From the Service de Microbiologie, Unité de
Virologie and
CNRS UPR 9051, Hôpital Saint-Louis, 1 avenue
Claude Vellefaux, 75475 Paris CEDEX 10, France, § INSERM
U380, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France, and ¶ Faculty of
Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsu-cho, Midori-ku, Yokohama 226, Japan
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ABSTRACT |
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The genetic expression of human B19 parvovirus is
only dependent on one promoter in vivo and in
vitro. This is the P6 promoter, which is located on the left side
of the genome and is a single-stranded DNA molecule. This led us to
investigate the regulation of the P6 promoter and the possible
resulting variability of the nucleotide sequence. After analysis of the
promoter region of 17 B19 strains, only 1.5% variability was found.
More exciting was the finding of mutations that were clustered around
the TATA box and defined a highly conserved region (nucleotides
113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo
cell extracts were found to protect this region, which contained a core
motif for Ets family proteins, with YY1 and Sp1 binding sites on either
side. Gel mobility shift assays performed with nuclear proteins from
HeLa and UT7/Epo cells identified DNA-binding proteins specific for
these sites. By supershift analysis, we demonstrated the binding of the
hGABP (also named E4TF1) protein to the Ets binding site and the
fixation of Sp1 and YY1 proteins on their respective motifs. In
Drosophila SL2 cells, hGABP and -
stimulated P6
promoter activity, and hGABP
/hGABP
and Sp1 exerted synergistic
stimulation of this activity, an effect diminished by YY1.
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INTRODUCTION |
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B19 parvovirus is the only member of the Parvoviridae family that is pathogenic for humans (1). It has been associated with a wide range of clinical symptoms and is responsible for erythema infectiosum in children and arthropathy in adults. B19 infections can be particularly severe, leading to hydrops fetalis during pregnancy, transient aplastic crisis in patients with underlying hemolytic diseases, or chronic bone marrow infection in immunocompromised patients (2). In vivo and in vitro, the infection of human bone marrow cells leads to the depletion of the immature erythroid progenitor cells, i.e. burst-forming unit erythroid and cluster-forming unit erythroid (3, 4). In the latter cells, replication occurs and results in cell cytotoxicity (5). However, despite such remarkable erythroid tropism, which is still unexplained, B19 infection can also impair megakaryocytopoiesis (6, 7). Whereas virus replication is responsible for the disruption of erythropoiesis, only viral transcription occurs in megakaryocytes. In these cells, the accumulation of the nonstructural protein NS1 seems to be responsible for cell lysis (8).
B19 virus, like other parvoviruses, is a nonenveloped icosahedral virus with a single-stranded DNA linear genome composed of 5596 nucleotides that encode one nonstructural protein (NS1), two structural proteins (VP1 and VP2), and several small polypeptides of unknown function (9-11). Both ends of the genome are composed of identical inverted repeat sequences of 383 nucleotides (12). The distal 365 nucleotides are imperfect palindromes that can form a hairpin structure. The transcription map of the B19 parvovirus has been determined in infected human bone marrow cells (9, 10). Its only known promoter, named P6 and located in the 5'-terminal region, directs the synthesis of up to nine viral transcripts (13, 14). Although the mRNAs encoding for the capsid proteins and the small polypeptides are spliced, the NS1 mRNA is not (10).
The regulation of the P6 promoter by viral or cellular proteins has not been extensively studied. In erythroid-permissive cells, this regulation might be preponderant. Thus, a recombinant adeno-associated virus, a defective parvovirus in which the P5 promoter has been substituted for the B19 P6 promoter, is able to replicate specifically and autonomously in erythroid cells (15). However, isolated in front of a reporter gene, the P6 promoter exhibits strong activity in many cell lines, as demonstrated after transfection (14, 16, 17). Like other parvoviruses, the nonstructural protein NS1 can up-regulate the P6 promoter (14, 18-20), but the exact mechanism of this up-regulation is not yet clear. The result of a recent study argues in favor of an indirect effect involving Sp1 and cAMP-response element binding proteins, as already demonstrated for other parvoviruses.1 The Sp1 transcription factor has been implicated in the regulation of the P6 promoter (22). Indeed, two GC box motifs located upstream of the TATA box have been implicated in the in vitro up-regulation of promoter transcription. The YY1 transcription factor also binds the P6 promoter to three different motifs (23), which results in a positive P6 promoter regulation.
