Abstract
DNA methylation contributes to the control of gene expression and plays an essential role in cellular physiology.
Well-defined patterns of DNA methylation are established and fixed during embryonic development, and changes
in these patternsmay be a contributing factor in developmental d...
Christina Dahl & Per Guldberg∗
Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen, Denmark; ∗ Author
for correspondence (e-mail: perg@cancer.dk; fax: +45-35257721)
Received 12 December 2002; accepted in revised form 10 March 2003
Key words: bisulfite, DNA methylation, 5-methylcytosine, PCR, review
Abstract
DNA methylation contributes to the control of gene expression and plays an essential role in cellular physiology.
Well-defined patterns of DNA methylation are established and fixed during embryonic development, and changes
in these patterns may be a contributing factor in developmental disorders, cancer and aging. Not least the possibility
of using DNA methylation as a marker for disease has created a strong need for techniques to detect and measure
DNA methylation. Different techniques provide information on DNA methylation at different levels, spanning
from genome-wide methylation content to methylation of single residues in specific genes. The limitations of
individual techniques strongly affect interpretation of data. In this review, we discuss some general themes in DNA
methylation analysis and outline the basic principles of current key techniques. We discuss the advantages and
disadvantages of these techniques, including potential artifacts and pitfalls, and suggest some overall guidelines
that may be instructive for a rational choice of methodology.
Introduction methylated, i.e., cytosines within CpG dinucleotides.
Furthermore, while CpGs in bulk DNA are usually
Approximately 1% of the bases in mammalian methylated, methylation is absent in CpG-rich regions
genomes are 5-methylcytosine. In contrast to the other (CpG islands) that are present near the 5′ end of 50–
four bases in the genome, 5-methylcytosine does not 60% of all genes. Overall, ∼4% of cytosines and
exist in the cellular environment as a free nucleotide. ∼75% of CpGs are methylated in genomic DNA
Rather, the addition of a methyl group to cytosine from normal human postnatal somatic tissues. The
occurs after DNA replication and is catalyzed by biological significance of this particular distribution
a group of DNA methyltransferase enzymes (Bestor of DNA methylation has become increasingly under-
2000). The distribution of 5-methylcytosine is not ran- stood through the unraveling of mechanisms by which
dom in the genome. Notably, with few possible excep- DNA methylation may contribute to the control of
tions, only cytosines that precede guanines can be gene transcription.
,234
Methylation is the predominant ‘epigenetic’ found in ICF syndrome, a rare disorder characterized
modification of DNA in mammalian genomes. The by immunodeficiency, facial anomalies, and hypo-
term epigenetic is used to denote that methylation methylation of the satellite DNA in juxtacentromeric
can modify the information content of DNA without heterochromatin of chromosomes 1 and 16 (Okano et
changing the primary nucleotide sequence. Hence, al. 1999).
DNA methylation does not alter the structure or In normal, fully differentiated cells, DNA methyla-
function of a gene, but provides information as to tion patterns are replicated with high fidelity during
where and when the gene should be expressed. There mitosis. However, a large body of data has shown that
is now substantial evidence that, in general, the these patterns can become altered and cause cellular
level of methylation within a gene’s promoter CpG functional abnormalities in the course of human
island correlates with its transcriptional silencing. disease and aging. The pathologic consequences of
Altering the binding of some transcription factors is altered DNA methylation have been most extensively
one possible mechanism by which 5-methylcytosine studied in cancer. Compared with normal cells, cancer
controls gene expression. An alternative model for cells frequently show genome-wide hypomethylation,
methylation-based gene silencing implicates a group hypermethylation of tumor suppressor genes, and
of proteins and protein complexes that specifically loss of genomic imprinting (Baylin et al. 1998).
bind to methyl-CpG and recruit histone deacetylates Recent studies have demonstrated increased rates of
to the sites of methylation (Wade 2001). The removal spontaneous mutations, deletions and rearrangements
of acetyl groups from the histones of the nucleosomal in cells lacking DNMT1, the DNA methyltransferase
core converts the open, transcriptionally competent responsible for maintaining DNA methylation patterns
chromatin structure into a closed structure that is after DNA replication (Chen et al. 1998). Accord-
inaccessible to the transcriptional machinery. A third ingly, loss of genomic methylation may represent an
possible mechanism of methylation-dependent gene early event in multistep carcinogenesis by providing
silencing involves DNA methyltransferases, which the premalignant cell with a mutator phenotype. In
have transcriptional repressor function and can recruit contrast, hypermethylation of CpG islands may cause
histone deacetylates to methylated DNA (Robertson gene silencing and can be functionally equivalent to
2002). mutation and deletion in activating tumor suppressor
In humans, methylation-dependent gene silencing genes (Jones and Laird 1999). Intriguingly, evidence is
has been implicated in inactivation of the X chromo- emerging that many of the changes observed in cancer
some in females, inactivation of the silent allele at cells are also present in aging cells in normal tissues. It
imprinted loci, and silencing of intragenomic para- has been proposed that these age-related methylation
sitic DNA elements (Costello and Plass 2001). Several changes are precursors for the aberrant methylation
lines of research have shown that the establishment patterns in cancer cells and thus may contribute to
and maintenance of well-defined methylation patterns the age-related increase in cancer risk (Ahuja and Issa
are required for normal embryonic development. First, 2000).
