REVIEW ARTICLE
DNA methylation-mediated nucleosome dynamics and
oncogenic Ras signaling
Insights from FAS, FAS ligand and RASSF1A
Samir K. Patra
1,
* and Moshe Szyf
2
1 Cancer Epigenetics Research, Kalyani, India
2 Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada
DNA methylation and chromatin modification and
remodeling are currently center stage in studies of the
epigenetic regulation of genome function in normal
physiology, disease states and development [1–25]. Sev-
eral isoforms of enzymes catalyzing both DNA and
histone modifications have been characterized. Con-
comitant with differentiation, cell-type-specific patterns
of DNA methylation and histone modification are gen-
erated and are believed to program cell-type-specific
physiological functions, including memory formation
in neurons [2,18,20]. These elaborate epigenetic
programs may be difficult to reverse and rebuild
during animal cloning procedures, because the signals
and mechanisms for gene-specific hypermethylation
and global demethylation patterns are not completely
understood [2]. In eukaryotes, the chromatin is orga-
nized as euchromatin and heterochromatin. Euchroma-
tin encompasses the majority of single-copy genes, it
replicates during early S phase and contains acetylated
histones. Heterochromatin is composed of long
Keywords

data implicate lipid rafts as the coordinators of signals emanating from the
cell membrane and are converging on the mechanisms linking DNA meth-
ylation and chromatin dynamics. This review focuses on some of these
recent advances and uses lipid-raft-facilitated Ras signaling as a paradigm
for understanding DNA methylation, chromatin dynamics and apoptosis.
Abbreviations
aSMAase, acid sphingomyelinase; DISC, death-inducing signaling complex; DNMTs, DNA methyltransferases; FADD, FAS-associated death
domain; FASL, FAS ligand; gld, generalized lymphoproliferative disorder; lpr, lymphoproliferative disorder; MAPK, mitogen activated protein
kinase; MBD, methyl-CpG-binding domain proteins; MGMT, O6-methylguanine methyltransferase; RESE, Ras epigenetic silencing effectors;
TNF, tumor necrosis factor.
FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5217
stretches of DNA repeats, replicates in late S phase
and contains lower levels of acetylated histones and
higher levels of DNA methylation [1–3,5–7,9,13–17,21–
24]. Cytosine methylation is implicated in controlling
transcription, maintaining genome stability, parental
imprinting and X chromosome inactivation [1,2,21,
24,25].
The DNA methylation-mediated repression of sev-
eral genes, including those encoding proteins involved
in cell-cycle regulation and apoptosis, is a major cause
of tumor development and cancer progression [1–9]. In
addition to the gene-specific hypermethylation of sev-
eral genes in many cancers, genomes of tumor cells are
globally hypomethylated and several genes critical for
tumor metastasis and progression are activated by
demethylation [2,3,6,11–16]. The enzymes and cofac-
tors responsible for demethylation in cancer cells are
currently unknown. It is known, however, that during
early development asymmetric DNA demethylation of

class of methyltransferases, DNMT3a and DNMT3b
are required for de novo methylation during embryonic
development [2,25], whereas DNMT3L cooperates with
the DNMT3 family to establish maternal imprints in
mice [29]. DNMT1 and DNMT3B interact among
themselves [30] and DNMT3A interacts with histone
methyltransferases SETDB1 in the promoters of
silenced gene during cancer development [16]. Catalytic
mechanisms of DNMTs involve the formation of a
covalent bond between a cysteine residue in the active
site of the enzyme and carbon 6 (C6) of cytosine in
DNA. The mechanisms involved have been described
recently [2,25,31–39]. Very recent data suggest that
DNMTs may also be involved in the deamination of
methylated cytosines to thymines [31,32]. The mis-
matched thymidine is then removed by base ⁄ nucleotide
excision repair resulting in repair to an unmethylated
cytosine [2]. This has been proposed to serve as a
mechanism for dynamic DNA methylation [31,32].
A different type of DNA methyltransferase is
O6-methylguanine DNA methyltransferase (MGMT;
EC 2.1.1.63). This enzyme does not methylate DNA
but is a DNA repair protein that removes mutagenic
and cytotoxic adducts from the O6 position of guan-
ine. O6-Methylguanine often mispairs with thymine
during replication. Following DNA replication this
would result in conversion of a guanine–cytosine (GC)
pair to an adenine–thymine (AT) pair. Thus, repairing
O6-methylguanine adducts is essential for the integrity
of the genome. Interestingly this DNA methyltransfer-

5218 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS
hydroxyl group attacking carbon 4, followed by elimi-
nation of ammonia will yield thymidine [2,25,34,46].
Epigenetic consequences of DNA
modifications – nucleosome dynamics
There is a bilateral relationship between DNA methyl-
ation and chromatin structure [2,16,49–58]. Promoters
of genes and important regulatory sequences are asso-
ciated with hyperacetylated histones, whereas silent
genes are associated with hypoacetylated histones.
Acetylated histones are associated with unmethylated
DNA and are rarely present in methylated DNA
regions [59]. In addition to histone acetylation, which
plays a critical role in gene regulation, other histone
modifications such as methylation, phosphorylation
and ubiquitination play a similar role in regulating
genome functions [49–56]. A combinatorial arrange-
ment of these modifications is believed to constitute a
‘histone code’. Methylation of DNA and deacetylation
of histones H3 and H4, combined with methylation of
K27 residue on the H3-histone tail in upstream regula-
tory regions leads to inactivation ⁄ repression of gene
expression, whereas selective acetylation of histones
H1, H3, H4, methylation of H3K4 and DNA deme-
thylation are associated with activation of transcrip-
tion [2,5,6,12,16,22,23] (Fig. 1).
How does DNA methylation signal for repression of
transcription? Repression of transcription may occur
through different mechanisms. One simple mechanism
is that DNA methylation interferes with the binding of

