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Mechanisms of Hormone Action |
Cincinnati Children's Hospital Medical Center and Department of Pediatrics, Division of Endocrinology, University of Cincinnati College of Medicine, 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, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence strongly suggest that the fusion of cytotrophoblast cells during trophoblast differentiation is due to the action of syncytin, a recently described, highly fusogenic membrane glycoprotein that can induce syncytium formation on interaction with the amino acid transporter/cell surface receptor (ASCT) [5]. Mi and coworkers [6] demonstrated that forskolin-induced syncytialization of BeWo choriocarcinoma cells causes a marked increase in syncytin expression that precedes syncytial formation and that the induction of syncytialization in these cells can be prevented by syncytin antiserum. In addition, Frendo and colleagues [7] showed that cell fusion and differentiation of normal human trophoblast cells in culture are markedly inhibited by specific syncytin antisense oligonucleotides.
Syncytin is encoded by an envelope gene of the human endogenous retrovirus-W (HERV-W) that has been incorporated into chromosome 7 of the human genome [8, 9]. The retrovirus genome typically contains three genes organized in the sequence gag-pol-env that are flanked by two long-terminal repeats (LTRs) (Fig. 1). The molecule sizes of gag, pol, env, and the LTR DNA sequences are usually about 2.0 kilobases (kb), 3.0 kb, 1.8 kb, and 0.2–1.5 kb, respectively (for reviews, see [8, 9]). Although most of the HERVs are defective because of mutational decay, many of them are transcriptionally active, and some HERVs still contain open reading frames (ORFs) for retroviral proteins. HERV-W contains a complete ORF coding for an env protein, but the ORFs for gal and pol are interrupted by frame shifts and stop codons [10]. HERV-W is highly expressed in human placenta, and weaker expression is noted in human testes [6]. Expression is not observed in other human tissues. In situ hybridization and immunohistochemical analyses indicate that the syncytin gene in the placenta is expressed in the syncytiotrophoblast cells [7, 11].
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Although syncytin has a critical role in the fusion of human cytotrophoblast cells, little is known about the mechanisms regulating the transcription of the syncytin gene. Yu et al. [12] recently reported that the transcription factor glial cells missing factor (GCM) enhances syncytin gene expression in BeWo and JEG3 choriocarcinoma cells. However, the precise mechanisms of regulation of expression of the syncytin gene remain unclear. In addition, the transcription initiation site and transcription factors involved in the regulation of syncytin promoter have not as yet been identified. In this study, we have isolated and characterized the syncytin gene promoter from human genomic DNA and have identified cis-regulatory elements that are critical for basal gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The syncytin mRNA sequence is located between nucleotides (nt) 34957 and 37837 of a 56-kb human bacterial artificial chromosome (BAC) clone (GenBank accession no. AC000064) that contains the HERV-W mRNA sequence previously described by Blond et al. [10] (Fig. 1). This 2.9-kb mRNA is derived from an 8.5-kb pre-mRNA by a splicing event [10]. Analysis of the 56-kb DNA sequence using the PROSCAN Version 1.7 suite of programs developed by Dr. Dan Pestridge (http://bimas.dcrt.nih.gov/molbio/proscan) revealed predicted promoters at several sites. The transcription start site was predicted at a TATA box at nt 28286, which is 5' of the LTR of the HERV-W gene [10]. The size of the syncytin gene was predicted to be 8500 base pairs (bp). A 2073-bp syncytin promoter fragment spanning from nt 26725 to 28796 was amplified by polymerase chain reaction (PCR) from human genomic DNA (kindly provided by Dr. David Repaske, Cincinnati Children's Hospital Medical Center, Cincinnati, OH) using the 5' primer 5'-ATC CAG ATG GCC TGA AGT AAC TGA-3', which corresponds to BAC clone sequence nt 26725–26748 and the 3' primer 5'CCA AGA TGG TAG CAG GCC GCT TCC-3', which corresponds to BAC clone sequence nt 28796–28771 bp (Fig. 1). The PCR product was gel purified and ligated into plasmid pCR2.1 to create pSYN(2073)-CR using a TA Cloning Kit (Invitrogen, New York, NY). The orientation and sequence of the construct were confirmed by DNA sequence analysis using the T7 Sequenase Quick Denature Plasmid Sequence Kit (USB Corp., Cleveland, OH).
