Published online February 18, 2004
Nucleic Acids Research, 2004, Vol. 32, No. 3 e31
Large-scale determination of the methylation status
of retrotransposons in different tissues using a
methylation tags approach
Konstantin Khodosevich*, Yuri Lebedev and Eugene D. Sverdlov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 16/10 Miklukho-Maklaya
St., 117997 Moscow, Russia
Received December 10, 2003; Revised and Accepted January 24, 2004
ABSTRACT
A technique for simultaneous determination of the
methylation status of numerous loci containing
retroelements (REs) is reported. It is based on the
observation that methylated and unmethylated
areas in the genome are usually extended,
and therefore the methylation of particular methyl-
sensitive restriction endonuclease recognition sites
might re¯ect the methylation status of DNA regions
around them. The method includes dot-blot hybridi-
zation of repeat ¯anking sequences arrayed on a
solid support with speci®cally ampli®ed ¯anking
regions of presumably unmethylated repeats. A
multitude of ¯anking regions of REs adjacent to
unmethylated restriction sites are ampli®ed simul-
taneously, providing a complex hybridization probe.
The technique thus allows the determination of the
methylation status of restriction sites, which serve
as tags of the methylation status of the surrounding
regions. The validity of the technique was con®rmed
by various means, including bisul®te sequencing.
The technique was successfully applied to the
identi®cation of methylation patterns of the regions
surrounding 38 human-speci®c HERV-K(HML-2)
long terminal repeats in cerebellum- and lymph
node-derived genomic DNAs. The described
technique can be readily adapted to the use of DNA
microarray technology.
INTRODUCTION
It is widely recognized that DNA methylation has profound
effects on gene expression. DNA methylation plays an
important role in embryonic development, X-chromosome
inactivation, imprinting, and suppression of repetitive
sequences in the genome (1). Approximately 80% of CpG
residues in the mammalian genome are methylated in most
adult tissues (2). CpG dinucleotides are distributed non-
randomly along DNA and clustered within so-called CpG
islands that are usually protected from methylation in normal
cells (3) and possibly are involved in tissue speci®city of gene
*To whom correspondence should be addressed. Tel: +7 095 330 6329; Fax: +7 095 330 6538; Email: kosthob@humgen.siobc.ras.ru
Nucleic Acids Research, Vol. 32 No. 3 ã Oxford University Press 2004; all rights reserved
DOI: 10.1093/nar/gnh035
expression, although this suggestion is still not reliably proven
(1). Even less is known about functions of CpG dinucleotides
located outside CpG islands. One of the important functions
usually assigned to methylation is suppression of the activity
of retroelements (REs), including that of long interspersed
elements (LINEs), short interspersed elements (SINEs) and
long terminal repeats (LTRs).
Due to evolutionary selection, REs are mostly located in
`inactive' genomic regions such as heterochromatin and are
heavily methylated. Moreover, it is quite possible that newly
integrated REs methylated by the cellular methylation
machinery become centers of spreading methylation over
surrounding genomic regions, thus suppressing their func-
tional activity (4). On the other hand, a multitude of REs were
`domesticated' by mammalian genomes and might be
involved in modulation of expression of resident genes (5,6).
The involvement of REs in regulation of gene expression has
been demonstrated in a number of studies (7±10). REs of this
group can be suggested to be active non-methylated regulatory
units located in active non-methylated genome areas.
Determination of the methylation status of REs and their
adjacent regions may be used to discriminate between
potentially active and inactive REs. Furthermore, insofar as
each type of RE represents a group of repetitive elements with
conserved sequence features, REs can be used as universal
genomic anchors allowing the design of easily detectable tags
of the methylation status of the loci where they are located.
In this work, we developed a new technique for detection of
tags of the methylation status of genomic loci containing
interspersed repeats, and successfully applied it to 38 LTRs of
human endogenous retroviruses (HERVs) belonging to the
most functionally active family, HERV-K(HML-2), and
located in different genomic loci (11±13). The methylation
patterns obtained in such a way for cerebellum- and lymph
node-derived genomic DNAs were compared and shown to be
different. The validity of this technique for determining the
methylation status was con®rmed by bisul®te sequencing and
PCR ampli®cation.
