DNA methylation and histone modifications: teaming up to
silence genes
François Fuks
DNA methylation, histone deacetylation, and methylation of
histone H3 at lysine 9 are the three best-characterized covalent
modifications associated with a repressed chromatin state.
Recent advances highlight an essential, intricate web of
interactions among these processes, generating a self-
reinforcing, self-perpetuating cycle of epigenetic events that
lead to long-term transcriptional repression. Histone
deacetylation and methylation at lysine 9 of H3 might also
contribute to the establishment of DNA methylation patterns, a
long-standing mystery in epigenetics. What’s more, recent
clues suggest a potential link between CpG methylation and
other histone modifications. A complex picture is emerging in
which DNA methylation and histone modifications work
hand-in-hand as parts of an epigenetic program that integrates
gene-silencing networks within the cell.
Addresses
Free University of Brussels, Faculty of Medicine, Laboratory of Molecular
Virology, 808 route de Lennik, 1070 Brussels, Belgium
Corresponding author: Fuks, François (ffuks@ulb.ac.be)
Current Opinion in Genetics & Development 2005, 15:490–495
This review comes from a themed issue on
Differentiation and gene regulation
Edited by Tony Kouzarides and Andrew J Bannister
Available online 10th August 2005
0959-437X/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2005.08.002
Introduction
DNA methylation, chromatin structure, and gene silen-
cing are interconnected in mammals. This has been
known for many years. Early studies revealed, for exam-
ple, that high levels of CpG methylation coincide with
heterochromatic regions. Also, upon integration into the
genome, in vitro methylated DNA was shown to associate
with a repressed chromatin structure. Other work
revealed that unmethylated CpG-island chromatin is
enriched in hyperacetylated histones [1]. The mechan-
isms underlying these observations have long remained
obscure, but a recent boom of new findings on how
chromatin structure regulates gene expression is paving
the way towards their elucidation.
Besides acetylation, histones undergo many post-transla-
tional modifications, such as methylation, phosphoryla-
tion, ubiquitination and ribosylation. Exciting recent
Current Opinion in Genetics & Development 2005, 15:490–495
discoveries have coalesced into the ‘histone code’ hypoth-
esis. According to this hypothesis, histone modifications,
acting alone or in specific combinations, provide binding
platforms for chromatin-associated proteins that initiate
or block gene transcription [2]. Among the histone mod-
ifications implicated in gene silencing, the best charac-
terized to date are histone deacetylation, and methylation
of histone H3 at lysine 9 (H3K9me1) [3]. It is increasingly
clear that, in various organisms, these modifications
work hand-in-hand with DNA methylation to repress
transcription [4,5].
This review, which focuses mainly on mammals, provides
a concise update on the emerging picture of how DNA
methylation, histone deacetylation, and H3K9 methyla-
tion contribute jointly to gene silencing. The possibility
that CpG methylation might ‘converse’ with other his-
tone modifications is also discussed.
DNA methylation, histone deacetylation and
H3K9 methylation: mutual boosting and
feedback loops
The existence of an epigenetic ‘conversation’ between
histones and DNA, involving cytosine methylation, his-
tone deacetylation, and H3K9 methylation, and leading to
transcriptional silencing, is now well established. What
remains unclear is the precise sequence of events.
On the one hand, there is evidence that DNA methylation
influences the histone modification pattern. For instance,
early observations showed that components of the DNA
methylation machinery (i.e. DNA methyltransferases
[DNMTs] and some methyl-CpG-binding domain
[MBD] proteins) recruit repressor complexes containing
histone deacetylases (HDACs) [6]. Other work using inhi-
bitors of DNA methylation and histone deacetylation
showed that CpG methylation is the dominant event that
seals transcriptional repression of hypermethylated genes
in cancer [7].
On the other hand, some studies suggest that histone
modification is a prerequisite for DNA methylation.
Observations made in fungi, plants and mammals high-
light methylation at lysine 9 of H3 as a kind of ‘beacon’ for
DNA methylation [8,9,10]. In mammals, DNA methyl-
transferases interact with Suv39h H3K9 methyltrans-
ferases [10,11], and loss of H3K9 methylation in
Suv39h-knockout embryonic stem cells decreases
Dnmt3b-dependent CpG methylation at major centro-
meric satellites [10]. In addition, H3K9 methylation and
silencing of the p16ink4a tumor suppressor gene can occur
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 DNA methylation and histone modifications: teaming up to silence genes Fuks 491
Glossary
Bisulfite sequencing: Bisulfite causes deamination of unmethylated
cytosines to uridine, thereby enabling discrimination between
unmethylated and methylated cytosine residues through sequence
analysis.
Euchromatin: Nuclear regions that generally contain decondensed
and transcriptionally active parts of the genome.
Imprinting: Mechanisms by which genes are selectively expressed