In this investigation, we first studied the genetic diversity of the B19 P6 promoter. A highly conserved region was characterized after sequencing 17 B19 strains. Within this region, a large sequence protected by erythroid or nonerythroid nuclear proteins was observed using in vitro footprinting analysis. For the first time, as far as we know, we demonstrated the presence of an Ets binding site (EBS)2 in the conserved protected region using electrophoretic mobility shift assays (EMSA). By supershift analysis, we characterized the binding of hGABP proteins, an Ets-related transcription factor so far not found to be involved in regulating a parvoviral promoter. In addition to the YY1 transcription factor described above, we demonstrated the fixation of the Sp1 factor to a GC box placed just downstream of the EBS. We then defined a 3-fold sequence composed of the YY1, Ets, and Sp1 binding sites. By transfection analysis of a Drosophila cell line, we studied the effect of the B19 P6 promoter regulation by YY1, hGABP, and Sp1 factors. We showed that Sp1 and hGABP activated transcription synergistically throughout this 3-fold sequence. This synergy was abolished by YY1. Of greater interest was the fact that we observed the same results with the P6 native promoter.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Reagents--
HeLa cells were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% heat-inactivated fetal calf serum (Eurobio).
UT7/Epo cells (24, 25) were maintained in -modified Eagle's medium
(Life Technologies) containing 10% fetal calf serum and 2 IU/ml
erythropoietin (Boehringer Mannheim). Drosophila
melanogaster SL2 cells (26) were grown in Schneider medium (Life
Technologies) supplemented with 10% fetal calf serum.
Oligonucleotides--
Oligonucleotides were synthesized and
purified by Genset (France). Complementary strands were phosphorylated
with T4 polynucleotide kinase (New England Biolabs), denatured at
88 °C for 2 min, and annealed at room temperature. A
HindIII restriction site was introduced at the end of each
oligonucleotide to facilitate radioactive labeling. The NFB probe
sequence was 5'-ACGTTACAAGGGACTTTCCGCTA-3' (27).
Plasmid Construction--
The pTK plasmid containing the 50 to
+55 region of the herpes simplex virus thymidine kinase (TK) promoter
linked to the luciferase gene was constructed as follows. The
XbaI/HincII fragment corresponding to the TK
promoter was obtained from pTK-50 (a gift from Dr. F. Thierry, Institut
Pasteur, Paris, France) (28). The pTK-50 plasmid was digested with
HincII and filled in with the Klenow fragment of DNA
polymerase I (New England Biolabs). After digestion with the
HindIII enzyme, the 105-bp TK fragment was inserted into the
pGL2 basic vector (Promega) at the NheI/HindIII (blunt made) sites to obtain the pTK-LUC plasmid. Oligonucleotides D to
G (see Fig. 2) were filled in with the Klenow fragment of DNA
polymerase I. They were then introduced into the pTK-LUC plasmid at the
SmaI site to obtain the pX-TK-LUC series of plasmids. The orientation and sequence of the recombinant constructs were verified by
DNA sequence analysis (T7 sequencing kit, Amersham Pharmacia Biotech).
Genetic Analysis-- Seventeen B19-PCR positive sera were collected between 1972 and 1995 in our laboratory. They were obtained from 4 blood donors and 12 patients with erythema, arthralgia, and acute or chronic anemia. One patient was asymptomatic. To amplify a 247-bp fragment corresponding to nucleotides 113-360 of the sequence published by Shade et al. (31), we used the primers 5'-AAATGACGTAATTGTCCGCCATCT-3' (nt 113-136) and 5'-AGCCCAGAAAGAAAGAGC-3' (nt 360-343). PCR was run for 30 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 52 °C, and 30 s at 72 °C using Taq polymerase (Boehringer) in a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2 in a 9600 Perkin-Elmer thermal cycler. For each serum sample, two PCR products were obtained by two independent amplifications. After purification of the 247-bp DNA fragment (Wizard DNA clean-up system; Promega), nucleotide sequences were directly determined by the enzymatic method of Sanger with Taq polymerase (fmol DNA sequencing system; Promega) using [35S]dATP. The primers used for the sequence reaction were the same as for the PCR step, thus allowing both the sense and nonsense DNA strands to be read. Each sequence was determined with two distinct PCR products.