MATERIALS AND METHODS
Genomic DNA isolation, restriction and adaptor ligation
Genomic DNA from human tissues was isolated using
proteinase K digestion and phenol extraction as described
e31 Nucleic Acids Research, 2004, Vol. 32, No. 3
Table 1. Oligonucleotides and primers used in hybridization probe
construction
Primers Structure (5¢±3¢)
A1A2 AGCAGCGAACTCAGTACAACAAGTCGACGCGTGCCC-
GGGCTGGT
A1 AGCAGCGAACTCAGTACAACA
A2 AGTCGACGCGTGCCCGGGCTGGT
a1 CGACCAGCCC
T1 CCACCTTACGAGAAACACCCACAG
T2 TGTTTCAGAGAGCACGGGGTTGGG
T3 CGAGAAACACCCACAGGTGTG
T4 TCTGATCTCTCTTGCTTTTCCCCAC
(14). A 1 mg aliquot of the genomic DNA from each tissue was
digested with 70 U of HpaII restriction enzyme (New England
Biolabs) at 37°C overnight in 100 ml of the reaction mixture.
The completeness of HpaII digestion was con®rmed by PCR
with primers to either side of four previously identi®ed non-
methylated CCGG sites located within exons, introns or
promoter regions of housekeeping genes (see table 1 of the
Supplementary Material available at NAR Online). The
digested DNAs were ethanol precipitated, dissolved in 10 ml
of sterile water and ligated to an excess of adaptor (2 mM) at
16°C overnight using T4 DNA ligase (Promega). To form the
adaptor, 300 pmol of each of the oligonucleotides A1A2 and
a1 (Table 1) were annealed in 20 ml of TM (10 mM Tris±HCl,
pH 7.8, 10 mM MgCl2) buffer. The ligation was terminated by
incubation at 65°C for 15 min. The ligates were then puri®ed
from excess oligonucleotides by passing them through a
QIAquick DNA Puri®cation Kit (Qiagen), and their 3¢ ends
were ®lled in by the Klenow fragment of DNA polymerase I
(Fermentas). Finally, the ligates were ethanol precipitated and
redissolved in 40 ml of sterile water.
Preparation of hybridization probes
Fractions of LTR-U5 and LTR-U3 ¯anking sequences were
ampli®ed using a PCR suppression approach (15). In the ®rst
round of suppressive PCR, A1 primer was used with T1 or T2
primers (Table 1). Primers T1 and T3, and T2 and T4 (Table 1)
correspond to the most conserved parts of the U5 and U3
regions of HERV-K(HML-2) LTRs, respectively. The PCR
mixture contained 10 ng of the ligate in 25 ml of PCR Buffer
for AdvantageÔ Taq (BD Biosciences, Clontech) containing
200 mM of each dNTP, 0.4 mM of each primer and 3 U of
Advantage Taq DNA polymerase (BD Biosciences,
Clontech). PCR was carried out in a thermal cycler (MJ
Research) as follows: 22 cycles at 95°C for 20 s, 65°C for 30 s,
72°C for 2 min. The PCR products were diluted 1000-fold and
re-ampli®ed with A2, T3 and T4 primers (Table 1) in the
second PCR round (17 cycles at 95°C for 20 s, 65°C for 30 s,
72°C for 2 min).
Hybridization membranes preparation
Primers against ¯anking sequences of 38 human-speci®c
HERV-K(HML-2) LTRs were designed using the `Gene
Runner (Version 3.00)' program (Hastings Software, Inc.).