from the paternal or maternal alleles.
before CpG methylation [12]. Such observations have led
investigators to propose that DNA methylation is a sec-
ondary event in gene silencing, and that only genes
silenced by other mechanisms are subject to CpG methy-
lation [6,13].
Extra pieces to the puzzle are also worth considering.
First, it appears that epigenetic information can flow from
histones to DNA through histone deacetylation and
subsequent DNA methylation. Transient transfection
studies on mammalian cells, for example, suggest that
histone deacetylation might, in some instances, dictate
DNA methylation [14]. Furthermore, HDAC inhibition
by trichostatin A causes specific cytosine hypomethyla-
tion in Neurospora and impairs CpG methylation of genes
coding for rRNA in mammals [15,16].
Second, there is evidence that DNA methylation might
exert a positive feedback on lysine methylation, thereby
reinforcing these two distinct layers of methylation. Bio-
chemical data suggest that methyl-CpG-binding-domain
proteins, such as MeCP2 (methyl-CpG-binding protein 2)
and MBD1, might favor H3K9 methylation in the vicinity
of the methylated genes that they regulate [17,18].
Although the identity of the H3K9 histone methyltrans-
ferase that binds to MeCP2 is still unclear, the SETDB1
enzyme binds to MBD1, and, in conjunction with the
chromatin-assembly factor CAF-1 (chromatin assembly
factor-1), might support stable inheritance of silenced
chromatin states at methylated DNA.
How does H3K9 methylation ‘translate’ into DNA
methylation or vice versa? This is not yet clear. The
identification of H3K9 histone methyltransferases, such
as SETDB1 and CLLD8, with potentially built-in
methyl-CpG-binding domains [19] suggests that these
histone-modifying enzymes might be directly recruited
to sites of CpG methylation. Further work is needed to
test this intriguing possibility. As for the recruitment of
DNA methyltransferases to sites bearing the H3K9
methylation mark, a possible ‘reader’ of this signal is
the HP1 (heterochromatin protein 1) adaptor molecule,
which is known to associate with mammalian DNA
methyltransferases [10,11]. HP1 is indeed indispensa-
ble to DNA methylation in Neurospora [20] but not in
Arabidopsis [21]. It will thus be interesting to elucidate
this matter in mammals.
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Taken together, the above data support a model in which
methylated DNA is part of an epigenetic program that
leads to transcriptional silencing and involves the physical
associations outlined in Figure 1. Methylation at lysine 9
of H3 by an H3K9 enzyme creates a foundation for an
adaptor molecule (possibly HP1), which would, in turn,
recruit DNA methyltransferases. The recruited DNA
methyltransferases would catalyze CpG methylation in
the region of the methylated lysine. Methyl-CpG-binding
domains would then bind to the methylated DNA. The
bound methyl-CpG-binding domains would attract
HDAC complexes, which, in turn, seem to be required
to prepare lysine 9 of H3 for methylation [22]. Further-
more, according to the model, methyl-CpG-binding
domains would also recruit H3K9 methyltransferases.
Thus, epigenetic information embodied in methylated
residues would flow from histone to DNA and back.
Variations of this model are possible to envisage. For
example, the well-described DNA methyltransferase–
HDAC interaction [6] might provide an alternative route
for delivery of the HDAC activity that paves the way for
H3K9 methylation.
Clearly, further study is needed to elucidate the precise
sequence of events leading to epigenetic silencing. None-
theless, the above model is appealing because it implies
that DNA methylation, histone deacetylation, and H3K9
methylation act in synergy to generate a self-reinforcing
epigenetic cycle that maintains and perpetuates a
repressed chromatin state. Such a cycle might be relevant
to situations in which genes need to be ‘locked’ into a
permanently silenced state, as with the case of imprinted
genes or of hypermethylated genes in cancer.
Although attractive, this model might not offer a com-
prehensive picture of the mechanisms by which CpG
methylation, histone deacetylation, and H3K9 methyla-
tion lead to gene silencing. It is worth stressing that these
epigenetic modification processes display a varying
degree of autonomy according to the context. For exam-
ple, DNA microarrays obtained after treatment of Arabi-
dopsis with trichostatin A and/or 5-azacytidine —
inhibitors of HDAC and DNA methyltransferases,
respectively — do not always yield redundant outcomes
for the studied responsive genes [23]. Other works show
that, in the mouse placenta, imprinting (see Glossary) of
several genes of distal chromosome 7 involves repressive
histone methylation independent of DNA methylation
[24,25].
Also to be borne in mind is the existence of three distinct
H3K9 methylation states: the mono-, di- and trimethy-
lated states — respectively denoted H3K9me1,
H3K9me2 and H3K9me3 in the ‘Brno nomenclature’
[26]. H3K9 histone methyltransferases display remark-
able specificity as regards the level of methylation
they promote [3]. As mentioned above, studies on
Current Opinion in Genetics & Development 2005, 15:490–495
492 Differentiation and gene regulation

