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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4059-4070
REVIEW ARTICLE
DNA Methylation
By
Rakesh Singal and
Gordon D. Ginder
From the Deparment of Medicine, Feist-Weiller Cancer Center and
Overton Brooks VA Medical Center, LSU Medical Center, Shreveport, LA;
and the Departments of Internal Medicine and Human Genetics, Massey
Cancer Center, Virginia Commonwealth University, Richmond, VA.
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INTRODUCTION |
SINCE 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 understand its significance (for review, see Weissbach1). DNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring (Fig 1). This reaction is
catalyzed by DNA methyltransferase in the context of the sequence
5'-CG-3', which is also referred to as a CpG dinucleotide.
It is the most common eukaryotic DNA modification and is one of the
many epigenetic (alteration in gene expression without a change in
nucleotide sequence) phenomena. Although extensive in plants and
mammals, the absence of detectable DNA methylation in some eukaryotes
such as Drosophila2 and Saccharomyces
cerevisiae3 has raised doubts about its significance in
normal development and tissue-specific gene expression. However, recent
studies showing abnormal development and embryonic lethality in
transgenic mice expressing decreased but not completely absent DNA
Methyltransferase (MTase) activity after DNA-MTase gene
knockout4 lends support to a critical role for DNA
methylation in developmental gene regulation. Others have proposed that
the control of intragenomic parasites is the primary function of DNA
methylation in mammalian cells.5
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| Fig 1.
Schematic representation of the biochemical pathways for
cytosine methylation, demethylation, and mutagenesis of cytosine and
5-mC.
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In this review, we will discuss the fundamental aspects of DNA
methylation and its role in transcription repression, neoplasia, and
transgene silencing and during development. In the interest of brevity,
the role of DNA methylation in genomic imprinting and X chromosome
inactivation are not discussed here, but have been reviewed recently
elsewhere.6-8
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DISTRIBUTION OF METHYLATED CYTOSINES AND CpG ISLANDS |
Eukaryotic genomes are not methylated uniformly but contain methylated
regions interspersed with unmethylated domains.9 During
evolution, the dinucleotide CpG has been progressively eliminated from
the genome of higher eukaryotes and is present at only 5% to 10% of
its predicted frequency.10-12 Cytosine methylation appears
to have played a major role in this process, because most CpG sites
lost represent the conversion through deamination of methylcytosines to
thymines. Approximately 70% to 80% of the remaining CpG sites contain
methylated cytosines in most vertebrates, including humans.10,12 These methylated regions are typical of the
bulk chromatin that represents the late replicating DNA with its
attendant histone composition and nucleosomal configuration and is
relatively inaccessible to transcription factors.13 In
contrast to the rest of the genome, smaller regions of DNA, called CpG
islands, ranging from 0.5 to 5 kb and occurring on average every 100 kb, have distinctive properties. These regions are unmethylated, GC rich (60% to 70%), have a ratio of CpG to GpC of at least 0.6, and
thus do not show any suppression of the frequency of the dinucleotide CpG.10,14 Chromatin containing CpG islands is generally
heavily acetylated, lacks histone H1, and includes a nucleosome-free
region.13 This so called open 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 5'
regulatory regions in the genome. Approximately half of all genes in
mouse and humans (ie, 40,000 to 50,000 genes) contain CpG
islands.10 These are mainly housekeeping genes that have a
broad tissue pattern of expression, but approximately 40% of genes
with a tissue-restricted pattern of expression are also
represented.14 Promoter region CpG islands are usually
unmethylated in all normal tissues, regardless of the transcriptional
activity of the gene. The main exceptions include nontranscribed genes
on the inactive X-chromosome and imprinted autosomal genes where one of
the parental alleles may be methylated.15 Tissue-specific
genes without CpG islands are variably methylated, often in a
tissue-specific pattern, and usually methylation is inversely
correlated with the transcriptional status of the genes (for review,
see Bird16 and Cedar17).
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DNA METHYLTRANSFERASES |
The enzymes that transfer methyl groups to the cytosine ring, cytosine
5-methyltransferases, or DNA methyltransferases (DNA-MTase) have been
characterized in a number of eukaryotes.18 The target site
for DNA-MTase in DNA is the dinucleotide palindrome CG (commonly referred to as CpG, with p denoting the phosphate group). The first
eukaryotic DNA-MTase gene cloned from mouse almost a decade ago19 is now referred to as Dnmt1. This gene is
highly conserved among eukaryotes. Dnmt1 orthologs have been
identified in various species, including humans
(DNMT1).20 Interestingly, Dnmt1 orthologs have not been found in organisms lacking DNA methylation, such as
Saccharomyces cerevisiae, Caenorhabditis elegans, and
Drosophila melanogaster.
Mammalian Dnmt1 methyltransferase has a high affinity for
hemimethylated substrates but is also capable of performing de novo methylation of unmethylated substrates in vitro. The de novo activity of mammalian Dnmt1 methyltransferase has been shown to be
stimulated by aberrant DNA structures21 and 5-mC
residues in single-stranded22 or
double-stranded DNA substrates.23 After it was found that the human and mouse Dnmt1 genes contain additional 5'
transcribed sequences with protein-encoding potential,24 it
became apparent that previous biochemical and cell culture assays had
relied on an incomplete Dnmt1 cDNA sequence that codes for a
truncated protein. Pradhan et al25 demonstrated that
full-length Dnmt1 MTase synthesized in Baculovirus was equally
active on unmethylated and hemimethylated substrates, in part by virtue
of an inhibitory effect of the additional N-terminal amino acids on
activity with hemimethylated DNA. These results are evidence that the
Dnmt1 MTase protein has inherent de novo methylating activity
that may be altered by protein-protein interactions and enhanced by
aberrant structures or 5-mC residues in the substrate DNA.
