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Just Accepted by Stress
Abnormal DLK1/MEG3 imprinting correlates with decreased HERV-K methylation after assisted reproduction and preimplantation genetic diagnosis
Eftychia Dimitriadou, Dimitrios Noutsopoulos, Georgios Markopoulos, Angeliki-Maria Vlaikou, Stefania Mantziou, Joanne Traeger-Synodinos, Emmanouel Kanavakis, George P. Chrousos, Theodore Tzavaras, Maria Syrrou*
doi: 10.3109/10253890.2013.817554
Abstract
Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an important role in retrotransposon silencing and genomic imprinting during mammalian development. Assisted reproduction technologies (ART) may be associated with increased stress and risk of epigenetic changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and imprinted genes. Here, we investigated whether the methylation status of HERV-K LTR retrotransposons and the imprinting signatures of the DLK1/MEG3, p57KIP2
and IGF2/H19 gene loci are linked during early human embryogenesis by examining trophoblast samples from ART pregnancies and preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status. Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to controls and this was associated with altered HERV-K methylation. No differences were detected in p57KIP2 and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using bioinformatics, we found that while the genome surrounding the p57KIP2 and IGF2/H19 genes differentially methylated regions had low coverage in transposable element sequences, the respective one of DLK1/MEG3 was characterized by an almost two-fold higher coverage. Moreover, our analyses revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that transposable elements may be affecting the establishment of genomic imprinting under stress conditions.
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Abnormal DLK1/MEG3 imprinting correlates with decreased HERV-K
methylation after assisted reproduction and preimplantation genetic diagnosis
Eftychia Dimitriadou1,§
, Dimitrios Noutsopoulos2,§,
*, Georgios Markopoulos1,§
, Angeliki-Maria Vlaikou1,
Stefania Mantziou1, Joanne Traeger-Synodinos
3, Emmanouel Kanavakis
3, George P. Chrousos
4,5,
Theodore Tzavaras1,§
, Maria Syrrou1,§,
*
1 Laboratory of General Biology, Medical School, University of Ioannina, 45 110 Ioannina, Greece.
2 Laboratory of Cellular and Molecular Neuroimmunology, Department of Biological Applications and
Technology, University of Ioannina, 45 110 Ioannina, Greece.
3 Laboratory of Medical Genetics, Medical School, National and Kapodistrian University of Athens,
"Aghia Sophia" Children's Hospital, Athens 11 527, Greece.
4 Division of Endocrinology and Metabolism, Medical School, National and Kapodistrian University of
Athens, "Aghia Sophia" Children’s Hospital, Athens 11 527, Greece.
5 Clinical Research Center, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
§These authors contributed equally to this work.
*Correspondence: D. Noutsopoulos, Laboratory of Cellular and Molecular Neuroimmunology,
Department of Biological Applications and Technology, University of Ioannina, 45 110 Ioannina, Greece.
Tel: 0030 26510 07371. Fax: 0030 26510 07064. E-mail: dnoutso@cc.uoi.gr.
M. Syrrou, Laboratory of General Biology, Medical School, University of Ioannina, 45 110 Ioannina,
Greece. Tel: 0030 26510 07612. Fax: 0030 26510 07863. E-mail: msyrrou@cc.uoi.gr.
Running head: HERV-K methylation, imprinting and ART
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Keywords: HERV-K retrotransposon, transposable element, methylation, genomic imprinting,
DLK1/MEG3, preimplantation genetic diagnosis, assisted reproduction, stress
Abstract
Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an
important role in retrotransposon silencing and genomic imprinting during mammalian development.
Assisted reproduction technologies (ART) may be associated with increased stress and risk of epigenetic
changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and
imprinted genes. Here, we investigated whether the methylation status of HERV-K LTR retrotransposons
and the imprinting signatures of the DLK1/MEG3, p57KIP2
and IGF2/H19 gene loci are linked during
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early human embryogenesis by examining trophoblast samples from ART pregnancies and
preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation
analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an
altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status.
Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to
controls and this was associated with altered HERV-K methylation. No differences were detected in
p57KIP2
and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using
bioinformatics, we found that while the genome surrounding the p57KIP2
and IGF2/H19 genes
differentially methylated regions had low coverage in transposable element sequences, the respective one
of DLK1/MEG3 was characterized by an almost two-fold higher coverage. Moreover, our analyses
revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the
DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD
conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that
transposable elements may be affecting the establishment of genomic imprinting under stress conditions.
