3.2.2.  The 5′LTR of placentally expressed HERVs display dissimilar methylation profiles in placenta tissues
We investigated and compared the 5′LTR methylation profiles of ERVWE1, ERVFRDE1 and ERV3 proviruses (schematized in Fig. 3A), in the same placenta-associated tissues as done for HERV-W LTRs. It appeared that all three 5′LTRs shared a common lack of methylation in villous placenta but under different modalities. In addition, their methylation was widely variable in the non-trophoblastic cells associated with the placenta.
Figure 3  CpG methylation envelope-coding HERV 5′LTRs in placenta-associated tissues. (A) Schematic representation of MaLR[LTR]–ERVWE1[5′LTR] (as in Fig. 2A), ERVFRDE1[5′LTR] and ERV3[5′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). Putative transcription factor binding sites proximal to or overlapping CpGs are indicated. Downstream horizontal bars are provirus internal sequence. (B–E) CpG methylation of (B) MaLR[LTR]–ERVWE1[5′LTR] (as in Fig. 1), (C) ERVFRDE1[5′LTR] and (D) ERV3[5′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 and CpGs with undetermined methylation state by gray circles. Global methylation percentage values in the U3 regions (highlighted in gray) as well as in the MaLR[LTR] (in parentheses) are given below the respective area for each sample.   In villous placenta, ERVWE1[5′LTR] had a global methylation level of 36.7%, and molecules were bimodally methylated, as mentioned above. An important amount of unmethylated molecules (7 out of 12 clones) was observed (Fig. 3B). In contrast, methylation of ERVFRDE1[5′LTR] was close to zero in the villous placenta, as only two CpGs were methylated in the whole sample (Fig. 3C). Finally, methylation of ERV3[5′LTR] reached a level of 23.5% and was distributed within three principal patterns (Fig. 3D). Thus, completely unmethylated molecules were found in high proportion (10 clones out of 17), completely methylated molecules were found in very low proportion (2 clones) and the remaining molecules were methylated only at the U3 area borders (5 clones). Although the methylation distribution of ERVWE1, ERVFRDE1 and ERV3 [5′LTRs] was undoubtedly different, an important amount of totally unmethylated clones was found for all three HERV 5′LTRs in agreement with their promoter function and activity in trophoblast.11
In placenta-associated non-trophoblastic cells, i.e. PBLs and placental fibroblasts, ERVWE1[5′LTR] was found heavily methylated. ERVFRDE1[5′LTR] was also essentially methylated in placental fibroblasts (77.9%), albeit CpGs 3, 5 and 8 were regularly unmethylated. In fetal cord blood cells on the contrary, the global methylation level of ERVFRDE1[5′LTR] was only 25.3%. This was due to a quasi-systematic but exclusive methylation on the CpGs neighboring the TATA box. This accurate methylation pattern was conserved in maternal blood cells, although an occasional methylation of the second CpG site slightly increased the methylation level up to 30.2%. ERVWE1 high methylation level in non-trophoblastic cells and ERVFRDE1 high methylation in placental fibroblasts are in agreement with the lack of expression previously established for ERVWE1 and ERVFRDE1 env mRNAs in these tissues.11,14,50 In spite of this, there is no trivial link between ERVFRDE1[5′LTR] low methylation level in PBLs and the described weak transcriptional activity of ERVFDRE1 env (200-fold less than in placenta).11 One explanation could be that essential transcription factors needed for ERVFRDE1 transcriptional activity are missing in PBL. Nonetheless, another explanation could be related to the precise and accurate methylation at only the CpGs closed to the TATA box. Such a local methylation was observed for the RANKL gene promoter and impaired transcriptional induction by disturbing the attachment of the TATA binding protein.56 Contrary to ERVWE1 and ERVFRDE1, methylation patterns of ERV3[5′LTR] in maternal blood cells and placental fibroblasts were highly similar to those found in term trophoblast. However, the proportion of each pattern and consequently the global methylation level varied depending on the sample type. Thus, the global methylation of the ERV3 5′LTR U3 region reached 37.5% in maternal blood (with 6 unmethylated clones out of 16) and 54.6% in placental fibroblasts (with 2 unmethylated clones out of 11). The variation in their relative proportion could explain the variable levels of ERV3 env transcription among human tissues in general and between placenta and PBL11 in particular. An exception to this trend of ERV3[5′LTR] generic methylation patterns was the finding of only one methylated CpG (equivalent to 1.9% of total methylation) in the fetal cord PBLs sample. This lack of methylation was all the more unexpected, since the LTRs of all the other HERVs analyzed in this study had a conserved methylation in fetal and adult PBLs. Whether this lack of methylation correlates with significant ERV3 env mRNA and protein levels in fetal PBLs deserves further investigation.
In conclusion, methylation status of these LTRs is globally in adequation with their respective promoter activity and could also be implicated in the modulation of their transcriptional level and consequently in the biological activity of their protein.