PMC:2725788 / 1686-7819 JSONTXT

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Introduction\nHuman endogenous retroviruses (HERVs) are constitutive elements of the human genome.1 Now transmitted exclusively in a Mendelian way, they derived through infectious retrovirus integration in the germ-line DNA and transmission to the offspring, millions of years ago. Different waves of retroviral endogenization and expansion by retrotranspositional events (copying mechanism) led to the formation of at least 31 phylogenic multicopy families of HERVs, corresponding to over 400 000 HERV copies, dispersed throughout the genome.2 Because of the accumulation of mutations, the contemporary HERV families consist of collections of heterogeneous HERV elements, ranging from full-length proviruses [gag, pol and env gene sequences flanked at each extremity by a long terminal repeat (LTR)] to isolated LTRs derived from inter-LTR recombination events.\nSome families or family members of HERVs are naturally expressed in particular physiological contexts and display physiological functions. This is not only the case in several LTRs active as alternative promoters of cellular genes in placenta3,4 but also in proviruses driving the expression of their own retroviral protein, e.g. ERVWE1, ERVFRDE1 and ERV3, three phylogenically unrelated proviruses highly active in the placenta. ERVWE1 belongs to the HERV-W family and contains gag and pol pseudogenes but its envelope gene ORF has been preserved (for review see Gimenez and Mallet5). This domesticated envelope, dubbed Syncytin-1, was demonstrated to be involved in trophoblast fusion occurring during placental development6 and was also recently proposed to exert anti-apoptotic function7 and to induce proliferation.8 The ERVFRDE1 provirus from the HERV-FRD family9 and the ERV3/envR provirus from the ERV3/HERV-R family10 also code for functional envelope proteins that probably play a role in placental development, i.e. Syncytin-2 and ERV3 Env, respectively.9,11,12 Syncytin-2, which is fusogenic9 and immunosuppressive,13 appears strictly expressed in placenta.11,14 In contrast, ERV3 Env, which inhibits cell proliferation15 and exhibits immunosuppressive properties,13 is transcribed at various levels in almost all investigated tissues.11,16,17 Except for these few examples of localized physiological activities, HERV elements appear essentially silent in somatic cells.\nHERV silencing is thought to be the result of the methylation of the LTRs (5′ LTR, 3′ LTR or solo LTR), which, as for infectious retroviruses, contain the regulatory elements and, in particular, the retroviral promoter in the LTR U3-subregion. This epigenetic process, which occurs in mammals on cytosines of 5′CpG dinucleotides, has the property to block the regulatory function of the targeted sequence.18,19 It is believed to have evolved primarily as a defense mechanism against the possible deleterious effects of retroelements and repeated sequences.20 Indeed, whole genome methylation studies showed that repeated elements, including HERVs, are globally methylated in normal somatic cells.21–23 Methylation-mediated repression of HERV LTRs activity was illustrated in vitro by methylation assays on functional HERV-K 5′LTRs.24 Conversely, demethylation-mediated derepression of copies from the HERV-K (HML-2) and HERV-E families was illustrated in cell culture by the use of the demethylating agent 5′-aza-deoxycytidine.25,26 In vivo HERV transcripts have been detected in numerous pathological situations and predominantly in autoimmune/inflammatory diseases26–30 and tumoral contexts.31–34 Methylation deregulations are strongly associated with tumors,22,23 and interestingly, tumoral transcriptional reactivations of HERVs have been associated with LTRs hypomethylation for some contexts when compared with normal tissues. This is the case of one HERV-H copy in gastrointestinal cancers,35 several HERV- K members in urothelial carcinomas36 and human primary testicular tumors25 and the HERV-W family in ovarian carcinomas.37\nThus, the impact of DNA methylation in the control of HERV elements seems obvious. However, HERV methylation status in physiological conditions has been less considered. HERV-E LTRs acting as alternative promoters of cellular genes in placenta were shown to be unmethylated in this tissue, but hypermethylated in blood where they do not exert promoter function.38 Likewise, the expression of the domesticated ERVWE1 locus has been correlated with hypomethylation of its 5′LTR in placenta in comparison with other tissues.39 Thus, HERV activity in physiological context seems related to a lack of methylation. The HERV-W family contains ∼80 full-length proviruses with flanking LTRs, 200 retrosequences with incomplete LTRs and 400 solitary LTRs.40,41 However, in contrast to ovarian carcinoma, expression of HERV-W family in placenta mainly results from the activity of the ERVWE1 locus.42 The high and specific placental expression of ERVWE1 has been attributed to the co-optation of its own retroviral promoter, localized in the 5′ LTR, with an LTR retrotransposon of MaLR (mammalian apparent LTR retrotransposon) type, acting as a trophoblast-specific enhancer (TSE).43–46 Still neither the methylation status of this enhancer LTR nor the methylation status of family related LTRs is known. Similarly, the methylation status of other proviruses producing envelope proteins, like ERVFRDE1 and ERV3, are unknown.\nGenerally, the features determining HERV methylation status are unknown. In addition, it remains poorly understood whether methylation not only can repress but also regulate HERV activity. Here, we asked whether ERVWE1 5′LTR methylation is related to the HERV family, the LTR role, the LTR-surrounding genomic context, the tissue or the implication of its envelope gene in a physiological process. For this purpose, we investigated and compared the methylation profiles of ERVWE1 5′LTR with ERVWE1 MaLR LTR enhancer, different HERV-W LTRs and the 5′LTRs from HERVs probably involved in placenta development and maintenance. Whether transcriptional activity and methylation were correlated was further addressed using model cell lines."}