JOURNAL OF VIROLOGY, Jan. 2005, p. 876–883 Vol. 79, No.

2
0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.2.876–883.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

CpG Methylation Directly Regulates Transcriptional Activity of the

Human Endogenous Retrovirus Family HERV-K(HML-2)
Laurence Lavie,† Milena Kitova,† Esther Maldener, Eckart Meese, and Jens Mayer*
Department of Human Genetics, University of Saarland, Hamburg, Germany
Received 23 June 2004/Accepted 12 August 2004

A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human
endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional
because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-
K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) tran-
scription remains poorly understood. We investigated in detail the role of CpG methylation on the transcrip-
tional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-
K(HML-2) proviral 5ⴕ LTRs are methylated at different levels in the cell line Tera-1. Methylation levels
correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of
HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5ⴕ LTR
reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5ⴕ LTR activity,
as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in
evolutionary old HERV-K(HML-2) 5ⴕ LTRs suggests that CpG methylation had already silenced HERV-
K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG meth-
ylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.

About 8% of the human genome is composed of sequences
with retroviral origins. The human endogenous retroviruses
(HERVs) stem from germ line infections of ancient exogenous
retroviruses. A large number of proviral elements and in par-
ticular the solitary long terminal repeats (LTRs) are present in
the human genome. Despite the long-time presence of HERV
LTRs in the human genome, several studies demonstrated
transcriptional activity of both proviral and solitary HERV
LTRs. Some HERV elements are involved in the transcription
of cellular genes (for instance, see references 10, 22, 26, and
31).
The HERV-K(HML-2) family displays a number of remark-
GCT, the regulation of the transcriptional activity of HERV-
K(HML-2), including cellular factors influencing that activity,
has hitherto been vaguely understood. An early study noted
upregulation of HERV-K(HML-2) expression in T47D cells
after treatment of cells with estradiol and progesterone (34). A
complex of at least three otherwise-unidentified proteins was
reported to bind to the 5⬘ portion of the LTR U3 region (2).
Furthermore, the transcription factor YY1 and other uniden-
tified proteins were shown to bind to an enhancer region within
the proviral LTR U3 region. However, the ubiquitous tran-
scription factor YY1 is not responsible for different activities in
cell lines expressing or not expressing HERV-K(HML-2) (25).

Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.

able features. First, despite their long-time presence in the
human genome, a number of proviruses still display open read-
ing frames for retroviral Gag, Prt, Pol, and/or Env proteins (3,
4, 29, 30, 40). Second, transcriptional activity of the HERV-
K(HML-2) family is strongly upregulated in human germ cell
tumors (GCTs) and GCT-derived cell lines, as opposed to
transcriptional silencing in non-GCT cell lines. Third, GCT
patients, particularly seminoma patients, display high antibody
titers against HERV-K(HML-2) Gag and Env proteins (6, 17,
37, 38). Fourth, HERV-K(HML-2) encodes the so-called Rec
protein, an additional splicing product from the proviral env
gene that associates with the promyelocytic zinc finger protein
and that may be involved in the development of GCT (5, 27,
42). Therefore, HERV-K(HML-2) can be considered a tumor
marker for GCT and is potentially involved in GCT develop-
ment.
Despite the possible involvement of HERV-K(HML-2) in
* Corresponding author. Mailing address: Department of Human
Genetics, Building 60, University of Saarland, Medical Faculty, 66421
Hamburg, Germany. Phone: 49 6841 1626627. Fax: 49 6841 1626186.
E-mail: jens.mayer@uniklinik-saarland.de.
† L.L. and M.K. made equal contributions.
Methylation of cytosines in CpG dinucleotides can have
profound impacts on gene expression. About 80% of the CpG
dinucleotides in the human genome are methylated, and meth-
ylation has been implicated, among other functions, in silenc-
ing of repetitive sequences (9, 21, 41). Methylation can repress
transcription when directly interfering with binding of se-
quence-specific transcription factors (20) or by an indirect
mechanism involving the methyl-CpG binding domain proteins
(16).
Transposable elements in genomic DNA are usually meth-
ylated, and cytosine methylation has been suggested to act as a
defense mechanism against such “intragenomic parasites”
(43). For example, the role of methylation in repressing tran-
scription of mouse intracisternal A-type particles is well doc-
umented (18, 19, 41). There is evidence that transcription of
HERVs is likewise influenced by methylation. For instance,
transcription of HERV sequences in systemic lupus erythem-
atosus may be due to methylation defects (33). Abrink et al. (1)
related upregulation of H-plk, a human Kruppel-related zinc
finger gene that is probably activated by an upstream ERV3
locus to tissue-specific demethylation of the corresponding
gene region in several human cell lines.

876
VOL. 79, 2005 CpG REGULATION OF HERV-K(HML-2) LTRs 877

TABLE 1. HERV-K(HML-2) proviral 5⬘ LTRs investigated in this studya

LTR Locus Chromosomal GenBank Transcripts
Location in April 2003 Freeze Or
designation no. localization accession no. in Tera-1

LTR-c5 2 5p13.3 chr5:30485437-30496686 AC025757.3 ⫺ ⫺

LTR-c6 3 6q14.1 chr6:78375915-78387115 AL590785.7 ⫺ ⫹
LTR-c7L 4 7p22.1 chr7:4329521-4354837 AC072054.10 ⫺ ⫹
LTR-c7C 5 7p22.1 chr7:4329521-4354837 AC072054.10 ⫺ ⫹
LTR-c10 7 10p14 chr10:7015479-7026716 AL392086.14 ⫺ ⫺
LTR-c11 8 11q22.1 chr11:101598996-101610238 AP000776.4 ⫹ ⫹
LTR-c12 9 12q14.1 chr12:58437090-58448318 AC025420.26 ⫹ ⫺
a
LTR designation, different construct names; locus numbers, provirus numbers with reference to Mayer et al. (28). The provirus location in the April 2003 Freeze
version of the Human Genome Browser (23), the GenBank accession number harboring a particular provirus, and the orientation (Or) within the GenBank entry (⫹,
⫺) are given. The column “Transcripts in Tera-1” indicates the transcriptional activity of proviral loci as previously determined (28).

