From the Division of Retroviral Gene Expression,
Research Program Applied Tumor Virology German Cancer Research
Center, Im Neuenheimer Feld 242, 69009 Heidelberg, Germany and
§ The State University of New York at Buffalo,
Department of Biochemistry, Buffalo, New York 14214
Received for publication, September 3, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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Gene expression of the internal and long terminal
repeat promoters of the spuma retrovirus is specifically activated by
the transactivator Bel1, the key regulator of viral gene expression. Bel1 directly binds to and activates DNA target sites of viral promoters and those of distinct cellular genes. To determine the contribution of cellular transcription factors to viral
transactivation, the viral internal promoter (IP) was analyzed by
transient expression, electrophoretic mobility shift assays),
and supershifts. Here we report that Bel1-mediated transactivation of
the full-length and shortened versions of the Bel1 response element
(BRE) were repressed by nuclear factor I (NFI). Electrophoretic
mobility shift assays using nuclear extracts from transfected 293T
cells revealed that different DNA-protein complexes consisting of DNA target sites of NFI and Bel1 proteins were formed. The specificity of
the repressor and transactivator DNA binding was shown by NFI- and
Bel1-specific antibodies that led to supershifts of the different nuclear protein-oligodeoxynucleotide complexes. The specificity of the
complexes was confirmed by using unlabeled, shortened, and mutated
IP.BRE oligodeoxynucleotides in competition experiments with the
authentic IP.BRE. Cotransfection of the infectious spumavirus DNA
genome with a human NFI-X1 expression plasmid into cell cultures greatly reduced the expression of viral structural and Bel1 proteins. These data demonstrate the relevance of NFI-mediated repression of
Bel1-driven transactivation in vivo.
Foamy viruses (FV)1 also
called spumaviruses belong to the orthoretroviruses and have
exceptional properties with respect to replication, pol expression, and
assembly (1, 2); for a review of the historical perspectives and the
apparent apathogenicity of FV, see Refs. 3 and 4. FV are unconventional
and complex retroviruses that code for transcriptional transactivators
(5, 6). The viral transactivators specifically recruit components of
the cellular transcription machinery to viral promoters so that
cellular transcription programs are specifically affected and even
reprogrammed in a way favorable for viral replication (7). A key
regulator of viral gene expression, the Bel1 transactivator of the
human foamy virus (HFV), recently renamed primate foamy virus) binds
directly to DNA target sites with no or low sequence conservation that
are located in both the 5'-long terminal repeat (LTR) and the internal
promoter (IP) (8-11). The IP is a strong early promoter that is
crucial for viral replication and critically dependent on Bel1
(11-13). Expression of Bel1 is initiated at the IP until threshold
levels of the Bel1 transactivator are reached which subsequently turn
on the 5'-LTR promoter to direct synthesis of virus structural proteins
(13-15). The transactivator Tas of simian foamy virus type 1 binds to
corresponding proximal and distal DNA target sites directly that are
not homologous to the Bel1 response element (BRE) of the IP (16). Bell
1 was shown to be a nuclear protein (17, 18) that contains at least two domains, a centrally located DNA binding region and a C-terminal activation domain (19). Bel1 has a molecular size of 36 kDa (20) and
can dimerize and multimerize (21). Alignments of Bel1 and Tas proteins
from different species result in a remarkably low degree of homology of
about 17-23% (22). The minimal IP.BRE of 27 bp is capable of directly
binding Bel1 and partial transactivation in vitro compared
with the full-length IP of 192 bp. High affinity DNA binding of Bel1
and full transactivation of the IP depend upon as yet unidentified
cellular factors.
Previously, a Kip2-BRE DNA element located within the second exon of
the human p57Kip2 gene was identified that
mediates a specific Bel1 transactivation (23). It is worth noting that
the Kip2-BRE primarily consists of direct repeats of three 14 mers that
partially overlap with the G base pattern conserved in both the IP.BRE
and Kip2-BRE (10, 23). The Kip2-BRE was bound by proteins in nuclear
extracts from 293T cells that had been transfected with Bel1 as shown
by EMSA and supershift experiments. A purified glutathione
S-transferase-Bel1 fusion protein was bound specifically to
Kip2-BRE, and the formation of the Bel1-Kip2-BRE complexes was
effectively blocked by the minimal viral IP.BRE oligonucleotide
(10, 23). Cotransfection with Sp1 expression plasmids enhanced the
Bel1-specific transactivation of Kip2-BRE to a limited
extent.2
In the present study we aimed to identify cellular transcription
factors and their DNA targets that either repress or activate Bel1-mediated transactivation of the viral internal promoter. We
analyzed the ability of cellular transcription factors to affect the
level of Bel1-driven transactivation with the clear focus on the core
IP element from Cell Culture, Plasmids, and Transfection--
Human 293T cells
were cultivated in Dulbecco's modified Eagle's medium supplemented
with 1% penicillin and streptomycin and 10% fetal calf serum.
