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DNA methylation and transcription in HERV (K, W, E) and LINE sequences remain unchanged upon foreign DNA insertions
Aim: DNA methylation and transcriptional profiles were determined in the regulatory sequences of the human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 elements and were compared between non-transgenomic and plasmid-transgenomic cells. Methods: DNA methylation profiles in the HERV (K, W, E) and LINE sequences were determined by bisulfite genomic sequencing. The transcription of these genome segments was assessed by quantitative real-time PCR. Results: In HERV-K, HERV-W and LINE-1.2 the levels of DNA methylation ranged between 75 and 98%, while in HERV-E they were around 60%. Nevertheless, the HERV and LINE-1.2 sequences were actively transcribed. No differences were found in comparisons of HERV and LINE1.2 CpG methylation and transcription patterns between non-transgenomic and plasmid-transgenomic HCT116 cells. Conclusion: The insertion of a 5.6 kbp plasmid into the HCT116 genome had no effect on the HERV and LINE-1.2 methylation and transcription profiles, although other parts of the HCT116 genome had shown marked changes. These repetitive sequences are transcribed, probably because the large number of HERV and LINE-1.2 elements harbor copies with non- or hypo-methylated long terminal repeat sequences.
Stefanie Weber1, Susan Jung2 & Walter Doerfler*,1,3 Institute of Clinical & Molecular Virology, University Erlangen-Nürnberg Medical School, 91054 Erlangen, Germany 2 Pediatric Research Center, University Erlangen-Nürnberg, 91052 Erlangen, Germany 3 Institute of Genetics, University of Cologne, 50674 Cologne, Germany *Author for correspondence: Tel.: +49 9131 852 6002 [email protected] 1
First draft submitted: 15 September 2015; Accepted for publication: 12 November 2015; Published online: 2 December 2015 Keywords: bisulfite genomic sequencing • comparisons of methylation and transcription between non-transgenomic and transgenomic cells • CpG methylation in HERV and LINE-1.2 DNA • human cell line HCT116 • human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 sequences • plasmid-transgenomic HCT116 cells • quantitative real-time PCR
Up to 8% of the human genome consists of endogenous retroviral sequences (HERVs) [1] . They are part of retroviral elements and retrotransposons comprising >30% of the human genome. Approximately 105 HERV elements represent incomplete ancient retrovirus-like genomes. There is no evidence that they can produce viral particles. HERVs are located at many sites in the human genome and in the genomes of some large DNA viruses [2] . Since their long terminal repeats (LTRs) contain potent regulatory elements they can modulate the activities of adjacent cellular genome segments [3] . Host cells have developed various defense mechanisms against the spread
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and activity of HERV or other foreign DNA sequences. Hypermethylation of HERV and LINE-1 genomes reflects the activity of these protective strategies [4] . The HERV-K sequences are among the most intact and transcriptionally active elements and are widely distributed in the human genome [5] . In human embryonic stem cells, HERV-H sequences are frequently transcribed into nuclear long noncoding nlncRNAs [6] . HERV-H transcriptional activity is thought to be a marker of pluripotency in human cells [7,8] . In human embryonic stem cells, HERVs are controlled by a number of factors including DNA methylation [9–11] .
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Short Communication Weber, Jung & Doerfler An inhibitor of DNA methylation, 5-aza-cytidine, can induce transcription of HERV-F sequences [12] . Altered transcription of the HERV-H, -K and -W elements can be associated with human diseases, like cancer, AIDS, autoimmune diseases and others [13] . HERVK transcription is markedly enhanced after infection with HIV-1 [14,15] or with human cytomegalovirus [16] . Genome-wide profiling of DNA methylation including HERV sequences has been reported for human cancer cells [17] . Epigenetic mechanisms, like DNA methylation, play an important role in the silencing of the HERV sequences. Nevertheless, at least some of the HERVs are still capable of being transcribed. In the human cell line HCT116, we have now analyzed the methylation profiles in the LTR’s and in some of the adjacent segments of the HERV-K (GenBank: AF074086.2), HERV-W (NCBI Reference Sequence: NM_014590.3), HERV-E (GenBank: AB062274.1) and the LINE-1.2 (GenBank: M80343.1) sequences by bisulfite sequencing. The analyzed genome segments are hyper-, but not completely, methylated. Transcripts of all these sequences have been identified in the total RNA of cell line HTC116. The aims of the present study are the following:
• Earlier work on adenovirus type 12 (Ad12)-transformed hamster cells with a single site of Ad12 DNA integration revealed extensive genomewide increases of CpG methylation in retroviral (intracisternal A particle) and also in unique gene sequences [18] . These observations raised the question of whether foreign DNA insertion at one genomic site might in general affect CpG methylation, preferentially in retroviral elements. The present investigation on human HERV and LINE elements was therefore designed to search for comparably wide-spread and substantial changes in DNA methylation and transcription patterns in the plasmid-transgenomic human cells; • Since HERVs and LINEs constitute major parts of the human genome, we have determined methylation and transcription patterns in parts of the HERV and LINE-1.2 sequences in human cell line HCT116. We also asked, how the high levels of DNA methylation could be reconciled with the transcriptional activities of these genome segments; • Our studies on the consequences of foreign DNA integration for the epigenetic stability of the
Non-transgenomic 1 Region 1: 771–1099 bp CpG 1
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Figure 1. DNA methylation levels of individual CpG dinucleotides in different parts of the HERV-K element were determined by the bisulfite sequencing method. In the methylation graphs, each square represents one CpG dinucleotide, in the unmethylated (white squares) or the methylated (black squares) state. Horizontally arranged symbols depict CpGs in one DNA molecule; vertically organized columns CpG pairs in different molecules which were analyzed as multimers for statistical significance. Percentage values for DNA CpG methylation were computed by counting the white and black squares (see Table 1). The methylation in the HERV-K elements from non-transgenomic control HCT116 clones (A) were compared with those from pC1–5.6 transgenomic cell clones ([B], see facing page). The generation of pC1–5.6-transgenomic clones [20] was described under methods. The analyzed regions 1, 2 and 3 and their locations in the HERV-K segment are shown in the map in ([C], see page overleaf). We chose this HERV-K locus for reproduction in (C), because it has been mapped most comprehensibly (GenBank accession number AF074086) [25] .
