REVIEW ARTICLE

DNA Methylation
By Rakesh Singal and Gordon D. Ginder

S INCE ITS FIRST recognition in 1948, the fifth base of

human DNA, 5-methylcytosine (5-mC) has generated
much interest and considerable controversy during attempts to
chromatin configuration may allow, or be a consequence of, the
interaction of transcription factors with gene promoters.14
The patterns of DNA methylation reflect two types of gene 58
understand its significance (for review, see Weissbach1). DNA regulatory regions in the genome. Approximately half of all
methylation in eukaryotes involves addition of a methyl group genes in mouse and humans (ie, 40,000 to 50,000 genes)
to the carbon 5 position of the cytosine ring (Fig 1). This contain CpG islands.10 These are mainly housekeeping genes
reaction is catalyzed by DNA methyltransferase in the context that have a broad tissue pattern of expression, but approxi-
of the sequence 58-CG-38, which is also referred to as a CpG mately 40% of genes with a tissue-restricted pattern of expres-
dinucleotide. It is the most common eukaryotic DNA modifica- sion are also represented.14 Promoter region CpG islands are
tion and is one of the many epigenetic (alteration in gene usually unmethylated in all normal tissues, regardless of the

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expression without a change in nucleotide sequence) phenom- transcriptional activity of the gene. The main exceptions include
ena. Although extensive in plants and mammals, the absence of nontranscribed genes on the inactive X-chromosome and im-
detectable DNA methylation in some eukaryotes such as printed autosomal genes where one of the parental alleles may
Drosophila2 and Saccharomyces cerevisiae3 has raised doubts be methylated.15 Tissue-specific genes without CpG islands are
about its significance in normal development and tissue-specific variably methylated, often in a tissue-specific pattern, and
gene expression. However, recent studies showing abnormal usually methylation is inversely correlated with the transcrip-
development and embryonic lethality in transgenic mice express- tional status of the genes (for review, see Bird16 and Cedar17).
ing decreased but not completely absent DNA Methyltransfer-
DNA METHYLTRANSFERASES
ase (MTase) activity after DNA-MTase gene knockout4 lends
support to a critical role for DNA methylation in developmental The enzymes that transfer methyl groups to the cytosine ring,
gene regulation. Others have proposed that the control of cytosine 5-methyltransferases, or DNA methyltransferases
intragenomic parasites is the primary function of DNA methyl- (DNA-MTase) have been characterized in a number of eukary-
ation in mammalian cells.5 otes.18 The target site for DNA-MTase in DNA is the dinucleo-
In this review, we will discuss the fundamental aspects of tide palindrome CG (commonly referred to as CpG, with p
DNA methylation and its role in transcription repression, denoting the phosphate group). The first eukaryotic DNA-
neoplasia, and transgene silencing and during development. In MTase gene cloned from mouse almost a decade ago19 is now
the interest of brevity, the role of DNA methylation in genomic referred to as Dnmt1. This gene is highly conserved among
imprinting and X chromosome inactivation are not discussed eukaryotes. Dnmt1 orthologs have been identified in various
here, but have been reviewed recently elsewhere.6-8 species, including humans (DNMT1).20 Interestingly, Dnmt1
orthologs have not been found in organisms lacking DNA
methylation, such as Saccharomyces cerevisiae, Caenorhabdi-
DISTRIBUTION OF METHYLATED CYTOSINES tis elegans, and Drosophila melanogaster.
AND CpG ISLANDS Mammalian Dnmt1 methyltransferase has a high affinity for
Eukaryotic genomes are not methylated uniformly but con- hemimethylated substrates but is also capable of performing de
tain methylated regions interspersed with unmethylated do- novo methylation of unmethylated substrates in vitro. The de
mains.9 During evolution, the dinucleotide CpG has been novo activity of mammalian Dnmt1 methyltransferase has been
progressively eliminated from the genome of higher eukaryotes shown to be stimulated by aberrant DNA structures21 and 5-mC
and is present at only 5% to 10% of its predicted frequency.10-12 residues in single-stranded22 or double-stranded DNA sub-
Cytosine methylation appears to have played a major role in this strates.23 After it was found that the human and mouse Dnmt1
process, because most CpG sites lost represent the conversion genes contain additional 58 transcribed sequences with protein-
through deamination of methylcytosines to thymines. Approxi-
mately 70% to 80% of the remaining CpG sites contain From the Deparment of Medicine, Feist-Weiller Cancer Center and
methylated cytosines in most vertebrates, including hu- Overton Brooks VA Medical Center, LSU Medical Center, Shreveport,
mans.10,12 These methylated regions are typical of the bulk LA; and the Departments of Internal Medicine and Human Genetics,
chromatin that represents the late replicating DNA with its Massey Cancer Center, Virginia Commonwealth University, Rich-
attendant histone composition and nucleosomal configuration mond, VA.
and is relatively inaccessible to transcription factors.13 In Submitted January 21, 1999; accepted March 26, 1999.
contrast to the rest of the genome, smaller regions of DNA, Supported by National Institutes of Health Grant No. DK29902 and
called CpG islands, ranging from 0.5 to 5 kb and occurring on by the Massey Cancer Center (G.D.G.) and the Feist-Weiller Cancer
Center (R.S.).
average every 100 kb, have distinctive properties. These regions
Address reprint requests to Gordon D. Ginder, MD, Professor of In-
are unmethylated, GC rich (60% to 70%), have a ratio of CpG to ternal Medicine and Human Genetics, Director, Massey Cancer Center,
GpC of at least 0.6, and thus do not show any suppression of the Virginia Commonwealth University, 401 College St, PO Box 980037,
frequency of the dinucleotide CpG.10,14 Chromatin containing Richmond, VA 23298-0037; e-mail: gginder@[Link].
CpG islands is generally heavily acetylated, lacks histone H1, r 1999 by The American Society of Hematology.
and includes a nucleosome-free region.13 This so called open 0006-4971/99/9312-0043$3.00/0

