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J. Biol. Chem., Vol. 277, Issue 51, 50062-50068, December 20, 2002
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§,,¶
From the Institute of Biological Chemistry, Academia
Sinica, Nankang, Taipei 115, Taiwan and the ¶ Graduate Institute
of Biochemical Sciences, National Taiwan University,
Taipei 106, Taiwan
Received for publication, September 11, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The human placental trophoblast cell can be
classified as either a cytotrophoblast or a syncytiotrophoblast.
Cytotrophoblasts can function as stem cells for the development of the
syncytiotrophoblast layer via cell fusion. An envelope gene
of the human endogenous retrovirus family W (HERV-W) called
syncytin is specifically expressed in the
syncytiotrophoblast layer. Syncytin is a fusogenic membrane protein;
therefore, it can mediate the fusion of cytotrophoblasts into the
syncytiotrophoblast layer, which is essential for pregnancy maintenance. GCMa is a placenta-specific transcription factor and is
required for placental development. To study the placenta-specific fusion mediated by syncytin, we tested whether GCMa is involved in this
process by regulating syncytin gene expression. In this report, we demonstrate that GCMa was able to regulate
syncytin gene expression via two GCMa-binding sites
upstream of the 5'-long terminal repeat of the
syncytin-harboring HERV-W family member in BeWo and JEG3
cells but not in HeLa cells. Furthermore, adenovirus-directed expression of GCMa enhanced syncytin gene expression and
syncytin-mediated cell fusion in BeWo and JEG3 cells but not in HeLa
cells. Therefore, the integration site of the
syncytin-harboring HERV-W family member in the human genome
is close to the functional GCMa-binding sites by which GCMa can
specifically transactivate syncytin gene expression in
trophoblast cells. Our results may help to explain the mechanism underlying the cell fusion event specific for syncytiotrophoblast formation.
The human placenta contains a specialized cell type called a
trophoblast, which is the first lineage to differentiate in embryo development and plays key roles during implantation and placentation. The human trophoblast cell can be further classified as
cytotrophoblasts and syncytiotrophoblasts. In the early gestation
stage, cytotrophoblast stem cells facing the maternal decidua
proliferate and fuse to form a syncytium, i.e. the
syncytiotrophoblast. Later on, vascular spaces called trophoblastic
lacunae appear in the syncytium around day 8-9. The cytotrophoblast
layer under the syncytium can rapidly proliferate into these spaces,
which results in the formation of the primary chorionic villi.
Subsequently, proliferation of the cytotrophoblasts, growth of
chorionic mesoderm (under the cytotrophoblast layer), and blood vessel
development transform the primary villi into secondary and tertiary
villi, which are composed of a core of mesenchyme cells surrounded by
an inner layer of cytotrophoblasts and an outer layer of multinucleate syncytiotrophoblasts (1, 2). The syncytiotrophoblast layer (syncytium)
transports nutrients and gases and produces hormones such as placental
lactogen and chorionic gonadotrophin, which are indispensable for the
further progression of pregnancy (1).
Recently, a membrane protein termed syncytin has been demonstrated to
mediate cell fusion of the human BeWo trophoblastic cell line (3).
Syncytin is an envelope protein of the newly identified human
endogenous retrovirus family W
(HERV-W)1 and is a
polypeptide of 538 amino acids (3, 4). After synthesis, a
post-translational cleavage is predicted to separate the syncytin polypeptide into surface protein and transmembrane protein. The latter
contains the membrane-spanning segment and a hydrophobic fusion domain.
In situ hybridization has demonstrated that
syncytin is specifically expressed in the
syncytiotrophoblast layer (3-5). These studies suggest that syncytin
can mediate fusion of cytotrophoblasts into the syncytiotrophoblast
layer, and the expression of syncytin is tightly regulated
in a temporal and spatial manner to maintain an integral and functional
syncytiotrophoblast layer.