Figure 1

Model for a self-reinforcing epigenetic cycle that might perpetuate a repressed chromatin state. A DNA methyltransferase (DNMT) bound to an
adaptor molecule such as HP1 would add a methyl group to DNA only on chromatin that is methylated at lysine 9 of histone H3 (H3K9). The
generation of methylated DNA by the DNMT would permit binding of methyl-CpG-binding domain (MBD) proteins to DNA. Some of these proteins
recruit histone deacetylase (HDAC) complexes in addition to H3K9 methyltransferases (H3K9 HMTs). Given that deacetylation of histone H3 at
lysine 9 is necessary for methylation to take place on this residue, H3K9 deacetylation would be followed by histone methylation, which, in turn,
might result in recruitment of the HP1 adaptor. Thus, epigenetic information embodied in residue methylation states would flow from histone
to DNA and back. This self-reinforcing cycle might be relevant to situations in which a gene or locus needs to be heritably ‘locked’ into a
repressed chromatin state; for example, at hypermethylated genes in cancer or at imprinted genes.

Suv39h-knockout embryonic stem cells indicate that
DNA methylation at major satellite repeats is directed
by Suv39h or, more precisely, by Suv39h-mediated tri-
methylation [10]. G9a appears to be the enzyme pre-
dominantly responsible in mouse embryonic stem cells
for the presence of H3K9me1 and H3K9me2 sites in
euchromatic domains (see Glossary) [27]. We might spec-
ulate that H3K9me1 and H3K9me2 are associated with
cytosine-methylated imprinted genes that are regulated
by G9a [28]. These few studies already underscore a new
dimension to be explored when unraveling the interplay
of DNA and H3K9 methylation: the plasticity of H3K9
methylation states.
Do other histone modifications ‘converse’
with DNA methylation?
Given the tight link between methylated CpG and chro-
matin structure, it seems likely that histone modifications
other than deacetylation and H3K9 methylation might
‘converse’ with methylated DNA. A candidate is methy-
lation of histone H3 at lysine 27 (H3K27), another epi-
genetic mark for transcriptional repression. This
modification is produced, at least in part, by the Polycomb
group (PcG) protein EZH2, an enzyme capable of form-
ing, with the EED and SUZ12 proteins, the Polycomb
repressive complexes 2 and 3 (PRC2/3) [29]. In addition
to their involvement in Hox gene silencing, the PRC2/3
complexes play a role in biological processes, such as
germ line development, X-inactivation and cancer metas-
tasis. Evidence is accruing that they are recruited to PcG
targets to impose a methyl mark on H3K27. Methylated
K27 serves as an anchorage point for the PRC1 complex,
the binding of which leads to transcriptional repression
[30] through mechanisms that are still poorly understood.
Are there reasons to suspect a link between PcG-
mediated H3K27 methylation and DNA methylation?
Evidence is rather scarce at this stage, but a recent
exciting study seems to point in this direction. Work
on Eed / mutant mice has revealed that Eed is essential

