Abstract
Terminal differentiation of villous cytotrophoblasts (CT) ends in formation of the multinucleated syncytiotrophoblast representing the fetal-maternal interface. Aberrations during this cell-fusion process are associated with Intrauterine Growth Restriction (IUGR), Preeclampsia (PE) and High Elevated Liver and Low Platelets (HELLP) Syndrome. Syncytin-1, the envelope gene of the human Endogenous Retrovirus ERVW-1, is one of the most important genes involved in cell-fusion and showed decreased gene expression during these pathological pregnancies. The aim of this study was to determine the methylation pattern of the entire promoter of ERVW-1 and to correlate these findings with the expression profile of Syncytin-1 in the placental syndromes. 14 isolated villous cytotrophoblasts from control (n = 3), IUGR (n = 3), PE (n = 3), PE/IUGR (n = 3) and HELLP/IUGR (n = 2) placentae were used to determine the mean methylation level (ML) for the ERVW-1 promoter region. ML rose significantly from 29% in control CTs to 49% in IUGR, 53% in PE, 47% in PE/IUGR and 64% in HELLP/IUGR indicating an epigenetic down-regulation of Syncytin-1 by promoter hypermethylation. DNA demethylation of the trophoblast like cell lines BeWo, JEG-3 and JAR with 5-AZA-2′desoxycytidine (AZA) showed an increased Syncytin-1 expression and fusion ability in all cell lines. Promoter activity of the 5′LTR could be inhibited by hypermethylation 42-fold using a luciferase based reporter-gene assay. Finally overexpression of the methyltransferases DNMT3a and LSH could be responsible for a decreased Syncytin-1 expression by promoter hypermethylation of ERVW-1. Our study linked decreased Syncytin-1 expression to an epigenetic hypermethylation of the entire promoter of ERVW-1. Based on our findings we are predicting a broad aberrant epigenetic DNA-methylation pattern in pathological placentae affecting placentogenesis, but also the development of the fetus and the mother during pregnancy.
Highlights
Epigenetics comprises changes in gene expression, without changing the DNA sequence
In the first trimester the 59LTR promoter region showed no CpG-methylation the methylation of the CpGs rose from 8.3% in the second trimester to 30% in term villous cytotrophoblasts (VCT) [49]. These findings support an epigenetic reprogramming during placentogenesis. The goal of this project was to unravel, if reduced expression of Syncytin-1 in PE, High Elevated Liver and Low Platelets (HELLP) and Intrauterine Growth Restriction (IUGR) cultured isolated trophoblasts was due to hypermethylation of the promoter region of ERVW-1 and if an aberrant expression of DNA-methyltransferases could be responsible for these changes
CTs from HELLP/IUGR had the most hypermethylated CpGs compared to control CTs, only CpG6 and 18 showed no differences to control CTs (Fig. 1; Table S1). These results suggest that reduced Syncytin-1 gene expression in pathological CTs could be due to hypermethylation of the 59LTR promoter region of ERVW-1 on chromosome 7q21.2
Summary
Epigenetics comprises changes in gene expression, without changing the DNA sequence. These effects are mediated by covalent attachment of chemical groups to DNA and its associated proteins and histones. Epigenetic marks are fixed after the cell has differentiated. In developmental stages as well as in some tumours, a broad epigenetic reprogramming takes place, which results to removing or changing of epigenetic marks [1,2]. In humans the methylation pattern of CpG-dinucleotides gives information about the activity of affected genes [3]. Hypermethylation of CpGs usually results in an inactivation of chromatin regions [4]. Responsible for placing epigenetic marks are DNA-methyltransferases (DNMT) such as DNMT1, DNMT3a and 3b [1]. Proteins which recognize and bind methylated CpGs by a Methyl-CpG-Binding Domain (MBD) are MBD1-4 and the methyl CpG-binding protein 2
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