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Figure 1. DNA methylation levels of individual CpG dinucleotides in different parts of the HERV-K element were determined by the bisulfite sequencing method (cont.).
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Figure 1. DNA methylation levels of individual CpG dinucleotides in different parts of the HERV-K element were determined by the bisulfite sequencing method (cont.).
genome [18,19] have recently been extended to a comparison of genome-wide CpG methylation and transcription patterns between non-transgenomic and pC1–5.6 plasmid-transgenomic HCT116 cell clones [20] . We have demonstrated marked alterations of cellular DNA methylation and transcription profiles in bacterial plasmid pC1–5.6-transgenomic human HCT116 cells in comparison to non-transgenomic cells [20] . As an extension of this previously published analysis, we have now found these patterns in the HERV-K, HERV-W, HERV-E and the LINE1.2 sequences to be identical in the same non-transgenomic and plasmid-transgenomic HCT116 cell clones. There is no evidence for hypermethylation of these elements as a consequence of foreign DNA insertions. Methods Standard techniques
The methods for the propagation of cell line HCT116 (ATCC Cat. No. CCL-247), for the isolation of its DNA and the bisulfite sequencing method [21,22] were described earlier [20,23] . Plasmid pC1–5.6-transgenomic HCT116 cell clones
Their generation and analysis were detailed elsewhere [20] . This 5648 bp plasmid contained the kanamycin/neomycin resistance gene under early SV40 promoter control. It also carried the bacterial kanamycin promoter and the nontranslated first exon with parts of the 5’-UTR and the CGG repeat of the human fragile X mental retardation gene 1 (FMR1) [20] . Total length of the FMR1 fragment was 2298 bps. HCT116 cells were transfected with pC1–5.6 by using the nucleofection protocol (amaxa/ Lonza, Cologne, Germany). Kanamycin resistant cell clones were selected and continuously maintained in the presence of 150 μg of Geneticin (G418) per ml culture
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medium. Persistence and integrated state of the plasmid were ascertained by kanamycin resistance of the clones and by PCR using primers inside the plasmid sequence. Chromosomal integration of pC1–5.6 was assessed by positioning one PCR primer in Alu sequences of the neighboring cellular DNA and the second primer within the plasmid sequence [20] . Bisulfite genomic sequencing
lists all primers used for bisulfite sequencing and for gene expression analyses. The primers selected for this study theoretically recognize many, possibly the majority, of all the HERV and LINE elements analyzed here. All DNA sequence determinations were performed in the sequencing facility of the Institute of Human Genetics, Universitätsklinikum Erlangen. Quantitative real-time PCR was described under Supplementary Methods [24] . Supplementary Table 1
Results CpG methylation profiles
The results of bisulfite sequencing analyses in the LTR-1, gag and LTR-2/gag regions of the HERV-K sequences in HCT116 cell clones ( Figure 1A , B ; map in Figure 1C) demonstrate that the CpG’s in these genome segments in non-transgenomic HCT116 cells were methylated to 82, 68 and 71%, respectively (Figure 1A & Table 1) . For the pC1.5.6 plasmid-transgenomic cells, the corresponding values were 87, 70 and 75% (Figure 1B & Table 1) . Similar results were found for the HERV-W sequences with methylation levels close to 100% in the three analyzed regions both in the non-transgenomic and the pC1–5.6 transgenomic HCT116 clones (Supplementary Figure 1A, B, respectively, Table 1; map in Supplementary Figure 1C). In the HERV-E sequences, only the 5’-LTR region was bisulfite sequenced.
