Reduced Syncytin-1 Expression Levels in Placental Syndromes Correlates with Epigenetic Hypermethylation of the ERVW-1 Promoter Region

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.

Evolutionary origins of the placental expression of chromosome 19 cluster galectins and their complex dysregulation in preeclampsia

“…The down-regulation of LGALS14 was confirmed by immunostainings of TMAs constructed from the same placentas and semi-quantitative immunoscorings (1.8-fold, p=4.6×10 −7 ) (Figure 4B). In accord with earlier placental and in vitro trophoblastic data [51,57–60,63,69,80,83,87], our results support that cAMP-induced, GCM1-driven trophoblast fusion is negatively impacted in preterm severe preeclampsia associated with SGA, while trophoblast differentiation - characterized here by CGB3 expression - is partially independent of trophoblast fusion and is not significantly impaired in preeclampsia. This finding confirms the results of a large set of placental microarray studies in preeclampsia [86,88] failing to detect the global alteration of the characteristic transcriptomic signature of villous trophoblast differentiation [50,52].…”

“…Since previously we found down-regulated placental LGALS13 expression in preterm preeclampsia [30], we were interested whether all placental Chr19 cluster galectins are differentially expressed in this syndrome, and if this supposed phenomenon may be related to an altered villous trophoblast fusion [57–60,63] or differentiation [64] program as shown or hypothesized before. Since our preterm preeclampsia placental microarray dataset [86] did not contain expression data for LGALS16 , we performed a qRT-PCR study for the investigated ten genes on placentas from women with preterm severe preeclampsia with SGA and preterm controls.…”

“…Defects in syncytiotrophoblast formation have been implicated in the development of preeclampsia. For example, the decreased placental expression of the molecular machinery of trophoblast fusion, including GCM1 transcription factor, fusogenic retroviral proteins and their receptors have been observed in severe preeclampsia [57–63]. It was even presumed that the whole villous trophoblastic differentiation program is severely disturbed in preeclampsia [64], a hypothesis that could not be confirmed by whole-genome transcriptomic studies [50,52].…”

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses

“…Some cancers lose ERV DNA methylation and aberrantly overexpress ERVs Cohen et al, 1988;Larsen et al, 2009;Larsson et al, 2007;Rycaj et al, 2014;Strick et al, 2007;Strissel et al, 2012;Wang-Johanning et al, 2001;Wang-Johanning et al, 2007) while others maintain silencing. Aza can induce specific ERV transcripts in melanoma, choriocarcinoma, and endometrial cancer cells (Laska et al, 2013;Ruebner et al, 2013;Stengel et al, 2010) (Strissel et al, 2012). Indeed, in initial testing, the ERVK subfamily (Wang-Johanning et al, 2003) transcripts increased 2.5-fold in the A2780 cell line upon Aza treatment (data not shown).…”

Molecular functions of human endogenous retroviruses in health and disease

“…Syncytin, the product of individual HERV-W (ERVW-1) proviral locus ERVW-1, binds to its extramembrane receptor SLC1A5/ ASCT-2/RDR/ATB(0) and initiates formation of trophoblast cell fusion, most likely via Cx43-mediated gap junctional intercellular communication [134]. The deficiencies in Syncytin expression, e.g., caused by hypermethylation of ERVW-1, were reported to be associated with various placental abnormalities [135]. As shown in cell culture experiments, Syncytin activity may be negatively regulated by an RNA-binding protein LIN28A, whose target downregulation, in turn, appeared to release Syncytin functionality and promoted fusion of cultured human trophoblast cells [136].…”