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Russian Journal of Bioorganic Chemistry, Vol. 30, No. 5, 2004, pp. 441–445. Translated from Bioorganicheskaya Khimiya, Vol.

30, No. 5, 2004, pp. 493–498.


Original Russian Text Copyright © 2004 by Khodosevich, Lebedev, Sverdlov.

The Tissue-Specific Methylation of Human-Specific Endogenous


Retroviral LTRs
K. V. Khodosevich1, Yu. B. Lebedev, and E. D. Sverdlov
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
Received July 23, 2003; in final form, October 27, 2003

Abstract—A possible involvement of retroelements in the epigenetic regulation of human gene expression was
considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus
family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched
regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results cor-
related with published data on the tissue-specific changes in the expression level of human genes adjacent to
the LTRs under study.

Key words: endogenous retroviruses, gene expression, human genome evolution, methylation

1 INTRODUCTION binding of specific proteins to the methylated CpG


The human genome contains dozens of thousands of dinucleotides apparently initiates changes in the tissue-
endogenous retroviruses and their long terminal repeats specific gene expression, which finally causes the inhi-
[1].2 These elements are potential regulatory elements, bition of expression of the gene containing this CpG
which include various transcription regulators: promot- island. It is considered that the change in the DNA
ers, enhancers, the elements of response to hormonal methylation status in various tissues occurs at extended
and polyadenylation signals. LTRs were shown to be genome regions, the so-called methylation blocks,
functional promoters of several human genes [2, 3]. rather than at single CpG sites [9]. The methylation-
The results of the experiments on the reporter gene induced LTR inactivation is presumed to protect cells
expression confirmed a pronounced tissue-specific pro- from the unregulated expression of many genes, which
moter and enhancer activities in the LTR elements of could take place due to the LTR promoter–enhancer
various families [4–8]. The activity of LTRs together activities. On the other hand, the demethylated status of
with their ability to be inserted near genes during the LTR may point to its functional activity. Therefore, the
evolution process and, in such a manner, to alter the study of the LTR methylation status is important to
regulation of these genes makes LTRs promising candi- judge on their functional status and possible role in the
dates for the role of factors affecting speciation. The regulation of adjacent genes.
LTR activity in genome depends on numerous factors, In this work, we tried for the first time to reveal the
the methylation status being probably one of them. changes in the methylation status of integration sites of
The methylation of cytosine bases in DNA and the the HERV-K LTR in various human tissues.
modifications of histones that determines the chromatin
compacting are considered to be two major mecha-
nisms of the epigenetic regulation of gene expression. RESULTS AND DISCUSSION
It is accepted that approximately 80% of CpG dinucle- We selected the group of single HERV-K (HML-2)
otides in the DNA of mammalian nonembryonic cells LTRs specific for human genome and, hence, possibly
are methylated [9]. At the same time, the CpG distribu- involved in the process of separation of evolutionary
tion in the genome is not random: the DNA sequence is lines of humans and chimpanzee for an analysis of the
relatively poor in CpG dinucleotides and most of them LTR methylation status in various tissues. The localiza-
are concentrated at small regions called CpG islands. tion of LTR sequences maximally close to several
These sites are usually demethylated in normal tissues, known or predicted genes was another selection crite-
and their methylation in tumor cells may cause the inhi- rion. Previously, we have identified and mapped
bition of expression of tumor-suppressor genes. The approximately 150 HERV-K (HML-2) LTRs that dis-
1 Corresponding author; phone: +7 (095) 330-6329; fax +7 (095)
tinguish human and chimpanzee genomes [10, 11]. An
330-6538; e-mail: kosthob@humgen.siobc.ras.ru analysis of gene surroundings showed that about 30%
2 Abbreviations: LTR, long terminal repeat; HERV-K (HML-2), regions of LTR human-specific integrations are located
human endogenous retrovirus of family K, subfamily HML-2. as close as 50 kbp from various human genes, including

1068-1620/04/3005-0441 © 2004 MAIK “Nauka /Interperiodica”