GCM (glial cell missing) was originally isolated from a
Drosophila melanogaster mutant line that produces additional
neurons at the expense of glial cells (6, 7). Currently, two
GCM-like genes (GCMa and GCMb) have
been reported in mouse, rat, and human (8, 9). Altogether these GCM
homologues form a novel family of transcription factors, which all
share sequence homology in the amino-terminal region that constitutes
the DNA-binding domain called the GCM motif. Although sequence homology
is less preserved outside the GCM motif, a transactivation domain (TAD)
has been identified in the extreme carboxyl terminus of GCM proteins
(10, 11). The optimal recognition sequence for the GCM motif is
5'-(A/G)CCC(T/G)CAT-3' or its 5'-ATG(A/C)GGG(T/C)-3' complement (10,
12). Drosophila GCM mRNA is transiently detected in
glial precursors and immature glial cells, except for mesectodermal
midline glia during a short period of gliogenesis within the central
nervous system (6, 7). In contrast, mouse GCMa mRNA is highly
expressed in the labyrinthine trophoblast cells (13). GCMa is required
for placental development because genetic ablation of mouse
GCMa leads to a failure of labyrinth layer formation and the
fusion of trophoblasts to syncytiotrophoblasts (14, 15).
To study the placenta-specific fusion mediated by syncytin, we tested
whether GCMa can activate the promoter activity of the long terminal
repeats (LTRs) of the syncytin-harboring HERV-W family
member to specifically drive syncytin expression in
trophoblast cells. In this study, we demonstrated that GCMa recognizes
two GCMa-binding sites (GBSs) in the upstream region of the 5'-LTR of
the syncytin-harboring HERV-W family member, activating
syncytin gene expression and consequently enhancing the
syncytin-mediated cell fusion. Our data help to explain the regulatory
mechanism underlying the placenta-specific trophoblastic fusion
mediated by syncytin.
Library Screening and Plasmid Constructs--
The human syncytin
and GCMa cDNAs were cloned by PCR using a human placental cDNA
library as template. The syncytin cDNA fragment was radiolabeled
and used to screen a
A GCMa cDNA fragment containing an amino-terminal HA epitope
sequence was subcloned into the pRcCMV plasmid (Invitrogen) to generate pCMVHAGCMa. Genomic fragments were subcloned into the pE1bCAT
reporter plasmid, which is derived from pCAT3-Basic (Promega, Madison,
WI) by insertion of the adenovirus E1B TATA box in front of
the bacterial CAT (chloramphenicol acetyltransferase) gene. For simplicity, the range of genomic fragments used for these constructs was based on the numbering of nucleotide (nt) residues in
the 083M05 BAC clone. Genomic fragments of nt 25468-30953 with deletions of GBS-(25538-25545), GBS-(28026-28033), or both were subcloned into pCAT3-Basic to generate deletion constructs
pCAT Cell Culture, Transfection, and Reporter Gene Assays--
The
mammalian cell lines used in this study were obtained from the American
Type Culture Collection (Manassas, VA). BeWo cells were grown at
37 °C in F-12K, 15% fetal bovine serum, 100 µg/ml streptomycin,
and 100 units/ml penicillin. JEG3 and HeLa cells were grown at 37 °C
in minimum Eagle's medium, 10% fetal bovine serum, and the same
antibiotics as mentioned above. BeWo or HeLa cells were transfected by
using the Geneporter system (GTS, San Diego, CA). CAT assays were
performed as described (16). The Student's t test was
performed to determine statistical significance for differences between
means of relative CAT activities. A p value of less than
0.05 was considered statistically significant. HAGCMa proteins in
transfected cells were subject to immunoblotting with an horseradish
peroxidase-conjugated rat monoclonal anti-HA antibody (Roche Molecular
Biochemicals). The membranes were stripped in 62.5 mM
Tris-HCl (pH6.7), 100 mM 2-mercaptoethaniol, and 2% SDS at
50 °C for 30 min and reprobed with a rabbit polyclonal anti-actin
antibody (Sigma).
Preparation of Recombinant GCMa Protein--
Sf9 cells
(Invitrogen) were maintained as suspension cultures at 28 °C
in Sf-900II SFM (Invitrogen), 0.125 µg/ml amphotericin B, 50 µg/ml
streptomycin, and 50 units/ml penicillin. A GCMa cDNA fragment with
a carboxyl-terminal FLAG epitope sequence was subcloned into the
pVL1392 transfer plasmid (BD Biosciences). The resultant construct was
cotransfected with Bsu36I-digested baculoviral genomic DNA
(Novagen, Madison, WI) into Sf-9 cells to generate recombinant GCMa-FLAG baculoviruses, which were used to express GCMa-FLAG proteins. GCMa-FLAG proteins were purified by the FLAG M2 monoclonal antibody affinity column (Sigma).