Mung Bean Nuclease Protection Analysis
Mung bean nuclease protection analyses were used to map the transcription start site of the syncytin gene. A 5' end-labeled single-stranded DNA probe was generated by PCR using a 475-bp PstI/XhoI fragment of pSYN(2073)-CR as template and a primer (5'-GTT GCC AGC TCG AAT GCC TG-3') that was complementary to the region from nt +51 to +70 of the human syncytin gene. The labeled single-stranded DNA was purified by G-50 Sepharose (Roche Molecular Biochemicals, Basel, Switzerland).
Poly(A)+ RNA (50 ng) from normal trophoblast or HepG2 cells that is used as control was denatured at 95°C for 5 min and then incubated at 68°C overnight in a reaction buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 75 mM KCl, 0.05 mM EDTA, 0.5 mM dithiothreitol [DTT], 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 2 µg poly[dI-dC], and 12% glycerol) containing 50 000 dpm of the labeled single strand DNA probe. The binding reaction was subjected to mung bean nuclease (75 units/reaction; Promega Corp., Madison, WI) digestion for 30 min at 37°C and then separated on 6% polyacrylamide gels. The exact transcription start site for the syncytin gene was determined by sequencing the 475-bp PstI/XhoI fragment of pSYN(2073)-CR using the same primer pair.
Construction of Luciferase Plasmids
The plasmid pGL3 basic (Promega) was used to construct pSYN-Luc plasmids containing syncytin promoter fragments. pSYN(-1519/+181)-Luc, the DNA fragment that contains the region from nt -1519 to +181 (relative to the transcription start site [+1]) of the 5'-flanking sequence of the human syncytin gene, was excised from pSYN(2073)-CR by double digestion with KpnI and XhoI and ligated into the KpnI and XhoI sites of pGL3 basic using T4 DNA ligase. Additional 5'-deletions of the syncytin promoter were made using specific restriction enzymes. Briefly, to create pSYN(-984/+181)-Luc, pSYN(-797/+181)-Luc, pSYN(-383/+181)-Luc, pSYN(-294/+181)-Luc, pSYN(-148/+181)-Luc, and pSYN(-49/181)-Luc, the pSYN(-1519/+181)-Luc plasmid was digested with ApaI, MscI, EcoRI, PstI, AvrII, and SstI, respectively. The linearized DNAs were further digested with KpnI and then separated by 1% agarose gel. The expected size fragments were purified from the agarose gel and recircularized with ligase after being blunted by T4 DNA polymerase. pSYN(-1519/-148)-Luc was created by removal of 3'-flanking DNA by AvrII and HindIII double digestion and then recircularized. pSYN(-1519/-797)-Luc was created by removal of 3'-flanking DNA by MscI and HindIII double digestion followed by recircularization. The orientation and sequence of each construct were confirmed by DNA sequencing analysis.
Site-Directed Mutagenesis
Mutations in the putative consensus binding sites for HAPF1, CBF, EcR, Oct, and transcriptional factor II D (TFIID, a TATA box site) in pSYN(-148/+180)-Luc were created using the Quick Change Site-Directed Mutagenesis Kit (Stratagene Inc., La Jolla, CA). The oligonucleotides used to create the mutations are as follows, with the mutated nucleotides underlined (the complementary strand sequence is not shown): HAPF1mt, 5'-CCC TAA GCC TAG CT
AGG TGA CCA CGT CC-3'; CBFmt, 5'-CTT AGC TCA CAC CTG A
A
T CAG AGA GCT CAC TA-3'; EcRmt, GAC CAA TCA GAG AGC T
C
A AAA TGC TAA TTA GG; Octmt, 5'-GCT CAC TAA AAT 
T AT TAG GCA AAG ACA GG-3'; and TATAmt, 5'-GAC AAC AAT CGG GA
T
AAC CCA GGC ATT CG-3'. Each of the mutated base pairs was confirmed by DNA sequencing.
Cell Culture
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 Cincinnati Children's Hospital Medical Center. Cytotrophoblast cells were isolated by enzymatic disaggregation and purified by negative CD-9 selection as described previously [13]. The cells were cultured with Eagle Dulbecco minimal essential medium containing 1.5 g/L sodium bicarbonate, 2 mM L-glutamine, and 10% fetal bovine serum (FBS). HepG2 human hepatoblastoma cells (ATCC HB-8065) were grown 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.
