Báo cáo khoa học: Epigenetics: differential DNA methylation in mammalian somatic tissues ppt

MINIREVIEW
Epigenetics: differential DNA methylation in mammalian
somatic tissues
Hiroki Nagase
1,2
and Srimoyee Ghosh
2
1 Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan
2 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA
Cytosine methylation of CpG dinucleotides is an
important epigenetic modification that has profound
roles in gene regulation, development and carcinogene-
sis [1,2]. Methylation of CpG clusters or CpG islands
within gene promoters can silence gene expression
[3,4]. Therefore, identifying changes in DNA methyla-
tion at CpG islands is expected to lead to a clearer
understanding of the differentiation of normal tissues
and the development of complex diseases including
cancer [5]. The DNA methylation pattern is somati-
cally heritable via the effect of the maintenance DNA
methyltransferase, DNMT1 [6]. The error rate of
maintaining DNA methylation is low ( 1% per divi-
sion) at human CpG sites [7]. During embryonic devel-
opment, both somatic and germ-cell DNA methylation
patterns are erased and then re-established during cell
differentiation. Once established, DNA methytlation
patterns are thought to be stable. Although it has been
reported that DNA methylation may play a role in
the regulation of tissue-specific gene expression [8,9],
differential DNA methylation patterns among adult
tissues were not confirmed until recently.

The flowering plant Arabidopsis thaliana, with muta-
tions in the cytosine–DNA-methyltransferase gene,
MET1, which led to a global reduction in cytosine
methylation, is viable, and the delay in its flowering
Keywords
cancer; CpG islands; differentially
methylated region; differentiation; DNA
methylation; epigenetics; mouse; restriction
landmark genomic scanning (RLGS); tissue-
specific DMR; Vi-RLGS
Correspondence
H. Nagase, Advanced Research Institute for
the Sciences and Humanities, Nihon
University, Nihon University Kaikan Daini
Bekkan, 12-5, Goban-cho, Chiyoda-ku,
Tokyo 102-8251, Japan
Fax: +81 3 3972 8337
Tel: +81 3 3972 8337
E-mail:
(Received 30 November 2007, revised 28
January 2008, accepted 11 February 2008)
doi:10.1111/j.1742-4658.2008.06330.x
Epigenetics refers to heritable phenotypic alterations in the absence of
DNA sequence changes, and DNA methylation is one of the extensively
studied epigenetic alterations. DNA methylation is an evolutionally con-
served mechanism to regulate gene expression in mammals. Because DNA
methyation is preserved during DNA replication it can be inherited. Thus,
DNA methylation could be a major mechanism by which to produce semi-
stable changes in gene expression in somatic tissues. Although it remains
controversial whether germ-line DNA methylation in mammalian genomes

is stably heritable, frequent tissue-specific and disease-specific de novo meth-
ylation events are observed during somatic cell development ⁄ differentia-
tion. In this minireview, we discuss the use of restriction landmark genomic
scanning, together with in silico analysis, to identify differentially methylat-
ed regions in the mammalian genome. We then present a rough overview
of quantitative DNA methylation patterns at 4600 NotI sites and more
than 150 differentially methylated regions in several C57BL ⁄ 6J mouse tis-
sues. Comparative analysis between mice and humans suggests that some,
but not all, tissue-specific differentially methylated regions are conserved. A
deeper understanding of cell-type-specific differences in DNA methylation
might lead to a better illustration of the mechanisms behind tissue-specific
differentiation in mammals.
Abbreviations
DMR, differentially methylated region; RLGS, restriction landmark genomic scanning; T-DMR, tissue-specific differentially methylated region;
Vi-RLGS, virtual-image restriction landmark genomic scanning; V-RLGS, virtual restriction landmark genomic scanning.
FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS 1617
time is observed only after several generations [10].
Embryos from DNA methyltransferase gene-deficient
mice, which have reduced levels of cytosine methyla-
tion, develop until the stage of 8.5 days, when many
tissues are already differentiated [11]. Furthermore,
analysis of DNA methylation patterns in genes known
to be expressed in a tissue-specific manner failed to
confirm a major role for DNA methylation in differen-
tiation [12,13]. Using microarray analysis, only five
genes expressed in a tissue-specific manner showed a
significant increase in expression level in an in vivo
system lacking DNA methylation [14]. These data
suggested that DNA methylation had no role in
regulating gene expression during development.