Plasmids pUC18, pCMV Construction of Eukaryotic IP-based luc Reporter
Plasmids--
Reporter constructs containing the HFV internal promoter
were constructed by PCR-mediated amplification of the defined promoter fragments. Sense primer was: IPs ( EMSAs, Supershifts, and Competition EMSAs--
EMSA experiments
were performed according to Soto et al. (26, 27) and
modified as described below. The probes used for EMSAs included the
IP.BRE (Fig. 1B, line 3). The oligodeoxynucleotides were
synthesized, annealed, and end-labeled using [ luc Expression Assays--
Plasmid pCMV Immunoblotting--
Cells were harvested 2 or 3 days after
transfection by lysis in 1% SDS, and the protein concentration was
determined using the DC protein assay (BioRad). Identical amounts of
proteins were separated by SDS-PAGE, blotted, reacted with monoclonal
serum directed against the HA epitope of the four different HA-tagged NFI proteins (Roche) or against hNFI-X1 protein (sc870X, Santa Crux),
or polyclonal sera directed against Bel1/Bet, Gag, or RNase H proteins
(23), and detected by enhanced chemiluminescence as described
previously (23).
Characterization of the Repressive Effects of Different NFI Family
Members on the Full-length Viral Internal Promoter--
The minimal
HFV IP has been reported to comprise 27 bp but longer promoter regions
showed higher activities when assayed by transient expression assays.
In the first step of our analysis, we decided to use a longer IP that
extends from
To assess expression levels of the Bel1 and NFI proteins, aliquots of
nuclear extracts from transfected 293T cells were analyzed on Western
blots. The results of immunoblotting showed that the expression levels
of the four mouse (m) NFI, hNF-X1, and Bel1 proteins were similar under
the conditions used (data not shown). This allowed us to compare the
level of repression of hNFI-X1 to that of the mNFI-X2 (bottom
lines in Fig. 2, A and B). Since this
comparison showed that minor differences in repression were not due to
differences in expression levels of the four mNFI and Bel1 proteins or
to differences of the NFI-X protein sequences (30), we conclude that
NFI-X1 and -X2 proteins from both species repressed Bel1-mediated
transactivation to similar degrees.
To determine more precisely the region of the IP that is responsible
for the NFI-repressive effects, shorter regions of the internal
promoter, two IP.BRE DNA fragments from positions
To further identify the region of the IP subject to repression by
NFI-X, the longer core IP.BRE DNA from EMSA of Nuclear Proteins from NFI-transfected Cells That Bind to
NFI DNA Target Sites of the Core IP.BRE (
Distinct bands were detected in nuclear extracts of cells transfected
with vectors expressing mNFI-A1, -B2, -C2, -X2, and hNFI-X1 proteins in
the absence of cotransfected Bel1 expression plasmid (Fig.
4A, lanes 4, 6, 8,
10, and 12 marked by vertical arrows
marked A1, B2, C2, X2, and
X1). After cotransfection with pbel1s, the four individual
NFI protein-DNA complexes gave rise to an additional set of complexes
with lower mobility (vertical arrows in Fig. 4A,
marked A1+ Bel1, C2+ Bel1, X2 +Bel1,
and X1+ Bel1). The partial shifts were concomitant with a
slight decrease in intensity of the DNA-Bel1 complexes. The
Bel1-DNA-NFI-X2 and -X1 complexes migrated as comparatively intense
bands (lanes 11 and 13). In contrast, the
NFI-B2-DNA complex became detectable only after longer exposure;
however, a band corresponding to a Bel1-DNA-NFI-B2 complex was not
observed (lane 7). Two additional bands observed in nuclear
extracts from transfected cells are considered to be unspecific
(lane 2) as reported previously (10, 23). These results
indicate that the different NFI-A1, -C2, -X2, and -X1 proteins bound
the core Bel1-IP.BRE DNA (
To confirm the specificity of the IP.BRE interaction with Bel1 and NFI
proteins, a polyclonal Bel1-specific antibody and a monoclonal directed
against the HA epitope of the four mNFI proteins was used in supershift
assays (Fig. 4B). The intense bands of the Bel1-DNA
complexes are marked by short arrows in lanes 3, 4, 9, and 10. The HA and Bel1
antibodies unrelated to each other served as specificity controls
(lanes 4 and 8). The EMSAs shown in Fig.