Cellular methylation was also reported to influence HERV-
K(HML-2) transcription. Treatment of the HERV-K(HML-
2)-expressing cell line Tera-1 with 5-azacytidine, a demethyl-
ating agent, further increased expression of HERV-K(HML-2)
Gag protein (14). Sequence regions flanking human-specific
HERV-K(HML-2) LTRs were reported to be differently meth-
ylated in cerebellum versus lymph node tissue (24). Hypom-
ethylation was also implicated to influence HERV-K(HML-2)
expression in urothelial cancer (11). Thus, methylation levels
seem to regulate transcription of HERV-K(HML-2) provi-
ruses. However, an indirect effect of methylation is conceivable
where general demethylation actually altered expression of
cellular activators or repressors involved in the transcriptional
regulation of these HERV elements.
We recently investigated HERV-K(HML-2) proviruses in
the human genome capable of producing Rec protein, which is
translated from a splicing product from the HERV-K(HML-2)
env gene. We identified four HERV-K(HML-2) proviruses
able to produce Rec mRNA, demonstrating transcriptional
activity of the respective promoters housed in the proviral 5⬘
LTRs. Rec transcripts from other proviral loci with potential to
produce Rec mRNA were not identified. The latter proviruses
therefore may be transcriptionally much less active, or they
(Roche) was used following the recommended conditions. PCR cycles were as
follows: 2 min at 94°C; 10 cycles, each cycle consisting 15 s at 94°C, 30 s at 55°C,
and 40 s at 72°C; 25 cycles, each cycle consisting of 15 s at 94°C, 30 s at 55°C, and
40 s at 72°C (plus a 20-s ramp per cycle); and finally 7 min at 72°C. After gel
electrophoresis, PCR products were eluted with a gel extraction kit (PeqLab).
The second round of PCR contained 1 ␮l of the DNA eluate. PCR conditions
were identical except for primer annealing at 60°C and the use of Taq polymerase
(Invitrogen). The various PCR primer sequences are available from the authors
on request. PCR products were subsequently cloned into the pGEM-T vector
(Promega), and single clones were sequenced with the SequiTherm Excel TM II
DNA Sequencing Kit-LC (Biozym) and an automated DNA sequencer (Licor
4000-L).
Plasmid generation. The reporter vectors pGL3-Basic and pCMV␤ were pur-
chased from Promega and Clontech, respectively. In construct pGL3-CMV,
luciferase expression is driven by the strong cytomegalovirus (CMV) early pro-
moter. To generate pGL3-CMV, we excised the CMV promoter region from
pCMV␤ by a EcoRI/SmaI restriction digestion. The blunted fragment was
cloned into the SmaI site of pGL3-Basic. Plasmids pLTR-c6, pLTR-c10, and
pLTR-c11 harbor HERV-K(HML-2) 5⬘ LTRs from different proviruses (Table
1) upstream from the luciferase reporter gene. We amplified respective 5⬘ LTRs
from human genomic DNA with a specific PCR primer located in an upstream
flanking cellular sequence and a universal reverse primer in the proviral gag gene.
Specific PCR primers were located between 32 and 85 bp upstream from the 5⬘
LTR. Due to a large amount of repetitive elements upstream from some of the
LTRs, a nested PCR approach was employed to generate constructs pLTR-c5,
pLTR-c7L, and pLTR-7C. Primer sequences included a HindIII site that was

Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.

