor DNA). (C) Different Ad serotypes induce E2F binding
to the E2F-1 promoter. Nuclear extracts from uninfected (Uninf.) A549
cells or A549 cells infected with Ad3, Ad9, or Ad12 were incubated with
the probe corresponding to the E2F-1 promoter. Lanes 1, 4, 7, and 10 show a binding reaction without the addition of specific competitor
DNA. In lanes 2, 5, 8, and 11, a 500-fold molar excess of unlabeled E2F
single binding site was added coincident with the probe. In lanes 3, 6, 9, and 12, a 500-fold molar excess of unlabeled E2F binding site was
added after a 60-min binding reaction. The binding reactions were
incubated an additional 15 min before loading on the gel. The
position of the Ad-induced E2F complex (Ad-E2F) is indicated on the
right.
|
|
To test the functional consequence of Ad induction of E2F binding to
the E2F-1 promoter, three different assays were used.
First, a reporter
vector containing the cellular E2F-1 promoter
linked to the CAT gene
was transfected into HepG2 cells alone
or with increasing amounts of
E4-6/7 expression vectors (Fig.
5). Two
E4-6/7 proteins were tested in this analysis: E4-6/7 wild
type
(E4-6/7-WT) and E4-6/7-F125P, a point mutant that contains
a single
amino change at position 125 (
41). E4-6/7-F125P was
previously shown to bind efficiently to E2F, but it is defective
for
E2F dimerization. This F125P mutant protein was analyzed to
distinguish
between two possible mechanisms of E2F activation
in this assay: relief
of Rb family protein repression via displacement
of Rb by E4-6/7
interaction with E2F and E2F-1 promoter transactivation
via E4-6/7
dimerization of E2F DNA binding activity. The E4-6/7-WT
and
E4-6/7-F125P mutant proteins were shown to be equally stable
when
expressed in vivo (
41). The level of CAT activity was taken
as a measure of promoter induction. These results showed that
E4-6/7-WT
induced expression from the E2F-1 promoter in HepG2
cells to a
significantly higher level that the dimerization mutant
E4-6/7-F125P.
We attribute transactivation by the mutant protein
to relief of Rb
repression and the additional increase in activation
observed with
E4-6/7-WT protein to the induction of E2F dimerization
and DNA binding.
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FIG. 5.
Transactivation of the E2F-1 promoter by the E4-6/7
protein in HepG2 cells. HepG2 cells were transfected with a reporter
vector containing the cellular E2F-1 promoter fused to CAT. The
reporter vectors were transfected alone () or with increasing
concentrations (2, 10, and 50 ng) of a vector expressing Ad2 E4-6/7-WT
or E4-6/7-F125P. Following transfection, the cells were maintained in
medium containing 0.5% serum for 24 h. The level of CAT activity
in cellular extracts was then measured. The level of expression of the
reporter vector alone is set at 1, and the results with E4-6/7
coexpression are indicated as fold activation relative to the basal
level. The results represent the average of five experiments. Error
bars indicate standard deviations.
|
|
In a second assay, quiescent REF52 cells were microinjected with a
plasmid containing the E2F-1 promoter region linked to
the GFP coding
region with or without coinjection of the E4-6/7-WT
expression vector
(Fig.
6). REF52 cells were used for this
experiment
since a majority of the cells establish quiescence under the
experimental
conditions used and they are readily microinjected. GFP
fluorescence
was taken as a measure of E2F-1 promoter activity. In
quiescent
cells, the E2F-1 promoter is repressed by Rb family-E2F bound
complexes, and little GFP activity was evident. In contrast, the
E4-6/7
protein significantly induced E2F-1 promoter activity to
a level
similar to that found with serum induction. The dimerization
mutant
E4-6/7-F125P was capable of transactivation of the E2F-1-GFP
reporter
vector under these experimental conditions (data not
shown). This
result is consistent with the ability of E4-6/7 to
displace Rb from E2F
and thus relieve E2F promoter repression
(Fig.
5), particularly under
conditions of growth arrest where
the majority of E2F is bound by Rb
family members. The nonlinear
response of a GFP fluorescence signal in
relation to GFP protein
levels precludes accurate quantitative
comparison of the level
of induction by E4-6/7-WT versus E4-6/7-F125P,
although we note
that fewer microinjected cells showed increased GFP
fluorescence
with E4-6/7-F125P in comparison to E4-6/7-WT. We conclude
from
these analyses that the E4-6/7 protein can induce stable E2F
binding
to the cellular E2F-1 promoter via an inverted E2F binding site
in vitro and that this induction of E2F binding by E4-6/7 correlates
well with transactivation of the E2F-1 promoter by E4-6/7 in vivo.
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FIG. 6.