Blood, Vol 93, No 12 (June 15), 1999: pp 4059-4070 4059

4060 SINGAL AND GINDER

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Fig 1. Schematic representation of the biochemi-
cal pathways for cytosine methylation, demethyl-
ation, and mutagenesis of cytosine and 5-mC.

encoding potential,24 it became apparent that previous biochemi- methylation-sensitive restriction enzymes. Singer-Sam et al32
cal and cell culture assays had relied on an incomplete Dnmt1 improved the sensitivity of methylation detection by combining
cDNA sequence that codes for a truncated protein. Pradhan et the use of methylation-sensitive restriction enzymes and poly-
al25 demonstrated that full-length Dnmt1 MTase synthesized in merase chain reaction (PCR). After cleaving the DNA with
Baculovirus was equally active on unmethylated and hemimeth- methylation-sensitive restriction endonucleases, eg, Hpa II,
ylated substrates, in part by virtue of an inhibitory effect of the PCR amplification with specific primers flanking the restriction
additional N-terminal amino acids on activity with hemimethyl- site will only occur if DNA cleavage has been prevented by
ated DNA. These results are evidence that the Dnmt1 MTase methylation. However, this method, like Southern-based ap-
protein has inherent de novo methylating activity that may be proaches, can only monitor CpG methylation in methylation-
altered by protein-protein interactions and enhanced by aberrant sensitive restriction sites. Also, a false-positive result may be
structures or 5-mC residues in the substrate DNA. obtained due to incomplete restriction enzyme digestion of
Evidence for an additional mammalian DNA-MTase capable cellular DNA.
of de novo methylation came from the experiments of Lei et Genomic sequencing protocols that have previously been
al.26 These investigators generated a null mutation of the Dnmt1 used to study DNA methylation use Maxam and Gilbert
gene through homologous recombination in mouse embryonic chemical cleavage reactions performed on genomic DNA33 with
stem cells. Dnmt1 null embryonic stem cells were viable and linker-mediated PCR (LMPCR) to enhance the signal.34 These
contained low but stable levels of methylcytosine and methyl- methods are based upon the fact that 5-mC is not cleaved during
transferase activity. Interestingly, integrated provirus DNA in the standard Maxam and Gilbert cytosine cleavage reaction.35
Moloney murine leukemia virus (MoMuLV)-infected homozy- Thus, 5-mC is identified in a sequencing gel by the lack of a
gous Dnmt1 knockout ES cells exhibited de novo methylation band that corresponds to a cleavage product of a cytosine
to a similar extent as in wild-type cells. Although a second degradation reaction. This assay is technically demanding and is
DNA-MTase has recently been identified in mouse (Dnmt2)27 subject to both false-positive and false-negative results.
and in humans (DNMT2),28 it is not yet clear if it is the long Frommer et al36 introduced a procedure based on bisulfite-
sought after de novo methyltransferase present in Dnmt1 induced oxidative deamination of genomic DNA under condi-
deficient ES cells. tions in which cytosine is converted to uracil and 5-mC remains
unchanged. The target sequence is then amplified by PCR using
TECHNIQUES TO STUDY DNA METHYLATION strand-specific primers. Upon sequencing of the amplified
Early techniques to study site-specific DNA methylation DNA, all uracil and thymine residues become detectable as
relied primarily on the inability of methylation-sensitive type II thymine and only 5-mC residues amplify as cytosines. This
restriction enzymes to cleave sequences containing one or more method, as shown in Fig 2, is presently the method of choice for
methylated CpG sites, combined with Southern hybridiza- the detailed analysis of 5-mC in any given genomic target
tion.29-31 This method requires large amounts of high molecular sequence.
weight DNA, detects methylation only if more than a few A number of rapid methods to detect 5-mC have been
percent of alleles are methylated, and only provides information developed based on the bisulfite deamination reaction in
about those CpG sites found within the recognition sequence of combination with PCR amplification. These are suitable for
DNA METHYLATION 4061

