MINI-REVIEW

Journal of
384
Cellular
Physiology
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 order to reestablish the methylation patterns so that they are
50 position of cytosine is methylated in a reaction catalyzed by not lost due to the inefficient activity of DNMT1 (Fig. 2).
DNA methyltransferases (DNMTs) with S-adenosyl-methionine Although much progress has been made in understanding the
as the methyl donor. In mammals, this modification occurs at role DNA methylation plays in controlling cellular processes,
CpG dinucleotides and can be catalyzed by three different there are still many details that are not fully understood.
enzymes, DNMT1, DMNT3a, and DNMT3b. DNA methylation Recently the DNA methylation field has been most focused
plays a role in the long-term silencing of transcription and in on trying to answer a few key questions: (1) What role does
heterochromatin formation. As an epigenetic modification, DNA methylation play in the silencing of genes? (2) How does
DNA methylation permits these silenced states to be inherited DNA methylation silence genes? (3) How is de novo DNA
throughout cellular divisions. methylation targeted? and (4) Why are CpG islands generally
In the context of DNA methylation, sequences within the not methylated in normal cells and what are the possible
genome can be classified into two different groups: CpG poor mechanisms that could lead to methylation of CpG islands
regions and CpG islands. CpG islands are defined as being during the development of cancer cells? This review surveys the
longer than 500 bp and having a GC content greater than 55% research that has been done in attempt to answer these
and an observed CpG/expected CpG ratio of 0.65 (Takai and questions.
Jones, 2002). CpG islands are often but not always found in
promoter regions and about 40% of genes contain CpG islands The Role of DNA Methylation
that are situated at the end of the 50 region (promoter,
untranslated region, and exon 1) (Jones and Baylin, 2002). The Silencing of genetic elements can be successfully initiated and
rest of the genome, such as the intergenic and the intronic retained by histone modifications and chromatin structure.
regions, is considered to be CpG poor. In healthy cells, CpG However, these modifications are easily reversible making
poor regions are usually methylated whereas CpG islands are them make poor gatekeepers for long-term silencing (Shi et al.,
generally hypomethylated, with a few exceptions including the 2004; Takenchi et al., 2006). Therefore, mammalian cells must
inactive X chromosome. During the development of cancer, possess an additional mechanism for prolong silencing of these
many CpG islands undergo hypermethylation while the CpG sequences. An important component of this process is DNA
poor regions become hypomethylated. This alteration in DNA methylation. DNA methylation is a stable modification that is
methylation pattern leads to changes in chromatin structure inherited throughout cellular divisions. When found within
causing the silencing of tumor suppressor genes and instability promoters, DNA methylation prevents the reactivation of
of the genome (Fig. 1) (Jones and Baylin, 2002). This change in silent genes, even when the repressive histone marks are
methylation pattern during cancer is similar to the pattern reversed (McGarvey et al., 2007). This allows the daughter cells
observed on the inactive X chromosome. The inactive to retain the same expression pattern as the precursor cells and
X chromosome is hypermethylated at CpG promoters, but is important for many cellular processes including the silencing
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 Contract grant sponsor: NIH;
methylation is catalyzed by DNMT3a and DNMT3b and is Contract grant number: R01 CA82422.
important for the establishment of methylation patterns in early Contract grant sponsor: TBM is supported by the American
embryos, during development, and during carcinogenesis Cancer Society Postdoctoral Fellowship;
Contract grant number: PF-07-100-01-GMC.
(Fig. 1) (Okano et al., 1999). In order to maintain these
methylation patterns set by de novo methylation, DNMT1 is *Correspondence to: Peter A. Jones, Department of Urology,
localized to the replication fork during cellular division and Biochemistry and Molecular Biology, Norris Comprehensive
conducts maintenance methylation (Leonhardt et al., 1992; Liu Cancer Center, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90033. E-mail: [email protected]
et al., 1998). However, DNMT1 has been shown to be
inefficient at maintaining the methylation of many CpG dense Received 13 June 2007; Accepted 15 June 2007
regions (Liang et al., 2002). Therefore, the de novo activities of DOI: 10.1002/jcp.21224
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 .
 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 repressed state of the corresponding genes and allows these
development. silent states to be inherited through every cell division (Chang
The mammalian genome is complex consisting of not only et al., 2006). Imprinting is important for determining which
coding material but also of transposons and other parasitic parental allele will be expressed. Imprinted genes are marked in
elements that have been acquired in the human genome over the gonads by DNA methylation of the imprinted control
time. These repetitive sequences make up much of the region (ICR) allowing for the daughter cells to retain the same
intergenic and intronic regions of DNA. Many of these mono-allelic expression as their parental origin (Jelinic and
repetitive elements contain long terminal repeat promoters Shaw, 2007).
which permit the transcription of these sequences (Kochanek The role DNA methylation plays in development has been a
et al., 1995; Yoder et al., 1997). Since the expression of these matter of debate. Although some germ-line and tissue specific
sequences can allow for the movement of the parasitic elements promoters have been shown to undergo DNA methylation
within the genome, these elements must be persistently during cellular differentiation, there are many developmental
silenced by DNA methylation in order to preserve the integrity genes which are not silenced by DNA methylation (Bird, 2002;
of the genome (Robertson and Wolffe, 2000). During Strichman-Almashanu et al., 2002; Fazzari and Greally, 2004).
tumorgenesis, however, these CpG poor regions become This leads many to question if DNA methylation really plays a
hypomethylated by an unknown mechanism. This substantial role in the developmental process. A recent study,
hypomethylation leads to the expansion of the parasitic however, has provided new evidence implicating DNA
elements, and may play a role in carcinogenesis (Wilson et al., methylation in the silencing of germline specific genes (Weber
2007). et al., 2007). By looking at global DNA methylation, this study
In addition to silencing repetitive elements, CpG methylation found a subset of genes that were methylated in fibroblasts but
in also an important constituent in X chromosome inactivation not in sperm. A large number of these differentially methylated
and in the establishment and maintenance of imprinted genes. genes were found to be germline specific. These results imply
Both X-inactivation and imprinting are forms of non-Medelian that somatic DNA methylation does contribute to
inheritance in which one allele becomes methylated leading to differentiation by repressing key genes in the germline and
mono-allelic expression. During embryogenesis one of the irreversibly forcing the cell on a path to differentiation (Weber
X chromosomes is inactivated in order to preclude the et al., 2007; Ziblerman, 2007).
expression of its genes. Methylation of CpG rich promoters Most of the research on DNA methylation and promoters
found within the inactive X chromosome stabilizes the has been focused on CpG islands and not CpG poor promoters.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