4B indicate that in the presence of nuclear extracts from 293T cells transfected with Bel1, the two Bel1-oligonucleotide complexes reacted with antibody directed against Bel1 that resulted in
a complete supershift to three bands (lane 5, short
arrows marked Bel1 +
The mobilities of the NFI-X2-DNA complexes (lanes 6,
8, and 9) were slightly slower than the Bel1-DNA
complexes in lanes 3, 4, 9, and
10). In contrast, the NFI-X2-DNA complex resulted in a
partial shift to a new position after cotransfection of the Bel1
expression plasmid (inset, lane 9). Clearly, the
ternary NFI-X2-DNA-Bel1 complexes were shifted by addition of either
anti-HA or anti-Bel1 antibodies (inset, lanes 9,
10, and 11). Thus, we defined the upper
band (lane 9) as a Bel1-DNA-NFI-X2 complex. The
uppermost band in lane 11 likely presents a DNA
complex of NFI-X2 and Bel1-anti-Bel1 that migrated more slowly than the
DNA complex of anti-HA-NFI-X2 and Bel1 (lane 10).
Similar EMSA evidence of NFI-Bel1-DNA ternary complexes was obtained
when the mNFI-C2 expression was used for transfection instead of
mNFI-X2 (data not shown).
To confirm the specificity of the EMSA assay and anti-HA and antiBel1
antibody supershifts, we carried out EMSA and supershift experiments
with a 32P-labeled FIB2.6 oligodeoxynucleotide. FIB 2.6 was
reported to bind specifically to NFI proteins with very high affinity
(31). The results of the binding assays in Fig. 4C clearly
show that with nuclear extracts from 293 cells cotransfected with Bel1
alone, FIB2.6 was incapable of binding to Bel1 (lanes 3-5).
Expression of Bel1 did not lead to any new band (lanes
9-11) even after reaction with Bel1-specific antibodies
(lane 11). Both proteins, mNFI-X2 and Bel1, were expressed
as confirmed by Western blotting (data not shown). The absence of any
band shift in lanes 9 and 11 indicates that
direct NFI-Bel1 protein interactions are not detected.
Competition EMSAs of Nuclear Extracts from NFI-X2-transfected Cells
Reacted with Various Double-stranded Oligodeoxynucleotides as
Competitors for Binding to the Core IP.BRE--
To determine
whether distinct oligonucleotides were able to act as competitors for
the formation of the different nuclear protein-IP.BRE complexes, the
following oligonucleotides were used: unlabeled core IP.BRE (
In an effort to further delineate the contributions of individual NFI
DNA binding sites to DNA-NFI complex formation, we used an unlabeled
27-bp-long IP.NFI DNA from
To determine the in vivo relevance of
NFI-dependent repression, the infectious pHSRV13 DNA
plasmid (12) was transfected with and without the hNFI-X1 expression
plasmid into two different cell lines. Immunoblotting of cellular
extracts revealed that pHSRV13-transfected cells expressed the viral
structural proteins Gag and Pol (Fig. 6,
A and B). The viral Gag proteins p70 and p68
migrated as a characteristic double band (marked by the
arrow in Fig. 6A) as reported previously
(11, 14), and their expression levels decreased upon cotransfection
with the NFI-X1 expression plasmid. The known HFV Pol proteins p127 and
p65 (11) were detected by an anti-RNase H serum (marked by two
arrows in B). In extracts from cells cotransfected with
hNFI-X1, the relative amounts of these Pol proteins were substantially
decreased (as marked by arrows in lanes 2,
4, and 6). Importantly, the cellular lysates expressed the 36-kDa Bel1 protein after transfection with pHSRV13 as determined with an anti-Bel1/Bet serum (Fig. 6C,
lanes 1 and 3).
In contrast, in cellular extracts from 293T cells cotransfected with
hNFI-X1 the relative amount of Bel1 protein strongly decreased
(Fig. 6C, compare lanes 1 and 3 with
lanes 2 and 4). Taken together, the results
indicate that in cell cultures that overexpress NFI-X1 proteins,
expression of Bel1 was down-regulated in agreement with our
transfection data.
