Tài liệu Báo cáo khoa học: DNA methylation-mediated nucleosome dynamics and oncogenic Ras signaling pptx

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

apoptosis; cancer; DNA methylation;
epigenetics; FAS; FAS ligand; H-Ras; K-Ras;
nucleosome dynamics; RASSF1A
Correspondence
S. K. Patra, Cancer Epigenetics Research,
Kalyani (B-7 ⁄ 183), Nadia, West Bengal, India
Fax: +91 332 582 8460
Tel: +91 943 206 0602
E-mail:
*Present address
Division of Biochemistry, Department of
Experimental Medicine, University of Parma,
Italy
(Received 5 June 2008, revised 8 August
2008, accepted 22 August 2008)
doi:10.1111/j.1742-4658.2008.06658.x
Cytosine methylation at the 5-carbon position is the only known stable
base modification found in the mammalian genome. The organization and
modification of chromatin is a key factor in programming gene expression
patterns. Recent findings suggest that DNA methylation at the junction of
transcription initiation and elongation plays a critical role in suppression
of transcription. This effect is mechanistically mediated by the state of
chromatin modification. DNA methylation attracts binding of methyl-
CpG-binding domain proteins that trigger repression of transcription,
whereas DNA demethylation facilitates transcription activation. Under-
standing the rules that guide differential gene expression, as well as tran-
scription dynamics and transcript abundance, has proven to be a taxing
problem for molecular biologists and oncologists alike. The use of novel
molecular modeling methods is providing exciting insights into the chal-
lenging problem of how methylation mediates chromatin dynamics. New

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

the paternal genome is observed just after fertilization
[2,17–19]. Similar to the global hypomethylation
observed in cancer, the enzymes responsible for this
demethylation are unknown.
Although the DNA methylation pattern is pro-
grammed during development, it remains highly sensi-
tive to both the chemical and social environment [10].
Studies have suggested that bioactive components of
food, both essential and nonessential nutrients, can
modify DNA methylation patterns in complex ways.
For example, consumption of a methyl-deficient diet
led to hypomethylation of specific CpG sites within
several oncogenes (such as c-myc,c-fos and c-H-Ras),
resulting in high expression of these genes [26]. Recent
studies have shown that tea catechins are effective
inhibitors of human DNA methyltransferase (DNMT)-
mediated DNA methylation in vitro, and re-expression
of a few genes in cultured cancer cells is observed in
response to tea catechins [27,28]. Thus, DNA methyla-
tion may be viewed as an interface between the envi-
ronment and the human genome. It stands to reason
that it might play critical role in several human pathol-
ogies, in particular age-related disease.
DNA methylation enzymes
In mammalian cells, DNA methylation is catalyzed
by two classes of DNMT. DNA-methyltransferase-1
(DNMT1; EC 2.1.1.37) is essential for maintaining
DNA methylation patterns in proliferating cells and is
also involved in establishing new DNA methylation
patterns; de novo methylation. Members of the second

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-

ase is regulated by DNA methylation. Hypermethyla-
tion of the MGMT promoter is associated with loss of
MGMT expression ⁄ function in many tumor types
[1,4,40]. MGMT hypermethylation is an example of
the emerging field of pharmacoepigenomics. The
impact of chemotherapy would be dependent on the
epigenetic state of cardinal genes such as MGMT
[1,41]. Knowing the state of methylation of critical
repair genes is critical for the proper planning of a
chemotherapeutic protocol.
Removal of the methyl group (
Me
C-DNA demethy-
lation) from critical positions in promoters is essential
for the transcription of many genes. A long line of
evidence suggests that active enzymatic
Me
C-DNA
demethylation occurs in nonreplicating cells to induce
the transcription of specific genes at distinct time
points [2]. The mechanisms of demethylation are
unknown and the enzymes involved are not firmly
established. One possible mechanism is through forma-
tion of a cytosine–Michael adduct ⁄ complex with
MBD2 protein but this would require a cofactor [2,42–
48] which is not known. The current notion of Michael
adduct chemistry is that in such types of complex the
SN2 mechanism would not occur. In principle,
water added across the 4C–5C double bond with the
DNA methylation-mediated nucleosome dynamics S. K. Patra and M. Szyf

