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Department of Pediatrics, University of Cincinnati College of Medicine and Division of Endocrinology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gene regulation, placenta, syncytiotrophoblast, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence strongly support a critical role for syncytin in human trophoblast differentiation. Syncytin is specifically expressed in the syncytiotrophoblast layer of the human placenta and appears to play a critical role in mediating cytotrophoblast cell fusion and syncytialization during trophoblast differentiation [1–3]. Syncytin mRNA and protein levels increase significantly during spontaneous in vitro differentiation of primary cultures of human cytotrophoblast cells and cAMP-induced differentiation of BeWo choriocarcinoma cells. The increase in syncytin gene expression is concomitant with the induction of cell fusion, and the cell fusion is markedly inhibited by specific syncytin antisense oligonucleotides [5]. Furthermore, overexpression of syncytin induces cell fusion in a variety of mammalian cell lines, including BeWo choriocarcinoma cells, COS, and 293 cells; and the induction of syncytialization in these cells can be prevented by syncytin antiserum [1].
Syncytin levels have been measured in placentas from women with normal placentas and various pathological conditions of pregnancy [6, 7]. Knerr and colleagues [8] showed that syncytin mRNA levels in normal human placentas are positively correlated with gestational age and placental weight. Syncytin mRNA levels are markedly reduced in placentas of preeclamptic patients [6, 7], suggesting that syncytin may be involved in the pathogenesis of preeclampsia.
Although syncytin has a pronounced effect on the fusion of human cytotrophoblast cells, little is known about the mechanisms regulating the transcription of the syncytin gene. We recently isolated the human syncytin gene promoter from genomic DNA and initiated studies of the cis- and trans-acting factors that are important for transcriptional regulation of syncytin gene expression [3]. We observed that mRNA initiation was TATA-box independent, with a NCA0A cap signal at the major transcription start site. A CCAAT motif located between nt–65 and nt–56 and an octamer protein-binding site located between nt–42 and nt–35 were found to be critical for basal promoter activity [3]. Two potential enhancer regions of the promoter at nt–1519 to –984 and nt–294 to nt–148 were required for maximal expression in normal trophoblast cells [3]. Prudhomme et al. [9] recently reported that the upstream regulatory element (from nt–436 to +1) of syncytin promoter contains a complex regulatory unit that may contribute to the tissue-specific expression of syncytin gene in the placenta. Yu et al. [10] recently reported that the transcription factor glial cells missing factor homolog 1 (GCM1, also known as GCMA) enhances syncytin gene expression in BeWo and JEG3 choriocarcinoma cells. However, the cis-DNA sequence(s) and trans-factor(s) involved in trophoblast-specific expression of the syncytin gene remain to be identified.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Luciferase reporter plasmids containing different size fragments of the syncytin promoter were prepared as previously described [3]. The pSYN(–294/+24)-Luc plasmid was constructed by amplifying the syncytin promoter region from nt–294 to nt+24 from pSYN(–1519/+181)-Luc [3] by PCR using the 5' primer GTC TTT GTG GCT ATG TGA CAT and the 3' primer CTG CTG TGC TCT CAG GCA ATA. The amplified fragment was then ligated into the SmaI site of pGL3-basic (Promega Corp., Madison, WI). Plasmids were also constructed that contain the nt–294/–148 region of the syncytin promoter subcloned upstream of the heterologous minimal promoters for SV40 and human ß-globin, using pGL3-promoter (Promega Corp.) and pGLOB-GL3 [11] vectors respectively. pSYN(–294/–148)/SV40-Luc was constructed by excising the syncytin fragment from pSYN(–294/–148)-Luc by AvrII and KpnI double digestion and ligating the fragment into a pGL3-promoter vector at the SmaI and KpnI sites. The pSYN(–294/–148)-/GLOB-Luc was constructed by ligating the fragment into pGLOB-GL3 at the SmaI and KpnI sites. The orientation and sequence of each construct was confirmed by DNA sequence analysis using the T7 Sequenase Quick Denature Plasmid Sequence Kit (USB Corp., Cleveland, OH). The expression vectors for mouse GATA2 and human GATA3 were kindly provided by Dr. James Douglas Engel [12] (Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI) and a plasmid for human SP1 was kindly provided by Dr. Jun Ma [13] (Department of Development Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH).