The present report indicates that NFI-mediated repression of
Bel1-driven transactivation and gene expression involves the interaction of NFI transcription factors with the retroviral internal promoter core IP.BRE. This report is the first that describes the
identification of a cellular transcription factor that negatively regulates Bel1-mediated transactivation of the core IP.BRE in vitro and in cell cultures. This promoter is transactivated by Bel1, the key regulator of viral expression (7, 11-14). In transiently transfected 293T cells, different NFI proteins specifically repressed the level of Bel1-driven transactivation as assessed by luc expression assays. The level of activation and repression was dependent on the
size of the promoter region. To gain insight into the mechanism of
repression, we focused on the core IP.BRE region from It is well known that NFI proteins can act as either activators or
repressors depending on the transcriptional context. The complex roles
of the NFI gene products in transcription and development have been
reviewed (33, 34). Expression of NFI proteins can modulate
transcription by either transactivation or repression. To account for
the multiple in vivo functions of NFI proteins, several
models have been put forward (33). As NFI proteins act as dimers, the
models include (i) recruitment of coactivators, corepressors, or RNA
polymerase components, (ii) displacement of activators, repressors, or
nucleosomes, and (iii) cooperative recruitment by specific NFI isoforms
of adjacent DNA binding proteins. Here we will focus the discussion on
repression. NFI proteins contain a highly conserved ~200-residue
N-terminal DNA binding and dimerization domain. The C-terminal domains
of the proteins harbor both repression and transactivation domains
including a Pro-rich domain (33).
There are multiple potential models to address the formation of the
different NFI-Bel1 complexes. The first is that IP.BRE DNA binds Bel1
and NFI-X2 proteins cooperatively so that the transcriptional factors
affect each other; i.e. once NFI protein is bound to its DNA
target site, it then affects subsequent Bel1 binding in a way that
repression of the Bel1-mediated activation is achieved. This would
likely require that the two binding proteins interact directly with
each other. Thus, the repressive effect of NFI would be due to affects
on Bel1 protein binding. However, our data provided no evidence for any
direct NFI-Bel1 interaction.
An alternative hypothesis is that the core IP.BRE DNA binds the Bel1
and dimeric NFI-X2 proteins directly but independently at distinct but
partially overlapping sites leading to a partial displacement of the
Bel1 activator, particularly when NFI proteins were overexpressed. The
presence of three different NFI half-sites within the core IP.BRE is in
agreement with this assumption as it is well known that NFI proteins
binds to half-sites as either homo- or heterodimers (35). However
again, our data did not indicate that NFI-X2 could displace Bel1 from
the full or minimal IP. BRE. A third model would predict that NFI-X2
binds to the promoter in the presence of Bel1 and recruits corepressor
molecules that abolish Bel1 activation of the promoter. This model
proposes as yet unidentified corepressors that NFI-X2 might recruit to the promoter. The identification of such corepressors will be an
important future task. Our data also revealed that one of the DNA-Bel1
complexes might contain unknown endogenous nuclear proteins as well as
endogenous NFI proteins (Fig. 5A). In supershift assays, this complex resulted in a partial supershift to a slowly migrating NFI-DNA-Bel1 complex indicating the composite nature of the DNA complexes and the possible presence of both activator and repressor proteins. The influence of other known or unknown cellular
transcription factors on Bel1 activation remains to be determined. We
can, however, rule out a role for C/EBP Further studies are needed to determine the complete complement of
endogenous transcription factors that influence Bel1-mediated activation of the IP.BRE.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
166 to
116. Our investigations revealed that
nuclear factor I (NFI) proteins negatively regulate spumaviral transactivation by binding to distinct core IP DNA target sites. Cotransfection of the infectious HFV DNA genome with a human NFI-X1 expression vector into two different human cell lines almost completely abolished viral gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal, pbel1s (24), pGL3-basic-HFV-IP (
192 to
1), pGL3-pro-IP.BRE
166 to
116 and
166 to
140 derivatives
(23) were transfected into 293T cells using the coprecipitation method
of Chen and Okayama (25). In general, 6 µg of plasmid DNA were
transfected into 293T cells grown in 6-cm Petri dishes. The human
fibrosarcoma cell line HT-1080 (ATCC number CCL-121) was propagated as
described by the supplier and transfected using LipofectAMINE plus (Invitrogen).
192 to
173),
5'-CGTCATGCTTTGGACTGGAC-3'; the antisense primer was: IPas (
1
to
22), 5'-GATAGATCTCAGCTTTTGCTCTTTCAATCTG-3'. PCR reactions
using sense primer IPs and antisense primer IPas were done with
Pfu polymerase (Stratagene) and pHSRV13 (12). PCR
reactions were carried out with the buffer recommended by supplier
supplemented for 1 min at 94 °C, 1 min at 56 °C, 2 min at
72 °C for 35 cycles. The resulting blunt-ended PCR amplicon was
digested with BglII and inserted into the BglII-
and Ecl136II-digested reporter plasmid pGL3 basic (Promega).