which facilitates the interaction of MBD proteins and
chromatin remodeling machines with the
Me
CpG sites.
The MBD–
Me
CpG complex then brings about deacety-
lation of histones H3 and H4 [2,22] by recruiting
class I histone deacetylases, which may be co-recruited
with DNA-topoisomerase II [62] (Fig. 1). Indeed, it
has been shown experimentally that methylation of
DNA brings about general deacetylation of histones
H3 and H4, prevents methylation at H3K4 and
induces methylation of H3 K9 [2,52–56]. Histone
H3K4 trimethylation is associated with transcription-
ally active genes [59,63–73]. MeCP2 has also been
shown to recruit the histone methyltransfaerase
SUV39 which targets H3 K9 [74]. Okitsu & Hsieh
observed a tight correlation between depletion of
H3K4Me2 and regions of DNA methylation, and pro-
posed that DNA methylation dictates a closed chroma-
tin structure devoid of H3K4Me2 [59]. The recent
discovery of histone demethylases has challenged the
originally held belief that histone methylation is static.
Histone demethylases specific for mono-, di- and
trimethylated histone H3K4 are now known and their
structures have been described [54,70–73,75–78]. It is
possible that the recently discovered histone demethy-
lase LSD1 also participates in maintaining methylated
regions of DNA devoid of H3K4 methylation [56,78].

gene expression affecting chromatin structure [2,6,22,59–71], because the presence of methyl groups on DNA affect the structure of DNA
and the interaction of other proteins and enzymes with local nucleosomes [2,60]. Methylation of DNA (
Me
CpG-) brings about a general hydra-
tion of DNA [61], which facilitates the methyl-CpG-sequence binding (MBD) proteins to recognize the
Me
CpG- sites in nucleosomes for
remodeling into a repressive complex. DNMT-
Me
CpG- influence deacetylation of histones H3 and H4 by recruiting class I histone deacety-
lases (HDACs); prevents methylation at H3K4, and induce methylation of H3 K9 in eukaryotes [56–69]. HDACs may be co-recruited with
DNA-topoisomerase II [62]. Histone H3K4 methylation is associated with transcriptionally active nucleosomes of chromatin in which K4 of
H3 are trimethylated, whereas H3 K27 methylation is associated with inactive chromatin [56,59,63–68,79]. Methylation of histones is revers-
ible and histone demethylases specific for di- and trimethylated histone H3K4 are discovered; for example, LSD1 represses transcription
through demethylation of H3K4 Me3 [72,75,77]. Okitsu & Hsieh [59] observed a tight correlation between the depletion of H3K4Me2 in the
regions of DNA methylation. Conversely, the level of H3K4Me2 remains high in the unmethylated DNA regions regardless of the presence
of RNA Pol II. It can be proposed that
Me
CpG- dictates a closed chromatin structure that is devoid of H3K4Me2 and inhibits transcription,
and that the presence of H3K4me2 marks an open chromatin structure that would permit transcription if all other conditions for active tran-
scription are fulfilled [56,58]. In early development, genomic methylation is erased and the somatic methylation pattern is re-established at
the time of implantation. The initiation of DNA demethylation-dependent nuclear processes is highly dependent on unfolding of chromatin
structure. In this context, acetylation of lysine ⁄ arginine of histone tails of H3 and ⁄ or H4 at the respective
Me
CpG-rich nucleosome depends
on histone acetyl transferases (HATs) [48–50]. In addition to methylation, H3 K9, H3 K14, H3 K23 and H3 K27 are also prone to acetylation,
whereas as H3 K18 is only acetylated [22,49–59,63–69]. This implies that nucleosome position is biased by the DNA sequence to facilitate
access to initiation factors and activators by hundreds of histone modification (deacetylation, demethylation and also phosphorylations at ser-
ine ⁄ threonine residues). Also, activation of specialized domains by removal of loosely associated mobile proteins, including HMG, HP and
H1, partly regulates the expression of independent genes modulating the access of the above factors [2,22]. Note: all the modifications men-

Despite profound improvements in our understanding
of the molecular and cellular mechanisms of action of
the Ras proteins, the expanding list of downstream
effectors and the complexity of the signaling cascades
that they regulate suggest that much remains to be
learnt [83]. The study of Ras proteins and their func-
tions in cell physiology has led to many insights not
only into tumorigenesis but also into many develop-
mental disorders [82–84].
Although Ras binds both GDP and GTP with very
high affinity, the GTP-bound form is active and the
GDP-bound form is inactive. The rate of intrinsic
nucleotide exchange and GTPase activity is very slow.
Ras–GDP predominates in resting cells, but when
Ras is activated, specific guanine nucleotide exchange
factors enhance nucleotide exchange, increasing the
Ras–GTP complex. Ras–GTP then activates down-
stream effectors such as Raf-1. GTPase-activating pro-
tein, however, causes the precipitation and
accumulation of inactive Ras–GDP within a cell and
may deregulate cellular physiology when overexpressed
[1,82–90]. The retroviral oncogene, V-Ras, encodes a
protein that differs from the C-Ras product by a point
mutation that maintains this Ras protein constitutively
active [83]. The Ras-related GTPase, Rho is required
for transmission of a proliferative signal by Ras. If
Rho is inhibited, the constitutively active Ras induces
the cyclin-dependent kinase inhibitor p21(Waf1) ⁄ Cip1,
which blocks entry into the DNA synthesis phase of
the cell cycle. Rho activity suppresses induction of

in the endoplasmic reticulum, K-Ras is transported to
the plasma membrane by a desorption–absortion
mechanism [92,94]. Ras detachment from lipid rafts
requires GTP hydrolysis [92,93]. H-Ras and N-Ras
are transported to different sub-compartments by
vesicular traffic, or by a nonvesicular pathway involv-
ing a constitutive deacylation–reacylation cycle
[1,83,92,95–97].
Inter-relationship of genetics and
epigenetics in Ras oncogenic signaling
There is a bilateral relationship between genetic and
epigenetic mechanisms in the activation of oncogenic
Ras signaling. Activation of K-Ras and H-Ras in
human cancers results in DNA hypermethylation
S. K. Patra and M. Szyf DNA methylation-mediated nucleosome dynamics
FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5221
of target genes [1,6,31,83,98]. Epigenetic deregula-
tion of critical repair genes can, however, increase the
rate of mutation of Ras genes (Fig. 2). For example,
silencing of the repair methyltransferase MGMT would
result in an increased rate of mutation of Ras and
other oncogenes. Figure 2 represents a scheme of how
the loss of MGMT expression would result in G to
A transitions in the K-Ras oncogene and in p53
[1,41,99]. Indeed, MGMT promoter hypermethylation
is significantly pronounced in tumours that also bear a
G to A mutation in p53 suggesting a link between
epigenetic and genetic events and apoptosis related
diseases [99].
Genetic activation of Ras would also cause a change

