CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues Magda Matoušková a , Jana Blažková a , Petr Pajer b , Adam Pavlíček a,1 , Jiří Hejnar a,⁎ a

Department of Cellular and Viral Genetics, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo námìstí 2, 16637 Prague 6, Czech Republic b Department of Molecular Virology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 16637 Prague, Czech Republic

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Syncytin-1 is a captive envelope glycoprotein encoded by one of human endogenous

Received 5 August 2005

retroviruses W. It is expressed exclusively in the placental trophoblast where it participates

Revised version received

in cell-to-cell fusion during differentiation of syncytiotrophobast. In other tissues, however,

6 December 2005

syncytin-1 expression must be kept in check because inadvertent cell fusion might be

Accepted 13 December 2005

dangerous for tissue organization and integrity. We describe here an inverse correlation

Available online 20 January 2006

between CpG methylation of syncytin-1 5′ long terminal repeat and its expression. Hypomethylation of the syncytin-1 5′ long terminal repeat in the placenta and in the

Keywords:

choriocarcinoma-derived cell line BeWo was detected. However, other analyzed primary

Syncytin-1

cells and cell lines non-expressing syncytin-1 contain proviruses heavily methylated in this

Human endogenous retroviruses W

sequence. CpG methylation of syncytin-1 is resistant to the effect of the demethylating agent

Trophoblast cells

5-azacytidine. The inhibitory role of CpG methylation is further confirmed by transient

Cell fusion

transfection of in-vitro-methylated syncytin-1 promoter-driven reporter construct.

Placenta

Altogether, we conclude that CpG methylation plays a principal role in the transcriptional

DNA methylation

suppression of syncytin-1 in non-placental tissues, and, in contrast, demethylation of the syncytin-1 promoter in trophoblast is a prerequisite for its expression and differentiation of multinucleated syncytiotrophoblast. © 2005 Elsevier Inc. All rights reserved.

Introduction Human endogenous retroviruses (HERVs) originate from ancient retroviral infections that entered the germ line and were fixed in populations of our ancestors. The human genome contains around 200 sequentially distinct HERV families [1,2]. The HERV-W family, one of the most extensively studied, comprises some 250 retrovirus sequences with striking prevalence of processed pseudogenes [3,4]. This family invaded the human and Old World monkeys lineage

approximately 25–40 million years ago and has been active for a short period of some five million years [5]. Most of HERV-Ws, like members of other HERV families, are compromised by large deletions and mutations so that only three of them retain ORFs longer than 1000 bp [3]. HERV-W envelope (env) sequences form a homogenous group and cluster together with HERV-F, HERV-H and ERV9 envs in phylogenetic alignment of their transmembrane (TM) subunits [6]. Despite the scarcity of intact ORFs within the HERV-W family, the defective ERVWE1 element localized at chromo-

⁎ Corresponding author. Fax: +42 223410955. E-mail address: [email protected] (J. Hejnar). 1 Current address: Genetic Information Research Institute, 1925 Landings Drive, Mountain View, CA 94043, USA. 0014-4827/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.12.010