Transfection Studies
Transient transfection studies of primary human trophoblast cells were performed in triplicates by the liposome method described by Cheng and Handwerger [13]. Briefly, cells were incubated in a humidified atmosphere of 5% CO2 at 37°C with plasmid-liposome complexes composed of 5 µg pSYN-Luc plasmid, 0.5 µg pRL-TK-Luc (Promega), and 10 µl freshly prepared liposomes. Five hours later, the cells were fed with complete medium. Luciferase assay was performed 48 h later with a dual luciferase assay kit (Promega). pRL-TK-Luc was cotransfected into the cells in order to normalize for transfection efficiency. The results represent the average of three independent transfection assays normalized to pRL-TK-Luc activity.
Isolation of mRNA from Primary Trophoblast and HepG2 Cells
Total RNA was isolated from normal placental cells or HepG2 cells using TRIzol reagent (Invitrogen, New York, NY), and poly(A)+ RNA was then purified from total RNA using Oligotex mRNA mini Kits (Qiagen Corp., Venlo, Netherlands).
Preparation of Nuclear Extract
Nuclear extracts were prepared as described by Cheng and Handwerger [13]. The protein concentration of each extract was determined by Bradford assay (Bio-Rad Laboratories Inc., Hercules, CA) using bovine serum albumin as a standard.
DNase I Footprinting
DNase I footprinting was performed as previously described by Cheng and Handwerger [14]. Briefly, a 5'-end-labeled probe of the human syncytin promoter region was generated by PCR using pSYN(-1519/+181)-Luc as template. The 5' primer was CTC TCT GGA GAG TGA ATT ACT GAG TCA CAT G, and the 3' primer was CTG CTG TGC TCT CAG GCA ATA GAT GAT TGG. The DNA binding reaction was performed on ice in a buffer containing 10 mM Tris pH 7.5, 5 mM MgCl2, 50 µM EDTA, 75 mM KCl, 12% glycerol, 0.5 mM DTT, and 0.2 mM PMSF. A nuclear extract prepared from normal trophoblast cells after 3 days of culture (35 µg), or BSA (20 µg, Sigma Corp., St. Louis, MO) was incubated with 2 µg of poly(dI-dC) (Roche) for 20 min. DNA probe (20 000 dpm) was added, and the incubation was continued for another 1 h. DNase I (Promega) digestion was then performed for 1.5 min at room temperature using different concentrations of DNase I (control DNA: 0.1, 0.2, and 0.3 units/reaction; nuclear extract: 2.5, 3.5, and 4.5 units/reaction), and the DNA fragments were separated on 6% polyacrylamide, 7 M urea sequencing gels. A G/A ladder of the same probe cleaved at guanine nucleotides with dimethyl sulfate and piperidine was used as a size marker. Protected regions were detected by comparing the digestion patterns with the trophoblast nuclear extract to that of control reactions using BSA.
Gel Mobility Shift Assays
Gel shift assays were performed as previously described by Cheng and Handwerger [13]. 32P-labeled synthetic double-stranded oligonucleotides were used. Oligonucleotide CBFwt (5'-CAC CTG ACC AAT CAG AGA GC-3' encompasses the CCAAT motif (nt -70/-51), and oligonucleotide Octwt (5'-ACT AAA ATG CTA ATT AGG CA-3') encompasses the Oct binding site (nt -48/-39). Binding reactions (24 µl) contained 20 mM Tris (pH 7.6), 50 mM KCl, 2 mM MgCl2, 1 mM DTT, 10% glycerol, 40 ng poly[d(I-C)], 50 000 cpm of 32P-labeled probe, and 5 µg nuclear extract protein. The binding reactions were incubated for 20 min at room temperature and then loaded onto a 5% polyacrylamide gel in 0.5x TBE (Tris-borate-EDTA). In competition assays, unlabeled probes were incubated with the nuclear extracts for 20 min before the addition of labeled probe. For supershift analysis, the nuclear extracts were incubated with antiserum before addition of radiolabeled probe. The antiserums to human CBF and Oct as well as various normal immunoglobulin Gs (IgGs) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and used according to the instructions provided. The normal serums used as control in the electrophoretic mobility shift assay (EMSA) experiments include normal rabbit IgG (catalog no. sc-2027), normal goat IgG (catalog no. sc-2028), and normal mouse IgG (catalog no. sc-2025). The antiserums used in the EMSA experiments include goat polyclonal IgG to CBF-B (C-18, catalog no. sc-7712x), rabbit polyclonal IgG to CBF-B (H-209, catalog no. sc10779), rabbit polyclonal IgG to NF-I (N-20, catalog no. sc-870x), goat polyclonal IgG to CDP (C-20, catalog no. sc-6327x), rabbit polyclonal IgG to C/EBPß (C-19, catalog no. sc-150x), rabbit polyclonal IgG to Oct-1 (C-21, catalog no. sc-232x), rabbit polyclonal IgG to Oct-2 (C-20, catalog no. sc 233x), mouse monoclonal IgG2b to Oct-4 (C-10, catalog no. sc5279x), and polyclonal IgG to Oct-6 (H-13, catalog no. sc-11660x).