Preparation of Nuclear Extracts and Proteins--
Nuclear
protein extractions were prepared as described previously (32).
Briefly, 107 cells were treated with 300 µl of lysis
buffer (50 mM Tris-HCl pH 7.9, 10 mM KCl, 0.2%
Nonidet P-40, 10% glycerol, 1 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 180 µg/ml
Na3VO4, and 1 mM dithiothreitol).
After centrifugation, the nuclei were treated for 30 min on ice with 30 µl of buffer containing 400 mM NaCl, 10 mM
KCl, 20% glycerol, 20 mM HEPES, pH 7.9, 1 mM
EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 180 µg/ml Na3VO4, and 1 mM dithiothreitol. After another centrifugation, the
supernatant was harvested as the nuclear protein extract and stored at
70 °C. Protein concentration was determined with protein assay
reagent (Bio-Rad).
In Vitro DNase I Footprinting Analysis-- Nuclear extracts of HeLa and UT7/Epo cells were used for this analysis. The P6 probe was prepared by PCR with a 3' 32P-labeled primer. A 226-bp fragment corresponding to nucleotides 78-304 of the P6 promoter sequence (31) was amplified from the BP06 plasmid using the primers 5'-ATTTCCTGTGACGTCATTTCCTG-3' (nt 78-100) and 5'-ACGCTCCGCCCATTTT AACCG-3' (nt 304-283). PCR was run for 35 cycles at 94 ° for 30 s, 56 °C for 30 s, and 68 °C for 2 min/cycle with Klen Taq polymerase, according to the manufacturer's instructions (Advantage-GC cDNA PCR kit; CLONTECH). The labeled fragment was purified by native agarose gel electrophoresis. About 0.5 ng of probe was incubated for 15 min at room temperature with 10 µg of nuclear extract in 25 µl of binding buffer (8 mM HEPES pH 7.9, 8% glycerol, 40 mM KCl, 0.08 mM EDTA, 0.08 mM phenylmethylsulfonyl fluoride, and 0.2 mM dithiothreitol). The probe was then digested for 2 min on ice with various concentrations of DNase I (Sigma) ranging from 20 to 160 µg/ml. The reaction was stopped by adding 250 µl of 0.05% SDS, 2.5 µM EDTA, and 300 µM NaCl. After phenol/chloroform extraction and ethanol precipitation, samples were loaded on an 8 M urea, 6% polyacrylamide sequencing gel. Autoradiography was performed for 2-8 days.
Electrophoretic Mobility Shift Assay--
The EMSA was performed
with nuclear extract or in vitro translated protein and the
D probe corresponding to the region spanning nucleotides 126-158 of
the P6 promoter sequence (see Fig. 2). This oligonucleotide probe was
labeled by filling in with the Klenow fragment of DNA polymerase I in
the presence of [-32P]dCTP. First, 2.5 µg of cell
extract or 1 µl of in vitro translated protein were
incubated for 10 min at room temperature in the binding buffer (4%
Ficoll, 20 mM HEPES, pH 7.5, 70 mM NaCl, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, and
0.01% Nonidet P-40) with 1 µg of poly(dI-dC)·poly(dI-dC)
(Amersham) and 0.5 µg of salmon sperm DNA. Next, either competitor or
antibodies were added when indicated and then 1 µl of the
32P-labeled probe (about 20,000 cpm). The preparation was
left to stand for 25 min at room temperature and then underwent
electrophoresis on 5% polyacrylamide gel in 0.5 × Tris
borate/EDTA buffer (45 mM Tris borate, 1 mM
EDTA). Lastly, the gel was dried and autoradiographed.