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Table 1. Percentage CpG methylation in different regions of the HERV-K, -W, -E and LINE-1.2 sequences. HERV and LINE segment analyzed for CpG methylation
Methylated (%)
HERV-K
Region 1: LTR1 771–1099 bp
Region 2: gag 1491–2044 bp
Region 3: LTR2/gag 9995–10548 bp
Non-transgenomic 1
81
76
66
Non-transgenomic 2
82
60
77
Mean
82
68
71
Transgenomic 1
100
74
76
Transgenomic 2
89
68
69
Transgenomic 3
68
71
83
Transgenomic 4
90
68
69
Transgenomic 5
87
68
75
Transgenomic 6
–
72
76
Mean
87 (p = 0.38)
70 (p = 1)
75 (p = 0.086)
HERV-W
Region 1: 16–442 bp
Region 2: 865–1252 bp
Region 3: 2121–2484 bp
Non-transgenomic 1
100
96
Non-transgenomic 2
95
100
98
Mean
95
100
97
Transgenomic 1
83
100
99
Transgenomic 2
72
–
97
Transgenomic 3
88
100
–
Transgenomic 4
79
–
99
Transgenomic 5
97
93
97
Transgenomic 6
89
100
–
Mean
85 (p = 0.57)
98 (p = 0.68)
98 (p = 0.47)
HERV-E
Region: LTR 138–544 bp
Non-transgenomic 1
52
Non-transgenomic 2
62
Non-transgenomic 3
59
Non-transgenomic 5
60
Non-transgenomic 6
62
Mean
59
Transgenomic 2
56
Transgenomic 3
58
Transgenomic 4
51
Transgenomic 5
50
Transgenomic 6
65
Percentage values were computed by counting white and black squares in Figure 1 & Supplementary Figures 1–3. There is no significant difference (see p-values between 0.38 and 1) in any of the DNA methylation profiles between pC1–5.6-transgenomic versus nontransgenomic cell clones. See maps in Figure 1 & Supplementary Figures 1–3.
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Table 1. Percentage CpG methylation in different regions of the HERV-K, -W, -E and LINE-1.2 sequences (cont.). HERV and LINE segment analyzed for CpG methylation
Methylated (%)
HERV-E (cont.)
Region: LTR 138–544 bp (cont.)
Transgenomic 7
51
Transgenomic 8
64
Mean
56 (p = 0.71)
LINE-1.2
Region: 5’-UTR 22–484 bp
Non-transgenomic 1
73
Non-transgenomic 2
84
Mean
78
Transgenomic 1
91
Transgenomic 2
78
Transgenomic 3
90
Transgenomic 4
66
Transgenomic 5
72
Transgenomic 6
94
Transgenomic 7
57
Transgenomic 8
75
Mean
78 (p = 1)
Percentage values were computed by counting white and black squares in Figure 1 & Supplementary Figures 1–3. There is no significant difference (see p-values between 0.38 and 1) in any of the DNA methylation profiles between pC1–5.6-transgenomic versus nontransgenomic cell clones. See maps in Figure 1 & Supplementary Figures 1–3.
Methylation levels were 59 and 56% in the nontransgenomic and transgenomic cell clones, respectively (Supplementary Figure 2A, B, Table 1; map in Supplementary Figure 2C). In the 5’-UTR of the LINE1.2 element, about 78% of CpG’s are methylated both in the non-transgenomic and the transgenomic cells (Supplementary Figure 3A, B, Table 1; map in Figure Supplementary Figure 3C). The p-values characterizing differences in the analyzed CpG methylation profiles in the HERV-K, HERV-W, HERV-E and LINE-1.2 sequences between non-transgenomic and plasmidtransgenomic clones, prove that there are no significant differences between the two cell types (Table 1) .
respectively. In HERV-K and HERV-W, the region 1 was analyzed for RNA transcription. Transcriptional activities were calculated relative to those of the cellular YWHAZ gene (NCBI Reference: NM_001135699.1). This gene is considered a suitable reference for normalization of gene expression [26] . The transcriptional activity of the GJC1 [17] (NCBI Reference Sequence: NM_005497.3) gene has been determined in Figure 2A. In conclusion, the analyzed HERV and LINE-1.2 segments are actively transcribed. There are no significant differences in transcription levels between non-transgenomic and transgenomic cells.
Transcription of HERV & LINE-1.2 sequences
Discussion & conclusion We conclude that CpG methylation levels in the analyzed segments of the HERV-K, HERV-W and LINE1.2 elements in cell line HCT116 range between 75 and 98%. In the analyzed region of HERV-E, methylation is lower at 56–59% only. The insertion of a 5.6 kbp plasmid transgenome into the genome of the studied cell clones has not detectably affected CpG methyla-
Although the HERV and LINE sequences are hypermethylated, they are actively transcribed in HCT116 cells as shown by reverse transcription of total RNA and quantitative real-time PCR amplification of the same HERV-K (Figure 2B), HERV-W (Figure 2C), LINE-1.2 (Figure 2D) and HERV- E (Figure 2E) regions studied in Figures 1 & Supplementary Figures 1–3,
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(Figure 1 & Supplementary Figure 1)
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tion (Figures 1 & Supplementary Figures 1–3) or transcription profiles (Figure 2) . There is no statistically significant difference in the DNA methylation profiles of transgenomic in comparison to non-transgenomic cell clones, as p-values range between 0.38 and 1 (Table 1) . In earlier work however, genome-wide transcription and methylation patterns in individual genes of the same pC1–5.6-transgenomic HCT116 cell clones have been found to be markedly altered in comparison to the non-transgenomic cells [20] . This study constitutes the positive control for the present investigation on the HERV and LINE sequences. Hence, the alterations in methylation and transcription patterns found in the previous study [20] did not extend into the HERV and LINE-1.2 segments studied here. Degree and location of epigenetic alterations might depend on the type and target location of the inserted foreign DNA plasmid. Of course, we cannot rule out the possibility of minor changes of DNA methylation in subpopulations of the HERV and LINE elements. However, the main reason for performing the HERV and LINE analyses was derived from our earlier observations on quite extensive changes in intracisternal A particle methylation patterns in an Ad12-transgenomic cell line which has been studied previously [18] . We pursue the notion that sites and extents of epigenetic effects upon foreign DNA integrations might be a function of the type of transgenome and its genomic location of insertion.