Evidence for an additional mammalian DNA-MTase capable of de novo
methylation came from the experiments of Lei et al.26 These
investigators generated a null mutation of the Dnmt1 gene through homologous recombination in mouse embryonic stem cells. Dnmt1 null embryonic stem cells were viable and contained low but stable levels of methylcytosine and methyltransferase activity. Interestingly, integrated provirus DNA in Moloney murine leukemia virus
(MoMuLV)-infected homozygous Dnmt1 knockout ES
cells exhibited de novo methylation to a similar extent as in wild-type
cells. Although a second DNA-MTase has recently been identified in
mouse (Dnmt2)27 and in humans
(DNMT2),28 it is not yet clear if it is the long
sought after de novo methyltransferase present in Dnmt1
deficient ES cells.
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TECHNIQUES TO STUDY DNA METHYLATION |
Early techniques to study site-specific DNA methylation relied
primarily on the inability of methylation-sensitive type II restriction
enzymes to cleave sequences containing one or more methylated CpG
sites, combined with Southern hybridization.29-31 This
method requires large amounts of high molecular weight DNA, detects
methylation only if more than a few percent of alleles are methylated,
and only provides information about those CpG sites found within the
recognition sequence of methylation-sensitive restriction enzymes.
Singer-Sam et al32 improved the sensitivity of methylation
detection by combining the use of methylation-sensitive restriction
enzymes and polymerase chain reaction (PCR). After cleaving the DNA
with methylation-sensitive restriction endonucleases, eg, Hpa
II, PCR amplification with specific primers flanking the restriction
site will only occur if DNA cleavage has been prevented by methylation.
However, this method, like Southern-based approaches, can only monitor
CpG methylation in methylation-sensitive restriction sites. Also, a
false-positive result may be obtained due to incomplete restriction
enzyme digestion of cellular DNA.
Genomic sequencing protocols that have previously been used to study
DNA methylation use Maxam and Gilbert chemical cleavage reactions
performed on genomic DNA33 with linker-mediated PCR (LMPCR)
to enhance the signal.34 These methods are based upon the
fact that 5-mC is not cleaved during the standard Maxam and Gilbert
cytosine cleavage reaction.35 Thus, 5-mC is identified in a
sequencing gel by the lack of a band that corresponds to a cleavage
product of a cytosine degradation reaction. This assay is technically
demanding and is subject to both false-positive and false-negative results.
Frommer et al36 introduced a procedure based on
bisulfite-induced oxidative deamination of genomic DNA under conditions in which cytosine is converted to uracil and 5-mC remains unchanged. The target sequence is then amplified by PCR using strand-specific primers. Upon sequencing of the amplified DNA, all uracil and thymine
residues become detectable as thymine and only 5-mC residues amplify as
cytosines. This method, as shown in Fig 2,
is presently the method of choice for the detailed analysis of 5-mC in
any given genomic target sequence.
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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
dinucleotides 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 associated 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
dinucleotides 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  15). Primers R and F indicate the sequence
of rho globin promoter used for designating internal primers and for
sequencing.
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A number of rapid methods to detect 5-mC have been developed based on
the bisulfite deamination reaction in combination with PCR
amplification. These are suitable for examining limited numbers of CpG
dinucleotides that are either found within or immediately adjacent to
the PCR primer sequences37,38 or within a restriction enzyme recognition sequence.39
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DNA METHYLATION AND TRANSCRIPTIONAL REPRESSION |
A role for DNA methylation in the differential regulation of gene
expression was hypothesized many years ago.40,41 The potential mechanism was suggested by a number of early observations in
which site-specific cytosine methylation within or adjacent to genes
was found to correlate with transcriptional
repression.31,42-45 Subsequently, this inverse relationship
between cytosine methylation and transcription has been observed in a
large number of genes, although not universally.
Despite the long held view that DNA methylation might act as a negative
regulator of transcription, the precise mechanism involved has remained
elusive. Numerous reports have shown the ability of promoter DNA
methylation to inhibit transcription of a wide variety of genes in in
vitro transfection assays, and in some cases, such methylation
corresponds to the inactive state of the gene under study in vivo
(reviewed in Bird16 and Razin and Cedar46).
Three possible mechanisms have been proposed to account for
transcriptional repression by DNA methylation, and each is shown in
Fig 3. The first mechanism involves direct
interference with the binding of specific transcription factors to
their recognition sites in their respective promoters. Several
transcription factors, including AP-2, c-Myc/Myn, the cyclic
AMP-dependent activator CREB, E2F, and NF- B, recognize sequences
that contain CpG residues, and binding to each has been shown to be
inhibited by methylation. In contrast, other transcription factors (eg,
Sp1 and CTF) are not sensitive to methylation of their binding
sites,47 and many factors have no CpG dinucleotide residues
in their binding sites.