may be inactive (28). In the present study, we investigated the
transcriptional potential of various proviral 5⬘ LTRs in more
detail, and examined the specific influence of CpG methylation
on those LTRs in the Tera-1 and the T47D cell lines. We find
that CpG methylation immediately affects HERV-K(HML-2)
transcriptional activity and that CpG methylation effectively
silences HERV-K(HML-2) LTRs. Yet, CpG methylation is
not entirely responsible for different transcriptional activities
observed between cell lines, e.g., CpG methylation does not
silence HERV-K(HML-2) expression in the T47D cell line.
MATERIALS AND METHODS
Bisulfite treatment of DNA. Genomic DNAs from Tera-1 cells and from T47D
cells were isolated by standard methods. To determine CpG methylation levels,
we employed the bisulfite sequencing method essentially as described by Fraga
and Esteller (12). To demonstrate the efficiency of bisulfite treatment, we added
10 ng of a BamHI-digested pBlueScript plasmid (Stratagene) to the digested
genomic DNAs before treatment. Plasmid and genomic DNAs were treated in
the same tube. Primers PBSME-1 and PBSME-2 were used to amplify a 134-bp
fragment of the vector on bisulfite-treated DNA as described by Goubely et al.
(15).
PCR amplification, cloning, and sequencing. We amplified, in total, five LTR
loci from bisulfite-treated DNA by nested PCR. The first round of PCR con-
tained 4 ␮l of treated DNA (⬃100 ng). The Expand High Fidelity PCR system
employed to clone PCR products into the pGL3-Basic vector previously linear-
ized with HindIII. The Expand High Fidelity PCR system (Roche) was used for
all amplifications. PCR primer sequences and PCR conditions are available from
the authors on request.
Cell lines and cell culture. Human teratocarcinoma cells (Tera-1) were cul-
tured in McCoy’s 5A medium (PAA). The human breast carcinoma cell line
T47D was cultured in RPMI 1640 medium (PAA). Cell culture media were
supplemented with L-glutamine, 10% fetal calf serum, and penicillin-streptomy-
cin (each at a concentration of 100 ␮g/ml).
Transfections and reporter gene assays. Cells were grown in 12-well plates to
60 to 80% confluency. We used FuGENE6 (Roche) for transfections, following
the manufacturer’s recommendations. Luciferase and ␤-galactosidase activities
were determined using the Luciferase and ␤-Galactosidase Assay Systems (Pro-
mega), following the manufacturer’s recommendations. Forty-eight hours after
transfection, cells were washed twice with 1⫻ phosphate-buffered saline and
were lysed in 1⫻ reporter lysis buffer, followed by two freeze-thaw cycles. Lu-
ciferase activities were measured with a Lumat LB 9507 luminometer (Berthold
Technologies). All reporter gene assays were performed at least in triplicate.
In vitro methylation. Two micrograms of reporter plasmid DNA were meth-
ylated with SssI methylase (CpG) (New England Biolabs) under recommended
conditions. Reaction mixtures contained 160 ␮M S-adenolsylmethionine (SAM)
and were incubated from 30 min to 4 h, followed by heat inactivation for 15 min
at 65°C. Negative-control reaction mixtures lacked SAM. To determine methyl-
ation levels, a 0.5-␮g aliquot from each reaction mixture was treated with the
methylation-sensitive restriction enzyme BstUI, and the resulting DNA frag-
ments were separated by gel electrophoresis and were visualized by ethidium
bromide staining.
878 LAVIE ET AL.
FIG. 1. Transcriptional activities of various HERV-K(HML-2) 5⬘
LTRs in Tera-1 (left) and T47D (right) cells, as determined by lucif-
erase reporter gene assays. LTR activities are given as percentages of
a positive control vector driven by a CMV promoter. Standard devia-
tions are indicated for each LTR. basic, a negative control vector that
lacked a promoter sequence. All tested LTRs were inactive in T47D
cells, shown on the right, solely to illustrate this finding.
RESULTS AND DISCUSSION
Promoter activity of HERV-K(HML-2) LTRs. We recently
investigated the HERV-K(HML-2) proviruses in the human
genome capable of producing the Rec mRNA splicing product,
and we investigated transcriptional activity of those proviruses.
We determined HERV-K(HML-2) proviral loci on human
chromosomes 6, 7, and 11 to be transcriptionally active in the
Tera-1 cell line. Transcripts from proviral loci on human chro-
mosomes 5, 10, and 12 were not among cloned and sequenced
reverse transcription-PCR (RT-PCR) products (Table 1) (28).
Thus, the latter are likely significantly underrepresented in, or
missing from, the Tera-1 cellular mRNA pool.
We asked whether different representation of proviral se-
quences in the cellular mRNA pool was due to different pro-
moter strength of the respective proviral 5⬘ LTRs. We PCR
amplified the complete 5⬘ LTR sequence, including short
stretches of upstream and downstream flanking sequence from
seven HERV-K(HML-2) proviral loci (Table 1). We cloned
the different LTRs harboring amplicons into a luciferase re-
porter vector and assayed their transcriptional activity in the
Tera-1 cell line. In the following discussion, we designate the
proviral 5⬘ LTR on human chromosome 5 as LTR-c5, for
instance. Because the HERV-K(HML-2.HOM) provirus, lo-
cated on human chromosome 7p22, is organized as a tandem
provirus sharing a central LTR between both proviruses (35),
we assayed the 5⬘ LTR as well as the central LTR of the
tandem arrangement (designated LTR-c7L and LTR-c7C).
When assaying the different 5⬘ LTRs in the Tera-1 cell line,
we found that all but one 5⬘ LTRs were transcriptionally active,
with activities ranging from 18.34 to 4.25% relative to the
activity of a luciferase control vector for which expression was
driven by a strong CMV promoter. LTR-c11 displayed the
highest activity and LTR-c6 the lowest activity. Only LTR-c10
activity was in the range of an empty reporter vector control.
LTR-c10 was therefore concluded to be transcriptionally inac-
tive (Fig. 1). Probably, the latter LTR is inactive because it is
J. VIROL.
TABLE 2. Number of C nucleotides and CpG sites analyzed in
various HERV-K(HML-2) 5⬘ LTR sequences employing bisulfite
genomic sequencinga
Total
Total C Total C Total CpG
Provirus CpG
in LTR analyzed in LTR
analyzed
LTR-c7L 240 106 19 9
LTR-c6 235 101 17 7
LTR-c11 234 104 17 9
LTR-c5 230 100 11 5
LTR-c12 227 95 15 7
a
Total numbers of C and CpG and respective sites analyzed within the various
proviral 5⬘ LTRs are listed. The proviruses are detailed in Table 1.
present in the genome for a longer time and has therefore
acquired more mutations than active LTRs (see below). Taken
together, the different representation of proviral transcripts in
the Tera-1 cellular mRNA pool is not due to strongly different
proviral 5⬘ LTR promoter activities. Other factors influencing
HERV-K(HML-2) transcriptional activity must be considered
to explain locus-specific transcription.
Differential CpG methylation of LTR sequences in Tera-1.
Previous data showed that hypomethylation of genomic DNA
boosted the overall HERV-K(HML-2) expression level (14).
However, the expression levels could have resulted from an
indirect effect. General demethylation might simply increase
expression of one or more cellular factors that then activate
HERV-K(HML-2) expression. We set out to elucidate
whether CpG methylation directly influences HERV-
K(HML-2) transcription.
In a first set of experiments, we investigated in detail the in
vivo methylation status of five out of seven HERV-K(HML-2)
proviral 5⬘ LTRs included in this study in the Tera-1 cell line,
employing the bisulfite genomic sequencing method (13). We
excluded LTR-c10 from the analysis, as it was shown to be
transcriptionally inactive (see above). We further excluded
Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.
LTR-c7C, because we did not succeed in obtaining specific
PCR products for that LTR. The lack of specific PCR product
was probably due to limitations of PCR product length in
bisulfite genomic sequencing that employs significantly modi-
fied DNA as template.
We amplified each 5⬘ LTR from bisulfite-treated genomic
Tera-1 DNA with specific PCR primers in the 5⬘ LTR up-
stream flanking region and a reverse primer located down-
stream from the 5⬘ LTR. The first PCR was followed by a
seminested PCR, employing forward primers in the flanking
region and a reverse primer in the 5⬘ LTR. By doing so, we
were able to examine the first 493 bp of the 567-bp U3 region
for each 5⬘ LTR.
We sequenced seven independent clones for each of the five
analyzed 5⬘ LTRs, analyzing between five and nine CpG sites
each (Table 2). We found significant methylation levels for
three 5⬘ LTRs. A total of 20 of 35 CpG sites were methylated
for LTR-c5, 23 of 63 sites were methylated for LTR-c11, and
35 of 49 CpG sites were methylated for LTR-c12. The overall
methylation level of LTR-c11 (36%) appeared lower than that
of LTR-c5 (57%) or that of LTR-c12 (71%). Several CpG sites
in incompletely methylated LTRs were found to be either
methylated or unmethylated. Bisulfite treatment efficiencies of
VOL. 79, 2005 CpG REGULATION OF HERV-K(HML-2) LTRs 879