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Fig 2. (A) Illustration of the steps involved in determining the
methylation status of cytosines in a known DNA sequence by the
bisulfite conversion method. (B) In vivo methylation of CpG dinucleo-
tides of rho promoter in 5-day and adult chicken erythroid cells using
bisulfite conversion technique. Arrows indicate methylated cytosines
and are only seen in adult erythroid cells. Positions indicated are
relative to the transcription start site. Cytosines that are not associ-
ated with CpG dinucleotides (sequence shown in [C]) have all been
converted to thymidines in both 5-day and adult erythroid cells. (C)
Graphic representation of the in vivo methylation of CpG dinucleo-
tides of rho promoter in 5-day and adult chicken erythroid cells using
bisulfite conversion technique. Arrows indicate methylated cytosines
and are clearly seen in (A) (data not shown for CpG dinucleotide at
position ⴚ15). Primers R and F indicate the sequence of rho globin
promoter used for designating internal primers and for sequencing.

examining limited numbers of CpG dinucleotides that are either of the gene under study in vivo (reviewed in Bird16 and Razin
found within or immediately adjacent to the PCR primer and Cedar46).
sequences37,38 or within a restriction enzyme recognition se- Three possible mechanisms have been proposed to account
quence.39 for transcriptional repression by DNA methylation, and each is
shown in Fig 3. The first mechanism involves direct interfer-
DNA METHYLATION AND TRANSCRIPTIONAL ence with the binding of specific transcription factors to their
REPRESSION recognition sites in their respective promoters. Several transcrip-
A role for DNA methylation in the differential regulation of tion factors, including AP-2, c-Myc/Myn, the cyclic AMP-
gene expression was hypothesized many years ago.40,41 The dependent activator CREB, E2F, and NF-␬B, recognize se-
potential mechanism was suggested by a number of early quences that contain CpG residues, and binding to each has
observations in which site-specific cytosine methylation within been shown to be inhibited by methylation. In contrast, other
or adjacent to genes was found to correlate with transcriptional transcription factors (eg, Sp1 and CTF) are not sensitive to
repression.31,42-45 Subsequently, this inverse relationship be- methylation of their binding sites,47 and many factors have no
tween cytosine methylation and transcription has been observed CpG dinucleotide residues in their binding sites.
in a large number of genes, although not universally. A second potential mechanism for methylation induced
Despite the long held view that DNA methylation might act silencing is through the direct binding of specific transcriptional
as a negative regulator of transcription, the precise mechanism repressors to methylated DNA. Two such factors, MeCP-1 and
involved has remained elusive. Numerous reports have shown MeCP-2 (methyl cytosine binding proteins 1 and 2), have been
the ability of promoter DNA methylation to inhibit transcription identified and shown to bind to methylated CpG residues in any
of a wide variety of genes in in vitro transfection assays, and in sequence context. Although in vertebrates DNA methylation
some cases, such methylation corresponds to the inactive state has been posited to inhibit transcription initiation, methylation
4062 SINGAL AND GINDER

has also been shown to block transcription elongation in naked DNA in a cell-free in vitro assay,60 suggesting that
Neurospora through a mechanism that may be mediated through chromatin formation is not necessary for its repressive action.
MeCP-1 and/or MeCP-2.48 A third mechanism by which methylation may mediate
MeCP-1 binds to DNA containing multiple symmetrically transcriptional repression is by altering chromatin structure.
methylated CpG sites, as opposed to hemimethylated CpGs, and Keshet et al64 transfected mouse L cells with M13 plasmid
manifests as a large complex on electrophoretic mobility shift constructs containing the human ␤-globin gene, as well as
assay.49 Repression of transcription from densely methylated several other eukaryotic genes, after enzymatic methylation.
genes can be mediated by MeCP-1, and cells deficient in Unmethylated DNA sequences, after integration and stable
MeCP-1 show much reduced repression of methylated genes.50 propagation in cell culture, were all detected in active chroma-
In a further study, it was demonstrated that sparse methylation tin, as measured by DNAse 1-sensitivity, in contrast to DNA
could repress transfected genes completely, but the inhibition sequences that were methylated in vitro before transfection that
was fully overcome by the presence in cis of an SV40 enhancer. were contained in DNAse1-resistant, transcriptionally inactive
However, densely methylated genes could not be reactivated by chromatin. Experiments using microinjection of certain methyl-
the strong enhancer. It was proposed that sparsely methylated ated and nonmethylated gene templates into nuclei have shown