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

transcriptional activators [24,25,53,55,59]. A second
mechanism involves recruitment of methyl-CpG-bind-
ing domain proteins (MBDs), such as MBD1, MBD2,
MBD3, MBD4 and MeCP2. MBDs recruit co-repres-
sor complexes to methylated genes, which include
histone-modifying enzymes such as histone deacetylas-
es and histone methyltransferases precipitating an inac-
tive chromatin structure [16,21,39,49,51,56]. This
mechanism provides an explanation for the correlation
between DNA methylation and inactive chromatin
configuration.
DNA methylation and histone modification act in
concert to program gene expression. Figure 1 presents
a model of the inhibition of gene expression by DNA
methylation. Cytosine methylation at the DNA
sequence d(GGCGCC)2 triggers an extended eccentric
double-helix structure called E-DNA. Like B-DNA,
E-DNA has a long helical rise and the base is perpen-
dicular to the helix axis. The 3¢-endo sugar conforma-
tion provides the characteristic deep major groove and
shallow minor groove of A-DNA [60]. Analysis of the
hydration pattern around methylated CpG sites in
crystal structures of A-DNA decamers at three high
resolutions (1.7, 2.15 and 2.2 A
˚
) reveals that the
methyl groups of cytosine residues are well hydrated
with a higher amount of Mg
2+
in their vicinity [61],

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].

DNA methylation immediately downstream of the
transcription start site has a dramatic impact on tran-
scription, affecting transcription elongation rather than
initiation. Recent findings suggest that DNA methyla-
tion at the junction of transcription initiation and
elongation is most critical in transcription suppression
and this effect is mechanistically mediated through
chromatin structure [53,56,59,78]. Although some
important ideas have been suggested in other studies
[64,68–70], it is still difficult to predict the effect of
methylated DNA segments on transcription because
differences in the size and position of the methylated
DNA regions may differentially affect transcription.
For example, although a methylated coding region
positioned 1 kb downstream of the promoter has little
impact on transcription initiation, as observed by
Lorincz et al. [65], the same methylated sequence might
have a much larger impact on transcription initiation if
positioned immediately downstream of the promoter
[54,64,68–70,78]. The context seems to be critical [56].
Recent data suggest that not only can DNA methyl-
ation direct the formation of inactive chromatin struc-
ture, but also that histone signals can direct DNA
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 5219
Fig. 1. Cytosine base in DNA – the amazing switch for the regulation of gene expression and chromatin remodeling. Cytosine, extended
from the sugar-phosphate backbone (black circles–pink lines) and expanded manifold beyond the scale, is the only base in mammalian chro-
mosomes which is stably modified by methylation at the carbon-5 position (formation of -
Me
CpG-) after replication. DNA methylation inhibits

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-

tioned here are not require for activation of a particular type of gene. Histone decoding, DNA modifications and the accessory factors are
predominantly dependent on the types of signals a cell receives for activation ⁄ repression of a specific gene or for a particular class of gene.
DNA methylation-mediated nucleosome dynamics S. K. Patra and M. Szyf
5220 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS
methylation. For example, methylation of tumor sup-
pressor genes in cancer usually occurs in regions of
DNA associated with H3-histone K27 methylation, a
suppressive histone mark [2,12,22,54]. The histone
methyltransferase EZH2 recruits the DNA methyl-
transferase to targets of EZH2 in the genome [1].
Thus, there is a bilateral relationship between DNA
methylation and inactive chromatin configuration.
DNA demethylation is also tightly associated with
chromatin structure; histone acetylation of H3 and H4
histone tails is a hallmark of active chromatin configu-
ration and transcriptionally active regions of the gen-
ome [22,48,52,53,55,57–59]. Hypomethylation of DNA
[2,11,14] is found in regions associated with hyperacet-
ylated histones, and pharmacological histone acetyla-
tion could induce DNA demethylation [48,79–81].
Thus, DNA demethylation, like DNA hypermethyla-
tion, has a bilateral relationship with chromatin modi-
fication.
Ras oncogenes and oncoproteins
Ras, Rho, Rab, Arf and Ran are the five major classes
of monomeric GTPases whose biological functions are
regulated by the Ras family GTPases. The cellular Ras
oncogene encodes a 21-kDa guanine nucleotide-bind-
ing protein, which plays a role in the regulation of
growth and differentiation in eukaryotic cells [1,82].