0.5 0.0
NonTransgenomic transgenomic HCT clones
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NonTransgenomic transgenomic HCT clones
In the present study, we had not intended to investigate epigenetic effects in the vicinity of the site of foreign DNA integration. This aspect was the subject of an earlier communication [27] . HERV and LINE-1.2 transcription is not completely silenced by LTR hypermethylation, possibly because some of the numerous integrated elements could have escaped de novo methylation in functionally decisive CpG dinucleotides, since CpG methylation is rarely 100% in these elements. The 5’-terminus of the 5’-LTRs in the HERV-E region has actually shown very sparse CpG methylation and might thus explain HERV-E transcriptional activity. The transcriptional activities in the non-transgenomic and the transgenomic cell clones showed no detectable differences. Future perspective We have continued to analyze possible alterations of DNA methylation and transcription patterns in mammalian cells in response to the insertion of foreign DNA into established genomes. In the same non-transgenomic and transgenomic cell clones, in which alterations of DNA methylation and transcription patterns had been documented in many parts of the genome in an earlier publication [20] , such differences were not observed in the HERV and LINE elements studied now. Location and level of changes in these patterns may depend on the site(s) of foreign DNA insertion(s). We plan to extend
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Figure 2. Comparisons of the transcriptional activities between non-transgenomic and transgenomic cell clones in the HERV and LINE segments. RNA transcription of GJC1 [17] (A), HERV-K (B), HERV-W (C), LINE-1.2 (D) and HERV-E (E) was quantified by real-time PCR and normalized to YWHAZ RNA expression [26] . Mean values and standard deviations of at least five clonal non-transgenomic and six transgenomic HCT cell lines were shown.
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Short Communication Weber, Jung & Doerfler our investigations and will study the role that insertion site and type of transgenome can play in interfering with the epigenetic stability of mammalian genomes. There is mounting evidence that epigenetic destabilizations of genomes are observed in many types of tumor cells. Hence, work on the regulation of epigenetic genome stability will be of profound future importance.
Author contributions
Supplementary data
Financial & competing interests disclosure
To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/ doi/full/10.2217/epi.15.109
This research was made possible by grants to W Doerfler from the Thyssen Foundation, Köln (Az. 10.07.2.138), from the Deutsche Forschungsgemeinschaft, Bonn (DO 165/281) and by support from the Rotary Club Weissenburg to S Weber. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Acknowledgements The authors are indebted to the Institute for Virology Erlangen University Medical School for their continued support of W Doerfler’s senior research group. The authors are grateful to the staff of the sequencing facility of the Institute of Human Genetics, Universitätsklinikum Erlangen for nucleotide sequence determinations.
S Weber performed all laboratory experiments, was involved in the planning of the project and interpretation of the data. S Jung planned and analyzed the real-time PCR data. W Doerfler initiated and designed the project, was involved in the interpretation of data and wrote the manuscript.
Executive summary • We have now extended and complemented a previously published study on genome-wide alterations of methylation and transcriptional patterns in human HCT116 cell clones. These cells were transgenomic for a 5.6 kbp bacterial plasmid which was most likely integrated at different genomic sites. The work described here was also prompted by the earlier observation of extensive increases in the methylation of intracellular A particle sequences (about 900 copies per cell) in Ad12-transgenomic hamster cells and the possibility that repetitive sequences might respond excessively to foreign DNA integration. • In 4.7% of the 28,869 gene segments analyzed, the transcriptional activities were upregulated (907 genes) or downregulated (436 genes) in plasmid-transgenomic cell clones in comparison to non-transgenomic control clones. Genome-wide methylation profiling was performed for >480,000 CpG sites. In comparisons of methylation levels in five transgenomic versus four non-transgenomic cell clones, 3791 CpGs were differentially methylated.
Results & discussion • As a corollary to our earlier study, it was of interest to investigate whether the alterations in transcriptional and methylation profiles in the same transgenomic HCT116 cell clones had affected also repetitive genome elements like the HERV and LINE-1.2 sequences. Such differences were not found. Apparently, in the cell clones selected for this investigation the HERV and LINE elements had not responded to foreign DNA insertions. • In addition, our present study provided a survey of the CpG modifications in the human endogenous viral sequences HERV-K, HERV-W, HERV-E and in LINE-1.2 whose methylation levels ranged between 60 and 98%. At least some of these elements were transcribed into RNA as determined by reverse transcription and PCR. Obviously, there are enough unmethylated control sequences to facilitate transcription of at least some of the tested elements into RNA.