They were synthesized and used for PCR ampli®cation of
human placenta DNA. Aliquots of the PCR products (50 ng
PAGE 2 OF 8
Table 2. Primers used in bisul®te sequencing
Primers Structure (5¢±3¢)
LTR11bisfor1 TATAAGTTGGATTTGTATTAGAGGATTTG
LTR11bisfor2 GGTTTAGAGTTTAGGAGTTTTGGTTGTTAG
LTR11bisrev1 CTTACTCACACTAAACCTAACAATAAATACTC
LTR11bisrev2 ACATCCCCAACCTACATCTCCCTC
LTR12bisfor1 GTAGGATATGAAGTATGGAGAATAGGTAAG
LTR12bisfor2 TATTTTTGTAGGGATGTATAGTGTTGGG
LTR12bisrev1 CACCTATAAACCATCCTTTACTATTATAAAC
LTR12bisrev2 AAAATCCTTTTACCTCCATTCATCTTC
LTR27bisfor1 TTTTGTTATTGAGAAGTATATAGTTTAGTGG
LTR27bisfor2 GGAAGAAGAGTTAATTGGTGGTATAAGG
LTR27bisrev1 AAATCTCTAATACCATTCCTCCTACC
LTR27bisrev2 ATAAAAATCCCAATATTAAAATCACATCC
The PCR primers were designed to be fully complementary to the
deaminated DNA strand and did not include CG dinucleotides. First round
ampli®cations (with primers for1 and rev1) were performed in 25 ml of PCR
buffer for Taq DNA polymerase (Promega) containing 200 mM of each
dNTP, 1.5 mM MgCl2, 0.4 mM of each primer and 1 U of Taq DNA
polymerase (Promega), with individual 3 ml beads in a thermal cycler (MJ
Research) as follows: 35 cycles at 95°C for 20 s, 63°C for 30 s, and 72°C
for 1.5 min. Ampli®cations using nested primers (for2 and rev2) were
performed using 1 ml of the PCR mixture after the ®rst round under PCR
conditions as follows: 22 cycles at 95°C for 20 s, 60°C for 30 s, and 72°C
for 1.5 min.
each) were then spotted and immobilized on nylon membranes
(Hybond N; Amersham) according to the manufacturer's
recommendations. The same amount of l phage DNA was
also spotted on the membranes as a negative hybridization
control.
Dot-blot hybridization
Hybridization probes were prepared by random labeling of the
second round suppressive PCR products (see Preparation of
hybridization probes). The DNA fragments dotted on the
membrane and the probes had the same sequences at their 5¢ or
3¢ termini, i.e. 25 and ~40 bp when ampli®ed from the LTR U3
or U5 terminal regions, respectively. To prevent cross-
hybridization between these sequences, the hybridization
mixture was supplemented with a 200-fold molar excess of
unlabeled competitive oligonucleotides corresponding to the
common parts of the spotted ¯anks. After random labeling,
probes were diluted, mixed with competitive oligonucleotides
(20 pmol each), denatured at 100°C for 5 min, immediately
chilled on ice and added to the hybridization mixture. Dot-blot
hybridization was performed at 68°C overnight in a buffer
containing 63 SSC, 53 Denhardt's, 0.5% SDS and 100 mg/ml
salmon sperm DNA. The membranes were washed under high
stringency conditions and exposed to X-ray ®lm (Renex,
Russia) for 2 days.
Ampli®cation of the second round PCR products
The results of dot-blot hybridizations were veri®ed by PCR to
check a correlation of signal intensity with the abundance of
the corresponding ¯anking DNA in the amplicon used as a
hybridization probe. PCR ampli®cations used T3 or T4 LTR
primers (Table 1) with the primers speci®c for particular LTR
¯anking sequences (primer sequences and PCR conditions are
shown in table 2 of the Supplementary Material), and the
second round PCR products as templates.
PAGE 3 OF 8 Nucleic Acids Research, 2004, Vol. 32, No. 3 e31

Figure 1. Scheme of the method. After restriction and adaptor ligation (stage 1), an unmethylated pool of RE ¯anks is selectively PCR ampli®ed (stage 2)
and hybridized (stage 3) with an (micro)array of the whole pool of the same RE-¯anking sequences. Methylated and non-methylated HpaII (HhaI) restriction
sites are shown by short vertical lines with black circles or without circles, respectively. REs and oligonucleotide adaptors are shown as green and
black-and-white boxes, respectively. Primer positions and orientations are indicated by arrows.