Transactivation of the E2F-1 promoter by the E4-6/7
protein in REF52 cells. Quiescent REF52 cells were microinjected with
the E2F-1-GFP reporter vector. Cells were coinjected with the
E2F-1-GFP vector and an empty CMV vector (a and b) or coinjected with
the E2F-1-GFP vector and an expression vector for the Ad2 E4-6/7
protein. In panels a and c, the cells were maintained in 0.5% serum;
panel b, the cells were treated with medium containing 20% FBS. GFP
activity was visualized 16 h after microinjection using a
fluorescein filter on an Axiovert 135 microscope (Zeiss).
|
|
In the third assay, endogenous E2F-1 protein levels were measured in
HeLa cells infected with Ad recombinants that express
different viral
E2F modulators. HeLa cells were used for this
experiment since they
contain a significant level of free E2F-1
activity, and thus relief of
Rb repression of E2F by the E4-6/7
protein should be minimized. HeLa
cells were infected with wild-type
Ad, an Ad that lacks the E1 region
but constitutively expresses
E4-6/7-WT, an Ad that lacks the E1 region
but constitutively expresses
E4-6/7-F125P, or an Ad that lacks E1 and
also contains a mutation
that disrupts the E4-6/7 protein. E4-6/7 and
endogenous E2F-1
protein levels were measured by immunofluorescence
using antibodies
specific to these products. E4-6/7 expression was
observed in
cells infected with viruses that express either E4-6/7-WT
or E4-6/7-F125P
(Fig.
7f to h), whereas
no signal was observed in cells infected
with a virus that lacks an
intact E4-6/7 reading frame (Fig.
7e).
Importantly, E4-6/7-WT but not
E4-6/7-F125P augmented the level
of endogenous E2F-1 activity to a
significant extent (compare
Fig.
7b and c with Fig.
7d). These results
confirm the transfection
and microinjection studies (Fig.
5 and
6) and
demonstrate that
E4-6/7-WT induced E2F-1 expression to a significantly
higher level
than the dimerization mutant E4-6/7-F125P, consistent with
transactivation
of the endogenous E2F-1 promoter. In related
experiments, the
viral E2a gene product DBP showed the same pattern of
induction
(data not shown), supporting the notion that the regulation
of
the E2a and E2F-1 promoter regions during infection is similar.
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FIG. 7.
Induction of E2F-1 protein levels by the E4-6/7 protein
in Ad-infected HeLa cells. HeLa cells were infected with wild-type
Adenovirus (expressing E1A and E4-6/7; b and f) or a recombinant that
lacks the E1 region and constitutively expresses E4-6/7-WT (c and g),
E4-6/7-F125P (d and h), or an E1-lacking virus that also contains a
mutation that disrupts the E4-6/7 protein (a and e). Cells were
harvested at 8 h after infection, and E2F-1 (a to d) and E4-6/7 (e
to h) were visualized by indirect immunofluorescence using antibodies
specific to these products.
|
|
| |
DISCUSSION |
The Ad E4-6/7 protein interacts directly with different members of
the E2F family and mediates the cooperative and stable binding of E2F
to a unique pair of binding sites in the Ad5 E2a promoter region
(16, 17, 26, 36, 48). This induction of E2F DNA binding
activity strongly correlates with increased E2a transcription when
analyzed in virus infection and transient expression assays (38,
39, 41, 42, 49). E2F heterodimers are typically found associated
with members of the Rb family of tumor suppressor proteins that repress
E2F activity. In resting cells, where very little free E2F activity is
present, these interactions are believed to preclude E4-6/7 binding
with E2F due to masking of the marked box region, a segment in E2F-1
required for binding to Rb and the E4-6/7 protein in vivo (7, 14,
19, 20, 43). Ad E1A proteins function to physically displace Rb
family proteins from E2Fs, thereby allowing association with E4-6/7
(8). The prevailing model of E4-6/7 protein function
proposes that E2F induction is an Ad-specific mechanism for recruiting
and sequestering E2F activity at a viral promoter region. The
experiments described in this report show that this model may not fully
represent the scope of E4-6/7 function during Ad infection.
Previous experiments with an Ad mutant that does not express a
functional E4-6/7 protein have shown that the mutant virus grows as
well as wild-type Ad5 in HeLa cells (15). Thus, while the
E4-6/7 protein augments E2a promoter activity via induction of E2F DNA
binding, this function of the E4-6/7 protein is not essential for Ad
growth in cultured cells. This appears to be due to the dominant nature
of the multifunctional E1A proteins exhibited in cultured cells. The
conservation of E2F induction by evolutionarily diverse Ad isolates
described in this report argues for a selective advantage for virus
growth provided by the E4-6/7 protein. While the precise nature of this
advantage is not yet certain, our results suggest that the induction of E2F binding to cellular, rather than viral, promoter regions may represent the basis for the selection of E4-6/7 activity. Whereas all
Ad isolates tested were capable of induction of E2F DNA binding activity, not all Ad E2a promoter regions recruit this induced E2F
activity. The Ad12 and Ad40 E2a promoter regions bind E2F via a single
binding site (Fig. 3C), yet the same promoter intervals very weakly
recruit dimerized (Ad-induced) E2F activity (Fig. 3B). The Ad3 E2a
promoter region binds E2F very weakly, even via a single binding site.
