MINI-REVIEW
Journal of
DNA Methylation: The Nuts and
Bolts of Repression
TINA BRANSCOMBE MIRANDA AND PETER A. JONES*
Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center,
Keck School of Medicine, University of Southern California, Los Angeles, California
DNA methylation is an epigenetic modification which plays an important role in chromatin organization and gene expression. DNA
methylation can silence genes and repetitive elements through a process which leads to the alteration of chromatin structure. The
mechanisms which target DNA methylation to specific sites in the genome are not fully understood. In this review, we will discuss the
mechanisms which lead to the long-term silencing of genes and will survey the progression that has been made in determining the targeted
mechanisms for de novo DNA methylation.
J. Cell. Physiol. 213: 384390, 2007. 2007 Wiley-Liss, Inc.
DNA methylation is a covalent modification in which the
50 position of cytosine is methylated in a reaction catalyzed by
DNA methyltransferases (DNMTs) with S-adenosyl-methionine
as the methyl donor. In mammals, this modification occurs at
CpG dinucleotides and can be catalyzed by three different
enzymes, DNMT1, DMNT3a, and DNMT3b. DNA methylation
plays a role in the long-term silencing of transcription and in
heterochromatin formation. As an epigenetic modification,
DNA methylation permits these silenced states to be inherited
throughout cellular divisions.
In the context of DNA methylation, sequences within the
genome can be classified into two different groups: CpG poor
regions and CpG islands. CpG islands are defined as being
longer than 500 bp and having a GC content greater than 55%
and an observed CpG/expected CpG ratio of 0.65 (Takai and
Jones, 2002). CpG islands are often but not always found in
promoter regions and about 40% of genes contain CpG islands
that are situated at the end of the 50 region (promoter,
untranslated region, and exon 1) (Jones and Baylin, 2002). The
rest of the genome, such as the intergenic and the intronic
regions, is considered to be CpG poor. In healthy cells, CpG
poor regions are usually methylated whereas CpG islands are
generally hypomethylated, with a few exceptions including the
inactive X chromosome. During the development of cancer,
many CpG islands undergo hypermethylation while the CpG
poor regions become hypomethylated. This alteration in DNA
methylation pattern leads to changes in chromatin structure
causing the silencing of tumor suppressor genes and instability
of the genome (Fig. 1) (Jones and Baylin, 2002). This change in
methylation pattern during cancer is similar to the pattern
observed on the inactive X chromosome. The inactive
X chromosome is hypermethylated at CpG promoters, but
contains less methylation than the active X chromosome at
CpG sites located downstream of the promoter (Fig. 1)
(Jones, 1999; Hellman and Chess, 2007).
Traditionally, DNA methylation has been divided into two
types: de novo and maintenance methylation. De novo
methylation is catalyzed by DNMT3a and DNMT3b and is
important for the establishment of methylation patterns in early
embryos, during development, and during carcinogenesis
(Fig. 1) (Okano et al., 1999). In order to maintain these
methylation patterns set by de novo methylation, DNMT1 is
localized to the replication fork during cellular division and
conducts maintenance methylation (Leonhardt et al., 1992; Liu
et al., 1998). However, DNMT1 has been shown to be
inefficient at maintaining the methylation of many CpG dense
regions (Liang et al., 2002). Therefore, the de novo activities of
DNMT3a and DNMT3b are also necessary in somatic cells in
2 0 0 7 W I L E Y - L I S S , I N C .
384
Cellular
Physiology
order to reestablish the methylation patterns so that they are
not lost due to the inefficient activity of DNMT1 (Fig. 2).
Although much progress has been made in understanding the
role DNA methylation plays in controlling cellular processes,
there are still many details that are not fully understood.
Recently the DNA methylation field has been most focused
on trying to answer a few key questions: (1) What role does
DNA methylation play in the silencing of genes? (2) How does
DNA methylation silence genes? (3) How is de novo DNA
methylation targeted? and (4) Why are CpG islands generally
not methylated in normal cells and what are the possible
mechanisms that could lead to methylation of CpG islands
during the development of cancer cells? This review surveys the
research that has been done in attempt to answer these
questions.
The Role of DNA Methylation
Silencing of genetic elements can be successfully initiated and
retained by histone modifications and chromatin structure.
However, these modifications are easily reversible making
them make poor gatekeepers for long-term silencing (Shi et al.,
2004; Takenchi et al., 2006). Therefore, mammalian cells must
possess an additional mechanism for prolong silencing of these
sequences. An important component of this process is DNA
methylation. DNA methylation is a stable modification that is
inherited throughout cellular divisions. When found within
promoters, DNA methylation prevents the reactivation of
silent genes, even when the repressive histone marks are
reversed (McGarvey et al., 2007). This allows the daughter cells
to retain the same expression pattern as the precursor cells and
is important for many cellular processes including the silencing
Contract grant sponsor: NIH;
Contract grant number: R01 CA82422.