An attractive hypothesis is that K-Ras or H-Ras sig-
nals originating from the membrane at different lipid
raft-anchored Ras pools would have distinct effects on
DNA methylation and demethylation [1,2,91,92], and
histone 3 phosphorylation and acetylation machineries
[55]. Interestingly, this relationship between lipid rafts,
Fig. 2. Epigenetic silencing of repair genes
affects genetics. O6-methylguanine DNA-
methyltransferase (MGMT) gene silencing
through promoter methylation demonstrates
how the loss of MGMT expression results
in G to A transitions of the K-Ras oncogene
(the most frequently mutated isoform of
Ras), and of p53 [1,5,6,41,99]. MGMT, a
DNA repair protein, removes mutagenic and
cytotoxic adducts from the O6 position of
guanine [41,183]. O6-methylguanine often
mispairs with thymine during replication,
and it results in conversion from a guanine–
cytosine (GC) pair to an adenine–thymine
(AT) pair if the adduct is not removed.
DNA methylation-mediated nucleosome dynamics S. K. Patra and M. Szyf
5222 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS
Ras and epigenetic states might be bilateral as well
because many lipid raft component encoding genes are
known to be regulated by DNA methylation
[2,91,110].
FAS, FAS ligand, FAS-associated death
domain and lipid raft-mediated FAS
signaling

has been shown that following activation of T cells,
the FAS receptor is rapidly induced. The interaction
between FAS and FASL induces cell death that occurs
in a cell-autonomous manner, similar to the classic
apoptotic sequence [117,118]. FAS activates caspase 3
by inducing the cleavage of the caspase zymogen to its
active subunits and by stimulating the denitrosylation
of its active site thiol [119].
Myc-induced apoptosis requires interaction between
FAS and FASL on the cell surface [120]. Hueber et al.
established the dependence of Myc on FAS signaling
for its potent cell killing activity [120,121]. The path-
way leading to apoptosis by FAS cross-linking with
FASL results in the formation of a death-inducing
signaling complex (DISC) composed of FAS, the signal
adaptor protein FADD, and procaspase 8 and 10, and
the caspase 8 ⁄ 10 regulator C-FLIP [91,122,123] Yeh
et al. [115] proposed that the interaction of FADD and
FAS through their C-terminal death domains unmasks
the N-terminal effector domain of FADD, allowing it
to recruit caspase 8 (CASP-8; 601763) to the FAS sig-
naling complex. This results in activation of a cysteine
protease cascade, which leads to cell death. Apoptosis
triggered by infection, radiation or chemotherapeutic
drugs is also mediated by FAS. This process involves
modification, placement in membrane, aggregation in
lipid rafts and internalization of the FAS–DISC com-
plex [91,124–130]. Internalization of FAS with either
lipid rafts or an endosomal compartment may deter-
mine which signaling pathways are involved. When

netic silencing of genes encoding DNA repair proteins
(for example, MGMT) may cause retention of
mutants as well as encourage neo-mutants [1–6,41].
The FAS apoptotic pathway is one of the most
promising targets of this process.
S. K. Patra and M. Szyf DNA methylation-mediated nucleosome dynamics
FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5223
FAS mutations in cancer and other
diseases
Several lines of evidence have highlighted that perhaps
all tumor cells express FAS, but in many cases the
gene is mutated encoding a nonfunctional protein.
Some cancers such as papillary thyroid carcinoma,
however, do express functional FAS [132]. Analysis of
the entire FAS coding region in micro-dissected biopsy
samples from 21 burn scar-related squamous cell carci-
nomas revealed somatic point mutations in all of the
splice sites from three patients [133]. The mutations
were located in all three domains of the protein: the
death domain, ligand-binding domain and transmem-
brane domain of the FAS gene. Analyses of the other
FAS alleles in tumors carrying the N239D and C162R
mutations indicated loss of heterozygosity, and expres-
sion of FAS was confirmed in all tumors with FAS
mutations [91,133].
In contrast to the situation in burn scar-related
squamous cell carcinoma, no mutations were detected
in 50 cases of conventional squamous cell carcinoma
[133]. This difference in mutation of the FAS gene is
interesting because burn scar-related squamous cell

thy, heightened autoimmunity and expanded popula-
tions of TCR-CD3(+)CD4())CD8()) lymphocytes,
resulting from defective FAS-mediated T-lymphocyte
apoptosis. While delineating the prognostic markers
for the disorder, Sneller et al. [137] further analyzed
one of the patients studied by Fisher et al., and
pointed out its resemblance to autosomal recessive
lpr ⁄ gld (generalized lymphoproliferative disorder)
mouse. The lpr and gld mice bear mutated genes for
FAS and FASL, respectively. The murine autosomal
recessive lpr phenotype is characterized by lymphade-
nopathy, hypergammaglobulinemia, multiple autoanti-
bodies and the accumulation of large numbers of
nonmalignant CD4, CD8 and T cells. Affected mice
usually develop a systemic lupus erythematosus-like
autoimmune disease, and a defect in the negative
selection of self-reactive T lymphocytes in the thymus.
The mouse lpr phenotype is identical to the phenotype
displayed by human patients bearing mutated FAS
[138,139].
Epigenetic downregulation of
apoptosis – the role of the Ras
signaling pathway
In addition to genetic mutations in FAS as discussed
above, FAS is silenced by epigenetic mechanisms in
several cancers. Several lines of evidence suggest that
the trigger for methylation of the FAS gene is activa-
tion of the Ras fi Raf fi MEK fi ERK fi Elk
signaling pathway [1,91,98]. Methylation of the FAS
gene is associated with loss of FAS expression in anti-