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somal position 7q21–22 contains an intact and full-length env gene [7], so called syncytin-1. The mature glycoprotein encoded by this gene displays cell-to-cell fusion capacity and induces differentiation of the placental syncytiotrophoblast in vitro and in vivo [8,9]. Gag and pol ORFs of ERVWE1 are interrupted by frame-shifts and stop codons [10]. Similarly, as in the envelope protein in exogenous viral particles, the TM subunit of syncytin-1 protein induces membrane fusion after binding of the surface subunit (SU) to its specific receptor. As to the receptor usage, HERV-Ws interfere with RD114 feline endogenous virus, baboon endogenous virus, simian retroviruses type D and avian reticuloendotheliosis virus and compete for the human sodium-dependent neutral amino acid transporter 2 (hASCT2) [11,12]. Furthermore, HERV-W env glycoproteins also efficiently use hASCT1 as a receptor [13]. Inhibition of syncytin-1 expression by specific anti-sense oligonucleotides leads to a decrease in the trophoblast fusion and differentiation in vitro [14]. Recently, other fusogenic retroviral envelope glycoproteins have been found to play a role in placentation. Out of 16 identified full-length HERV env genes, one belonging to the HERV-FRD family and dubbed syncytin-2 was shown to be specifically expressed in the placenta, to induce cell-to-cell fusion and to confer infectiveness on lentiviral pseudotypes [15,16] in addition to syncytin-1. Env protein of the single-copy HERV ERV3 is also specifically expressed in syncytiotrophoblast. Since it is truncated at the 3′ end by a stop codon in the transmembrane domain, it is a soluble protein that might be involved in the development of the placenta acting as a regulatory or immunosuppressive factor instead of inducing cell-to-cell fusion. This protein, however, is not essential because at least 1% of the fertile population contain another stop codon within its coding sequence [17]. Two other fulllength env genes, envV and envP(b), belonging to newly characterized HERV families HERV-V and HERV-P(b), respectively, do not fulfil the criteria for sensu stricto syncytins because envV does not display any fusogenic activity and envP (b) is not specifically expressed in placenta [18]. Two retroviral env genes, syncytin-A and -B, were also identified in the mouse genome. They are specifically expressed in placenta, display fusogenic activity and their orthologous loci are conserved in the family Muridae [19]. The transcriptional activity of HERV-W elements was found to be high in most human tissues analyzed by a retrovirus-specific microarray and real-time RT-PCR [20]. Expression of syncytin-1, however, is tightly restricted to the trophoblast, particularly to the multinucleated syncytiotrophoblast layer of the placenta [8,9,14], although weak expression was observed in testes [9]. In the process of placenta morphogenesis, cytotrophoblastic cells serve as precursors for the syncytiotrophoblast differentiation. The syncytiotrophoblast layer is necessary for normal physiological functions of the placenta, i.e. hormone production and gas/substrate exchange between the maternal and fetal blood circulations. Altered cytotrophoblast fusion during placentogenesis and hypertensive disorders in pregnancy such as preeclampsia and HELLP syndrome is often accompanied by deregulation of syncytin-1 expression or its incorrect localization [21,22]. Reduced syncytin-1 expression correlated with insufficient or late syncytiotrophoblast differentiation in Down's syndrome

pregnancies [23]. The human choriocarcinoma cell line BeWo has been widely used as a relevant model of trophoblast differentiation. These cells can be induced to cell fusion with forskolin, whereas a rabbit polyclonal antibody raised against syncytin-1 inhibits heterologous fusion between BeWo and COS cells [9]. The promoter region and transcription initiation site of syncytin-1 were localized within the 5′ long terminal repeat (LTR) of ERVWE1 provirus by deletion analysis and reporter assay for the basal promoter activity [24]. Mutagenesis of this promoter region together with DnaseI footprint analysis revealed that the CCAAT motif and the octamer protein binding site are critical for transcriptional regulation of syncytin-1. Another study [25] showed that a transcription factor called Glial Cell Missing a (GCMa) binds to two GCMabinding sites upstream of the 5′ LTR and enhances syncytin-1 expression in BeWo and JEG3 choriocarcinoma cells. Conversely, mutation of the ecdysone receptor response element slightly increases basal promoter activity, suggesting that this nuclear hormone receptor is a negative regulator of the syncytin-1 gene [24]. Similarly, a correlation between the decreased level of oxygen in placenta and insufficient expression of syncytin-1 was also detected in BeWo cells [26]. However, the precise mechanisms of syncytin-1 gene regulation remain unclear. Of particular importance is the question of transcriptional suppression of syncytin-1 in non-placental tissues, where its inadvertent expression could be dangerous given the almost ubiquitous expression of hASCT receptors. Usually, methylation of cytosines in CpG dinucleotides has profound impacts on promoter activity. As well as with genes, the transposable elements integrated in the genomic DNA are subverted to this host defense mechanism, which protects the cell against intragenomic parasites [27]. The methylation status of both exogenous and endogenous retroviruses correlates with the level of their expression [28–30], and the promoter activities of in-vitro-methylated LTRs are dramatically reduced when measured in transient reporter assays [30– 32]. Transcription of hypomethylated HERVs, particularly HERV-Ks, has been implicated in tumors [33] and systemic lupus erythematosus [34]. Elevated HERV transcription in placenta and other reproductive tissues [17,20,35], associated even with budding of retroviral particles, corresponds well with a decreased level of methylation in the trophoblast ([36], for the review). There are, however, individual HERV loci or HERV subfamilies that reveal exclusive expression in nonplacental tissues and organs [20,35]. In addition to the syncytin1-mediated cell-to-cell fusion, HERV-encoded envelope glycoproteins may play other beneficial roles, such as suppression of maternal immunoreactivity against the fetus [37] and competition with infectious exogenous retroviruses transferred from maternal blood circulation [38]. On the other hand, syncytin-1 can pseudotype human immunodeficiency virus type 1 (HIV-1) [39] and, due to the particular expression of hASCT1 in liver and brain [40], could broaden the range of HIV1 infectiveness. A high transcriptional activity of hypomethylated HERVs in reproductive tissues is necessary for their efficient amplification, retrotransposition, penetration into the germ line and subsequent genetic fixation. The aforementioned data suggest that high expression of HERV-Ws in placenta is enabled by the hypomethylation of