A second potential mechanism for methylation induced silencing is
through the direct binding of specific transcriptional repressors to
methylated DNA. Two such factors, MeCP-1 and MeCP-2 (methyl cytosine
binding proteins 1 and 2), have been identified and shown to bind to
methylated CpG residues in any sequence context. Although in
vertebrates DNA methylation has been posited to inhibit transcription initiation, methylation has also been shown to block transcription elongation in Neurospora through a mechanism that may be
mediated through MeCP-1 and/or MeCP-2.48
MeCP-1 binds to DNA containing multiple symmetrically methylated CpG
sites, as opposed to hemimethylated CpGs, and manifests as a large
complex on electrophoretic mobility shift assay.49 Repression of transcription from densely methylated genes can be
mediated by MeCP-1, and cells deficient in MeCP-1 show much reduced
repression of methylated genes.50 In a further study, it
was demonstrated that sparse methylation could repress transfected genes completely, but the inhibition was fully overcome by the presence
in cis of an SV40 enhancer. However, densely methylated genes
could not be reactivated by the strong enhancer. It was proposed that
sparsely methylated genes form an unstable complex with MeCP-1 that
prevents transcription when the promoter is weak. This complex can be
disrupted by a strong promoter, thereby allowing the methylated gene to
be transcribed.51
We have recently shown that a complex with electrophoretic mobility
similar to MeCP-1 forms efficiently with the methylated but not with
unmethylated embryonic rho-globin gene promoter sequences, and the
complex can be detected using nuclear extracts from the same primary
avian erythroid cells in which methylation-mediated transcriptional
inhibition was demonstrated.52 These results, in
conjunction with the demonstration of a role for methylation in
silencing rho-globin gene transcription in vivo in normal adult avian
erythroid cells,53,54 suggest a role for MeCP-1 or a similar complex in developmental silencing of embryonic globin genes
during normal erythropoiesis.
A component of the MeCP-1 complex, PCM1, has a methyl-CpG binding
domain (MBD) and two cysteine-rich domains (CXXC) that are found in animal DNA methyltransferases and in the mammalian HRX proteins, MLL and ALL-1. Although the functional significance of the
CXXC domain is not known, there is evidence that it is a part of a
transcriptional repression domain.55,56 PCM1 has been shown
to repress transcription in vitro in a methylation-dependent manner.57
MeCP-2 is more abundant than MeCP-1 in the cell and is able to bind to
DNA containing a single methylated CpG pair.58 MeCP-2, like
DNA methyltransferase, is dispensable for the viability of embryonic
stem cells, but is essential for embryonic development.59 MeCP-2 has two domains: a methyl-CpG binding domain that is essential for chromosomal localization and a transcriptional repressor domain (TRD) that can inhibit transcription from a promoter at a distance, suggesting that MeCP-2 interacts with the transcriptional machinery or
the initiation complex.60 Recently, a region of MeCP-2 that localizes with the TRD was shown to associate with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo was relieved by the
deacetylase inhibitor trichostatin A, suggesting that two global
mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP-2.61,62 However, in
some instances DNA methylation has been shown to play a dominant role over histone deacetyloses in transcriptional
repression.53,54 It has been proposed that MeCP-2 might
contribute to the assembly of a more stable repressive chromatin
structure.63 However, MeCP-2 can repress transcription of
naked DNA in a cell-free in vitro assay,60 suggesting that
chromatin formation is not necessary for its repressive action.
A third mechanism by which methylation may mediate transcriptional
repression is by altering chromatin structure. Keshet et al64 transfected mouse L cells with M13 plasmid constructs
containing the human  -globin gene, as well as several other
eukaryotic genes, after enzymatic methylation. Unmethylated DNA
sequences, after integration and stable propagation in cell culture,
were all detected in active chromatin, as measured by DNAse
1-sensitivity, in contrast to DNA sequences that were methylated in
vitro before transfection that were contained in DNAse1-resistant,
transcriptionally inactive chromatin. Experiments using microinjection
of certain methylated and nonmethylated gene templates into nuclei have
shown that methylation inhibits transcription only after chromatin is
assembled.65 Even a strong transcriptional activator,
GAL4-VP16, cannot counteract the effect of chromatin once it has
assumed the inactive state induced by DNA methylation.65
Therefore, in addition to stabilizing the inactive state, methylation
also prevents activation by blocking the access of transcription
factors.63,66 Whether this chromatin effect of methylation
is mediated solely by MeCP-2-associated histone deacetylase remains to
be determined.
One important issue regarding DNA methylation and transcriptional
silencing has been whether methylation is a primary control mechanism
or a secondary effect of gene activity. In the case of some genes with
sparse CpG nucleotides, gain of methylation occurs after
transcriptional silencing67; in other cases, loss of
methylation occurs after transcriptional activation.68 At the same time, in other systems it has been shown that despite optimum
nuclear conditions for transcription, including DNAseI-sensitive chromatin, transcription can be tightly repressed by CpG
methylation.52,53 It appears that methylation, particularly
of CpG-rich genes, may serve as a locking off mechanism that may follow
or precede other events that turn a gene off, but that once in place
can prevent activation despite an optimum nuclear environment for transcription.
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DNA DEMETHYLATION DURING DEVELOPMENT AND TISSUE-SPECIFIC
DIFFERENTIATION |
A critical aspect of the overall regulatory role of DNA methylation is
the process of demethylation. Using methylation-sensitive restriction
enzymes to monitor the general level of DNA methylation, it was shown
that, during early development, a dramatic reduction in methylation
levels occurs in the preimplantation embryo.69 This is
followed by a wave of de novo methylation involving most CpG residues
but leaving the CpG islands unmethylated at the time of
implantation.70 After implantation, most of the genomic DNA is methylated, whereas tissue-specific genes undergo demethylation in
their tissues of expression.46 Thus, a means for both
global and tissue- or gene-specific removal of methylcytosines from DNA must exist.
