MINIREVIEW
Epigenetics: the study of embryonic stem cells by
restriction landmark genomic scanning
Naka Hattori* and Kunio Shiota
Laboratory of Cellular Biochemistry, Animal Resource Sciences ⁄ Veterinary Medical Sciences, University of Tokyo, Japan
Differentiation of a specific cell type involves the
establishment of a precise epigenetic profile comprised
of genome-wide epigenetic modifications such as DNA
methylation and histone modification. Because epi-
genetic modifications in gene areas regulate transcrip-
tional activity, the epigenetic profile of the cell reflects
the transcriptome of the cell, at least partially. DNA
methylation is a major component of epigenetic modi-
fication in mammals [1,2]. The DNA methylation pro-
file at tissue-specific differentially methylated regions
(originally named tissue-dependent and differentially
methylated regions: T-DMRs) in one cell type is differ-
ent from others and represents a unique property of
the cell [3,4]. However, the precise mechanism behind
formation of the epigenetic profile, including the DNA
methylation profile during development, remains to be
elucidated.
A wide range of methods has been developed for
qualitative and quantitative DNA methylation assays
[5]. Although methods based on microarray technology
are undoubtedly useful and promising for analyzing
whole-genome profiles of DNA methylation, as well as
histone modifications [4], restriction landmark genomic
scanning (RLGS), which is based on 2D electrophore-
sis in combination with methylation-sensitive restric-
tion enzymes [6], is still a powerful method for DNA

epigenetic modifications.
Abbreviations
Dnmt, DNA methyltransferase; EB, embryoid body; ED, epigenetic distance; EG cell, embryonic germ cell; ES cell, embryonic stem cell;
RLGS, restriction landmark genomic scanning; T-DMR, tissue-specific differentially methylated region or tissue-dependent and differentially
methylated region; TS cell, trophoblast stem cell; Vi-RLGS, virtual image restriction landmark genomic scanning.
1624 FEBS Journal 275 (2008) 1624–1630 ª 2008 The Authors Journal compilation ª 2008 FEBS
methylation analysis. Although RLGS requires a larger
genomic sample than is necessary for microarray-based
methods, it has advantages for analyzing genome-wide
methylation states: (a) it is a highly reproducible quan-
titative method; (b) genomic DNA is not amplified,
thus limiting or avoiding detection bias; (c) it detects
unmethylated landmarks in the genome and keeps out
repeated sequences that are usually highly methylated;
and (d) it targets predominantly CpG islands by using
restriction enzymes that have recognition sites with
high CG contents, such as NotI. Moreover, virtual
image RLGS (Vi-RLGS), a recently developed soft-
ware simulating RLGS in silico using genomic
sequences, overcomes the difficulty in identifying
sequences of RLGS fragments [7].
One of the most important advances in develop-
mental biology and cell biology is the establishment
of embryonic stem (ES) cells, which maintain the
ability to form all types of cells in the body, and can
differentiate into a variety of cell types in vitro [8].
The use of ES cells in epigenetic studies enables us to
analyze how epigenetic profiles change during devel-
opmental processes and the effects on epigenetic
regulators of fetal exposure to chemical agents. In

from developing embryos revealed the uniqueness of
the epigenetic profile in ES cells. In contrast to ES
cells, which maintain the ability to differentiate into all
cell types of the embryo proper [10], trophoblast stem
(TS) cells originate from the trophectoderm of blast-
ocysts and can differentiate only into placental cells
in vivo and in vitro [11]. Differentiation of cells from
the early blastomere stage to the blastocyst stage is
accompanied by a change in the epigenetic profile that
directs the differentiation pathway to either the
embryo proper or the placenta. Thus, a significant dif-
ference between ES and TS cells is likely to be
observed by comparing their epigenetic profiles. Analy-
sis by RLGS revealed that DNA methylation profiles
at T-DMRs are totally different between ES and TS
cells [9]. Compared with TS cells, 20 genomic loci were
methylated and 57 loci were demethylated in ES cells,
supporting the idea that a bifurcation of the epigenetic
profile exists before development of the blastocyst.
Embryonic germ (EG) cells are known to have simi-
lar characteristics to ES cells with respect to differenti-
ation and proliferation capabilities, despite their
different origins [12,13]. It was demonstrated that glo-
bal gene-expression profiles of ES and EG cells were
indistinguishable [14]. However, analysis of DNA-
methylation profiles by RLGS revealed a significant
difference between ES and EG cells [9]. Among 1500
genomic loci in the RLGS profile, 49 (3%) were found
to be methylated differentially in ES and EG cells,
indicating that ES and EG cells can be distinguished