Introduction
The dynamic interrelation between genome and epigenome has a central role in the regulation of
gene expression, which is fundamental for normal differentiation during mammalian development. The
epigenetic modifications involving, amongst others, DNA methylation and histone modifications can alter
cell physiology and homeostasis in response to intrinsic and environmental signals (Jaenisch and Bird
2003). DNA methylation is established during development, maintained in adult somatic cells in a highly
orchestrated manner (Szyf 2010) and is essential in several processes, such as X chromosome
inactivation, genomic imprinting and retrotransposon silencing (Chen and Riggs 2011).
Genomic imprinting is an important epigenetic regulatory mechanism resulting in monoallelic
gene expression related to parental origin (Bartolomei and Tilghman 1997). At present, approximately 80
human imprinted genes have been reported, while more than 150 have been predicted by computational
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studies (Piedrahita 2011; Wilkins and Úbeda 2011). Of all mechanisms involved in genomic imprinting,
DNA methylation is the best studied and is established in CpG-rich domains called differentially
methylated regions (DMR). DNA methylation is catalysed by DNA methyltransferases (DNMTs), while
its maintenance is performed by either DNMTs or trans-acting factors, such as MBD3, PGC7/Stella,
ZFP57 and KAP1 (Bartolomei 2009). It should be noted that we used the term "DNA methylation" for
covalent modifications of mammalian DNA occurring via the 5-methylation or 5-hydroxymethylation of
cytosine typically in the context of the CpG dinucleotide. This is in order to be consistent with primary
publications, as other DNA modifications involved in DNA demethylation have been recently identified
(Inoue et al. 2011). In general, genomic imprints are erased in embryonic germ cells and re-established
afterwards during gametogenesis and after fertilization (Reik and Walter 2001). Imprinted genes are
essential for the correct regulation of both placental and fetal growth (Wilkins and Úbeda 2011; Radford
et al. 2011) and their epigenetic deregulation can lead to fetal growth abnormalities as well as imprinting-
associated changes and disorders (Wilkins and Úbeda 2011).
Transposable elements (TEs) are repetitive genetic elements that constitute over two thirds of the
human genome (de Koning et al. 2011). Retrotransposons, which represent the major class of TEs
(Cordaux and Batzer 2009), may be mobilized through a RNA-intermediate, which upon its conversion
into cDNA by an endogenous reverse transcriptase, is integrated into new genomic sites. They may affect
cell functions, which range from local instability to large-scale structural variation, driving genome
evolution and altering genetically and/or epigenetically gene expression (Cordaux and Batzer 2009;
Goodier and Kazazian 2008). To regulate retrotransposon RNA expression and mobilization activity, cells
have developed different defense mechanisms of which DNA methylation is a major one (Goodier and
Kazazian 2008). Retrotransposons are generally silenced, as ~90% of methylated cytosine residues in
human DNA lie within retrotransposons (Yoder et al. 1997). Their transcriptional activity however is less
restrained in proliferating germ and stem cells (Georgiou et al. 2009; Garcia-Perez et al. 2007), as well as
during development and embryogenesis (Kano et al. 2009). Retrotransposon activity can be induced by
environmental factors and, particularly, stress (Capy et al. 2000). Although controlled retrotransposon
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activity might be beneficial for the cell (Capy et al. 2000), the deregulated state may cause monogenic
genetic diseases (e.g. haemophilia, cystic fibrosis, Duchenne muscular dystrophy), and has been linked to
multifactorial diseases (e.g. cancer, autoimmune diseases) (Cordaux and Batzer 2009; Goodier and
Kazazian 2008).
Human Endogenous Retroviruses (HERVs) are long terminal repeat (LTR) retrotransposons that
constitute ~8.3% of the human genome and are derived from germline-integrated proviruses that have
undergone endogenization (Bannert and Kurth 2004). HERV-K is the evolutionarily youngest and more
active subfamily of HERVs consisting of 91 proviruses, which retained functional full-length open
reading frames (ORFs) coding for gag, prt, pol and env, as well as 941 solitary LTRs (Bannert and Kurth
2004; Subramanian et al. 2011). HERV-K10, the prototype of HERV-K genome retrotransposon is
homologous to the mouse LTR retrotransposon Intracisternal A-Particle (IAP) (Ono et al. 1986). The
transcriptional activity of HERV-Ks is directly regulated by CpG methylation (Lavie et al. 2005).
Notably, HERV-K is transcriptionally induced under stress conditions (Cho et al. 2008), while its
transcripts are detected in human oocytes, lymphocytes and cancer cells (Georgiou et al. 2009).