The mechanism underlying the process of demethylation has not been
fully elucidated. In some cases, demethylation could be a passive
process, ie, inhibition of methylation after DNA
replication.45 Despite the tight coordination of DNA
methylation and replication that has been demonstrated in mammalian
cells,71 the mechanism of methylation inhibition in the
presence of DNA replication remains to be defined. The existence of an
active demethylation process not involving DNA replication in mammalian
cells has been supported by a number of observations (reviewed in
Szyf72). In transient transfection assays using myoblast
cells, a methylated  -actin gene was shown to have transcriptional
activity similar to unmethylated template secondary to an active
demethylation process.73 Using transient transfection
assays, Szyf et al74 showed that an in vitro methylated
SK-plasmid bearing no sequence homology to eukaryotic genes became
fully demethylated between 1 and 2 days after tranfection into the
mouse embryonal carcinoma cell line P19. This demethylating activity
was not sequence-selective and was independent of DNA replication.74 During B-cell development, both the heavy
and light chain Ig genes undergo demethylation.75 Specific
cis-acting elements, including both the intronic and 3'
enhancers in the case of the light chain gene, have been shown to
direct demethylation.75,76 Further analysis of the intronic
enhancers suggested the involvement of the NF-B family of proteins
as trans-acting factors in inducing demethylation. Both
cis-acting elements and trans-acting factors, therefore, appear to direct the demethylation machinery to its target locus.
The biochemical mechanism underlying the process of demethylation
remains unclear. However, extracts from chicken embryos have been
demonstrated to have sequence nonspecific demethylating activity in
vitro.77 This enzymatic activity was limited to demethylation of hemimethylated DNA and involved removal of the methylated cytosine and subsequent repair of the resulting apyrimidinic acid residue.78 In the case of rat myoblasts, demethylation activity is performed by an active component whose activity is to
excise methylated nucleotides from the DNA template and replace them
with an unmodified form.79 Interestingly, in the presence of a myoblast extract, demethylation of the -actin but not of the
adenine phosphoribosyl transferase (APRT) CpG island fragment was
observed. Although this demethylating activity was initially felt to be
dependent on an RNA component, subsequent investigation of a more
purified active fraction demonstrated complete resistance of the
activity to RNAse treatment.80
It has long been supposed that direct removal of the methyl group by
breaking the carbon-to-carbon bond of 5-mC would be so energetically
unfavorable as to be an unlikely event. However, a gene encoding an
enzyme capable of directly removing the methyl group from methylated
CpG-containing DNA in cell free reactions as well as in transfected
cells has been reported recently.81 If the corresponding
endogenous gene product can be shown to have the same activity, this
will provide a remarkable new avenue for studying the control of
demethylation during normal differentiation and development as well as
in oncogenesis.
It has been proposed that the core demethylase in cells is kept in an
inactive form by interaction with a protein inhibitor. Demethylation
could then come about by the removal or modification of the inhibitor.
Such an inhibitor could either affect the entire genome resulting in a
pattern similar to that seen in the early preimplantation embryo or be
confined to the specific sequence influenced by local
cis-acting elements and trans-acting factors, as in the
case of the Ig gene example discussed above.82
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DNA METHYLATION AND CANCER |
A role for DNA methylation in oncogenesis has been hypothesized for
many years. Numerous studies have suggested aberrations in DNA
methyltransferase activity in tumor cells.83-85 Transformed cells often have increased total DNA-MTase activity, widespread loss of
methylation from normally methylated sites, and more regional areas of
hypermethylated DNA.86 The potential contribution of DNA
methylation to oncogenesis appears to be mediated by one or more of the
following mechanisms that are shown in Fig
4.
Signature C T mutation in cancer cells.
The high mutation rate of cytosine residues within the dinucleotide
CpG, the target site of mammalian DNA-MTase, can be accounted for by an
increased rate of cytosine to thymine transitions, which are, in
turn, a consequence of hydrolytic deamination of
5-mC87 (Fig 1). This mechanism of mutation was first
recognized in prokaryotic systems.88
Unmethylated cytosine can also undergo deamination to yield uracil, but
the well-characterized Uracil-DNA glycosylase efficiently repairs G:U
but not G:T mismatch.89 However, DNA-MTase may block this
repair, contributing to CUT transition. DNA-MTase
may also mediate 5-mCT transition at CpG dinucleotides under
conditions that lead to increased DNA-MTase expression or decreased
cellular S-adenosylmethionine levels.90
A striking example of how this process may lead to oncogenesis is shown
by the tumor-suppressor gene p53. Mutations in the p53 tumor-suppressor
gene occur in more than 50% of human solid tumors.91 An
estimated 24% of these mutations are CT transitions at CpG
dinucleotides, suggesting that DNA methylation may contribute to these
mutations.92
DNA hypomethylation in cancer.
Decreased levels of overall genomic methylation are common findings in
tumorigenesis.93 This decrease in global methylation appears to begin early and before the development of frank tumor formation.94,95 Apart from the overall genomic
hypomethylation, specific oncogenes have been observed to be
hypomethylated in human tumors. A good inverse correlation between
methylation and gene expression was observed in the antiapoptotic bcl-2
gene in B-cell chronic lymphocytic leukemia96 and for the
k-ras proto-oncogene in lung and colon
carcinomas.97
Hypermethylation of tumor-suppressor genes.