Transfection and Luciferase Assay-- One day before transfection, SL2 cells were seeded at 2 × 106 cells/35-mm well. They were co-transfected with the indicated amount of expression plasmid and 1 µg of reporter plasmid using the calcium phosphate coprecipitation method (33). The total amount of DNA was kept constant at 10 µg by adding nonrecombinant expression plasmid (control plasmid). After the addition of DNA, the plates were left undisturbed until the time of harvest 48 h later. Cells were then washed once with phosphate-buffered saline, and luciferase activity was measured in a luminometer (Lumat LB 9501, Berthold), as described previously (20). To normalize the luciferase assay, the total protein concentration was evaluated for each cell lysate (protein assay reagent, Bio-Rad).
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RESULTS |
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Genetic Diversity Analysis-- As stated above, the 17 B19 PCR-positive sera collected between 1972 and 1995 in our laboratory were obtained from 4 blood donors and 12 patients with erythema, arthralgia, and acute or chronic anemia. One patient was asymptomatic. To analyze the nucleotide composition of the P6 promoter, we amplified a 247-bp fragment including the TATA box. This fragment corresponded to nucleotides 113-360 of the sequence published by Shade et al. (31). For each serum, two PCR products were sequenced independently. Twenty nucleotide modifications (18 substitutions, 1 deletion, and 1 insertion) were observed in comparison with the reference sequence (31) given an average of 1.5% of variation (data not shown). However, these mutations were not equally distributed on the P6 promoter and allowed two regions to be distinguished. All the mutations were situated between nucleotides 210 and 340, and a highly conserved region comprising nucleotides 113-210 was observed on the 5' side of the amplified promoter. Even though this conserved region corresponded to the palindromic sequence of the hairpin terminus indispensable for parvovirus replication, we could not rule out the possibility that this region has an important role in regulating transcription, as recently suggested.1 Hence, we examined the cellular factors interacting with the conserved sequence comprising nucleotides 113-210 of the B19 P6 sequence.
Protection Analysis of the Highly Conserved P6 Region--
The
ability of cellular proteins to bind the highly conserved P6 region was
explored using in vitro DNase I footprinting assays. The
double-stranded P6 probe covered nucleotides 78-304 of the sequence
previously described (31). Only one extremity of the double strand
probe was labeled alternatively and incubated with nuclear extracts
from epithelial HeLa and erythroid UT7/Epo cells. A large protected
region was observed within the conserved region with the nuclear
extract from UT7/Epo cells (Fig. 1). By
comparison with a DNA ladder, the protected region covered the
nucleotides 129-146 (220/
203) on the sense strand and nucleotides
129-150 (
220/
199) on the nonsense one (Fig. 1). The same footprint
was observed with the extract from HeLa cells (data not shown). In the
protected region, a YY1 binding site was described previously between
nucleotides
220 and
212 (23) and an Sp1 binding site between
nucleotides
200 and
195 (16, 22). Between these two protected
regions, we noticed for the first time as far as we know, an Ets family
consensus binding site CCGGAAGT located between nucleotides
208 and
201 (Fig. 2). These results suggested that nuclear proteins from HeLa or UT7/Epo cells were able to protect
the nucleotide sequence spanning nucleotides 129-150 (
220/
199) in
the highly conserved region of the P6 promoter.
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Sp1, YY1, and GAPB Bind to the Conserved Part of the P6
Region--
To confirm the binding of eukaryotic proteins to
nucleotides 129-150 (220/
199) of the conserved region, we
performed EMSA with the radiolabeled D probe (Fig. 2) corresponding to
the binding sites of YY1 (
220/
212), EBS (
208/
201), and Sp1
(
200/
195). When the D probe was incubated with nuclear extracts
from HeLa and UT7/Epo cells, it generated seven retarded complexes
(Fig. 3A). The migration of
six of these complexes was the same in each cell type, whereas the
migration of the complex V was more delayed with HeLa proteins than
with UT7/Epo proteins (compare lanes 2 and 8).
All these seven complexes proved to be specific, since competition with
a 30-fold excess of an unrelated sequence (the NF
B region of the
HIV-1 long terminal repeat) did not affect their formation (Fig.
3A, lanes 4 and 10). This was
confirmed by a competition assay with the cold D probe, which inhibited nucleoprotein complex formation (lanes 3 and 9).
