PMC:2725788 / 44331-51486 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2725788","sourcedb":"PMC","sourceid":"2725788","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2725788","text":"3.3. Association between HERV LTRs methylation and transcriptional activity\nTo investigate whether a systematic correlation exists between LTRs methylation status and transcriptional activities, we conducted paralleled transcription and methylation analyses in various cell lines, namely BeWo (choriocarcinoma), U937 (monocytes), 85HG66 (astrocytoma) and OVCAR-3 (ovarian carcinoma). Although the situation appears complex (Fig. 5), we identified essentially four situations.\nFigure 5 LTR promoter methylation and derived transcriptional activity. LTR-derived transcriptional activity in cell lines is represented by histograms, and the associated methylation profiles of the U3 promoter region of the LTRs are represented underneath. Real-time qPCR values were normalized by the geometric mean of HPRT and 18S housekeeping genes average and are expressed in copy number/1000 cells or 12.5 ng total RNA (numbers on the top of each bar). The associated methylation profiles were determined by bisulfite sequencing PCR. Each sample result originates from the same conversion reaction by bisulfite. Each line represents an independent molecule. Methylated CpGs are schematized by black circles and unmethylated CpGs by white circles. Percentage values express the global methylation level in the U3 promoter region. MaLR[LTR] methylation percentages are shown in parentheses. First, there is a strong correlation between transcription and methylation. Thus, ERVWE1 and ERVFRED1 env transcription in placental BeWo cells was correlated with their 5′LTR fully unmethylated status. Similarly, faint transcription (below 2 copies/10 cell) was mostly associated with a high and homogeneous methylation of LTRs (e.g. ERVWE1[5′LTR] in U397 and ERVFRDE1[5′LTR] in OVCAR-3).\nSecond, comparison of ERVWE1 LTRs and HW_12[solo LTR] suggests that the absence of expression is highly correlated with a huge methylation for functional LTRs (ERVWE1), in contrast to the HW_12[solo LTR] which displays a significant amount of unmethylated U3 regions in 85HG66 and OVCAR-3 cells. This LTR, which exhibits extremely heterogeneous methylation status, is probably not functional; even if we cannot exclude that the lack of related transcription could be attributed to transcription factors paucity.\nThird, comparison of ERVFRDE1 and HW_4 in 85HG66 suggests different processes impairing the activity of these functional 5′LTRs. ERVFRDE1 lack of expression is surely due to dedicated transcriptional activators deficit rather than methylation as several molecules are unmethylated. In contrast, HW_4 lack of expression could be due to the systematic methylation of the CpGs at the 5′LTR U3/R boundary.\nThe fourth situation, concerning ERV3[5′LTR], is more complex. In U937 cells, the env region was very poorly expressed, although the 5′LTR had, so to say, no methylated CpGs. Yet, this permissive methylation is compatible with the induction of ERV3 env upon U937 stimulation with differentiating agents such as retinoic acid.65 In contrast, in 85HG66, BeWo and to a lesser extent in OVCAR-3, we observed a high expression level of the env region, but meanwhile a strong methylation level of ERV3[5′LTR]. Analysis of ERV3 env containing mRNA using the USCS genome browser (http://genome.ucsc.edu/cgi-bin/hgTracks, chr7:64088622–64104466) suggests the existence of an alternative initiation site in addition to the published initiation site in the ERV3[5′LTR].12 The mRNA AK295189, which has 99.8% identity with the genomic sequence, notably supports this. It encompasses the full-length ERV3 env region, but not the retroviral sequence ahead, and starts about 7240 bp upstream from the ERV3[5′LTR] transcription start site. It is thus conceivable that in the cell lines 85HG66, BeWo and possibly in OVCAR-3, the observed transcription is related to such an alternate mRNA