In addition to point mutations or gene deletions, transcriptional
repression by hypermethylation of promoter sequences suggests an
alternative means for the inactivation of tumor-suppressor genes in
cancer. This may result from the increased DNA-MTase levels that have
been demonstrated in various cancers83 or it could occur as
a result of some other transient event that silences tumor-suppressor
gene transcription. The retinoblastoma gene (Rb) was the first classic
tumor-suppressor gene in which CpG island hypermethylation was
detected. Three of 21 sporadic cases in one study98 and 5 of 32 sporadic cases in another study99 had hypermethylated
CpG islands at the 5' end of the Rb gene. Subsequently, it was
also shown that in vitro methylation of the promoter region of Rb
directly blocked promoter activation.100
Inactivation of the von Hippel-Lindau (VHL) gene by somatically
acquired mutations in one copy of the VHL gene along with loss of the
second allele has been implicated as the initiating event in
spontaneous cases of clear-cell renal carcinoma.101 Herman
et al102 found hypermethylation of the VHL gene CpG island and concomitant lack of expression in 5 of 26 cases of the sporadic form of clear cell renal carcinoma that had no VHL intragenic mutations. Treatment of a clear cell renal carcinoma cell line with
5-deoxyazacytidine, a potent inhibitor of DNA-MTase, resulted in
demethylation and expression of a previously silent VHL gene.
One of the most important cell cycle regulatory proteins is p16 (also
known as MTS-1 for major tumor suppressor 1, INK4a for INhibitor of cyclin-dependent Kinase 4a,
and CDKN2a for Cyclin-Dependent Kinase
iNhibitor 2a). The major biochemical effect of p16
is to halt cell-cycle progression at the G1/S boundary, and the loss of
p16 function may lead to cancer progression by allowing unregulated cellular proliferation.103 A common genetic alteration in
tumor cell lines and to a lesser extent in primary tumors is the loss of heterozygosity at chromosome 9p21, which contains both the related
p16 and p15 genes. Among most solid tumors studied, a CpG island in the
5' region of p16 gene has been found to be frequently methylated,
and treatment of cell lines carrying a hypermethylated p16 allele with
5-azacytidine results in transcriptional activation of the
gene.104,105 The presence of either coding region deletions or promoter hypermethylation of p16 correlate inversely with the presence of Rb gene mutations in multiple tumor types (reviewed in
Baylin et al86). Interestingly, in some instances, p16
promoter hypermethylation may be the sole inactivating event for both
alleles of the gene and may be the only lesion associated with loss of the cyclin D-Rb pathway.86 In colon cancer, despite the
lack of allelic loss of the 9p region, homozygous deletions of the p16
gene, or Rb gene mutations,106 30% to 40% demonstrate
hypermethylated p16 alleles.105 Hypermethylation-mediated
inactivation of the p16 gene has been demonstrated in brain, breast,
colon, head and neck, and non-small-cell lung cancer and in high grade
non-Hodgkin's lymphoma (reviewed in Baylin et
al86).
P15 (INK4b) gene is an inhibitor of the cyclin-dependent kinases CDK4
and CDK6 and appears to play a role in transforming growth factor-
(TGF-)-mediated growth inhibition responses.107 Methylation-mediated inactivation of the p15 gene occurs predominantly in hematopoietic neoplasms such as acute myelogenous leukemia, acute
lymphoblastic leukemia, and Burkitt's lymphoma.86
Recently, hypermethylation of the p15 gene has been demonstrated in
myelodysplastic syndromes108 and hypermethylation of both
p15 and p16 has been found in multiple myeloma.109
Induction of chromosomal instability.
Lengauer et al110 introduced exogenous CpG-rich sequences
in the form of a retrovirally contained -galactosidase gene into 10 colon cancer cell lines. Five of 10 cell lines failed to express the
-gal gene, and these lines were deficient in mismatch activity repair (MMR), whereas other cell lines competent for mismatch repair (MMR+) expressed the gene. MMR cell lines were found to be methylation proficient (MET+), and MMR+ cell lines were methylation deficient (MET) based on Southern blot analysis and
5-azacytidine-induced reactivation of the -gal gene. It was
proposed that in mismatch repair proficient colon cells a methylation
defect (MET-) directly facilitates the gain and loss of whole
chromosomes, leading to the genomic instability necessary for the
development and progression of cancer. In contrast, MMR cells
have normal methylation proficiency and develop the required genomic
instability by the alternative pathway of mismatch repair
deficiency.111 Consistent with a role for hypomethylation
in chromosomal instability were the findings by Chen et
al112 in analyzing embryonic stem (ES) cells nullizygous for the DNA-MTase gene, Dnmt I. Gene deletions of selectable marker genes due to mitotic recombination or chromosomal loss were detected at
a much higher frequency in the Dnmt 1-deficient ES cells compared with
wild-type control cells.
