3.1.  Methylation of HERV-W LTRs in placenta and placenta-associated tissues

3.1.1.  Description of the investigated HERV-W LTRs
ERVWE1/Syncytin-1 (Ch7q21.2; AC007566) transcription is regulated by two distinct elements, i.e. the ERVWE1 5′LTR U3 promoter region and an adjacent upstream TSE. This enhancer is located within a MaLR solo LTR within which the ERVWE1 provirus integrated ∼25–40 millions years ago.43,46 We chose to analyze together the methylation status of these two co-opted but unrelated LTRs involved in Syncytin-1 regulation, further referred to as MaLR[LTR]–ERVWE1[5′LTR]. As a minimal model to test the family related methylation process hypothesis, three other HERV-W LTRs were studied: ERVWE1[3′LTR], HW_4[5′LTR] and HW_12[solo LTR]. ERVWE1[3′LTR] is the 3′LTR of ERVWE1 provirus and represents topographically and phylogenetically the closest HERV-W LTR relative to ERVWE1[5′LTR]. HW_4[5′LTR] is an another functional (data not shown) 5′LTR belonging to a HERV-W provirus located on chromosome 4 (Ch4p13; AC024 022). HW_12[solo LTR] is a full-length solitary HERV-W LTR localized on chromosome 12 (Chr12q24.32; AC005868), and a cDNA overlapping the 51 bp at the 3′ end of the U5 region was found to have 100% identity with this part of the U5 region (BC038735). These HERV-W LTRs exhibit common but also specific CpG sites. An alignment of the investigated LTR U3 regions with highlighted CpG location is presented in Fig. 1. About 200 bp of the upstream env region from the ERVWE1[3′LTR] and ∼100 bp of the upstream region from the HW_4[5′LTR], which contains a MER110 type LTR, were co-amplified with the respective HERV-W LTRs as controls of genomic environment methylation.
Figure 1  Alignment of HERV-W LTR U3 promoter. ERVWE1[5′LTR] and ERVWE1[3′LTR] U3 region allelic forms were previously determined on DNA amplified from 24 individuals (e.g. -132A. individual 132, allele A), i.e. 48 sequences.45 HW_12[solo LTR] and HW_4[5′LTR] allelic forms derive from the annotated genome sequence (-A, reference allele; -B, alternative allele; SNP ID, rs6489086). Proportion of each allelic form is given in hooks (e.g. [3], 3 of the 48 sequences hold this allelic form; [nd], not determined). CAAT box, TATA box and transcription binding sites are indicated. Effective transcription factor binding sites are underlined; putative transcription factor binding sites are marked by interrupted lines. CpG sites are indicated by gray background, their position in the U3 region is given relative to ERVWE1 5′LTR sequence.

3.1.2.  Methylation unequally affects ERVWE1 5′LTR and other HERV-W LTRs
The methylation profiles of ERVWE1 and related HERV-W LTRs were investigated in villous placenta and in non-trophoblastic cells composing the placenta, i.e. placental fibroblasts, fetal blood cells and maternal blood cells (Fig. 2).
Figure 2  CpG methylation of HERV-W LTRs in placenta-associated tissues. (A) Schematic representation of MaLR[LTR]–ERVWE1[5′LTR], ERVWE1[env-3′LTR], HW_4[5′LTR] and HW_12[solo LTR] analyzed regions. LTR regions are represented by boxes and CpG dinucleotides by circles on vertical bars. The U3 region (light gray) constitutes the retroviral promoter, transcription starts at the U3/R boundary (arrow). For each LTR, CAAT and TATA boxes as well as putative and effective transcription factor binding sites proximal to or containing CpG are indicated. Symbols above CpGs point out conserved CpGs between the different LTRs, asterisk indicates CpGs conserved in all three LTRs. TSE, trophoblast-specific enhancer of ERVWE1 provirus, located in the 5′part of the MaLR LTR (white box at the 5′ end); env, env-3′UTR region of ERVWE1 provirus. The MER114 LTR located upstream from HW_4[5′LTR] is also represented (hatched box). (B–E) CpG methylation of (B) MaLR[LTR]–ERVWE1[5′LTR], (C) ERVWE1[env-3′LTR], (D) HW_4[5′LTR] and (E) HW_12[solo LTR]. Methylation was determined by bisulfite sequencing PCR in villous trophoblast of term placenta, related fetal and maternal blood cells and in placental fibroblasts from chorionic villi of a first trimester placenta. Each sample result originates from the same conversion reaction. Each line represents an independent clone as determined by methylation and/or conversion differences. Methylated CpG are schematized by black circles, unmethylated CpGs by white circles, the mutated CpG (SNP) in HW_12 is represented by a cross and CpGs with undetermined methylation state by gray circles. Global methylation percentages in the U3 regions (highlighted in gray) as well as in the upstream regions (MaLR LTR, env, MER114 LTR) are given below the respective area for each sample (in parentheses for non-U3 regions).   The phylogenetically related HERV-W LTRs schematized in Fig. 2A were found highly methylated in a majority of the investigated normal tissues (Fig. 2B–E). In blood particularly, the high global methylation of the HERV-W LTRs (from 85% to 98.2% of methylated CpGs) was similar to the methylation range observed for various LTRs of another HERV family, namely HERV-E.38 In placental fibroblast, all LTRs were also highly methylated (76.9–93.9% of methylated CpGs) except for HW_12[solo LTR] whose methylation level was found at its lowest (38.5%).
The villous placenta appeared to be the tissue with the lower level of methylation for all the LTRs except HW_12[solo LTR]. In spite of it, both the methylation levels and the methyl-group distribution within the molecules were different between the LTRs. Thus, CpG methylation level was low for ERVWE1[5′LTR] and HW_4[5′LTR] (∼40%), whereas it was relatively high for ERVWE1[3′LTR] (∼70%) and even higher for HW_12[solo LTR] (∼80%). Methyl-group distribution on ERVWE1[5′LTR] was bimodal, as molecules were either unmethylated (7 out of 12 clones) or densely methylated. This feature, first described by Matouskova et al.,39 appeared to be specific to ERVWE1 promoter. The methylation defaults on the other HERV-W LTRs were more diffusely distributed. Thus, some molecules were found repeatedly unmethylated on definite CpG sites, solely or together with contiguous CpGs (e.g. the first CpG of ERVWE1[3′LTR] and the second CpG of HW_4[5′LTR]), but no molecule was found fully unmethylated. On the contrary, some other CpG sites were found constitutively methylated (e.g. the first CpG of HW_4[5′LTR], the last CpG of ERVWE1[3′LTR] and the last two CpGs of HW_12[solo LTR]).
Interestingly in this tissue, the CpG site located at the end of the ERVWE1[5′LTR] ER binding site and conserved in ERVWE1[3′LTR] and HW_4[5′LTR] was not methylated in approximately the same proportion of clones in both LTRs (e.g. in 7 out of 10 clones and 9 out of 10 clones, respectively). Likewise, the CpG located before the TATA box, conserved in ERVWE1[3′LTR] and HW_12[solo LTR], was not methylated in 4 out of 10 clones and 4 out of 12 clones. However, other conserved CpGs did not have the same pro-rata of methylation default (e.g. the CpG at the U3/R border was not methylated in 2 out of 10 clones of ERVWE1[3′LTR] and 7 out of 10 of HW_4[5′LTR] and none of HW_12[solo LTR]), indicating that the ER binding site and TATA-box-associated CpGs might have particular methylation-targeting features.
These results demonstrate that LTRs from the same phylogenetic lineage can be unequally methylated within the same cellular type, suggesting that methylation establishment on HERV sequences might not be family dependent.