ART involves in vitro manipulation of gametes, such as in vitro fertilization (IVF) and
intracytoplasmic sperm injection (ICSI), and embryo culture and related procedures (Piedrahita 2011).
ART accounts for 1-3% of births in developed countries (Shiota and Yamada 2009) and its wide clinical
application has led to the development of methods testing for genetic defects, such as PGD. ART
procedures take place in a critical time-window, during which DNA methylation patterns are initiated,
and may be associated with increased parental stress during pregnancy compared to natural conceptions
(Kanaka-Gantenbein et al. 2010). More specifically, epidemiological data have raised some concern
whether ART may increase the risk of epigenetic changes leading to genomic imprinting disorders, either
during ART procedures and/or during the resulting pregnancies following ART (Kanaka-Gantenbein et
al. 2010; Lucifero et al. 2004a).
Retrotransposon methylation may influence imprinted genes, as exemplified by the mouse LTR
retrotransposon IAP at the Agouti locus (Michaud et al. 1994). On the other hand, HERVs respond to
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stress signals (Cho et al. 2008), and stress might cause epigenetic errors in imprinted genes (Jirtle and
Skinner 2007). Based on these data, we asked whether LTR retrotransposon methylation status and
genomic imprinting signatures are linked following ART-PDG, a condition likely associated with stress
(Kanaka-Gantenbein et al. 2010). Here, we investigated the methylation status of HERV-Ks – an active
HERV subfamily (Bannert and Kurth 2004) – and three well-known imprinted domains DLK1/MEG3,
IGF2/H19 and p57KIP2
widely used in methylation analysis studies (Wilkins and Úbeda 2011). Our results
showed that following ART-PGD HERV-K methylation changes are correlated with abnormal imprinting
of the DLK1/MEG3 locus, which is characterized by a higher coverage in TEs sequences and KAP1-
binding sites residing within retrotransposons, compared to the IGF2/H19 and p57KIP2
loci.
Methods
Samples
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We used chorionic villi samples, collected in the Laboratory of Medical Genetics (University of
Athens), after informed consent of the participants who were undergoing prenatal diagnosis after PGD.
The samples were divided in two groups: i) samples taken after ICSI and PGD (ART-PGD) and ii)
matched samples after natural conceptions, as controls. The confidentiality and anonymity of all
participants were ensured by coded references and appropriate safeguards of the data stored. The study
was approved by the Ioannina University Hospital Ethics Committee (560/2005).
DNA extraction and bisulfite modification
DNA was extracted from the samples using a commercially available DNA extraction kit
(QIAamp DNA extraction Mini Kit, Qiagen). Genomic DNA (1-2 μg) derived from each sample was
bisulfite modified (Sigma), as previously described (Manning et al. 2000). The modification took place at
55°C in the dark after an overnight (15-17 h) incubation. Subsequently, DNA was purified using the
Wizard DNA clean-up system (Promega), followed by neutralization with ammonium acetate (Sigma)
and glycogen (Invitrogen) and precipitation with 70% ethanol (Sigma). Finally, DNA was diluted in
double distilled water and stored at -80°C until use.
Methylation-specific PCR (MS-PCR) and statistical analysis
Methylation analysis was performed by methylation-specific PCR (MS-PCR), using specific
primers sets for the methylated and unmethylated alleles under conditions shown in Figure 1. In
particular, we used previously described primers for the analysis of DMR methylation status of IGF2/H19
(Poon et al. 2002), p57KIP2
(Li et al. 2002) and DLK1/MEG3 (Murphy et al. 2003) (Figure 1A). For the
methylation analysis of HERV-K retrotransposons, we designed primer sets for the methylated and
unmethylated allele based on the sequence of HERV-K10 CpG island spanning from 981 to 1085 bp in
relation to the transcriptional start site (Ono et al. 1986) using MethPrimer software (Figure 1A and B).
HERV-K primer sequences were tested and the exact genomic positions were retrieved using the UCSC
in silico PCR tool (Kent et al. 2002). All primer sets were designed to amplify the methylated and
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unmethylated sequences in a way that distinguishes the amplicons by size and the positivity or negativity
of the samples on the presence or absence of the PCR products, respectively. As a negative control,
reactions without template DNA were performed. MS-PCR products were fractionated in 2% agarose gel
and photographed under UV light. All samples were analyzed in triplicate (n=3). The statistical analysis
of MS-PCR experiment data was performed using the chi-square test. p-values < 0.05 were considered
statistically significant.