Electrophoretic Mobility Shift Assay and DNase I Footprinting
Analysis--
The electrophoretic mobility shift assay (EMSA) was
performed as described (16) with minor modifications. End-labeled DNA fragments or oligonucleotide probes were incubated with 20 ng of
GCMa-FLAG proteins in a binding reaction buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 0.05 mM
ZnCl2, 4 mM spermidine, 0.05% Nonidet P-40, 5 mM dithiothreitol, 10% glycerol, 0.25 µg poly(dI-dC) and
7.5 µg of bovine serum albumin. After incubation, the reaction
mixtures were analyzed by electrophoresis on 5% nondenaturing
polyacrylamide gels in running buffer (25 mM Tris-HCl, pH
8.5, 190 mM glycine, 1 mM EDTA) at 4 °C. Two
oligonucleotides, dGCMa
(5'-ACTTCTGTCCCTCATGGCCAGT-3') and pGCMa
(5'-TTCTGGGATGAGGGCAAAACG-3'), were synthesized. A
mutant pGCMa oligonucleotide, Mut
(5'-TTCTGGGATGATAGCAAAACG-3'), was also synthesized as a
negative control. Antiserum against human GCMa was induced in guinea
pigs using a His-tagged GCMa recombinant protein expressed in
BL21(DE3). 1 µl of antiserum or normal serum was used for supershift
experiments. DNase I footprinting analysis was performed essentially as
described by Chen et al. (17).
Chromatin Immunoprecipitation (ChIP) Assay--
Approximately
3 × 107 BeWo cells transfected with 20 µg of
pCMVHAGCMa were subject to a ChIP assay as described by Boyd and Farnham (18). HAGCMa-DNA complexes were immunoprecipitated by a rat
monoclonal anti-HA antibody (Roche Molecular Biochemicals) and Protein
A-agarose beads (Oncogene, Boston, MA). Specific sequences of regions
upstream of the 5'-LTR of HERV-W in the immunoprecipitates were
detected by PCR with specific primers. PCR products were analyzed on
2% MetaPhor agarose gels (FMC, Rockland, ME). Sequences of primers are
5'-CTCAGTCCGGCTTACAGTTTCGTTC-3' and 5'-GAATAAGACGGCCTTCTGACCCTTC-3' for
region 22473-22371; 5'-GGCGTCAGATCCCATTACTCTAGG-3' and
5'-AATAGAATGGGCCTGTGAGGCTGG-3' for region 25461-25686;
5'-GCCCATTTCGATTGTAACATCTGCCAC-3' and 5'-GCAAGATAATTGCTGTATCTCCAGGC-3'
for region 27800-28064.
Construction of Ad-HAGCMa--
Recombinant HAGCMa adenoviruses
(Ad-HAGCMa) were generated in CRE8 cells by cotransfection of the
linearized transfer vector (pAdlox-HAGCMa) and the
RNA was isolated using RNeasy reagents (Qiagen, Hilden, Germany).
RNA (20 µg) were assayed for Northern analysis using human GCMa or
Fusion Assay and Fluorescence Microscopy--
293 cells were
transfected with the red fluorescent protein plasmid pDsRed1-N1
(Clontech) 24 h before cell fusion assay.
HeLa, JEG3, or BeWo Cells were transduced with Adlox or Ad-HAGCMa. Four hour post-infection, cells were trypsinized, and 8 × 105 infected cells and 1 × 106
transfected 293 cells were cocultured onto a 60-mm culture dish. After
another 30 h at 37 °C, cell fusions were examined under an
Olympus microscope (Tokyo, Japan) equipped with a cooled
charge-coupled device camera (DP50). Images were prepared for
presentation using Adobe Photoshop® 6.0.