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genes form an unstable complex with MeCP-1 that prevents that methylation inhibits transcription only after chromatin is
transcription when the promoter is weak. This complex can be assembled.65 Even a strong transcriptional activator, GAL4-
disrupted by a strong promoter, thereby allowing the methylated VP16, cannot counteract the effect of chromatin once it has
gene to be transcribed.51 assumed the inactive state induced by DNA methylation.65
We have recently shown that a complex with electrophoretic Therefore, in addition to stabilizing the inactive state, methyl-
mobility similar to MeCP-1 forms efficiently with the methyl- ation also prevents activation by blocking the access of
ated but not with unmethylated embryonic rho-globin gene transcription factors.63,66 Whether this chromatin effect of
promoter sequences, and the complex can be detected using methylation is mediated solely by MeCP-2–associated histone
nuclear extracts from the same primary avian erythroid cells in deacetylase remains to be determined.
which methylation-mediated transcriptional inhibition was dem- One important issue regarding DNA methylation and tran-
onstrated.52 These results, in conjunction with the demonstra- scriptional silencing has been whether methylation is a primary
tion of a role for methylation in silencing rho-globin gene control mechanism or a secondary effect of gene activity. In the
transcription in vivo in normal adult avian erythroid cells,53,54 case of some genes with sparse CpG nucleotides, gain of
suggest a role for MeCP-1 or a similar complex in developmen- methylation occurs after transcriptional silencing67; in other
tal silencing of embryonic globin genes during normal erythro- cases, loss of methylation occurs after transcriptional activa-
poiesis. tion.68 At the same time, in other systems it has been shown that
A component of the MeCP-1 complex, PCM1, has a methyl- despite optimum nuclear conditions for transcription, including
CpG binding domain (MBD) and two cysteine-rich domains DNAseI-sensitive chromatin, transcription can be tightly re-
(CXXC) that are found in animal DNA methyltransferases and pressed by CpG methylation.52,53 It appears that methylation,
in the mammalian HRX proteins, MLL and ALL-1. Although particularly of CpG-rich genes, may serve as a locking off
the functional significance of the CXXC domain is not known, mechanism that may follow or precede other events that turn a
there is evidence that it is a part of a transcriptional repression gene off, but that once in place can prevent activation despite an
domain.55,56 PCM1 has been shown to repress transcription in optimum nuclear environment for transcription.
vitro in a methylation-dependent manner.57
MeCP-2 is more abundant than MeCP-1 in the cell and is able DNA DEMETHYLATION DURING DEVELOPMENT
to bind to DNA containing a single methylated CpG pair.58 AND TISSUE-SPECIFIC DIFFERENTIATION
MeCP-2, like DNA methyltransferase, is dispensable for the A critical aspect of the overall regulatory role of DNA
viability of embryonic stem cells, but is essential for embryonic methylation is the process of demethylation. Using methylation-
development.59 MeCP-2 has two domains: a methyl-CpG sensitive restriction enzymes to monitor the general level of
binding domain that is essential for chromosomal localization DNA methylation, it was shown that, during early development,
and a transcriptional repressor domain (TRD) that can inhibit a dramatic reduction in methylation levels occurs in the
transcription from a promoter at a distance, suggesting that preimplantation embryo.69 This is followed by a wave of de
MeCP-2 interacts with the transcriptional machinery or the novo methylation involving most CpG residues but leaving the
initiation complex.60 Recently, a region of MeCP-2 that local- CpG islands unmethylated at the time of implantation.70 After
izes with the TRD was shown to associate with a corepressor implantation, most of the genomic DNA is methylated, whereas
complex containing the transcriptional repressor mSin3A and tissue-specific genes undergo demethylation in their tissues of
histone deacetylases. Transcriptional repression in vivo was expression.46 Thus, a means for both global and tissue- or
relieved by the deacetylase inhibitor trichostatin A, suggesting gene-specific removal of methylcytosines from DNA must
that two global mechanisms of gene regulation, DNA methyl- exist.
ation and histone deacetylation, can be linked by MeCP-2.61,62 The mechanism underlying the process of demethylation has
However, in some instances DNA methylation has been shown not been fully elucidated. In some cases, demethylation could
to play a dominant role over histone deacetyloses in transcrip- be a passive process, ie, inhibition of methylation after DNA
tional repression.53,54 It has been proposed that MeCP-2 might replication.45 Despite the tight coordination of DNA methyl-
contribute to the assembly of a more stable repressive chroma- ation and replication that has been demonstrated in mammalian
tin structure.63 However, MeCP-2 can repress transcription of cells,71 the mechanism of methylation inhibition in the presence
DNA METHYLATION 4063