Contract grant sponsor: TBM is supported by the American
Cancer Society Postdoctoral Fellowship;
Contract grant number: PF-07-100-01-GMC.
*Correspondence to: Peter A. Jones, Department of Urology,
Biochemistry and Molecular Biology, Norris Comprehensive
Cancer Center, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90033. E-mail: jones_p@ccnt.usc.edu
Received 13 June 2007; Accepted 15 June 2007
DOI: 10.1002/jcp.21224
 DNA METHYLATION 385

Fig. 1. Chromatin structure of CpG islands and CpG poor regions in healthy cells and during cancer. In healthy cells, CpG islands are generally
hypomethylated. This allows for an open chromatin structure. However, the CpG poor regions found in repetitive elements within the intergenic
and intronic regions of the genome are methylated and thereby maintain a closed chromatin structure. In cancer and on the inactive
X chromosome many CpG islands become methylated, forcing these regions into a closed chromatin structure. When CpG islands located within
promoters are methylated, the corresponding genes are persistently silenced. In contrast, the CpG poor regions become hypomethylated allowing
for an open chromatin structure.

of repetitive elements, X-inactivation, imprinting, and
development.
The mammalian genome is complex consisting of not only
coding material but also of transposons and other parasitic
elements that have been acquired in the human genome over
time. These repetitive sequences make up much of the
intergenic and intronic regions of DNA. Many of these
repetitive elements contain long terminal repeat promoters
which permit the transcription of these sequences (Kochanek
et al., 1995; Yoder et al., 1997). Since the expression of these
sequences can allow for the movement of the parasitic elements
within the genome, these elements must be persistently
silenced by DNA methylation in order to preserve the integrity
of the genome (Robertson and Wolffe, 2000). During
tumorgenesis, however, these CpG poor regions become
hypomethylated by an unknown mechanism. This
hypomethylation leads to the expansion of the parasitic
elements, and may play a role in carcinogenesis (Wilson et al.,
2007).
In addition to silencing repetitive elements, CpG methylation
in also an important constituent in X chromosome inactivation
and in the establishment and maintenance of imprinted genes.
Both X-inactivation and imprinting are forms of non-Medelian
inheritance in which one allele becomes methylated leading to
mono-allelic expression. During embryogenesis one of the
X chromosomes is inactivated in order to preclude the
expression of its genes. Methylation of CpG rich promoters
found within the inactive X chromosome stabilizes the
repressed state of the corresponding genes and allows these
silent states to be inherited through every cell division (Chang
et al., 2006). Imprinting is important for determining which
parental allele will be expressed. Imprinted genes are marked in
the gonads by DNA methylation of the imprinted control
region (ICR) allowing for the daughter cells to retain the same
mono-allelic expression as their parental origin (Jelinic and
Shaw, 2007).
The role DNA methylation plays in development has been a
matter of debate. Although some germ-line and tissue specific
promoters have been shown to undergo DNA methylation
during cellular differentiation, there are many developmental
genes which are not silenced by DNA methylation (Bird, 2002;
Strichman-Almashanu et al., 2002; Fazzari and Greally, 2004).
This leads many to question if DNA methylation really plays a
substantial role in the developmental process. A recent study,
however, has provided new evidence implicating DNA
methylation in the silencing of germline specific genes (Weber
et al., 2007). By looking at global DNA methylation, this study
found a subset of genes that were methylated in fibroblasts but
not in sperm. A large number of these differentially methylated
genes were found to be germline specific. These results imply
that somatic DNA methylation does contribute to
differentiation by repressing key genes in the germline and
irreversibly forcing the cell on a path to differentiation (Weber
et al., 2007; Ziblerman, 2007).
Most of the research on DNA methylation and promoters
has been focused on CpG islands and not CpG poor promoters.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

386 MIRANDA AND JONES

Fig. 2. De novo and maintenance methylation. During preimplantation development the genome undergoes a wave of demethylation, which
removes most of the DNA methylation. After implantation of the embryo, during development, and during carcinogenesis new methylation
patterns are set by de novo methylation. During replication the methylation patterns must be maintained. In order to accomplish this, the
maintenance methyltransferase DNMT1 methylates the hemimethylated DNA after strand synthesis. However, DNMT1 is inefficient at copying
some of the methylation patterns. Therefore, the de novo activities of DNMT3a and DNMT3b are necessary in order to reestablish the methylation
patterns so that they are not lost due to the inefficient activity of DNMT1.

About 60% of human genes contain non-CpG rich promoters
and although these promoters are CpG poor they are not
necessarily methylated in healthy cells (Takai and Jones, 2002).