Statistical Analysis
Statistical difference between two groups was determined by t-test, and multiple comparisons were performed by one-way ANOVA or repeated measures ANOVA together with post hoc pairwise comparisons. The values were expressed as mean ± SD, and P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify the promoter that drives expression of syncytin, 2073 bp of the 5'-flanking region of syncytin gene were amplified from genomic DNA and cloned into the PCR2.1 vector. A detailed restriction map of the 5'-flanking region of the syncytin gene is shown in Figure 1. The sequence of the 2073-bp flanking region, including about 1.5 kb of promoter region and 0.5 kb of transcription region, is shown in Figure 2. Computer analysis of the 5'-flanking region using the TRANSFAC program (http://transfac.gbf.de/TRANSFAC) revealed a putative CCAAT motif located -65 to -56 bp upstream from the transcription start site. Putative regulatory elements, including HAPF1, CBF, EcR, and Oct, were present within 148 bp upstream from the transcription initiation site, and DNA binding sites for SOX5, TBP, c-Myb, MEP1/MTF1, and YY1 were located 150 bp downstream from the transcription start site.
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Determination of the Transcription Start Site
Since the transcripts of HERV-W (including syncytin mRNA sequence) are derived from a single locus by alternative usage of splicing signals [10], the transcription initiation site of the syncytin gene should be localized to the 5'-LTR region. The transcription initiation site of the syncytin gene was mapped by mung bean nuclease protection with a probe that extended to nt -294. Two protected bands were observed when the poly(A)+ RNA from primary human trophoblast cells was used, which located the transcription start position to adenine residues. The major initiation site was located 56 bp downstream from the putative CCAAT motif (beginning at nt 28244 of BAC clone sequence), and the minor site was located 52 bp downstream from the putative CCAAT motif (beginning at nt 28240 of BAC clone sequence) (Fig. 3, lane 1). No protected band was observed using poly(A)+ RNA from HepG2 cells, which do not express syncytin (Fig. 3, lane 2). Since the syncytin gene is specifically expressed in trophoblast cells but not in HepG2 cells, we concluded that the major transcriptional start site of syncytin gene is located at nt 56 downstream from the putative CCAAT motif.
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Deletion Analysis of the Syncytin Promoter in Primary Human Trophoblast Cells
To delineate the sequences essential for transcription of the human syncytin gene, a series of 5'-deletion constructs was transiently transfected into primary human trophoblast cells. As shown in Figure 4, luciferase activity of the primary human trophoblast cells transiently transfected with pSYN(-1519/+181)-Luc was 150-fold greater than that of cells transfected with the promoterless control vector (pGL3B-Luc). Deletion of the 5'-flanking region to nt -984 (pSYN[-984/+181]-Luc) caused a 30% decrease in luciferase activity, suggesting that one or more DNA element(s) located between nt -1519 to -984 is required for maximal expression of syncytin gene. Further deletion of the 5'-flanking region to nt -294 had no significant effect on luciferase activity. However, further deletions to nt -148 and -49 caused additional decreases in luciferase activity of 50% and 95%, respectively, suggesting that an element(s) between nt -294 and -49 is required for optimal gene expression. 3'-Deletion of nt -148 to +181 almost completely abolished luciferase activity (Fig. 4), suggesting that the 148-bp fragment proximal to the transcription start site is required for basal promoter activity.