FIG. 2. CpG site methylation status of U3 regions of five HERV-K(HML-2) proviral 5⬘ LTRs in Tera-1 cells, as determined by bisulfite
sequencing. The upper part of the figure depicts the examined LTR U3 region, including PCR primer locations for amplification of LTRs from
the human genome. Primers FP2 were specific for flanking cellular sequences, while primer RP2 was specific for the LTR sequence. The different
proviral 5⬘ LTRs examined are indicated on the left. Methylated CpG sites are indicated by solid circles, and unmethylated CpG sites are indicated
by open circles. Thus, LTR-c7L and LTR-c6 are unmethylated, while the others are partially methylated. Note that methylation patterns for the
latter LTRs are variable. Methylation levels for CpG sites as well as other C nucleotides are summarized as percentages on the right side of the
figure. Positive and negative RT-PCR results indicate transcriptional activity of a particular provirus in Tera-1 cells, and fractional amounts of
RT-PCR products from respective proviruses, as determined previously (28), are given in parentheses.

99%, as determined from the internal control, refute the hy- LTR-c11, could explain lower representation of corresponding
pothesis that incomplete methylation seen for these LTRs is transcript in the Tera-1 cell line.
due to an experimental artifact. In contrast to the results with Taken together, these findings strongly support the idea that

Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.