Methylation of CpG islands has been shown to have a direct
effect on the silencing of genes; however, no strong correlation
has been established yet for CpG poor promoters. Many CpG
poor promoters have been shown to be methylated in cell
lineages, suggesting that methylation of CpG poor promoters
may play a role in development and differentiation (Futscher
et al., 2002; Hattori et al., 2004; Blelloch et al., 2006). This link
has yet to be clearly established and calls for further research in
this area.
Regulation of Transcription by DNA Methylation
DNA methylation has been correlated with transcriptional
silencing for over 20 years. However, it has not been until
recently that the mechanisms by which DNA methylation
inhibits transcription have begun to be uncovered. Numerous
processes by which DNA methylation can influence
transcription have been proposed. One model suggests that
DNA methylation can directly impede the binding of
transcriptional factors to their target sites, thus prohibiting
transcription. Most of the other proposed mechanisms are
based on the idea that methylation of CpG sequences can alter
chromatin structure by effecting histone modifications and
nucleosome occupancy within the promoter regions of genes.
Many transcription factors are targeted to CG-containing
sequences and methylation of CpG sites within these sequences
have been shown to prevent the binding of these proteins to
these sites (Comb and Goodman, 1990; Prendergast et al.,
1991). Most of the evidence for this mechanism comes from
studies done on c-myc and CTCF. c-Myc is a transcription
factor that is involved in the regulation of the cell growth and
differentiation. Studies using gel-shift assays have shown that
DNA methylation excludes c-Myc from binding to its consensus
site (Prendergast et al., 1991). CTCF is most known for the role
it plays in imprinting at the H19/If2 locus. CTCF acts to silence
the maternal copy of the Igf2 gene by binding to a site between
the enhancer and promoter. At the paternal locus, however,

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

 DNA METHYLATION
methylation of the CTCF binding site prevents CTCF binding
and allows for activation of Igf2 (Bell and Felsenfeld, 2000).
Although this is an important mechanism, relatively few factors
whose binding is affected by methylation have been identified.
Furthermore, methylated DNA has been shown to be
transcribed in the absence of chromatin or methyl-binding
proteins (MBPs, Kass et al., 1997). This suggests that there must
be additional mechanisms involving chromatin structure by
which DNA methylation can silence genes.
DNA methylation can prevent transcriptional activation by
interfering with the propagation of active chromatin marks.
Genes are poised for transcriptional activation by the
methylation of H3K4 (histone H3 lysine 4) in a reaction that is
catalyzed by a family of H3K4 methyltransferases (Ruthenburg
et al., 2007). Some of these H3K4 methyltransferases have been
shown to be targeted to sites which contain a local
concentration of CpG dinucleotides (Voo et al., 2000; Birke
et al., 2002; Ayton et al., 2004; Lee and Skalnik, 2005).
Methylation of these sites prevents binding of the these
methyltransferases thereby impeding these genes from being
primed for activation (Voo et al., 2000; Birke et al., 2002; Ayton
et al., 2004; Lee and Skalnik, 2005). In addition, recent evidence
has shown DNA methylation prevents the incorporation of
H3K4me2 and H3K4me3 (Okitsu and Hsieh, 2007) on
episomes. This mechanism allows a way for DNA methylation
to safeguard the silent state of genes.
In addition to directly inhibiting transcriptional factors from
binding, DNA methylation also recruits MBPs that specifically
bind to methylated CpGs. This family of proteins consists of five
members all containing a homologous methyl-CpG-binding
domain (Sansom et al., 2007). In addition, a non-homologous
protein called Kaiso has also been shown to bind a methylated
CGCG motif (Sansom et al., 2007). MBD1, MBD2, MBD3,
MeCP2, and Kaiso have all been shown to play a role in
methylation-dependent silencing of transcription. How these
proteins repress transcription is not fully understood.
However, it has been shown that MBPs can bind repressors and
histone deacetylases which may lead to an inactive chromatin
structure (Jones et al., 1998; Nan et al., 1998; Harikrishnan et al.,
2005).