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DNA methylation and transcription in HERV (K, W, E) and LINE sequences remain unchanged upon foreign DNA insertions
Aim: DNA methylation and transcriptional profiles were determined in the regulatory sequences of the human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 elements and were compared between non-transgenomic and plasmid-transgenomic cells. Methods: DNA methylation profiles in the HERV (K, W, E) and LINE sequences were determined by bisulfite genomic sequencing. The transcription of these genome segments was assessed by quantitative real-time PCR. Results: In HERV-K, HERV-W and LINE-1.2 the levels of DNA methylation ranged between 75 and 98%, while in HERV-E they were around 60%. Nevertheless, the HERV and LINE-1.2 sequences were actively transcribed. No differences were found in comparisons of HERV and LINE1.2 CpG methylation and transcription patterns between non-transgenomic and plasmid-transgenomic HCT116 cells. Conclusion: The insertion of a 5.6 kbp plasmid into the HCT116 genome had no effect on the HERV and LINE-1.2 methylation and transcription profiles, although other parts of the HCT116 genome had shown marked changes. These repetitive sequences are transcribed, probably because the large number of HERV and LINE-1.2 elements harbor copies with non- or hypo-methylated long terminal repeat sequences.
Stefanie Weber1, Susan Jung2 & Walter Doerfler*,1,3 Institute of Clinical & Molecular Virology, University Erlangen-Nürnberg Medical School, 91054 Erlangen, Germany 2 Pediatric Research Center, University Erlangen-Nürnberg, 91052 Erlangen, Germany 3 Institute of Genetics, University of Cologne, 50674 Cologne, Germany *Author for correspondence: Tel.: +49 9131 852 6002 [email protected] 1
First draft submitted: 15 September 2015; Accepted for publication: 12 November 2015; Published online: 2 December 2015 Keywords: bisulfite genomic sequencing • comparisons of methylation and transcription between non-transgenomic and transgenomic cells • CpG methylation in HERV and LINE-1.2 DNA • human cell line HCT116 • human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 sequences • plasmid-transgenomic HCT116 cells • quantitative real-time PCR
Up to 8% of the human genome consists of endogenous retroviral sequences (HERVs) [1] . They are part of retroviral elements and retrotransposons comprising >30% of the human genome. Approximately 105 HERV elements represent incomplete ancient retrovirus-like genomes. There is no evidence that they can produce viral particles. HERVs are located at many sites in the human genome and in the genomes of some large DNA viruses [2] . Since their long terminal repeats (LTRs) contain potent regulatory elements they can modulate the activities of adjacent cellular genome segments [3] . Host cells have developed various defense mechanisms against the spread
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and activity of HERV or other foreign DNA sequences. Hypermethylation of HERV and LINE-1 genomes reflects the activity of these protective strategies [4] . The HERV-K sequences are among the most intact and transcriptionally active elements and are widely distributed in the human genome [5] . In human embryonic stem cells, HERV-H sequences are frequently transcribed into nuclear long noncoding nlncRNAs [6] . HERV-H transcriptional activity is thought to be a marker of pluripotency in human cells [7,8] . In human embryonic stem cells, HERVs are controlled by a number of factors including DNA methylation [9–11] .
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Short Communication Weber, Jung & Doerfler An inhibitor of DNA methylation, 5-aza-cytidine, can induce transcription of HERV-F sequences [12] . Altered transcription of the HERV-H, -K and -W elements can be associated with human diseases, like cancer, AIDS, autoimmune diseases and others [13] . HERVK transcription is markedly enhanced after infection with HIV-1 [14,15] or with human cytomegalovirus [16] . Genome-wide profiling of DNA methylation including HERV sequences has been reported for human cancer cells [17] . Epigenetic mechanisms, like DNA methylation, play an important role in the silencing of the HERV sequences. Nevertheless, at least some of the HERVs are still capable of being transcribed. In the human cell line HCT116, we have now analyzed the methylation profiles in the LTR’s and in some of the adjacent segments of the HERV-K (GenBank: AF074086.2), HERV-W (NCBI Reference Sequence: NM_014590.3), HERV-E (GenBank: AB062274.1) and the LINE-1.2 (GenBank: M80343.1) sequences by bisulfite sequencing. The analyzed genome segments are hyper-, but not completely, methylated. Transcripts of all these sequences have been identified in the total RNA of cell line HTC116. The aims of the present study are the following:
• Earlier work on adenovirus type 12 (Ad12)-transformed hamster cells with a single site of Ad12 DNA integration revealed extensive genomewide increases of CpG methylation in retroviral (intracisternal A particle) and also in unique gene sequences [18] . These observations raised the question of whether foreign DNA insertion at one genomic site might in general affect CpG methylation, preferentially in retroviral elements. The present investigation on human HERV and LINE elements was therefore designed to search for comparably wide-spread and substantial changes in DNA methylation and transcription patterns in the plasmid-transgenomic human cells; • Since HERVs and LINEs constitute major parts of the human genome, we have determined methylation and transcription patterns in parts of the HERV and LINE-1.2 sequences in human cell line HCT116. We also asked, how the high levels of DNA methylation could be reconciled with the transcriptional activities of these genome segments; • Our studies on the consequences of foreign DNA integration for the epigenetic stability of the
Non-transgenomic 1 Region 1: 771–1099 bp CpG 1
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Figure 1. DNA methylation levels of individual CpG dinucleotides in different parts of the HERV-K element were determined by the bisulfite sequencing method. In the methylation graphs, each square represents one CpG dinucleotide, in the unmethylated (white squares) or the methylated (black squares) state. Horizontally arranged symbols depict CpGs in one DNA molecule; vertically organized columns CpG pairs in different molecules which were analyzed as multimers for statistical significance. Percentage values for DNA CpG methylation were computed by counting the white and black squares (see Table 1). The methylation in the HERV-K elements from non-transgenomic control HCT116 clones (A) were compared with those from pC1–5.6 transgenomic cell clones ([B], see facing page). The generation of pC1–5.6-transgenomic clones [20] was described under methods. The analyzed regions 1, 2 and 3 and their locations in the HERV-K segment are shown in the map in ([C], see page overleaf). We chose this HERV-K locus for reproduction in (C), because it has been mapped most comprehensibly (GenBank accession number AF074086) [25] .
