PMC:2725788 / 21549-26454 JSONTXT

{"project":"2_test","denotations":[{"id":"19561344-17617638-25963816","span":{"begin":2522,"end":2524},"obj":"17617638"},{"id":"19561344-16427621-25963817","span":{"begin":3326,"end":3328},"obj":"16427621"}],"text":"3.1.2. Methylation unequally affects ERVWE1 5′LTR and other HERV-W LTRs\nThe 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).\nFigure 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%).\nThe 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]).\nInterestingly 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.\nThese 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."}