Bioinformatics
The in-silico program RepeatMasker (http://www.repeatmasker.org/) was used to measure the
percentage coverage of TEs sequences within the DLK1/MEG3, IGF2/H19 and p57KIP2
loci. We used the
UCSC genome browser (Kent et al. 2002) to extract sequences from the human genome (hg19),
encompassing the imprinted gene as well as the genomic region upstream of the DMR of interest and
having a total size of approximately 126Kb in all three cases. Specifically, the nucleotide sequences
analyzed were: i) chr14:101,201,234 – 101,327,360 for the DLK1/MEG3 locus, ii) chr11:2,003,437 –
2,129,448 for the IGF2/H19 locus and iii) chr11:2,897,414 – 3,024,995 for the p57KIP2
locus.
Data mining of KAP1 Chip-seq peaks in DLK1/MEG3, IGF2/H19 and p57KIP2
loci, respectively,
was performed using the UCSC table browser (Karolchik et al. 2004). Data were extracted from a table
with Chip-Seq data from the ENCODE project (wgEncodeRegTfbsClusteredV2), containing, among
others, the datasets for KAP1-binding sites (UCSC accession numbers wgEncodeEH001779,
wgEncodeEH001776 and wgEncodeEH001779). Retrotransposon-associated KAP1-binding sites were
found by intersecting KAP1 Chip-seq peaks and Repeatmasker data tracks in the UCSC table browser.
Peaks were viewed and analysed in the UCSC Genome browser (Kent et al. 2002).
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Results
Alteration of HERV-K methylation status following ART-PGD compared to natural conceptions
It has been suggested that endogenous retroviruses are permanently inactivated during embryonic
development (Rowe and Trono 2011). However, HERV-K methylation status during early human
embryogenesis remains unknown so far. To examine this, we investigated the methylation status of
HERV-Ks in human trophoblast samples from pregnancies initiated after ICSI and PGD and from
matched control samples after natural conceptions, hereafter referred to as PGD and controls,
respectively.
To study HERV-K methylation, we designed two sets of primers based on the sequence of CpG
island of HERV-K10 (Ono et al. 1986) and the specificity of the designed primers was checked using the
UCSC in silico PCR tool (Kent et al. 2002). Our analysis showed that they specifically amplified HERV-
K members located in seventeen different chromosomal positions. It should be noted that almost all
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HERV-Ks amplified were characterized by human-specificity and polymorphisms, while many of them
bore full-length ORFs (data not shown), indicating that our primers can detect active HERV-K sequences.
By analyzing control samples, we found that 54 out of 60 samples (90%) exhibited total
methylation, while the remaining 6 control samples (10%) presented a pattern of both methylated and
unmethylated alleles. In PGD samples, on the other hand, we found that 26 out of 35 samples (74.3%)
showed a totally methylated pattern, while 9 out of 35 (25.7%) had both methylated and unmethylated
alleles. The differences observed between PGD samples and controls corresponded to a statistically
significant alteration of HERV-K methylation (p<0.042) (Table 1).
These data show that HERV-Ks are usually methylated in trophoblast samples deriving from
natural conceptions, while their methylation status is altered following ART-PGD.
Altered HERV-K methylation is correlated with abnormal imprinting of DLK1/MEG3, but not
IGF2/H19 and p57KIP2
We next investigated the methylation status of DLK1/MEG3, p57KIP2
and IGF2/H19
imprinted genes in the two groups of samples to address whether genomic imprinting signatures
are affected following ART-PGD.
DLK1/MEG3 methylation analysis showed that all control samples exhibited both
methylated and unmethylated alleles, as expected. In contrast, we found a statistically significant
alteration of DNA methylation pattern in PGD samples (p<0.001). While 25 out of 35 samples
(71.5%) showed the expected pattern of both methylated and unmethylated alleles, 9 samples
(25.7%) showed hypomethylation and 1 sample (2.8%) revealed hypermethylation, and hence a
disturbed methylation status (Benetatos et al. 2010) (Table 2A). Notably, in 8 out of 10 PGD
samples (80%) with abnormal DLK1/MEG3 imprinting, altered methylation of HERV-K was
detected as well. Specifically, all these samples (samples AF-139, AF-141, AF-195, AF-210,
AF-221, 04-C44, 05-C50 and 05-C61) exhibited hypomethylation of DLK1/MEG3 and a hemi-
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methylated HERV-K pattern (Table 3). As regards p57KIP2
and IGF2/H19, we found that all
samples of either the control or the PGD group expressed a pattern of both methylated and
unmethylated alleles, thus indicating no difference on the establishment of imprints (Tables 2B,
2C and 3).