the LTRs discussed above, we found that LTR-c7L and
LTR-c6 were completely unmethylated in Tera-1 (Fig. 2).
Thus, methylation levels of the different examined HERV-
K(HML-2) LTRs vary significantly in the Tera-1 cell line.
Some LTRs appear significantly methylated, while others are
completely unmethylated. When LTR methylation levels are
compared to previously observed transcriptional activities (28),
there is a good inverse correlation between methylation and
transcriptional activity. The previously observed lack of
HERV-K(HML-2) transcripts from proviruses on human
chromosomes 5 and 12 in Tera-1 cells (28) could be explained
by methylation-mediated 5⬘ LTR promoter silencing, as
LTR-c5 and LTR-c12 were found to be methylated. In con-
trast, the proviruses on chromosomes 6 and 7 could be tran-
scriptionally active in Tera-1 cells because of the observed
complete lack of methylation for the corresponding LTR-c6
and LTR-c7. For LTR-c11, only 1 RT-PCR clone from the
corresponding proviral locus was isolated, in contrast to 15
clones from the corresponding LTR-c7L provirus (28). Al-
though LTR-c11 is about 60% transcriptionally more active
than LTR-c7L (Fig. 1), the Tera-1 RNA pool contains signif-
icantly less transcript from LTR-c11 than from LTR-c7L. In-
complete methylation of LTR-c11, and thus partial silencing of
CpG methylation of HERV-K(HML-2) LTRs directly regu-
lates transcription of HERV-K(HML-2) proviruses in the
Tera-1 cell line.
Influence of methylation on HERV-K(HLM-2) LTRs. To
further substantiate the direct influence of CpG methylation
on the transcriptional activity of HERV-K(HML-2) 5⬘ LTRs,
we examined promoter activities of in vitro-methylated LTRs.
We methylated CpG sites in luciferase reporter constructs
harboring LTR-c5, LTR-c11, and LTR-c12, employing the
CpG-specific SssI methylase. Methylation levels of plasmid
DNAs were assessed by BstUI restriction enzyme digestions,
with BstUI sensitive towards methylated CpG within the rec-
ognition sequence. We observed increasing methylation levels
for SssI incubation times ranging from 30 min to 4 h. Plasmid
DNAs were fully methylated after 4 h of incubation (Fig. 3A).
We assayed transcriptional activities of the different in vitro-
methylated LTR reporter constructs in Tera-1 cells. We ob-
served that transcriptional activity negatively correlated with
methylation levels. Increasing methylation levels continuously
decreased transcriptional activity of the examined 5⬘ LTRs.
Fully methylated constructs displayed only about 5.5% activity,
compared to unmethylated constructs (Fig. 3B). Hence, meth-
ylated CpG sites in HERV-K(HML-2) 5⬘ LTRs strongly inhibit
880 LAVIE ET AL.
FIG. 3. Transcriptional activity of in vitro-methylated HERV-
K(HML-2) 5⬘ LTRs in Tera-1 cells. (A) Different methylation levels of
in vitro-methylated reporter constructs. Shown here are in vitro meth-
ylation reactions of reporter plasmids that were terminated after 60
and 240 min. Plasmid DNAs were subsequently treated with methyl-
ation-sensitive BstUI restriction enzyme, and the fragments were elec-
trophoresed. Note that BstUI partially digests plasmid DNA incubated
for 60 min while plasmid incubated for 240 min is completely pro-
tected, indicating incomplete and complete methylation levels, respec-
tively.-SAM, control reaction mixtures incubated for similar time pe-
riods but lacking SAM substrate. Thus, BstUI is able to completely
digest these plasmid DNAs. (B) Effect of different CpG methylation
levels on transcriptional activity of HERV-K(HML-2) 5⬘ LTRs. Three
different LTRs with transcriptional activity were examined. Incubation
times for in vitro methylation reactions are given in minutes. Tran-
scriptional activities are given as percentages of a CMV-driven positive
control vector. basic, a promoter-lacking negative control vector.
transcriptional activity of HERV-K(HML-2) proviral loci.
These findings further corroborate that CpG methylation plays
an important and direct role in the transcriptional regulation
of the HERV-K(HML-2) family. Furthermore, partial tran-
scriptional silencing of the LTR-c11 due to incomplete meth-
ylation levels in the Tera-1 cell line (see above) is corrobo-
rated. Partially in vitro-methylated LTRs display trans-
criptional activity, yet clearly lower activities than unmeth-
ylated LTRs. At this point, it remains to be elucidated
whether methylated CpG dinucleotides inhibit binding of
one or several transcription factors or whether methylated
CpGs recruit methyl-CpG binding proteins.
J. VIROL.
Evidence for CpG methylation in old HERV-K(HML-2)
LTRs. Zsiros et al. (44) noted a significantly lower frequency of
CpG dinucleotides in the reverse transcriptase portion of
HERV-K(HML-2) proviruses. This finding suggested previous
methylation of CpG sites, eventually resulting in fading of
CpGs because of higher mutation rates of methylated cytosines
(8).
We likewise found evidence for previous CpG methylation
in HERV-K(HML-2) 5⬘ LTR sequences. Notably, the tran-
scriptionally inactive LTR-c10 displays several mutated CpG
sites compared to transcriptionally active LTRs (Fig. 4). Ob-
viously, that LTR accumulated more mutations and therefore
is probably evolutionarily older than the other LTRs. A higher
evolutionary age of LTR-c10 is also indicated from phyloge-
netic analysis (data not shown). In the light of several mutated
CpG sites and higher mutation rates of methylated cytosines, it
can be concluded that LTR-c10 used to be methylated. Further
evidence comes from evolutionarily older HERV-K(HML-2)
proviruses. The so-called HERV-K(OLD) proviruses are an-
cestral, yet very similar in sequence, to the “modern” HERV-
K(HML-2) proviruses. HERV-K(OLD) proviruses were
formed in germ cells approximately 28 million years ago (36).
We compared 5⬘ LTR sequences in modern HERV-
K(HML-2) proviruses with 5⬘ LTR sequences in HERV-
K(OLD) proviruses that we previously identified in the human
genome (unpublished data). Similar to the LTR-c10 sequence,
HERV-K(OLD) 5⬘ LTRs commonly displayed mutated CpG
dinucleotides. In particular, (former) CpG sites in the 5⬘ por-
tion of the LTRs appeared mutated (Fig. 4). We found that 94
of 129 CpG sites (73%) mutated to either TG or CA, which are
expected mutations when methylated cytosine is converted to
thymine. Thus, there is strong evidence that HERV-K(OLD)
proviruses used to be methylated. In combination with above-
described silencing of HERV-K(HML-2) LTRs by CpG meth-
ylation, we conclude that CpG methylation, at that point in
evolution, was employed to silence HERV-K(OLD) proviruses
after their integration into the germ line 28 million years ago,
Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.
just as CpG methylation at present represses the expression of
modern HERV-K(HML-2) proviruses. During evolution, CpG
methylation appears to have been a universal mechanism to
silence HERV-K(HML-2) proviral loci.
HERV-K(HML-2) inactivity in T47D cells is not due to CpG
methylation. In the course of the study of HERV-K(HML-2)
Rec mRNA expression in the Tera-1 cell line (28), Rec mRNA
expression in the breast cancer cell line T47D was also exam-
ined. However, no RT-PCR products were obtained from
T47D cells under experimental conditions identical to the one
for the Tera-1 cell line (J. Mayer and M. Sauter, unpublished
results). Hence, transcription levels of Rec-producing proviral
loci are significantly lower in T47D cells than in Tera-1 cells, or
HERV-K(HML-2) proviruses are transcriptionally inactive in
T47D. We examined in more detail the observed cell type
specificity. We assayed the promoter activity of the seven 5⬘
LTR luciferase reporter constructs in the T47D cell line. In
contrast to the Tera-1 cell line, none of the different reporter
constructs displayed promoter activities in T47D cells (Fig. 1).
We furthermore determined methylation levels for three
HERV-K(HML-2) 5⬘ LTRs (LTR-c6, LTR-c7L, and LTR-
c11) in T47D cells. We observed methylation levels of 71 and
44% for LTR-c6 and LTR-c11, respectively. The overall meth-
VOL. 79, 2005 CpG REGULATION OF HERV-K(HML-2) LTRs 881