Promoter Analysis of the syncytin Gene--
Syncytin is encoded by
the envelope (env) gene of an HERV-W family
member with a genomic configuration of
5'-LTR-gag-pro-pol-env-LTR-3' (Fig. 1A) (4). To investigate
the placenta-specific expression of syncytin, promoter
analysis was performed to identify potential elements and transcription
factors that could enhance the LTR promoter activity of the
syncytin-harboring HERV-W family member. For simplicity, we
refer to the syncytin-harboring HERV-W family member as
HERV-W for the rest of this report. A
The role of the placenta-specific transcription factor GCMa in the
expression of syncytin gene was investigated, because GCMa is known to play an important role in murine placental development (14,
15). Transient expression experiments of the promoter constructs were
performed in BeWo cells. As shown in Fig. 1C, the CAT
activity directed by pE1bCAT-(25468-30953) is higher than those of the
other constructs examined. In addition, a statistically significant
3.3-fold transactivation by GCMa on pE1bCAT-(25468-30953) was observed
when it was cotransfected with pCMVHAGCMa. This up-regulation was not
due to a differential expression of HAGCMa proteins in transfected
cells because comparable amounts of HAGCMa proteins were detected (Fig.
1C). When the pE1bCAT- (25468-30953) was divided into
pE1bCAT-(25468-28066) and pE1bCAT-(28067-30953), transcriptional activation by HAGCMa was not observed with either construct.
pE1bCAT-(28067-30953) contains the 5'-LTR; therefore, these results
suggest that potential GBSs are present in nucleotides 25468-30953,
which, in conjugation with 5'-LTR, can be up-regulated by HAGCMa. The
positive effect of pCMVHAGCMa on pE1bCAT-(25468-30953) was also
observed in another human trophoblastic cell line, JEG3 (data not
shown). The optimal recognition sequence for the DNA binding domain of
Drosophila GCM is 5'-(A/G)CCC(T/G)CAT-3' or its
5'-ATG(A/C)GGG(T/C)-3' complement (10, 12). Indeed, close scrutiny of
GBS(s) in nucleotides 25468-30953 revealed two potential GBSs
(25538-25545 and 28026-28033) upstream of the 5'-LTR of HERV-W. The
sequences for GBS-(25538-25545) and GBS-(28026-28033) are TCCCTCAT
and ATGAGGGC, respectively.
Analysis of the Binding Elements of GCMa in the syncytin
Promoter--
To test whether GCMa directly binds the two potential
GBSs, we performed EMSA with radiolabeled (25488-25587) and
(27978-28077) DNA fragments and a recombinant GCMa-FLAG protein (Fig.
2A). GCMa-FLAG specifically
bound to radiolabeled (25488-25587) and (27978-28077) DNA fragments
(Fig. 2B, lanes 2 and 8) because the
unlabeled oligonucleotide pGCMa, consisting of the GBS-(28026-28033)
at a 100-fold excess, competed with complex formation (Fig.
2B, lanes 3 and 9). However, a pGCMa
mutant oligonucleotide, Mut, containing mutated nucleotide residues in
GBS-(28026-28033) could not compete with complex formation (Fig.
2B, lanes 4 and 10). Furthermore, the
GCMa antiserum, but not the control serum, was able to supershift the
DNA-protein complex (Fig. 2B, lanes 5,
6, 11, and 12). These results suggest that GBSs are existent in (25488-25587) and (27978-28077) DNA fragments.
To localize the binding sites of GCMa in (25488-25587) and
(27978-28077) DNA fragments, DNase I footprinting analyses were performed using GCMa-FLAG and the radiolabeled probes of the two fragments. As shown in Fig. 2C, the regions protected by
GCMa-FLAG in both fragments encompass the GBS core sequence and some
immediate 5'-flanking nucleotides (Fig. 2C). We further
verified the footprinting results by EMSA using GCMa-FLAG and labeled
dGCMa and pGCMa oligonucleotides spanning the GCMa-FLAG-protected
regions GBS-(25538-25545) and GBS-(28026-28033), respectively (Fig.
2D). Specific complexes were observed in lanes without pGCMa
and dGCMa competitor oligonucleotides and in lanes with the Mut
negative control oligonucleotide (Fig. 2D, lanes
2, 5, 9, and 12). A supershifted
complex was only observed using the GCMa antiserum (Fig. 2D,
lanes 7 and 14). Taken together, the EMSA and
footprinting results suggest that GCMa specifically recognizes
GBS-(25538-25545) and GBS-(28026-28033) in the 5'-flanking region of
HERV-W 5'-LTR.