Song et al. [15] reported 150 tissue-specific differen-
tially methylated regions (T-DMRs) in the mouse
genome using restriction landmark genomic scanning
(RLGS) in conjunction with virtual-image restriction
landmark genomic scanning (Vi-RLGS) and confirmed
at least 14 T-DMRs by bisulfite sequencing. Some of
the confirmed T-DMRs exhibited a tissue-specific
expression pattern that is consistent with methylation
status and may play a role in tissue differentiation
[15]. Subsequent studies reported the existence of fre-
quent tissue-specific methylation events in mice [16,17]
and humans [9,18–20]. Thus, the extent of DNA meth-
ylation appears to change in a systematic way during
mammalian development.
DNA methylation status may be influenced by
environmental exposure [2,20,21]. In gastric mucosa,
Helicobacter pylori infection potently induces the meth-
ylation of several CpG islands [21]. Young monozy-
gotic twins are essentially indistinguishable in their
epigenetic markings, whereas older monozygous twins
exhibit remarkable differences in overall content and
genomic distribution of 5-methylcytosine DNA and
histone acetylation [22]. Thus, the previous hypothesis
that DNA methylation patterns acquired during devel-
opment in mammals were stable in adult somatic cells
can be discarded in favor of the accumulated evidence
of frequent appearances of differentially methylated
genomic regions in various tissue environments.
RLGS method for the mammalian
genome

RLGS is a method for the 2D display of end-labeled
DNA restriction fragments [23]. This method involves
digestion of high molecular mass genomic DNA with a
‘landmark’ enzyme. The landmark enzyme, such as the
methylation-sensitive enzyme NotIorAscI, determines
the sites of the genome that will be labeled by filling in
enzyme half-sites with radioactive nucleotides. Because
the NotI recognition site contains two CpGs, and
> 90% of the NotI sites are thought to lie within
CpG islands, RLGS (with NotI and similar restriction
enzymes) displays the DNA methylation status of the
CpG islands and associated regions [23]. For example,
when comparing normal and cancer RLGS profiles,
spot loss due to methylation occurs because the methy-
lated NotI site is not cut by the enzyme and is there-
fore not labeled. By contrast, spot gain in cancer
indicates ‘demethylation’ of a NotI site which was
methylated in normal tissues. Using methylation-
sensitive NotI as a landmark,  1500–2000 spots
(end-labeled restriction fragments) can be resolved on
a single gel. These methods have been used to identify
imprinted sites, aberrant methylated sites in cancers
and epigenetic remodeling of mammalian tissues
[23–25].
The lack of a PCR step and hybridization in the
RLGS procedure provides an important advantage
over other methods in identifying aberrant DNA meth-
ylation. RLGS profiles are quantitative, and the sensi-
tivity is such that methylation can be reliably detected
when > 40% of the alleles are methylated. This level

of sensitivity ensures that the major demethylation
events present in the sample could be detected. By con-
trast, other approaches, such as bisulfite sequencing or
chromatin ⁄ methyl-cytosine precipitation, would allow
the detection of very rare methylation events (false
positive) or the omission of a partial DNA methyla-
tion (false negative), due to the involvement of array
hybridization, PCR amplification and affinity precipi-
tation.
Computational approaches for RLGS
(Vi-RLGS)
Despite its clear importance and successes, RLGS has
some potential drawbacks, the most significant of
which is the difficulty of cloning individual spots
[26,27]. This is critically important because the
sequence of the altered RLGS spot must be deter-
mined in order to identify the affected gene. Clearly,
with the availability of the genome sequence for many
organisms, it has become possible to use this informa-
tion to identify specific restriction fragments within ge-
nomes and produce in silico size fractionations [28–30].
Several in silico analyses that have been made in this
sense include the Virtual Genome Scan (http://dot.
ped.med.umich.edu:2000/VGS/index.html) [30], in silico
digests [28], Vi-RLGS [29] and virtual restriction land-
mark genomic scanning (Conime by R. Wenger; http://
www.cse.ohio-state.edu/wenger/research/conime/contact.
html). These tools help to create a more complete map
DNA methylation in the mammalian genome H. Nagase and S. Ghosh
1618 FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS

of genomic sites that are either methylated or demethy-
lated in current human and mouse genomic DNA
sequence data.
Application of Vi-RLGS to the
C57BL
⁄
6J mouse genome
We applied the Vi-RLGS software directly to the mouse
genome using a NotI–PstI–PvuII combination. Examin-
ations of a sample field from C57BL ⁄ 6J liver DNA
identified  1460 unmethylated spots in real RLGS,
compared with 2170 spots in the same field of the vir-
tual pattern. The vast majority of the ‘extra spots’ in
the virtual profile in the mouse are, in fact, derived
from repetitive sequences, which would be expected to
be methylated and absent in the real profile [15].
This method has been applied to the mouse genome
using six different tissues (testis, brain, colon, kidney,
liver and muscle) [15]. The methylation status of
 4600 genomic sites fell into one of the following
three categories: constitutively methylated, constitu-
tively demethylated and methylated in a tissue-specific
manner. The frequency of T-DMRs is estimated to be
 5% (836 ⁄ 15 500 CpG islands) in the mouse genome
[15]. This estimate may be different because RLGS is
strongly biased by the genomic location of the NotI
sites. DNA methylation profiling of human chromo-
somes 6, 20 and 22 suggested that many T-DMRs are
not in CpG islands [31], but recent global human
genome searches have identified  700 T-DMRs for

human promoter regions, many of which are included
in CpG islands [9,19,20]. However, when the two
recent human global analyses are compared, 283 gene
promoters are identified as ‘testis-specific’ DMRs in
one analysis [19] and 104 in another [20]. Among these
gene promoters, only 18 were concurrently identified
as ‘testis-specific’ DMRs by both studies [19,20].
Although potential sources of contradictions in these
two publications may be the differences in type of tis-
sue, DNA purification and methodology used, most of
recent methodologies are not accurately quantitative.
A reproducible method for quantitative DNA methyla-
tion detection is needed for the in vivo study, in which
DNAs are often prepared from a mixed cell popula-
tion.
The application of a quantitative whole-genome
methylation analysis by RLGS to the mouse genome
provides evidence for specific differences in the DNA
methylation patterns during development, differentia-
tion, aging and in diseases such as cancer. The
Vi-RLGS application, together with a sophisticated
image-matching and registration program and spot
intensity analysis program may provide a new analyti-
cal tool to measure the global methylation patterns of
cell population in specific tissue environments [32,33]
(G. E. Bove and P. Rogersen, University at Buffalo,
NY, USA; unpublished data). Analysis of deposited
numbers of previously performed RLGS images may
prove to be a treasure chest for understanding the
methylation patterns of each tissue or disease state.