form. Alternatively although uncommon, we cannot exclude that the ERV3 promoter contains methylation-dependent transcription factor binding sites, as recently described for Epstein–Barr virus.66\nERVWE1 is the only known HERV with a juxtaposed enhancer. This TSE is part of a MaLR LTR, and interestingly, all the MaLR CpG sites are located within the TSE. We were thus interested to know the extent to which methylation of the TSE could influence ERVWE1 transcriptional activity. Progressively 5′deleted MaLR[LTR]–ERVWE1[5′LTR] reporter constructs (simplified ‘TSE-U3’, ‘U3 full-length’ and ‘U3 minimal’ promoters, see Fig. 6A) were gradually methylated, transfected in BeWo cell and analyzed for luciferase activity (Fig. 6B). The TSE-U3 promoter activity was the maximal among the three promoter constructs in unmethylated state, but dropped dramatically when methylated. Thus, after 30 min of methylation (incomplete methylation), we observed a 5.4-fold decrease in comparison with only 3- and 1.8-fold for the U3 full-length and minimal promoters. Still, in this state, the TSE-U3 promoter activity remained the highest with 18.4% of the maximal activity, compared with only 10% for the TSE-deleted construct. Further increase in the methylation time to complete methylation reduced the activities to about the same level, i.e. 12.5% for TSE-U3 promoter, 8.5% for the full-length U3 promoter and 7.6% for the minimal U3 promoter. In line with the observed conjoint methylation status of the MaLR[LTR] and ERVWE1[5′LTR], these results underline the importance of the total absence of methylated CpGs on both LTRs to obtain maximal transcription and, conversely the need of a concomitant methylation of both LTRs for an efficient repression.\nFigure 6 Effect of CpG methylation on MaLR[LTR]–ERVWE1[5′LTR] promoter strength. (A). Schematic representation of TSE-U3, U3 full-length and U3 minimal promoter constructs. Numbering starts from the first position in ERVWE1 5′LTR. Effective and putative transcription factor binding sites associated with the MaLR[LTR] TSE region (Sp-1, c-myb, GCMA, GATA), with the ERVWE1[5′LTR] U3 5′-subdomain (GATA, Pit-1a, ERalpha, Sp-1, Ap-2 and Oct-1), and CAAT and TATA boxes are indicated by black boxes. CpGs are depicted by circles on vertical bars. (B). Relative promoter activities in BeWo cells following in vitro methylation. pGL-LTR firefly luciferase plasmids were methylated in vitro during 30, 60 and 240 min, with and without -SAM. On the basis of restriction profiles with BstUI enzyme -SAM controls were, as expected, not methylated, and methylation level did not evolve anymore after 60 min indicating a similar methylation at 60 and 240 min. Plasmid methylated during 30 and 240 min and -SAM controls were transfected in BeWo b30 choriocarcinoma cells, which contain all the necessary factors for MaLR[LTR]–ERVWE1[5′LTR] promoter activity. Firefly luciferase activities were normalized to the activity of the co-transfected pRL-TK Renilla luciferase plasmids. The mean and standard deviation from at least three independent experiments are shown and expressed as a percentage of the maximal activity. Promoter activity of the vector backbone represents \u003c0.3% of the larger TSE-U3 construct and \u003c1.3% of the U3 minimal promoter construct (not shown).\n\n4","divisions":[{"label":"Title","span":{"begin":6,"end":76}},{"label":"Figure caption","span":{"begin":477,"end":1376}},{"label":"Figure caption","span":{"begin":5595,"end":7154}}],"tracks":[{"project":"2_test","denotations":[{"id":"19561344-8707424-25963852","span":{"begin":3006,"end":3008},"obj":"8707424"},{"id":"19561344-2884330-25963853","span":{"begin":3439,"end":3441},"obj":"2884330"},{"id":"19561344-19325883-25963854","span":{"begin":4044,"end":4046},"obj":"19325883"}],"attributes":[{"subj":"19561344-8707424-25963852","pred":"source","obj":"2_test"},{"subj":"19561344-2884330-25963853","pred":"source","obj":"2_test"},{"subj":"19561344-19325883-25963854","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ece5","default":true}]}]}}