PMC:2725788 / 30042-44330 JSONTXT

{"project":"2_test","denotations":[{"id":"19561344-15450384-25963828","span":{"begin":344,"end":346},"obj":"15450384"},{"id":"19561344-2884330-25963829","span":{"begin":658,"end":660},"obj":"2884330"},{"id":"19561344-15507602-25963830","span":{"begin":661,"end":663},"obj":"15507602"},{"id":"19561344-12970426-25963831","span":{"begin":4002,"end":4004},"obj":"12970426"},{"id":"19561344-12970426-25963832","span":{"begin":4890,"end":4892},"obj":"12970426"},{"id":"19561344-16714059-25963833","span":{"begin":4893,"end":4895},"obj":"16714059"},{"id":"19561344-15993705-25963834","span":{"begin":4896,"end":4898},"obj":"15993705"},{"id":"19561344-12970426-25963835","span":{"begin":5093,"end":5095},"obj":"12970426"},{"id":"19561344-17008384-25963836","span":{"begin":5516,"end":5518},"obj":"17008384"},{"id":"19561344-12970426-25963837","span":{"begin":6182,"end":6184},"obj":"12970426"},{"id":"19561344-16427621-25963838","span":{"begin":8706,"end":8708},"obj":"16427621"},{"id":"19561344-12620933-25963839","span":{"begin":8882,"end":8884},"obj":"12620933"},{"id":"19561344-11253064-25963840","span":{"begin":9056,"end":9058},"obj":"11253064"},{"id":"19561344-16411991-25963841","span":{"begin":10452,"end":10454},"obj":"16411991"},{"id":"19561344-16411991-25963842","span":{"begin":10592,"end":10594},"obj":"16411991"},{"id":"19561344-16411991-25963843","span":{"begin":10665,"end":10667},"obj":"16411991"},{"id":"19561344-11854637-25963844","span":{"begin":10668,"end":10670},"obj":"11854637"},{"id":"19561344-15533715-25963845","span":{"begin":11523,"end":11525},"obj":"15533715"},{"id":"19561344-18215254-25963846","span":{"begin":11571,"end":11573},"obj":"18215254"},{"id":"19561344-17008384-25963847","span":{"begin":12586,"end":12588},"obj":"17008384"},{"id":"19561344-8372456-25963848","span":{"begin":13019,"end":13021},"obj":"8372456"},{"id":"19561344-7716822-25963849","span":{"begin":13022,"end":13024},"obj":"7716822"},{"id":"19561344-8372456-25963850","span":{"begin":13671,"end":13673},"obj":"8372456"},{"id":"19561344-7716822-25963851","span":{"begin":13674,"end":13676},"obj":"7716822"}],"text":"3.2. 5′LTR methylation of HERVs coding for proteins associated with placenta morphogenesis\n\n3.2.1. Description of the placentally expressed HERV included in the study\nERVFRDE1 Env (Syncytin-2) and ERV3 Env together with ERVWE1 Env (Syncytin-1) are the three retroviral envelope proteins exhibiting a probable function in placenta development.55 Syncytin-2 and ERV3 Env are, respectively, encoded by ERVFRDE1, a provirus belonging to the HERV-FRD family (HERV-FRD_6p24.1; AL136139) and the provirus ERV3 belonging to the ERV3/HERV-R family (HERV-R_7q11.2; AC073210). LTR borders and transcription start sites have already been published for ERVWE1 and ERV3.12,43 ERVFRDE1 LTR borders limits were determined using the RepeatMasker software (www.repeatmasker.org). ERVFRDE1[5′LTR] U3/R boundary, delineating the transcription start site, was located at nt +492 or nt +504 from the [5′LTR] 5′ end, using four human placental full-length or 5′ end containing mRNAs displaying more than 99% identity with ERVFRDE1 genomic sequence (BC068585, AK123938, AL543429 and CR626159).\n\n3.2.2. The 5′LTR of placentally expressed HERVs display dissimilar methylation profiles in placenta tissues\nWe 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.\nFigure 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\nIn 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.\nIn 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.\n\n3.2.3. Pregnancy-associated 5′LTR methylation changes in HERV coding for envelope proteins in cytotrophoblasts\nERVWE1, ERVFRDE1 and ERV3 env expression is localized in cytotrophoblastic cells of the placenta and is temporally regulated during pregnancy, according to a dynamic specific to each envelope gene. We further investigate whether changes in promoter methylation of the 5′LTR of these HERVs may contribute to the observed transcriptional variations. Analyses were carried out in purified cytotrophoblasts from one normal first trimester placenta (placenta 1.1), trisomic 21 (T-21) second trimester placentas (placentas 2.1 and 2.2) and normal term placentas (placentas 3.1, 3.2 and 3.3) (for clarity, results from one sample for each time of gestation are shown in Fig. 4).\nFigure 4 CpG methylation dynamics of envelope-coding HERV 5′LTRs in cytotrophoblasts during pregnancy. (A) Schematic representation of the MaLR[LTR]–ERVWE1[5′LTR], ERVFRDE1[5′LTR] and ERV3[5′LTR] analyzed regions. (B–D) CpG methylation of (B) MaLR[LTR]–ERVWE1[5′LTR], (C) ERVFRDE1[5′LTR] and (D) ERV3[5′LTR]. Methylation was determined by bisulfite sequencing PCR in cytotrophoblasts (CT) at different times of gestation. One sample is represented here for each trimester i.e. CT of first trimester placenta from legally induced abortion (placenta 1.1), second trimester placenta from trisomy 21-affected pregnancy (T21, placenta 2.2) and term placenta from healthy mother (placenta 3.3). Each sample result originates from the same conversion reaction. Each line represents an independent molecule. Methylated CpGs are schematized by black circles and unmethylated CpGs by white circles. The bimodal methylation pattern previously observed for the ERVWE1[5′LTR] domain39 and herein shown to equally affect the associated MaLR[LTR] TSE in term villous placenta could have reflected an imprinted