tumors. In neuroblastomas and neurobalstoma cell
lines, which are resistant to apoptosis induced by
TRAIL, CASP-8 and the FLIP gene, and in tissues
adjacent to tumors the CASP-8 gene is hypermethyla-
ted [145]. The FLIP protein is a negative regulator of
CASP-8, and the methylation of CASP-8 and FLIP
genes is somewhat correlated [91,125,145].
Mechanisms of silencing of FAS in
response to Ras activation
How does Ras activation cause methylation and epige-
netic silencing of FAS and other apoptosis-related
genes? Activated Ras epigenetically silences FAS
expression in mouse NIH3T3 cells [143], and in human
K-Ras transformed cell line, HEC1A [98]. Twenty-
eight ‘Ras epigenetic silencing effector’ (RESE) genes
were discovered in a genome-wide functional screen
[98]. Nine RESEs were found to be bound to different
regions of the FAS promoter in K-Ras-transformed
NIH3T3 cells [98], whereas in nontransfected NIH3T3
cells only one RESE (NPM2) was associated with the
FAS promoter. It was therefore proposed that these
nine RESEs were recruited to specific regions of FAS
promoter in response to expression of oncogenic
K-Ras and are involved in the recruitment of DNMT1
and other chromatin modifiers to the promoter, result-
ing in DNA methylation and epigenetic silencing. In
support of this hypothesis, knockdown of any of the
28 RESEs in K-Ras-transformed NIH3T3 cells
resulted in an absence of DNMT1 on the FAS
promoter, demethylation of the FAS promoter and

several putative tumor-suppressor genes are located at
chromosome 3p21 [83,150–152]. RASSF1 produce
eight transcripts, A–H, derived from alternative splic-
ing and promoter usage [152–154]. The RASSF1 gene
contributes to the spatiotemporal regulation of mitosis
through a number of regulatory mechanisms that
cooperate to restrict the activity of APC ⁄ C to a spe-
cific period in the cell cycle [153–155]. Mechanistic
roles for RASSF1A in inducing apoptosis in cancer
cells and solid tumors are emerging [156–158].
RASSF1A function was missing in a variety of solid
tumors and cancer cell lines, including small cell lung
cancer and prostate [150–156]. DNA methylation of
the CpG island promoter sequence of RASSF1A was
implicated in its silencing [16,155]. RASSF1A is the
most frequently methylated gene in both primary
tumors and cell lines and in a group of nine genes
mapped in 175 primary pediatric tumors and 23 tumor
cell lines. RASSF1A methylation was tumor specific
and absent in adjacent nonmalignant tissues [157].
RASSF1A gene silencing is also associated with aber-
rant methylation and histone deacetylation in a variety
of other cancers [158–163]. RASSF2 methylation and
inactivation is a consequence as well of K-Ras-induced
oncogenic transformation [164]. Apart from Ras-regu-
lated methylation of RASSF1A, Ras and RASSF1A
have direct physical interaction in cellular physiology
[155,160].
Lipid raft facilitated Ras signaling and
chromatin modification

[129,130,176] and induces DNA demethylation-medi-
ated expression of genes [177,178]. The inhibitory
effect of (-)-epigallocatechin gallate on activation of
the epidermal growth factor receptor is shown experi-
mentally to be brought about by altering the lipid
order of rafts in HT29 colon cancer cells [169]. Small
drugs, including edelfosine, perifosine, ether lipid
ET-18-OCH(3) and aplidine alter cytoskeleton-medi-
ated FAS and FASL concentrations in lipid rafts.
These rafts form apoptosis-promoting clusters in can-
cer chemotherapy [170–175]. Thus, there is a relation-
ship between lipid rafts, Ras–MAPK signaling and the
response to chemotherapeutic agents. Activation of
Ras in lipid rafts triggers DNA methylation and the
silencing of repair and apoptotic genes. The FAS gene
is repressed by DNA methylation transduced by
H-Ras and K-Ras signaling that is facilitated by lipid
rafts, on the one hand [98,144] (Fig. 3A), and radia-
tion ⁄ chemotherapy can cause demethylation-induced
FAS expression, on the other hand (Fig. 3B). Eventu-
ally, demethylation also results in activation of the
gene encoding acid sphingomyelinase (aSMAase).
Translocation of the increased levels of aSMAase to
membrane lipid rafts [128,129] lead to the transfor-
mation of the proliferative cholesterol raft to a
death-inducing ceramide raft and is associated with
FAS–DISC internalization and eventual cell death [91].
It is therefore important whether radiation ⁄ drug-
induced changes in lipid raft composition transmit the
altered Ras-MAPK signal that causes eventual FAS

Ase. Translocation of FAS and aSMAse to membrane domain lipid rafts affects lipid-raft composition. Hydrolysis of sphingomyeline by aSM-
Ase produces ceramide. This in situ produced ceramide displaces cholesterol from cholesterol rafts transforming them into ceramide rafts
[91], which predominantly transmit death signals [128–130,172–179]. All the components in the figures are not drawn to the same scale. For
example, lipid rafts in membranes are drawn as circles many fold larger than the membrane leaflet.
S. K. Patra and M. Szyf DNA methylation-mediated nucleosome dynamics
FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5227
suggesting that induction of repair enzymes would lead
to hypersensitization to chemotherapeutic agents.
Conclusion and perspectives
We have discussed data supporting DNA methylation-
mediated chromatin dynamics and described how one
of the signals for reversible DNA methylation is trans-
mitted by Ras oncoproteins. We also developed the
hypothesis that the Ras signal for DNA methylation
emanates from the membrane and is coordinated by
lipid rafts. One or more components of the potent cell
killing machinery, including FAS, FASL, FADD and
RASSF1 genes are often repressed by DNA methyla-
tion in carcinogenesis in response to activation of the
Ras signaling pathway. The silencing of repair genes
by methylation has consequences for the genetic integ-
rity of cells, as well as for the responsiveness of cells to
chemotherapeutic agents. There is a bilateral relation-
ship between genetic lesions and epigenetic aberrations.
Epigenetic silencing of repair genes such as MGMT
could lead to elevated levels of Ras mutations, and
Ras-activating mutations turn on downstream signal-
ing, resulting in epigenetic silencing. There is also a
bilateral relationship between chemotherapeutic agents
and epigenetic states. Chemotherapeutic drugs can