Bisul®te sequencing using primers T3 or T4 (Table 1) and primers corresponding to

RE ¯anks were sequenced using agarose beads by a modi®ed
method for bisul®te-based cytosine methylation analysis, as
described (16). Lymph node-derived DNA was digested with
EcoRI restriction endonuclease, denaturated and then treated
with bisul®te to convert all unmethylated cytosines into
uracils. This reaction did not affect methylated cytosine
residues. For each conversion, 100 ng of lymph node DNA
was taken. The converted DNA fragments were subjected to
two rounds of PCR ampli®cation using a nested primer
approach (Table 2) and then cloned into a TA cloning vector
(Promega). Four clones for each RE-¯anking fragment were
sequenced. The cytosine residues converted with bisul®te are
identi®ed in the course of sequencing as Ts.
PCR of genomic DNA digested by HpaII
Cerebellum- and lymph node-derived DNAs were digested as
described above in Genomic DNA isolation, restriction and
adaptor ligation. The digested DNAs were then PCR ampli®ed
LTR-¯anking sequences (primer sequences are available on
request). The PCR mixture contained 10 ng of the digested
DNA in 25 ml of PCR buffer for Taq DNA polymerase
(Promega) containing 200 mM of each dNTP, 0.4 mM of each
primer and 1 U of Taq DNA polymerase (Promega). PCR was
carried out in a thermal cycler (MJ Research) as follows: 28
cycles at 95°C for 20 s, 61°C for 30 s, and 72°C for 1 min.
RESULTS
Technique rationale
The technique we have developed is aimed at whole-genome
identi®cation of DNA methylation patterns of interspersed
repeats (interspersed repeat-containing loci) and is based on
the DNA array technique that can be easily adapted for DNA
microchip technology. The technique uses two basic proper-
ties of interspersed repeats: (i) characteristic conservative
sequence features that can be used for selective simultaneous
e31 Nucleic Acids Research, 2004, Vol. 32, No. 3
Figure 2. Dot-blot hybridization with cerebellum (A and B) and lymph
node (C) DNA-derived probes. LTRs are numbered from left to right, and
from top to bottom. In (A) and (C), position A1 corresponds to LTR1, A2
to LTR2, etc., B1 to LTR13, etc., C1 to LTR25, etc., D1 to LTR37, and D2
to LTR38. Spots D3 in (A) and (C) and A11 in (B) correspond to 50 ng of
phage l DNA. Designation of spots in (B) corresponds to that in (A). Rows
A and B in (B) represent the same materials spotted in duplicate.
isolation of ¯anking sequences for all REs; and (ii) different
¯anking sequences that allow reliable discrimination of
individual RE integration sites. Thus, REs can be considered
natural repetitive universal markers of a multitude of genomic
loci. Here we used these markers for evaluation of the
methylation status of genomic regions surrounding HERV-K
LTRs that were integrated in the human genome after the
divergence of the human and chimpanzee lineages. The
principles of the approach used are schematized in Figure 1.
The sequences representing ¯anks of REs were immobi-
lized on a solid support as a DNA array. These RE-¯anking
sequences can be prepared by any available technique
(chemical synthesis, PCR ampli®cation, etc.). In the present
work, we used ¯anks of HERV-K(HML-2) LTRs prepared by
selective PCR ampli®cation (Fig. 1).
A pool of genomic RE-¯anking regions was prepared by
selective PCR ampli®cation with one of the primers repre-
senting a universal RE-speci®c sequence designed from the
consensus of the RE type under investigation. The other
primer was also universal but targeted at the arti®cial adaptors
attached to the termini of the restriction fragments obtained by
digestion of the genomic DNA with a methyl-sensitive
restriction endonuclease. The labeled amplicon was used for
hybridization with the array (Fig. 1, stage 3). Such a
hybridization is supposed to reveal only those spots on the
array that correspond to non-methylated RE-¯anking restric-
tion sites.