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their promoters and that the expression of syncytin-1 may be controlled by CpG methylation as well. In the present study, we therefore investigated the methylation of 5′ LTR of syncytin1 in different tissues and cell lines, the stability of 5′ LTR methylation pattern and the sensitivity of 5′ LTR promoter activity to in vitro methylation. We conclude that CpG methylation of syncytin-1 LTR efficiently blocks its transcription in non-placental tissues and that demethylation is a prerequisite for the basal promoter activity in trophoblast cells.

Materials and methods

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round of PCR contained ∼25 ng of DNA. The Taq polymerase (Takara) was used following the manufacturer's recommendation with 5 μM MgCl2, 0.9 M betain and 0.9% DMSO [42]. Conditions for PCR were as follows: 95°C for 1 min, 52°C for 2 min, 72°C for 1 min (30 cycles). The second round of PCR started with 1 μl of the first round PCR product, and the PCR conditions were identical. PCR products were subsequently cloned using the pGEM-T vector cloning system (Promega). Individual PCR clones were sequenced using the AmpliTaq®FS L′ Big Dye Terminator sequencing kit (Applera) with universal pUC/M13 forward and reverse primers. Only PCR clones with at least 95% conversion of Cs outside CpGs were taken into account. At least nine fully converted PCR clones were analyzed from each PCR.

Cell cultures and tissue samples HeLa cells were grown in F-12 and MEM-D mixed 1:1 (Sigma) supplemented with 1% NaHCO3, 5% calf serum, penicillin– streptomycin mix (100 μg/ml each) and 2.5 mg/ml amphotericin B. Choriocarcinoma BeWo cells, purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK), were grown in the same medium supplemented with 10% fetal calf serum and 300 μg/ml glutamine. To induce syncytin-1 expression in BeWo cells, 40 μM forskolin was added for 48 h to 50% confluent cultures. Primary human skin fibroblasts were derived from skin obtained after breast reduction surgery from the Clinic of Plastic Surgery, Faculty Hospital Královské Vinohrady, Prague. They were cultured in Eagle's H-MEM, supplemented with 1% NaHCO3, a mix of nonessential amino acids, 0.12 g/l natrium pyruvate, 10% calf serum, penicillin– streptomycin mix (100 μg/ml each) and 2.5 mg/ml amphotericin B. Samples of placental tissues with a prevalence of chorionic villi were obtained from Gynecologic Clinic, Faculty Hospital Královské Vinohrady, Prague. Breast cancer cells from invasive ductal carcinomas were obtained from the Department of Gynecology and Obstetrics of the Third Medical Faculty, Charles University in Prague. Human ethics approval was granted by the Committee for Ethics, Handling the Recombinant DNA and Clinical Research at the Institute of Molecular Genetics, and informed consents were obtained from the patients. DNA samples of peripheral blood mononuclear cells (PBMC) from ICF (immunodeficiency, centromeric instability, facial anomalies) syndrome patients were a kind gift from Keith Robertson, Epigenetic Gene Regulation and Cancer Section, NCI, NIH, Bethesda.

Treatment of HeLa cells with 5-azacytidine (azaC) and trichostatin A (TSA) HeLa cells were grown up to approximately 70% confluency and then cultivated at indicated concentrations of azaC (Sigma) and TSA (Sigma). After 48 h, the treated cells were harvested for DNA analysis.

SssI methyl-accepting assay We have used the slightly modified protocol of Wu et al. [43]. Analyzed samples of genomic DNA were digested to completion overnight with 5 U HindIII per μg of DNA. DNA concentration was determined spectrophotometrically and then verified on 1% agarose gel. Three hundred nanograms of digested DNA was incubated with 4 U of SssI methylase (CpG methylase, New England Biolabs), 2 μM S-adenosyl-L-[methyl3 H]methionine ([3H]SAM, 81 Ci/mmol, Amersham) and 1.5 μM non-radioactive S-adenosylmethionine (SAM, New England Biolabs) in the buffer supplied by the manufacturer at 37°C for 17 h. The reactions were stopped by adding 0.4 μl 32 mM SAM and spotted onto glass microfiber filters (Whatman). The filters were dried, washed twice in 10 ml of 5% trichloride acetate acid for 10 min and twice in 10 ml of 70% ethanol for 10 min, dried again and placed in a scintillation vial containing 4 ml of scintillation mixture. The scintillation was counted for 1 min, and the results were normalized to the value of a control mock-demethylated sample.

