Journal of Medicine and Medical Science Vol. 2(3) pp. 696-713, March 2011
Available online @http://www.interesjournals.org/JMMS
Copyright © 2011 International Research Journals
Review
Epigenetics and its role in ageing and cancer
Abhimanyu K. Jha*, Shailesh Kumar*, Mohsen Nikbakht, Vishal Sharma, Jagdeep Kaur§
Department of Biotechnology, Panjab University, Chandigarh, India
*These authors contributed equally to this work
Accepted 21 March 2011
Epigenetics refers to the change in gene expression without the change in the sequence of the gene.
Epigenetics includes alternate phenotypic states that are not based on differences in genotype, but are
generally stably maintained during cell division and are potentially reversible. A much more expanded
view of epigenetics involves multiple mechanisms interacting to collectively establish alternate states
of chromatin structure, histone modification, associated protein composition and transcriptional
activity. Chromatin structure is not fixed. Instead, chromatin is a dynamic identity and is subject to
extensive developmental, environmental and age-associated remodeling. In some cases, this
remodeling appears to counter the ageing and age-associated diseases, such as cancer, and extend
organismal lifespan. Advances in our understanding of chromatin structure, histone modification,
transcriptional activity, promoter hypermethylation and global hypomethylation resulted in an
increasingly integrated and expanded view of epigenetics. The study of epigenetics reveals how
patterns of gene expression are passed from one cell to its descendants, how gene expression
changes during the differentiation of one cell type into another, and how environmental factors can
change the way genes are expressed. There are far-reaching implications of epigenetic research for
human biology and diseases, including our understanding of cancer and ageing. Due to these
developments, epigenetic therapy is expanding to include combinations of histone deacetylase
inhibitors and DNA methyltransferase inhibitors. This review encompasses the different types of
epigenetic changes, the interplay among them and the implications of these epigenetic changes in
relation to cancer and ageing. Besides this, the development and perspective of epigenetic therapy
has also been discussed in brief.
Keywords: Epigenetics, histone deacetylase inhibitors, DNA methyltransferase inhibitors, cancer, histone
modification, promoter hypermethylation.
INTRODUCTION
The central dogma of molecular biology states that the
information embedded in the linear nucleotide sequence
of DNA contains coding information for RNA and protein,
as well as regulatory sequences that control the biology
of DNA itself. The word "epigenetics" was coined by the
developmental biologist C. H. Waddington in 1942
(Waddington et al., 1942) The Greek prefix "epi-" in the
word "epigenetics" implies features that are "on top of" or
"in addition to" genetics. Waddington proposed the
epigenetic model to describe, the interaction of genes
within a multicellular organism with their surroundings to
produce a particular phenotype and this hypothesis was
known as Conrad hypothesis. Holliday and Pugh
proposed in 1975 that covalent chemical DNA
§
Corresponding author Email: jagsekhon@yahoo.com
modifications, including methylation of cytosine-guanine
(CpG) dinucleotides, were the molecular mechanisms
behind Conrad’s hypothesis. Because all cells within an
organism inherit the same DNA sequences, the process
of cellular differentiation depend on pattern of epigenetic
rather than genetic inheritance. Robin Holliday defined
epigenetics as "the study of the mechanisms of temporal
and spatial control of gene activity during the
development of complex organisms" (Holliday et
al.,1996). Thus, the word "epigenetic" can be used to
describe any aspect other than DNA sequence that
influences the development of an organism. The modern
usage of the word "epigenetics" usually
refers to
heritable traits that do not involve changes to the
underlying DNA sequence.
Epigenetic regulation mediates adaptation to the
environment by the genome lending plasticity that
Jha et al. 697
translates into the presenting phenotype, particularly
under “mismatched” environmental conditions (Godfrey
et
al., 2007). Epigenetic inheritance involves the
transmission of information not encoded in DNA
sequences from cell to daughter cell or from generation
to generation. Covalent modifications of the DNA or its
packaging histones are responsible for transmitting
epigenetic information. Epigenetic modifications, such as
acetylation, phosphorylation, methylation, ubiquitination,
and ADP ribosylation, of the highly conserved core
histones, H2A, H2B, H3, and H4, influence the genetic
potential of DNA. The enormous regulatory potential of
histone modification is illustrated in the vast array of
epigenetic markers found throughout the genome. In
addition, the modification of histones can cause a region
of chromatin to undergo nuclear compartmentalization
and, as such, specific epigenetic markers are
nonrandomly distributed within interphase nuclei (Andrulis
et al., 1998). But it can also be an important determinant
of cellular senescence and organism ageing (Wilson et
al.,1983; Issa 2003; Fraga et
al., 2005 ). Now
epigenetics in broad sense has changed to epigenomics
that include the higher order of chromosome
organization, Nucleosome formation and modification of
histone tail like methylation, acetylation, phosphorylation
and DNA methylation. Epigenomics is defined as a
genome-wide approach to study epigenetics. This term
encompasses whole-genome studies of epigenetic
processes and the identification of the DNA sequences
that specify where the epigenetic processes are
targeted.The central goal of epigenomics is to define the
DNA sequence features that in turn direct epigenetic
processes.