In our case, we used HERV-K(HML-2) LTR-speci®c
primers, and the amplicon/hybridization probe thus contained
PAGE 4 OF 8
¯anks of HERV-K(HML-2) LTRs obtained by selective PCR
ampli®cation of the genomic DNA (Fig. 1, stage 2) digested
with a methyl-sensitive endonuclease, such as HpaII or HhaI
(Fig. 1, stage 1). Therefore, the selection resulted in only those
genomic fragments bordered by the nearest to the LTR non-
methylated restriction sites.
To make the ampli®cation of the target fragments as
speci®c as possible, we used the PCR suppression effect
through ligation of special PCR suppressive adaptors to the
restriction fragments (Fig. 1, stage 1). This modi®cation
prevents PCR ampli®cation of the fragments lacking REs,
with the adaptors on both termini, but does not preclude the
ampli®cation of RE-containing fragments by PCR initiated
from sites within REs.
Comparative analysis of the distribution of methylation
sites neighboring the LTRs in human cerebellum and
lymph node
The genomic DNAs isolated from cerebellum and lymph node
and digested with methyl-sensitive restriction endonuclease
HpaII were ligated to the suppressive adaptors (see Materials
and Methods). The ®rst round of the PCR-suppressive
selective ampli®cation was followed by nested primer PCR
to improve the selectivity. The amplicons obtained were
labeled with 32P and used as hybridization probes.
Individual ¯anking regions of 38 human-speci®c HERV-K
(HML-2) LTRs were prepared by PCR ampli®cation using
primers against the outermost parts of the LTR (T3 or T4,
Table 1) and a unique region of the adjacent genomic ¯ank,
respectively (primer sequences are available on request). All
the 38 ¯anking sequences obtained were dotted on Hybond-N
nylon membranes. One of the identical membranes was then
hybridized with a human cerebellum DNA-derived probe, and
the other with a probe prepared from human lymph node
DNA.
Figure 2 shows the resulting hybridizations of the human-
speci®c LTR-¯anking sequences with the human cerebellum
and lymph node DNA-derived probes. The results of the
hybridizations were used to identify the methylation status of
CpG dinucleotides within various HpaII cleavage sites in the
LTR-¯anking sequences. Although a similar number of
positive hybridization signals (approximately 15) was
observed with the cerebellum (Fig. 2A) and lymph node
(Fig. 2B) probes, the intensities and patterns of the signals
were signi®cantly different (see Table 3). The differences in
intensities might be due to different methylation levels of
various HpaII sites that lead to the different abundance of the
corresponding fragments in the hybridization probes. Some of
the HpaII sites in the LTR-¯anking regions have the same
methylation status in both tissues. For example, the HpaII site
adjacent to LTR28 (C4 position in Fig. 2) seems to be non-
methylated in both cerebellum and lymph node. In contrast,
the HpaII site adjacent to LTR2 (A2 position in Fig. 2) is
apparently heavily methylated in both tissues. Some loci have
a different methylation status in these tissues. In particular, the
LTR18-adjacent region (B6 position in Fig. 2) is more
methylated, and the ¯ank of LTR27 (C3 position in Fig. 2) is
less methylated in lymph node than in the cerebellum-derived
DNA.
The hybridization signals did not depend on probe or dotted
DNA lengths or on the presence of various repetitive elements
PAGE 5 OF 8 Nucleic Acids Research, 2004, Vol. 32, No. 3 e31

Table 3. Probe and dotted DNA characteristics for analyzed LTRs

Accession no. Length of probe fragments (bp); Length of dotted fragments (bp); Repeats within ¯ank sequences Hybr signal
positions in corresponding positions in corresponding for dotted fragments
accession no. accession no.