Methylation of CpG sites within promoters may also affect
nucleosome occupancy at the transcriptional start sites of
genes. This in turn could effect transcriptional activation of
these genes. Nucleosome occupancy has been shown to
decrease the binding of transcription factors and RNA
polymerase II (Li et al., 2007). Experimental studies suggest that
promoters contain nucleosome free regions at their
transcriptional start sites which allow for the binding of
transcriptional activators and RNA polymerase II to the DNA
(Gal-Yam et al., 2006; Ozsalak et al., 2007; Lin et al., personal
communication). Evidence suggests that DNA methylation can
affect nucleosome occupancy. The first experimental data in
support of this claim came from in vitro studies which showed
that methylation of CpG sites could affect nucleosome
positioning at particular sequences (Davey et al., 1997). Since
then, studies of the MGMT and MLH1 promoters have shown
that DNA methylation does affect nucleosome occupancy in
these nucleosome free regions in vivo (Patel et al., 1997;
Lin et al., personal communication). The mechanism by which
DNA methylation dictates nucleosome occupancy is not fully
understood. It has been shown that MeCP2 binds to Brahma, a
catalytic subunit of the chromatin remodeling complex SWI/
SNF (Harikrishnan et al., 2005). This interaction may allow for
the targeting of the chromatin-remodeling complex to
methylated CpG sites, resulting in changes of nucleosome
occupancy. In addition, DNMT3a has been shown to interact
with the chromatin remodeler hSNF2h, suggesting that
chromatin remodelers may be directly targeted to these sites
by DNA methyltransferases (Geiman et al., 2004).
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
387
Regulation of De Novo Methylation
In somatic cells between 70% and 90% of CpG dinucleotides
found in the genome are methylated. In healthy cells most of this
methylation occurs at CpG poor regions dispersed throughout
the genome whereas most CpG islands are unmethylated
(Jones and Baylin, 2002). Given the existence of both
methylated and unmethylated CpG sites there must be
mechanisms within the cell which control these patterns of
methylation.
One mechanism by which DNA methyltransferases may be
targeted to particular sites within the genome is by recognizing
specific chromatin structures. Traditionally chromatin has been
divided into two different states: an active euchromatic state
and an inactive heterochromatic state. Euchromatin makes up a
large portion of the genome and is usually defined by di- and
trimethylation of lysine 4 on histone H3 and acetylation of the
histones H3 and H4. Euchromatin represents a flexible state
where genes can be kept on or turned off (Kouzarides, 2007). In
contrast, heterochromatin is a compact structure involved in
mitosis and in the protection of chromosome ends. In
mammals, heterochromatin is marked by either trimethylation
of lysine 27 on histone H3, trimethylation of lysine 9 on histone
H3 or methylation of lysine 20 on histone H4. In mammals
H3K9me3 is preferentially localized to pericentrometric
heterochromatin whereas H3K27me3 is involved in homeotic
gene silencing and marks the inactive X chromosome
(Kouzarides, 2007). Since DNA methylation occurs at
heterochromatic regions, these histone modifications, either
individually or together, make probable targets for the DNA
methyltransferases.
For many years it has been suspected that chromatin
structure could recruit DNA methyltransferases to specific
loci. DNA methyltransferases have been shown to interact with
the histone methyltransferases SUV39, which is responsible for
methylation of H3K9, and EZH2, which catalyzes H3K27
methylation (Fuks et al., 2003; Vire et al., 2006). These
interactions have been proven to be important for DNA
methylation of heterochromatin and EZH2 targeted genes. In
both Neurospora and Arabidopsis, mutations in the histone
H3K9 methyltransferase caused significant loss of genomic
DNA methylation and in mammalian cells SUV39 has been
shown to be required for Dnmt3b-dependent DNA
methylation at pericentric repeats (Jackson et al., 2002;
Lehnertz et al., 2003; Tamaru et al., 2003). Knockdown studies
of the polycomb methyltransferase EZH2 show that EZH2 is
necessary for the CpG methylation of EZH2 targeted genes
(Vire et al., 2006). DNA methyltransferases have also been
shown to interact with HP1, a protein that specifically binds to
methylated lysine 9 on histone H3, and evidence supports a
model in which HP1 is responsible for recruiting the DNA
methyltransferases to loci marked by H3K9me3 (Smallwood
et al., 2007). Further proof that methylation of H3K9 and
H3K27 may target DNA methylation comes from genome wide
studies showing that these histone modifications precede DNA
methylation. These studies have shown that when DNA
methylation is inhibited there is no effect on the methylation of
H3K9 or H3K27 in repeat sequences or CpG islands
(McGarvey et al., 2006). Taken together, these results suggest
that trimethylation of H3K9 and H3K27 are important markers
of DNA methylation.
Recently this has been proposed that the occurrence of
H3K9me3/H3K9me2 and H3K27me3 within the same region of
the genome serve as a signal for DNA methylation of CpG
islands within promoters (Ohm and Baylin, 2007). The need of
dual silencing marks to signal DNA methylation was first
discovered in plants. Non-CpG methylation was shown to be
targeted only to loci that contained both H3K27me3 and
H3K9me3 (Lindroth et al., 2004). Since then it has been shown
388 MIRANDA AND JONES

Fig. 3. A proposed model for long-term silencing of CpG islands. A: This state represents an active promoter. In this state genes are actively
transcribed. The active chromatin marks H3K4me2 and H3K4me3 (represented by a green oval) are present at this time. This active conformation
is not a target of DNMTs. B: In the permissive state genes are not being actively expressed however they are in a conformation which poises them for
transcription. This state also contains the active histone marks H3K4me2 and H3K4me3 and is also not a target for DNA methylation. C: During the
susceptible state H3K4me is removed by lysine demethylases. The nucleosomes now contain the silencing chromatin marks H3K9me3, H3K9me2,
and H3K27me3. It is not yet established if these marks are found together or independently of one another. This state does not necessarily undergo
DNA methylation, but is susceptible to DNA methylation. There may be other factors involved that causes DNA methylation of this configuration.
