Vol 13, Issue 5, 868-874, May 2003
LETTER
Fidelity of the Methylation Pattern and Its Variation in the Genome
Toshikazu Ushijima1,2,
Naoko Watanabe1,
Eriko Okochi1,
Atsushi Kaneda1,
Takashi Sugimura1 and
Kazuaki Miyamoto1
1Carcinogenesis Division, National Cancer Center Research
Institute, Chuo-ku, Tokyo 104-0045, Japan
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ABSTRACT
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The methylated or unmethylated status of a CpG site is copied
faithfully from parental DNA to daughter DNA, and functions as a
cellular memory. However, no information is available for the fidelity
of methylation pattern in unmethylated CpG islands (CGIs) or its
variation in the genome. Here, we determined the methylation status of
each CpG site on each DNA molecule obtained from clonal populations of
normal human mammary epithelial cells. Methylation pattern error rates
(MPERs) were calculated based upon the deviation from the methylation
patterns that should be obtained if the cells had 100% fidelity in
replicating the methylation pattern. Unmethylated CGIs in the promoter
regions of five genes showed MPERs of 0.018–0.032 errors/site/21.6
generations, and the fidelity of methylation pattern was calculated as
99.85%–99.92%/site/generation. In contrast, unmethylated CGIs
outside the promoter regions showed MPERs more than twice as high
(P < 0.01). Methylated regions, including a CGI in the
MAGE-A3 promoter and DMR of the H19 gene, showed much
lower MPERs than unmethylated CGIs. These showed that errors in
methylation pattern were mainly due to de novo methylations in
unmethylated regions. The differential MPERs even among unmethylated
CGIs indicated that a promoter-specific protection mechanism(s) from de
novo methylation was present.
[Supplemental material is available online at www.genome.org.]
CpG methylation is known to serve as cellular
memory, and is involved in various biological processes, such as
tissue-specific gene expression, genomic imprinting, and X chromosome
inactivation (Jones and Takai 2001 ; Bird 2002; Futscher et al. 2002;
Strichman-Almashanu et al. 2002). These important functions of
methylations are based upon the fact that the methylated or
unmethylated status of a CpG site is faithfully inherited. The
methylated status of a CpG site is inherited upon DNA replication by
the function of maintenance methylase, represented by DNA
methyltransferase 1, which is located at replication forks and
methylates hemimethylated CpG sites into fully methylated CpG sites
(Leonhardt et al. 1992; Araujo et al. 1998; Hsu et al. 1999). The
unmethylated status of a CpG site is inherited by not being methylated
upon DNA replication or any other occasions. Unmethylated CpG sites
generally cluster to form a CpG island (CGI), and most CGIs are kept
unmethylated (Gardiner-Garden and Frommer 1987; Bird 2002).
Methylations of CGIs in promoter regions are known to cause
transcriptional silencing of their downstream genes by changing
chromatin structures and blocking transcription initiation (Bird 2002;
Richards and Elgin 2002). There are limited numbers of CGIs that are
normally methylated (normally methylated CpG islands; NM-CGIs) (De Smet
et al. 1999; Futscher et al. 2002). CpG sites outside CGIs, especially
those in repetitive sequences, are also normally methylated (Bird
2002).
To keep the methylation pattern, maintenance of both methylated and
unmethylated statuses of CpG sites during DNA replication is necessary.
However, the fidelity of the methylation pattern has been analyzed only
for the maintenance of the methylated status (Wigler et al. 1981; Otto
and Walbot 1990; Pfeifer et al. 1990). The fidelity in maintaining the
methylated status of an exogenously introduced DNA was shown to be 94%
per generation per site by Southern blot analysis (Wigler et al. 1981).
The fidelity in maintaining the methylated status of a CGI in the 5'
region of the PGK1 gene, which was derived from the inactive X
chromosome, was estimated to be 98.8%–99.9% per site per generation
by the ligation-mediated PCR method after chemical cleavage of DNA
(Pfeifer et al. 1990).
Normally unmethylated regions might show different fidelities from
normally methylated regions. Even among the unmethylated CGIs, the
fidelities of their methylation pattern have been suggested to be
different according to their location against a gene promoter.