442 KHODOSEVICH et al.

0 20 40 60 80 100 120 140 160


kbp
14q23.3

LTR1

My015 GPX2 FNTB


9q34.13
LTR2

MGC12921 MGC10526
1p31.2
LTR3

PDE4B
2p23.3

LTR4

NRBP KCP3 AL110218 FRCP1 GCKR


11q13.3
LTR5

GSTP1 FLJ90834 BC018644 ACY-3 ALDH3B2


NDUFV1 16p12.2

LTR6

UBPH NDUFAB1 FLJ21816 MGC3248

Fig. 1. The scheme of locus structures containing the LTRs under study. The chromosome regions with the corresponding locus
names at the right sides are designated with arrows. The arrow direction corresponds to the locus orientation toward the telomer.
Open rectangles on the arrows correspond to the CpG island positions. Fine arrows under the locus schemes designate the transcrip-
tion direction of the genes, whose names are given beneath them. The exon positions are marked with black vertical rectangles on
the arrows. Light arrows above the gene schemes show the LTR positions and U3–U5 directions of LTRs. The LTR numeration
corresponds to that in the text and the tables. A scale in kbp is given above the locus and gene schemes.

12 LTRs localized in intron regions [12, 13]. An addi- place. If the site is methylated, no cleavage will take
tional analysis of this LTR group using Human Genome place, and the DNA will be amplified. The DNA prep-
Draft Browser programs (see the Experimental section) arations from various tissues, including cerebellum,
allowed us to choose six sequences located in various liver, kidney, lymph node, stomach, and lung, were
gene-enriched loci of human genome. used for the methylation analysis.
The loci containing the LTRs under study are sche- The completeness of the DNA hydrolysis was
matically presented in Fig. 1. Three LTRs, arbitrarily checked using the control locus-specific PCR proce-
designated as LTR4, LTR5, and LTR6, are localized in dures. Four primer pairs corresponding to the unique
2p23.3, 11q13.3, and 16p12.2 loci containing four to genomic sequences flanking the HpaII recognition sites
six genes with short intergenic regions at the DNA sites in constitutively demethylated fragments of the house-
less than 100 kbp long. The LTR3 is localized in the last hold genes WT1, RB1, BRCA1, and CDKN1A were
intron of the gene of the type 4B cAMP-specific phos- used for this PCR procedure. The absence of the PCR
phodiesterase. LTR1 and LTR2 are located in inter- product after the amplification of the HpaII-treated
genic regions in the immediate vicinity to CpG islands. DNA and the simultaneous formation of the PCR prod-
Five of the six loci under study were shown to have an uct on a template of the initial DNA indicated that the
increased density of the DNA fragments enriched with restriction proceeded completely.
CpG repeats (Fig. 1). All the LTRs used in this study contained a single
The methylation status of the LTRs under study was HpaII recognition site. We carried out the PCR proce-
determined using the PCR amplification of the frag- dure with the primers corresponding to the genomic
ments of genomic DNA hydrolyzed with the methyla- regions flanking the studied LTRs, in order to analyze
tion-sensitive restriction endonuclease HpaII (Fig. 2a). the methylation degree of this site in various human tis-
The method is based on the use of the primer pairs sues (see, e.g., Fig. 2b). In this case, the PCR fragment
(Table 1) between the binding sites of which the HpaII of the expected length (1780 bp) was obtained by the
site is located in DNA. If the site is not methylated, the amplification of the HpaII-digested genomic DNA
DNA will be cleaved, and no amplification will take from liver, cerebellum, lymph node, and lung cells,

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004


THE TISSUE-SPECIFIC METHYLATION 443

(a) 4for 4for

LTR 4rev LTR 4rev


Treatment with HpaII and PCR
with primers 4for and 4rev

4for

LTR 4rev
No cleavage Cleavage
A full-size PCR product No amplification

(b) 1 2 3 4 5 6 M

bp
2000
1780 bp 1500
1000
750
500

250

Fig. 2. Determination of the methylation status of single LTRs. (a) Experimental scheme. Black and open circles show methylated
and nonmethylated HpaII-restriction sites, respectively, in the LTRs under study. (b) An example of the PCR amplification of the
AL110218 gene fragment from the LTR4-containing 2p23.3 locus. Electrophoretic separation in 1% agarose gel of the PCR prod-
ucts with primers 4for and 4rev (LTR4, Table 1) after the amplification of liver, cerebellum, lymph node, kidney, stomach, and lung
DNA (lanes 1, 2, 3, 4, 5, and 6, respectively). M, the DNA length marker “1 kb DNA Ladder” (Sibenzyme, Russia). The arrow
shows the PCR amplification products. Amplification did not take place in control experiments with constitutively expressing genes
WT1, RB1, BRCA1, and CDKN1A after digestion with HpaII (data not shown).