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)

Bisulfite cytosine methylation analysis DNA samples for bisulfite analysis were isolated by phenolchloroform extraction and digested by HindIII restriction endonuclease. Bisulfite treatment of DNA was performed according to Hájková et al. [41]. We amplified the syncytin-1 U3 5′ LTR together with a short unique sequence upstream to the LTR as a 285 bp DNA fragment encompassing five CpGs from bisulfite-treated DNA by semi-nested PCR: forward external primer 5′-TTGGATAGTGAATATAGATA-3′ (nucleotides −92 to −72 upstream to the LTR), forward internal primer 5′-AGATATAGTAATTATTTTGT-3′ (nucleotides −23 to −3 upstream to the LTR), and reverse primer 5′-CAAAAAAAAACTACTATTAC-3′ (nucleotides 242 to 262 of the LTR). The first

The presence of syncytin mRNA was detected by RT-PCR using β-actin as a standard. Total RNA was extracted from cultured cells with TriReagent (Sigma) according to the manufacturer's protocol. RNA samples were treated with DNaseI (Roche) before RT-PCR to remove any contaminant DNA for 15 min in the M-MLV reverse transcriptase buffer (Promega). One microgram of RNA was reversely transcribed into cDNA using oligo(dT)12–18 primer and M-MLV reverse transcriptase (Promega) according to the manufacturer's protocol. The primers used in the subsequent RT-PCR were as follows: syncytin-1 forward, 5′-AGGAGCTTCGAAACACTGGA-3′ and backward, 5′-GTGAGCTAAGTTGCAAGCCC3′ [26]; β-actin, forward 5′-CA CCATGTACCCTGGCATTG-3′ and

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backward 5′-GCCGGACTCGTCATACTCCT-3′ [35]. The expected sizes of the PCR products were 494 bp for syncytin-1 and 190 bp for β-actin. cDNA samples were used for PCR amplification as follows: 90 s 95°C, 30 cycles each consisting of 15 s 95°C, 45 s 60°C, 50 s 72°C, and finally 2 min 72°C for syncytin-1, and 90 s 95°C, 30 cycles each consisting of 15 s 95°C, 15 s 55°C, 20 s 72°C, and finally 2 min 72°C for β-actin. The amount of template cDNA was determined experimentally so that the reference gene gives about the same amount of PCR product. PCR products were separated on a 1% agarose gel. As a negative control for the absence of exogenous DNA contamination, reactions run in the absence of the reverse transcriptase revealed no amplified product (data not shown).

was replaced with standard cultivation medium 24 h posttransfection. Cell lysates were prepared 48 h after transfection using lysis buffer containing 150 mM NaCl, 20 mM Tris pH 7.5, 1% Triton X-100 and 2 mM EDTA. Luciferase activity was measured in the mix of 10 μl of lysate and 30 μl of luciferin solution (Promega) using luminometer DLReady™ Promega, detection system Berthold. To measure the β-gal activity, the mixture of 30 μl of cell lysate, 200 μl of 100 mM phosphate buffer, 3 μl of 100 mM magnesium buffer, 1 μl of β-mercaptoethanol and 66 μl of 0.4% onitrophenyl β-D-galactopyranoside (Sigma) solution was prepared. This mixture was incubated in 37°C for 10–20 min, and then the reaction was stopped by adding 500 μl of 1 M Na2CO3. The β-gal activity was measured spectrophotometrically at the wavelength 420 nm (Beckham DU 640).

Construction of the pLTRluc reporter vector We amplified the whole 5′ LTR of syncytin-1 from genomic DNA of placental origin using the Expand Long Template PCR System (Roche) according to manufacturer's instructions with following primers: forward 5′-TTCAACATCCATTCCAACACCACC-3′ (nucleotides −202 to −179 upstream to the LTR) and reverse 5′-CTGAGTCTTAAGTCCGGTGGCAC-3′ (nucleotides 913 to 936 from the beginning of the LTR). In order to discriminate between the LTR of syncytin-1 and LTRs of other HERV-Ws present in the human genome, the forward primer is complementary to the unique sequence immediately flanking the 5′ LTR of ERVWE1. PCR cycles were as follows: 5 min 95°C; 20 cycles, each consisting of 1 min 95°C, 2 min 60°C, 1 min 72°C; 19 cycles, each cycle consisting of 1 min 95°C, 2 min 60°C, 1 min 72°C plus a 20 s ramp per cycle; finally 8 min 72°C. The blunted PCR fragment 1138 bp in length was cloned into the SmaI site of pBluescript (Stratagene). To generate pLTRluc, we cleaved the resulting plasmid with NotI, blunted the NotI ends and digested again with HindIII. This DNA fragment was used to replace the HindIII–SmaI promoter region of the luciferase reporter vector pGL3-promoter (Promega). In this way, the luciferase reporter gene in resulting pLTRluc was driven by the 5′ LTR of the human syncytin-1 gene.