Possible mechanisms of epigenetic inheritance
The best understood sequence-independent inheritance
mechanism is that of DNA methylation, in which the
maintenance methyltransferase DNMT1 specifically
recognizes semi-methylated DNA and methylates the
remaining strand. About 4% of the cytosines are usually
methylated in mammalian genomic DNA and it has been
shown that this methylation is essential for mouse
development (Li et al., 1992). In addition, it has also been
shown that DNA methylation plays an essential role in
several epigenetic phenomena, including genomic
imprinting, X-chromosome inactivation, and retro-element
silencing.
DNA methylation is known to interplay with other
chromatin marks, such as histone modifications (Jones,
1998). Accurate transmission of the histone code through
cell generations presents a paradox, because
nucleosomes are not deposited in a semi-conservative
manner during replication. Rather, ‘old’ histones are
distributed randomly between the DNA molecules and the
‘gaps’ are filled with freshly synthesized (unmodified)
histones, leading to a dilution of chromatin marks. It has
been suggested that the chromatin code can then be
reinstated by chromatin modifiers that are recruited to the
remaining marks (Henikoff et al., 2004). It has also been
proposed that the timing of locus replication might have a
role in the maintenance of epigenetic states (Zhang et al.,
2002). Nucleosome disruption and replacement are
crucial activities that maintain epigenomes, but these
highly dynamic processes have been difficult to study. A
recent study described a direct method for measuring
nucleosome turnover dynamics genome-wide. It
concluded that nucleosome turnover is most rapid over
active gene bodies, epigenetic regulatory elements, and
replication origins in Drosophila cells. Nucleosomes
turnover is faster at sites for trithorax-group than
polycomb-group protein binding, suggesting that
nucleosome turnover differences underlie their opposing
activities and challenging models for epigenetic
inheritance that rely on stability of histone marks. The
results
establish a general strategy for studying
nucleosome dynamics and uncover nucleosome turnover
differences across the genome that are likely to have
functional importance for epigenome maintenance, gene
regulation, and control of DNA replication (Deal et al.,
2010). This is in contrast to another study which has
clearly observed that silent histone modifications within
large heterochromatic regions are maintained by copying
modifications from neighboring preexisting histones
without the need for H3-H4 splitting events (Xu et al.,
2010). Semiconservative DNA replication ensures the
faithful duplication of genetic information during cell
divisions. However, how epigenetic information carried by
histone modifications propagates through mitotic
divisions remains elusive. To address this question, the
DNA replication dependent nucleosome partition pattern
should be clarified.
Epigenetic mechanisms play a fundamental role in the
interpretation of genetic information (Li et al., 2002).
Depending on its particular epigenetic modification
pattern, a gene can be expressed or silenced. Epigenetic
modifications thus represent an integral mechanism for
the control of complex gene expression patterns. One
prominent example for an epigenetic control mechanism
is the covalent modification of histones (Jenuwein et al.,
2001). Epigenetic processes are natural and essential to
many organism functions. Till now many types of
epigenetic processes have been identified which include
histone modifications, like acetylation, phosphorylation,
ubiquitination and methylation and DNA methylation. The
main types of epigenetic changes have been mentioned
in Figure 1.
The epigenome and histone modifications
The work of Kornberg in 1974 proposed that chromatin is
a repeating unit of 8-histone proteins and 200 bps of DNA
wrapped around it. Later on the Nucleosome model
698 J. Med. Med. Sci.
became the center of all molecular biology research to
understand the script of life. Nucleosome is made up of
four kinds of basic proteins –H1, H2A, H2B, H3 and H4.
The amino acid sequencing of H4 reveals that this protein
is highly conserved among species. The precise
organization of chromatin is critical for many cellular
processes, including transcription, replication, repair,
recombination and chromosome segregation. Dynamic
changes in chromatin structure are directly influenced by
post-translational modifications of the amino-terminal tails
of the histones (Luger et al., 1998). Packaging of DNA
sequences of promoters region in nucleosomes prevents
the initiation of transcription by bacterial and eukaryotic
RNA polymerases in vitro (Knezetic et al.,1986,
Lorch,1987). Nucleosomes exert a similar inhibitory effect
upon transcription in vivo: turning off histone synthesis by
genetic means in yeast, and consequent nucleosome
loss, turns on transcription of all previously inactive genes
tested (Han et al., 1988).
The four core histones are present in equal amounts in
the cell, whereas H1 is half as abundant as the other
histones. This is in consistency with the finding that only
one molecule of H1 is associated with each nucleosome
(which contains two copies of each core histone). As they
are closely associated with the negatively-charged DNA
molecule, the histones have a high content of positivelycharged amino acids. It has been observed that greater
than 20% of the residues in each histone are either lysine
or
arginine.
Histone
lysine
methyltransferase
G9a/KMT1C mediates H1.4K26 mono- and dimethylation
in vitro and in vivo and thereby provides a recognition
surface for the chromatin-binding proteins HP1 and
L3MBTL1 (Trojer et al., 2009). Histone proteins are
subjected to four major kinds of posttranslational
modifications like methylation of arginine and lysine
residues, phosphorylation of serine, lysine acetylation
and lysine ubiquitination (Kouzarids, 2007). The types of
histone modifications have been shown in Table no.1.
Different types of histone modifications at different sites
are shown in Figure 2.