include and discuss in this article.
References
1 Patra SK (2008) Ras regulation of DNA methylation
and cancer. Exp Cell Res 314, 1193–1201.
2 Patra SK, Patra A, Rizzi F, Ghosh TC & Bettuzzi S
(2008) Demethylation of (cytosine-5-C-methyl) DNA
and regulation of transcription in the epigenetic path-
ways of cancer development. Cancer Metast Rev 27,
315–334.
3 Patra SK, Patra A, Zhao H & Dahiya R (2002)
DNA-methyltransferase and demethylase in human
prostate cancer. Mol Carcinogen 33, 163–167.
4 Patra SK, Patra A & Dahiya R (2001) Histone
deacetylase and DNA-methyltransferase in human
prostate cancer. Biochem Biophys Res Commun 287,
705–713.
5 Brown SE, Fraga MF, Weaver ICG, Berdasco M &
Szyf M (2007) Variations in DNA methylation pat-
terns during the cell cycle of HeLa cells. Epigenomics
2, 54–65.
6 Jones PA & Baylin SB (2007) The epigenomics of
cancer. Cell 128, 683–692.
7 Li LC, Okino ST & Dahiya R (2004) DNA methylation
in prostate cancer. Biochim Biophys Acta 1704, 87–102.
8 Ehrlich M (2006) Cancer-linked DNA hypomethyla-
tion and its relationship to hypermethylation. Curr
Top Microbiol Immunol 310, 251–274.
9 Lopez-Serra L, Ballestar E, Fraga MF, Alaminos
M, Setien F & Esteller M (2006) A profile of
methyl-CpG binding domain protein occupancy of

´
PE, Riggs AD &
Pfeifer GP (2006) The histone methyltransferase SET-
DB1 and the DNA methyltransferase DNMT3A
interact directly and localize to promoters silenced in
cancer cells. J Biol Chem 281, 19489–19500.
17 Reik W (2007) Stability and flexibility of epigenetic
gene regulation in mammalian development. Nature
447, 425–432.
18 McLay DW & Clarke HJ (2003) Remodeling the
paternal chromatin at fertilization in mammals.
Reproduction 125, 625–633.
19 Haaf T (2006) Methylation dynamics in the early
mammalian embryo: implications of genome repro-
gramming defects for development. Curr Top Micro-
biol Immunol 310, 13–22.
20 Miller CA & Sweatt JD (2007) Covalent modification
of DNA regulates memory formation. Neuron 53,
857–869.
21 Klose RJ & Bird AP (2006) Genomic DNA methyla-
tion: the mark and its mediators. Trends Biochem Sci
31, 89–97.
22 Bernstein BE, Meissner A & Lander ES (2007) The
mammalian epigenome. Cell 128, 669–681.
23 Richards EJ & Elgin S (2002) Epigenetic codes for
heterochromatin formation and silencing: rounding up
the usual suspects. Cell 108, 489–500.
24 Fraga MF, Ballestar E, Paz EF, Ropero S, Setien F,
Ballestar ML, Heine-Suner D, Cigudosa JC, Urioste
M, Benitez J et al. (2005) Epigenetic differences arise

ron C,
Jurkowska RZ, Carmouche RP, Ibberson D, Barath P,
Demay F, Reid G et al. (2008) Cyclical DNA methyl-
ation of a transcriptionally active promoter. Nature
452, 45–50.
32 Kangaspeska S, Stride B, Me
´
tivier R, Polycarpou-
Schwarz M, Ibberson D, Carmouche RP, Benes V,
Gannon F & Reid G (2008) Transient cyclical
methylation of promoter DNA. Nature 452, 112–
115.
33 Reither S, Li F, Gowher H & Jeltsch A (2003) Cata-
lytic mechanism of DNA-(cytosine-C5)-methyltransfe-
rases revisited: covalent intermediate formation is not
essential for methyl group transfer by the murine
Dnmt3a enzyme. J Mol Biol 329, 675–684.
34 Christman JK (2002) 5-Azacytidine and 5-aza-
2¢-deoxycytidine as inhibitors of DNA methylation:
mechanistic studies and their implications for cancer
therapy. Oncogene 21, 5483–5491.
35 Erlanson D, Dhen L & Verdin GL (1993) Enzymatic
DNA methylation through a locally unpaired inter-
mediate. J Am Chem Soc 115, 12583–12584.
36 Santi DV, Garrett CE & Barr PJ (1983) On the mech-
anism of inhibition of DNA-cytosine methyltransfer-
ase by cytosine analogs. Cell 33, 9–10.
37 Wilson VL & Jones PA (1983) Inhibition of DNA
methylation by chemical carcinogens in vitro. Cell 32,
239–246.

dine induced cytotoxicity in human breast cancer
cells. J Biol Chem 272, 32260–32266.
44 Bhattacharya SK, Ramchandani S, Cervoni N & Szyf
M (1999) A mammalian protein with specific demeth-
ylase activity for
m
CpG DNA. Nature 397, 579–783.
45 Cedar H & Verdine GL (1999) Gene expression: the
amazing demethylase. Nature 397, 568–569.
46 Smith SS (2000) Gilbert’s conjecture: the search for
DNA (cytosine-5) demethylase and the emergence of
new functions for eukaryotic DNA (cytosine-5) meth-
yltransferases. J Mol Biol 302, 1–7.
47 Kress C, Thomassin H & Grange T (2001) Local
DNA demethylation in vertebrates: how could it be
performed and targeted? FEBS Lett 494, 135–140.
48 Dong E, Guidotti A, Grayson DR & Costa E
(2007) Histone hyperacetylation induces demethyla-
tion of reelin and 67 kDa glutamic acid decar-
boxylase promoters. Proc Natl Acad Sci USA 104,
4676–4681.
49 Sakamoto Y, Watanabe S, Ichimura T, Kawasuji M,
Koseki H, Baba H & Nakao M (2007) Overlapping
roles of the methylated DNA-binding protein MBD1
and polycomb group proteins in transcriptional
repression of HOXA genes and heterochromatin foci
formation. J Biol Chem 282, 16391–16400.
50 Sarraf SA & Stancheva I (2004) Methyl-CpG binding
protein MBD1 couples histone H3 methylation at
lysine 9 by SETDB1 to DNA replication and chroma-