Additional experimental support for the hypothesis linking DNA
methylation to chromosomal integrity comes from earlier studies by
Feinberg et al,113 who demonstrated an average of 8% and
10% reduction in genomic 5-mC content in colon adenomas and
adenocarcinomas, respectively, with no significant
difference between benign and malignant tumors. Interestingly, three
patients with the highest 5-mC content in their normal colon appeared
to have Lynch syndrome (HNPCC), which was subsequently shown to have
the MMR phenotype.113 In a recent study, the
occurrence of genome-wide undermethylation, retroviral element
amplification, and chromosome remodeling in an interspecific mammalian
hybrid (Macropus eugenii × Wallabia bicolor) was
demonstrated. Atypically extended centromeres of Macropus
eugenii-derived autosomes in the hybrid were composed primarily of
unmethylated, amplified retroviral elements not detectable in either
parental species, indicating that the failure of DNA methylation to
occur and resultant mobile-element activity in the cell hybrids could
facilitate rapid karyotypic evolution.114
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ALTERED DNA-METHYLTRANSFERASE ACTIVITY IN CANCER |
Several studies in the last few years have demonstrated an increase in
DNA-MTase activity in neoplastic cells. Kautiainen and
Jones83 examined the levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines. In all but 2 cases, the extractable methyltransferase activity was fourfold to
3,000-fold higher in tumorigenic than in nontumorigenic cells.83 Increased DNA-MTase activity has been reported in
colon cancers versus normal mucosa from the same patient.84
Laird et al85 bred normal mice heterozygous for deletion of
the DNA-MTase gene (and having 50% of the DNA-MTase activity compared
with wild-type) with mice having a mutant allele of the APC
gene.85 When combined with loss of the other APC allele,
this mutation results in progressive formation of adenomas throughout
the gastrointestinal tract of newborn animals. Intestinal adenomas were
reduced by 50% in the offspring mice with both the APC and DNA-MTase
gene mutations and were essentially eliminated when treatment with
5-deoxyazacytidine was combined with DNA-MTase allelic deletion.
The mechanism underlying the increased DNA-MTase activity in neoplastic
cells has not yet been elucidated. MacLeod et al115 proposed that the activation of the ras signal transduction
pathway causes increased activity by interacting with AP-1 sites in the presumed DNA-MTase promoter region in a murine adrenocortical tumor
cell line. However, this apparent promoter region of the DNA-MTase gene
was subsequently found to lie far downstream of the transcription start
site.24,116 It has also been demonstrated that
overexpression of the c-fos gene results in cellular transformation through increased DMTase activity.117 Further studies are
required to understand the regulation of the DNA-MTase genes in normal and neoplastic cells.
The mechanisms responsible for maintaining normal and abnormal
methylation patterns in normal and neoplastic cells remain unclear. A
recent study demonstrated the binding of DNA-MTase to proliferating
cell nuclear antigen (PCNA), an auxiliary factor for DNA replication
and repair. This binding occurred in intact cells at foci of newly
replicated DNA and did not alter DNA-MTase activity. A peptide derived
from the cell cycle regulator p21(WAF1) was shown to disrupt the
DNA-MTase-PCNA interaction, suggesting that p21(WAF1) may regulate
methylation by blocking access of DNA-MTase to PCNA. The extent of
expression of DNA-MTase and p21(WAF1) were found to be inversely
related in both SV40-transformed and nontransformed
cells.118 Based on these findings, it has been proposed
that, in normal cells, the p21 protein negatively regulates DNA-MTase-PCNA interaction in early S phase and protects CpG islands from methylation, whereas diminished effects of p21 in late S phase
result in the targeting of DNA-MTase to methylated DNA. In cancer
cells, loss of p21 function allows DNA-MTase-PCNA interaction in early
S phase, possibly facilitating aberrant increased methylation of CpG
islands, whereas no change in decreased relative targeting of DNA-MTase
to late S phase foci results in loss of normal
methylation.15 This type of mechanism could explain the
apparent paradox posed by the observation of both excessive and
deficient DNA methylation during tumorigenesis.
| |
CLINICAL AND THERAPEUTIC IMPLICATIONS OF DNA METHYLATION |
Just as the vertebrate globin genes were among the first examples of an
association between DNA methylation and transcriptional silencing,31,43,44,119 so too were they the first target
for clinical intervention based on drugs that affect
methylation.120-122 Treatment with 5-azacytidine, an
irreversible inhibitor of DNA-MTase, was shown to increase expression
of the fetal  -globin gene in nonhuman primates and subsequently in
patients with -thalassemia and sickle cell anemia. Because of its
mutagenicity and the observation that other S-phase active cytotoxic
agents that do not inhibit DNA methylation could induce similar
increases in -globin gene expression,123-125
5-azacytidine has not been widely used for this application. These
experiences point to the limitations of attempting to alter gene
expression through the use of global DNA methylation inhibitors that
also possess other potent cellular effects and emphasize the need for a
more complete understanding of the specificity of DNA methylation and
demethylation control.
The recent advances in understanding of altered DNA methylation in
cancer discussed above also have potential clinical implications. Because methylation of many involved genes may represent a process specific to neoplastic cells, this change may be a sensitive index of
micrometastases.86 It may also be of some prognostic value in certain situations. For instance, methylation of the abl
promoter in chronic myeloid leukemia has been associated with disease
of long-standing duration, most likely associated with a higher
probability of imminent blast transformation.126
The increased DNA-MTase activity seen in multiple cancers has prompted
targeting of the inhibition of this enzyme as an anticancer strategy.