Cerebellum Lymph node

LTR1 AC044819 1170 (130 901±131 973) 280 (130 901±131 181) 1±250 bp HERV7 ± ±
LTR2 AC025420 1910 (46 613±48 529) 120 (46 613±46 736) 1±180 bp L2 ± ±
LTR3 AC010267 1430 (106 778±108 212) 130 (106 778±106 909) 1±90 bp MSTA ± ±
LTR4 AC002508 430 (33 657±34 088) 300 (33 657±33 952) 20±150 bp MLTJ ± ±
LTR5 AL354855 490 (71 833±72 321) 210 (71 833±72 042) 120±250 bp L1 ± +
LTR6 AC012146 990 (24 102±25 089) 70 (24 102±24 171) No repeats ± ±
LTR7 AL671681 1120 (167 436±168 552) 100 (167 436±167 534) 1±120 bp MLT1A1 + +
LTR8 AL139421 530 (69 211±69 744) 820 (69 211±70 031) No repeats ± +
LTR9 AL592220 320 (93 880±94 203) 270 (93 933±94 203) 1±60 bp AluSg ± ±
LTR10 AL359701 520 (61 942±62 464) 650 (61 817±62 464) 1±700 bp HERV9 + ±
LTR11 AC021987 1100 (20 313±21 412) 50 (21 360±21 412) 1±20bp LTR26 + +
LTR12 AC074117 530 (154 755±155 289) 160 (155 132±155 289) No repeats ± ±
LTR13 AL139404 600 (60 431±61 027) 50 (60 977±61 027) 1±50 bp LTR49 ± ±
LTR14 AC084028 50 (141 209±141 258) 150 (141 209±141 356) 1±120 bp MLT1A1 ± ±
LTR15 AL162412 1610 (66 382±67 997) 210 (67 791±67 997) 150±250 bp AluSx ± ±
LTR16 AL139090 580 (1536±2115) 280 (1536±1816) No repeats + ±
LTR17 AC024884 170 (43 103±43 169) 40 (43 103±43 144) No repeats ± ±
LTR18 AC000389 180 (9548±9731) 680 (9052±9731) No repeats + +
LTR19 AC113425 240 (41 008±41 248) 202 (41 046±41 248) No repeats ± +
LTR20 AC021294 1410 (145 000±146 412) 200 (146 216±146 412) 1±200 bp L2 ± ±
LTR21 AC091895 1080 (102 415±103 493) 210 (102 415±102 625) 1±240 bp L1PA13 + ±
LTR22 AL162723 210 (19 165±19 370) 130 (19 240±19 370) 1±50 bp MSTA ± ±
LTR23 AC099661 1590 (149 214±150 807) 160 (150 643±150 807) No repeats ± +
LTR24 AC074019 950 (111 321±112 273) 330 (111 947±112 273) 1±300 bp L1MC ± ±
LTR25 AL359644 550 (93 908±94 457) 40 (94 417±94 457) No repeats ± ±
LTR26 AP000812 1020 (74 214±75 234) 400 (74 838±75 234) 1±40 bp MER61 + +
LTR27 AC002400 380 (109 107±109 490) 330 (109 107±109 437) 1±150 bp L2 + +
LTR28 U47924 410 (97 456±97 869) 660 (97 206±97 869) 250±500 bp AluSg + +
LTR29 AP001591 700 (52 405±53 106) 110 (52 405±52 510) 70±150 bp MER58A + +
LTR30 Z84493 90 (8120±8210) 520 (7689±8210) No repeats + +
LTR31 AC002350 850 (45 768±46 614) 260 (45 768±46 027) No repeats + +
LTR32 AC073898 900 (78 284±79 177) 150 (79 029±79 177) No repeats ± +
LTR33 Z80898 310 (6071±6380) 270 (6071±6337) 1±250 bp L1PA1 + +
LTR34 AC007326 1120 (35 759±36 874) 770 (36 100±36 874) 1±150, 450±550 bp AluJ, ± ±
150±450 bp ERV1
LTR35 AC023074 1060 (119 305±120 365) 330 (120 037±120 365) 120±160 bp L1PA8A ± +
LTR36 AC006432 1810 (10 078±11 894) 410 (10 078±10 490) No repeats + ±
LTR37 AL109763 210 (72 566±72 773) 670 (72 566±73 233) 1±700 bp L2 + +
LTR38 AP001631 120 (108 235±108 355) 110 (108 235±108 345) No repeats + ±

in these DNAs (see Table 3). This ®nding suggests that the
signal intensity correlates with the abundance of the corres-
ponding ¯ank DNA in the amplicon used as the hybridization
probe. To verify this correlation, seven LTR ¯anks were PCR
re-ampli®ed using speci®c primers with the amplicon as a
template. The amount of each individual ¯ank DNA was
estimated by the minimal number of PCR rounds suf®cient to
visualize the band on electrophoregrams of the PCR products.