which confirmed the methylated state of LTR in these lated in the liver, cerebellum, lymph node, and lung
tissues. On the contrary, LTR4 from kidney and stom- cells and is methylated in the kidney and stomach cells.
ach DNA was not methylated, since the locus-specific This LTR is located in the region enriched with genes
amplification product was only obtained using the and surrounded with NDUFAB1, UBPH, and
HpaII-untreated genomic DNA (data not shown). MGC3248 genes, as well as with some other genes with
All the six LTRs under study were similarly ana- unknown functions (Fig. 1). The gene NDUFAB1
lyzed (see Table 2). One can see that most of the LTRs encodes another NADH: ubiquinone–oxidoreductase
are methylated in various human tissues, except for cer- subunit [16]. The mitochondrial respiration is one of
ebellum. This agrees with the common opinion that a the main intracellular processes supplying the cell with
cell reduces the harm done by new integrations of energy. The differential methylation of LTR5 and LTR6
mobile elements using the methylation as one of the integrated near the genes, whose products provide the
ways of their repression [14]. However, four of the six mitochondrial respiration, as well as the demethylated
studied LTRs have different methylation status in vari- status of both LTRs in liver cells, where the expression
ous tissues, which may indicate their involvement in the of these genes is enhanced [17], may indicate the
functioning of the loci in which they are located. involvement of these LTRs in the expression regulation
of the genes NDUFAB1 and NDUFV1.
LTR5 is located at a distance of 3 kbp from the first
exon of the gene NDUFV1 (Fig. 1), which encodes the LTR3 in the locus 1p31.2 is methylated in all the tis-
most important subunit of NADH:ubiquinone–oxi- sues except cerebellum. It is situated in the last intron
doreductase [14]. This enzyme is also called complex I of the PDE4B gene of the type 4B cAMP-specific phos-
of the mitochondrial respiratory chain. This subunit phodiesterase. The isozymes PDE4 (A, B, C, and D)
contains a binding site for NADH, and mutations in the participate in the processes of mood control, vomiting
gene encoding this protein cause considerable distur- reflex, and signal transduction from olfactory receptors
bances in the NADH:ubiquinone–oxidoreductase func- [18]. In each of the previously investigated brain parts,
tioning [15]. The LTR localized in this locus is not only isozyme PDE4, specific for this part, prevails. It
methylated in the cells of all the studied tissues, except has previously been shown that isozyme PDE4B pre-
for liver cells. LTR6 in the locus 16p12.2 is not methy- vails in the cerebral cortex, hippocampus, and cerebel-

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004


444 KHODOSEVICH et al.

Table 1. The PCR primers corresponding to the genome regions flanking the HERV-K LTR under study
LTR Accession number* Primer Primer sequence 5' 3' (length, nt)
LTR1 AL139022 1for G T GGT AGAAACAT GAGCT GT CCA (23)
1rev C ACT T AAGACCCCT CT AAGACT G (23)
LTR2 AL354855 2for C ACGT GT GGACAT AAGCAACC(21)
2rev G CT GAGT CGT CCT GAT T CT T G (21)
LTR3 AL391820 3for G CAGCAT T GT CAT CT T GGG (19)
3rev T GCACCCT T GCCT T T CC (17)
LTR4 AC074117 4for C CCAT ACCT GGCT T CGAT G (19)
4rev C CAGT GGCT GAT GGACACAC (20)
LTR5 AP003385 5for C AT ACAAGCT GGAT CT GCACC (21)
5rev A T CGT GCCT T ACT CACACT GG(21)
LTR6 AC008870 6for C AT CT GCT T GACACAGT AGGT AT G (24)
6rev G T CCCAAT AT T AAGAT CACAT CCT (24)
* The accession numbers of the nucleotide sequences of human genome fragments containing the corresponding LTRs and registered in
the GeneBank international database are given.

