© 2013 Informa UK Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.

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.

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.

1

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

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.

2

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

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.

3

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

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.

4

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

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.

5

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

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.

6

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

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.

7

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

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.

8

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).

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.

9

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

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.

10

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-

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.

11

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

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.

12

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.

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.

13

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.

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.

14

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

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.

15

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

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.

16

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

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.

17

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.

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.

18

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.

References

Bannert N, Kurth R. 2004. Retroelements and the human genome: New perspectives on an old relation.

Proc Natl Acad Sci U S A 101 Suppl 2:14572−14579.

Bartolomei MS, Tilghman SM. 1997. Genomic imprinting in mammals. Annu Rev Genet 31:493−525.

Bartolomei MS. 2009. Genomic imprinting: employing and avoiding epigenetic processes. Genes Dev

23(18):2124−2133.

Benetatos L, Hatzimichael E, Dasoula A, Dranitsaris G, Tsiara S, Syrrou M, Georgiou I, Bourantas KL.

2010. CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia

and myelodysplastic syndromes. Leuk Res 34(2):148−153.

Benetatos L, Hatzimichael E, Londin E, Vartholomatos G, Loher P, Rigoutsos I, Briasoulis E. 2013. The

microRNAs within the DLK1-DIO3 genomic region: involvement in disease pathogenesis. Cell Mol Life

Sci 70(5):795–814.

Capy P, Gasperi G, Biémont C, Bazin C. 2000. Stress and transposable elements: co-evolution or useful

parasites? Heredity (Edinb) 85(Pt 2):101−106.

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.

19

Chen ZX, Riggs AD. 2011. DNA methylation and demethylation in mammals. J Biol Chem

286(21):18347−18353.

Cho K, Lee YK, Greenhalgh DG. 2008. Endogenous retroviruses in systemic response to stress signals.

Shock 30(2):105−116.

Cordaux R, Batzer MA. 2009. The impact of retrotransposons on human genome evolution. Nat Rev

Genet 10(10):691−703.

Cowley M, de Burca A, McCole RB, Chahal M, Saadat G, Oakey RJ, Schulz R. 2011. Short interspersed

element (SINE) depletion and long interspersed element (LINE) abundance are not features universally

required for imprinting. PLoS One 6(4):e18953.

de Koning AP, Gu W, Castoe TA, Batzer MA, Pollock DD. 2011. Repetitive elements may comprise over

two-thirds of the human genome. PLoS Genet 7(12):e1002384.

Garcia-Perez JL, Marchetto MC, Muotri AR, Coufal NG, Gage FH, O'Shea KS, Moran JV. 2007. LINE-1

retrotransposition in human embryonic stem cells. Hum Mol Genet 16(13):1569−1577.

Georgiou I, Noutsopoulos D, Dimitriadou E, Markopoulos G, Apergi A, Lazaros L, Vaxevanoglou T,

Pantos K, Syrrou M, Tzavaras T. 2009. Retrotransposon RNA expression and evidence for

retrotransposition events in human oocytes. Hum Mol Genet 18(7):1221−1228.

Georgiou I, Syrrou M, Pardalidis N, Karakitsios K, Mantzavinos T, Giotitsas N, Loutradis D, Dimitriadis

F, Saito M, Miyagawa I, Tzoumis P, Sylakos A, Kanakas N, Moustakareas T, Baltogiannis D,

Touloupides S, Giannakis D, Fatouros M, Sofikitis N. 2006. Genetic and epigenetic risks of

intracytoplasmic sperm injection method. Asian J Androl 8(6):643−673.

Goodier JL, Kazazian HH. 2008. Retrotransposons revisited: the restraint and rehabilitation of parasites.

Cell 135(1):23−35.

Hosoki K, Kagami M, Tanaka T, Kubota M, Kurosawa K, Kato M, Uetake K, Tohyama J, Ogata T,

Saitoh S. 2009. Maternal uniparental disomy 14 syndrome demonstrates prader-willi syndrome-like

phenotype. J Pediatr 155(6):900–903.e1.

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.

20

Inoue A, Shen L, Dai Q, He C, Zhang Y. 2011. Generation and replication-dependent dilution of 5fC and

5caC during mouse preimplantation development. Cell Res 21(12):1670−1676.

Jaenisch R, Bird A. 2003. Epigenetic regulation of gene expression: how the genome integrates intrinsic

and environmental signals. Nat Genet 33(Suppl):245−254.

Jirtle RL, Skinner MK. 2007. Environmental epigenomics and disease susceptibility. Nat Rev Genet

8(4):253−262.

Kanaka-Gantenbein C, Sakka S, Chrousos GP. 2010. Assisted reproduction and its neuroendocrine

impact on the offspring. Prog Brain Res 182:161−174.

Kano H, Godoy I, Courtney C, Vetter MR, Gerton GL, Ostertag EM, Kazazian HH Jr. 2009. L1

retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism. Genes Dev

23(11):1303−1312.

Karolchik D, Hinrichs AS, Furey TS, Roskin KM, Sugnet CW, Haussler D, Kent WJ. 2004. The UCSC

Table Browser data retrieval tool. Nucleic Acids Res 32(Database issue):D493−D496.

Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The human

genome browser at UCSC. Genome Res 12(6):996−1006.

Lavie L, Kitova M, Maldener E, Meese E, Mayer J. 2005. CpG methylation directly regulates

transcriptional activity of the human endogenous retrovirus family HERV-K(HML-2). J Virol

79(2):876−883.

Li Y, Nagai H, Ohno T, Yuge M, Hatano S, Ito E, Mori N, Saito H, Kinoshita T. 2002. Aberrant DNA

methylation p57(KIP2) gene in the promoter region in lymphoid malignancies of B-cell phenotype. Blood

100(7):2572−2577.