Histone Methylation
Histone methylation was first discovered more than thirty
five years ago but very little was known about the impact
of this biological phenomenon. The initial study was
stressed on the pattern of methylation and its
maintenance and relation with the kind of histone. It has
been revealed that methylation of specific lysine residues
in histone tails function as a stable epigenetic mark that
directs particular biological functions, ranging from
transcriptional regulation to heterochromatin assembly .
There are three different types of methylation forms
which occurs in lysine (me1,me2 and me3). There are
three modifications, K9me3, K20me3 and K27me3, which
are associated with repressed chromatin in many
organisms. High-resolution ChIP seq in different
mammalian cells gives a genome-wide snapshot of the
modifications and reveals distinct patterns that reflect
chromosome organization (Barski, 2007; Mikkelsen
2007).
Their interaction with different histone binding proteins
play a key role in transducing the pattern of modifications
into a functional outcomes (Taverna et al., 2007 ).
Previous studies showed that lysines 4, 9, 27 and 36 of
H3 and lysine 20 in H4 can be mono, di or trimethylated
(Van Holde, 1989). Histone lysine methylation appears to
be a rather static process, and consequently it is
generally viewed as an epigenetic mark rather than as a
flexible regulatory signal (Jenuwein, 2001). In addition to
lysines, certain arginines in histones H3 and H4 can be
methylated (Davie et al., 2002) and this methylation can
be correlated to gene activation (Bauer et. al., 2002).
Several other histone methyltransferases (HMTs) have
been characterized (Zhang et al., 2001) and it has
become evident that specific methylation patterns
correlate with gene activity. H3-K9 methylation has been
observed to be primarily associated with heterochromatin
(Noma et al., 2001; Lachner et al., 2001) whereas H3-K4
methylation (in higher eukaryotes) is observed in
transcriptionally active regions(Noma et al., 2001; Strahl
et al.,1999).
Histone proteins assemble into nucleosomes, which
function as DNA packaging units as well as
transcriptional regulators. Methylation at lysine 9 (Lys-9)
on histone H3 has recently been shown to be a marker of
heterochromatin from yeast to mouse(Jenuweinet et
al.,2002; Schneider et al., 2002). Lys-9 methylation is
recognized by heterochromatin-associated proteins, such
as HP-1, and is required to maintain heterochromatin.
Studies have shown that modifications of histone H3 also
contribute to euchromatin gene silencing by switching
between Lys-9 acetylation and methylation (Taverna et
al., 2007). Another histone modification, methylation of
histone H3 Lys-4, localizes to sites of active transcription,
and this modification may stimulate transcription. These
different combinations of histone modifications at different
residues may act synergistically or antagonistically to
alter gene expression (Roh et al., 2006).
H3 Lys-9 methylation is also closely related to DNA
methylation and acts as an epigenetic mark of silencing
in the tumor suppressor genes. Recent reports show that
H3 Lys-9 methylation can be regulated by Suv39h-HP1independent pathways and occurs in facultative
heterochromatin on the inactive X chromosome (Boggs et
al. 2002). Methylation of histone H4 at lysine 20 (K20)
has been implicated in transcriptional activation, gene
silencing, heterochromatin formation, mitosis, and DNA
repair. Even in newly synthesized histones, 90% of
histone get methylated in 2 to 3 cell cycles and it can be
concluded that it is required for normal mitosis and cell
cycle progression, K20 methylation proceeds normally
even in the colchicines treated cells (Pesavento et al.,
Jha et al. 699
Table 1. Modifications of histones
Modification
Acetylation
ADP-ribosylation
Histone (s) modified
H3, H4
Core histone
Methylation
Phosphorylation
H3, H4
H1
H3
Ubiquitination
H2A, H4
H2A and H2B
Effects/function
Activation of gene
Local
disruption
of
chromatin
structure to facilitate DNA repair
Repression of transcription
Chromatin condensation
Gene
activation,
chromatin
condensation,
Nucleosome assembly
Description of chromatin structure to
facilitate transcription
Figure 1. Main types of epigenetic changes
2008).
Histone modification maps as ageing marks: Histone
modifications also have a defined profile during ageing
and cell transformation. For example, the trimethylation of
H4-K20, which is enriched in differentiated cells (Biron et
al., 2004) increases with age (Prokocimer et al., 2006)
and is commonly reduced in cancer cells. The increase of
trimethylated H4-K20 in aged-like cells has been
associated with defects in the nuclear lamina (Shumaker
et al., 2006).
Histone Acetylation
Acetylation neutralises the positive charge on the amino
group of lysine and acts as a binding site for proteins
containing a bromodomain (Guenther et al., 2007). There
is a direct link between histone acetylation and active
chromatin. It has been demonstrated directly that
transcriptionally active genes carry acetylated core
histones (Hebbes et al., 1988).