The DNA-MTase inhibitors, 5-azacytidine and 5-azadeoxycytidine, have
been used clinically for the treatment of patients with myelodysplastic syndromes or leukemia.127,128 Because, as noted, these
agents have effects other than inducing demethylation, have many side effects, and need to be administered by continuous infusion, efforts to
develop novel DNA-MTase inhibitors are clearly warranted. In one such
approach, Ramchandani et al129 demonstrated that
intraperitoneal injection of DNA-MTase antisense oligonucleotides
reduced the level of DNA-MTase and inhibited the growth of Y1
adrenocortical carcinoma in syngeneic LAF mice.
| |
THE ROLE OF DNA METHYLATION IN EVOLUTION |
Two hypotheses have been proposed for the evolutionary role of DNA
methylation. The first hypothesis is derived from the perspective of
gene numbers and biologic complexity.12 The size of the
genome in free-living organisms has increased from a few thousand genes in prokaryotes (eg, Escherichia coli with 4,000 genes) to 7,000 to 25,000 genes in nonvertebrate eukaryotes and 50,000 to 100,000 genes
in vertebrates. Bird12 has proposed that, if
the number of tissue-specific genes is to increase during evolution,
the efficiency of gene repression must be high. This repression, or transcriptional noise reduction, in eukaryotes has been attributed to
two features (the nuclear envelope and histones) that are present in
eukaryotes but not in prokaryotes. Vertebrates, in turn, have several
fold more genes than the nonvertebrate eukaryotes, and one possible
additional repression mechanism for vertebrates could be DNA
methylation. A comparison of methylation patterns in invertebrates versus vertebrates shows some important differences. In the
invertebrates, methylation of cytosine occurs at only a minor fraction
of the CpG dinucleotides in the genome and in some cases, such as
Drosophila, cannot be detected at all. It is likely that, in
the vast majority of eukaryotes, DNA methylation functions as part of a
system that silences potentially damaging DNA elements such as
transposons, viral genomes, etc. However, vertebrates have the bulk of
their DNA methylated, except for CpG islands. Bird12
proposes that DNA methylation in vertebrates provides a novel layer of
global repression, further reducing the transcriptional noise and
thereby allowing vertebrates to accumulate and selectively use the
extra genes that are crucial to their development.
The alternative theory proposed is that cytosine methylation in mammals
is a nuclear host-defense system that evolved primarily to counter the
threats posed by endogenous parasitic mobile genetic elements.5 Cytosine methylation inactivates the promoter of most viruses and transposons, including retroviruses and Alu elements, and such sequences are methylated in the DNA of differentiated cells.
In fact, the large majority of 5-mCs in the genome lie within these
elements. Demethylating drugs have been shown to activate transcription
of endogenous transposons.130 Also, cloned, unmethylated
human L1 elements transpose at a higher rate in transfected human
cells,131 and this transposition rate is far in excess of
the rate of the identical but methylated endogenous elements. In an
interspecific mammalian hybrid involving Macropus eugenii and
Wallabia bicolor, the occurrence of genome-wide
undermethylation, retroviral element amplification, and chromosome
remodeling has been demonstrated.114 Yoder et
al5 also argue that there is little compelling evidence for
a role for reversible promoter methylation in developmental gene
control. However, our laboratory has shown that methylation does appear
to play a role in specific developmental gene control.52-54
| |
METHYLATION AND FOREIGN GENE SILENCING |
In both cultured cells transfected with foreign DNA and transgenic
organisms, the newly integrated foreign DNA frequently becomes de novo
methylated.132 It has been proposed that de novo methylation constitutes a cellular defense mechanism to silence integrated foreign DNA or genes.132
Evidence over the past several years suggests that DNA methylation is
involved in the inactivation of virally introduced genes in vivo. The
MoMuLV has been shown to be completely inactive in embryonic stem cell
and embryonic carcinoma cell lines, and the inactivity is accompanied
by de novo methylation of the proviral sequences.133,134
Orend et al135 have shown that, upon integration, de novo
methylation spreads from the center of the integrated collinear viral
DNA. Methylation of specific sites in the adenoviral promoter results
in promoter inactivation.136 Herpes virus also undergoes de
novo methylation in mammalian cells.137 Epstein-Barr virus
DNA has been found to be methylated in the normal lymphocytes of
healthy volunteers.138
In transduced murine hematopoietic cells, transcriptional inactivation
of the proviral MoMuLV-LTR was shown to be associated with methylation
of the proviral-LTR.139 We have recently demonstrated that
an in vitro-methylated retroviral LTR fragment containing 13 CpG
dinucleotides was able to compete in binding assays for an MeCP complex
that has a mobility similar to MeCP-1,140 although the role
of methylation in LTR silencing remains unresolved.
Methylation-mediated inactivation of foreign gene expression in
specific cell types has important therapeutic and pharmacological implications in that inhibition of the methylation of therapeutically introduced genes might enhance gene therapy significantly by preventing transcriptional silencing.
| |
CONCLUSION |
DNA methylation clearly plays an important regulatory role in
vertebrates, as evidenced by its vital role in embryonic development. Whether the primary evolutionary role of DNA methylation is through transcriptional silencing or as a host defense system against endogenous and exogenous parasitic sequence elements remains to be
fully determined. However, there is substantial evidence that DNA
methylation plays a critical role in silencing specific genes during
development and cell differentiation. The intrinsic mutagenicity of
5-mC, activation of proto-oncogenes through hypomethylation, transcriptional inactivation of tumor-suppressor genes through hypermethylation, and defects in chromosomal segregation due to failure
of de novo methylation may all contribute to neoplasia. Selective
modulation of DNA methylation may therefore have important clinical
implications for the prevention and treatment of cancer. To develop
safe and effective strategies for therapeutic alteration of DNA
methylation, the factors that regulate the specificity of both the
methylation and demethylation processes must be more fully understood.