The resulting reporter construct was designated pGL3-basic-HFV-IP.
Separately, pGL3-promoter plasmids were constructed. The core IP.BRE
(
166 to
116),
5'-AGGCCACTGGTTGCGGAAGAAAGATTGAGCTTTGAGCCACGACTGCCAA-3' (or the IP.BRE
from
166 to
140) was mixed with the corresponding antisense
oligodeoxynucleotides, heat-denatured, annealed, and inserted into
Ecl136II-digested luc reporter plasmid pGL3-promoter in the
sense orientation. These plasmids were designated pGL3-pro-IP.BRE-166 to
116 or
160 to
140, respectively.
-32P]ATP
(3000 Ci/mmol, Amersham Biosciences) with T4 polynucleotide kinase (New
England Biolabs). The labeled probe was purified by electrophoresis on
a 15% polyacrylamide gel. Nuclear extracts were prepared as described
(28, 29). Protein concentrations were determined with the DC protein
assay (BioRad). The binding reaction was carried out with 2 µg of
nuclear extracts that were preincubated for 5 min at room temperature
in a volume of 40 µl containing 10 mM Tris-HCl (pH 7.5),
50 mM NaCl, 2.5 mM MgCl2, 1 mM EDTA, 1.0 mM dithiothreitol, 1.0 mg/ml
bovine serum albumin, 0.2 mM phenylmethylsulfonyl fluoride,
25 ng/µl poly(dA-dT)·poly(dA-dT) (Amersham Biosciences) as
indicated. Labeled DNA probe, 20,000 cpm, were added and incubated for
30 min at 25 °C. For competition experiments, unlabeled competitor
oligodeoxynucleotides were added in 1000-fold molar excess at the
preincubation period (29). Polyclonal antibody against Bel1 (23) and
monoclonal anti-hemagglutinin (HA) antibody (Roche Molecular
Biochemicals) used in supershift assays were added at a 1:80 dilution
or 5 ng/µl respectively 30 min after addition of the labeled probe
and further incubated for 1 h at 4 °C. Oligodeoxynucleotides
FIB2.6, (Fig. 1B) and AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3' were
used in competition EMSAs (30). The DNA protein complexes were resolved
in a 5.5 or 6.5% non-denaturing polyacrylamide gel, dried, and exposed
for 4 h or overnight to Kodak BioMax MR1 films.
gal directing
-galactosidase expression from the CMV-IE promoter was used for
normalization of transfection efficiency. luc reporter gene assays were
performed and quantified as described (23) using a Luminoskan TL Plus
luminometer (Labsystems, Frankfurt, Germany). Cells were harvested
24 h after transfection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
192 to the internal transcriptional start site of HFV
(Fig. 1A). To analyze
Bel1-mediated transactivation of the pGL3-HFV-IP, cotransfections with
and without the eukaryotic expression plasmid pbel1s DNA were carried
out. Since we had identified putative NFI binding sites within the IP,
we tested the ability of NFI transcription factors to affect expression
from the IP. The effect of human NFI-X1 (hNFI-X1) was determined by
comparing the results of the normalized luc reporter assays in the
presence and absence of hNFI-X1 expression (Fig. 2A). These data show that
hNFI-X1 repressed Bel1-driven transactivation by about 10-fold.
Since vectors expressing the products of the four known human NFI genes
were not available, this analysis was extended using well characterized
vectors that express products of the four murine NFI genes. The results
shown in Fig. 2B indicated that the four different NFI
proteins, NFI-A1, -B2, -C2, and -X2, each inhibited the Bel1-mediated
transactivation of the full-length promoter 4- to 6-fold (lower
panel, Fig. 2B). In contrast, transfection of the four
different NFI family members separately or alone did not affect the
basal activity of the full-length IP.BRE (upper panel, Fig.
2B).
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Fig. 1.
Diagram of the foamy viral internal
promoter. A, the internal promoter is shown from
position 192 to the
1 cap site determined previously (12). The core
IP.BRE from position
166 to
116 is in BOLD FACE and
underlined; the minimal IP.BRE (from
166 to
140) of 27 bp is marked by broken underlining, and the IP.NFI from
142 to
116 by solid underlining; six predicted NFI
half-sites are marked by overlining and numbered;
NFI half-sites s1, s2, and s7 (identified here) are boxed
and were analyzed in more detail (see "Results"). Locations
of the TATA box and cap site are shown; the rectangular
arrow indicates the direction of Bel1 transcription. B,
nucleotide sequences of oligodeoxynucleotides representing NFI
half-sites (underlined) and IP.BRE mutants used; mutated
bases in NFI sites are boxed and in italics.