Taken together, our data show that altered HERV-K methylation is differentially
associated with the methylation status of imprinted loci, correlating with abnormal imprinting of
DLK1/MEG3 but having no correlation with that of p57KIP2
and IGF2/H19.
DLK1/MEG3 is characterized by a high coverage in TEs sequences and KAP1-binding
sites lying within retrotransposons
The differential methylation status of DLK1/MEG3, p57KIP2
and IGF2/H19 observed prompted us
to investigate for other factors that possibly affect the establishment of imprinting signatures. Given that
the DMR surrounding genome sequence has a fundamental role in the establishment of genomic
imprinting (Reinhart et al. 2002), we measured the coverage in TEs sequences within the aforementioned
imprinted loci using the in-silico program RepeatMasker.
Since MEG3 DMR encompasses nucleotides of both a 90Kb intergenic region and the promoter
of MEG3 (Murphy et al. 2003), we examined both regions of the imprinted gene cluster DLK1/MEG3.
Our analysis revealed that DLK1/MEG3 gene cluster was characterized by coverage in TEs sequences that
amounted to 37.37% (Figure 2A). More specifically, we found a significant load in Short Interspersed
Nuclear Elements (SINEs) and Long Interspersed Nuclear Elements (LINEs) sequences, corresponding to
percentage values of 14.05% and 13.16%, respectively. Furthermore, 7.10% of the examined region was
covered by LTR retrotransposon and 3.06% by DNA transposon sequences (Figure 2A).
In contrast, respective analyses for p57KIP2
and IGF2/H19 revealed that both loci had a lower
content in TEs sequences compared to DLK1/MEG3. Thus, analysis of IGF2/H19 revealed that 21.60% of
the nucleotide sequence examined was constituted of TEs. The coverage in SINE, LINE, LTR
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retrotransposon and DNA transposon sequences was 4.95%, 8.29%, 5.39% and 2.87%, respectively
(Figure 2B). Analysis of p57KIP2
revealed an abundance in TEs sequences of 25.03%, almost similar to
that of IGF2/H19 (Figure 2C). In p57KIP2
, abundance in SINE sequences was slightly lower than
DLK1/MEG3 (13.80%). Of note, a significant difference was observed in the coverage in LINE, LTR
retrotransposon and DNA transposon sequences, which were significantly lower than the IGF2/H19, with
values of 6.97%, 2.09% and 2.18%, respectively.
The different coverage in TEs sequences of the three imprinted loci prompted us to get further
insights into their possible contribution to the imprinting changes previously observed. Given that KAP1
is essential for DNA methylation of endogenous retroviruses (ERVs) (Rowe et al. 2013) and possibly
involved in the establishment of imprinting during embryogenesis (Strogantsev and Ferguson-Smith
2012), we examined DLK1/MEG3, p57KIP2
and IGF2/H19 imprinted loci for presence of KAP1-binding
sites. Bioinformatic analysis of the DLK1/MEG3 locus revealed the existence of four KAP1-binding sites.
Interestingly, three out of four sites resided within retrotransposon sequences. Two of them (site 1 and 2)
were located at the boundary of two neighboring LINE and LTR retrotransposons and composed by part
of their sequences. More precisely, site 1 (chr14: 101,222,006 – 101,222,455) bore sequences of the
MLT1A0 member of LTR retrotransposon ERVL-MaLR family and the L1MB8 LINE, while site 2
(chr14: 101,228,449 – 101,228,898) those of the L1MB8 LINE and the ERV3-16A3_I-int ERV-L LTR
retrotransposon. Site 3 (chr14: 101,274,622 – 101,275,071) was partially composed by sequences of the
FLAM_A member of the Alu family of SINE retrotransposons (Figure 3A). Respective analyses of the
p57KIP2
and IGF2/H19 loci revealed one KAP1-binding site not residing within retrotransposon sequences
in the former (Figure 3B) and absence of KAP1-binding sites in the latter (Figure 3C), respectively.
Collectively, these results show that while the abundance of TEs in p57KIP2
and IGF2/H19 loci is
low, the DLK1/MEG3 locus is characterized by a higher coverage in TEs sequences and the presence of
KAP1-binding sites lying within retrotransposons.