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

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

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

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

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

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

Another mechanism by which DNA methyltransferases can silencing mechanism by which DNA methylation leads to the
be targeted to promoters is by repressors. Evidence for this recruitment of chromatin remodelers possibly through MBPs.
comes from studies done on the PML-RAR fusion protein and These chromatin remodelers would then affect the
on Myc. These studies showed that the oncogenic PML-RAR nucleosome occupancy within promoters leading to chromatin
fusion protein can induce gene hypermethylation and silencing compaction and long-term silencing of genes (Fig. 3D,E).
by recruiting DNA methyltransferases to target promoters Further studies will need to be done to show that there is a link
(Di Croce et al., 2002). In addition, the Myc protein was shown between DNA methylation of promoters and nucleosome
to associate with DNMT3a and recruit DNMT3a to the p21cip1 occupancy on a global scale.
promoter. This recruitment results in the silencing of the The mechanism that targets DNA methylation to particular
p21cip1 promoter (Brenner et al., 2005). sites is still not understood. Many studies have linked various
RNAi has also been shown to play a role in targeting DNA histones modifications with the DNA methylation status of
methylation. In plants RNAi-mediated silencing can result in de genes. These results propose a model in which inactive marks
novo methylation of genes (Matzke and Birchler, 2005). Cell on histones play a role in dictating which CpG sites will become
culture studies have shown that RNAi-mediated silencing can methylated. When CpG islands contain active histone marks,
lead to de novo methylation (Morris et al., 2004). There are they are not targets of DNA methylation, however, when these
contradicting results, however, Murchison et al. (2005) and active marks are lost and the inactive H3K27me3 and H3K9me3
further research need to be done to show that this mechanism marks are acquired, these sites now become susceptible to
occurs in mammals. 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.
Aberrant DNA Methylation and Cancer Given that DNA methylation plays a prominent role in
cancer development, it is important for us to fully understand
Early indications that DNA methylation may be involved in
the targeting mechanisms used by the cell to target the DNA
cancer came with the discovery of global hypomethylation in
methyltransferases to specific loci. It is imperative that we have
tumors (Riggs and Jones, 1983). Since then it has been shown
a complete grasp on how DNA methylation leads to the
that although the genome as a whole is hypomethylated in
silencing. Only then we will be able to develop better therapies
cancer, many CpG islands are hypermethylated (Jones and
for the prevention and treatment of cancer.
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 Acknowledgments
has perplexed the field for decades is how this change in
distribution of methylation comes about? Unfortunately, We thank Mark Miranda and Connie Cortez for proofreading
without fully understanding the targeting mechanisms for DNA the manuscript.
methylation, this question is difficult to answer.
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JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP