2.  Materials and methods

2.1.  Biological samples
Placental tissues from the first trimester legally induced abortions (8–12 weeks of gestation) were obtained from the Department of Obstetrics and Gynecology at the Broussais, Saint Vincent de Paul and Cochin Hospitals. Second trimester placental tissues were collected at the time of termination of pregnancy at 15 and 25 weeks of gestation (in weeks of amenorrhea) in trisomy 21-affected pregnancies. Fetal Down syndrome was diagnosed by karyotyping of amniotic fluid cells. The indication of amniocentesis was the maternal age. Term placentas were obtained after elective cesarean section from healthy mothers near term with uncomplicated pregnancies. With written informed consent of the pregnant woman, the following samples were collected at term: a fragment of the placenta, umbilical cord fetal blood and maternal blood. The placenta biopsy sample was collected at a depth of 1 cm and at a distance of 8 cm from the edge of the placenta, with the maternal side facing upward. The use of these biological samples was approved by local ethical committee. Villous cytotrophoblastic cells were isolated by sequential enzymatic digestion of chorionic villi from the first trimester, second trimester and term placenta and purified on a discontinuous percoll gradient, as described previously.47–49 These cells were positively stained for cytokeratin 7 at 95%, indicating the cytotrophoblastic origin of the cells. Placental fibroblasts were isolated by prolonged enzymatic digestion from first trimester chorionic villi as in Malassine et al.50 Fibroblastic cells (1.25 × 105 cells/mL) were plated on 35-mm plastic dishes (TPP, Switzerland) and cultured for 48 h, and the culture medium was changed daily. These cultured fibroblasts were characterized using immunocytochemistry. It was found that 98% of the cells were vimentin positive and also positive for a monoclonal antibody against specific fibroblast antigen (clone ASO2, Dianova, Hamburg, Germany). Owing to the limited amount of cells, only DNA was extracted.

2.2.  Cell lines
Human choriocarcinoma cells BeWo b30 were cultured in F-12K medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) (Invitrogen). Human ovarian carcinoma cells OVCAR-3 were cultured in the RPMI 1460 medium supplemented with 20% FCS (LGC Promochem, Molsheim, France). Human astrocytoma cells 85HG66 were cultured in the DMEM medium (Invitrogen) supplemented with 10% FCS (Invitrogen). Monocytoid cells U937 were cultured in the RPMI 1640 medium containing 10% FCS. All media were supplemented with antibiotics, i.e. 100 IU of penicillin/mL, 100 µg of streptomycin/mL and 0.25 µg/mL amphotericin B (Fungizone) (Invitrogen). Cells were maintained in culture until confluence before DNA and RNA extractions.

2.3.  Sodium bisulfite modification of genomic DNA
Genomic DNA from confluent cell lines, blood samples and villous placenta section was isolated using the Nucleospin®Blood or Tissue kit following the manufacturer's instructions (Macherey-Nagel). Genomic DNA from cytotrophoblasts and placental fibroblasts was obtained following the overnight proteinase K lysis and phenol extraction procedure.51 To perform methylation studies, genomic DNA was submitted to sodium bisulfite treatment. This procedure converts all methyl-free cytosines into uracils, whereas methylated cytosines remain unchanged. For each sample, 1 µg of genomic DNA was mixed with 3 µg of carrier pUC18 DNA in a final volume of 100 µL. The mixture was heated for 10 min at 65°C, and the DNA denatured in 20 µL NaOH 3N for 30 min at 37°C. To convert unmethylated cytosines, denatured DNA was incubated in 50 µL hydroquinone 0.1M and 350 µL sodium bisulfite 3M for 16 h at 50°C. Modified DNA was then purified using Wizard DNA Clean-up System (Promega) and eluted in 50 µL of sterile water. DNA modification was completed by incubation in 5 µL NaOH 3N for 15 min at room temperature. DNA was washed by precipitation for 30 min at −20°C in 10 µL of ammonium acetate 5M and 200 µL of absolute ethanol, followed by centrifugation at 13 000 rpm at 4°C for 10 min. An additional wash was realized by centrifugation in 500 µL ethanol 70% for 5 min. The DNA pellet was eluted in 30 µL of sterile water.

2.4.  DNA methylation analyses
Strand-specific primers were designed to specifically amplify the HERV LTR copy of interest on bisulfite modified DNA by nested PCR (Table 1). First-round PCR was accomplished in a 50 µL mixture containing 3 µL of modified DNA, 0.66 µM of each primer, 1 mM of each dNTP, 1 U of DyNAzyme polymerase (Fynnzyme), 1× Mg-free DyNAzyme buffer (Fynnzyme) and 1.5 mM MgCl2 (Fynnzyme). PCR cycling conditions were: 5 min at 94°C; 35 cycles as follows: 1 min at 94°C, 2 min at a temperature depending on the primer set (Table 1), 1 min at 72°C; and a final extension at 72°C for 5 min. Nested PCR was performed on 1 µL of the first PCR products with internal primers in the same reaction mixture conditions. Nested PCR cycling conditions were 5 min at 94°C; 30 cycles as follows: 1 min at 94°C, 1 min at a temperature depending on a primer set (Table 1), 1 min at 72°C; and a final extension at 72°C for 5 min. PCR fragments were gel-extracted, cloned and sequenced using plasmid forward and reverse primers. To determine CpG methylation, and cytosine conversion rate, the sequence of each clone was aligned and compared with the in silico modified reference sequence (with all cytosines converted into thymines independently of their context). CpG positions were located and the methylation status at each CpG site observed. To prevent false-positive interpretation, only sequences with more than 95% of converted bases (all cytosines out of CpG context) were taken in analyses. Independence of the sequences was determined based either on their within-sample unique methylation pattern and/or on unique non-conversion of cytosines out of CpG context. For some samples, conversion rate was too high to determine the molecular independence of sequences based on unconverted cytosines. In these cases, more than 10 sequences were systematically analyzed to ensure methylation pattern representativity, and results were confirmed on other samples.
Table 1  Primer sets used for determination of HERV LTRs methylation   aNucleotides in bold and lowercase letters represent changes in primers relative to the genomic sequence due to bisulfite treatment, as follows: t is C and a is G in the untreated sequence. The position relative to the LTR start is given in parentheses.