Site-Directed Mutagenesis
Olignucleotide-directed mutagenesis of the SP1-, ETS2-, MYB- (v-myb myeloblastosis viral oncogene homolog [avian]), and GATA1- and GATA2-binding sites in pSYN(–294/+24)-Luc was performed using the Quick Change Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). Each of the mutated base pairs was confirmed by DNA sequencing. The sequences of the double-stranded oligonucleotides used for site-directed mutagenesis assay are listed in Table 1.
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Cell Cultures
Cell cultures were performed as previously described in our laboratory [14]. Third-trimester placentas were obtained from women with normal pregnancies and deliveries. The protocol for obtaining placentas was approved by the Human Investigation Committees of the University of Cincinnati and the Children's Hospital Medical Center. Cytotrophoblast cells were isolated by enzymatic disaggregation and purified by negative CD-9 selection and grown in Dulbecco modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). JEG3 human choriocarcinoma cells (ATCC HTB-36) were grown in Eagle MEM with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. BeWo human choriocarcinoma cells (ATCC CCL-98) were grown in F-12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 10% FBS. HepG2 human hepatoma cells (ATCC HB-8065) were cultured in Eagle MEM with 2.0 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. CV-1 monkey kidney cells (ATCC CCL-70) were cultured in Eagle MEM with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. Schneider line 2 (S2) cells (kindly provided by Dr. Jun Ma) were grown at 25°C in Shields and Schneider insect cell medium (Invitrogen Life Technologies Inc., New York, NY) supplemented with 10% FBS.
Transfection Studies
Transient transfection studies of BeWo, JEG3, HepG2, and CV-1 cells were performed in triplicate using six-well plates by the liposome method described by Cheng et al. [11]. Each well contained 5 µg of reporter plasmid and 0.5 µg of pRL-TK-Luc (Promega Corp.) as well as 1 µg of expression plasmids DNA or filler plasmid DNA. S2 cells were transfected with Lipofectamine reagent (Invitrogen Life Technologies Inc.). S2 cells were pelleted and washed twice with Schneider medium. Then 5 x 105 cells were resuspended in 100 µl of Schneider medium. For each transfection, 10 µl of Lipofectamine and 6.5 µg of plasmid DNA were separately added into tubes containing 150 µl of Schneider medium. Lipofectamine and DNA in medium were then mixed and immediately added to resuspended S2 cells. After 2–4 h of incubation at room temperature, cells were precipitated and washed twice with Schneider medium. Cells were then resuspended in 2 ml of complete Schneider medium, plated, and kept at 25°C. The cells were harvested 48 h after transfection. Cell extracts were prepared using luciferase cell lysis reagent (Promega Corp.). The results represent the average of three independent transfection assays normalized to pRL-TK-Luc using a dual luciferase reporter assay system (Promega Corp.).
Preparation of Nuclear Extract
Nuclear extracts were prepared from human trophoblast cells that were cultured for 3 days and from cell lines as previously described by Cheng et al. [3]. The protein concentrations of the extracts were determined by a Bradford assay (Bio-Rad Laboratories Inc., Hercules, CA) using bovine serum albumin as a standard. The nuclear extracts were aliquoted and stored at –80°C.
DNase I Footprinting
DNase I footprinting was performed as previously described [3]. A 5'-end-labeled probe of a 362-base pair (bp) syncytin promoter region were generated by PCR using pSYN(–1519/+181)-Luc as template with the 5' primer ACT CTC TGG AGA GTG AAT TAC TGA GTC ACA TG and the 3' primer CTG CTG TGC TCT CAG GCA ATA GAT GAT TGG. Protected regions were detected by comparing the digestion patterns with that of control reactions using BSA in place of nuclear extracts.