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Discussion
This study provides experimental evidence for a correlation between altered HERV-K
methylation and abnormal imprinting of the DLK1/MEG3 locus in human trophoblast samples from ART-
PGD compared to normal conceptions. The correlation between HERV-K LTR retrotransposon and
imprinted gene methylation status under stress conditions may uncover a possible role of TEs in the
establishment of a normal imprinting pattern of certain imprinted genes, such as DLK1/MEG3.
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We showed that HERV-K retrotransposons are usually totally methylated during early human
embryonic development. Our results are in line with the current viewpoint, based on studies performed
solely in mice (Rowe and Trono 2011). Unexpectedly, we found a number of samples in the control group
(10%) revealing both methylated and unmethylated alleles. This could be attributed to possible inter-
individual variations of LTR DNA methylation, as already documented for the mouse LTR
retrotransposon IAP (Waterland and Jirtle 2003). Thus, the hemi-methylated pattern of HERV-K in
natural conception samples might be due to the effect of intrinsic or extrinsic/environmental stress and/or
other hitherto unknown factors, which lead to a relaxation of epigenetic control during early pregnancy.
One key finding was the significant alteration of HERV-K methylation status following ART-
PGD, documented by a hemi-methylated pattern. This epigenetic perturbation could be due to stress
elicited during ART-PGD as the result of distinct events occurring during the preimplantation and/or
postimplantation stages. First, an altered gene expression has been reported in human preimplantation
embryos attributed to stress conditions, with Rb being one of the affected genes (Wells et al. 2005).
Notably, a correlation between Rb pathway and HERV-K expression has been reported (Li et al. 2010).
Thus, HERV-K epigenetic deregulation might be the result of Rb altered expression. Second, reactive
oxygen species (ROS) in ART culture media can potentially activate HERV-K, since it has been
suggested that oxidative stress might establish a hypomethylation status of certain endogenous retrovirus
loci (Cho et al. 2008). Third, ovarian hyperstimulation may lead to production of supraphysiological
serum estradiol levels (Santos et al. 2010), known to activate HERV-K (Ono et al. 1987). Fourth, it
cannot be excluded that ART, as an extended exposure to the in vitro environment, and PGD, as an
invasive method (Georgiou et al. 2006), may lead to epigenetic relaxation during preimplantation and/or
postimplantation embryonic development.
The other interesting finding of our study was the observation of a differential
methylation status between DLK1/MEG3, and p57KIP2
or IGF2/H19 following ART-PGD. While
the imprinting status of p57KIP2
and IGF2/H19 was not disturbed in the PGD group, in agreement
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with other results in IVF and ICSI samples (Tierling et al. 2010), we found a significant
alteration of DLK1/MEG3 methylation. It has been suggested that two fundamental cis-acting
elements are required for the establishment of an imprint on a gene: i) a DMR, which is
necessary but not sufficient alone, and ii) the surrounding genome sequence (Reinhart et al.
2002). In view of the differential methylation observed, we were challenged to evaluate genomic
features of the imprinted loci studied, such as the load in TEs sequences of their DMR
surrounding genome. Strikingly, our findings corroborated the above suggestion since following
ART-PGD a disturbed methylation status was only detected in an imprinted gene characterized
by a high coverage in TEs sequences in its DMR surrounding genome. Remarkably,
DLK1/MEG3 was characterized by a higher load in sequences of all the TE families and, in
total, approximately 1.5- and 1.7-fold higher coverage than those of p57KIP2
and IGF2/H19,
respectively. It should be mentioned that these findings are combatible with a previous study
showing that SINE depletion and LINE abundance at imprinted loci are not features universally
required for imprinting (Cowley et al. 2011). Moreover, another feature that differed between the
imprinted loci studied was the presence of KAP1-binding sites located within retrotransposons
solely in DLK1/MEG3, the only locus characterized by disturbed imprinting. Important recent
work documented that KAP1 shapes DNA methylation at ERV-containing loci in early
development (Rowe et al. 2013). Therefore, we suggest that the abundance in TEs sequences of
the imprinted gene DMR surrounding genome, as well as the presence of KAP1-binding sites
lying within those elements, may represent locus-specific features affecting the establishment of
DNA methylation under stress.
We found that the vast majority of PGD samples with abnormal imprinting of
DLK1/MEG3 was accompanied by a disturbed methylation pattern of HERV-K. Two lines of
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evidence agree with our results. First, it was previously proposed that the epigenetic regulation of
LTR retrotransposons and imprinted genes shares similarity due to the repeat-like nature of the
imprinted gene DMRs (Lucifero et al. 2004b). Second, it was documented that DMR-associated
genomic imprinting in mammals can originate from the repression of retrotransposons by DNA
methylation (Suzuki et al. 2007). Based on these, it is tempting to propose that the regulation of
HERV-K retrotransposon methylation and genomic imprinting of loci with certain features of the
DMR surrounding genome might be common under stress conditions during early human
embryogenesis.