In human T cells, there are more than 45,000
acetylation islands rich in H3K9acK14ac, many of which
correspond to transcriptional regulatory elements,
particularly enhancers and promoters of active genes
(Brownell et al., 1996; Kuo et al.,1998). Acetylation of
lysine residues on histones H3 and H4 leads to the
formation of an open chromatin structure. Histone H4
acetylation distinguishes coding regions of the human
genome from heterochromatin in a differentiationdependent but transcription independent manner. It has
been concluded that nucleosomes containing acetylated
H4 are scattered infrequently and possibly randomly
through coding and adjacent regions and are essentially
absent from heterochromatin. Induction of differentiation
of HL-60 cells by exposure to dimethylsulfoxide or 12-otetradecanoylphorbol 13-acetate (TPA) did not alter the
level of H4 acetylation within either the c-MYC or c-FOS
genes or other coding regions, but did induce a transient
increase in H4 acetylation within centric heterochromatin
(Fukuda et al., 2006; O'Neill et al., 1996). Histone
acetylation and phosphorylation are highly dynamic
700 J. Med. Med. Sci.
Figure 2. Different types of histone modifications at different sites.
processes with rapid turn-over rates. The
connection between acetylation and transcription
was established by the demonstration that yeast
Gcn5 protein, a positive transcriptional regulator
of many genes, has HAT activity (Parekh et al.,
1999) and stimulation of transcription by Gcn5
requires the HAT activity (Grunstein, 1997).
Histone acetylation by promoter-associated
transcription factors is localized. For example,
increased acetylation of H3 and H4, attributed to
p300/CBP, was found upon viral infection in two to
three nucleosomes surrounding the interferon-b
promoter (Struhl, 1998). Histone acetylation also
plays an important role in the activity-dependent
regulation of sulfiredoxin and sestrin 2 which are
neuroprotective antioxidant enzymes that reduce
hydroperoxides and protect neurons against
oxidative stress (Soriano et al., 2009).
Jha et al. 701
Histone Deacetylation
The connection between acetylation and transcription is
further shown by the fact that deacetylation can cause
repression. It
has been discovered that many
coactivators are HATs , proteins originally identified as
corepressors which have now been shown to possess
deacetylase activity (Grozinger et al., 1999 ; SantosRosa et al., 2005).
The deacetylation–repression connection was most
clearly demonstrated by the isolation of a human histone
deacetylase, HDAC1, whose sequence was highly similar
to that of a yeast negative regulatory protein Rpd3. Many
additional deacetylases have been identified in yeast and
human cells (Nowak et al., 2002).
Histone acetylation tends to open up chromatin
structure. Accordingly, histone acetyltransferase (HATs)
tend to be transcriptional activators whereas histone
deacetylases (HDACs) tend to be repressors. Many HAT
genes are altered in some way in a variety of cancers
(Cheung et al., 2000). For instance, the p300 HAT gene
is mutated in a number of gastrointestinal tumours
(Swarthout et al., 2009). On the other hand, alteration of
HDAC genes in cancer seems to be far less common.
However, despite of low incidence of genetic mutation in
cancer, HDAC inhibitors are performing well in the clinic
as anti-cancer drugs. Recent studies have shown that
HDAC
inhibition
lead
to
Ubiquitin-Dependent
Proteasomal Degradation of DNA Methyltransferase 1 in
Human Breast Cancer Cells (Zhou et al., 2008).
Histone Phosphorylation
H1 histones play an important role in regulating higher
order structure of chromatin and are potential regulators
of gene expression. Phosphorylation of H1 on serine and
threonine in their amino and carboxyl terminal tails occurs
in vivo and alters their interaction with DNA. H1
phosphorylation destabilizes higher order chromatin
structure which is thought to allow accessory factors to
participate in replication, mitotic condensation, and gene
activation. For instance, it has been observed that
histone H3 thr 45 phosphorylation is a replicationassociated post-translational modification in S. cerevisiae
(Baker et al., 2010). Quiescent fibroblasts treated with
epidermal growth factor undergo rapid serine 10
phosphorylation which is coincident with the induction of
early response genes such as c-FOS. This
phosphorylation is catalyzed by the Rsk-2 kinase. The
mechanism by which phosphorylation contributes to
transcriptional activation is not well understood. The
addition of negatively charged phosphate groups to
histone tails neutralizes their basic charge and is
considered to reduce their affinity for DNA. Furthermore,
it has been observed that several acetyltransferases
increased HAT activity on serine 10-phosphorylated
substrates and mutation of serine 10 decreases
activation of mGcn5-regulated genes (Clayton et al.,
2000 ; Cheung et al., 2000). Thus, phosphorylation may
contribute to transcriptional activation through the
stimulation of HAT activity on the same histone tail.
Indeed, phosphoacetylation of histone H3 on c-fos and cjun associated nucleosomes has been demonstrated
upon gene activation (De Souza et al., 2000).
Phosphorylation of H2A has also long been correlated
with mitotic chromosome condensation, and in this case
also serine 10 appears to play an important role. For
instance, mutation of serine 10 Tetrahymena histones
causes abnormal chromosomal condensation and
defective chromosome separation during anaphase (Hsu
et al., 2000).
Phosphorylation of histone H3 is also known to occur
after activation of DNA-damage signaling pathways. A
conserved motif (ASQE, in the single-letter amino-acid
code) found in the carboxyl terminus of yeast H2A and
the mammalian H2A variant H2A.X is rapidly
phosphorylated upon exposure to DNA-damaging agents
(Downs et al., 2000; Rogakou et al., 1999)
Serine 139 has been identified as the site for this
modification, and its phosphorylation in response to
damage is dependent on the phosphatidylinositol-3-OH
kinase Mec1 in yeast. Mec1 dependent serine 139
phosphorylation is apparently required for efficient nonhomologous end-joining repair of DNA. This suggests
that phosphorylation mediates an alteration of chromatin
structure, which in turn facilitates repair. H3S10 and
H3S28 are phosphorylated at mitosis which is a crucial
part of the cell cycle. Any misregulation here is often
associated with cancers. Indeed, the Aurora kinases that
perform this H3 phosphorylation are implicated in cancer
(Zhang et al., 2005).