It remains possible that the E4-6/7 proteins from diverse Ad serotypes
such as 3, 12, and 40 have evolved stable interactions with E2F and
other distinct transcription factors in their promoter regions. While
we can not preclude this possibility, experiments to test this idea
using matched infected-cell nuclear extracts and E2a promoter regions
of different Ad serotypes did not reveal any novel Ad-induced DNA
binding complexes (data not shown).
Our data show that adenovirus infection induces the binding of E2F to
an inverted configuration of binding sites in the cellular E2F-1
promoter. This induction of E2F binding observed in vitro (Fig. 4B and
C) correlates well with the observed transactivation of this promoter
by E4-6/7 expression in vivo (Fig. 5 to 7). The results from both
transient expression assays (Fig. 5 and 6) support the notion that
E4-6/7 can effectively modulate transcription from E2F-responsive
promoter elements. However, the reliability of making direct inferences
about endogenous promoter activity based solely on heterologous
transient expression assays prompted our examination of endogenous
E2F-1 activity in Ad-infected cells (Fig. 7). Indeed, we found a
striking induction of E2F-1 protein levels following expression of
E4-6/7-WT protein but not the dimerization mutant E4-6/7-F125P,
supporting the idea that this increase is due to transactivation of the
endogenous cellular E2F-1 promoter. The transactivation of the cellular
E2F-1 promoter by the E4-6/7 protein appears to complement the relief
of repression of this promoter, driven by E1A protein-mediated release
of Rb-repressive complexes from endogenous E2F activity. In addition,
the ability of E4-6/7-F125P to transactivate E2F-1 promoter expression
(Fig. 5) likely reflects the capacity of this protein to displace Rb from E2F-1 via competition for overlapping binding regions, i.e., the
marked box region of E2F-1 (19, 21, 43). The inverted configuration of E2F binding sites found in the E2F-1 promoter that
serve as a target for E4-6/7-induced E2F DNA binding activity may
represent a fortuitous arrangement of these elements. Indeed, while a
number of cellular promoter regions carry multiple E2F binding sites,
this is the only cellular promoter region that we have identified that
contains the inverted configuration of E2F sites and that stably binds
Ad-induced E2F activity. It is possible that a subset of cellular
E2F-responsive promoter regions carry such inverted E2F binding sites
and that these sites may be targets of specific E2F protein complexes.
Perhaps the E4-6/7 protein has evolved as an analogue of a cellular
gene product which serves the same function. Considering the centrol
role that the E2F-1 gene product plays in the regulation of cell cycle
progression, a novel regulatory circuit may control its expression.
Finally, why have only certain Ad serotypes evolved inverted E2F
binding sites that are capable of recruiting Ad-induced E2F activity?
Ad2 and Ad5 were originally isolated from long-term cultures of adenoid
tissue. A large proportion of the cell mass of adenoids consists of
lymphoid cells. Group C viruses (Ad1, Ad2, Ad5, and Ad6) establish
persistent infections of both lymphoid and monocyte cultures in vitro
(6, 52). In addition, DNA sequences of group C Ad have been
isolated from peripheral blood lymphocytes from healthy individuals
(24). While not yet proven, these results have suggested
that group C Ad isolates establish persistent infections of circulating
lymphocytes. The stable binding of E2F-E4-6/7 complexes to the viral
E2a promoter region may help to mediate viral DNA replication in these
cells via activated expression of the replication genes driven by the
E2a promoter. When free E2F activity is available in late
G1 and early S phase, the resident Ad in a latent infection
may need an active mechanism to recruit E2F to the viral E2a promoter.
In this situation, limiting viral genomes are in competition with
numerous cellular E2F binding sites for E2F activity. This may contrast
with lytic infection where amplified viral DNA may readily compete for
limiting concentrations of cellular transcription factors to allow for
efficient E2a transcription. This very speculative possibility awaits
further investigation.
| |
ACKNOWLEDGMENTS |
The first two authors contributed equally to this work.
We thank Mike Hayman, Todd Miller, Nancy Reich, Peter Tegtmeyer,
and our laboratory colleagues for many helpful discussions, and we
thank Gia Feeney and Tina Philipsberg for excellent technical help. We
thank Bayar Thimmappaya for the E2a-CAT vector, Peggy Farnham for the
E2F-1 promoter clone, and Sergei Zolotukhin for the GFP clone.
This research was supported by Public Health Service grant CA28146 from
the National Cancer Institute to D.B. and P.H. R.J.O., J.S., and
L.J.T. were supported by NIH training grant CA09176.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics and Microbiology, School of Medicine, State
University of New York, Stony Brook, NY 11794. Phone: (631) 632-8813. Fax: (631) 632-8891. E-mail: phearing{at}ms.cc.sunysb.edu.
Present address: Department of Anatomy, Howard Hughes Medical
Institute, University of California, San Francisco, CA 94143.
| |
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Journal of Virology, March 2000, p. 2084-2093, Vol. 74, No. 5
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