Current Opinion in Genetics & Development 2005, 15:490–495 www.sciencedirect.com

 DNA methylation and histone modifications: teaming up to silence genes Fuks 493

for the silencing of a subset of autosomal imprinted loci. Figure 2

Significantly, in these mice, de-repression of the paternal
alleles of these loci correlates with demethylation of
specific CpGs in the differentially methylated regions
of the imprinted genes [31]. It is too early to propose a
mechanism for the influence of EED on DNA methyla-
tion patterns, but a possibility is that EED, and perhaps
other components of the PRC2/3 complexes, might con-
trol CpG methylation through direct physical contact with
DNA methyltransferases. In support of this hypothesis,
recent studies using chromatin immunoprecipitation
(ChIP) coupled to CpG-island microarrays have identi-
fied a cohort of target genes that bind EZH2, EED and
SUZ12 and appear to be regulated by the H3K27 methyl-
transferase activity of the PRC2/3 complexes [32]. Inter-
estingly, several of these PRC2/3-repressed genes Potential link between DNA methylation and PcG-mediated H3K27
associate with DNA methyltransferases and are silenced methylation. Although limited at this stage, preliminary evidence ([31],
by DNA methylation (E Viré, C Brenner et al., unpub- E Viré, C Brenner et al., unpublished) points to a possible mechanistic
connection between methylated DNA and methylation at lysine 27 of
lished). Hence, although highly speculative, the hypoth-
histone H3 (H3K27), catalyzed by the Polycomb repressive complexes
esis that transcriptional silencing might be brought about 2 and 3 (PRC2/3). These complexes comprise, in part, EZH2, EED
through a direct link between PcG-mediated H3K27 and SUZ12 proteins. A DNA methyltransferase (DNMT) might interact
methylation and DNA methylation (Figure 2) deserves (dashed lines) with EZH2, EED and/or SUZ12. The interplay between
future study. these proteins would result in methylation of DNA and lysine 27 of
histone H3, and consequent transcriptional repression. As in the
case of the link between DNA and H3K9 methylation, the ‘conversation’
H3 is not the only histone to undergo lysine-residue between methylated CpG and H3K27 methylation might be bidirectional.
methylation. Histone H4, in particular, is methylated at

Figure 3

Model of how DNA methylation might be linked to trimethylated K20 of histone H4. Recent evidence indicates that loss of H4K20me3 at
constitutive heterochromatin in certain cancer cells, possibly involving the Suv4–20h enzymes, is associated with hypomethylation of DNA at
these sequences [36]. These data suggest that in normal cells, DNA methyltransferase (DNMT) might interact with Suv4–20h. This interaction
might be direct or through the HP1 protein (dashed lines), because this adaptor binds to both DNMT [11] and Suv4–20 h [34]. Such associations
would lead to concomitant H4K20me3 and methylation of DNA repeat sequences. In cancer cells, the interaction of Suv4–20h, HP1 and DNMT
would be disrupted — by mutation, translocation, an inappropriate expression level, or defective post-translational modification of one of the partners.
This would result in the observed DNA hypomethylation and decrease in H4K20 trimethylation.