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

p21(Waf1) ⁄ Cip1 by Ras, thus overcoming the block on
entry into the S phase of the cell cycle. Cells lacking
p21(Waf1) ⁄ Cip1 activity do not require Rho for the
induction of DNA synthesis by activated Ras [1,83].
Lipid rafts and Ras signaling
Palmitoylation of N-Ras, K-Ras-4A and H-Ras, but
not K-Ras-4B, in their C-terminal hypervariable
regions is commonly required for their membrane relo-
cation. After relocation to the membrane, H-Ras and
K-Ras-4A are translocated to lipid rafts; however,
K-Ras-4B remains in the non-raft portion of the mem-
brane [83]. Hypervariable regions are responsible for
targeting the isoforms to different microdomains in
membrane. Ras proteins localize to different plasma
membrane microdomains, lipid rafts, formed by segre-
gation of lipids based on their dissimilar biophysical
properties [83,91]. A comprehensive model of how Ras
proteins are clustered for amplification, internalized
and transmit their signals has recently been proposed
[1]. Eisenberg et al. [92], employing fluorescence recov-
ery after photobleaching, demonstrated coupling
between membrane domains (rafts) in the external and
internal leaflets of the plasma membrane and showed
that this coupling modulated transbilayer signal trans-
duction.
Ras circulates between the Golgi, the endoplasmic
reticulum and the plasma membranes. H-Ras local-
ized in the membranes of the endoplasmic reticulum
and Golgi apparatus is activated by epidermal growth
factor [1,91–93]. After post-translational modification

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

in the state of methylation of several genes. Constitu-
tive activation of Ras induces DNMT1 expression at
the transcriptional level through activation of cJUN
[100–102]. The excess of unscheduled DNMT levels
would target certain genes for hypermethylation. It is
believed that promoters marked by H3-K27 methyla-
tion are targets of hypermethylation perhaps through
recruitment of DNMTs by EZH2 [103,104]. Indeed,
targeting the Ras signaling pathway by drugs such as
methotrexate and inhibitors of ERK ⁄ mitogen activated
protein kinase (MAPK) decreases DNA methylation in
malignant hematologic diseases and colon cancer cells
indicating a causal relationship between Ras signaling
and DNA methylation [105–108].
Ras activation would affect the DNA-methylation
state and chromatin dynamics in the other direction as
well. Expression of v-H-Ras in mouse embryonal P19
cells resulted in genome-wide demethylation of certain
genes, including a skeletal muscle-specific gene, adrenal
cortex (c21)-specific gene, ubiquitous genes and exo-
genously introduced sequences [100,109]. Hence, DNA
demethylase might be a potential downstream effector
of Ras signaling [1,2]. Also, stimulation of the Ras–
MAPK pathway leads to chromatin modification by
histone H3 serine 10 and 28 phosphorylation in an
acetylation-dependent and -independent fashion [55].
In summary, activation of the Ras-signaling pathway
would trigger the methylation aberrations and histone
modifications that are a hallmark of cancer: regional
DNA hypermethylation and global hypomethylation.