D: In this state the DNA has become methylated by DNMTs. This state is not easily reversible. E: DNA methylation recruits MBPs, chromatin
remodelers, and HDACS which causes a compact chromatin structure. This allows for the long-term silencing of the gene.

that H3K9me3/H3K9me2 and H3K27me3 are found in genes
that are commonly hypermethylated in cancer, suggesting a
similar mechanism may occur in mammals within CpG islands
(Ohm and Baylin, 2007). Recent studies have suggested that
adult stem cells, which contain the bivalent histone marks
H3K4me2 and H3K27me3 at many CpG islands, can loose the
H3K4me2 mark and gain the H3K9me3 and H3K9me2 marks.
Once this occurs the dual marked CpG island is targeted for
DNA methylation and the stem cell is on its way to becoming
cancerous (Ohm and Baylin, 2007).
Although these studies show convincing evidence that
certain modifications are needed for DNA methylation, these
studies do not prove that these modifications are the targeting
factors for DNA methylation. Data have been collected from
only a few genes and genome wide studies will need to be done
in order to determine if there is a global correlation between
DNA methylation and these silencing histone modifications. In
addition, there have been cases where H3K9me3 and
H3K27me3 are present at loci where there is no DNA
methylation (Lewis et al., 2004; Umlauf et al., 2004). Therefore,
methylation of H3K9me3, H3K9me2, and H3K27me3 may
predispose sequences for DNA methylation but there may be
additional mechanisms needed to target the DNA
methyltransferases to these sites.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

 DNA METHYLATION
Another mechanism by which DNA methyltransferases can
be targeted to promoters is by repressors. Evidence for this
comes from studies done on the PML-RAR fusion protein and
on Myc. These studies showed that the oncogenic PML-RAR
fusion protein can induce gene hypermethylation and silencing
by recruiting DNA methyltransferases to target promoters
(Di Croce et al., 2002). In addition, the Myc protein was shown
to associate with DNMT3a and recruit DNMT3a to the p21cip1
promoter. This recruitment results in the silencing of the
p21cip1 promoter (Brenner et al., 2005).
RNAi has also been shown to play a role in targeting DNA
methylation. In plants RNAi-mediated silencing can result in de
novo methylation of genes (Matzke and Birchler, 2005). Cell
culture studies have shown that RNAi-mediated silencing can
lead to de novo methylation (Morris et al., 2004). There are
contradicting results, however, Murchison et al. (2005) and
further research need to be done to show that this mechanism
occurs in mammals.
Aberrant DNA Methylation and Cancer
Early indications that DNA methylation may be involved in
cancer came with the discovery of global hypomethylation in
tumors (Riggs and Jones, 1983). Since then it has been shown
that although the genome as a whole is hypomethylated in
cancer, many CpG islands are hypermethylated (Jones and
Baylin, 2007). Hypermethylation of CpG islands leads to
silencing of genes, including many tumor suppressors, thereby
contributing to the process of tumorgenesis. One question that
has perplexed the field for decades is how this change in
distribution of methylation comes about? Unfortunately,
without fully understanding the targeting mechanisms for DNA
methylation, this question is difficult to answer.
Many hypotheses have been proposed to explain why certain
genes are methylated in cancer. One Darwinian theory suggests
that particular genes become methylated in tumors because
inactivation of these genes provides a selective growth
advantage for the cells (Esteller, 2005). Another hypothesis
suggests that genes which are under the control of PcG are
more vulnerable to DNA methylation. Recently it has been
shown that PcG marked genes are 20 times more likely to be
methylated in cancer. This idea has been reviewed in detail
elsewhere (Ohm and Baylin, 2007). In addition, it has been
suggested that the DNMTs may be aberrantly targeted to
specific promoters by fusion proteins (Di Croce et al., 2002).
Aberrantly targeting of DNMTs by fusion proteins has been
found to occur in a limited number of cases and is therefore
probably not a general phenomenon in cancer.
Conclusion
Much progress has been made in our understanding of the basic
mechanisms of DNA methylation, yet there are still many
questions left unanswered. Though DNA methylation has
definitively been shown to play a role in X-inactivation,
imprinting and silencing of repetitive sequences, the role of
DNA methylation in development is still an area of debate.