For all of the investigated LTRs, the number of PCR cycles
was found to be in a good reciprocal correlation with the
intensities of the corresponding hybridization signals on the
array. The results demonstrated that the intensity of the
hybridization signals was mostly proportional to the content of
the corresponding product in the hybridization probe
(amplicon).
To con®rm the technique reproducibility, we repeated the
hybridization using independently prepared probes and ®lters.
In all cases, the relative intensities of the hybridization signals
were in good accord with those presented in Figure 2.
Figure 2A and B demonstrates the hybridization reproduci-
bility as well as the total reproducibility of relative intensities
of the signals in independent experiments.
PCR ampli®cation through HpaII sites con®rms the
methylation status determined by the DNA array
The methylation status of some HpaII sites was additionally
veri®ed by PCR through them using primers hybridizing on
either side of the sites and DNA digested with HpaII. In this
case, the DNA fragments with an internal methylated HpaII
site are supposed to be ampli®ed, while the ampli®cation of
the fragments with an unmethylated site will fail because such
sites are not protected from digestion with HpaII.
As an example, Figure 3 presents PCR ampli®cations for
three arbitrarily chosen LTR ¯anks. It can be seen that the
HpaII sites adjacent to LTR30 and LTR28 are unmethylated in
both lymph node and cerebellum DNAs, while the ¯ank of
e31 Nucleic Acids Research, 2004, Vol. 32, No. 3 PAGE 6 OF 8

Figure 3. PCR through HpaII sites of interest. (A) Cerebellum- and lymph node-derived DNAs were digested by the methyl-sensitive restriction endonuclease
HpaII and ampli®ed with primers speci®c to either side of the HpaII site (e.g. P1 and P2, or P3 and P2, where P2 is the primer against the LTR sequence).
Methylated or unmethylated CpG/CCGG sites are designated by ®lled and empty circles, respectively. (B) Gel electrophoresis of the PCR products after
ampli®cation for the LTR28 (lanes 1±3), LTR30 (lanes 4±6) and LTR14 (lanes 7±9) ¯anks through adjacent HpaII sites. Lanes 2, 5 and 8, and 3, 6 and 9
represent PCR products generated by ampli®cation of HpaII-digested DNAs from cerebellum and lymph node, respectively. Lanes 1, 4 and 7 correspond to
the ampli®ed DNA fragments from native placenta. Lane 10, length marker.

Figure 4. Results of bisul®te sequencing. For each of three LTR-¯anking sequences, four clones were sequenced. The methylation results obtained for each
of the clones are schematically presented at the bottom of the ®gure as lines of circles. Filled (black) and empty circles designate methylated and
unmethylated CpGs, respectively.

LTR14 is methylated in both tissues. These and our previous
data on LTR methylation analysis (17) are in good accord with
the results of the DNA array hybridization.