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

FAS-triggered apoptosis is another critical process
which is an effector of Ras signaling and is tightly
associated with epigenetic deregulation. The 36 kDa
cell surface cytokine receptor, FAS (TNFRSF6 ⁄ FAS ⁄
APT1 ⁄ APO1 ⁄ CD95, OMIM 134637) contains a
16-amino acid signal sequence followed by a mature
protein of 319 amino acids that contains a solo trans-
membrane domain and two specialized functional
domains; a FAS death domain and a FAS ligand
(FASL) binding domain [111]. FAS-associated death
domain (FADD, OMIM 602457) protein is the univer-
sal adaptor-protein for apoptosis. This FADD medi-
ates signaling of all known death domain-containing
members of the tumor necrosis factor (TNF) receptor
superfamily [112]. The FADD gene contains two exons
and spans  3.6 kb [113]. Northern blot analysis
revealed that FADD was expressed as a 1.6-kb mRNA
in many fetal and adult tissues [114]. The death
domain of FADD is 25–30% identical to those of
FAS and the TNF receptor, TNFR1 (OMIM 191190).
Natural ligands, cognate agonist antibodies and inter-
actions of FADD with FAS at their respective death
domains trigger apoptosis through FAS and TNFRI.
High expression of FADD in mammalian cells induces
apoptosis, which can be blocked by Crma, a Pox-virus
gene product that also blocks FAS-induced apoptosis
[115,116].
The FAS protein shows structural homology with a
number of cell surface receptors, including TNFR1
and the low-affinity nerve growth factor receptor. It

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

internalization of FAS is blocked, the receptor cannot
induce apoptosis and instead remains fully engaged,
most probably in activating nonapoptotic ⁄ proliferative
pathways [112,115,125,127,131].
Inter-relation of genetic and epigenetic
alterations in cancer
It is now evident, as discussed above, that changes
occurring in cancer cells, including chromosomal
instability, an increased propensity for mutation, acti-
vation of oncogenes, silencing of tumor suppressor
genes and inactivation of DNA repair systems are a
result of both genetic and epigenetic abnormalities.
The correlation between the status of -CpG-island
hypermethylation and ⁄ or mutations in critical genes
shows that, for virtually every tumor type, both gene-
specific hypermethylation and distinct genetic altera-
tions over time are major driving forces in neoplastic
development. But naturally occurring mutations of
specific genes in somatic cells are infrequent, because
under normal conditions maintenance of genomic
integrity is guarded by a complex array of DNA
monitoring and repair enzymes. Karyotypic order is
also guaranteed by other molecular guards, such as
cell-cycle check-points that operate at critical times
during mitotic division. Together, these systems
ensure that mutations are rare events, so rare indeed
that the multiple mutations known to be present in
tumor cells, which are necessary for cancer progres-
sion, are low probability events within a normal
human life span. However, during oncogenesis epige-

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

carcinoma is usually more aggressive than conven-
tional squamous cell carcinoma. It was therefore sug-
gested that somatic mutations in the FAS gene may
contribute to the development and ⁄ or progression of
burn scar-related squamous cell carcinoma. The fol-
lowing mutations in the gene encoding FAS were iden-
tified: a 957A-to-G transition resulting in an N239D
substitution in the FAS death domain; a 547A-to-G
transition resulting in an N102S substitution in the
FAS ligand-binding domain; a 726T-to-C transition
resulting in a C162R substitution in the FAS trans-
membrane domain [133]. Zhang et al. [134] genotyped
1000 Han Chinese lung cancer (211980) patients
and 1270 controls for two functional polymorphisms
in the promoter regions of the FAS and FASL
genes, -1377G-to-A (134637.0021) and -844T-to-C
(134638.0002), respectively. Compared with noncarri-
ers, there was an increased risk of developing lung
cancer for carriers of either the FAS -1377AA or the
FASL -844CC genotype; carriers of both homozygous
genotypes had a more than fourfold increased risk
[134]. Their results further support the concept that
the inactivation of FAS- and FASL-triggered apopto-
sis pathway plays an important role in human carcino-
genesis [91,135].
A heterozygous mutation in the FAS gene in five
unrelated children (134637.0001–134637.0005), with a
rare autoimmune lymphoproliferative (lpr) syndrome
was identified by Fisher et al. [136]. The disease is
characterized by massive nonmalignant lymphadenopa-