Methylation of CGIs in promoter regions almost always leads to
transcriptional silencing while that of CGIs outside promoter regions
does not (Gonzalgo et al. 1998; Jones 1999). Considering the cellular
expense in maintaining methylation pattern, a cell could sacrifice the
fidelity of methylation pattern for CGIs outside promoter regions. In
addition, by recent genomic scanning techniques for methylation changes
(Ushijima et al. 1997; Toyota et al. 1999; Costello et al. 2000; Jones
and Baylin 2002), aberrant methylations of CGIs in cancers are observed
in a nonrandom manner (Toyota et al. 1999; Costello et al. 2000; Kaneda
et al. 2002a; Kaneda et al. 2002b). It is indicated that CGIs outside
promoter regions were more frequently methylated than those in promoter
regions (Nguyen et al. 2001; Takai et al. 2001; Kaneda et
al. 2002a; Asada et al. 2003).
Here, we analyzed the methylation status of each CpG site on each DNA
molecule by the bisulfite sequencing technique (Clark et al. 1994) in
six clonal populations of normal human mammary epithelial cells
(HMECs), for CGIs in the promoter regions, CGIs outside the promoter
regions, and CpG sites outside CGIs. By analyzing the deviation from
the most common two patterns, MPERs, which reflected the fidelity in
replicating both methylated and unmethylated statuses, were measured.
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RESULTS
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Preparation of HMECs
A single HMEC in its log phase was plated, and expanded to
1.4 x 106 to 1.5 x 106 cells (Fig.
1). Plating efficiency during the two
transfers of plates was 67 ± 0.9(mean ± SE)%. Based on these
values, the number of cells that should have been produced at the time
of harvest was calculated as 3.2 x 106
(1.4 x 106/0.67/0.67). This value predicted that each cell
harvested underwent 21.6 generations from the initial single cell.
Doubling time was 48 h.
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Figure 1. Strategy of cell culture. A single HMEC was inoculated in a well by
limiting dilution, and the cell was expanded up to approximately
106 cells. Based on the plating efficiencies during the two
transfers and the actual final cell count, the number of cells that
should have been produced at the time of harvest and the number of
generations observed were calculated. DNA was extracted from the final
cells, and used for bisulfite sequencing. Six independent cultures were
performed.
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Gene Selection and Their Expression Levels
Methylation statuses were determined by bisulfite sequencing for
CGIs in the promoter regions of the E-cadherin,
p41-Arc, SIM2, 3-OST-2, and Cyclophilin
A genes; CGIs in the downstream exon/introns of the
E-cadherin, p41-Arc, and SIM2 genes; CpG
sites outside CGIs of the E-cadherin and p41-Arc
genes; a NM-CGI of the MAGE-A3 gene; and differentially
methylated region (DMR) of the H19 gene (Fig.
2A). The former five genes
were selected because they had CGIs in the downstream exon/introns that
met a strict criterion of CGIs, regions of DNA of >500 bp with a
G+C  55%, and observed CpG/expected CpG of 0.65 (Takai and Jones
2002). The MAGE-A3 gene and the DMR of the H19 gene
were selected as a representative NM-CGI and a region critically
involved in genomic imprinting, respectively. By quantitative RT-PCR
analysis, their expression levels were shown to range from almost none
(SIM2 and MAGE-A3) to very high
(E-cadherin), with p41-Arc, 3-OST-2 and
Cyclophilin A being intermediate (Fig. 2B).
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Figure 2. Structures and expressions of the genes analyzed. (A)
Schematic representation of the genomic regions analyzed. Regions
analyzed by bisulfite sequencing are shown by closed boxes, and
designations A–L correspond to panels in Fig. 3. CGI-P: a CGI in the
promoter regions; CGI-outside: a CGI outside the promoter regions;
Non-CGI: CpG sites outside CGIs; and DMR: differentially methylated
region. All panels are drawn to the same scale. (B) Expression
levels of the seven genes in HMECs.
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Establishment of How to Measure MPERs
The CGI in the promoter region of the E-cadherin gene (Fig.