FIG. 4. Occurrence of CpG sites in 5⬘ LTRs belonging to the HERV-K(HML-2) and the HERV-K(OLD) families (see the text for details).
HS consensus is a previously reported consensus sequence for human-specific HERV-K(HML-2) LTRs (7). The entire 5⬘ LTR sequence is shown
for each LTR. Each sequence was analyzed for the presence of CpG sites (triangles), as well as apparently mutated CpG sites (X’s). TATA boxes
are furthermore indicated for each LTR, when they could be identified. For each LTR, the 5⬘-most 620 bp were analyzed. Note that HERV-
K(OLD) 5⬘ LTRs frequently display mutated CpG sites, indicating former CpG methylation.

ylation level of LTR-c11 (44%) appeared lower than that of to fully explain transcriptional activity of HERV-K(HML-2)
LTR-c6. On the other hand, no CpG methylation was found proviruses in particular cell types, such as the T47D cells that
for LTR-c7L (Fig. 5). In contrast to the Tera-1 cell line, LTR- were examined in this study. In combination with the above-

Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.

c7L is transcriptionally inactive in T47D, although it is likewise described transcriptional inactivity of LTR reporter constructs
unmethylated. Thus, CpG methylation levels are not sufficient in T47D cells, one can conclude that either cellular activators