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-

gen-specific cytotoxic T cells [140]. There is evidence
for involvement of DNA methylation in silencing of
FAS–FASL signaling and loss of apoptosis [141].
Silencing of FASL and TRAIL-R1, TRAIL-R2 and
Caspase-8 expression by DNA methylation has been
linked to resistance of small cell lung cancer cells to
FASL and TRAIL induced apoptosis [142]. FAS pro-
moter methylation in prostatic and bladder carcinomas
and respective cell lines correlates with downregulation
of FAS expression [143].
H-Ras is linked to the silencing of FAS-triggered
apoptosis through DNA methylation. Peli et al. [144]
reported that oncogenic H-Ras downregulated FAS
by DNA methylation. It was suggested that the
phosphatidylinositol 3-kinase pathway was involved in
mediating this effect of RAS. The involvement of
phosphatidylinositol 3-kinase points to the possibility
that some of the known anti-apoptotic effects of
PKB ⁄ Akt kinase may be mediated, at least in part, by
the downregulation of FAS expression through DNA
DNA methylation-mediated nucleosome dynamics S. K. Patra and M. Szyf
5224 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS
methylation [144]. It remains to be seen whether this
effect of the Ras signaling pathway on DNA methyla-
tion is brought about by increase of DNMT1 as pre-
viously reported [102] or through activation of
factor(s) that recruits DNMT to specific targets such
as apoptosis.
The epigenetic downregulation of apoptosis path-
ways involves additional genes in various types of

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

induction of FAS expression.
What are the biochemical and cellular functions of
other RESEs? Among the 28 RESE proteins discov-
ered using a functional genomics approach there
are transcriptional activators and repressors (CTCF,
EID1, E2F1, RCOR2 and TRIM66 ⁄ TIF1D),
sequence-specific DNA-binding proteins (SOX14,
ZCCHC4 and ZFP345B), histone methyltransferases
(DOT1L, EZH2 and SMYD1), histone deacetylase
(HDAC9), histone chaperones (ASF1A and NPM2),
DNMT1 and several Polycomb group proteins (BMI1,
EED and EZH2). Several recent studies have linked
Polycomb proteins to abnormal DNA methylation and
gene silencing [1,146–148]. It is surprising that one of
the nuclear RESEs is BAZ2A ⁄ TIP5, previously known
to be involved only in repression of RNA polymer-
ase I-directed ribosomal gene transcription [149]. A
number of RESEs were substantially upregulated at
the transcriptional or post-transcriptional level in
K-Ras-transformed NIH3T3 cells compared with non-
transformed NIH3T3 cells, explaining at least in part,
how K-Ras activates this silencing pathway (Fig. 3A).
Treatment of K-Ras NIH3T3 cells with the demethy-
lating drug 5-aza-CdR resulted in FAS re-expression,
supporting the hypothesis that FAS is regulated by
DNA methylation in Ras-transformed cells [98].
Epigenetic inactivation of the RAS
effector homolog RASSF1
Genes encoding human RAS effector homolog,
RASSF1 (OMIM, 605082) family proteins, along with

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

Clustering of raft-associated receptors, like epidermal
growth factor receptor, facilitates the early step of
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 5225
DNA methylation-mediated nucleosome dynamics S. K. Patra and M. Szyf
5226 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS
H-Ras conversion to an activated, GTP-loaded state.
GTP hydrolysis releases the raft-resident H-Ras
[83,90–93] eliciting downstream signals for DNA meth-
ylation-mediated repression of cell-cycle control and
pro-apoptotic genes (for example, FAS) [1–4]. Activa-
tion of the Ras–MAPK pathway stimulates histone
H3, S10 and S28 phosphorylation and nucleosome
remodeling and gene expression ⁄ repression by acetyla-
tion-dependent and -independent mechanisms [165–
168]. The location of the serine 10 residue in close
proximity to other modifiable amino acids in the his-
tone H3 tail suggests a possible interaction between
phosphorylation of serine 10 and methylation and ⁄ or
acetylation of lysine 9 and lysine 14 [165,168]. Visuali-
zation with indirect immunofluorescence shows most
foci of phosphorylated H3 S28 did not co-localize with
foci of H3 phosphorylated on S10 or S10 and K14,
suggesting that these phosphorylation events act inde-
pendently [166,167].
Lipid rafts are not just modulators of the epigenetic
response to chemotherapy [169–175], but also targets
of chemotherapeutic drugs known to block DNA
methylation [27,28]. Radiation therapy also modulates
both the size and composition of lipid rafts