In vitro DNA methylation pLTRluc reporter vector was methylated in vitro by incubating 10 μg of plasmid DNA with 10 U of Sss1 methylase (CpG methyltransferase, New England Biolabs) in the recommended buffer containing 160 μM SAM for 2 h at 37°C. To confirm that the methylation was complete, we digested the methylated plasmid by a methyl-sensitive restriction enzyme HpaII and compared the reaction product with the HpaII digest of the non-methylated plasmid DNA.

DNA transfection and reporter expression assays The methylated and mock-methylated pLTRluc constructs were transfected into BeWo and HeLa cells. The cells were grown on 35 mm Petri dishes to 50% or 70% confluence, respectively, and 1 μg of plasmid DNA was applied using the Fugene 6 lipofection reagent (Roche) at a DNA/reagent mixture ratio of 1:3 in OPTIMEM (Invitrogen). Equal amount of plasmid pCMVβgal (Stratagene) bearing the CMV promoter-driven β-galactosidase (β-gal) was cotransfected together with pLTRluc, and β-gal activity was measured to normalize the transfection efficiency. OPTI-MEM

Results CpG methylation of the syncytin-1 in human tissues and cell lines As transcription of vertebrate genes, endogenous retroviruses and other retroelements are often regulated by CpG methylation, we have compared the level of methylation within the syncytin-1 regulatory region in the placenta, in other human tissues and in cultured cells. Using the technique of genomic sequencing after bisulfite treatment, we assayed the CpG methylation pattern of all five CpG dinucleotides within the 5′ U3 region of the ERVWE1 provirus bearing the syncytin-1 gene (Fig. 1a). In order to distinguish ERVWE1 5′ LTR from the 3′ LTR, as well as other members of the HERV-W family, we have shown the external forward and internal forward primers for the semi-nested PCR into the unique sequence adjacent to the 5′ LTR (Fig. 1a). The nucleotide sequence of PCR products confirms that we have selectively amplified the 5′ LTR of ERVWE1. In two samples of term placentas, the sequences obtained were either heavily methylated or almost unmethylated (Fig. 1b). This bimodal methylation pattern is probably caused by the heterogeneity of the tissue sample. We suggest that unmethylated sequences come from the trophoblast or syncytiotrophoblast cells, whereas the methylated sequences come from blood cells and connective cells. In contrast, among a representative number of sequences obtained from skin fibroblasts, we have not found any unmethylated clone, with most of them being fully methylated (Fig. 1c). Furthermore, we analyzed the methylation pattern of syncytin-1 5′ LTR in HeLa cells, mammary carcinoma cells and PBMC from ICF syndrome patients. Although these cells display a decrease in the overall genome methylation level due to their transformed character or a lack in Dnmt3b activity, we have not found any decrease in methylation density within the syncytin-1 5′ U3 region, and most of the clones were fully methylated (Fig. 1c).

CpG methylation and transcription of the syncytin-1 in human trophoblastic cell line BeWo Because of the substantial heterogeneity of placental biopsy, we have chosen the human choriocarcinoma-derived cell line BeWo for further studies on the methylation of syncytin-1. This

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Fig. 1 – CpG methylation status of the U3 region of ERVWE1 5′ LTR. (a) Schematic representation of the examined ERVWE1 provirus bearing the env gene (syncytin-1) and of its 5′ LTR U3 region. Arrows show location of PCR primers. Vertical bars with ellipses represent CpG dinucleotides (C in positions 32, 70, 86, 214 and 235) within the U3 region. (b) CpG methylation status of 5′ U3 region as detected by bisulfite sequencing in two samples of placental tissue. (c) CpG methylation status of the same sequence in non-placental human cells and a tumor sample: primary skin fibroblasts, cervical carcinoma HeLa cell line, tissue sample of a breast carcinoma and peripheral blood mononuclear cells from patients with ICRF syndrome. Numeric representation shows number of methylated CpG/number of all CpG and the percentage in parenthesis. Methylated CpG dinucleotides are depicted by solid circles, unmethylated CpG sites are indicated by open circles.