Gel Mobility Shift Assays
Gel shift assays were performed essentially as previously described [3]. The binding reaction contained the appropriate radiolabeled probes and 5 mg nuclear extract from BeWo cells. The sequences of the double-stranded oligonucleotides used for gel mobility shift assays are listed in Table 2. For competition assays, the 100-fold molar excess of unlabeled oligonucleotides were incubated with the nuclear extracts for 20 min before the addition of labeled probe. For supershift analysis, nuclear extracts were incubated with appropriate antiserum before addition of the radiolabeled probe. The antiserums to human SP1, GATA, and normal immunoglobulin Gs (IgGs) were purchased from Santa Cruz Biotechnology, Inc. and used according to the instructions provided. The normal serums used as control in the electrophoretic mobility assay (EMSA) experiments included normal rabbit IgG, normal goat IgG, and normal mouse IgG. The antiserums used in the EMSA experiments included goat polyclonal IgG to GATA1, rat monoclonal IgG2
to GATA1, goat polyclonal IgG to GATA2, mouse monoclonal IgG1 to GATA2, goat polyclonal IgG to GATA3, mouse monoclonal IgG1 to GATA3, goat polyclonal IgG to GATA4, and goat polyclonal IgG to SP1. The binding reactions were resolved in a 5% nondenaturing polyacrylamide gel.
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Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from BeWo cells, JEG3 cell, normal placental cells, and HepG2 hepatoma cells. First-strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Invitrogen Life Technologies Inc.) and oligo dT according to the manufacturer's protocol. Primer sequences used for detection of GATA1, GATA2, GATA3, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts are listed in Table 3.
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Reverse transcription-polymerase chain reaction (RT-PCR) of the GATA1, GATA2, GATA3, and GAPDH mRNAs yielded products with the predicted sizes of 130, 94, 100, and 192 bp, respectively. The PCR products were electrophoresed on 2% agarose gels at 225 V for 2.5 h, and the gels were stained with Gelstar Nucleic Acid Gel Stain (Cambrex Bio Science Rockland, Inc., Rockland, ME) for 30 min. The signals were detected using a Kodak ID V3.6 Imaging System (Kodak Scientific Imaging Systems, New Haven, CT).
Real-Time Quantitative PCR
Real-time quantitative PCR was performed on a Mx3000 Real-time PCR System (Stratagene Inc.) using a standard procedure with SYBR Green Master Mix (Stratagene Inc.). The oligonucleotides for detection of GATA2 and -3 are listed in Table 3. Electrophoresis of the real-time PCR products by 2% agarose gel electrophoresis yielded single bands of the predicted sizes. In each instance, the amount of RT-PCR product for the gene of interest was normalized to the amount of GAPDH in the same sample.
Statistical Analysis
Statistical differences between sample means were determined by ANOVA followed by post hoc multiple comparison testing using the Neuman-Keuls test. Values are expressed as mean ± SEM, and P < 0.05 is considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To delineate the putative DNA sequences that are important in the regulation of syncytin gene expression, transient transfection studies were performed in two trophoblast cell lines (BeWo and JEG3 human choriocarcinoma cells) that express syncytin during differentiation and two nontrophoblast cell lines (HepG2 and CV-1 cells) that do not express syncytin. The reporter construct pSYN (–1519/ +181)-Luc supported high levels of reporter expression in all four cell lines that were 100- to 200-fold higher than the promoterless vector (pGL3-basic) (Fig. 1, A and B). Progressive deletions of the promoter from nt–1519 to nt–294 did not significantly reduce promoter activity in any of the cell types. However, deletion of the 146 bp of the 5'-flanking region from nt–294 to –148 resulted in a 70% decrease in luciferase activity in the trophoblast cells but not in nontrophoblast cells. Further removal of sequences to nt–49 abolished transcriptional activity in both the trophoblast (Fig. 1A) and nontrophoblast cell lines (Fig. 1B). Taken together, these data suggest that the 146-bp fragment between nt–294 and nt–148 confers trophoblast-specific expression and that the 148-bp fragment proximal to the transcription start site is required for basal promoter activity.