Histone Ubiquitination
Modification of the N- and C-terminal tails of histones is
thought to occur in patterns, which recruits specific
effector proteins that alter chromatin structure and
regulate gene expression. The addition and removal of
histone modifications are thought to have antagonistic
effects. Cul4–Ddb1, a Ring H2 ubiquitin ligase, plays an
important role in many vital cellular processes including
DNA replication, DNA repair and transcription (Saha et
al., 2006)
Of the four core histones, H2A and H2B have long
been known to be modified by ubiquitin conjugation.
Ubiquitination of histone H2B (uH2B) on lysine 120
(K120) in humans (Becker et al., 2002) and lysine 123 in
yeast(O’Connel et al., 2007) has been correlated with
increase in methylation of lysine 79 (K79) of histone H3
by K79-specific methyltransferase. Regulators of H2A
and H2B ubiquitylation play roles in gene silencing (ubH2A) or in transcription initiation and elongation (ub-H2B)
702 J. Med. Med. Sci.
(Oselu et al., 2006).
In addition to participating in the cellular response to
DNA damage, CUL4-mediated histone ubiquitination may
regulate other aspects of chromatin function, including
heterochromatin silencing (West et al., 1980). It has been
proposed that uH2B may induce H3 K79 methylation
directly, either by altering chromatin structure and
therefore nucleosomal accessibility, or through the
recruitment of enzymatic function (Robzyk et al., 2000).
Compared with other histone modifications, ubiquitination
involves the addition of a relatively large molecule that is
two-thirds the mass of an individual histone. Due to the
large size of this molecule, it has been proposed that
ubiquitination should have an impact on chromatin
structure. Chromatin fibers reconstituted with ubH2A
molecules have similar properties to the control
chromatin with regard to folding and sedimentation
(Wang et al., 2006).
Sumoylation
Small ubiquitin-related modifier (SUMO) is the best
characterized member of a growing family of ubiquitin-like
proteins involved in posttranslational modifications. In
mammals, there are three members of the SUMO protein
family: SUMO-1, SUMO-2 (SMT3a), and SUMO-3
(SMT3b), which are implicated in partly overlapping, yet
distinct functions (Sun et al., 2002). SUMO is covalently
attached to other proteins through the activities of an
enzyme cascade (E1–E2–E3) similar to that for
ubiquitination. There is only one known SUMO-activating
enzyme, E1 (a heterodimer of SAE1 and SAE2) and only
one known SUMO-conjugating enzyme, E2 (UBC9).
It has been proposed that histone sumoylation acts as
a component of the group of modifications that appear to
govern chromatin structure and function to regulate
transcriptional repression and gene silencing (Jason et
al., 2002). In Drosophila polytene chromosomes, the
SUMO moiety was detected in many euchromatic sites
and the chromocenter (Tatham et al., 2001) suggesting
that histone (chromatin) sumoylation plays a role in both
euchromatic
transcriptional
repression
and
heterochromatic gene silencing. It is reported that
pontin, a component of chromatin-remodeling complexes,
is SUMO-modified, and that SUMOylation of pontin is an
active control mechanism for the transcriptional
regulation of pontin on androgen-receptor target genes in
prostate cancer cells (Kim et al., 2007).
It has been hypothesized that SUMOylation is involved
in the regulation of p53 in both protein stability and
function by direct modification of p53 or in an indirect way
via modulating the stability of MDM2. It has long been
identified that p53 underwent SUMOylation on lysine 386
that increase transcriptional activity of p53 (Schreiber et
al.,2002) and a necessary factor for full apoptotic activity
of p53 (Lehembre et al., 2000).
As SUMO E2, Ubc9 are only conjugating enzyme in
SUMOylation so correlated with their involvement in
cancer development and tumorigenesis. The increased
expression of Ubc9 has been reported in several human
ovarian cancer cell lines such as PA-1 and OVCAR-8 as
well as in ovarian tumor tissues (Gostissa et al., 1999)
human lung adenocarcinomas (Muller et al., 2000) and
metastastic prostate cancer cell line, LNCaP
(Mo et al., 2005).
Chromatin remodeling
Chromatin represents an important regulatory entity that
provides a means of maintaining genome stability (e.g. by
suppressing uncontrolled recombination or transposon
mobilization) and allows the integration of multiple
endogenous and exogenous signals at the single-gene
level. Although a wide variety of proteins cooperate in
reorganizing chromatin structure in response to these
signals, the basic mechanisms seem to involve the
covalent modification of histone tails and changes of
nucleosome positioning that utilize ATP. Most of the key
regulator complexes identified to date contain either one
or both of these activities. These include not only protein
complexes that are involved in the short-term regulation
of gene activity but also components that affect long-term
regulation and epigenetic inheritance, such as the Sir2
family of HDACs and various members of the Polycomb
and Trithorax groups of proteins. Recent studies show
that TF2I is involved in chromatin remodeling during
embryonic stem cell differentiation but yet the exact role
of these factors are yet to be revealed (Aleksandr et al.,
2009). Dynamic chromatin remodeling utilizes several
basic
mechanisms,
including
covalent
histone
modifications, ATP-dependent chromatin remodeling,
utilization of histone variants and DNA methylation to
alter the accessibility of DNA. These mechanisms work
either independently or in tandem to allow optimal
chromatin remodeling for efficient transcriptional
regulation, DNA replication and DNA-damage repair.