2.5.  Real-time qRT–PCR
HERV LTR-derived transcript-specific primer sequences were determined using Oligo 6 software (MBI, Cascade, USA) or taken from published work (Table 2). Systems specificity was checked by in silico PCR on the human genome (UCSC Genome Browser: http://genome.ucsc.edu/cgi-bin/hgPcr; reference assembly: March 2006).
Table 2  Primer sets used for analyses of HERV LTR-derived transcriptional activity   aFrom De Parseval et al.11   Total RNA was isolated using the RNeasy Midi Kit including an on-column DNAse treatment in accordance with the manufacturer's instructions (Qiagen, Hilden, Germany). RNA quantity was measured at OD260 in a spectrophotometer, and RNA quality was assessed with a Bioanalyzer capillary electrophoresis device (Agilent, Palo Alto, USA). For each sample, 5 µg of total RNA was converted into cDNA in a final volume of 20 µL with random hexamer primers using ThermoScript RT–PCR System for first-strand cDNA synthesis (Invitrogen) following the manufacturer's recommendations. Reverse-transcriptase-free reactions were carried out simultaneously to verify the absence of contaminating genomic DNA.
Real-time PCRs were performed with the Stratagene Mx3005P quantitative PCR system (Stratagene, La Jolla, CA, USA) using the SYBR (with dissociation curve) option. Each quantitative PCR was performed on 10 µL of 100-fold diluted cDNA, in a PCR mix containing 0.2 µM of individual target primers, 10 µL of 2× Brilliant SYBR Green QPCR Master Mix (Stratagene) and water to reach a volume of 25 µL. Housekeeping genes were analyzed in the same experiment as the target transcripts. The experiments were performed in duplicate and included RT-minus controls. Following a 10 min denaturation at 95°C, gene amplification was performed as follows: 40 cycles at 95°C for 30 s, 55°C for 1 min and 72°C for 30 s. Melting curve acquisition was performed within a 55–95°C range, and resulting amplicons were sequenced and analyzed to check for false-positive detection. 18S and HPRT were the most stable housekeeping genes among the samples (variation less than a factor of 4.05 and 4.25, respectively). The geometric mean of their expression average was calculated and used to normalize for differences in the amount of total RNA added to the reaction. The relative quantification of transcripts was then calculated with respect to an external standard calibration curve established using serial dilutions of purified genomic DNA amplification products. The copy number of LTR-derived transcripts was related to 1000 cells, corresponding to ∼12.5 ng of total RNA.

2.6.  In vitro methylation of ERVWE1 promoter constructs and luciferase assays
To investigate the influence of methylation on the different regulatory regions of the ERVWE1 promoter activity, we used the previously described reporter plasmid pGL3-basic firefly luciferase HW-67/310 (TSE-U3), HW-1/310 (U3 full-length) and HW-90/310 (minimal promoter) constructs.43 Three micrograms of each pGL3-LTR vectors were methylated in a reaction mix containing 4 U of SssI CpG methylase, 1× NEBuffer 2 and 160 µM S-adenosyl-methionine (SAM) in a final volume of 20 µL (New England Biolabs). The methylation reactions were realized at 37°C and stopped after 30, 60, 120 and 240 min by heat inactivation of SssI at 65°C for 20 min. The negative control (no methylation) underwent the same treatment lacking SAM. To determine the methylation level of each reaction, 500 ng aliquots were digested with 0.5 U BstUI methylation-sensitive endonuclease (New England Biolabs) in 1× NEBuffer for 1 h at 60°C, and restriction profiles were analyzed after electrophoresis on a 1% agarose gel. BeWo b30 choriocarcinoma cells at 60–70% confluence were seeded in 12-well plates. Twenty-four hours later, cells were co-transfected with 0.7 µg of pGL3-LTR firefly luciferase plasmid either methylated or not (-SAM) and 35 ng of pRL-TK Renilla luciferase plasmid for internal standardization (Kit Dual-Luciferase® Reporter Assay System, Promega) by use of Lipofectamine Plus (GibcoBRL-Life Technologies, Invitrogen) as recommended by the manufacturer. Cells were lysed 24 h after transfection with passive lysis buffer (Promega). Firefly and Renilla luciferase activities were measured by using the Dual-Luciferase® Reporter Assay 1000 System (Promega) according to the manufacturer's instructions. Promoter activities were normalized to the internal Renilla luciferase control, i.e. (Firefly luciferase activity/Renilla luciferase activity) × 100. All reporter gene assays were performed in triplicate.