cell line serves as an in vitro model of trophoblast induction and differentiation and was previously used for demonstration of the fusogenic capacity of syncytin-1 [9] as well as for transient transfection assays of syncytin-1 promoter regulation [25]. We analyzed the CpG methylation pattern within the U3 region of syncytin-1 LTR in the BeWo cells using the aforementioned PCR primers (Fig. 1a). In contrast to the samples of placental tissue, we have not found any heavily methylated sequences; out of nine clones, only three contained a single methylated CpG, and six clones were completely unmethylated (Fig. 2a). We therefore conclude that the syncytin-1 promoter region is unmethylated in trophoblastic cells and any further demethylation cannot occur during the differentiation from cytotrophoblast to syncytiotrophoblast.

In order to see whether the level of CpG methylation within the U3 region of syncytin-1 5′ LTR correlates with the transcription of syncytin-1, we detected specific transcripts using RT-PCR in HeLa and BeWo cells. As expected and in accordance with previous studies [25], we observed syncytin1-specific transcripts in uninduced BeWo cells but not in HeLa cells (Fig. 2b). This correlation suggests that CpG methylation of the syncytin-1 U3 region might be a potent suppressor of its transcriptional activity. We have not observed any spontaneous fusogenic activity in BeWo cells used for this assay. Only after stimulation of BeWo cells with forskolin, the cell-to-cell fusions occur (not shown) and the level of syncytin-1-specific transcripts increased (Fig. 2b).

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PCR. After 30 cycles of PCR, we did not detect any syncytin-1 transcript in HeLa cells (Fig. 3b). The increase in PCR cycle number to 35 led to the detection of a very low amount of syncytin-1 transcript in mock-demethylated and in mediumdemethylated HeLa cells (data not shown). This probably represents an accidental background transcriptional activity of syncytin-1, which, however, does not correlate with the decrease of CpG methylation.

CpG methylation suppresses the transcriptional activity of syncytin-1 LTR in vitro

Fig. 2 – CpG methylation status and expression of syncytin-1 in choriocarcinoma BeWo cell line. (a) CpG site methylation status of ERVWE1 5′ U3 region in BeWo cells. Numeric representation shows number of methylated CpG/number of all CpG and the percentage in parenthesis. Methylated CpG sites are indicated by solid circles, unmethylated CpG sites are indicated by open circles. (b) RT-PCR products amplified with syncytin-1-specific primers in BeWo cells, in forskolin-stimulated BeWo cells (BeWoF) and in HeLa cells. β-actin was used as an internal standard. M represents 100 bp ladder with indicated fragment sizes.

Stability of CpG methylation of the syncytin-1 in non-placental cells If CpG methylation ensures transcriptional suppression of syncytin-1 and prevents improper cell-to-cell fusion in somatic tissues, it should be very stable and resistant to accidental fluctuations. We therefore analyzed the stability of the CpG methylation level within the U3 region of syncytin-1 5′ LTR in HeLa cells treated with azaC and TSA, inhibitors of DNA methylation and histone acetylation, respectively. We applied two different concentrations of azaC alone or together with TSA. The higher concentrations of both agents severely decreased the proliferation of HeLa cells, indicating that we have reached the maximum of tractable genome demethylation. The effectiveness of whole-genome demethylation was checked by methylaccepting assay using the SssI methyltransferase. Low concentration of azaC alone had no effect on the U3 5′ LTR methylation level, although we detected a substantial decrease in total genome methylation. Higher concentration of azaC alone and particularly in combination with TSA resulted in further decrease in total CpG methylation, but only a slight decrease in methylation of the analyzed U3 region. A small fraction of analyzed sequences were found to be demethylated, but the majority of them remained untouched (Fig. 3a). Furthermore, it seems that the third CpG dinucleotide is less resistant than others to the azaC/ TSA-induced CpG demethylation. Furthermore, we tested whether the mild demethylation of HeLa cells releases the block in the transcription of syncytin-1. RNA from azaC- and TSA-treated HeLa cells and from BeWo cells, as a positive control, was isolated and subverted to RT-

The tight negative correlation between CpG methylation in the U3 region of syncytin-1 LTR and transcriptional activity suggests that methylation could be the mechanism of transcriptional suppression. In order to assess the negative influence of U3 CpG methylation, we have fused together the complete syncytin-1 5′ LTR and the luciferase reporter gene. In this way, we have created a new reporter vector pLTRluc. We methylated all CpG sites in pLTRluc in vitro with the CpG-specific SssI methylase and measured the promoter activity after transient transfection of methylated and mockmethylated pLTRluc DNA into HeLa and BeWo cells. Both HeLa and BeWo cell lines displayed high luciferase activity after transfection by the mock-methylated reporter vector; in-vitro-methylated pLTRluc vector, however, induced only weak luciferase activity in both cell lines. We conclude that CpG methylation suppresses transcription of syncytin-1 and the non-methylated U3 region of syncytin-1 is transcriptionally active in HeLa cells in the absence of placental-specific transcription factors. During the 2-day transient transfection assay, we did not observe any cell fusions either in HeLa or in BeWo cells.