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494 Differentiation and gene regulation
lysine 20 (H4K20). In a similar fashion to H3K9 and
H3K27 methylation, H4K20 methylation is associated
with silent chromatin [33,34]. And, again, the residue
can undergo mono-, di- or trimethylation (me1, me2
and me3, respectively) [3]. In particular, H4K20me3
methylation by the newly identified Suv4–20h histone
methyltransferases is a hallmark of pericentric hetero-
chromatin [34,35]. A very recent and elegant study,
based notably on mass spectrometry, has demonstrated
a loss of H4K20me3 in certain cancer cells [36].
Whether this loss involves the Suv4–20 h enzymes
remains to be formally proven. In the same cancer cells,
the loss of H4K20me3 appeared to occur in the vicinity of
pericentromeric repeats that display an overall reduction
in DNA methylation [36]. Although this loss of both
H4K20me3 and methylated DNA in pericentric hetero-
chromatin might be coincidental, it might, on the con-
trary, be meaningful. For instance, contact between DNA
methyltransferases and Suv4–20h enzymes is an imagin-
able mechanism for concomitant marking of repeat-rich
sequences in pericentric chromatin by both methylated
CpG and trimethylated H4K20. In cancer cells, this
contact would somehow be disrupted, resulting in the
observed overall hypomethylation and loss of H4K20
trimethylation in pericentric heterochromatin (Figure 3).
The hypothetical contact could be direct or through the
HP1 adaptor protein, which can bind directly to both DNA
methyltransferases [11] and Suv4–20 h enzymes [34].
Conclusions
Epigenetics has reached a new level of maturity over the
past two years, with many findings highlighting the inti-
mate link between DNA methylation and histone mod-
ifications. Thanks to these discoveries, we might have
begun to resolve the long-standing mystery of how CpG
methylation patterns are established. Histone deacetyla-
tion and H3K9 methylation appear to pave the way for
CpG methylation. In addition, evidence suggests that the
DNA methylation machinery teams up with histone
deacetylation and H3K9 methylation to generate a self-
propagating cycle that promotes long-term transcriptional
repression. Yet our understanding of the interplay
between these epigenetic modifications is far from com-
plete. A major challenge is to untangle the mutual rein-
forcements of repression and the different states of
covalent histone and DNA modification required to
silence specific genomic regions in specific cases of epi-
genetic regulation. Comprehensive and comparative ana-
lyses of mammalian epigenomes are emerging [35].
They should yield invaluable insights into these matters.
An intriguing question is whether DNA methylation
‘communicates’ with other histone modifications besides
deacetylation and H3K9 methylation. Exciting recent
findings described in this review point to a possible
mechanistic link with H3K27 and H4K20me3 methyla-
tion. Further studies are eagerly awaited to see if the links
Current Opinion in Genetics & Development 2005, 15:490–495
between CpG methylation and H3K27 in addition to
H4K20me3 methylation are strong enough to support
the weight of the proposed tentative models (Figures 2
and 3).
There is tremendous ferment in epigenetics. We might
have just begun to lift the veil on the link between
histone and DNA modifications. The past few years have
seen many exciting discoveries, and, undoubtedly, many
more are in store. Surely this is a field that is bound to
thrive and sparkle in the coming years.
Acknowledgements
We thank Adrian Bird and Luciano Di Croce for helpful comments
on the manuscript. We thank Manel Esteller for sharing unpublished
results, and apologize for not citing all relevant references, owing to
space constraints. Our work is supported by funds from the ‘Fédération
Belge contre le Cancer’, Fonds National de la Recherche Scientifique,
‘FB Assurances’ and the ‘Action de Recherche Concertée de la
Communauté Française de ’elgique’.
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chromatin. Mol Cell 2002, 9:1201-1213.
34. Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G,
Reinberg D, Jenuwein T: A silencing pathway to induce H3-K9
and H4-K20 trimethylation at constitutive heterochromatin.
Genes Dev 2004, 18:1251-1262.
35. Martens JH, O’Sullivan RJ, Braunschweig U, Opravil S, Radolf M,
 Steinlein P, Jenuwein T: The profile of repeat-associated
histone lysine methylation states in the mouse epigenome.
EMBO J 2005, 24:800-812.
Using directed and array-based ChIP, the authors analyzed the distribu-
tion of repressive histone methylation states across the repetitive com-
plement of the mouse genome. Distinct chromatin modification patterns
are observed between the tandem repeats and the various interspersed
repeats. In addition, a comparison of four distinct epigenomes revealed a
surprising variability of repetitive histone lysine methylation marks at
interspersed repeats. This study adds important findings to the emerging
notion of epigenome plasticity.
36. Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J,
 Schotta G, Bonaldi T, Haydon C, Ropero S, Petrie K et al.: Loss of
acetylation at Lys16 and trimethylation at Lys20 of histone H4
is a common hallmark of human cancer. Nat Genet 2005,
37:391-400.
This elegant study showed for the first time the loss of mono-acetylation
at H4K16 and trimethylation at H4K20 in tumor cells. These reductions
occur in repetitive DNA sequences in association with the global loss of
DNA methylation. Future studies should address the temporal order of
events and the molecular mechanisms involved.
Current Opinion in Genetics & Development 2005, 15:490–495