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Footprinting Analysis of the Proximal Syncytin Promoter
To identify putative transcription factor binding sites on the proximal promoter region of the syncytin gene, DNase I footprinting analysis was performed using a probe that contains the region between nt -252 and +110 of the syncytin promoter and a nuclear extract derived from normal trophoblast cells that had been cultured for 3 days. As shown in Figure 5, three protected regions (FP1, FP2, and FP3) were observed. FP1 spans approximately 45 bp (nt -159 to -115) and contains five hypersensitive sites, FP2 spans approximately 60 bp (nt -78 to -19) and contains one hypersensitive site, and FP3 spans approximately 65 bp (nt -6 to +59) and contains one hypersensitive site. Computer analysis (http://transfac.gbf.de/TRANSFAC) revealed that FP1 contains a putative consensus element for HAPF1. FP2 contains putative transcription factor-binding sites for CBF, EcR; and Oct protein. FP3 contains a Sox5 binding site and a putative TATA box.
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Site-Directed Mutagenesis of Syncytin Proximal Promoter Region
To determine which of the putative consensus binding sites in FP1, FP2, and FP3 are required for basal expression, the HAPF1, CBF, EcR, Oct, and TATA box were mutated by site-directed mutagenesis. The promoter activities of the mutated constructs were compared with the wild-type construct in transfection studies in primary human trophoblast cells. As shown in Figure 6, mutation of the HAPF1 binding site in FP1 or the TATA box in FP3 had no effect on basal promoter activity. However, mutation of the CCAAT motif and the Oct binding site in FP2 resulted in decreases of 88% and 76%, respectively. Mutation of the EcR binding site in FP2 results in a 2-fold increase of basal promoter activity.
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EMSA Analyses of the Binding Activities in the CCAAT Box and the Oct Site
Taken together, the deletion analyses and site-directed mutation assays strongly suggested that the CCAAT motif and the Oct binding site in FP2 are critical for syncytin basal promoter activity. In order to demonstrate whether nuclear protein factors can bind specifically to the CCAAT motif and Oct binding site, gel shift assays were performed with oligonucleotides containing either a CCAAT motif (CBFwt) or an Oct binding site (Octwt) and a nuclear extract from normal trophoblast cells. As shown in Figure 7A, two major DNA-protein complexes (complexes A and B) were formed when the 32P-labeled CBFwt oligonucleotide probe was reacted with trophoblast nuclear extract (lane 1). Formation of the two complexes was completely prevented when the reaction was performed in the presence of 100-fold excess unlabeled wild-type probe (lane 2) but not in the presence of 100-fold excess oligonucleotide containing a mutated CBF binding site (lane 3). The A and B complexes were supershifted by specific CBF antiserums (lanes 4 and 5) but not supershifted by antiserums either to C/EBP (lane 6), NF-I (lane 7), or CDP (lane 8), indicating that transcription factor CBF can bind to the CCAAT motif. Lanes 9 to 11 were used as controls that contained a normal rabbit IgG (lane 9), normal goat IgG (lane 10), and normal mouse IgG (lane 11). Figure 7B shows that a specific DNA-protein complex is formed when a 32P-labeled Octwt oligonucleotide probe is reacted with nuclear extract from normal trophoblast cells (lane 12). The formation of this complex was completely prevented by an unlabeled wild-type Oct oligonucleotide (lane 13) but not by an oligonucleotide containing a mutated Oct binding site (lane 14). The complex formed by Octwt oligonucleotide and the nuclear extract is supershifted by a specific Oct1 antiserum (lanes 15 and 16) but is not supershifted by antiserums to Oct2 (lane 17), Oct4 (lane 18), and Oct6 (lane 19). Lanes 20 to 22 indicate that normal rabbit IgG (lane 20), normal goat IgG (lane 21), and normal mouse IgG (lane 22) do not cause a supershift.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HERV-W initiator region alone is not sufficient to direct significant transcription (Fig. 2). Functional analysis of the 5'-flanking region of the syncytin gene in normal trophoblast cells defined a basal core region that is located within the proximal 148 bp from the start site. Deletion of the basal core region almost abolished promoter activity. DNase I footprint analysis of nt -252 to +110 of syncytin gene using nuclear extracts from trophoblast cells protected three regions, FP1–FP3. Sequence analysis revealed that the FP1 site contains a putative HAPF1 binding site. Site-directed mutagenesis of the putative HAPF1 site had no effect on promoter activity in transfected normal trophoblast cells, indicating that this site is not necessary for transcriptional regulation of the syncytin gene. However, site-directed mutagenesis of the Oct binding site in FP2 decreased syncytin basal promoter by 70%; and mutagenesis of CCAAT motif in FP2 almost abolished syncytin promoter activity, indicating that the transcriptional regulation of the syncytin gene in trophoblast is highly dependent on the CCAAT motif and Oct site (Fig. 5). In contrast, the EcR response element in FP2 may act as a repressor of syncytin gene expression in trophoblast cells since site-directed mutagenesis of the EcR response element resulted in a 2-fold increase of basal promoter activity. A putative TATA box in FP3 that is located in nt +40 bp downstream from start site has little or no role in the transcription regulation of syncytin gene since site-directed mutagenesis of the TATA box has no effect on basal promoter activity.