2746–2757.
60 Vargason JM, Eichman BF & Ho PS (2000) The
extended and eccentric E-DNA structure induced by
cytosine methylation or bromination. Nat Struct Biol
7, 758–761.
61 Mayer-Jung C, Moras D & Timsit Y (1998) Hydra-
tion and recognition of methylated CpG steps in
DNA. EMBO J 17, 2709–2718.
62 Tsai S-H, Valkov N, Yang W-M, Gump J, Sullivan D
& Seto E (2000) Histone deacetylases interacts directly
with DNA topoisomerase II. Nat Genet 26, 349–353.
63 Schu
¨
beler D, Lorincz MC, Cimbora DM, Telling A,
Feng Y-Q, Bouhassira EE & Groudine M (2000)
Genomic targeting of methylated DNA: influence of
methylation on transcription, replication, chromatin
structure and histone acetylation. Mol Cell Biol 20,
9103–9112.
64 Hashimshony T, Zhang J, Keshet I, Bustin M &
Ceder H (2003) The role of DNA methylation in set-
ting up chromatin structure during development.
Nat Genet 34, 187–192.
65 Lorincz MC, Dickerson DR, Schmitt M & Groudine
M (2004) Intragenic DNA methylation alters chroma-
tin structure and elongation efficiency in mammalian
cells. Nat Struct Mol Biol 11, 1068–1075.
66 Irvine RA, Lin IG & Hsieh C-L (2002) DNA methyl-
ation has a local effect on transcription and histone
acetylation. Mol Cell Biol 22, 6689–6696.

homolog LSD1. Cell 119, 941–953.
73 Kubicek S & Jenuwein T (2004) A crack in histone
lysine methylation. Cell 119, 903–906.
74 Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP & Kouza-
rides T (2003) The methyl-CpG-binding protein
MeCP2 links DNA methylation to histone methyla-
tion. J Biol Chem 278, 4035–4040.
75 Stavropoulos P, Blobel G & Hoelz A (2006) Crystal
structure and mechanism of human lysine-specific
demethylase-1. Nat Struct Mol Biol 13, 626–632.
76 Li H, Ilin S, Wang W, Duncan EM, Wysocka J, Allis
CD & Patel DJ (2006) Molecular basis for site-specific
read-out of histone H3K4me3 by the BPTF PHD
finger of NURF. Nature 442, 91–95.
77 Taverna SD, Li H, Ruthenburg AJ, Allis CD & Patel
DJ (2007) How chromatin-binding modules interpret
histone modifications: lessons from professional
pocket pickers. Nat Struct Mol Biol 14, 1025–1040.
78 Vakoc CR, Sachdeva MM, Wang H & Blobel GA
(2006) Profile of histone lysine methylation across
transcribed mammalian chromatin. Mol Cell Biol 26,
9185–9195.
79 D’Alessio AC, Weaver IC & Szyf M (2007) Acetyla-
tion-induced transcription is required for active DNA
demethylation in methylation-silenced genes. Mol Cell
Biol 27, 7462–7474.
80 Cervoni N, Detich N, Seo SB, Chakravarti D & Szyf M
(2002) The oncoprotein Set ⁄ TAF-1beta, an inhibitor of
histone acetyltransferase, inhibits active demethylation
of DNA, integrating DNA methylation and transcrip-

state acetate and hydrogen peroxide in NIH3T3 cells.
Biochem Biophys Res Commun 311, 1026–1033.
88 Bland KI, Konstadoulakis MM, Vezeridis MP &
Wanebo HJ (1995) Oncogene protein co-expression –
value of Ha-ras, c-myc, c-fos and p53 as prognostic
discriminants for breast carcinoma. Ann Surg 221,
706–720.
89 Fukano T, Sawano A, Ohba Y, Matsuda M & Miy-
awaki A (2007) Differential Ras activation between
caveolae ⁄ raft and non-raft microdomains. Cell Struct
Funct 32, 9–15.
90 Hancock JF (2003) Ras proteins: different signals
from different locations. Nat Rev Mol Cell Biol 4,
373–384.
91 Patra SK (2008) Dissecting lipid raft facilitated cell
signaling pathways in cancer. Biochim Biophys Acta
1785, 182–206.
92 Eisenberg S, Shvartsman DE, Ehrlich M & Henis YI
(2006) Clustering of Raft-associated proteins in the
external membrane leaflet modulate internal leaflet
H-Ras diffusion and signaling. Mol Cell Biol 26,
7190–7200.
93 Parton RG & Hancock JF (2004) Lipid rafts and
plasma membrane microorganization: insights from
Ras. Trends Cell Biol 14, 141–147.
94 Gomez GA & Daniotti JL (2005) H-Ras dynamically
interacts with recycling endosomes in CHO-K1 cells:
involvement of Rab5 and Rab11 in the trafficking of
H-Ras to this pericentriolar endocytic compartment.
J Biol Chem 280, 34997–35010.