ART procedures may be associated with increased stress compared with spontaneous
conception and take place in a time frame critical for the re-establishment of DNA methylation
patterns (Kanaka-Gantenbein et al. 2010). The functional consequences of the differential
methylation observed at HERV-Ks are unknown so far. The imprinting defects of DLK1/MEG3
have been associated with uniparental disomy of chromosome 14 (Murphy et al. 2003) and
Prader-Willi syndrome-like phenotype (Hosoki et al. 2009) as well as several types of cancer
(Benetatos et al. 2010; Benetatos et al. 2013). However, MEG3 function remains poorly
understood (Benetatos et al. 2013). Given that ART may be linked to epigenetic risks as well as
having some impact on the health of the offspring in later adult life (Kanaka-Gantenbein et al.
2010), the differential methylation observed might have long-term effects. HERVs are affected
by stress signals and their deregulation has been associated with multifactorial diseases (Cho et
al. 2008; Goodier and Kazazian 2008; Cordaux and Batzer 2009). Moreover, imprinted genes are
possible targets of disease-causing epigenetic errors induced by stress (Jirtle and Skinner 2007).
It is widely accepted that stress-related DNA methylation alterations leading to human diseases
might involve changes in networks of genes (Szyf 2009). In our opinion, HERVs could be
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important components of such networks regulating gene expression. Following early life stress,
the disturbed methylation of HERVs might serve as mark of a life-long lasting epigenetic
"memory". Upon stress stimuli in adult life, this "memory" can be reactivated leading to HERV
deregulation, further responsible for: i) the induction of genome instability and ii) alterations in
gene expression and, particularly, that of imprinted genes. Thus, HERVs and imprinted genes
might participate in a stress-driven gene network, involved in the transgenerational inheritance of
stress and the pathogenesis of multifactorial diseases.
In the present work, by conducting a qualitative analysis, we provide evidence of a correlation
between HERV-K and imprinted gene methylation status following ART-PGD. A limitation of our study
is the lack of direct nucleotide sequencing of the MS-PCR results. Future studies will shed more light in
the factors conferring epigenetic lability in response to stress during human embryogenesis and future
quantitative analyses are essential to determine the methylation profile of retrotransposons and imprinted
genes and whether stress-induced changes in methylation are associated with different mRNA expression
patterns in the early life stages. In addition, functional studies may help to establish whether epigenetic
modifications of KAP1-binding sites within the DMR surrounding genome have a direct effect on KAP1
binding and imprinted gene expression. Finally, recent technological advances can provide the required
data to map long-range intra- and inter-chromosomal interactions and gain insights into the locus-specific
features that are important for the establishment of genomic imprinting.
Overall, our results document a correlation between altered HERV-K methylation and abnormal
imprinting under conditions likely associated with stress during early human embryogenesis. Further
studies will reveal the interwoven structural and functional roles of TEs in the establishment of genomic
imprinting.
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Acknowledgements
The authors thank the patients for their participation in the study. This work was supported by
institutional funds of University of Ioannina. We also thank Prof. Ioannis Georgiou (Genetics and IVF
Unit, Department of Obstetrics and Gynecology, Medical School, University of Ioannina, Ioannina,
Greece) for the critical reviewing of the manuscript.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of
the paper.
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Figure Captions
Figure 1. Primers and conditions used for methylation analysis by MS-PCR.
(A) List of primers for the methylated and unmethylated alleles and thermal conditions used for
methylation analysis by MS-PCR. MF and MR, forward and reverse primers specific to bisulfite
converted methylated DNA; UF and UR, forward and reverse primers specific to bisulfite converted
unmethylated DNA; U/M R, reverse primer specific to bisulfite converted unmethylated and methylated
DNA.
(B) Schematic representation of HERV-K primers designed. In relation to the transcriptional start site of
HERV-K10, primers are complementary to the following positions: HERV-K10-UF, 959 to 983 bp;
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HERV-K10-MF, 958 to 983 bp; HERV-K10-UR and HERV-K10-MR, 1036 to 1060 bp. HERV-K10
NCBI accession number is indicated in parenthesis.