Discussion The studies presented here demonstrate that the syncytin-1 promoter is sensitive to CpG methylation. We have also found this sequence to be densely methylated in all tissues and cell cultures with the exception of terminal placenta and trophoblastic cell line BeWo. We therefore believe that CpG methylation of the syncytin-1 promoter sequences plays a role in transcriptional suppression of this gene normally expressed exclusively in the trophoblast and, to a much lesser extent, in testes [9]. The regulation of the syncytin-1 transcription has been studied in terms of transcription factors and their binding sites, hormonal level and oxygen level in the placenta. GCMa is the best candidate for the role of master activator of syncytin-1 because it interacts with two GCMa-binding sites upstream from the 5′ LTR and increases the level of syncytin-1 transcripts in BeWo and JEG3 choriocarcinoma cells ∼4- and 3-fold, respectively [25]. Furthermore, it is specifically synthesized in the developing placenta [44]. However, overexpression of GCMa fails to induce transcription of endogenous syncytin-1 in HeLa cells, whereas basal promoter activity of syncytin-1 5′ LTR can be detected in choriocarcinoma cell lines [25]. Therefore, there must be some mechanism suppressing the syncytin-1 expression in

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Fig. 3 – Impact of the DNA methyltransferase and histone deacetylase inhibitors azaC and TSA, respectively, on the CpG methylation and the expression of syncytin-1 in HeLa cells. (a) Methylation pattern of the syncytin-1 5′ U3 region in HeLa cells demethylated with increased concentrations of azaC and/or TSA. Micromolar concentrations of azaC and TSA are indicated on the right. Numeric representations of CpG methylation show numbers of methylated CpG/numbers of all CpG. The relative decrease of CpG methylation in comparison with mock-treated HeLa cells is given in parenthesis. The relative extents of total genome demethylation as estimated by the increase of methyl acceptance are given on the right side of the figure. Methylated CpG sites are indicated by solid circles, unmethylated CpG sites are indicated by open circles. (b) Expression of syncytin-1 was determined by RT-PCR with syncytin-1 env-specific primers in HeLa cells demethylated with increasing concentrations of azaC and TSA as indicated below. BeWo cells were used as a positive control of syncytin-1 expression, β-actin mRNA levels were used as an internal loading control. M, 100 bp ladder with indicated fragment sizes.

non-trophoblastic cells and protecting the organism from inadvertent cell-to-cell fusion outside of the placenta. At least one example of pathogenic non-placental expression of syncytin-1 has been shown. Antony et al. [45] detected the syncytin-1 protein in astrocytes and activated glial cells in patients with multiple sclerosis. Cheng et al. [24] describe the ecdyson receptor response element as a negative cis-regulatory sequence within the syncytin-1 5′ LTR. The suppressive effect of this element is, however, weak and does not explain the absence of syncytin-1 mRNA in most of tissues. Smallwood et al. [46] suggest that there is another mechanism of syncytin-1 expression at the level of splicing. They observed significantly decreased syncytin-1 protein synthesis at late pregnancies in comparison to early placentas. Concomitantly, the abundance of testis-specific non-spliced syncytin-1 transcript of 8.0 kb increased at the expense of spliced placental transcript of 3.1 kb. Even this competition

between two mRNA species is not strong enough to represent an efficient control of syncytin-1 expression in non-trophoblastic cells. Provided that HERVs and other transposable elements are transcriptionally suppressed by methylation, recently shown, e.g. in the HERV-K family [30], and that placenta where the syncytin-1 is expressed contains hypomethylated tissues derived from trophoblast and primitive endoderm ([36] for the review), we set out to analyze the CpG methylation pattern of syncytin regulatory sequences and its influence on syncytin-1 expression. In accordance with our assumption, we have detected hypomethylated or completely unmethylated syncytin-1 promoter in placenta, although only in part of the obtained sequences. We have detected syncytin-1 mRNA in choriocarcinoma-derived BeWo cell line and found complete or nearly complete demethylation of promoter sequences in all cases. On the contrary, HeLa cells containing heavily methylated syncytin-1 do not