of DNA replication remains to be defined. The existence of an DNA METHYLATION AND CANCER
active demethylation process not involving DNA replication in A role for DNA methylation in oncogenesis has been
mammalian cells has been supported by a number of observa- hypothesized for many years. Numerous studies have suggested
tions (reviewed in Szyf72). In transient transfection assays using aberrations in DNA methyltransferase activity in tumor cells.83-85
myoblast cells, a methylated ␣-actin gene was shown to have Transformed cells often have increased total DNA-MTase
transcriptional activity similar to unmethylated template second- activity, widespread loss of methylation from normally methyl-
ary to an active demethylation process.73 Using transient ated sites, and more regional areas of hypermethylated DNA.86
transfection assays, Szyf et al74 showed that an in vitro The potential contribution of DNA methylation to oncogenesis
methylated SK-plasmid bearing no sequence homology to appears to be mediated by one or more of the following
eukaryotic genes became fully demethylated between 1 and 2 mechanisms that are shown in Fig 4.
days after tranfection into the mouse embryonal carcinoma cell Signature C=T mutation in cancer cells. The high muta-
line P19. This demethylating activity was not sequence- tion rate of cytosine residues within the dinucleotide CpG, the
selective and was independent of DNA replication.74 During target site of mammalian DNA-MTase, can be accounted for by
B-cell development, both the heavy and light chain Ig genes an increased rate of cytosine to thymine transitions, which are,

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undergo demethylation.75 Specific cis-acting elements, includ- in turn, a consequence of hydrolytic deamination of 5-mC87 (Fig
ing both the intronic and 38 enhancers in the case of the ␬ light 1). This mechanism of mutation was first recognized in
chain gene, have been shown to direct demethylation.75,76 prokaryotic systems.88
Further analysis of the intronic enhancers suggested the involve- Unmethylated cytosine can also undergo deamination to
ment of the NF-␬B family of proteins as trans-acting factors in yield uracil, but the well-characterized Uracil-DNA glycosylase
inducing demethylation. Both cis-acting elements and trans- efficiently repairs G:U but not G:T mismatch.89 However,
acting factors, therefore, appear to direct the demethylation DNA-MTase may block this repair, contributing to C=U=T
machinery to its target locus. transition. DNA-MTase may also mediate 5-mC=T transition
The biochemical mechanism underlying the process of at CpG dinucleotides under conditions that lead to increased
demethylation remains unclear. However, extracts from chicken DNA-MTase expression or decreased cellular S-adenosylmethio-
embryos have been demonstrated to have sequence nonspecific nine levels.90
demethylating activity in vitro.77 This enzymatic activity was A striking example of how this process may lead to oncogen-
limited to demethylation of hemimethylated DNA and involved esis is shown by the tumor-suppressor gene p53. Mutations in
removal of the methylated cytosine and subsequent repair of the the p53 tumor-suppressor gene occur in more than 50% of
resulting apyrimidinic acid residue.78 In the case of rat myo- human solid tumors.91 An estimated 24% of these mutations are
blasts, demethylation activity is performed by an active compo- C=T transitions at CpG dinucleotides, suggesting that DNA
nent whose activity is to excise methylated nucleotides from the methylation may contribute to these mutations.92
DNA template and replace them with an unmodified form.79 DNA hypomethylation in cancer. Decreased levels of over-
Interestingly, in the presence of a myoblast extract, demethyl- all genomic methylation are common findings in tumorigen-
ation of the ␣-actin but not of the adenine phosphoribosyl esis.93 This decrease in global methylation appears to begin
transferase (APRT) CpG island fragment was observed. Al- early and before the development of frank tumor formation.94,95
though this demethylating activity was initially felt to be Apart from the overall genomic hypomethylation, specific
dependent on an RNA component, subsequent investigation of a oncogenes have been observed to be hypomethylated in human
more purified active fraction demonstrated complete resistance tumors. A good inverse correlation between methylation and
of the activity to RNAse treatment.80 gene expression was observed in the antiapoptotic bcl-2 gene in
It has long been supposed that direct removal of the methyl B-cell chronic lymphocytic leukemia96 and for the k-ras
group by breaking the carbon-to-carbon bond of 5-mC would be proto-oncogene in lung and colon carcinomas.97
so energetically unfavorable as to be an unlikely event. How- Hypermethylation of tumor-suppressor genes. In addition
ever, a gene encoding an enzyme capable of directly removing to point mutations or gene deletions, transcriptional repression
the methyl group from methylated CpG-containing DNA in cell by hypermethylation of promoter sequences suggests an alterna-
free reactions as well as in transfected cells has been reported tive means for the inactivation of tumor-suppressor genes in
recently.81 If the corresponding endogenous gene product can be cancer. This may result from the increased DNA-MTase levels
shown to have the same activity, this will provide a remarkable that have been demonstrated in various cancers83 or it could
new avenue for studying the control of demethylation during occur as a result of some other transient event that silences
normal differentiation and development as well as in oncogen- tumor-suppressor gene transcription. The retinoblastoma gene
esis. (Rb) was the first classic tumor-suppressor gene in which CpG
It has been proposed that the core demethylase in cells is kept island hypermethylation was detected. Three of 21 sporadic
in an inactive form by interaction with a protein inhibitor. cases in one study98 and 5 of 32 sporadic cases in another
Demethylation could then come about by the removal or study99 had hypermethylated CpG islands at the 58 end of the Rb
modification of the inhibitor. Such an inhibitor could either gene. Subsequently, it was also shown that in vitro methylation
affect the entire genome resulting in a pattern similar to that of the promoter region of Rb directly blocked promoter
seen in the early preimplantation embryo or be confined to the activation.100
specific sequence influenced by local cis-acting elements and Inactivation of the von Hippel-Lindau (VHL) gene by
trans-acting factors, as in the case of the ␬ Ig gene example somatically acquired mutations in one copy of the VHL gene
discussed above.82 along with loss of the second allele has been implicated as the
4064 SINGAL AND GINDER

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Fig 3. Proposed mechanisms
of transcriptional repression me-
diated by cytosine methylation.