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Figure 1. DNA methylation levels of individual CpG dinucleotides in different parts of the HERV-K element were determined by the bisulfite sequencing method (cont.).
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Figure 1. DNA methylation levels of individual CpG dinucleotides in different parts of the HERV-K element were determined by the bisulfite sequencing method (cont.).
genome [18,19] have recently been extended to a comparison of genome-wide CpG methylation and transcription patterns between non-transgenomic and pC1–5.6 plasmid-transgenomic HCT116 cell clones [20] . We have demonstrated marked alterations of cellular DNA methylation and transcription profiles in bacterial plasmid pC1–5.6-transgenomic human HCT116 cells in comparison to non-transgenomic cells [20] . As an extension of this previously published analysis, we have now found these patterns in the HERV-K, HERV-W, HERV-E and the LINE1.2 sequences to be identical in the same non-transgenomic and plasmid-transgenomic HCT116 cell clones. There is no evidence for hypermethylation of these elements as a consequence of foreign DNA insertions. Methods Standard techniques
The methods for the propagation of cell line HCT116 (ATCC Cat. No. CCL-247), for the isolation of its DNA and the bisulfite sequencing method [21,22] were described earlier [20,23] . Plasmid pC1–5.6-transgenomic HCT116 cell clones
Their generation and analysis were detailed elsewhere [20] . This 5648 bp plasmid contained the kanamycin/neomycin resistance gene under early SV40 promoter control. It also carried the bacterial kanamycin promoter and the nontranslated first exon with parts of the 5’-UTR and the CGG repeat of the human fragile X mental retardation gene 1 (FMR1) [20] . Total length of the FMR1 fragment was 2298 bps. HCT116 cells were transfected with pC1–5.6 by using the nucleofection protocol (amaxa/ Lonza, Cologne, Germany). Kanamycin resistant cell clones were selected and continuously maintained in the presence of 150 μg of Geneticin (G418) per ml culture
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medium. Persistence and integrated state of the plasmid were ascertained by kanamycin resistance of the clones and by PCR using primers inside the plasmid sequence. Chromosomal integration of pC1–5.6 was assessed by positioning one PCR primer in Alu sequences of the neighboring cellular DNA and the second primer within the plasmid sequence [20] . Bisulfite genomic sequencing
lists all primers used for bisulfite sequencing and for gene expression analyses. The primers selected for this study theoretically recognize many, possibly the majority, of all the HERV and LINE elements analyzed here. All DNA sequence determinations were performed in the sequencing facility of the Institute of Human Genetics, Universitätsklinikum Erlangen. Quantitative real-time PCR was described under Supplementary Methods [24] . Supplementary Table 1
Results CpG methylation profiles
The results of bisulfite sequencing analyses in the LTR-1, gag and LTR-2/gag regions of the HERV-K sequences in HCT116 cell clones ( Figure 1A , B ; map in Figure 1C) demonstrate that the CpG’s in these genome segments in non-transgenomic HCT116 cells were methylated to 82, 68 and 71%, respectively (Figure 1A & Table 1) . For the pC1.5.6 plasmid-transgenomic cells, the corresponding values were 87, 70 and 75% (Figure 1B & Table 1) . Similar results were found for the HERV-W sequences with methylation levels close to 100% in the three analyzed regions both in the non-transgenomic and the pC1–5.6 transgenomic HCT116 clones (Supplementary Figure 1A, B, respectively, Table 1; map in Supplementary Figure 1C). In the HERV-E sequences, only the 5’-LTR region was bisulfite sequenced.