Double-stranded oligodeoxynucleotide FIB2.6 was taken from (31).
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Fig. 2.
Repression of Bel1-mediated transactivation
by different NFI plasmids as determined by luc reporter assays.
A, the pGL3-basic HFV-full-length internal promoter (HFV-IP
from 192 to
1, see Fig. 1) was cotransfected with plasmids pbel1s
into 293 T cells with and without human NFI-X1 (32). B, four
different murine NFI plasmids were separately cotransfected with
pbel1s; luciferase activity was determined as described previously
(23). The data represent the mean ± S.D. for three experiments
shown by error bars.
166 to
140 and
166 to
116 were cloned into the corresponding luc reporter plasmid
pGL3-pro (Fig. 1A, broken and solid
underlining, respectively). When the IP.BRE was shortened to the
minimal IP.BRE of 27 bp encompassing the region from
166 to
140,
the level of NFI-mediated repression of Bel1-driven
transactivation decreased to a relatively very low level compared with
the full-length internal promoter (Figs. 2 and
3A).
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Fig. 3.
Repressive effects of Bel1-mediated
transactivation by human NFI-X1 after cotransfection with pbel1s and
two pGL3-pro-IP.BRE plasmids of different promoter/enhancer lengths as
determined by luc assays. A, the pGL3-pro-IP.BRE
plasmid from 166 to
140 was cotransfected with the Bel1 expression
plasmid pbel1s into 293T cells with and without hNFI-X1 (32). In
panel B, the pGL3-pro-IP.BRE from
166 to
116 was used;
after cotransfection with pbel1s and hNFI-X1, luciferase activity was
measured.
166 to
116 was cloned into
the pGL3-pro plasmid and cotransfected with pbel1s and phNFI-X1, and
luciferase activity was subsequently determined. Bel1-dependent transactivation of the core IP was about
7-fold higher on the longer core IP.BRE compared with the
minimal IP.BRE and was repressed to a much greater extent (Fig.
3A and B). When luc reporter assays were done
with the core IP as template and the four mNFI plasmids, similar levels
of transactivation and repression were obtained after cotransfection
with Bel1 (data not shown). We reasoned that the difference in the
level of repression between the two different length promoters might be
due to a putative additional NFI site 2 (s2) located close to the
3'-end of the IP.BRE at position
122 to
118 (Fig. 1). As the NFI-s2
GCCAA is a perfect consensus NFI half-site, we decided to subject the core IP.BRE from
166 to
116 to a more refined analysis of the repressive effect of NFI to gain more insight into the functional role
of this region.
166 to
116)--
To
determine whether the longer core IP.BRE region is recognized by NFI
proteins, EMSAs were carried out. Nuclear extracts from 293T cells
transfected with vectors expressing NFI proteins, with or without Bel1,
were prepared and incubated with the 32P-labeled IP.BRE
(
166 to
116) oligodeoxynucleotide. In pilot experiments, we had
observed that synthetic polymers poly(dA-dT)·poly(dA-dT) instead of
poly(dI-dC)·poly(dI-dC) markedly improved the detection of the
different DNA-nuclear protein complexes. In nuclear extracts from
Bel1-transfected cells, one intense and broad and another minor and
slower migrating, Bel1-IP.BRE bands were clearly detectable (Fig.
4A, lanes 3,
5, 7, 9, 11, and
13, long arrow marked Bel1). The minor
Bel1-IP.BRE band of low intensity likely presents a complex consisting
of Bel1, DNA, and an unknown endogenous nuclear protein migrating close
to the NFI-C2-Bel1-DNA band (see below).
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Fig. 4.
EMSA of nuclear proteins from NFI- and
Bel1-transfected 293T cells that bind to DNA target sites of the core
IP.BRE from 166 to
116. A, the core IP.BRE
oligonucleotide from
166 to
116 (see Fig. 1) was used. EMSAs were
carried out with nuclear extracts from 293T cells transfected with the
Bel1 expression plasmid pbel1s or cotransfected with one of five
different NFI plasmids as indicated. The oligonucleotide IP.BRE probe
was labeled with [
-32P]ATP and incubated with 2 µg
of nuclear extract and 25 ng/µl poly(dA-dT)·poly(dA-dT) and
processed as described under "Experimental Procedures."