ATP-dependent chromatin-remodeling enzymes and
their functions
ATP-dependent chromatin-remodeling enzymes, which
are highly conserved in organisms from yeast to humans,
are similar to the SNF2 (sucrose non-fermenting 2) family
of DNA translocases and all of them contain a catalytic
ATPase subunit (Doniels et al., 2002). These ATPase
machineries utilize the energy of ATP hydrolysis to
mobilize nucleosomes along DNA, evict histones off DNA
or promote the exchange of histone variants, which in
turn modulate DNA accessibility and alter nucleosomal
structures (Kim et. al., 2006). Based on distinct domain
structures, there are four well-characterized families of
Jha et al. 703
mammalian chromatin remodeling ATPases: (a)
SWI/SNF (switching defective/ sucrose non-fermenting)
family: SWI/SNF remodeling complexes primarily
disorganize and reorganize nucleosome positioning to
promote accessibility for transcription-factor binding and
gene activation. However, they also promote
transcriptional- repressor binding and gene repression
under certain conditions (Martens et al., 2003) (b) ISWI
(imitation SWI) family: The ISWI remodeling complexes
primarily organize and order nucleosome positioning to
induce repression (Ooi et al., 2006) although they also
mediate transcriptional activation and transcriptional
elongation (Corona et al., 2004 ; Badenhorst et al., 2002;
Morillon et. al., 2003) (c) NuRD (nucleosome remodeling
and deacetylation)/ Mi-2/CHD (chromodomain, helicase,
DNA binding) family NuRD/Mi-2/CHD remodeling
complexes primarily mediate transcriptional repression in
the nucleus (Shimono et al., 2005). However, they are
also involved in transcriptional activation of rRNA in the
nucleolus. (d) INO80 (inositol requiring 80) family (Bao et.
al., 2007). The INO80 remodeling complexes appear to
have both activating and repressive effects for a specific
set of genes (Jonsson et al., 2004 ; Hassan et al., 2002).
Both members of the SWI/SNF family of ATPases has
BRM and BRG1 (BRM/SWI2-related gene 1), contain a
C-terminal bromodomain that binds to acetylated histone
tails (Hassan et al., 2002). ISWI family members, SNF2H
and SNF2L, have a SANT (SWI3, ADA2, NCOR and
TFIIIB’ DNA-binding domains) and a SLIDE (SANT-like
ISWI) domain that mediate interaction with unmodified
histone tails and linker DNA (Boyer et al., 2004).
NuRD/Mi-2/CHD family members, CHDs 1–5, have
unique tandem chromodomains that specifically
recognize methylated histone tails (Flanagan et al.,
2005). Different chromatin remodeling complexes have
been mentioned in Table 2 (taken and modified after
getting permission from the author and the publisher)
(Kornberg et al., 1999).
Besides HAT activities, ATP-dependent chromatin
remodeling complexes have also been recently
implicated in DNA repair processes. Demonstration was
made a few years ago that Cockayne syndrome B protein
(CSB) was required for coupling NER to transcription.
CSB, a DNA-dependent ATPase of the SWI2/SNF2
family, has been shown to remodel chromatin substrates
in vitro (Citterio et al., 2000). It has also been concluded
that mammalian SWI/SNF complexes prevent DNA
damage-induced apoptosis in part by facilitating efficient
repair and thereby ensuring timely elimination of
unrepaired DSBs that could otherwise lead to excessive
prolongation of p53 activation (Park et al., 2009). It has
been found that ATP dependent chromatin remodeling is
highly affected by Inositol Polyphosphates. It has also
been demonstrated that mutations in genes encoding
inositol polyphosphate kinases that produce IP4, IP5, and
IP6 impair transcription in vivo. These results provide a
link between inositol polyphosphates, chromatin
remodeling and gene expression (Shen et al., 2003).
According to a recent study, structural studies of the RSC
chromatin-remodeling complex prompts a proposal for
the remodeling mechanism: RSC binding to the
nucleosome releases the DNA from the histone surface
and initiates DNA translocation (through one or a small
number of DNA base pairs); ATP binding completes
translocation and ATP hydrolysis resets the system.
Binding energy thus plays a central role in the remodeling
process. RSC may disrupt histone-DNA contacts by
affecting histone octamer conformation and through
extensive interaction with the DNA. Bulging of the DNA
from the octamer surface is possible, and twisting is
unavoidable, but neither is the basis of remodeling (Lorch
et al., 2010).
DNA Methylation
DNA methylation occurs in bacteria, fungi, plants and
animals, however its role varies widely among different
organisms. DNA methylation might have evolved to
protect bacterial genomes from invasion by foreign DNA.