Li Z, Sheng T, Wan X, Liu T, Wu H, Dong J. 2010. Expression of HERV-K correlates with status of

MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells. Cancer Invest 28(10):1031−1037.

Lucifero D, Chaillet JR, Trasler JM. 2004a. Potential significance of genomic imprinting defects for

reproduction and assisted reproductive technology. Hum Reprod Update 10(1):3−18.

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.

21

Lucifero D, Mann MR, Bartolomei MS, Trasler JM. 2004b. Gene-specific timing and epigenetic memory

in oocyte imprinting. Hum Mol Genet 13(8):839−849.

Manning M, Lissens W, Bonduelle M, Camus M, De Rijcke M, Libaers I, Van Steirteghem A. 2000.

Study of DNA methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no

imprinting defects. Mol Hum Reprod 6(11):1049−1053.

Michaud EJ, van Vugt MJ, Bultman SJ, Sweet HO, Davisson MT, Woychik RP. 1994. Differential

expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced

by parental lineage. Genes Dev 8(12):1463−1472.

Murphy SK, Wylie AA, Coveler KJ, Cotter PD, Papenhausen PR, Sutton VR, Shaffer LG, Jirtle RL.

2003. Epigenetic detection of human chromosome 14 uniparental disomy. Hum Mutat 22(1):92−97.

Ono M, Kawakami M, Ushikubo H. 1987. Stimulation of expression of the human endogenous retrovirus

genome by female steroid hormones in human breast cancer cell line T47D. J Virol 61(6):2059−2062.

Ono M, Yasunaga T, Miyata T, Ushikubo H. 1986. Nucleotide sequence of human endogenous retrovirus

genome related to the mouse mammary tumor virus genome. J Virol 60(2):589−598.

Piedrahita JA. 2011. The role of imprinted genes in fetal growth abnormalities. Birth Defects Res A Clin

Mol Teratol 91(8):682−692.

Poon LL, Leung TN, Lau TK, Chow KC, Lo YM. 2002. Differential DNA methylation between fetus and

mother as a strategy for detecting fetal DNA in maternal plasma. Clin Chem 48(1):35−41.

Radford EJ, Ferrón SR, Ferguson-Smith AC. 2011. Genomic imprinting as an adaptative model of

developmental plasticity. FEBS Lett 585(13):2059−2066.

Reik W, Walter J. 2001. Genomic imprinting: parental influence on the genome. Nat Rev Genet

2(1):21−32.

Reinhart B, Eljanne M, Chaillet JR. 2002. Shared role for differentially methylated domains of imprinted

genes. Mol Cell Biol 22(7):2089−2098.

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.

22

Rowe HM, Friedli M, Offner S, Verp S, Mesnard D, Marquis J, Aktas T, Trono D. 2013. De novo DNA

methylation of endogenous retroviruses is shaped by KRAB-ZFPs/KAP1 and ESET. Development

140(3):519−529.

Rowe HM, Trono D. 2011. Dynamic control of endogenous retroviruses during development. Virology

411(2):273−287.

Santos MA, Kuijk EW, Macklon NS. 2010. The impact of ovarian stimulation for IVF on the developing

embryo. Reproduction 139(1):23−34.

Shiota K, Yamada S. 2009. Intrauterine environment-genome interaction and children’s development (3):

Assisted reproductive technologies and developmental disorders. J Toxicol Sci 34 Suppl 2:SP287−SP291.

Strogantsev R, Ferguson-Smith AC. 2012. Proteins involved in establishment and maintenance of

imprinted methylation marks. Brief Funct Genomics 11(3):227−239.

Subramanian RP, Wildschutte JH, Russo C, Coffin JM. 2011. Identification, characterization, and

comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses.

Retrovirology 8:90.

Suzuki S, Ono R, Narita T, Pask AJ, Shaw G, Wang C, Kohda T, Alsop AE, Marshall Graves JA, Kohara

Y, Ishino F, Renfree MB, Kaneko-Ishino T. 2007. Retrotransposon silencing by DNA methylation can

drive mammalian genomic imprinting. PLoS Genet 3(4):e55.

Szyf M. 2009. Early life, the epigenome and human health. Acta Paediatr 98(7):1082−1084.

Szyf M. 2010. DNA methylation and demethylation probed by small molecules. Biochim Biophys Acta

1799(10−12):750−759.

Tierling S, Souren NY, Gries J, Loporto C, Groth M, Lutsik P, Neitzel H, Utz-Billing I, Gillessen-

Kaesbach G, Kentenich H, Griesinger G, Sperling K, Schwinger E, Walter J. 2010. Assisted reproductive

technologies do not enhance the variability of DNA methylation imprints in human. J Med Genet

47(6):371−376.

Waterland RA, Jirtle RL. 2003. Transposable elements: targets for early nutritional effects on epigenetic

gene regulation. Mol Cell Biol 23(15):5293−5300.

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.

23

Wells D, Bermúdez MG, Steuerwald N, Malter HE, Thornhill AR, Cohen J. 2005. Association of

abnormal morphology and altered gene expression in human preimplantation embryos. Fertil Steril

84(2):343−355.

Wilkins JF, Úbeda F. 2011. Diseases associated with genomic imprinting. Prog Mol Biol Transl Sci

101:401−445.

Yoder JA, Walsh CP, Bestor TH. 1997. Cytosine methylation and the ecology of intragenomic parasites.

Trends Genet 13(8):335−340.

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;

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.

24

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.

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.

25

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.

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.

26

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.

27

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

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.

28

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

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.

29

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.

30

05-C50 M/U U M/U M/U

05-C51 M M/U M/U M/U

05-C61 M/U U M/U M/U

06-C5 M M/U M/U M/U

06-C24 M M/U M/U M/U

06-C31 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.