Transactivation of HERV-W 5'-LTR by GCMa Depends on the Two GBSs
and Is Cell Type-dependent--
Transient expression
experiments were performed in BeWo cells, using GBS-deletion promoter
constructs, to test whether transactivation of HERV-W 5'-LTR by GCMa
depends on GBS-(25538-25545) and GBS-(28026-28033) (Fig.
3A). As shown in Fig.
3B, transcriptional activation by HAGCMa of both
pCAT In Vivo Interaction between GCMa and GBSs Upstream of the HERV-W
5'-LTR--
Because GBS-(25538-25545) and GBS-(28026-28033) could be
functionally transactivated by GCMa, we further tested whether GCMa interacts with both sites in vivo by means of a ChIP assay.
BeWo cells were transfected with pCMVHAGCMa, and the DNA-HAGCMa
complexes were immunoprecipitated by anti-HA antibody for PCR analysis
(Fig. 4A). Positive signals
were detected when regions 25461-25686 and 27800-28064 were amplified
by PCR (Fig. 4B). These two regions encompass
GBS-(25538-25545) and GBS-(28026-28033), respectively. No signal was
detected when a more distal upstream region, 22473-22731, was
amplified. These results suggest that GCMa associates with the two GBSs
in the 5'-flanking region of HERV-W 5'-LTR in the nuclei of BeWo
cells.
GCMa Transactivates syncytin Gene Expression--
We next tested
whether the expression of GCMa increases the synthesis of syncytin
proteins. A recombinant HAGCMa adenovirus, Ad-HAGCMa, and an empty
recombinant adenovirus, Adlox, were generated. Expression of HAGCMa in
BeWo, JEG3, or HeLa cells transduced with Ad-HAGCMa was analyzed by
Northern and Western analyses at different time points. As shown in
Fig. 5A, Northern analyses
revealed that HAGCMa transcripts in HeLa and BeWo cells were detected
from 16 h to at least 72 h post-transduction. In JEG3 cells,
HAGCMa transcripts were detected from 24 to 72 h
post-transduction. Correspondingly, increasing levels of HAGCMa protein
were detected in the transduced cells (Fig. 5A,
lower panel). To investigate the effect of HAGCMa on syncytin expression, ribonuclease protection assays were
performed to specifically detect the syncytin transcripts in
Ad-HAGCMaC-transduced cells at 24 or 48 h post-transduction. In
comparison with untransduced cells, the level of syncytin
transcripts in transduced JEG3 and BeWo cells at 48 h
post-transduction increased ~4.2- and 3.4-fold, respectively (Fig.
5B). Interestingly, no syncytin transcript was
detected in transduced HeLa cells in the presence of a higher level of
the HAGCMa protein. Western analyses on the syncytin proteins in BeWo
and HeLa cells, transduced with Ad-HAGCMa or Adlox, were performed at
40 h post-transduction. As shown in Fig. 5C, HeLa cells
transduced with the control virus Adlox or Ad-HAGCMa did not reveal any
signals for syncytin proteins (lanes 3 and 4).
However, two bands of 75 and 200 kDa were observed in
Ad-HAGCMa-transduced BeWo cells (lane 2). The
band of 75 kDa may represent the syncytin precursor, whereas the band
of 200 kDa may represent trimeric syncytin (5). The endogenous syncytin
proteins in the Adlox-transduced BeWo cells were detected after a
longer exposure (data not shown). The results of ribonuclease
protection assays and Western analyses also suggest that GCMa
transactivates syncytin gene expression in a cell
type-dependent manner.
Cell fusion assays were performed to investigate the effect
of GCMa-activated syncytin expression on cell fusion. 293 cells expressing red fluorescent protein were cocultured with Adlox- or
Ad-HAGCMa-transduced cells and examined under fluorescence microscopy
30 h after coculture. In comparison with Adlox-transduced cells
(Fig. 6, A-C), fusion events
were significantly increased in Ad-HAGCMa-transduced BeWo and JEG3
cells (Fig. 6, E and F). No fusion events were
observed in Ad-HAGCMa-transduced HeLa cells (Fig. 6D). Taken
together, our study indicates that GCMa up-regulates syncytin gene expression via two GBSs upstream of the HERV-W
5'-LTR and consequently enhances syncytin-mediated cell fusion.