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Table 1. Percentage CpG methylation in different regions of the HERV-K, -W, -E and LINE-1.2 sequences. HERV and LINE segment analyzed for CpG methylation
Methylated (%)
HERV-K
Region 1: LTR1 771–1099 bp
Region 2: gag 1491–2044 bp
Region 3: LTR2/gag 9995–10548 bp
Non-transgenomic 1
81
76
66
Non-transgenomic 2
82
60
77
Mean
82
68
71
Transgenomic 1
100
74
76
Transgenomic 2
89
68
69
Transgenomic 3
68
71
83
Transgenomic 4
90
68
69
Transgenomic 5
87
68
75
Transgenomic 6
–
72
76
Mean
87 (p = 0.38)
70 (p = 1)
75 (p = 0.086)
HERV-W
Region 1: 16–442 bp
Region 2: 865–1252 bp
Region 3: 2121–2484 bp
Non-transgenomic 1
100
96
Non-transgenomic 2
95
100
98
Mean
95
100
97
Transgenomic 1
83
100
99
Transgenomic 2
72
–
97
Transgenomic 3
88
100
–
Transgenomic 4
79
–
99
Transgenomic 5
97
93
97
Transgenomic 6
89
100
–
Mean
85 (p = 0.57)
98 (p = 0.68)
98 (p = 0.47)
HERV-E
Region: LTR 138–544 bp
Non-transgenomic 1
52
Non-transgenomic 2
62
Non-transgenomic 3
59
Non-transgenomic 5
60
Non-transgenomic 6
62
Mean
59
Transgenomic 2
56
Transgenomic 3
58
Transgenomic 4
51
Transgenomic 5
50
Transgenomic 6
65
Percentage values were computed by counting white and black squares in Figure 1 & Supplementary Figures 1–3. There is no significant difference (see p-values between 0.38 and 1) in any of the DNA methylation profiles between pC1–5.6-transgenomic versus nontransgenomic cell clones. See maps in Figure 1 & Supplementary Figures 1–3.
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Table 1. Percentage CpG methylation in different regions of the HERV-K, -W, -E and LINE-1.2 sequences (cont.). HERV and LINE segment analyzed for CpG methylation
Methylated (%)
HERV-E (cont.)
Region: LTR 138–544 bp (cont.)
Transgenomic 7
51
Transgenomic 8
64
Mean
56 (p = 0.71)
LINE-1.2
Region: 5’-UTR 22–484 bp
Non-transgenomic 1
73
Non-transgenomic 2
84
Mean
78
Transgenomic 1
91
Transgenomic 2
78
Transgenomic 3
90
Transgenomic 4
66
Transgenomic 5
72
Transgenomic 6
94
Transgenomic 7
57
Transgenomic 8
75
Mean
78 (p = 1)
Percentage values were computed by counting white and black squares in Figure 1 & Supplementary Figures 1–3. There is no significant difference (see p-values between 0.38 and 1) in any of the DNA methylation profiles between pC1–5.6-transgenomic versus nontransgenomic cell clones. See maps in Figure 1 & Supplementary Figures 1–3.
Methylation levels were 59 and 56% in the nontransgenomic and transgenomic cell clones, respectively (Supplementary Figure 2A, B, Table 1; map in Supplementary Figure 2C). In the 5’-UTR of the LINE1.2 element, about 78% of CpG’s are methylated both in the non-transgenomic and the transgenomic cells (Supplementary Figure 3A, B, Table 1; map in Figure Supplementary Figure 3C). The p-values characterizing differences in the analyzed CpG methylation profiles in the HERV-K, HERV-W, HERV-E and LINE-1.2 sequences between non-transgenomic and plasmidtransgenomic clones, prove that there are no significant differences between the two cell types (Table 1) .
respectively. In HERV-K and HERV-W, the region 1 was analyzed for RNA transcription. Transcriptional activities were calculated relative to those of the cellular YWHAZ gene (NCBI Reference: NM_001135699.1). This gene is considered a suitable reference for normalization of gene expression [26] . The transcriptional activity of the GJC1 [17] (NCBI Reference Sequence: NM_005497.3) gene has been determined in Figure 2A. In conclusion, the analyzed HERV and LINE-1.2 segments are actively transcribed. There are no significant differences in transcription levels between non-transgenomic and transgenomic cells.
Transcription of HERV & LINE-1.2 sequences
Discussion & conclusion We conclude that CpG methylation levels in the analyzed segments of the HERV-K, HERV-W and LINE1.2 elements in cell line HCT116 range between 75 and 98%. In the analyzed region of HERV-E, methylation is lower at 56–59% only. The insertion of a 5.6 kbp plasmid transgenome into the genome of the studied cell clones has not detectably affected CpG methyla-
Although the HERV and LINE sequences are hypermethylated, they are actively transcribed in HCT116 cells as shown by reverse transcription of total RNA and quantitative real-time PCR amplification of the same HERV-K (Figure 2B), HERV-W (Figure 2C), LINE-1.2 (Figure 2D) and HERV- E (Figure 2E) regions studied in Figures 1 & Supplementary Figures 1–3,
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(Figure 1 & Supplementary Figure 1)
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tion (Figures 1 & Supplementary Figures 1–3) or transcription profiles (Figure 2) . There is no statistically significant difference in the DNA methylation profiles of transgenomic in comparison to non-transgenomic cell clones, as p-values range between 0.38 and 1 (Table 1) . In earlier work however, genome-wide transcription and methylation patterns in individual genes of the same pC1–5.6-transgenomic HCT116 cell clones have been found to be markedly altered in comparison to the non-transgenomic cells [20] . This study constitutes the positive control for the present investigation on the HERV and LINE sequences. Hence, the alterations in methylation and transcription patterns found in the previous study [20] did not extend into the HERV and LINE-1.2 segments studied here. Degree and location of epigenetic alterations might depend on the type and target location of the inserted foreign DNA plasmid. Of course, we cannot rule out the possibility of minor changes of DNA methylation in subpopulations of the HERV and LINE elements. However, the main reason for performing the HERV and LINE analyses was derived from our earlier observations on quite extensive changes in intracisternal A particle methylation patterns in an Ad12-transgenomic cell line which has been studied previously [18] . We pursue the notion that sites and extents of epigenetic effects upon foreign DNA integrations might be a function of the type of transgenome and its genomic location of insertion.