Fig 4. Models for the differ-

ent mechanisms through which
cytosine methylation can pro-
mote oncogenesis.
DNA METHYLATION 4065

initiating event in spontaneous cases of clear-cell renal carci- instability necessary for the development and progression of
noma.101 Herman et al102 found hypermethylation of the VHL cancer. In contrast, MMR⫺ cells have normal methylation
gene CpG island and concomitant lack of expression in 5 of 26 proficiency and develop the required genomic instability by the
cases of the sporadic form of clear cell renal carcinoma that had alternative pathway of mismatch repair deficiency.111 Consistent
no VHL intragenic mutations. Treatment of a clear cell renal with a role for hypomethylation in chromosomal instability
carcinoma cell line with 5-deoxyazacytidine, a potent inhibitor were the findings by Chen et al112 in analyzing embryonic stem
of DNA-MTase, resulted in demethylation and expression of a (ES) cells nullizygous for the DNA-MTase gene, Dnmt I. Gene
previously silent VHL gene. deletions of selectable marker genes due to mitotic recombina-
One of the most important cell cycle regulatory proteins is tion or chromosomal loss were detected at a much higher
p16 (also known as MTS-1 for major tumor suppressor 1, frequency in the Dnmt 1-deficient ES cells compared with
INK4a for INhibitor of cyclin-dependent Kinase 4a, and wild-type control cells.
CDKN2a for Cyclin-Dependent Kinase iNhibitor 2a). The Additional experimental support for the hypothesis linking
major biochemical effect of p16 is to halt cell-cycle progression DNA methylation to chromosomal integrity comes from earlier
at the G1/S boundary, and the loss of p16 function may lead to studies by Feinberg et al,113 who demonstrated an average of 8%

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cancer progression by allowing unregulated cellular prolifera- and 10% reduction in genomic 5-mC content in colon adenomas
tion.103 A common genetic alteration in tumor cell lines and to a and adenocarcinomas, respectively, with no significant differ-
lesser extent in primary tumors is the loss of heterozygosity at ence between benign and malignant tumors. Interestingly, three
chromosome 9p21, which contains both the related p16 and p15 patients with the highest 5-mC content in their normal colon
genes. Among most solid tumors studied, a CpG island in the 58 appeared to have Lynch syndrome (HNPCC), which was
region of p16 gene has been found to be frequently methylated, subsequently shown to have the MMR⫺ phenotype.113 In a
and treatment of cell lines carrying a hypermethylated p16 recent study, the occurrence of genome-wide undermethylation,
allele with 5-azacytidine results in transcriptional activation of retroviral element amplification, and chromosome remodeling
the gene.104,105 The presence of either coding region deletions or in an interspecific mammalian hybrid (Macropus eugenii ⫻
promoter hypermethylation of p16 correlate inversely with the Wallabia bicolor) was demonstrated. Atypically extended cen-
presence of Rb gene mutations in multiple tumor types (re- tromeres of Macropus eugenii-derived autosomes in the hybrid
viewed in Baylin et al86). Interestingly, in some instances, p16 were composed primarily of unmethylated, amplified retroviral
promoter hypermethylation may be the sole inactivating event elements not detectable in either parental species, indicating
for both alleles of the gene and may be the only lesion that the failure of DNA methylation to occur and resultant
associated with loss of the cyclin D-Rb pathway.86 In colon mobile-element activity in the cell hybrids could facilitate rapid
cancer, despite the lack of allelic loss of the 9p region, karyotypic evolution.114
homozygous deletions of the p16 gene, or Rb gene mutations,106
30% to 40% demonstrate hypermethylated p16 alleles.105 ALTERED DNA-METHYLTRANSFERASE
Hypermethylation-mediated inactivation of the p16 gene has ACTIVITY IN CANCER
been demonstrated in brain, breast, colon, head and neck, and Several studies in the last few years have demonstrated an
non–small-cell lung cancer and in high grade non-Hodgkin’s increase in DNA-MTase activity in neoplastic cells. Kautiainen
lymphoma (reviewed in Baylin et al86). and Jones83 examined the levels of DNA methyltransferase in
P15 (INK4b) gene is an inhibitor of the cyclin-dependent nuclei from 9 tumorigenic and 9 nontumorigenic cell lines. In
kinases CDK4 and CDK6 and appears to play a role in all but 2 cases, the extractable methyltransferase activity was
transforming growth factor-␤ (TGF-␤)–mediated growth inhibi- fourfold to 3,000-fold higher in tumorigenic than in nontumori-
tion responses.107 Methylation-mediated inactivation of the p15 genic cells.83 Increased DNA-MTase activity has been reported
gene occurs predominantly in hematopoietic neoplasms such as in colon cancers versus normal mucosa from the same patient.84
acute myelogenous leukemia, acute lymphoblastic leukemia, Laird et al85 bred normal mice heterozygous for deletion of the
and Burkitt’s lymphoma.86 Recently, hypermethylation of the DNA-MTase gene (and having 50% of the DNA-MTase activity
p15 gene has been demonstrated in myelodysplastic syn- compared with wild-type) with mice having a mutant allele of
dromes108 and hypermethylation of both p15 and p16 has been the APC gene.85 When combined with loss of the other APC
found in multiple myeloma.109 allele, this mutation results in progressive formation of adeno-
Induction of chromosomal instability. Lengauer et al110 mas throughout the gastrointestinal tract of newborn animals.
introduced exogenous CpG-rich sequences in the form of a Intestinal adenomas were reduced by 50% in the offspring mice
retrovirally contained ␤-galactosidase gene into 10 colon cancer with both the APC and DNA-MTase gene mutations and were
cell lines. Five of 10 cell lines failed to express the ␤-gal gene, essentially eliminated when treatment with 5-deoxyazacytidine
and these lines were deficient in mismatch activity repair was combined with DNA-MTase allelic deletion.
(MMR⫺), whereas other cell lines competent for mismatch The mechanism underlying the increased DNA-MTase activ-
repair (MMR⫹) expressed the gene. MMR⫺ cell lines were ity in neoplastic cells has not yet been elucidated. MacLeod et
found to be methylation proficient (MET⫹), and MMR⫹ cell al115 proposed that the activation of the ras signal transduction
lines were methylation deficient (MET⫺) based on Southern pathway causes increased activity by interacting with AP-1 sites
blot analysis and 5-azacytidine–induced reactivation of the in the presumed DNA-MTase promoter region in a murine
␤-gal gene. It was proposed that in mismatch repair proficient adrenocortical tumor cell line. However, this apparent promoter
colon cells a methylation defect (MET-) directly facilitates the region of the DNA-MTase gene was subsequently found to lie
gain and loss of whole chromosomes, leading to the genomic far downstream of the transcription start site.24,116 It has also
4066 SINGAL AND GINDER