[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

demethylation and expression, or whether radiation ⁄
drug-induced DNA repair-based FAS expression,
post-translation FAS modification and simultaneous
translocation with aSMAase to raft domains cause
changes of lipid raft composition (compare Fig. 3A,B).
One therapeutic implication of the complex relation-
ship among lipid rafts, RAS signaling and epigenetic
modulation is that it might be possible to identify new
strategies to combine inhibitors of Ras-mediated epige-
netic silencing of cell cycle and damage repair genes
and chemotherapeutic agents [179–191]. By under-
standing the pathway leading from membrane lipid
rafts to DNA hypermethylation it would be possible to
dissect points which could be targeted by pharmaco-
logical inhibitors. For example, mouse embryonic stem
cells have been shown to be hypersensitive to apoptosis
triggered by DNA damaging agents due to the high
activity of E2F1-regulated mismatch repair [190,191],
Fig. 3. Epigenetic signaling for proliferation or death emanating from the plasma membrane microdomains. (A) Lipid raft-dependent (H-Ras
and K-Ras-4A) and -independent (K-Ras-4B) signaling. A model scheme proposed for the modulation of transbilayer signaling by clustering of
raft protein H-Ras, in which external clustering (antibody or ligand mediated) enhances the association of internal leaflet proteins with the
stabilized clusters, promoting either enhancement or inhibition of signaling. Clustering raft-associated epidermal growth factor receptor pro-
teins may facilitate the early step, whereby H-Ras is converted to an activated, GTP-loaded state but inhibit the ensuing step of downstream
signaling via the MEK ⁄ ERK pathway. GTP hydrolysis releases the raft-resident H-Ras [83,90–93] which ensures one of the downstream pro-
liferative signals for DNA methylation-associated repression of cell-cycle control and pro-apoptotic genes (e.g. FAS) [1–4]. (B) Radiation- or
drug-induced activation of genes. Chemotherapeutic drugs like aplidine, edelfosin and green tea catechins (for example, EGCG) affect mem-
brane raft composition, on the one hand [91,172–178] and the DNA-methylation status [26–28] of many genes (e.g. FAS and CLU), on the
other hand. EGCG can bind to GpC-rich regions of DNA. Radiation can damage the respective genes, thereby inviting repair enzymes [2].
This facilitates repair-based DNA demethylation [2,47] and the induction of genes for transcription. For exampe, FAS gene is known to be
repressed by DNA methylation, for which a signal is transduced by H-Ras (Fig. 3A) and radiation can induce FAS in association with aSM-

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

cause demethylation and activation of repair genes,
whereas methylation or demethylation would alter
responsivity to chemotherapeutic agents. In contrast to
genetic alteration, epigenetic marks are reversible and
thus the epigenetic consequences of Ras-mediated FAS
modulations could be targeted therapeutically. The sig-
nals for aberrant DNA methylation by H-Ras and
K-Ras 4A and 4B may be transmitted through differ-
ent locations in membranes. We suggest that future
studies with Ras-regulated chromatin dynamics and
DNA modification should focus on the following
issues: (a) dissection of lipid raft-dependent and -inde-
pendent RAS signaling for DNA methylation; (b)
determine the relationship between chromatin activa-
tion and nucleosome opening in response to radiation
and chemotherapeutic drugs, and signals from plasma
membrane microdomains to the nucleus; and (c) search
for gene-specific extinguishers of Ras-triggered aber-
rant DNA demethylation and hypermethylation.
Acknowledgements
This review is dedicated (by SKP) to Dr MK Pal,
retired professor of biochemistry, University of Kaly-
ani and Dr D Chattopadhyay, professor of biochemis-
try, molecular biology and biotechnology, University
of Calcutta, India. Work from the laboratory of MS is
supported by grants from the National Cancer Insti-
tute of Canada and the Canadian Institute of Health
Research. MS is a fellow of the Canadian Institute for
Advanced research. We apologise for many other
important contributions that we have not been able to

include and discuss in this article.
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