Exciting new evidence has aroused implication of DNA
methylation in silencing of germline specific genes, however,
more work needs to be done in this area to establish whether
or not this methylation is a cause of differentiation and not a
result of it.
In addition, there is still more left to learn about the
mechanism by which DNA methylation silences genes. There is
strong evidence supporting the notion that DNA methylation
prevents binding of some transcription factors to their targeted
sites, but this seems limited to a subset of factors and genes and
is not itself a global mechanism. Recent evidence suggests a
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
389
silencing mechanism by which DNA methylation leads to the
recruitment of chromatin remodelers possibly through MBPs.
These chromatin remodelers would then affect the
nucleosome occupancy within promoters leading to chromatin
compaction and long-term silencing of genes (Fig. 3D,E).
Further studies will need to be done to show that there is a link
between DNA methylation of promoters and nucleosome
occupancy on a global scale.
The mechanism that targets DNA methylation to particular
sites is still not understood. Many studies have linked various
histones modifications with the DNA methylation status of
genes. These results propose a model in which inactive marks
on histones play a role in dictating which CpG sites will become
methylated. When CpG islands contain active histone marks,
they are not targets of DNA methylation, however, when these
active marks are lost and the inactive H3K27me3 and H3K9me3
marks are acquired, these sites now become susceptible to
DNA methylation (Fig. 3). Whether or not this is all that is
needed for DNA methylation to occur or if there is another
layer of defense is still a matter of debate.
Given that DNA methylation plays a prominent role in
cancer development, it is important for us to fully understand
the targeting mechanisms used by the cell to target the DNA
methyltransferases to specific loci. It is imperative that we have
a complete grasp on how DNA methylation leads to the
silencing. Only then we will be able to develop better therapies
for the prevention and treatment of cancer.
Acknowledgments
We thank Mark Miranda and Connie Cortez for proofreading
the manuscript.
Literature Cited
Ayton PM, Chem EH, Cleary ML. 2004. Binding to nonmethylated CpG DNA is essential for
target recognition, transactivation, and myeloid transformation by an MLL oncoprotein.
Mol Cell Biol 24:1047010478.
Bell AC, Felsenfeld G. 2000. Methylation of a CTCF-dependent boundary controls imprinted
expression of the Igf2 gene. Nature 405:482485.
Bird A. 2002. DNA methylation patterns and epigenetic memory. Genes Dev 16:621.
Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K, Titgemeyer F, Slany RK. 2002. The MT
domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminated
against methylation. Nucleic Acids Res 30:958965.
Blelloch R, Wang Z, Meissner A, Pollard S, Smith A, Jaenisch R. 2006. Reprogramming
efficiency following somatic cell nuclear transfer is influenced by the differentiation and
methylation state of the donor nucleus. Stem Cells 24:20072013.
Brenner C, Replus R, Didelot C, Loriot A, Vire E, De Smet C, Gutierrez A, Danovi D, Bernard
D, Boon T, Pelicci PG, Amati B, Kouzarides T, de Launoit Y, Di Croco L, Fuks F. 2005. Myc
represses transcription through recruitment of DNA methyltransferase corepressor.
EMBO J 24:336346.
Chang SC, Tucker T, Thorogood NP, Brown CJ. 2006. Mechanisms of the X-chromosome
inactivation. Front Biosci 11:852866.
Comb M, Goodman HM. 1990. CpG methylation inhibits proenkephalin gene expression and
binding of the transcription factor AP-2. Nucleic Acid Res 18:39753982.
Davey C, Pennings S, Allan J. 1997. CpG methylation remodels chromatin structure in vitro.
J Mol Biol 267:276288.
Di Croce LD, Raber VA, Corsaro M, Fazi F, Farelli M, Faretta M, Fuks F, Lococo F, Kouzarides
T, Nervi C, Minucci S, Pelicci PG. 2002. Methyltransferase recruitment and DNA
hypermethylation of target promoters by an oncogenic transcription factor. Science
295:10791082.
Esteller M. 2005. Aberrant DNA methylation as a cancer-inducing mechanism. Annu Rev
Pharmol Toxicol 45:629656.
Fazzari MJ, Greally JM. 2004. Epigenomics: Beyond CpG islands. Nat Rev Genet 5:446455.
Fuks F, Hurd PJ, Deplus R, Kouzarides T. 2003. The DNA methyltransferases associate with
HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 31:23052312.
Futscher BW, Oshiro MM, Wozniak RJ, Holtan N, Hanigan CL, Duan H, Domann FE. 2002.
Role for DNA methylation in the control of cell type specific maspin expression. Nat Genet
31:175179.