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The 146-bp Fragment Between nt–294 and nt–148 Acts as a Trophoblast-Specific Enhancer in the Heterologous Genes
To examine whether the 146-bp fragment acts as a tissue-specific enhancer, the fragment was subcloned directly upstream of a minimal heterologous simian virus 40 (SV40) promoter and a minimal human ß-globin promoter linked to a luciferase gene. In the trophoblast cell lines, the promoter activities of the chimeric genes were 4-fold (BeWo cells) and 6-fold (JEG3 cells) greater than the activities of the minimal SV40 and ß-globin promoters (Fig. 2, A and B). However, identical experiments performed in the nontrophoblast cells demonstrated that the promoter activities of the chimeric gene plasmids were nearly identical to the activities of the minimal promoters (Fig. 2, C and D). Taken together, these data support the hypothesis that the region of the syncytin promoter between nt–294 and nt–148 acts as a trophoblast-specific enhancer.
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DNase I Footprinting of the Syncytin-Enhancer Element
To assess whether the syncytin-enhancer region interacts with transcription factors in trophoblast cells (BeWo) and nontrophoblast cells (HepG2), DNase I footprinting analyses were performed with these nuclear extracts using a 362-bp fragment of the syncytin gene (nt–338/+24) that includes the enhancer region. As shown in Figure 3A, nuclear extracts of BeWo cells but not HepG2 cells protected six regions (FP1–FP6) of the fragment. Both FP5 and FP6 are located downstream of the 146 bp of syncytin enhancer region. FP1 spans approximately 7 bp (nt–270 to –264) and FP2 spans 9 bp (nt–259 to –251). FP3 spans 29 bp (nt–239 to –211) and contains five hypersensitive sites, and FP4 spans 40 bp (nt–204 to –165) and contains three hypersensitive sites. Computer analysis revealed that FP3 contains consensus SP1- and an ETS2 protein-binding sites and FP4 contains GATA- and MYB-binding sites. FP3 also contains a binding site for GCM1 that has previously been shown to be important for the induction of syncytin gene expression in BeWo chorciocarcinoma cells [10]. The sequence of the syncytin enhancer element (nt–294 to –148) is presented in Figure 3B with the footprinting regions underlined. The GATA-binding site in FP4 differs from the consensus GATA motif by the downstream (TTATCT) in an inverted orientation (Table 2). Ko and Engel [15] reported that this DNA sequence organization of a double site in an inverted orientation produced the highest affinity for GATA protein binding in vitro.
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Mutational Analysis of the Enhancer
To determine which footprinted regions of the enhance region are important for the regulation of syncytin gene expression, site-directed mutagenesis of the putative SP1-, ETS2-, GATA-, and MYB-binding sites were performed and the promoter activities of the mutated constructs were compared with the wild-type construct. Mutations of the ETS2 site in FP3 and the MYB site in FP4 did not affect syncytin promoter activity. However, mutations of the SP1-binding site on FP3 and the GATA-binding site in FP4 inhibited promoter activity in the trophoblast cell lines by 30–40% and 70–80%, respectively (Fig. 4, A and B). However, identical experiments performed in the nontrophoblast cell lines demonstrated that mutagenesis of the SP1- and GATA-binding sites had no effect on enhancer activity (Fig. 4, C and D). Taken together, the results from the deletion and site-directed mutagenesis analyses strongly suggest that the SP1 motif (FP3) and GATA-binding sites in FP4 are critical for syncytin-enhancer activity.