Based on those efforts and a considerable number of
RLGS experiments, a rough draft overview of quantita-
tive DNA methylation patterns with a quantitative
DNA measurement of the C57BL ⁄ 6J mouse genome
has been created by using the Vi-RLGS in silico analy-
sis. Figure 1 shows a draft NotI methylation map of
C57BL ⁄ 6J strain based on two RLGS profiles of NotI–
PstI–PvuII and NotI–PvuII–PstI enzyme combinations.
Table 1 is a preliminary distribution pattern on each
chromosome of T-DMRs, constitutively methylated
and constitutively non- or partially methylated NotI
sites using a virtual-image RLGS analysis (note that
for most NotI sites the methylation pattern has yet to
be confirmed by other methods). Interestingly, consid-
ering the gene-poor regions [34], a significantly high
number of T-DMRs are located in gene-rich genomic
region, while non-T-DMRs are located in both (Fig. 2).
In addition, a relatively high percentage of NotI sites in
T-DMRs are located in the non-promoter region
(exons, introns and 3’ regions). This may suggest that
T-DMRs are likely to modify gene expression through
transcriptional regulation or may have other functions
that are unrelated to transcription. However, the func-
tional relationships between T-DMRs and regulation
mechanisms involved in tissue differentiation are
unknown. Intragenic DNA methylation is known to be
capable of altering the chromatin structure and elonga-
tion efficiency in mammalian cells, depleting RNA
polymerase II exclusively in the methylated region [35].
It has been suggested that the methylation of Alu ele-

ments could suppress transcription and contribute to
differential gene expression [36–38]. A study of trans-
genic mice demonstrated that the epigenetic modifica-
tion of transgenes under the control of the mouse
mammary tumor virus LTR conferred a tissue-depen-
dent influence on the transcription of the transgenes
[39]. Recent evidence suggested that there are related
regulation mechanisms between micro RNA and epige-
netics [40]. Although it has been reported that 95% of
mammalian genomes are transcribed and have some
functional means [41], the localization bias of T-DMRs
may suggest that DNA methylation is a critical tissue-
specific regulation mechanism and that it modifies
RNA transcription. The T-DMR located in gene-poor
regions may also facilitate the identification of previ-
ously unidentified regulatory mechanisms.
H. Nagase and S. Ghosh DNA methylation in the mammalian genome
FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS 1619
RLGS for cancer study
RLGS has been used to study the degree of hypome-
thylation as well as to identify the targets of hyperme-
thylation in many different types of cancerous tissues
[24,42]. These studies reveal that CpG island methyla-
tion in cancers is non-random and shows both inter-
and intratumor heterogeneity. Furthermore, certain
Fig. 1. NotI methylation map of the C57BL ⁄ 6J genome. The methylation status of NotI sites is plotted on 19 mouse autosomal chromo-
somes. Constitutively methylated, constitutively unmethylated or tissue-specific methylated sites are indicated by red bars (left-hand side of
the chromosome), green bars (right-hand side of the chromosome) and blue bars (left-hand side of the chromosome), respectively. Vi-RLGS
analysis was performed in conjunction with duplicate RLGS analyses of six tissues (liver, muscle, kidney, colon, testis and brain) using
Mouse Aug. 2005 (mm7) assembly, and then the methylation pattern of each spot of RLGS autographs were analyzed.

DNA methylation in the mammalian genome H. Nagase and S. Ghosh
1620 FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS
RLGS fragments were found to be methylated in many
different cancers, whereas others were methylated
exclusively in one. In the set of tumors described by
Costello et al. [24], gliomas had 34 RLGS loci methy-
lated in > 40% of the samples (n = 14), colon tumors
had 23 (n = 8) and medulloblastomas had eight
(n = 22). Even if the associated gene is not known or
ever found to be associated with cancer, given that
they are methylated at such high frequency, methyla-
tion of these loci could be used as biomarkers. Rarely
known tumor-suppressor genes have been shown to be
methylated at a frequency of > 40% of tumors, even
using highly sensitive PCR-based techniques [43]. One
remarkable exception to this is the GSTP1 gene, which
was shown to be hypermethylated in 40 ⁄ 42 human
prostate cancers [44] and shows promising signs of
becoming an excellent biomarker for prostate cancer
[45]. A quantitative analysis such as RLGS provides
an opportunity to scan the genome for frequent targets
of methylation in a mixed cell population such as
tumor DNA. This may have even more potential than
existing DNA methylation biomarkers. In addition,
studying DNA methylation for loci whose primary
route of inactivation is through CpG island methyla-
tion, rather than through genetic disruption, may be
more likely to result in the identification of effective
therapeutic targets.
Conclusion