lum [19]. The PDE4B gene expression is higher in of neighboring genes. Possibly, the LTRs are involved
human cerebellum than in the macaque and rat cere- in the expression regulation of these genes. The LTR
bella [18]. In addition, the PDE4B gene is transcribed involvement in the primate molecular evolution is cur-
in peripheral blood T cells in two variants: as a full-size rently being widely discussed [22–24]. Along with
and as a last exon-lacking copies [20, Human Genome other factors, a certain role in the human evolution
Draft Browser]. Possibly, this LTR somehow partici- could be played by the effect of demethylated human-
pates in this regulation. specific LTRs on the expression of adjacent genes
The LTRs methylated in all the studied tissues are involved in mitochondrial respiration, mood control,
also found; for example, LTR1 in the 14q23.3 locus in signal transduction from olfactory receptors, etc.
the promoter region of the FNTB gene (the β subunit of
farnesyltransferase). Most probably, such LTRs are EXPERIMENTAL
functionally unimportant for these tissues. However,
the demethylation of these LTRs cannot be excluded Isolation of human genomic DNA from various
under certain conditions (for example, at heat shock), tissues was carried out by the procedure of isolation of
as it takes place in the case of other retroelements, for high-molecular DNA using proteinase K and phenol
example, Alu [21], followed by the activation of their [25].
regulatory elements. Nucleotide sequences and their analysis. The LTR
These results obtained are the first step toward anal- genomic surroundings were analyzed using the pro-
ysis of the functional significance of LTR methylation gram packets of Draft Human Genome Browser
status. The LTR methylation/nonmethylation status (http://genome.ucsc.edu/goldenPath/hgTracks.html),
mostly correlates with the lack/existence of expression RepeatMasker (http://ftp.genome.washington.edu/),
and BLAST2 (http://www.ncbi.nlm.nih.gov/BLAST/).
The primers were selected using the Gene Runner (Ver-
Table 2. The methylation status of the human-specific LTRs sion 3.00) program.
in various tissues
Genomic DNA was digested with HpaII
Cere- Lymph Stom- restrictase in an incubation mixture (100 µl) contain-
LTR Liver Kidney Lungs ing genomic DNA (1 µg, HpaII (30 U, NEB, United
bellum node ach
States), 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM
LTR1 + + + + + + dithiothreitol (pH 7.0 at 25°ë). The mixture was incu-
LTR2 + + + + + + bated at 37°ë for 16 h, and the enzyme was inactivated
LTR3 + – + + + + by incubation at 65°C for 15 min.
LTR4 + + + – – + Genomic PCR. The human genomic DNA treated
LTR5 + – – – – – and untreated with restriction endonuclease HpaII were
used as templates (the HpaII-untreated DNA was added
LTR6 – – – + + –
to control the PCR process). A Taq DNA polymerase
* (+), methylated; (–), nonmethylated. (0.5 U, Institute of Bioorganic Chemistry, Russia) was

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004


THE TISSUE-SPECIFIC METHYLATION 445

added into the PCR incubation mixture containing a 9. Jaenisch, R. and Bird, A., Nat. Genet., 2003, vol. 33,
buffer (50 mM KCl, 10 mM Tris-HCl, and 0.1% Triton pp. 245–254.
X-100, pH 9.0), 1.5 mM MgCl2 0.125 mM dNTPs, 10. Buzdin, A., Khodosevich, K., Mamedov, I., Vinogra-
0.4 µM primers, and genomic DNA (10 ng). The total dova, T., Lebedev, Y., Hunsmann, G., and Sverdlov, E.,
volume of the reaction mixture was 25 µl. The PCR Genomics, 2002, vol. 79, pp. 413–422.
(28 cycles) was carried out as follows: 95°ë for 20 s; 11. Mamedov, I., Batrak, A., Buzdin, A., Arzumanyan, E.,
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ACKNOWLEDGMENTS dov, I., Lebedev, Y., Hunsmann, G., and Sverdlov, E.,
We thank V.K. Potapov and N.V. Skaptsova for the Genomics, 2003, vol. 81, pp. 149–156.
synthesis of oligonucleotide. 14. Kidwell, M.G. and Lisch, D., Proc. Natl. Acad. Sci. USA,
1997, vol. 94, pp. 7704–7711.
The work was supported by the Russian Foundation
for Basic Research, project no. 02-04-48614; INTAS, 15. Schuelke, M., Smeitink, J., Mariman, E., Loeffen, J.,
Plecko, B., Trijbels, F., Stockler-Ipsiroglu, S., and van
project 01-0759; and Russian Ministry of Industry, Sci- den Heuvel, L., Nat. Genet., 1999, vol. 21, pp. 260–261.
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16. Triepels, R., Smeitink, J., Loeffen, J., Smeets, R., Bus-
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