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Gel Shift and Supershift Studies of the Enhancer
To investigate whether nuclear proteins bind specifically to the SP1- and GATA-binding sites, gel shift assays were performed using oligonucleotides containing either a SP1 motif (SP1wt) or a GATA-binding site (GATAwt) and nuclear extracts from BeWo cells. As shown in Figure 5, two major DNA-protein complexes (complexes A and B) were identified when 32P-labeled SP1wt oligonucleotide probe was incubated with BeWo cell nuclear extract (lane 1). Formation of the two complexes was completely prevented when the reaction was performed in the presence of a 100-fold excess of unlabeled wild-type oligonucleotide (lane 2) but not in the presence of a 100-fold excess of an oligonucleotide containing a mutated SP1-binding site (lane 3). The complexes were supershifted by a SP1 antiserum (lanes 4 and 5), indicating that SP1 binds to the SP1 motif. Normal rabbit IgG, however, did not cause a supershift (lane 6).
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Figure 6 shows that a specific DNA-protein complex was formed when a 32P-labeled GATAwt oligonucleotide probe was incubated with a nuclear extract from BeWo cells (lane 1). The formation of the complex was prevented by incubation of the reaction mixture with excess unlabeled oligonucleotide corresponding to the GATA-binding site in FP4 (lane 2) and an oligonucleotide containing a consensus GATA motif (Santa Cruz) (lane 3). Oligonucleotide containing mutated GATA-binding sites, however, did not compete for binding (lanes 4 and 5). No supershift bands were observed when GATA1 (lanes 6 and 7) or GATA4 (lane 12) antisera were added to the incubation mixtures. However, the complex formed by GATAwt oligonucleotide and the nuclear extract was supershifted by GATA2 (lanes 8 and 9) and GATA3 antisera (lanes 10 and 11). No supershift occurred when normal goat IgG (lane 13), normal rabbit IgG (lane 14), or normal mouse IgG (lane 15) was used in place of the GATA2 and GATA3 antisera. Taken together, the DNase I footprinting analysis and supershift assays strongly suggest that GATA2 and GATA3 bind to the syncytin enhancer in trophoblast cells.
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SP1 and GATA2 and -3 Function as Activators of the Syncytin Enhancer
To determine whether SP1 modulates human syncytin promoter activity, S2 cells, which are deficient in Sp-related proteins [16], were used. S2 cells were cotransfected with the wild-type syncytin promoter or its mutant and increasing concentrations of pAct-SP1 expression vector. As shown in Figure 7, pAct-SP1 stimulated syncytin wild-type promoter activity in a dose-dependent manner. The pAct-SP1 at 1 µg/well stimulated syncytin promoter activity by 20-fold. However, a mutant reporter construct that lacked the SP1-binding site (pSP1mt-Luc) was stimulated by SP1 by only 2-fold. These data suggest that SP1 is an activator of the syncytin enhancer element.
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HepG2 cells, which do not express GATA proteins, were used to investigate the functional requirement of GATA2 and -3 for syncytin expression. HepG2 cells were cotransfected with the syncytin construct and either the pCMV-mGATA2, pCMV-hGATA3, or pCMV empty vector. As shown in Figure 8A, both GATA2 and -3 stimulated syncytin wild-type promoter activity in a dose-dependent manner. Maximal stimulation by GATA2 and -3 were 3- and 5-fold, respectively. The specificity of these responses was determined using the mutant syncytin promoter constructs that eliminated the GATA-binding site. As shown in Figure 8B, mutations of the two GATA-binding sites in the syncytin promoter abolished the induction of promoter activity by overexpression of GATA2 and GATA3.
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Analysis of GATA2 and GATA3 Expression Patterns
Experiments were performed to determine whether GATA family members are expressed in trophoblast cells and whether GATA mRNA levels change during trophoblast differentiation. As seen in Figure 9, GATA2 and GATA3 mRNAs are expressed in trophoblast cells (BeWo, JEG3, and primary trophoblast cells) but not in HepG2 cells. In contrast, GATA1 mRNA is not detectable in the trophoblast cells and HepG2 cells.