The recent technical revolution in epigenetic detection
is providing a clearer understanding of epigenetic
Table 1. Preliminary distribution pattern of T-DMRs, on each chromosome showing constitutively methylated and constitutively non- or par-
tially methylated NotI sites identified by virtual-image RLGS analysis system.
Chromosome 12345678910111213141516171819Total
Constitutively
unmethylated
regions
224 342 184 275 271 174 252 218 228 207 316 154 171 129 143 138 174 125 124 3849
% 78.0 88.4 81.1 91.1 86.9 80.2 86.0 85.8 86.0 83.8 89.0 86.5 81.4 79.1 85.6 81.7 84.1 82.2 87.3 85.9
Constitutively
methylated
regions
58 37 35 17 29 32 28 25 31 36 23 15 35 29 18 25 25 22 14 534
% 20.2 9.6 15.4 5.6 9.3 14.7 9.6 9.8 11.7 14.6 6.5 8.4 16.7 17.8 10.8 14.8 12.1 14.5 9.9 11.8
T-DMRs 5881012111311641694566854151
% 1.7 2.1 3.5 3.3 3.8 5.1 4.4 4.3 2.3 1.6 4.5 5.1 1.9 3.1 3.6 3.6 3.9 3.3 2.8 3.3
Total 287 387 227 302 312 217 293 254 265 247 355 178 210 163 167 169 207 152 142 4534
% 6.2 8.4 4.9 6.6 6.8 4.7 6.4 5.5 5.8 5.4 7.7 3.9 4.6 3.5 3.6 3.7 4.5 3.3 3.1
Physical
length Mb
195 181 158 154 150 150 139 127 124 130 121 115 114 118 103 97 92 90 60 2596
% 7.5 7.0 6.1 5.9 5.8 5.8 5.4 4.9 4.8 5.0 4.7 4.4 4.4 4.5 4.0 3.7 3.5 3.5 2.3
10
0
20
30
40
50
60

70
80
90
100
non-CpG promoter
non-CpG exon
non-CpG intron
non-CpG 3’
non-CpG junk
CpG promoter
CpG exon
CpG Intron
CpG 3’
CpG junk
CpG islands
non-CpG islands
Junk
non-junk
%
NotI unmethylated regions
NotI T -DMRs
Fig. 2. Genomic regions of unmethylated
NotI sites and NotI sites showing tissue-
specific differentially methylated regions
(T-DMRs). A bar graph indicates the percent
of genomic distribution of NotI sites sepa-
rately analyzed between those located
within constitutively unmethylated regions
(white bars) and those in T-DMRs (gray
bars) at each indicated genomic region.

Intergenic region (Junk), intragenic region
(promoter, exon, 3¢ regions; non-Junk), CpG
island and non-CpG island were evaluated
by UCSC genome browser (mm8).
H. Nagase and S. Ghosh DNA methylation in the mammalian genome
FEBS Journal 275 (2008) 1617–1623 ª 2008 The Authors Journal compilation ª 2008 FEBS 1621
marks in the genomes of every mammalian cell type,
even though genome-wide quantitative DNA methyla-
tion analysis is yet to be completely established.
RLGS, together with in silico analysis, is a useful tech-
nique for comparing genome-wide DNA methylation
patterns between tissues or disease states. The RLGS
analysis has provided evidence of frequent T-DMRs in
mammalian genomes and epigenetic control of tissue-
specific RNA transcription modification.
Acknowledgements
We thank C. J. Kemp and W. A. Held for his critical
reading. This work is supported by the Nihon Univer-
sity Multidisciplinary Research Grant for 2006, the
Academic Frontier Project for 2006 Project for Private
Universities: matching fund subsidy from MEXT,
National Cancer Institute Grant CA102423 and the
National Cancer Institute Center Support Grant
CA16056 (to Roswell Park Cancer Institute).
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