Arrows in lanes 3, 5, 7,
9, 11, and 13 mark the Bel1
protein-DNA complexes at distinct positions. B, supershift
EMSA of the DNA protein complexes reacted with either the monoclonal
antibody (anti-HA 5 ng/µl) against the HA-tagged NFI or polyclonal
antiserum against Bel1 at 1:80 dilution. The core IP.BRE
oligodeoxynucleotide from
166 to
116 (see Fig. 1) was used.
Conditions were as described in panel A. Minus signs
indicate absence of pbel1s or NFI-plasmids. Inset presents
enlargement of upper part of lanes 9-11. C, the
32P-labeled FIB2.6 oligonucleotide was used as probe.
Nuclear extracts from cotransfected cells were used as described in
A and supershifts were done as described in
B.
166 to
116) together with Bel1. To assess
the presence of other potential protein-IP.BRE complexes, different
conditions with varying buffers and copolymers were used. No evidence
for any other complexes than those detected before and shown in Fig.
4A was found.
Bel1, Fig. 4B) as reported
previously (10, 23). Incubation with antibody against the HA epitope
located at the N terminus of the NFI-X2 protein resulted in a
supershift of the original NFI-X2-oligonucleotide complex to positions
marked by the long arrow NFI-X2+
HA (lane 7,
inset, lane 9 versus
10).
166 to
116), FIB2.6 (Fig. 1B) shown previously to strongly bind
NFI in vitro (31) and an unrelated AP-1 site (sequence under
"Experimental Procedures") in the presence of excess of
poly(dA-dT)·poly(dA-dT). In nuclear extracts from Bel1-transfected
cells one major and a minor DNA-Bel1 complex were formed (Fig.
5A, lanes 3,
5, and 6). The data shown in Fig. 5A
revealed that unlabeled core IP.BRE (
166 to
116) functioned as
effective competitor for both the major and minor DNA-Bel1 complexes
(lanes 4 and 12) almost as well as for the
DNA-NFI-X2 complexes (lanes 8 and 12). It is
noteworthy that the unlabeled 26-bp FIB2.6 oligonucleotide effectively
competed for the NFI-X2-DNA complex (lanes 9 and
13) but not for Bel1-DNA complex (lane 5). Interestingly, the proposed ternary NFI-X2-Bel1-DNA complex was effectively competed out by FIB2.6 (Fig. 5A, lane
13 versus lane 11). FIB2.6 partially blocked the
formation of the minor IP.BRE-Bel1 complex (lanes 5 and
13). These results indicate that most or all of the upper
band in lane 11 contained a NFI protein. By analogy, the
modest decrease in intensity of the slowest migrating DNA-Bel1* complex
suggests that an endogenous NFI protein may be part of this complex
(lanes 5 and 13 compared with lanes 3 and 6). These data are in agreement with the results
obtained in the supershift assay (Fig. 4B, lane
10). The unrelated AP-1 oligonucleotide did not act as competitor
for either of the nuclear protein-DNA complexes as anticipated. The
data argue for a specific interaction of the NFI DNA binding sites s1
and s2 of the IP.BRE with NFI proteins.
View larger version (52K):
[in a new window]
Fig. 5.
EMSA of nuclear extracts from NFI- and
Bel1-transfected cells and reacted with various oligonucleotides as
competitors for binding to the core IP.BRE and NFI sites.
A, the oligonucleotide IP.BRE 166 to
116 was labeled
with [
-32P]ATP and used as probe. The probe was
incubated with 2 µg of nuclear extract and 1000 molar excess of
unlabeled competitors IP.BRE
166 to
116 (lanes 4,
8, and 12); the strongly NFI-binding
oligonucleotide FIB2.6 (lanes 5, 9, and
13) and an unrelated AP-1 oligonucleotide (lanes
6, 10, and 14). Arrows mark the
different DNA-nuclear protein complexes. B, The IP.BRE
oligonucleotide from
166 to
116 was labeled with
[
-32P]ATP and used as probe. Nuclear extracts from
mNFI-X2-transfected 293T cells were treated as described in
A and incubated with excess of various unlabeled
oligonucleotides as competitors as indicated (sequences in Fig.
1B).
142 to
116 as competitor. In addition,
three IP.BRE mutants, DNAs of five different NFI sites, and two IP.BREs
were used tested for competition (for sequences, see Fig. 1,
A and B). The core IP.BRE was used as labeled
probe. As shown in Fig. 5B, lane 6, the core
IP.BRE was capable of inhibiting formation of the DNA-NFI-X2 complex.