3A), the non-CGI region of the
p41-Arc gene (Fig. 3F), the CGI in the promoter region of the
MAGE-A3 gene (Fig. 3K), and the DMR of the H19 gene
(Fig. 3L) were found to contain two major populations of clones. The
two major populations were considered to represent the methylation
pattern of the two alleles in the original single cell. The methylation
patterns of the two major populations were different from each other in
the six cultures, which indicated that the HMECs before cloning had
diverse patterns of methylation, but the patterns were relatively
conserved during the culture from a single cell to approximately
106 cells. Therefore, we measured the number of errors in the
methylation pattern based upon the culture from a single cell to
approximately 106 cells. An MPER of a region in a culture was
calculated from the number of errors in methylation pattern as
described in Methods, and an average MPER of the region was calculated
from the six MPERs obtained for the six cultures.
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Figure 3. Distribution of unmethylated and methylated CpG sites shown by
bisulfite sequencing. Unmethylated and methylated CpG sites are shown
by open and closed circles, respectively. (A)–(C) A
CGI in the promoter region, a CGI outside the promoter region and CpG
sites in non-CGIs of the E-cadherin gene.
(D)-(F) A CGI in the promoter region, a CGI outside
the promoter region and CpG sites in non-CGIs of the p41-Arc
gene. (G), (H) A CGI in the promoter region and a CGI
outside the promoter region of the SIM2 gene. (I) A
CGI in the promoter region of the 3-OST-2 gene. (J) A
CGI in the promoter region of the Cyclophilin A gene.
(K) A CGI in the promoter region of the MAGE-A3 gene,
which is normally methylated. (L) A CGI in the differentially
methylated region of the H19 gene.
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To examine the effect of an arbitrary selection of the "original
methylation pattern" in ambiguous cases, a permutation test was
performed for the CGI in the E-cadherin promoter region of
HMEC10. One of the clones #5–#14 (Fig. 3A) was hypothesized as one of
the original methylation pattern, and the number of errors in the
methylation pattern was calculated. The numbers ranged from 18–22, and
these values were expected to result in the average MPER ranging from
0.022–0.023. Similar permutation tests were performed for the CGI in
exon 2 of the E-cadherin gene of HMEC12 and HMEC15. The
numbers of errors in methylation pattern ranged from 13–16 for HMEC12
and from 12–15 for HMEC15, and these values were expected to result in
the average MPER ranging from 0.050–0.058. These showed that arbitrary
selection of the original methylation pattern in ambiguous cases does
not seriously affect the resultant average MPER.
The efficiency of bisulfite conversion was examined by analyzing DNA
with no methylation in the CGIs in the promoter region and exon 2 of
the E-cadherin gene. In the CGI in the promoter region, none
of the 600 cytosines at CpG sites (30 CpG sites per clone, 20 clones
analyzed) remained unconverted, showing that unconversion rate was
almost 0 in this region under our experimental condition. In the CGI in
exon 2, one of 483 cytosines at CpG sites (23 CpG sites per clone, 21
clones analyzed) remained unconverted, showing that the unconversion
rate was 0.0021. These values showed that the MPERs in CGIs in the
promoter regions are 10-fold more than the unconversion rates.
MPERs and Fidelities of Methylation Pattern in the Genome
The average MPERs obtained for each region are summarized in Table
1. Unmethylated CGIs in the promoter
regions showed MPERs between 0.018 and 0.032 errors/site/21.6
generations. In contrast, CGIs outside promoter regions showed
significantly higher MPERs, ranging from 0.037 to 0.091
(P < 0.01 or 0.005). MPERs in the CGIs outside the promoter
regions were more than twice as high as those in the promoter regions
of the same genes.
NM-CGI of the MAGE-A3 gene and methylated alleles of the DMR
of the H19 gene showed MPERs of 0.002 and 0.007, respectively.
Any genomic regions that were normally methylated, whether or not they
were in CGIs, showed significantly lower MPERs than those unmethylated.
This was particularly clear when the MPER of the allele methylated at
DMR of the H19 gene was compared with that of the other
unmethylated allele.