FIG. 5. CpG site methylation status of U3 regions of three HERV-K(HML-2) proviral 5⬘ LTRs in T47D cells, as determined by bisulfite
sequencing. Please refer to the legend to Fig. 2 for details.
882 LAVIE ET AL.
or repressors involved in HERV-K(HML-2) transcription are
missing or present, respectively, in T47D cells. It is currently
unclear what cellular factors trigger HERV-K(HML-2) activ-
ity. Those factors remain to be identified in future studies.
We show in the present study that CpG methylation directly
influences transcription of HERV-K(HML-2) sequences. Pro-
viral 5⬘ LTRs are efficiently silenced by CpG methylation. Our
findings have immediate implications for the understanding of
upregulated HERV-K(HML-2) expression observed in GCTs.
It was previously reported that the genome of seminoma tumor
cells is hypomethylated, including CpG dinucleotides (39). An-
other study showed general hypomethylation in ovarian carci-
noma (32). Therefore, changes in the CpG methylation pattern
towards CpG hypomethylation, which very likely also concern
CpGs located in HERV-K(HML-2) 5⬘ LTRs, can directly re-
sult in transcriptional activation of HERV-K(HML-2) 5⬘
LTRs. CpG methylation as a repressing factor for HERV-
K(HML-2) transcriptional activity is removed from the 5⬘
LTRs. Presence of thus-far-unidentified cellular (transcrip-
tion) factors in seminoma cells could then enable expression of
HERV-K(HML-2). This may also apply to other tissues. For
instance, Khodosevich et al. recently reported a number of
human-specific HERV-K(HML-2) LTRs that appeared to be
located in differentially methylated genomic regions, when
compared among each other and between cerebellum and
lymph node tissue (24). So, with appropriate methylation levels
and in the presence of required transcription factors, particular
HERV-K(HML-2) LTRs may be active in other tissues as well.
While our study demonstrates the direct role of CpG methyl-
ation in HERV-K(HML-2) transcriptional regulation, the cel-
lular (transcription) factors contributing to high HERV-
K(HML-2) expression remain to be elucidated in future
studies.
ACKNOWLEDGMENTS
This study was supported by grants from the Deutsche Forschungs-
gemeinschaft to J.M. (Ma2298/2-1) and E.M. (Me917/16-1).
REFERENCES
1. Abrink, M., E. Larsson, and L. Hellman. 1998. Demethylation of ERV3, an
endogenous retrovirus regulating the Kruppel-related zinc finger gene H-
plk, in several human cell lines arrested during early monocyte development.
DNA Cell Biol. 17:27–37.
2. Akopov, S. B., L. G. Nikolaev, P. P. Khil, Y. B. Lebedev, and E. D. Sverdlov.
1998. Long terminal repeats of human endogenous retrovirus K family
(HERV-K) specifically bind host cell nuclear proteins. FEBS Lett. 421:229–
233.
3. Barbulescu, M., G. Turner, M. I. Seaman, A. S. Deinard, K. K. Kidd, and J.
Lenz. 1999. Many human endogenous retrovirus K (HERV-K) proviruses
are unique to humans. Curr. Biol. 9:861–868.
4. Berkhout, B., M. Jebbink, and J. Zsiros. 1999. Identification of an active
reverse transcriptase enzyme encoded by a human endogenous HERV-K
retrovirus. J. Virol. 73:2365–2375.
5. Boese, A., M. Sauter, U. Galli, B. Best, H. Herbst, J. Mayer, E. Kremmer, K.
Roemer, and N. Mueller-Lantzsch. 2000. Human endogenous retrovirus
protein cORF supports cell transformation and associates with the promy-
elocytic leukemia zinc finger protein. Oncogene 19:4328–4336.
6. Boller, K., O. Janssen, H. Schuldes, R. R. Tonjes, and R. Kurth. 1997.
Characterization of the antibody response specific for the human endoge-
nous retrovirus HTDV/HERV-K. J. Virol. 71:4581–4588.
7. Buzdin, A., S. Ustyugova, K. Khodosevich, I. Mamedov, Y. Lebedev, G.
Hunsmann, and E. Sverdlov. 2003. Human-specific subfamilies of HERV-K
(HML-2) long terminal repeats: three master genes were active simulta-
neously during branching of hominoid lineages. Genomics 81:149–156.
8. Cooper, D. N., and M. Krawczak. 1989. Cytosine methylation and the fate of
CpG dinucleotides in vertebrate genomes. Hum. Genet. 83:181–188.
9. Costello, J. F., and C. Plass. 2001. Methylation matters. J. Med. Genet.
38:285–303.
J. VIROL.
10. Dunn, C. A., P. Medstrand, and D. L. Mager. 2003. An endogenous retro-
viral long terminal repeat is the dominant promoter for human ␤1,3-galac-
tosyltransferase 5 in the colon. Proc. Natl. Acad. Sci. USA 100:12841–12846.
11. Florl, A. R., R. Lower, B. J. Schmitz-Drager, and W. A. Schulz. 1999. DNA
methylation and expression of LINE-1 and HERV-K provirus sequences in
urothelial and renal cell carcinomas. Br. J. Cancer 80:1312–1321.
12. Fraga, M. F., and M. Esteller. 2002. DNA methylation: a profile of methods
and applications. BioTechniques 33:632
13. Frommer, M., L. E. McDonald, D. S. Millar, C. M. Collis, F. Watt, G. W.
Grigg, P. L. Molloy, and C. L. Paul. 1992. A genomic sequencing protocol
that yields a positive display of 5-methylcytosine residues in individual DNA
strands. Proc. Natl. Acad. Sci. USA 89:1827–1831.
14. Gotzinger, N., M. Sauter, K. Roemer, and N. Mueller-Lantzsch. 1996. Reg-
ulation of human endogenous retrovirus-K Gag expression in teratocarci-
noma cell lines and human tumours. J. Gen. Virol. 77:2983–2990.
15. Goubely, C., P. Arnaud, C. Tatout, J. S. Heslop-Harrison, and J. M. De-
ragon. 1999. S1 SINE retroposons are methylated at symmetrical and non-
symmetrical positions in Brassica napus: identification of a preferred target
site for asymmetrical methylation. Plant Mol. Biol. 39:243–255.
16. Hendrich, B., and A. Bird. 1998. Identification and characterization of a
family of mammalian methyl-CpG binding proteins. Mol. Cell. Biol. 18:
6538–6547.
17. Herbst, H., M. Sauter, C. Kuhler-Obbarius, T. Loning, and N. Mueller-
Lantzsch. 1998. Human endogenous retrovirus (HERV)-K transcripts in
germ cell and trophoblastic tumours. APMIS 106:216–220.