In contrast, the minimal IP.BRE (
166-140), IP.BRE mutant 1, and
oligonucleotides representing the NFI DNA binding sites s1-s6 did not
block formation of the DNA-NFI-X2 complexes (lanes 4,
9-13 versus 3). Intriguingly, the IP.NFI (lane 8) was the more effective competitor pointing
to an essential role of NFI-s2 or another site that resides in the flanking sequences. A role of NFI-s2 was revealed by the ability of IP.BRE.M2 (lane 6) to compete with the IP.BRE. IP.BRE.M2
contains a mutation in the NFI-s2, which resulted in a detectable
competition although slightly less effective than that of the native
core IP.BRE (Fig. 5B). To more precisely determine the roles
of the potential NFI sites s2 and s7, a double mutant, IP.BRE mutant 3 that contains an additional mutation at s7, was used as competitor (Fig. 1B). It is revealing that the double mutant had almost
completely lost its ability to act as competitor (Fig. 5B,
lane 7) when compared with IP.BRE
166 to
116 (lane
5). We conclude that the NFI-s2 and -s7 sites play more important
roles than the NFI-s1 DNA binding site.
View larger version (39K):
[in a new window]
Fig. 6.
Repression of Bel1 and viral structural
protein expression by transfection of the infectious pHSRV13 DNA and
cotransfection with the NFI-X1 expression plasmid into two cell lines
as determined by immunoblotting. A, Western blot
analysis of HFV Gag expression after transfection of pHSRV13 into
either 293T cells (lanes 1-4) or HT-1080 cells (lanes
5 and 6). Cellular lysates were harvested after 48 or
72 h after transfection with pHSRV13 (lanes 1-6) or
cotransfection with hNFI-X1 (lanes 2, 4, and
6) as indicated. All antisera were used at a dilution of
1:1000. Polyclonal anti-Matrix serum was reacted with the
nuclear extract; arrow marks the characteristic double HFV
Gag band. B, Western blotting as described in A
except that a polyclonal anti-RNase H serum was used. C,
Western blotting as described in A except that a polyclonal
anti-Bel1/Bet antiserum was used; arrow marks the 36-kDa
Bel1 protein and asterisk the abundantly expressed viral Bet
protein of 56 kDa. D, Western blotting as described in
A except that a polyclonal antibody against human NFI-X was
used. Arrow marks the NFI-X1 proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
166 to
116
that allowed us to define the roles of NFI and Bel1 proteins in DNA
binding by carrying out EMSAs, supershifts, and competition EMSAs.
Several groups have reported that Bel1 binds its target sequence
directly (10, 16, 23). The minimal IP.BRE of 27 bp seems to be the one
with a high DNA binding affinity compared with the corresponding LTR
BRE (10). Our data show that the minimal IP.BRE of 27 bp was
transactivated by Bel1 to a limited extent, but it did not efficiently
bind NFI proteins. Accordingly, the levels of NFI-mediated repression
were relatively low when the minimal IP.BRE was used. In contrast, the
core IP.BRE possessed both higher transactivation activity and stronger
NFI binding activity as assessed by luc assays and competition EMSAs.
Thus, the functional and DNA binding properties of NFI are consistent for both IP.BREs. Apparently, the minimal region for optimal
NFI repressor activity resides within the core IP.BRE.
that neither activated nor
repressed Bel1-mediated transactivation of the
IP.BRE.3
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ACKNOWLEDGEMENTS |
---|
We thank Jennifer Reed for critically reading the manuscript, Peter Angel for helpful discussions, Ulrich Bernard, Doris Apt, and Christian Trautwein for advice and reagents. These studies were supported in part by the National Institutes of Health Grant DK 58401 (to R. M. G).
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We dedicate this article to Harald zur Hausen on the occasion of his retirement as head of the German Cancer Research Center (DKFZ) with gratitude and appreciation.
¶ To whom correspondence should be addressed: Div. of Retroviral Gene Expression, Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, INF 242, 69009 Heidelberg, Germany. Tel.: 49-6221-424611; Fax: 49-6221-424865; E-mail: r.m.fluegel@dkfz-heidelberg.de.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M208963200
2 K. Kido and R. M. Flügel, unpublished observation.
3 K. Kido, H. Bannert, R. M. Gronostajski, and R. M. Flügel, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: FV, foamy virus; BRE, Bel1 response element; EMSA, electrophoretic mobility shift assay; HFV, human foamy virus; IP, internal promoter; luc, luciferase; NFI, nuclear factor I; HA, hemagglutinin; m, mouse; h, human; LTR, long terminal repeat; Kip2, cyclin-dependent kinase inhibitor 2.
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REFERENCES |
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