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During in vitro differentiation of human cytotrophoblast cells, GATA2 mRNA levels increase more than 16-fold during the first 3 days of differentiation when the cells are fusing to form a syncytium (Fig. 10). The levels of GATA2 mRNA subsequently remain relatively constant for the remainder of the 7-day culture period. GATA3 mRNA levels exhibit a similar expression pattern, increasing about 8-fold during the first 3 days of differentiation (Fig. 10). Syncytin mRNA levels increase about 4-fold during the first 3 days of differentiation, reach a plateau from Day 3 to Day 6 and then begin to decrease once cell fusion is complete (Fig. 10). The observations that GATA2 and -3 are expressed in human placenta with expression patterns during trophoblast cell differentiation that parallel the expression of the syncytin gene further suggest that GATA2 and -3 may be involved in regulation of trophoblast cell differentiation in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNase I footprint analyses indicated that the single SP1- and single GATA-binding sites in the enhancer region are protected by nuclear extracts of trophoblast cells. Mutation of the two sites significantly decreased syncytin promoter activity in transient transfection studies, indicating that the SP1- and GATA-binding sites in the enhancer region are also critical for basal and cell-specific expression of the syncytin gene in trophoblast cells. Results from other experiments indicated that overexpression of SP1 and GATA2 and -3 could significantly stimulate syncytin promoter activity in transfected cell lines, suggesting that SP1 and GATA2 and -3 function as trans activators of syncytin gene expression in vivo.
The Sp family contains four members, SP1 to Sp4 [17]. SP1 and Sp3 compete for a similar binding site [18–21], and the ratio of the two transcription factors has been shown to regulate the relative rates of transcription of several genes. Sp2 is least similar to the other Sp family members, and very little is known about its function. Sp4 has a narrow expression profile restricted to brain, epithelial tissues, testis, and developing teeth [22]. Gel shift and supershift analyses showed that the SP1 motif in the enhancer region binds SP1. Experiments to study binding of other SP1 family members were not performed. Because the complex formed by the SP1 binding site and nuclear extracts from trophoblast cells was not completely supershifted by an antiserum to SP1, it is possible that a component of the shifted complex reflects an interaction with other members of the SP1 family.
The GATA family in mammals is composed of six members (GATA1 to -6) [23–27]. The family members have two highly conserved zinc finger domains, C-X2-C-X17-C-X2-C that recognize and bind with high affinity to the DNA consensus motif WGATAR [15, 28]. Gel shift and supershift analyses showed that the GATA motif in the enhancer region binds GATA2 and -3 but not GATA1 and GATA4. The levels of GATA2 and GATA3 mRNAs during in vitro trophoblast differentiation were very similar to that of syncytin mRNA. All three mRNAs increased markedly during the first 3 days of differentiation as the cells fused to form a multinucleated syncytium. The GATA2 and GATA3 mRNA levels remained at relatively high levels for the remainder of the 7-day culture, while syncytin mRNA levels began to decrease on the last day.
Numerous studies have shown that the expression of cell type-specific genes is tightly controlled by the combined action of multiple transcription factors that interact with specific DNA sequences [29, 30]. Transcription factors may also bind coactivators and repressors that enhance or repress transcriptional activity. Because the SP1- and GATA-binding sites within the syncytin enhancer region were not protected by nuclear extracts of HepG2, it is very likely that the absence of syncytin expression in these cells is due, at least in part, to the fact that the cells do not express GATA2 and GATA3.
SP1 is expressed in most tissues, and targeted inactivation of the gene in the mouse results in retarded growth of the embryos, which die early during gestation [31]. Despite the ubiquitous expression of SP1 protein, it was proposed that cell-type-specific transcription can be regulated through its interaction with others transcription factor(s)/ cofactor(s) [32]. Many trophoblast genes, including hCG- and -ß genes, are controlled by SP1, which, in some cases, acts cooperatively with other transcription factors [33, 34]. For example, AP-2 (also known as TFAP2A, transcription factor AP-2 [activating enhancer binding protein 2 alpha]), which is expressed in trophoblast cells but not in HepG2 cells, can interact with SP1 to regulate expression of the hCG- and -ß genes in trophoblast cells [34]. Furthermore, syncytium-specific CYP2C19 (cytochrome P450, family 2, subfamily C, polypeptide 19) expression was also shown to be controlled by SP1, AP-2, and GCM1 [35].