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DISCUSSION
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It was first demonstrated here that the fidelity of replicating
methylation patterns of CGIs in the promoter regions is significantly
higher than that of CGIs outside the promoter regions. It was also
demonstrated here that methylated genomic regions show much higher
fidelity than unmethylated genomic regions. These showed that
maintenance methylation of hemimethylated CpG sites into fully
methylated CpG sites at DNA replication was highly reliable, while
unmethylated CpG sites tended to be methylated by de novo methylation.
It is well-known that exogenous DNA is exposed to a de novo methylation
pressure (Doerfler et al. 2001; Bird 2002), and a similar methylation
pressure seems to be working on the endogenous DNA. To maintain the
unmethylated status of CGIs, protection mechanisms from the de novo
methylation pressure seem to be necessary. Since the MPERs were
significantly lower in CGIs in the promoter regions than in CGIs
outside the promoter regions, the presence of a protection mechanism(s)
specific to the promoter regions, in addition to a mechanism(s) common
to all CGIs, was indicated. Although the details of the mechanisms are
still unknown, binding of transcriptional factors, such as Sp1, has
been indicated as a promoter-specific mechanism (Han et al. 2001).
The differential fidelities in replicating methylation patterns of CGIs
in the promoter regions and those outside indicated that aberrant
methylation of CGIs would occur at different rates depending upon their
locations. This will be important when tumors are analyzed for the CGI
methylator phenotype (CIMP), which are considered to be caused by
molecular defects that allow accumulation of aberrant CGI methylations
(Toyota et al. 1999). The differential fidelities shown here suggest
that there are two types of CIMP, one due to a defect(s) in the
protection mechanisms common to all CGIs and the other due to a
defect(s) in the protection mechanisms specific to CGIs in the promoter
regions. Actually, a correlation between the CIMP and the diffuse-type
histology was clearly observed in gastric cancers when CGIs in the
promoter regions were used for CIMP analysis (Kaneda et al. 2002b),
while it was unclear when CGIs outside the promoter regions were used.
In order for an impaired fidelity in maintaining a methylation pattern
to exert any biological effect, methylation statuses of multiple CpG
sites in a CGI must be altered. A significant increase of MPERs would
be necessary for this, and quantitative analysis of MPERs in cells with
suspected increase of MPERs is necessary. DMR of the H19 gene
had a polymorphism at nt. 391 (nt. 8217; GenBank accession no.
AF125183), and this served to distinguish the two alleles clearly. The
G-allele was methylated in all of the six cultures, and the T-allele
was unmethylated. The methylation patterns of the T-alleles were
similar in HMEC11 and HMEC15, but were essentially variable among the
six cultures. This indicated that, although the original cells in
HMEC11 and HMEC15 might have had a common ancestral cell, methylation
patterns in a tissue alter significantly during a human life
span.
Future clarification of what protection mechanisms are involved and how
they are impaired in various diseases will contribute to understanding
of aging (Ahuja et al. 1998; Issa et al. 2001) and various pathological
conditions.
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METHODS
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Cell Culture and DNA/RNA Extraction
HMECs were purchased from Clonetics, and cultured in MEBM
(Clonetics). HMECs are known to have a stable normal diploid karyotype
(Stampfer and Bartley 1988; Berthon et al. 1992). A single cell in its
log phase was plated in a well of a 96-well plate, and inoculation of a
single cell was confirmed by observing stochastic distribution of
positive wells in the plate and a single colony in a positive well.
Cells were transferred serially to a well of a 12-well plate and to a
10 cM dish. When the cells were subconfluent, they were collected, and
high molecular weight genomic DNA was extracted by serial extraction
with phenol/chloroform and ethanol precipitation. Culturing and DNA
extraction was performed for six progenitor single cells. Plating
efficiencies were measured by parallel plating of 300 cells and
observing their viability. The number of cell generations observed was
calculated from the plating efficiencies and the final cell count.
Sodium Bisulfite Modification and Sequencing
Sodium bisulfite modification was performed according to previous
reports (Clark et al. 1994; Rein et al. 1997). Genomic DNA was
restricted with BamHI restriction enzyme (New England
Biolabs), and 500 ng of the restricted DNA was denatured in 0.3 N NaOH.