Experiments involving competition between the D probe and the cold A,
B, C probes were used to link binding sites of the YY1, EBS, or Sp1
transcription factors with the cell protein complexes. The formation of
complexes I and II was specifically inhibited by the presence of the
cold C probe corresponding to the Sp1 binding site (lanes 7 and 13). The intensity of complex I was not the same with
the two cell protein extracts, as the HeLa extract produced a stronger
signal than the UT7/Epo, suggesting that different concentrations of the proteins were involved in this complex. Complexes III, IV, and V
disappeared when the B probe corresponding to the EBS was used
(lanes 6 and 12). Several complexes are usually
observed for the EBS since different Ets proteins recognize the same
DNA sequence (34). Nevertheless, complex V did not have the same mobility pattern with the two nuclear extracts, suggesting the binding
of two different members of the Ets family. Finally, the A probe
corresponding to the YY1 binding site inhibited the formation of
complexes I, VI, and VII (lanes 5 and 11).
Complex I formation was also inhibited by competition with the C probe
(lanes 7 and 13), suggesting that its formation
involved the binding of the two proteins YY1 and Sp1 to the D probe.
The two other complexes, VI and VII, probably corresponded,
respectively, to the binding of the complete and the truncated forms of
the YY1 protein, as previously suggested (35, 36).
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Effect of Overexpression of YY1, hGABP, and Sp1 on
Transcription--
The transcriptional effect of the YY1, hGABP, and
Sp1 factors on the 3-fold sequence was evaluated by transfection. We
isolated the wild-type 3-fold sequence (nt 223/
192, corresponding
to oligonucleotide D in Fig. 2) or the sequences mutated on
the three different sites (oligonucleotides E, F,
G in Fig. 2) upstream of the minimal promoter of the herpes
simplex virus TK and the gene encoding the firefly luciferase. The
resulting constructs, pD-TK-LUC, pE-TK-LUC, pF-TK-LUC, and pG-TK-LUC
were co-transfected with expression vectors for Sp1, YY1, hGABP
, and
-
(pPac-Sp1, pPac-YY1, pPac-GABP
, and pPac-GABP
, respectively)
into D. melanogaster SL2 cells, which are devoid of
endogenous Sp1 and YY1 (29). After 48 h of incubation, luciferase
activity was then estimated in cell extracts. Luciferase activity
levels were calculated in relation to the activity in cells into which
only the reporter plasmid was transfected. The results illustrated in
Fig. 4A show that Sp1 protein
activated LUC expression in a dose-dependent manner from
the pD-TK-LUC plasmid harboring the wild-type 3-fold sequence, and that
this activation was impaired by a mutation that suppressed the binding
of Sp1 (Fig. 4A, compare lanes 1-3 with
lane 4). On the other hand, YY1 protein had no significant effect on pD-TK-LUC transcription (Fig. 4A, lanes
5-7). When, the effect of hGABP proteins on the activity of the
3-fold sequence was similarly investigated, no activation was observed
when each subunit was present alone (Fig. 4B, lanes
1-3). In addition, transcription was activated when hGABP
was
coexpressed with hGABP
1 or
2 (lanes 4-9).
Nevertheless, the formation of an increased amount of
subunits
reduced transcription activation. Such an effect was not observed with
another viral promoter, the adenovirus E4 promoter (30). All these
results indicate that Sp1 and hGABP complexes are able to transactivate
the 3-fold sequence in SL2 cells. The YY1 transcription factor had no
effect on transcription whatever the concentration of expression
plasmid used.
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Synergistic Activation by hGABP and Sp1--
To observe the
possible functional interplay between the Sp1, YY1, and hGABP factors,
we performed co-transfection experiments in SL2 cells using the
pD-TK-LUC plasmid, the reporter plasmid construction pP6-LUC, which
included the entire promoter region (nt 102-480) and the expression
plasmids for the factors. When the reporter plasmid pD-TK-LUC was used,
as depicted in Fig. 5A, the
concomitant production of Sp1 and hGABP +
1 resulted in reporter
gene levels that were seven to ten times higher than those achieved by
each factor alone (compare lanes 1, 3, and
5). This effect was not observed when Sp1 and YY1 or YY1 and
hGABP were co-expressed (Fig. 5A, lanes 4 and
6). Nevertheless, when the plasmid expressing YY1 was added
to the Sp1 and hGABP plasmids, the synergistic effect of Sp1 and hGABP
was 2 times inhibited (78-fold activation, lane 5 versus
32-fold activation, lane 7). Similar results were obtained
when using a plasmid expressing hGABP
2 for co-transfection (data not
shown). When we transfected the pP6-LUC reporter plasmid, we observed
the same results as with pD-TK-LUC construct (Fig. 5B).