been demonstrated that overexpression of the c-fos gene results The increased DNA-MTase activity seen in multiple cancers
in cellular transformation through increased DMTase activity.117 has prompted targeting of the inhibition of this enzyme as an
Further studies are required to understand the regulation of the anticancer strategy. The DNA-MTase inhibitors, 5-azacytidine
DNA-MTase genes in normal and neoplastic cells. and 5-azadeoxycytidine, have been used clinically for the
The mechanisms responsible for maintaining normal and treatment of patients with myelodysplastic syndromes or leuke-
abnormal methylation patterns in normal and neoplastic cells mia.127,128 Because, as noted, these agents have effects other
remain unclear. A recent study demonstrated the binding of than inducing demethylation, have many side effects, and need
DNA-MTase to proliferating cell nuclear antigen (PCNA), an to be administered by continuous infusion, efforts to develop
auxiliary factor for DNA replication and repair. This binding novel DNA-MTase inhibitors are clearly warranted. In one such
occurred in intact cells at foci of newly replicated DNA and did approach, Ramchandani et al129 demonstrated that intraperito-
not alter DNA-MTase activity. A peptide derived from the cell neal injection of DNA-MTase antisense oligonucleotides re-
cycle regulator p21(WAF1) was shown to disrupt the DNA- duced the level of DNA-MTase and inhibited the growth of Y1
MTase-PCNA interaction, suggesting that p21(WAF1) may adrenocortical carcinoma in syngeneic LAF mice.
regulate methylation by blocking access of DNA-MTase to