0.5 0.0
NonTransgenomic transgenomic HCT clones
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NonTransgenomic transgenomic HCT clones
In the present study, we had not intended to investigate epigenetic effects in the vicinity of the site of foreign DNA integration. This aspect was the subject of an earlier communication [27] . HERV and LINE-1.2 transcription is not completely silenced by LTR hypermethylation, possibly because some of the numerous integrated elements could have escaped de novo methylation in functionally decisive CpG dinucleotides, since CpG methylation is rarely 100% in these elements. The 5’-terminus of the 5’-LTRs in the HERV-E region has actually shown very sparse CpG methylation and might thus explain HERV-E transcriptional activity. The transcriptional activities in the non-transgenomic and the transgenomic cell clones showed no detectable differences. Future perspective We have continued to analyze possible alterations of DNA methylation and transcription patterns in mammalian cells in response to the insertion of foreign DNA into established genomes. In the same non-transgenomic and transgenomic cell clones, in which alterations of DNA methylation and transcription patterns had been documented in many parts of the genome in an earlier publication [20] , such differences were not observed in the HERV and LINE elements studied now. Location and level of changes in these patterns may depend on the site(s) of foreign DNA insertion(s). We plan to extend
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Figure 2. Comparisons of the transcriptional activities between non-transgenomic and transgenomic cell clones in the HERV and LINE segments. RNA transcription of GJC1 [17] (A), HERV-K (B), HERV-W (C), LINE-1.2 (D) and HERV-E (E) was quantified by real-time PCR and normalized to YWHAZ RNA expression [26] . Mean values and standard deviations of at least five clonal non-transgenomic and six transgenomic HCT cell lines were shown.
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Short Communication Weber, Jung & Doerfler our investigations and will study the role that insertion site and type of transgenome can play in interfering with the epigenetic stability of mammalian genomes. There is mounting evidence that epigenetic destabilizations of genomes are observed in many types of tumor cells. Hence, work on the regulation of epigenetic genome stability will be of profound future importance.
Author contributions
Supplementary data
Financial & competing interests disclosure
To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/ doi/full/10.2217/epi.15.109
This research was made possible by grants to W Doerfler from the Thyssen Foundation, Köln (Az. 10.07.2.138), from the Deutsche Forschungsgemeinschaft, Bonn (DO 165/281) and by support from the Rotary Club Weissenburg to S Weber. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Acknowledgements The authors are indebted to the Institute for Virology Erlangen University Medical School for their continued support of W Doerfler’s senior research group. The authors are grateful to the staff of the sequencing facility of the Institute of Human Genetics, Universitätsklinikum Erlangen for nucleotide sequence determinations.
S Weber performed all laboratory experiments, was involved in the planning of the project and interpretation of the data. S Jung planned and analyzed the real-time PCR data. W Doerfler initiated and designed the project, was involved in the interpretation of data and wrote the manuscript.
Executive summary • We have now extended and complemented a previously published study on genome-wide alterations of methylation and transcriptional patterns in human HCT116 cell clones. These cells were transgenomic for a 5.6 kbp bacterial plasmid which was most likely integrated at different genomic sites. The work described here was also prompted by the earlier observation of extensive increases in the methylation of intracellular A particle sequences (about 900 copies per cell) in Ad12-transgenomic hamster cells and the possibility that repetitive sequences might respond excessively to foreign DNA integration. • In 4.7% of the 28,869 gene segments analyzed, the transcriptional activities were upregulated (907 genes) or downregulated (436 genes) in plasmid-transgenomic cell clones in comparison to non-transgenomic control clones. Genome-wide methylation profiling was performed for >480,000 CpG sites. In comparisons of methylation levels in five transgenomic versus four non-transgenomic cell clones, 3791 CpGs were differentially methylated.
Results & discussion • As a corollary to our earlier study, it was of interest to investigate whether the alterations in transcriptional and methylation profiles in the same transgenomic HCT116 cell clones had affected also repetitive genome elements like the HERV and LINE-1.2 sequences. Such differences were not found. Apparently, in the cell clones selected for this investigation the HERV and LINE elements had not responded to foreign DNA insertions. • In addition, our present study provided a survey of the CpG modifications in the human endogenous viral sequences HERV-K, HERV-W, HERV-E and in LINE-1.2 whose methylation levels ranged between 60 and 98%. At least some of these elements were transcribed into RNA as determined by reverse transcription and PCR. Obviously, there are enough unmethylated control sequences to facilitate transcription of at least some of the tested elements into RNA.
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