Thus, we found a synergistic activation of the P6 promoter by Sp1 and
hGABP
+
1 (compare lanes 1 and 3 with
lane 5 in Fig. 5B). We also observed that this activation was inhibited by the YY1 transcription factor (compare lanes 5 and 7 in Fig. 5B). All these
results indicate that Sp1 and the dimer formed by hGABP
and hGABP
cooperate in the activation of the 3-fold sequence formed in SL2 cells
by the binding sites Sp1, EBS, and YY1. Furthermore, this cooperation
seemed to be partially inhibited by the YY1 protein. Nevertheless,
these effects were weaker when the 3-fold sequence was situated in the
promoter context (compare Fig. 5, A and B),
possibly due to the involvement of other factors that might regulate
other regions of the P6 promoter. The fact that the synergistic effect
between Sp1 and hGABP was only partially affected by the mutation of
one of their binding sites (data not shown) could suggest a mechanism
involving protein-protein interaction. Nevertheless, this hypothesis is
actually under investigation in our laboratory.
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DISCUSSION |
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Contrary to other parvoviruses whose genetic expression is
controlled by two functional promoters located on the left and the
middle of the genome, only one promoter has been described for the
human parvovirus B19. We therefore attempted to analyze its variability
by PCR amplification and sequencing of the promoter region of 17 B19
virus strains. Our data show an average variation of 1.5%, in
agreement with previous results.1 The mutations were
clustered around the TATA box, and a highly conserved region was
observed in the area proximal of the P6 promoter. This region was
located between nucleotides 113 and 210. When it was deleted, there was
a 90% loss in transcriptional activity.1 Therefore we
decided to examine the cellular factors that interact with this region.
By DNA footprinting assays on both strands, a part of the conserved
region was seen to be protected by nuclear extracts from the
nonerythroid HeLa cell line or the erythroid UT7/Epo cell line.
Previous footprinting experiments showed that this region interacts
with the proteins of HeLa and CEM cells (16, 22). Here, we noticed that
an 8-nucleotide EBS site CCGGAAGT (208/
201) is located between the
binding sites YY1 (
220/
212) and Sp1 (
200/
195). An EBS was
described earlier in the P4 promoter of the minute virus of mice and
was also located immediately upstream from the GC box that binds the
Sp1 transcription factor (37). Using EMSA with synthetic
oligonucleotidic probes and competition assays with the corresponding
probes, we confirmed the binding of YY1 previously observed by Momoeda
et al. (23). Binding was also detected at this site for Sp1,
contrarily to the findings of Liu et al. (16). The results
were similar whether the extract used was from HeLa or UT7/Epo cells.
Supershift analysis allowed us to establish that the Ets motif at
nucleotides
208/
201 in the conserved B19 promoter region is
recognized by hGAPB
, a ubiquitously expressed Ets protein. In
addition, antibodies confirmed that HeLa and UT7/Epo binding complexes
contain proteins that are immunologically related to both hGABP
and
hGABP
. The complexes produced by the hGABP proteins were specific,
as evaluated by probe competition. hGABP (also named E4TF1) is indeed
composed of three distinct polypeptides: hGABP
(60 kDa), hGABP
1
(53 kDa), and hGABP
2 (47 kDa), all of which are required for high
affinity DNA binding (
subunit) and transcriptional activation (
and
subunits) (38, 39). hGABP binds to a purine-rich cis-regulatory
element required for the VP16-mediated activation of herpes simplex
virus immediate early gene and regulates adenovirus E4 gene
transcription (39, 40). We therefore investigated the possible
involvement of hGABP in the regulation of the B19 promoter. hGABP was
shown to activate a 3-fold sequence comprising YY1-GABP-Sp1 binding sites with the TK minimal promoter in Drosophila SL2 cells;
this activation was also found with the P6 promoter. It is noteworthy that our GABP binding site was immediately adjacent to the SP1 site.