X, Kuban RJ, Ungethu
¨
m U, Krapfenbauer U, Herzel
HP, Scha
¨
fer R et al. (2006) Oncogenic H-Ras
suppresses clusterin expression through promoter
hypermethylation. Oncogene 25, 4890–4903.
102 MacLeod AR, Rouleau J & Szyf M (1995) Regulation
of DNA methylation by the Ras signaling pathway.
J Biol Chem 270, 11327–11337.
103 Caretti G, Di Padova M, Micales B, Lyons GE &
Sartorelli V (2004) The polycomb Ezh2 methyltrans-
ferase regulates muscle gene expression and skeletal
muscle differentiation. Gene Dev 18, 2627–2638
(erratum appears in Gene Dev 19, 768).
104 Vire
´
E, Brenner C, Deplus R, Blanchon L, Fraga M,
Didelot C, Morey L, Van Eynde A, Bernard D,
Vanderwinden JM et al. (2006) The Polycomb group
protein EZH2 directly controls DNA methylation. Nat-
ure 439, 871–874 (erratum appears in Nature 446, 824).
105 Morgan MA, Ganser A & Reuter CW (2007) Target-
ing the RAS signaling pathway in malignant hemato-
logic diseases. Curr Drug Target 8, 217–235.
106 Diaz-Flores E & Shannon K (2007) Targeting onco-
genic Ras. Gene Dev 21, 1989–1992.
107 Philips MR (2004) Methotrexate and Ras methylation:
a new trick for an old drug? Sci STKE 225, pe13.

115 Yeh W-C, de la Pompa JL, McCurrach ME, Shu H-B,
Elia AJ, Shahinian A, Ng M, Wakeham A, Khoo W,
Mitchell K et al. (1998) FADD: essential for
embryo development and signaling from some,
but not all, inducers of apoptosis. Science 279,
1954–1958.
116 Zhang J, Cado D, Chen A, Kabra NH & Winoto A
(1998) Fas-mediated apoptosis and activation-induced
T-cell proliferation are defective in mice lacking
FADD ⁄ Mort1. Nature 392, 296–300.
117 Brunner T, Mogil RJ, LaFace D, Yoo NJ, Mahboubi
A, Echeverri F, Martin SJ, Force WR, Lynch DH,
Ware CF et al. (1995) Cell-autonomous Fas
(CD95) ⁄ Fas-ligand interaction mediates activation-
induced apoptosis in T-cell hybridomas. Nature 373,
441–444.
118 Ju S-T, Panka DJ, Cui H, Ettinger R, El-Khatib M,
Sherr DH, Stanger BZ & Marshak-Rothstein A
(1995) Fas(CD95) ⁄ FasL interactions required for pro-
grammed cell death after T-cell activation. Nature
373, 444–448.
119 Mannick JB, Hausladen A, Liu L, Hess DT, Zeng M,
Miao QX, Kane LS, Gow AJ & Stamler JS (1999)
Fas-induced caspase denitrosylation. Science 284,
651–654.
120 Hueber A-O, Zornig M, Lyon D, Suda T, Nagata S
& Evan GI (1997) Requirement for the CD95 recep-
tor-ligand pathway in c-Myc-induced apoptosis.
Science 278, 1305–1309.
121 Hueber A-O (2000) CD95: more than just a death

128 Rotolo JA, Zhang J, Donepudi M, Lee H, Fuks Z &
Kolesnick R (2005) Caspase-dependent and -indepen-
dent activation of acid sphingomyelinase signaling.
J Biol Chem 280, 26425–26434.
129 Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden
D, Rafii S, Haimovitz-Friedman A, Fuks Z &
Kolesnick R (2003) Tumor response to radiotherapy
regulated by endothelial cell apoptosis. Science 300,
1155–1159.
130 Gulbins E & Kolesnick R (2003) Raft ceramide in
molecular medicine. Oncogene 22, 7070–7077.
131 Miacznska M, Pelkmans L & Zerial M (2004) Not
just a sink: endosomes in control of signal transduc-
tion. Curr Opin Cell Biol 16, 400–406.
132 Arscott PL, Stokes T, Myc A, Giordano TJ, Thomp-
son NW & Baker JR Jr (1999) Fas (CD95) expression
is up-regulated on papillary thyroid carcinoma. J Clin
Endocrin Metab 84, 4246–4252.
133 Lee SH, Shin MS, Kim HS, Park WS, Kim SY, Jang
JJ, Rhim KJ, Jang J, Lee HK, Park JY et al. (1999)
Somatic mutations of Fas (Apo-1 ⁄ CD95) gene in
cutaneous squamous cell carcinoma arising from a
burn scar. J Invest Dermatol 114, 122–126.
134 Zhang X, Miao X, Sun T, Tan W, Qu S, Xiong P,
Zhou Y & Lin D (2005) Functional polymorphisms in
cell death pathway genes FAS and FASL contribute
to the risk of lung cancer. J Med Genet 42, 479–484.
135 Green DR & Ferguson TA (2001) The role of Fas
ligand in immune privilege. Nat Rev Mol Cell Bio 2,
917–924.

Hirsch I, Van Lint C & Collette Y (2006) Active
transcription of the human FASL ⁄ CD95L ⁄ TNFSF6
promoter region in T lymphocytes involves chro-
matin remodeling: role of DNA methylation and
protein acetylation suggest distinct mechanisms of
transcriptional repression. J Biol Chem 281, 14719–
14728.
142 Hopkins-Donaldson S, Ziegler A, Kurtz S, Bigosch C,
Kandioler D, Ludwig C, Zangemeister-Wittke U &
Stahel R (2003) Silencing of death receptor and
caspase-8 expression in small cell lung carcinoma cell
lines and tumors by DNA methylation. Cell Death
Differ 10, 356–364.
143 Santourlidis S, Warskulat U, Florl AR, Maas S, Pulte
T, Fischer J, Mu
¨
ller W & Schulz WA (2001) Hyper-
methylation of the tumor necrosis factor receptor
superfamily 6 (APT1, Fas, CD95 ⁄ Apo-1) gene pro-
moter at rel ⁄ nuclear factor kappaB sites in prostatic
carcinoma. Mol Carcinogen 32, 36–43.
144 Peli J, Schro
¨
ter M, Rudaz C, Hahne M, Meyer C,
Reichmann E & Tschopp J (1999) Oncogenic Ras
inhibits Fas ligand-mediated apoptosis by
downregulating the expression of Fas. EMBO J 18,
1824–1831.
145 van Noesel MM, van Bezouw S, Vou
ˆ