regulation of ERVWE1, as was suggested previously.57 This process is regularly mediated by the methylation of one of the two alleles in a parent-of-origin manner and often related to genes involved in placenta morphogenesis.58 However, the bimodal methylation was not clearly observed at all stages of pregnancy. Thus, in the first trimester placenta, the MaLR[LTR]–ERVWE1[5′LTR] was not methylated at all (placenta 1.1 Fig. 4B). Among the two second trimester samples derived from T-21 placentas, placenta 2.1 also showed no methylation at all (not shown), whereas placenta 2.2 presented one fully methylated clone out of 12 (Fig. 4B). In contrast, in all the three CT samples derived from term placenta, the bimodal methylation pattern was clearly observed though with a significant sample-to-sample variation in the proportion of the two methylation patterns. More precisely, the number of methylated clones was 1 out of 12 in placenta 3.1 (not illustrated), 5 out of 12 in placenta 3.2 (not illustrated) and 6 out of 14 in placenta 3.3 (Fig. 4B), respectively. In short, the global amount of methylated CpGs in the MaLR[LTR]–ERVWE1[5′LTR] region varied from 0% in the first trimester placenta to 33.3% at term (as observed in the term placental villi sample). The absence of stability in the bimodal repartition of molecules methylation throughout gestation argues against the proposed imprinting hypothesis. In contrast, the apparent increased frequency of hypermethylated sequences from first trimester to term is consistent with the decrease in ERVWE1 env expression observed between 37 and 40 weeks of gestation.59 In addition, the sample-to-sample variations are compatible with the inter-individual variation observed for the ERVWE1 expression level.59 Consequently, ERVWE1 env mRNA down-regulation in placental dysfunction59,60 could be related to an early remethylation of ERVWE1 promoter. The discovery of a single atypical clone exhibiting a methylated MaLR[LTR] and an unmethylated ERVWE1[5′LTR] in villous placenta (Fig. 3B) may reflect a ‘targeted’ remethylation process that would deserve further investigation.\nContrary to ERVWE1, ERVFRDE1[5′LTR] was stably unmethylated in normal first and term cytotrophoblasts as well as in second trimester T-21 cytotrophoblasts, as overall methylation in the samples did not exceed 5.7% (0%, 5.7% and 2.7% in placenta 1.1, 2.2 and 3.3, respectively, see Fig. 4C; and 1.3%, 5.6% and 3.3% in placenta 2.1, 3.1 and 3.3, respectively, not illustrated). This stable unmethylation allows a constitutive transcriptional activity but cannot account for transcriptional modulations. Thus, ERVFRDE1 env mRNA progressive decrease along gestation61 and down-regulation in T-21 affected placenta62 might preferentially be linked to a modulation of the transcription factor pool rather than to alteration of the LTR methylation.\nERV3 [5′LTR] methylation levels [investigated in one representative CT sample for each stage of pregnancy, i.e. placentas 1.1, 2.2 and 3.3 (Fig. 4D)] were somewhat same during all gestation. Hence, in the U3 region, 29.2% of the CpGs were methylated in the first trimester, 18.2% in the second trimester and 25.4% at term. Still, as in ERVWE1, ERV3[5′LTR] methylation pattern did change. However, these changes were not progressive but more drastically linked to the gestation stage, and differences were more particularly obvious between the first and the second trimester. Indeed and interestingly, ERV3[5′LTR] methylation profile in the first trimester was very similar to the one observed for ERVFRDE1[5′LTR] in blood, i.e. precisely and systematically associated with the TATA box. The fact that this precise promoter methylation has been found for the conventional RANKL gene,56 and herein for two unrelated HERVs (ERV3 and ERVFRDE1, see Fig. 3C), might indicate an important regulation mechanism and would have to be studied further. ERV3 expression in placenta along gestation has been described sparsely. However, under the same hypothesis as proposed above for ERVFRDE1, this methylation pattern could correlate with the faint or absent ERV3 env expression in the first trimester highly proliferative CTs.63,64 In the second trimester CT sample, we could observe lower methylation of these CpGs leading to an unmethylated U3 region in 5 of the 11 sequences. In contrast, a new methylation occurred on the first CpG downstream from the LTR on all clones, which was never methylated in the first trimester. Finally, three methylation patterns were observed in term CTs, which were similar to those of the term villous placenta sample. Thus, over the 15 clones analyzed, 8 were completely unmethylated in the U3 area, whereas the others were methylated along the whole U3 region or only on the first and last CpGs. Correlating with ERV3 env highest expression,63,64 the maximal number of unmethylated ERV3[5′LTR] U3 sequences was found at this stage of pregnancy.\nAltogether, these results support the hypothesis that the transcriptional activity of these domesticated proviruses is epigenetically controlled and that methylation might be further involved in the developmentally associated regulation of, at least, ERVWE1 env and ERV3 env transcriptional levels in placenta and possibly of ERV3 env in PBLs. Whether the epigenetic picture observed in the second trimester reflects the 15–25 weeks of amenorrhea or the T-21 pathological context deserves further investigations.\n"}