Thus, bacteria have devised a way to distinguish their
own DNA from that of an invader; in the bacterial genome
the sequence contains the methylated base, whereas in
foreign DNA the same sequence is unmethylated and
therefore digested by the restriction endonuclease (Sims
et al., 2005). DNA methylation in mammalian cells
occurs at the 5-position of cytosine within the CpG
dinucleotide. CpG islands are the sites present in the
promoter of most of the tumor suppressor genes, and
hypermethylation at CpG island leads to silencing of the
expression of these genes. The CpG islands have the
following important characteristics: (i) G+C content of
0.50 or greater
(ii) observed to expected CpG
dinucleotide ratio of 0.60 or greater and (iii) both
occurring within a sequence window of 200 bp or greater.
CpG dinucleotides methylation in mammals represent
the target for the covalent modification of DNA (Bao et
al., 2007). This methyl group protrudes from the cytosine
nucleotide into the major groove of the DNA and it
displaces transcription factors that normally bind to the
DNA (Kumar et al., 1994 ; Kim et al., 2003). Cell-typespecific
cytosine
methylation
and
histone-tail
modifications could contribute to the differences in gene
expression patterns between cell types. CpG island
definition based on sequence composition identifies
these elements at the promoter sites of approximately
half of the genes in the human genome (Ioshikhes et al.,
2000) most of which are expressed in most or all tissues,
hence they have been designed as ‘housekeeping’
genes. The dynamic nature of cytosine methylation
becomes especially evident during tumorigenesis in
which methylation is decreased genome-wide, whereas
the CpG islands at promoters of tumour-suppressor
genes acquire methylation, which leads to their silencing
704 J. Med. Med. Sci.
and subsequent tumour progression. Hypermethylation is
also linked to chromosomal instability, a common
phenomenon in human tumours (Lengauer et al., 1998)
which has been observed in mice with hypomethylated
genomes due to engineered methyltransferase
deficiencies (Gaudet et al., 2003). Although CpG islands
account for only about 1% of the genome and for 15% of
the total genomic CpG sites, these regions contain over
50% of the unmethylated CpG dinucleotides. There are
about 45,000 CpG islands, most of which reside within or
near the promoters or first exons of genes and are
unmethylated in normal cells, with the exception of CpG
islands on the inactive X chromosome in females (Arber
et al., 1969).
CpG sites have been shown to act as hot-spots for
germline mutations, contributing to 30% of all point
mutations in the germ line and for acquired somatic
mutations that lead to cancer. For example, methylated
CpG sites in the TP53 coding region contribute to as
many as 50% of all inactivating mutations in colon cancer
and 25% of cancers (Hendrich et al., 1999).
Cellular DNA methylation patterns is established by a
complex interplay of at least three independent DNA
methyltransferases: DNMT1, DNMT3A and DNMT3B.
The first methyltransferase to be discovered was DNMT1.
Pioneering work has established that DNMT1 has a 10–
40-fold preference for hemimethylated DNA (Pradhan et
al.,1999; Pradhan et al., 1997). By providing both
enhanced transcriptional control and protection against
mutation, the methyl-CpG binding proteins could have
facilitated the expansion of the methylated DNA
compartment within the evolving vertebrate genome.
MBD2 and MBD3 are the only vertebrate methyl-CpG
binding proteins and in mammals, MBD2 and MBD3
genes have an identical genomic structure, differing only
in the sizes of their introns, and they encode proteins that
are 70% identical (Baylin et al., 2002).
The DNMT protein motif is evolutionarily ancient,
occurring in all known DNA methyltransferases from
bacteria to plants and humans (Baylin et al., 2000). The
animals, in which DNA methylation is predominantly
associated with transcriptional repression, the presence
or absence of DNA methylation and of the DNMTs varies,
as does the apparent use of DNA methylation within
animal genomes (Bird et al., 2002).
CpG islands are associated with at least half of all
cellular genes and are normally methylation-free. Dense
methylation of cytosine residues within islands results in
strong and heritable transcriptional silencing. Such
silencing normally occurs almost solely at genes subject
to genomic imprinting or to X chromosome inactivation.
Aberrant methylation of CpG islands associated with
tumor suppressor genes has been proposed to contribute
to carcinogenesis (Antequera et al., 1993). In addition to
carcinogensis and genomic imprinting, DNA methylation
has also been found to regulate memory formation and
synaptic plasticity in the adult rat hippocampus (Miller
et al., 2008). The understanding of chromatin with
respect to the components that specify for states of gene
expression is growing rapidly, and this knowledge is
establishing a base from which abnormal as well as
normal gene expression events can be understood. In
this regard, an especially active field in cancer research
is concerned with patterns of aberrant gene promoter
hypermethylation that have been associated with loss of
transcription of a growing list of genes in virtually every
type of human cancer (Greenblatt et al., 1994).