18. Hojman-Montes de Oca, F., J. Lasneret, L. Dianoux, M. Canivet, R. Ravi-
covitch-Ravier, and J. Peries. 1984. Regulation of intracisternal A particles
in mouse teratocarcinoma cells: involvement of DNA methylation in tran-
scriptional control. Biol. Cell 52:199–204.
19. Hsiao, W. L., S. Gattoni-Celli, and I. B. Weinstein. 1986. Effects of 5-aza-
cytidine on expression of endogenous retrovirus-related sequences in C3H
10T1/2 cells. J. Virol. 57:1119–1126.
20. Iguchi-Ariga, S. M., and W. Schaffner. 1989. CpG methylation of the cAMP-
responsive enhancer/promoter sequence TGACGTCA abolishes specific
factor binding as well as transcriptional activation. Genes Dev. 3:612–619.
21. Jaenisch, R., and A. Bird. 2003. Epigenetic regulation of gene expression:
how the genome integrates intrinsic and environmental signals. Nat. Genet.
33(Suppl.):245–254.
22. Kapitonov, V. V., and J. Jurka. 1999. The long terminal repeat of an endog-
enous retrovirus induces alternative splicing and encodes an additional car-
boxy-terminal sequence in the human leptin receptor. J. Mol. Evol. 48:248–
251.
23. Kent, W. J., C. W. Sugnet, T. S. Furey, K. M. Roskin, T. H. Pringle, A. M.
Zahler, and D. Haussler. 2002. The human genome browser at UCSC.
Genome Res. 12:996–1006.
24. Khodosevich, K., Y. Lebedev, and E. D. Sverdlov. 2004. Large-scale deter-
mination of the methylation status of retrotransposons in different tissues
using a methylation tags approach. Nucleic Acids Res. 32:e31.
25. Knossl, M., R. Lower, and J. Lower. 1999. Expression of the human endog-
enous retrovirus HTDV/HERV-K is enhanced by cellular transcription fac-
tor YY1. J. Virol. 73:1254–1261.
Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.
26. Landry, J. R., A. Rouhi, P. Medstrand, and D. L. Mager. 2002. The opitz
syndrome gene mid1 is transcribed from a human endogenous retroviral
promoter. Mol. Biol. Evol. 19:1934–1942.
27. Magin, C., R. Lower, and J. Lower. 1999. cORF and RcRE, the Rev/Rex and
RRE/RxRE homologues of the human endogenous retrovirus family
HTDV/HERV-K. J. Virol. 73:9496–9507.
28. Mayer, J., S. Ehlhardt, M. Seifert, M. Sauter, N. Muller-Lantzsch, Y. Me-
hraein, K. D. Zang, and E. Meese. 2004. Human endogenous retrovirus
HERV-K(HML-2) proviruses with Rec protein coding capacity and tran-
scriptional activity. Virology 322:190–198.
29. Mayer, J., E. Meese, and N. Mueller-Lantzsch. 1997. Chromosomal assign-
ment of human endogenous retrovirus K (HERV-K) env open reading
frames. Cytogenet. Cell Genet. 79:157–161.
30. Mayer, J., E. Meese, and N. Mueller-Lantzsch. 1997. Multiple human en-
dogenous retrovirus (HERV-K) loci with gag open reading frames in the
human genome. Cytogenet. Cell Genet. 78:1–5.
31. Medstrand, P., J. R. Landry, and D. L. Mager. 2001. Long terminal repeats
are used as alternative promoters for the endothelin B receptor and apoli-
poprotein C-I genes in humans. J. Biol. Chem. 276:1896–1903.
32. Menendez, L., B. B. Benigno, and J. F. McDonald. 2004. L1 and HERV-W
retrotransposons are hypomethylated in human ovarian carcinomas. Mol.
Cancer 3:12.
33. Okada, M., H. Ogasawara, H. Kaneko, T. Hishikawa, I. Sekigawa, H. Hashi-
moto, N. Maruyama, Y. Kaneko, and N. Yamamoto. 2002. Role of DNA
methylation in transcription of human endogenous retrovirus in the patho-
genesis of systemic lupus erythematosus. J. Rheumatol. 29:1678–1682.
34. Ono, M., M. Kawakami, and H. Ushikubo. 1987. Stimulation of expression
of the human endogenous retrovirus genome by female steroid hormones in
human breast cancer cell line T47D. J. Virol. 61:2059–2062.
35. Reus, K., J. Mayer, M. Sauter, D. Scherer, N. Muller-Lantzsch, and E.
Meese. 2001. Genomic organization of the human endogenous retrovirus
VOL. 79, 2005
HERV-K(HML-2.HOM) (ERVK6) on chromosome 7. Genomics 72:314–
320.
36. Reus, K., J. Mayer, M. Sauter, H. Zischler, N. Muller-Lantzsch, and E.
Meese. 2001. HERV-K(OLD): ancestor sequences of the human endoge-
nous retrovirus family HERV-K(HML-2). J. Virol. 75:8917–8926.
37. Sauter, M., K. Roemer, B. Best, M. Afting, S. Schommer, G. Seitz, M.
Hartmann, and N. Mueller-Lantzsch. 1996. Specificity of antibodies directed
against Env protein of human endogenous retroviruses in patients with germ
cell tumors. Cancer Res. 56:4362–4365.
38. Sauter, M., S. Schommer, E. Kremmer, K. Remberger, G. Dolken, I. Lemm,
M. Buck, B. Best, D. Neumann-Haefelin, and N. Mueller-Lantzsch. 1995.
Human endogenous retrovirus K10: expression of Gag protein and detection
of antibodies in patients with seminomas. J. Virol. 69:414–421.
39. Smiraglia, D. J., J. Szymanska, S. M. Kraggerud, R. A. Lothe, P. Peltomaki,
and C. Plass. 2002. Distinct epigenetic phenotypes in seminomatous and
nonseminomatous testicular germ cell tumors. Oncogene 21:3909–3916.
CpG REGULATION OF HERV-K(HML-2) LTRs 883
40. Tonjes, R. R., F. Czauderna, and R. Kurth. 1999. Genome-wide screening,
cloning, chromosomal assignment, and expression of full-length human en-
dogenous retrovirus type K. J. Virol. 73:9187–9195.
41. Walsh, C. P., J. R. Chaillet, and T. H. Bestor. 1998. Transcription of IAP
endogenous retroviruses is constrained by cytosine methylation. Nat. Genet.
20:116–117.
42. Yang, J., H. P. Bogerd, S. Peng, H. Wiegand, R. Truant, and B. R. Cullen.
1999. An ancient family of human endogenous retroviruses encodes a func-
tional homolog of the HIV-1 Rev protein. Proc. Natl. Acad. Sci. USA
96:13404–13408.
43. Yoder, J. A., C. P. Walsh, and T. H. Bestor. 1997. Cytosine methylation and
the ecology of intragenomic parasites. Trends Genet. 13:335–340.
44. Zsiros, J., M. F. Jebbink, V. V. Lukashov, P. A. Voute, and B. Berkhout.
1999. Biased nucleotide composition of the genome of HERV-K related
endogenous retroviruses and its evolutionary implications. J. Mol. Evol.
48:102–111.
Downloaded from https://journals.asm.org/journal/jvi on 18 April 2024 by 49.190.43.138.