The fusogenic activity of the syncytin protein has been
demonstrated in a variety of primate cell lines including BeWo, COS, HeLa, and 293 (3, 5). In situ hybridization has revealed that the expression of the syncytin gene is restricted to
the syncytiotrophoblast layer in human placenta (3). These observations suggest that syncytin can mediate fusion of cytotrophoblasts into the
syncytiotrophoblast layer. Strict regulation of
syncytin gene expression is important in maintaining an
integral syncytiotrophoblast layer, because the overexpression of
syncytin in cultured cells causes extensive cell fusion and leads to
cell death.2
In this study, we identified GCMa as a transactivator for the
trophoblast-specific expression of the syncytin gene.
Several lines of evidence support this conclusion. First, GCMa
associated with GBS-(25538-25545) and GBS-(28026-28033) in the
5'-flanking region of HERV-W 5'-LTR in vivo based on ChIP
analysis. Second, the expression of GCMa specifically increased the
levels of syncytin transcripts and proteins in trophoblastic cells.
Third, syncytin-mediated cell fusion was increased after GCMa
expression. Interestingly, expression of the syncytin gene
was not detected in HeLa cells expressing a high level of GCMa protein.
This suggests that regulation of syncytin expression by GCMa
is cell type-dependent or that other placenta-specific
factors may be involved in the trophoblast-specific expression of
syncytin gene.
Mutation analysis revealed that nucleotide residues 2, 3, 6, 7, and 8 in the optimal GCM recognition sequence (5'-ATG(A/C)GGG(T/C)-3') are
important for interaction with Drosophila GCM and mouse GCMa (12). We demonstrated that two GCMa binding sites in the 5'-flanking region of HERV-W 5'-LTR were responsive to GCMa. The proximal site,
GBS-(28026-28033), is 34 bp upstream of the 5'-LTR, and its sequence
matches the optimal binding sequence perfectly. The distal site,
GBS-(25538-25545), is 2522-bp upstream of the 5'-LTR, and its sequence
has a mismatch in position 8 in comparison to the optimal binding
sequence. Correspondingly, GBS-(25538-25545) has a lower binding
efficiency with GCMa in EMSA (Fig. 2D, lanes 9-11). Deletion of GBS-(25538-25545) had less of an effect than deletion of GBS-(28026-28033) on the transcriptional activation by
GCMa, suggesting that the two sites may contribute differentially to
the promoter activity (Fig. 3B). In fact, it has been shown that there are at least five GCM-binding sites in the 5'-flanking region of Drosophila GCM gene, each contributing
differentially to the promoter activity of the GCM gene
(20).
Our Western analyses detected the syncytin precursor proteins and their
trimers in Ad-HAGCMa-transduced BeWo cells (Fig. 5C). Blond
et al. (5), using a mouse monoclonal anti-syncytin antibody, have also detected syncytin precursor proteins and their trimers in
transient expression experiments. It is possible that the efficiency of
post-translational cleavage of syncytin protein is too low to produce a
detectable level of surface protein for our Western analyses. Further
investigations into the biosynthesis of syncytin protein and
syncytin-mediated cell fusion may help to clarify this possibility.
GCMa is a placenta-specific transcription factor required for placental
development (14, 15). In addition, GCMa proteins have been
immunolocalized in human syncytioblasts and cytotrophoblasts (21). In
this study, two functional GBSs were identified upstream of the HERV-W
5'-LTR due to the integration of HERV-W in the human genome. We found
that GCMa recognizes these two functional GBSs, induces the
trophoblast-specific expression of the syncytin gene, and
consequently enhances syncytin-mediated trophoblastic fusion. These
events could ensure the formation of an integral syncytiotrophoblast layer only in the placenta. A recent clinical survey on the expression of syncytin in human placentas has revealed a lower syncytin mRNA level in patients with placental dysfunction, including preeclampsia and hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome (22). Because syncytin is a target gene of GCM, this
warrants an investigation into the role played by GCMa in the etiology of preeclampsia and HELLP syndrome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DASH II human genomic library (Stratagene, La
Jolla, CA). A genomic clone, L13, covering the entire proviral genome
of the syncytin-harboring HERV-W family member was isolated
and used to build promoter constructs (Fig. 1A). The human
BAC clone, 083M05 (GenBankTM accession no. AC000064), was
used to isolate more distal genomic regions upstream and downstream of
the proviral genome of the syncytin-harboring HERV-W family
member (Fig. 1A).
d-(25468-30953), pCATp-(25468-30953), or
pCATdp-(25468-30953), respectively.