Likewise, understanding the factors involved in DNA methylation-induced
gene silencing will facilitate attempts to selectively affect gene
expression. Recent studies have linked two global mechanisms of gene
regulation, DNA methylation, and histone
deacetylation.61,62 Further investigations are necessary to
understand the complex links between the methyltransferases, demethylases, methyl cytosine binding proteins, histone acetylation, and the transcriptional activity of genes.
| |
ACKNOWLEDGMENT |
The authors acknowledge the helpful assistance of Catharine W. Tucker
in preparing their manuscript and thank Dr Steven Snyder for comments.
| |
FOOTNOTES |
Submitted January 21, 1999; accepted March 26, 1999.
Supported by National Institutes of Health Grant No. DK29902 and by the
Massey Cancer Center (G.D.G.) and the Feist-Weiller Cancer Center
(R.S.).
Address reprint requests to Gordon D. Ginder, MD, Professor of
Internal Medicine and Human Genetics, Director, Massey Cancer
Center, Virginia Commonwealth University, 401 College St, PO Box
980037, Richmond, VA 23298-0037; e-mail: gginder{at}mcc1.mcc.vcu.edu.
| |
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De novo methylation of an embryonic globin gene during normal development is strand specific and spreads from the proximal transcribed region
Blood,
December 1, 2001;
98(12):
3441 - 3446.
[Abstract]
[Full Text]
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D. L. Carlone and D. G. Skalnik
CpG Binding Protein Is Crucial for Early Embryonic Development
Mol. Cell. Biol.,
November 15, 2001;
21(22):
7601 - 7606.
[Abstract]
[Full Text]
[PDF]
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J. L. Gabrilove
Hematologic Malignancies: An Opportunity for Targeted Drug Therapy
Oncologist,
October 1, 2001;
6(2008):
1 - 3.
[Full Text]
[PDF]
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R. Singal, J. van Wert, and M. Bashambu
Cytosine Methylation Represses Glutathione S-Transferase P1 (GSTP1) Gene Expression in Human Prostate Cancer Cells
Cancer Res.,
June 1, 2001;
61(12):
4820 - 4826.
[Abstract]
[Full Text]
[PDF]
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Z. Chen, A. C. Karaplis, S. L. Ackerman, I. P. Pogribny, S. Melnyk, S. Lussier-Cacan, M. F. Chen, A. Pai, S. W.M. John, R. S. Smith, et al.
Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition
Hum. Mol. Genet.,
March 1, 2001;
10(5):
433 - 443.
[Abstract]
[Full Text]
[PDF]
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S.-i. Mizuno, T. Chijiwa, T. Okamura, K. Akashi, Y. Fukumaki, Y. Niho, and H. Sasaki
Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia
Blood,
March 1, 2001;
97(5):
1172 - 1179.
[Abstract]
[Full Text]
[PDF]
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R. Singal, J. vanWert, M. Bashambu, S. A. Wolfe, D. C. Wilkerson, and S. R. Grimes
Testis-Specific Histone H1t Gene Is Hypermethylated in Nongerminal Cells in the Mouse
Biol Reprod,
November 1, 2000;
63(5):
1237 - 1244.
[Abstract]
[Full Text]
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E. H. Estey
How I treat older patients with AML
Blood,
September 1, 2000;
96(5):
1670 - 1673.
[Full Text]
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A PETRONIS and R PETRONIENE
Epigenetics of inflammatory bowel disease
Gut,
August 1, 2000;
47(2):
302 - 306.
[Full Text]
[PDF]
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D. W. Lee, K. Zhang, Z.-Q. Ning, E. H. Raabe, S. Tintner, R. Wieland, B. J. Wilkins, J. M. Kim, R. I. Blough, and R. J. Arceci
Proliferation-associated SNF2-like Gene (PASG): A SNF2 Family Member Altered in Leukemia1
Cancer Res.,
July 1, 2000;
60(13):
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[Abstract]
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Q. Dang, J. Auten, and I. Plavec
Human Beta Interferon Scaffold Attachment Region Inhibits De Novo Methylation and Confers Long-Term, Copy Number-Dependent Expression to a Retroviral Vector
J. Virol.,
March 15, 2000;
74(6):
2671 - 2678.
[Abstract]
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N. C. Gorin, E. Estey, R. J. Jones, H. I. Levitsky, I. Borrello, and S. Slavin
New Developments in the Therapy of Acute Myelocytic Leukemia
Hematology,
January 1, 2000;
2000(1):
69 - 89.
[Abstract]
[Full Text]
[PDF]
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S. A. Kuismanen, M. T. Holmberg, R. Salovaara, P. Schweizer, L. A. Aaltonen, A. de la Chapelle, M. Nystrom-Lahti, and P. Peltomaki
Epigenetic phenotypes distinguish microsatellite-stable and -unstable colorectal cancers
PNAS,
October 26, 1999;
96(22):
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[Abstract]
[Full Text]
[PDF]
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R. Singal, S. Z. Wang, T. Sargent, S. Z. Zhu, and G. D. Ginder
Methylation of Promoter Proximal-transcribed Sequences of an Embryonic Globin Gene Inhibits Transcription in Primary Erythroid Cells and Promotes Formation of a Cell Type-specific Methyl Cytosine Binding Complex
J. Biol. Chem.,
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[Abstract]
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C. P. Kalberer, R. Pawliuk, S. Imren, T. Bachelot, K. J. Takekoshi, M. Fabry, C. J. Eaves, I. M. London, R. K. Humphries, and P. Leboulch
From the Cover: Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta -globin in engrafted mice
PNAS,
May 9, 2000;
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[Abstract]
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TOP
INTRODUCTION
DISTRIBUTION OF METHYLATED...