3.1.3.  Methylation of ERVWE1 LTRs and associated MaLR LTR suggests a regional control
By definition, ERVWE1[5′LTR] and ERVWE1[3′LTR] belong to the same proviral unit, but appeared differentially methylated in villous placenta. This unmethylation of the 5′LTR corresponds with its promoter function in placenta, as reported previously.39 The 3′LTR methylation in return may prevent a local competition with ERVWE1 5′LTR for promoter activity as well as a 3′LTR-derived transcriptional interference with the nearby antisense-oriented ODAG/GATAD1 gene.46 Such a differential methylation has been observed for HTLV-152 and HIV-1 proviruses during viral latency.53 For these two retroviruses, however, methylation favored repression of the 5′LTR promoter activity as the 5′LTR was methylated and the 3′LTR was not. This suggests different methylation features for exogenous/pathogenic and endogenous/domesticated proviruses but possibly a conserved strategy to prevent methylation spreading on both proviral LTRs, like the use of boundary elements as hypothesized for HTLV-1.52 The CTCF protein is a factor able to create boundaries between methylated and unmethylated genomic domains.54 Interestingly, two domains containing potential binding sites for CTCF were identified within ERVWE1 provirus (www.essex.ac.uk/bs/molonc/spa.htm), respectively, downstream from the 5′ LTR and upstream from the 3′ LTR region that we found methylated. Further investigations are required to ascertain CTCF functional relevance in the context of proviruses. Nevertheless, it appears that methylation might be regionally controlled and linked either to the genomic environment or to the LTR function.
The upstream flanking sequence of three out of the four HERV-W LTRs was amplified along with the respective LTRs. Except for one clone in the villous placenta tissue, the MaLR[LTR] directly upstream from ERVWE1 presented the same methylation profiles as the ERVWE1[5′LTR], i.e. either unmethylated, partially methylated or highly methylated (for methylation values, see Fig. 2B). In contrast, the CpG sites contained within the ∼200 bp of the env gene co-amplified with ERVWE1[3′LTR] appeared systematically highly methylated, including those in the villous placenta (with 92.5% of methylation level), despite a lower methylation (68%) of the 3′LTR in this tissue (Fig. 2C). Finally, methylation levels between HW_4[5′LTR] and the upstream MER114[LTR] part were similar in PBL and placental fibroblasts, but in the villous placenta, HW_4[5′LTR] methylation level was much lower (40%) than the MER114 (70%) (Fig. 2D). Thus, except for ERVWE1[5′LTR] and the upstream MaLR[LTR], there was no clear correlation between the methylation of the HERV-W LTRs and their flanking sequence. Reiss et al.38 also recently concluded that the hypomethylation of HERV-E LTRs acting as placenta promoters of cellular genes, when compared with random LTRs, cannot be explained by a difference in methylation of their flanking sequences, irrespective of its nature—repeated element or not.
The MaLR[LTR] and the ERVWE1[5′LTR] elements have been earlier demonstrated to be co-opted and to specifically control ERVWE1 placental expression.43,46 Thus, although belonging to distinct LTR types, their shared unmethylation status in placenta could be linked to their involvement in the regulation of Syncytin-1 transcription. Likewise, the heavy methylation of both LTRs in non-placenta cells supports a repression process outside placenta. Overall, these results are compatible with ERVWE1 env regulation as a bona fide gene.45