Figure 2. Abundance in transposable element sequences within DLK1/MEG3, IGF2/H19 and p57KIP2
imprinted loci.
Histograms indicate the percentage coverage in SINE, LINE, LTR retrotransposon, DNA transposon as
well as the total one in transposable element sequences within (A) DLK1/MEG3, (B) IGF2/H19 and (C)
p57KIP2
imprinted loci. The tables, below each histogram, show the percentage values of the coverage of
SINE, LINE, LTR retrotransposon, DNA transposon and total transposable element sequences within the
aforementioned regions.
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Figure 3. Bioinformatic analysis for KAP1-binding sites within DLK1/MEG3, p57KIP2
and IGF2/H19
loci.
KAP1-binding sites were viewed and analyzed in the UCSC Genome Browser. (A) The snapshot, in the
upper part, shows in the first track the data from the analysis for KAP1-binding sites within DLK1/MEG3
locus. KAP1-all denotes the total KAP1-binding sites found, while KAP1 the retrotransposon-associated
KAP1-binding sites, respectively. The next two tracks show the genes and the CpG islands contained in
the genomic region, respectively. The Repeating Elements cluster shown in the last track summarizes the
set of transposable element sequences from RepeatMasker. In the lower part, the snapshots, named site 1,
2 and 3, indicate the individual retrotransposon-associated KAP1-binding sites within DLK1/MEG3 locus.
(B) and (C) The snapshots show the data from the respective analyses for p57KIP2
and IGF2/H19 loci,
respectively.
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Table 1. Methylation analysis of HERV-K among PGD and control samples.
Methylated Unmethylated Methylated/
Unmethylated Total p-value
Control 54 (90%) 0 (0%) 6 (10%) 60 (100%) 0.042
PGD 26 (74.3%) 0 (0%) 9 (25.7%) 35 (100%)
Data indicate the number of the samples and the respective frequencies (in parentheses). p-value derived following analysis with the chi-square test of data
deriving from the MS-PCR experiments.
Table 2. Methylation analysis of (A) DLK1/MEG3, (B) p57KIP2 and (C) IGF2/H19 DMRs among PGD and control samples.
A
Methylated Unmethylated Methylated/
Unmethylated Total p-value
Control 0 (0%) 0 (0%) 40 (100%) 40 (100%) 0.001
PGD 1 (2.9%) 9 (25.7%) 25 (71.4%) 35 (100%)
B
Methylated Unmethylated Methylated/ Total
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Unmethylated
Control 0 (0%) 0 (0%) 40 (100%) 40 (100%)
PGD 0 (0%) 0 (0%) 35 (100%) 35 (100%)
C
Methylated Unmethylated Methylated/
Unmethylated Total
Control 0 (0%) 0 (0%) 40 (100%) 40 (100%)
PGD 0 (0%) 0 (0%) 35 (100%) 35 (100%)
Data indicate the number of the samples and the respective frequencies (in parentheses). p-value derived following analysis with the chi-square test of data
deriving from the MS-PCR experiments.
Methylation analysis was carried out in 40 out of 60 samples of control group due to the limited DNA amount of samples.
Table 3. Total results of methylation analyses performed in PGD samples.
PGD samples ID HERV-K DLK1/MEG3 IGF2/H19 p57KIP2
AF-138 M M/U M/U M/U
AF-139 M/U U M/U M/U
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AF-141 M/U U M/U M/U
AF-150 M M M/U M/U
AF-168 M M/U M/U M/U
AF-173 M M/U M/U M/U
AF-180 M/U M/U M/U M/U
AF-189 M M/U M/U M/U
AF-193 M M/U M/U M/U
AF-195 M/U U M/U M/U
AF-200 M M/U M/U M/U
AF-206 M M/U M/U M/U
AF-210 M/U U M/U M/U
AF-211 M M/U M/U M/U
AF-216 M M/U M/U M/U
AF-218 M M/U M/U M/U
AF-219 M M/U M/U M/U
AF-220 M M/U M/U M/U
AF-221 M/U U M/U M/U
AF-223 M U M/U M/U
AF-225 M M/U M/U M/U
AF-226 M M/U M/U M/U
04-C44 M/U U M/U M/U
04-C78 M M/U M/U M/U
04-C89 M M/U M/U M/U
05-C39 M M/U M/U M/U
05-C39 M M/U M/U M/U
05-C41 M M/U M/U M/U
05-C48 M M/U M/U M/U
Stre
ss D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Oxf
ord
on 0
6/27
/13
For
pers
onal
use
onl
y.