(40.2%) and brain of adult mice (48.6%) but higher in
kidney (53.7%). A similar change was observed in the
in vitro differentiation system; methylation levels were
low (39.6%) in EBs and higher (41.3–44.4%) in three
independently developed teratomas derived from ES
cells or EBs. The number of methylated loci in the
profiles of teratomas was less than that of the somatic
tissues, probably because the teratomas still contained
a significant number of undifferentiated proliferating
cells, or all cells in teratomas were not fully differenti-
ated yet. Because the methylation status of T-DMRs
partially corresponds with the transcriptional status of
the neighboring gene, identifying differentially methyl-
ated genomic loci in ES cells, EBs and teratomas is
expected to provide information about genes that are
responsible for the developmental process.
Potential of ES cells in embryotoxicological
studies
Embryonic exposure to chemical agents or medicine
may have deleterious effects on proper embryogenesis,
especially during the early developmental stages. Such
agents may influence embryos at genetic, transcriptional
and protein levels. It is also conceivable that epigenetic
alterations occur with exposure of embryos to these
agents, because epigenetic profiles are established
actively in developing embryos. Differentiation of ES
cells into EBs has been studied as an in vitro model of
normal and abnormal mammalian development [16].
Because differentiation from ES cells to EBs is accom-
panied by changes in DNA methylation profiles at

sequences.
Analysis of the DNA methylation profile for
therapeutic use of human ES cells in regenerative
medicine
The potential use of human ES cells in the field of
regenerative medicine has been discussed previously,
and differentiation of human ES cells into various tis-
sues has been investigated [8]. Several lines of human
ES cells were established, and differences between these
ES cell lines with respect to karyotypic stability [21]
and expression profiles [22] have been investigated. It
ICM
TE
PGC
TS cells
ES cells EG cells
4977
Placental cells Embr
y
onic cells
Fig. 1. Epigenetic distances between ES cells and other stem cells
derived from developing embryos. ES cells derived from the inner
cell mass (ICM) of blastocysts and EG cells derived from the pri-
mordial germ cells (PGCs) in developing genital ridges can develop
into cells of the embryo proper, after they are injected into blast-
ocysts to form chimeras. By contrast, TS cells derived from the
trophectoderm (TE) of blastocysts contribute only to placenta.
Although there is an apparent ED between ES cells and EG cells,
the ED of TS cells to ES cells (77) is greater than that of EG cells
to ES cells (49), confirming the similarity of EG cells to ES cells.

perform gene targeting at specific chromosomal loci
and to investigate gene function [28]. In addition,
knockout mice have been generated to study the devel-
opmental role of the gene by germline transmission of
a targeted allele. Genetic manipulations of many epige-
netic regulators, including Dnmts [29–33] and histone
methylases [34,35], have been reported. Genome-wide
DNA methylation analysis of ES cells deficient in epi-
genetic regulators will assist in revealing the mecha-
nism for maintaining DNA methylation in T-DMRs,
as well as the interplay between DNA methylation and
other epigenetic modifications.
Mechanism for maintaining DNA methylation
at T-DMRs
Based on studies regarding the properties of Dnmts, it is
widely accepted that Dnmt1 is a maintenance DNA
methyltransferase and Dnmt3a ⁄ 3b are de novo DNA
methyltransferases in vivo [36]. Dnmt3a and Dnmt3b
have no preference for hemimethylated DNA [37], and a
transgene of Dnmt3a, but not of Dnmt1, to Drosophila
exhibited de novo methylation activity [38], indicating
that Dnmt3a ⁄ 3b function in de novo DNA methylation,
but not in maintenance DNA methylation. However,
following these studies, it was still unclear how Dnmt1
and Dnmt3a ⁄ 3b are involved in DNA methylation
in T-DMRs, thereby establishing DNA methylation
profiles of cells, and whether Dnmt3a ⁄ 3b have any role
in maintenance DNA methylation in T-DMRs.
We demonstrated cooperation of Dnmt1 and either
Dnmt3a or Dnmt3b in the maintenance of DNA meth-

absent. Consequently, Dnmt1-deficient ES cells seem to
have partial DNA methylation maintenance activity,
which is provided by the re-methylating actions of
Dnmt3a ⁄ Dnmt3b (Fig. 2). Dnmt3a and Dnmt3b
appear to function both as maintenance and as de novo
methyltransferases in gene areas, and thus are crucial
for the establishment of the DNA methylation profile
during development.
Analyzing the interplay between DNA
methylation and histone methylation
Chromatin structure, which is affected by DNA meth-
ylation and histone modification, is closely associated
N. Hattori and K. Shiota Epigenetic study of embryonic stem cells
FEBS Journal 275 (2008) 1624–1630 ª 2008 The Authors Journal compilation ª 2008 FEBS 1627
with the transcriptional activity of genes. During mam-
malian development, the epigenetic profile is not estab-
lished solely by one particular epigenetic regulator, but
rather by the interplay of epigenetic regulators [41,42].
The relationship between DNA methylation and other
epigenetic modifications can be examined by genome-
wide DNA methylation analysis using ES cells defi-
cient in epigenetic regulators. Growing evidence has
indicated that histone lysine methylation can direct
DNA methylation in many organisms [43]. G9a is a
euchromatin-localized histone methylase that catalyzes
the methylation of histone H3 at Lys9 and Lys27
(H3–K9 and H3–K27) [44], which are often found in
heterochromatic regions and in transcriptionally inac-
tive loci of the genome [45]. RLGS analysis of G9a-
deficient ES cells revealed a direct interaction between

ation and other epigenetic modifications through
identification of DNA methylation profiles by RLGS
or other genome-wide analysis methods.
Acknowledgements
We thank M. Higgins for reviewing the original manu-
script. This work was supported by the Program for
Promotion of Basic Research Activities for Innovative
Biosciences (PROBRAIN).
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