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express any mRNA detectable by RT-PCR. Treatment of HeLa cells with high doses of DNA methylation and histone deacetylase inhibitors affected the syncytin-1 methylation just slightly, and it had no effect on the syncytin-1 expression. Furthermore, we have not observed any significant hypomethylation of syncytin-1 in otherwise hypomethylated tumor cells [47] and cells from ICF syndrome patients. These altogether show that the hypermethylation of the syncytin-1 promoter is very stable because such treatment efficiently demethylates and transcriptionally activates most sequences examined [48]. This resistance of syncytin-1 methylation to the DNA methyltransferase inhibitor strongly supports its role in transcriptional suppression. The lack of CpG methylation of the syncytin-1 5′ LTR in trophoblast could be explained by either demethylation, active or non-specific, or persistence of the unmethylated state as a remnant of unmethylated genome during the early embryogenesis. Anyway, there must be some sort of antimethylation protection because the DNA in terminal placenta is methylated, at least to some extent [36]. Promoters of cellular genes as well as proviral LTRs are protected from CpG methylation by the presence of CpG islands [49,50], but this is not the case of syncytin-1. The transcriptional suppression of syncytin-1 is very strong; we have not detected any syncytin-1 mRNA by RT-PCR in HeLa cells after treatment with azaC and TSA (Fig. 3b), and Yu et al. [25] failed to induce its transcription by overexpression of GCMa. As the transcriptional suppression of in-vitro-methylated syncytin-1 promoter is rather leaky in BeWo as well as in HeLa cells (Fig. 4), we have to take account of DNA methylation-precipitated chromatin remodeling as a mechanism strengthening gene silencing. In this regard, the transiently transfected plasmid vector does not simulate the chromosomal context exactly. Anyway, our results show that no placental-specific transcription factors are necessary for the basal syncytin-1 promoter activity. Both mononuclear cytotrophoblast cells and BeWo cells prior to induction by GCMa transcribe syncytin-1 mRNA just four times less than cells induced to syncytiotrophoblast differentiation, whereas protein products are hardly detectable without induction [14,25]. This indicates that posttranscriptional events are involved in down-regulation of syncytin-1 expression in human placenta. Cheynet et al. [51] described in BeWo cells the glycosylation of gPr73, a precursor protein of syncytin-1, which is responsible for the homotrimerization and proteolytic maturation into gp50 and gp24 subunits. These steps require cellular factors since the human genome does not encode any HERV-W pro gene within the translatable ORF [4,7]. In addition, regulation of syncytin-1 expression might be imposed at the level of mRNA stability/degradation or during translation. To investigate the regulation properly, a quantitative test of the mRNA level, run-on and run-off assays should be performed. We conclude that CpG methylation suppresses the syncytin-1 expression, and that methylation of syncytin-1 promoter sequence protects the non-placental cells from accidental syncytia formation or inflammatory challenge. Demethylation of the syncytin-1 promoter is a prerequisite for its basal

Fig. 4 – Effect of CpG methylation on the transcriptional activity of ERVWE1 5′ LTR in HeLa and BeWo cells. The complete ERVWE1 5′ LTR was fused with the luciferase reporter into pLTRluc. This vector was in-vitro-methylated with SssI methyltransferase, and 1 μg of plasmid DNA was transfected transiently into HeLa and BeWo cells grown on 35 mm Petri dishes. The transcriptional activity of mock-methylated (black columns) and methylated (grey columns) constructs was measured as luciferase activity 2 days after transfection. Error bars correspond to SE calculated from four parallels in HeLa and from six parallels in BeWo. *P b 0.05; **P b 0.01.

transcriptional activity but not sufficient for syncytialization. This basal activity can be further enhanced by placentaspecific transcriptional factors during syncytiotrophoblast differentiation.

Acknowledgments We would like to thank Zdena Vernerová for providing us with the samples of terminal placentas, Keith Robertson for DNA from ICF patients, Eva Matoušková for human skin fibroblasts and the sample of breast carcinoma and Lubomíra Fričová for the help with methyl-accepting assay. We appreciate very much the technical assistance provided by Věra Hoserová and Iva Rybková. This work was supported by the grant No. GA204/ 05/0939 awarded by the Grant Agency of the Czech Republic, grant No. IAA5052207 awarded by the Grant Agency of the Academy of Sciences of the Czech Republic and project

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AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic. REFERENCES

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