for a 3p21.3 tumor suppressor gene. Oncogene 16,
3151–3157.
151 Dammann R, Li C, Yoon J-H, Chin PL, Bates S &
Pfeifer GP (2000) Epigenetic inactivation of a RAS
association domain family protein from the lung
tumour suppressor locus 3p21.3. Nat Genet 25, 315–
319.
152 Lerman MI & Minna JD (2000) The 630-kb lung
cancer homozygous deletion region on human
chromosome 3p21.3: identification and evaluation of
the resident candidate tumor suppressor genes. Cancer
Res 60, 6116–6133.
153 Agathanggelou A, Cooper WN & Latif F (2005) Role
of the Ras-association domain family 1 tumor
suppressor gene in human cancers. Cancer Res 65,
3497–3508 (erratum appears in Cancer Res 65, 5480).
154 Donninger H, Vos MD & Clark GJ (2007) The
RASSF1A tumor suppressor. J Cell Sci 120, 3163–
3172.
155 Pfeifer GP & Dammann R (2005) Methylation of the
tumor suppressor gene RASSF1A in human tumors.
Biochemistry (Moscow) 70, 576–583.
156 Kawamoto K, Okino ST, Place RF, Urakami S,
Hirata H, Kikuno N, Kawakami T, Tanaka Y,
Pookot D, Chen Z et al. (2007) Epigenetic modi-
fications of RASSF1A gene through chromatin
remodeling in prostate cancer. Clin Cancer Res 13,
2541–2548.
157 Harada K, Toyooka S, Maitra A, Maruyama R, Toy-
ooka KO, Timmons CF, Tomlinson GE, Mastrangelo

Mol Cell 27, 962–975.
164 Akino K, Toyota M, Suzuki H, Mita H, Sasaki Y,
Ohe-Toyota M, Issa JP, Hinoda Y, Imai K & Tokino
T (2005) The Ras effector RASSF2 is a novel tumour-
suppressor gene in human colorectal cancer. Gastroen-
terology 129, 156–169.
165 Cheung P, Tanner KG, Cheung WL, Sassone-Corsi P,
Denu JM & Allis CD (2000) Synergistic coupling of
histone H3 phosphorylation and acetylation in
response to epidermal growth factor stimulation. Mol
Cell 5, 905–915.
166 Strelkov IS & Davie JR (2002) Ser-10 phosphoryla-
tion of histone H3 and immediate early gene expres-
sion in oncogene-transformed mouse fibroblasts.
Cancer Res 62, 75–78.
167 Dunn KL & Davie JR (2005) Stimulation of the
Ras-MAPK pathway leads to independent phosphory-
lation of histone H3 on serine 10 and 28. Oncogene
24, 3492–3502.
168 Nowak SJ & Corces VG (2004) Phosphorylation of
histone H3: a balancing act between chromosome
condensation and transcriptional activation. Trends
Genet 20, 214–220.
169 Adachi S, Nagao T, Ingolfosson HI, Maxfield FR,
Andersen OS, Kopelovich L & Weinstein IB (2007)
The inhibitory effect of (-)-epigallocatechin
gallate on activation of the epidermal growth
factor receptor is associated with altered lipid order
in HT29 colon cancer cells. Cancer Res 67, 6493–
6501.

176 Wang Y & Li G (2006) ING3 Promotes UV-induced
apoptosis via Fas ⁄ caspase-8 pathway in melanoma
cells. J Biol Chem 281, 11887–11893.
177 Lieberman MW, Beach LR & Palmiter RD (1983)
Ultraviolet radiation-induced metallothionein-I gene
activation is associated with extensive DNA demethy-
lation. Cell 35, 207–214.
178 Privat E & Sowers LC (1996) Photochemical deamina-
tion and demethylation of 5-methylcytosine. Chem
Res Toxicol 9, 745–750.
179 Ehmann F, Horn S, Garcia-Palma L, Wegner W,
Fiedler W, Giehl K, Mayr GW & Ju
¨
cker M (2006)
Detection of N-RAS and K-RAS in their active
GTP-bound form in acute myeloid leukemia without
activating RAS mutations. Leukemia Lymphoma 47,
1387–1391.
180 Roos WP, Batista LF, Naumann SC, Wick W, Weller
M, Menck CF & Kaina B (2007) Apoptosis in malig-
nant glioma cells triggered by the temozolomide-
induced DNA lesion O6-methylguanine. Oncogene 26,
186–197.
181 Roos WP, Christmann M, Fraser ST & Kaina B
(2007) Mouse embryonic stem cells are hypersensitive
to apoptosis triggered by the DNA damage O(6)-
methylguanine due to high E2F1 regulated mismatch
repair. Cell Death Differ 14, 1422–1432.
182 Fenton RG, Hixon JA, Wright PW, Brooks AD &
Sayers TJ (1998) Inhibition of Fas (CD95) expression

3792–3795.
188 Reifenberger J, Knobbe CB, Sterzinger AA, Blaschke
B, Schulte KW, Ruzicka T & Reifenberger G (2004)
Frequent alterations of Ras signaling pathway genes
in sporadic malignant melanomas. Int J Cancer 109,
377–384.
189 Irimia M, Fraga MF, Sanchez-Cespedes M & Esteller
M (2004) CpG-island promoter hypermethylation of
the Ras-effector gene NORE1A occurs in the context
of a wild-type K-Ras in lung cancer. Oncogene 23,
8695–8699.
190 DeGregori J & Johnson DG (2006) Distinct and over-
lapping roles for E2F family members in transcrip-
tion, proliferation and apoptosis. Curr Mol Med 6,
739–748.
191 Hallstrom TC, Mori S & Nevins JR (2008) An E2F1-
dependent gene expression program that determines
the balance between proliferation and cell death.
Cancer Cell 13, 11–22.
S. K. Patra and M. Szyf DNA methylation-mediated nucleosome dynamics
FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5235