Gal-Yam EN, Jeong S, Tanay A, Egger G, Lee AS, Jones PA. 2006. Constitutive nucleosome
depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule
footprinting. PLoS Genet 2:e160.
Geiman TM, Sankpal UT, Robertson AK, Zhao Y, Zhao Y, Robertson KD. 2004. DNMT3b
interacts with hSNF2h chromatin remodeling enzyme, HDACs1 and 2, and components of
the histone methylation system. Biochem Biophys Res Commun 318:544555.
Harikrishnan KN, Chow MZ, Bakev EK, Pal S, Bassal S, Brasacchio D, Wang L, Craig JM, Jones
PL, Sif S, El-Osta A. 2005. Brahma links the SWI/SNF chromatin-remodeling complex with
MeCP2-dependent transcriptional silencing. Nat Genet 37:254264.
Hattori N, Nishino K, Ko YG, Ohgane J, Tanaka S, Shiota K. 2004. Epigenetic control of mouse
Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J Biol Chem
279:1706317069.
Hellman A, Chess A. 2007. Gene body-specific methylation on the active X chromosome.
Science 315:11411143.
390 MIRANDA AND JONES
Jackson JP, Lindroth AM, Cao X, Jacobsen SE. 2002. Control of CpNpG DNA methylation by
the KRYPTONITE histone H3 methyltransferase. Nature 416:556560.
Jelinic P, Shaw P. 2007. Loss of imprinting and Cancer. J Pathol 211:261268.
Jones PA. 1999. The DNA methylation paradox. TIG 15:3437.
Jones PA, Baylin SB. 2002. The fundamental role of epigenetic events in cancer. Nat Rev Genet
3:415428.
Jones PA, Baylin SB. 2007. The epigenomics of cancer. Cell 128:683692.
Jones PC, Veenstra GJ, Wade PJ, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe
AP. 1998. Methylated DNA and MeCP2 recruit histone deacetylases to repress
transcription. Nat Genet 19:187191.
Kass SU, Landsberger N, Wolffe AP. 1997. DNA methylation directs a time-dependent
repression of transcription initiation. Curr Biol 7:157165.
Kochanek S, Renz D, Doerfler W. 1995. Transcriptional silencing of human Alu sequences
and inhibition of protein binding in the box B regulatory elements by 50 -CG-30 methylation.
FEBS Lett 360:115120.
Kouzarides T. 2007. Chromatin modifications and their function. Cell 128:693705.
Lee J-H, Skalnik DG. 2005. CpG binding (CXXC) finger protein 1 is a component of the
mammalian set1 histone H3-lys4 methyltransferase complex, the analogue of the yeast
set1/compass complex. J Biol Chem 280:4172541731.
Lehnertz B, Yoshihide U, Derijck AAHA, Braunschweig U, Perez-Burgos L, Kubicek S, Chen
T, Li E, Jenuwein T, Peters AHFM. 2003. SUV39h-mediated histone H3 lysine methylation
directs DNA methylation to major satellites repeats at pericentric heterochromatin. Curr
Biol 13:11921200.
Leonhardt H, Page AW, Weier H-U, Bestor TH. 1992. A targeting sequence directs DNA
methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71:865873.
Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, Walter J, Higgins M, Feil R, Reik W. 2004.
Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation
independent of DNA methylation. Nat Genet 36:12911295.
Li B, Carey M, Workman JC. 2007. The role of chromatin during transcription. Cell 128:707
719.
Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, Laird PW, Jones PA. 2002.
Cooperativity between DNA methyltransferases in the maintenance methylation of
repetitive elements. Mol Cell Biol 22:480491.
Lindroth AM, Shultis D, Jasencakova Z, Fuchs J, Johnson L, Schubert D, Patnaik D, Pradhan S,
Goodrich J, Sschubert I, Jenuwein T, Khorasanizadeh S, Jacobsen SE. 2004. Dual histone H3
methylation marks at lysine 9 and 27 required for interaction with chromolase3. EMBO J
23:42864296.
Liu Y, Oakeley EJ, Sun L, Jost J-P. 1998. Multiple domains are involved in the targeting of the
mouse DNA methyltransferase to the DNA replication foci. Nucleic Acids Res 26:1038
1045.
Matzke MA, Birchler JA. 2005. RNAi-mediated pathways in the nucleus. Nat Rev Genet 6:24
35.
McGarvey KM, Fahrnerl A, Greene E, Martens J, Jenuwein T, Baylin SB. 2006. Silenced tumor
suppressor genes reactivated by DNA demethylation do not return to a fully euchromatic
state. Cancer Res 66:35413549.
McGarvey KM, Greene E, Fahrner JA, Jenuwein T, Baylin SB. 2007. DNA methylation and
complete transcriptional silencing of cancer genes persist after depletion of EZH2. Cancer
Res 67:50975102.