Lee and coworkers [17] recently analyzed transcriptional regulatory elements in a 3'-LTR fragment of HERV-W that was synthesized from genomic DNA by PCR using primers from the consensus sequence of the 3'-LTR of HERV-W reported by Blond et al. [10]. Transfection studies of the 3'-LTR in HeLa cells revealed that the TATA box determined cell-type specificity. An Oct-1 binding site was important as a silencer, and a C/EBP site was important as an enhancer. These studies clearly demonstrate striking differences between the 3'-LTR and the 5'-LTR.
EMSA studies indicate that several transcription factors bind to the FP2 region. Gel supershift analysis showed that CBF binds specifically to the CCAAT motif and that Oct1 specifically binds to the Oct binding site (Fig. 7). The importance of these binding sites for syncytin promoter activity was further indicated by functional analyses in transfected normal trophoblast cells with the mutant Oct binding site and CCAAT motif constructs (Fig. 6). The Oct DNA binding sequence is required for both ubiquitous and tissue-specific expression of various genes [18, 19]. Oct1 is expressed in all cell types so far examined, while the expression of the other Oct family members is more restricted [18]. Oct1 but not Oct2, Oct4, or Oct6 binds to the Oct binding site of the syncytin promoter. However, the possibility that other members of Oct family can bind to the syncytin promoter is not excluded. Many eukaryotic promoters possess a transcriptional regulatory element that contains the pentanucleotide sequence CCAAT between 50 and 80 bp upstream of the initiation site [20]. Several CCAAT-binding proteins, including CBF, CDP, NF-I, and C/EBP, can bind to the CCAAT motif [20–24]. CBF has been shown to regulate the transcription of various promoters by cooperative interactions with other transcription factors, such as AP-1 [25, 26] and Sp1 [27, 28] and its family members [29]. The proximity of the EcR response element and the Oct binding site near the CCAAT motif in the human syncytin promoter suggests that Oct and EcR (perhaps a steroid hormone receptor in mammalian cells) may cooperate with CBF in the regulation of human syncytin gene expression. However, functional interactions between CBF and Oct or other transcription factors in transcriptional regulation of the syncytin gene remain to be determined.
Several studies indicate that cAMP stimulates syncytin gene expression in human choriocarcinoma BeWo cells [6]. Although the mechanism by which cAMP mediates syncytin gene expression is unknown, the absence of a cAMP-response element (CRE) (TGACGT[A/C]A) [30] in the proximal 2.0 kb of the syncytin promoter strongly suggests that the transactivation of the syncytin gene by cAMP is not mediated by the binding of CRE-binding protein (CREB). The syncytin promoter region contains three AP-1 consensus sequences. Since cAMP can increase the DNA binding activity of AP-1 complexes containing JunB, JunD, and Fos [31], the effect of cAMP may be mediated via the AP-1 binding sites. The proximal syncytin promoter also contains five potential binding sites for Sp1, a zinc-finger nuclear protein that specifically binds to GC-rich DNA sequences [32]. Since the binding of Sp1 to its binding site is dependent on phosphorylation of Sp1 by protein kinase A [32–34], transactivation of syncytin gene by cAMP may also be mediated in part by inducing the binding of Sp1 to the syncytin promoter. The regulatory region of syncytin gene contains five potential AP-2 consensus elements that act as basal transcription enhancers but are also responsive to cAMP [13, 35, 36].