5 genomic DNA
(19). For a control, an empty recombinant adenovirus (Adlox) was
generated using a linearized pAdlox and 5 genomic DNA. Ad-HAGCMa and
Adlox were grown and amplified in CRE8 cells for two consecutive
cycles. Cells in culture plates were transduced with Ad-HAGCMa or Adlox at an multiplicity of infection of 100 or 200 at 37 °C for 90 min.
After that, the virus was removed, and fresh culture media were added
and incubated for an additional time until analysis.
-actin cDNA probes. To detect syncytin transcripts, 15 µg of
RNA from Ad-HAGCMa-transduced cells were analyzed by ribonuclease protection assays using the RPA III kit (Ambion, Austin, TX). The
syncytin riboprobe contains nucleotides 417-821 relative to the
translation start site. A kit-provided -actin riboprobe was used as
an internal control. The protected syncytin bands were quantified by
BAS-1500. HAGCMa and syncytin proteins in transduced cells were subject
to immunoblotting with an anti-HA antibody and a syncytin antibody,
respectively. Antiserum against syncytin was induced in guinea pigs
using a His-tagged surface protein (amino acids 21-215) expressed in BL21(DE3).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DASH II genomic clone (L13)
containing the HERV-W genome was isolated using the syncytin cDNA
probe (Fig. 1A). A human BAC clone, 083M05, which encompasses the L13 clone, was used together with L13 to build a series
of promoter constructs covering genomic regions up to 14.8-kb upstream
of the 5'-LTR and 5.1-kb downstream of the 3'-LTR of HERV-W (Fig. 1,
A and B).
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Fig. 1.
Promoter analysis of the syncytin
gene. A, schematic representation of the human
BAC clone 083M05, the DASH II clone L13, and HERV-W. The numbers
above and underneath 083M05 denote the positions of the numbered
nucleotides. B, schematic representation of promoter
constructs used in this study. The genomic fragment inserted in pE1bCAT
is indicated on the left by a horizontal
filled bar, denoting its location in the BAC
clone 083M05 as listed in panel A. The numbers on
the right indicate the ranges of the genomic fragments in
the BAC clone 083M05. C, promoter analysis for the
syncytin gene. BeWo cells were transfected with 0.5 µg of
the indicated promoter construct in the absence (black
bar) or presence (gray bar) of 0.5 µg of pCMVHAGCMa. Mean values and S.E. obtained from six independent
transfection experiments are provided. Asterisks denote
statistically significant differences (**, p < 0.01) between mock- and expression plasmid-transfected groups. HAGCMa
proteins in the total cell lysate of mock- and pCMVHAGCMa-transfected
groups were analyzed by Western analysis with a horseradish
peroxidase-conjugated rat monoclonal anti-HA antibody. As a loading
control, actin proteins in the lysate were detected with a rabbit
polyclonal anti-actin antibody.
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Fig. 2.
Analysis of GCMa binding to GBSs in the
5'-flanking region of HERV-W 5'-LTR. A, GCMa-FLAG
proteins were affinity purified from Sf-9 cells infected by a
recombinant GCMa-FLAG baculovirus. Approximately 0.2 µg of purified
GCMa-FLAG was analyzed by SDS-PAGE and detected by Coomassie Blue R-250
staining. B, EMSA of GCMa-FLAG and radiolabeled genomic
fragments containing potential GBSs. Ab, antibody.