The denatured DNA was sulfonated in a solution of 3.1 M
NaHSO3 (pH 5.3) and 0.5 mM hydroquinone, which underwent 15
cycles of denaturation at 95°C for 30 sec and incubation at 50°C
for 15 min. The sample was desalted with the Wizard DNA clean-up system
(Promega), and desulfonated by treatment in 0.3 N NaOH at room
temperature for 5 min. The DNA sample was ethanol-precipitated with
ammonium acetate, and dissolved in 20 µL of TE buffer. For bisulfite
sequencing, 1 µL of the DNA solution was used for PCR with the
primers common for methylated and unmethylated DNA sequences (See
Supplementary Table at www.genome.org). PCR products were cloned
into pGEM-T Easy Vector (Promega), and 10–18 clones from each sample
were cycle-sequenced using T7 and Sp6 primers with a BigDye Terminator
kit (PE Biosystems) and an ABI automated DNA sequencer (PE Biosystems).
To measure unconversion rates during the bisulfite modification, a
completely unmethylated DNA was prepared by PCR of a 6,629-bp fragment,
which covered CGIs in the promoter region and exon 1 of the
E-cadherin gene, with primers shown in the Supplementary Table
and LA-Taq (Takara). The PCR solution contained 1M betaine, and the PCR
was performed for 30 cycles consisting of 10-sec denaturation at 94°C
and 15-min annealing/extension at 72°C. The PCR product was purified,
and added to the rat genomic DNA at an equimolar concentration. In the
same manner with the samples, the rat genomic DNA with the PCR product
was restricted with BamHI, modified with bisulfite, and
sequenced.
Quantitative Reverse-Transcription-PCR
cDNA was synthesized from 3 µg of DNase-treated total RNA in 20
µL with oligo (dT)12–18 primer and SuperScript II reverse
transcriptase (Life Technologies). One µL of the cDNA solution was
amplified in a solution that contained SYBR Green PCR Core Reagents
(Applied Biosystems) and 200 nM of primers. Real-time PCR analysis was
performed using an iCycler iQ detection system (Bio-Rad Laboratories),
with a PCR condition of 40 cycles of denaturation at 94°C for 30 sec,
annealing at a specified temperature for 30 sec, and extension at
72°C for 30 sec. The sequences of the primers and annealing
temperature are listed in the Supplementary Table. The absence of
nonspecific amplification was confirmed by electrophoresing the PCR
products in agarose gels. The number of cDNA molecules was quantified
by comparing amplification of an unknown sample to those of standard
samples that contained 101–107 copies of the gene.
The amount of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) of each cDNA solution was also quantified,
and the amount of a gene of interest was normalized to the amount of
GAPDH.
Calculation of MPER and Fidelity, and Statistical Analysis
To calculate MPERs, clones sequenced for each region were
classified by their methylation patterns. The most and second most
prevalent patterns were determined. When the second most prevalent
patterns were present in multiple, a pattern that would minimize the
number of deviations in the remaining clones from the original two
patterns was regarded as an original pattern. By counting the number of
deviations from the original two patterns (numbers shown to the right
of each clone in Fig. 3), the total number of methylation pattern
errors in each clone was calculated (shown for each culture). To obtain
a MPER of a culture, the total number of methylation errors was divided
by the total number of CpG sites examined in the culture. Fidelity of
methylation pattern (F: %/site/generation) was calculated
from MPERs (M: error/site/21.6 generations) by an equation:
M=1-F21.6.
The MPERs of two regions were statistically compared using a
t-test.
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Acknowledgements
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The authors are grateful to Dr. Nathalie McDonell for her critical
reading of the manuscript. This study was supported by a Grant-in-Aid
for Human Genome, Tissue Engineering and Food Biotechnology; and a
Grant-in-Aid for the Second Term Comprehensive 10-year Strategy for
Cancer Control from the Ministry of Health, Labour and Welfare.
The publication costs of this article were defrayed in part by payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
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Footnotes
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2 Corresponding author. 
E-MAIL tushijim{at}ncc.go.jp; FAX 81-3-5565-1753.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.969603.
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Received November 5, 2002;
accepted in revised format February 26, 2003.
13:868-874 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00
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ABSTRACT
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