Morris KV, Chan SW, Jacobsen SE, Looney DJ. 2004. Small interfering RNA-induced
transcriptional gene silencing in human cells. Science 305:12891292.
Murchison EP, Partridge JF, Tam OH, Cheloufi S, Hannon GJ. 2005. Characterization of
Dicer-deficient murine embryonic stem cells. Proc Natl Acad Sci USA 102:1213512140.
Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. 1998.
Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone
deacetylase complex. Nature 393:386389.
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
Ohm JE, Baylin SB. 2007. Stem cell chromatin patterns: An instructive mechanism for DNA
hypermethylation. Cell Cycle 9:10401043.
Okano M, Bell DW, Haber DA, Li E. 1999. DNA methyltransferases DNMT3a and DNMT3b
are essential for de novo methylation and mammalian development. Cell 99:247257.
Okitsu CY, Hsieh C-L. 2007. DNA methylation dictates H3K4 methylation. Mol Cell Biol
27:27462757.
Ozsalak F, Song JS, Liu XS, Fisher DE. 2007. High-throughput mapping of the chromatin
structure of human promoters. Nat Biotechnol 25:244248.
Patel SA, Graunke DM, Pieper RO. 1997. Aberrant silencing of the CpG Island-containing
human O6-methylguanine DNA methyltransferase gene is associated with the loss of
nucleosome-like positioning. Mol Cell Biol 17:58135822.
Prendergast GC, Lawe D, Ziff EB. 1991. Association of Myn, the murine homolog of max, with
c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation. Cell
65:395407.
Riggs AD, Jones PA. 1983. 5-Methylcytosine, gene regulation, and cancer. Adv Cancer Res
40:130.
Robertson KD, Wolffe AP. 2000. DNA methylation in health and disease. Nat Rev Genet
1:1119.
Ruthenburg AJ, Allis CD, Wysocka J. 2007. Methylation of lysine 4 on histone H3: Intricacy of
writing and reading a single epigenetic mark. Mol Cell 25:1530.
Sansom OJ, Maddison K, Clarke AR. 2007. Mechanisms of disease: Methyl-binding domain
proteins as potential therapeutic targets in cancer. Nat Clin Pract Oncol 4:305315.
Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y. 2004.
Histone demethylation mediated by the nuclear amine oxidase homolog L SD1. Cell
119:941953.
Smallwood A, Esteve PO, Pradhan S, Carey M. 2007. Functional cooperation between HP1
and DNMT1 mediates gene silencing. Genes Dev 21:11691178.
Strichman-Almashanu LZ, Lee RS, Onyango PO, Periman E, Flam F, Frieman MB, Feinberg AP.
2002. A genome-wide screen for normally methylated human CpG islands that can identify
novel imprinted genes. Genome Res 12:543554.
Takai D, Jones PA. 2002. Comprehensive analysis of CpG islands in human chromosomes 21
and 22. Proc Natl Acad Sci USA 99:37403745.
Takenchi T, Watanabe Y, Takano-Shimizu T, Kondo S. 2006. Roles of jumonji and jumonji
family genes in chromatin regulation and development. Dev Dyn 235:24492459.
Tamaru H, Zhang X, McMillen D, Singh PB, Nakayama J, Grewal SI, Allis CD, Cheng X, Selker
EU. 2003. Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in
Neurospora crassa. Nat Genet 34:7579.
Umlauf D, Goto Y, Cao R, Cerqueira F, Wagschal A, Zhang Y, Feil R. 2004. Imprinting along
the Kcne1 domain on mouse chromosome 7 involves repressive histone methylation and
recruitment of polycomb group complexes. Nat Genet 36:12961300.
Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard
D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F. 2006. The
polycomb group protein EZH2 directly controls DNA methylation. Nature 439:871874.
Voo KS, Carlone DL, Jacobsen BM, Flodin A, Skalnik DG. 2000. Cloning of a mammalian
transcriptional activator that binds unmethylated CpG motifs and shares CXXC domain
with DNA methyltransferases, human thrithorax, and methyl-CpG binding domain
protein 1. Mol Cell Biol 20:21082121.
Weber M, Hellmann I, Stadler MB, Ramos L, Paabos S, Rebhan M, Schubeler D. 2007.
Distribution, silencing potential and evolutionary impact of promoter DNA methylation in
the human genome. Nat Genet 39:457466.
Wilson AS, Power BE, Molloy PL. 2007. DNA hypomethylation and diseases. Biochim Biophys
Acta 1775:138162.
Yoder JA, Walsh CP, Bestor TH. 1997. Cytosine methylation and the ecology of intragenomic
parasites. TIG 13:335340.
Ziblerman D. 2007. The human promoter methylome. Nat Genet 39:442443.