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PCNA. The extent of expression of DNA-MTase and p21(WAF1) THE ROLE OF DNA METHYLATION IN EVOLUTION
were found to be inversely related in both SV40-transformed Two hypotheses have been proposed for the evolutionary role
and nontransformed cells.118 Based on these findings, it has of DNA methylation. The first hypothesis is derived from the
been proposed that, in normal cells, the p21 protein negatively perspective of gene numbers and biologic complexity.12 The
regulates DNA-MTase-PCNA interaction in early S phase and size of the genome in free-living organisms has increased from
protects CpG islands from methylation, whereas diminished a few thousand genes in prokaryotes (eg, Escherichia coli with
effects of p21 in late S phase result in the targeting of 4,000 genes) to 7,000 to 25,000 genes in nonvertebrate eukary-
DNA-MTase to methylated DNA. In cancer cells, loss of p21 otes and 50,000 to 100,000 genes in vertebrates. Bird12 has
function allows DNA-MTase-PCNA interaction in early S proposed that, if the number of tissue-specific genes is to
phase, possibly facilitating aberrant increased methylation of increase during evolution, the efficiency of gene repression
CpG islands, whereas no change in decreased relative targeting must be high. This repression, or transcriptional noise reduc-
of DNA-MTase to late S phase foci results in loss of normal tion, in eukaryotes has been attributed to two features (the
methylation.15 This type of mechanism could explain the nuclear envelope and histones) that are present in eukaryotes
apparent paradox posed by the observation of both excessive but not in prokaryotes. Vertebrates, in turn, have several fold
and deficient DNA methylation during tumorigenesis. more genes than the nonvertebrate eukaryotes, and one possible
additional repression mechanism for vertebrates could be DNA
CLINICAL AND THERAPEUTIC IMPLICATIONS methylation. A comparison of methylation patterns in inverte-
OF DNA METHYLATION brates versus vertebrates shows some important differences. In
Just as the vertebrate globin genes were among the first the invertebrates, methylation of cytosine occurs at only a minor
examples of an association between DNA methylation and fraction of the CpG dinucleotides in the genome and in some
transcriptional silencing,31,43,44,119 so too were they the first cases, such as Drosophila, cannot be detected at all. It is likely
target for clinical intervention based on drugs that affect that, in the vast majority of eukaryotes, DNA methylation
methylation.120-122 Treatment with 5-azacytidine, an irreversible functions as part of a system that silences potentially damaging
inhibitor of DNA-MTase, was shown to increase expression of DNA elements such as transposons, viral genomes, etc. How-
the fetal ␥-globin gene in nonhuman primates and subsequently ever, vertebrates have the bulk of their DNA methylated, except
in patients with ␤-thalassemia and sickle cell anemia. Because for CpG islands. Bird12 proposes that DNA methylation in
of its mutagenicity and the observation that other S-phase active vertebrates provides a novel layer of global repression, further
cytotoxic agents that do not inhibit DNA methylation could reducing the transcriptional noise and thereby allowing verte-
induce similar increases in ␥-globin gene expression,123-125 brates to accumulate and selectively use the extra genes that are
5-azacytidine has not been widely used for this application. crucial to their development.
These experiences point to the limitations of attempting to alter The alternative theory proposed is that cytosine methylation
gene expression through the use of global DNA methylation in mammals is a nuclear host-defense system that evolved
inhibitors that also possess other potent cellular effects and primarily to counter the threats posed by endogenous parasitic
emphasize the need for a more complete understanding of the mobile genetic elements.5 Cytosine methylation inactivates the
specificity of DNA methylation and demethylation control. promoter of most viruses and transposons, including retrovi-
The recent advances in understanding of altered DNA ruses and Alu elements, and such sequences are methylated in
methylation in cancer discussed above also have potential the DNA of differentiated cells. In fact, the large majority of
clinical implications. Because methylation of many involved 5-mCs in the genome lie within these elements. Demethylating
genes may represent a process specific to neoplastic cells, this drugs have been shown to activate transcription of endogenous
change may be a sensitive index of micrometastases.86 It may transposons.130 Also, cloned, unmethylated human L1 elements
also be of some prognostic value in certain situations. For transpose at a higher rate in transfected human cells,131 and this
instance, methylation of the abl promoter in chronic myeloid transposition rate is far in excess of the rate of the identical but
leukemia has been associated with disease of long-standing methylated endogenous elements. In an interspecific mamma-
duration, most likely associated with a higher probability of lian hybrid involving Macropus eugenii and Wallabia bicolor,
imminent blast transformation.126 the occurrence of genome-wide undermethylation, retroviral
DNA METHYLATION 4067

element amplification, and chromosome remodeling has been must be more fully understood. Likewise, understanding the
demonstrated.114 Yoder et al5 also argue that there is little factors involved in DNA methylation-induced gene silencing
compelling evidence for a role for reversible promoter methyl- will facilitate attempts to selectively affect gene expression.
ation in developmental gene control. However, our laboratory Recent studies have linked two global mechanisms of gene
has shown that methylation does appear to play a role in specific regulation, DNA methylation, and histone deacetylation.61,62
developmental gene control.52-54 Further investigations are necessary to understand the complex
links between the methyltransferases, demethylases, methyl
METHYLATION AND FOREIGN GENE SILENCING cytosine binding proteins, histone acetylation, and the transcrip-
In both cultured cells transfected with foreign DNA and tional activity of genes.
transgenic organisms, the newly integrated foreign DNA fre-
quently becomes de novo methylated.132 It has been proposed ACKNOWLEDGMENT
that de novo methylation constitutes a cellular defense mecha- The authors acknowledge the helpful assistance of Catharine W.
nism to silence integrated foreign DNA or genes.132 Tucker in preparing their manuscript and thank Dr Steven Snyder for
Evidence over the past several years suggests that DNA comments.

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methylation is involved in the inactivation of virally introduced
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