GATA transcription factors have been shown to regulate differentiation, growth, and survival of a wide range of cell types (for reviews, see [36–38]). The genes encoding GATA factors are transcriptionally regulated in a tissue-restricted manner, and the various proteins are believed to regulate distinct subsets of target genes. GATA1 is expressed in erythroid, megakaryocytic, and mast cells, as well as in the testis [24]. GATA2 is expressed in hematopoietic stem and progenitor cells, endothelial cells, and other cells, including trophoblast cells [12, 23]. GATA3 is expressed in T lymphocytes, placenta, kidney, adrenal gland, and nervous system [12, 26]. GATA4, GATA5, and GATA6 are commonly transcribed in various mesoderm- and endoderm-derived tissues [27]. Despite the unique expression patterns and developmental functions of GATA family members, considerable interplay exists among these factors [24, 25].
GATA2 and GATA3 are implicated in developmental regulation in a variety of cell types [12, 13, 39]. Their mRNAs and proteins have also been localized in the murine placenta to giant trophoblast cells; and their mRNAs peak during midgestation (Day 10), coincident with the peak in placental lactogen I mRNA [39, 40]. In in vivo studies, murine placentas lacking GATA2 or GATA3 exhibit reduced placental lactogen I and proliferin gene expression, with GATA2 null placentas having greater reductions in proliferin [12].
Our findings that the enhancer region binds SP1 and GATA1 and that mutations in each site dramatically impair its transcriptional activity suggest coorperativity between these factors. Indeed, cooperativity between SP1 and members of the GATA family has also been observed for the mouse AMY1A (amylase, alpha 1A; salivary) gene in K562 cells [41] and human CYP2C18 (cytochrome P450, family 2, subfamily C, polypeptide 18) gene in JEG cells [42]. Therefore, the synergism and possible interactions between these factors need to be analyzed.
GCM1 has been reported earlier to bind to the FP3 region of the syncytin enhancer and regulate syncytin-mediated BeWo and JEG cell fusion [10]. Currently, two GCM-like genes (GCM1 and GCM2) have been reported in mouse and human. GCMs encode transcription factors with a new type of zinc-containing DNA-binding domain, a so-called GCM domain that recognizes the DNA sequence 5'-ATGCGGGT-3' [43]. Although GCM1 regulates target genes by binding directly to octameric GC-rich DNA-binding site, it can also activate target genes in combination with interacting transcription factors, like Pix2 [43]. Because FP3 contains both Sp1- and GCM-binding sites, transcription factors Sp1 and GCM1 may synergize to regulate syncytin gene expression in trophoblast cells.
In our earlier studies of syncytin promoter in trophoblast cells, we noted that ubiquitous transcription factors CEBPZ (CCAAT/enhancing binding protein zeta) and POU2F1 (POU domain, class 2, transcription factor 1, also known as OCT-1) that bind, respectively, to a CCAAT motif and a POU2F1-binding site of the proximal promoter play an essential role for basal expression of the syncytin gene [3]. It will be of interest to define other factors and/or cofactors that function in conjunction with GATA and CEBPZ or POU2F1 to regulate the syncytin gene.
In summary, we have identified a 146-bp trophoblast-specific enhancer element on the 5'-flanking region of the syncytin gene that contains a functional SP1-binding site and a GATA motif that are important for basal and cell-specific gene expression. We have also shown that GATA2 and GATA3 mRNAs are expressed during trophoblast differentiation with patterns similar to that of syncytin, suggesting that GATA2 and GATA3 may play critical roles in the regulation of placenta development in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Stuart Handwerger, Division of Endocrinology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. FAX: 513 636 7486; stuart.handwerger{at}cchmc.org
Received: 13 January 2005.
First decision: 2 February 2005.
Accepted: 27 April 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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