Two enhancer regions, one between nt -1519 and -984 and another between nt -294 and -148, were identified by 5'-deletion assay in transfected trophoblast cells. Sequence analysis of the enhancer region (nt -1519 to -984) reveals many putative cis elements, including two c-Myb binding sites, one NF-IL6 element, three AP-2 binding sites, three Sp1 binding sites, two c-Ets2 binding sites, and many other response elements. The second enhancer region (nt -294 to -148) contains a glucocorticoid receptor (GR) element half-site and a progesterone receptor (PR) response element half-site as well as binding sites for transcription factors Sp-1, AP-2, c-Ets2, c-Myb, and GATA-1. Since NF-IL6, AP-2, c-Ets2, c-Myb, and GATA-1 are involved in cell proliferation and differentiation [36–40], these two enhancer regions may contribute to spatial and temporal expression of syncytin gene in trophoblast cells. More detailed studies will be required to delineate precisely the specific cis-acting elements involved in syncytin gene expression.
In summary, we have isolated and characterized the 5'-flanking promoter region of the syncytin gene. The proximal 148-bp region upstream from the transcription initiation site contains a CCAAT motif and an Oct binding site that are required for basal expression. Since CBF and Oct bind specifically to these regulatory elements, these transcription factors may play a critical role in transcription of syncytin gene. Two enhancer regions in the 5'-flanking region of the gene may also be involved in cell-specific expression. An understanding of the transcription factors and cis-acting elements involved in regulation of the human syncytin gene expression should facilitate a better understanding of the regulation of syncytin expression in physiologic and pathologic states of trophoblast syncytialization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: You-Hong Cheng, Division of Endocrinology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039. FAX: 513 636 5960; youhong.cheng{at}cchmc.org
Received: 23 September 2003.
First decision: 19 October 2003.
Accepted: 4 November 2003.
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REFERENCES |
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gene that contains a critical Ets1 binding site. J Clin Endocrinol Metab 2003 88:3305-3311This article has been cited by other articles:
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  M. Chang, D. Mukherjea, R. M. Gobble, K. A. Groesch, R. J. Torry, and D. S. Torry Glial Cell Missing 1 Regulates Placental Growth Factor (PGF) Gene Transcription in Human Trophoblast Biol Reprod, May 1, 2008; 78(5): 841 - 851. [Abstract] [Full Text] [PDF]
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 H. Ishikawa, V. Fencki, E. E. Marsh, P. Yin, D. Chen, Y.-H. Cheng, S. Reisterd, Z. Lin, and S. E. Bulun CCAAT/Enhancer Binding Protein {beta} Regulates Aromatase Expression via Multiple and Novel Cis-Regulatory Sequences in Uterine Leiomyoma J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 981 - 991. [Abstract] [Full Text] [PDF]
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 X.-S. Wang, Z. Zhang, H.-C. Wang, J.-L. Cai, Q.-W. Xu, M.-Q. Li, Y.-C. Chen, X.-P. Qian, T.-J. Lu, L.-Z. Yu, et al. Rapid Identification of UCA1 as a Very Sensitive and Specific Unique Marker for Human Bladder Carcinoma. Clin. Cancer Res., August 15, 2006; 12(16): 4851 - 4858. [Abstract] [Full Text] [PDF]
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 M. Caceres, NISC Comparative Sequencing Program, and J. W. Thomas The Gene of Retroviral Origin Syncytin 1 is Specific to Hominoids and is Inactive in Old World Monkeys J. Hered., March 1, 2006; 97(2): 100 - 106. [Abstract] [Full Text] [PDF]
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Y.-H. Cheng and S. Handwerger A Placenta-Specific Enhancer of the Human Syncytin Gene Biol Reprod, September 1, 2005; 73(3): 500 - 509. [Abstract] [Full Text] [PDF]
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 S. Prudhomme, G. Oriol, and F. Mallet A Retroviral Promoter and a Cellular Enhancer Define a Bipartite Element Which Controls env ERVWE1 Placental Expression J. Virol., November 15, 2004; 78(22): 12157 - 12168. [Abstract] [Full Text] [PDF]
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