elimination of any of the three known DNA methyl- The central role of DNA methylation in main-
transferases, Dnmt1, Dnmt3a and Dnmt3b, is lethal taining cellular function and the recognition that
in mice (Okano et al. 1999; Li et al. 1992). Second, changes in DNA methylation patterns may have broad
a number of human disorders are associated with implications for human health have created a strong
genetic abnormalities involving chromosomal regions need for techniques to reliably detect and measure
that are subject to genomic imprinting, includ- methylation in DNA from diverse sources. In this
ing Beckwith-Wiedemann syndrome, Prader-Willi review, we describe different ways of analyzing DNA
syndrome, and Angelman syndrome (Paulsen and methylation and outline the basic principles of current
Ferguson-Smith 2001). Third, two human syndromes techniques (listed in Table 1), with emphasis on tech-
have been identified that are caused by inherited niques for gene-specific analysis of DNA methylation.
defects in genes affecting the DNA methylation Each of these techniques is limited in the information
machinery. Rett syndrome, an X-linked, neuropsychi- content of the data it generates, which strongly affects
atric disorder with onset in early childhood, is caused interpretation of data.
by loss-of-function mutations in the gene encoding
methyl-CpG binding protein MeCP2 (Amir et al.
1999), and mutations in the DNMT3B gene have been
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Table 1. Techniques described in this review, grouped according to the dimensions in which they can resolve DNA methylation.
Initial treatment of PCR-
Application Technique genomic DNA based Key references
Analysis of genome- HPLC Enzymatic hydrolysis No Kuo et al. 1980
wide methylation HPCE Enzymatic hydrolysis No Fraga et al. 2002
content TLC Enzyme digestion No Schmitt et al. 1997
SssI acceptance assay None No Wu et al. 1993
Chloroacetaldehyde assay Bisulfite treatment No Oakeley et al. 1999
Immunochemical analysis Denaturation/depurination No Oakeley et al. 1997
Qualitative analysis MSRE-Southern Enzyme digestion No Southern 1975
of single CpGs MSRE-PCR Enzyme digestion Yes Singer-Sam et al. 1990
Methylation-specific PCR Bisulfite treatment Yes Herman et al. 1996
COBRA Bisulfite treatment Yes Xiong and Laird 1997
Direct bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
Cloned bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
Quantitative analysis Ms-SnuPE Bisulfite treatment Yes Gonzalgo and Jones 1997
of single CpGs MethyLight Bisulfite treatment Yes Eads et al. 2000
COBRA Bisulfite treatment Yes Xiong and Laird 1997
Direct bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
Analysis of allelic Direct bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
methylation Cloned bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
heterogeneity MethyLight Bisulfite treatment Yes Eads et al. 2000
MS-MCA Bisulfite treatment Yes Worm et al. 2001
MS-DGGE Bisulfite treatment Yes Aggerholm et al. 1999
MS-SSCA Bisulfite treatment Yes Maekawa et al. 1999
MS-DHPLC Bisulfite treatment Yes Baumer et al. 2001
MSO Bisulfite treatment Yes Gitan et al. 2002;
Adorjan et al. 2002
Random screening for RLGS Enzyme digestion No Costello et al. 2000
altered methylation MCA-RDA Enzyme digestion Yes Toyota et al. 1999
loci MS-AP-PCR Enzyme digestion Yes Gonzalgo et al. 1997
DMH Enzyme digestion Yes Huang et al. 1999
Analysis of genome-wide methylation content High-performance liquid chromatography (HPLC)
and related techniques
Data on genome-wide methylation content in a pool
of DNA is usually generated by determining the
For many years, HPLC has been the preferred tech-
ratio between 5-methylcytosine and cytosine. Current
nique for quantitative determination of the global
standard procedures involve complete enzymatic
levels of DNA methylation (Kuo et al. 1980). Accord-
hydrolysis of DNA, followed by high-resolution
ing to standard procedures, total genomic DNA is
separation to obtain the total base composition of the
hydrolyzed to deoxyribonucleotides (base + deoxyri-
genome. Alternative techniques have been developed,
bose + phosphate) using a combination of deoxyribo-
including assays based on the use of bacterial DNA
nuclease I and nuclease P1. Following hydrolysis,
methyltransferase or 5-methylcytosine specific anti-
deoxyribonucleotides are further converted into
bodies.
deoxyribonucleosides (base + deoxyribose) by treat-
ment with alkaline phosphatase, and the products
are then separated by standard reverse-phase HPLC.
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