Ets-related transcription factors such as hGABP are often found in
large complexes with other transcription factors (41-47). For example,
Ets-1 and Sp1 interact to activate synergistically the human T-cell
lymphotrophic virus long terminal repeat (29). In addition, Sp1
activity is known to be modulated by factors that recognize the DNA
elements flanking or overlapping a GC box (48, 49). In the present
work, Sp1 transactivated the 3-fold sequence and the P6 promoter and
displayed synergistic activation with hGABP. Similarly, by
co-transfection experiments using also Drosophila SL2 cells,
the P4 promoter of minute virus of mice was found to be transactivated
synergistically by Ets-1, the prototype member of the Ets family of
transcription factors, and the Sp1 factor that binds to a GC box
flanking the EBS motif (37). In our study, the mutations of the GABP
and Sp1 sites suggest that the combined synergistic effect of the
corresponding transcription factors seems to incriminate DNA binding
but also protein interactions. Whatever the precise mechanism under
investigation, this cooperation was partially inhibited by YY1 protein.
In adeno-associated virus, YY1 was found to act as a repressor of
transcription from the adeno-associated virus P5 promoter, which is
relieved by EIA proteins (21). For B19 parvovirus, the positive effect
of YY1 on transcription was described by Momoeda et al. in
HeLa cells but was very weak, i.e. 1.3-1.9-fold above basal
transcription (23). We did not find that YY1 had any effect on the
3-fold sequence or the P6 promoter in SL2 cells. This difference may be
due to the type of cells transfected.
The present study demonstrated, for the first time as far as we know,
that the specific DNA-binding proteins for the CCGGAAGT motif of the
human B19 parvovirus promoter is very likely to be hGABP, as indicated
by the following results. (i) The DNA protein complex detected in the
gel shift assay was abolished by the competition assay, (ii) antibodies
against GABP and -
subunits supershifted this complex, and (iii)
the combination of in vitro translated hGABP
and -
proteins produced a complex with essentially the same mobility as that
produced by the HeLa or UT7/Epo cell extract. Lastly, our results
clearly demonstrated that in nonerythroid cells, hGABP proteins,
ubiquitously expressed Ets protein, stimulate the expression of the
human B19 parvovirus promoter. The precise mechanism of the synergy
exerted by hGABP and Sp1, which is diminished by YY1, is currently
under investigation.
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ACKNOWLEDGEMENTS |
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We would like to thank C. Rahuel (INSERM U91,
INTS, Paris, France) for help with footprinting assays. We are also
grateful to J. Ghysdael (Institut Curie, Orsay, France) for the
generous gift of pPac plasmids and to T. Shenk (Princeton University,
NJ) for the generous gift of YY1 cDNA. Thanks are due to F. Moreau-Gachelin (Institut Curie, Paris, France), T. Brown (Pfizer), and
S. T. Smale (UCLA school of Medicine, Los Angeles, CA) who,
respectively, contributed kind gifts of antibodies against PU-1 and
Spi-B, GABP and GABP
, and Ets-1 and Elf-1 and to F. Thierry
(Institut Pasteur, Paris, France) for the gift of pTK-50 plasmid.
Lastly, we are indebted to the Laboratoire Photographique
d'Hématologie for photographic work and to M. C. Daudon for
typing the manuscript.
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FOOTNOTES |
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* 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.
** To whom correspondence should be addressed. Tel.: 33 1 42 49 94 93; Fax: 33 1 42 49 92 00; E-mail: fr.morinet{at}chu-stlouis.fr.
1 Gareus, R., Gigler, A., Hemauer, A., Leruez-Ville, M., Morinet, F., Wolf, H., and Modrow, S. (1998) J. Virol. 72, 609-616.
2 The abbreviations used are: EBS, Ets binding site; EMSA, electrophoretic mobility shift assays; TK, thymidine kinase; bp, base pair(s); PCR, polymerase chain reaction; nts, nucleotides.
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REFERENCES |
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