Con®rmation of the correlation of the methylation status
of HpaII sites and neighboring CpGs by bisul®te
sequencing
Three arbitrarily chosen LTR ¯anks were taken for bisul®te
sequencing (Table 2). CpG dinucleotides within the ¯ank of
LTR12 were found to be predominantly methylated, whereas
the LTR11 ¯ank was unmethylated in lymph node-derived
DNA. The results shown in Figure 4 allow one the following
conclusions to be drawn. (i) For all the sequences analyzed,
the results on the methylation status of HpaII sites obtained
with all the techniques used (¯ank DNA arrays, PCR
ampli®cation of HpaII-digested genomic DNAs, and bisul®te
sequencing) agreed well with each other. (ii) The methylation
status of HpaII sites in all three studied cases coincided with
the methylation status of the nearest CpG dinucleotides, thus
demonstrating that methylation follows the cooperative prin-
ciple due to which methylated and unmethylated CpGs are
clustered. This feature of methylation was previously reported
as `methylation spread' (4). Due to this property, the
methylation status of a particular CpG within such a cluster
PAGE 7 OF 8
is characteristic of the whole cluster. Accordingly, a
methylated/unmethylated recognition site of a methylation-
sensitive restriction endonuclease within an extended region
can serve as a tag of the methylation status of this region.
DISCUSSION
Although REs are often considered inert components of the
genome, there is evidence of their participation in genome
functioning (6,13,18±21). However, genome-wide analysis of
the functional status of LTRs is still at the very beginning and
restricted to some individual LTRs chosen more or less on a
random basis. The novel technique described here allows one
to perform systematic genome-wide analysis of the methyla-
tion status of CpG sites neighboring LTRs. These sites might
serve as tags of methylation of extended regions harboring the
LTRs. In turn, the methylation status of a genomic region may
re¯ect its functional status. A rationale for such a suggestion is
a widely discussed methylation spread effect (4), i.e. an ability
of methylation to spread over adjacent genome regions. The
results obtained here with bisul®te sequencing are in agree-
ment with this effect. Indeed, Figure 4 clearly demonstrates
clustering of methylated and unmethylated CpGs. Thus we
can consider HpaII sites with their methylation status as tags
of the methylation status of the wider surrounding region.
The technique described here allowed us to show that some
human-speci®c LTRs are differentially methylated in human
tissues, their methylation status being probably linked with the
expression of neighboring genes. The technique can be used
for analysis of methylation patterns of any low and medium
copy number REs of genomes. In particular, it might be useful
for studying certain subgroups of such important human
genome constituents as ERVs, SINEs and LINEs. Although
these RE families are very abundant in the human genome,
being represented by ~4.5 3 105, 1.6 3 106 and 8.7 3 105
individual elements (22), respectively, each family consists of
a lot of less abundant subfamilies ampli®ed during various
evolutionary periods. Using our technique, each subfamily can
be analyzed separately. The youngest HERV, L1 and Alu
subfamilies comprise from tens to hundreds of members (23±
25). Some of these recently inserted REs are still not ®xed in
the human species, and it would be interesting to estimate their
impact on the functioning of the host genome.
The technique can easily be applied to any family of repeats
providing that their copy number is not too high, because the
main restriction of the method is the complexity of
hybridization probes prepared by selective PCR ampli®cation.
High complexity of the probes results in weak hybridization
signals.
There are also some limitations to the technique regarding
tissue samples due to their heterogeneity that leads to
heterogeneous methylation of DNA isolated from different
cells of one and the same tissue. However, positive
hybridization signals always mean the presence of unmethy-
lated sites in a given sample. Sorting of cells using, for
example, a cell sorter might improve the results.
We hope that the technique described here will be helpful
for a better understanding of the role of REs in genome
functioning and molecular evolution. It is an ef®cient and
inexpensive tool for genome-wide analysis of methylation
Nucleic Acids Research, 2004, Vol. 32, No. 3 e31
pro®les and therefore for studying epigenetic effects of REs
that is still a black box of genome functioning analysis.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
The authors thank Boris O. Glotov for critical reading of the
manuscript and valuable comments. The work was supported
by INTAS 01-0759 and the Russian Foundation for Basic
Research 01-04-48900 and 2006.200054 grants, and by the
Physico-Chemical Biological Program of the Russian
Academy of Sciences.
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