Comp, the presence or absence ( 
) of unlabeled pGCMa or Mut
oligonucleotide in a 100-fold (100×) excess. NPGS, normal
guinea pig serum. The arrow and arrowhead
indicate the GCMa-FLAG-DNA complex and its supershifted complex,
respectively. C, DNase I footprinting analysis of GBSs in
the 5'-flanking region of HERV-W 5'-LTR. Two genomic fragments (nt
25488-25587 and nt 27978-28077) in the HERV-W 5'-flanking region were
asymmetrically radiolabeled. The labeled DNA probe was incubated with
GCMa-FLAG protein, digested with DNase I, and analyzed on 8%
polyacrylamide-urea gels. The vertical lines on the
right denote the protected regions of which sequences are
listed and underlined on the left. G+A, Maxam and Gilbert G
plus A sequencing reaction of the probe. B, bound or
protected probe. F, free probe. D,
oligonucleotides pGCMa and dGCMa, derived from the footprinting
analysis, were radiolabeled as probes for GCMa-FLAG in EMSA.
d-(25468-30953) and pCATp-(25468-30953) was observed;
however, this activation was lower than that observed with
pCAT-(25468-30953). Moreover, transactivation was abolished in
pCATdp-(25468-30953). In contrast, no transcriptional activation by
HAGCMa of these constructs was observed in HeLa cells. These results
suggest that transactivation of HERV-W 5'-LTR by GCMa depends on the
two GBSs and is cell type-dependent.
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Fig. 3.
Transactivation of GCMa depends on the
presence of GBSs in the 5'-flanking region of HERV-W 5'-LTR.
A, schematic representation of the mutant promoter
constructs. The deleted site is indicated by X. B, BeWo or HeLa cells were transfected with 0.5 µg of the
indicated promoter construct in the absence or presence of 0.5 µg of
pCMVHAGCMa. Mean values and S.E. were obtained from seven and three
independent transfection experiments for BeWo and HeLa cells,
respectively. Asterisks denote statistically significant
differences (**, p < 0.01) between mock- and
expression plasmid-transfected groups. HAGCMa and actin proteins in the
transfected cells were analyzed as in Fig. 1C.
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Fig. 4.
In vivo interaction between GCMa
and the GBSs in the 5'-flanking region of HERV-W 5'-LTR.
A, schematic representation of the GBSs, HERV-W, and primer
sets used in a ChIP assay. B, HAGCMa interacts with
GBS-(25538-25545) and GBS-(28026-28033) in vivo. ChIP
assays of BeWo cells transfected with pCMVHAGCMa were performed for
genomic fragments covering nt 25461-25686 (containing
GBS-(25538-25545)), nt 27800-28064 (containing GBS-(28026-28033)),
and nt 22473-22731. Immunoprecipitates without input chromatin
(mock) or in the absence of antibody were used as
controls. Input chromatin represents a portion of the
sonicated chromatin prior to immunoprecipitation.
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Fig. 5.
Adenovirus transduction of GCMa induces
syncytin expression. A, Northern (NB) and
Western (WB) analyses of HAGCMa expression in
Ad-HAGCMa-transduced cells at the indicated time post-transduction
(p.t.). B, HA-GCMa transactivates
syncytin expression in trophoblastic cells. The syncytin
transcripts in Ad-HAGCMa-transduced cells were detected by ribonuclease
protection assays using a syncytin-specific riboprobe. A -actin
riboprobe was used as an internal control. C, Western
analyses of HAGCMa and syncytin proteins in BeWo or HeLa cells
transduced with Adlox or Ad-HAGCMa 40 h post-transduction. As a
loading control, the blot was reprobed with an actin antibody.
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[in a new window]
Fig. 6.
Expression of GCMa increases
syncytin-mediated cell fusion. 293 cells transiently
expressing red fluorescent protein were cocultured with cells
transduced with Adlox (A-C) or Ad-HAGCMa (D-F).
After 30 h, cell fusions were examined under a fluorescence
microscope. Bar, 120 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENT |
|---|
We thank Dr Hsou-min Li for critical reading of this manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the National Science Council (91-2311-B-001-043) and Academia Sinica, Taiwan (to H. C.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
011-886-2-27855696 ext. 6090; Fax: 011-886-2-27889759; E-mail:
hwchen@gate.sinica.edu.tw.
Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M209316200
2 P. Chen and H. Chen, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: HERV-W, human endogenous retrovirus family W, LTR, long terminal repeat; GBS, GCMa-binding site; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase; nt, nucleotide(s); EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; Ad, adenovirus.
| |
REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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