Several mechanisms have been proposed to account
for transcriptional repression by DNA methylation. The
first mechanism involves direct interference with the
binding of specific transcription factors to their recognition
sites in their respective promoters. Several transcription
factors, including AP-2, c-Myc/Myn, the cyclic AMPdependent activator CREB, E2F and NFkB, recognize
sequences that contain CpG residues, and binding of
each has been shown to be inhibited by methylation
(Baylin et al., 1998). The second mode of repression
involves a direct binding of specific transcriptional
repressors to methylated DNA. Hypomethylation is the
second kind of methylation defect that is observed in a
wide variety of malignancies (Jones et al., 1999). It is
common in solid tumors such as metastatic
hepatocellular cancer, cervical cancer, prostate tumors,
and also in hematologic malignancies such as B-cell
chronic
lymphocytic
leukemia.
The
global
hypomethylation seen in a number of cancers, such as
breast, cervical, and brain, show a progressive increase
with the grade of malignancy (Kim et al., 1994). A
mutation of DNMT3b has been found in patients with
immunodeficiency, centromeric instability, and facial
abnormalities, which causes the instability of the
chromatin (Okano et. al., 1999). Hypomethylation has
been hypothesized to contribute to oncogenesis by
activation of oncogenes such as cMYC and H-RAS or by
activation of latent retrotransposons (Alves et al., 1996)
or by chromosome instability (Tuck-Muller et al., 2000).
Much attention in the methylation field has focussed on
CpG islands, primarily because of the propensity of such
sequences to become aberrantly hypermethylated in
tumours, resulting in the transcriptional silencing of the
associated gene (Kochanek et al., 1995; Jones et al.,
1998).
Tumor cells have less methylation than normal cells,
and this loss appears to occur primarily from parasitic
and repetitive DNAs, which are usually heavily
methylated.The connection between CpG methylation
and transcriptional silencing in vertebrates has been
recognized for more than two decades (Tate et al., 1993).
The local cytosine methylation of a particular sequence
could directly interfere with transcription-factor binding
(Wade et al., 1999). Chromatin assembly facilitates the
repression of methylated DNA. Methyl-CpG binding
proteins, including MECP2, associate with co-repressor
complexes that include histone deacetylases (Nan et al.,
Jha et al. 705
1998).
Two important additional links between DNA
methylation and chromatin structure have recently come
to light. First, DNMT1 forms a complex with Rb, E2F1,
and HDAC1 and represses transcription from E2F
responsive promoters (Robertson et al., 2000). The
second link between chromatin structure and methylation
comes from patients with mutations in a putative ATPdependent chromatin-remodelling factor of the SNF2
family, termed ATR-X (Gibbons et al., 2000).
a lower percentage of methylated CpG than other
vertebrates (20%), shares the same global methylation
distribution and has a similar density of methylation
(Tweedie et al., 1997). This is significant because
methylation density is a factor in methylation dependent
silencing. In all the substantially methylated invertebrates
tested the distribution of methylated bases is quite
different from that of vertebrates. Rather than global
distribution with increased distances between methylated
sites, the genome is separated into alternating
compartments of methylated and unmethylated DNA
(Reik et al., 2001).
Methyltransferase recognition sequences
Methylation in most animals occurs at cytosines within
the sequence CpG with additional low levels of non-CpG
methylation reported in some species. Plants are
additionally methylated extensively
at CpNpG
sequences. However there are exceptions to these
general rules: CpT was recently identified as the
preferred
recognition
sequence
for
Drosophila
methylation (Lyko et al., 2004) and non-CpG methylation
is common in methylated fungi (Selker, 1997).
Methylation changes during development
Methylation patterns can also get altered during the
course of development. For instance in mammals there is
loss of methylation in early development and then the
pattern is
established again (Egger et al., 2004)
whereas methylation in Drosophila is only present during
early development. Changes also occur locally with loss
of methylation at some sites in some tissues; such local
demethylation often correlates with expression.
Methylation levels
DNA methylation and ageing
The percentage of methylated cytosines varies
substantially between species from no detectable
methylation (e.g. the nematode Caenorhabditis elegans,
the flat worm Schistosoma mansoni and the yeast
species,
Schizosaccharomyces
pombe
and
Saccharomyces cerevisiae) to very high levels in typical
vertebrates (60–90% of all CpGs methylated) and most
plants. It is assumed that methylation has been lost in
some lineages, but the details of which and when remain
incomplete because of the scarcity of data. All
invertebrates tested have either no methylation or some
intermediate level of methylation. Direct comparison
between species is complicated by the fact that there are
several different ways to estimate methylation levels.
These methods differ in sensitivity. To complicate matters
further, some methods test only a subset of cytosines
and the levels of methyl cytosine are alternatively
reported as the percentage of all bases, percentage of all
cytosines, or fraction of the subset of sites tested
(Jablanka et al., 1995).
Distribution of methylated sites
Vertebrate genomes are globally methylated i.e.
methylated cytosines are found over the entire genome
for short (nearly 1-kb) stretches. This unmethylated DNA,
the CpG island fraction, accounts for around 1% of the
genome and frequently coincides with promoter regions.
It is interesting to note that the lamprey, although having
The previous studies found a pattern of low global DNA
methylation levels in many aged mammalian tissues.The
great fidelity with which DNA methylation patterns in
mammals are inherited after each cell division is ensured
by the DNA methyltransferases (DNMTs). However, the
ageing cell undergoes a DNA methylation drift. Early
studies showed that global DNA methylation decreases
during ageing in many tissue types and it was
subsequently observed that mammalian fibroblasts
cultured to senescence increasingly lost DNA methylation
(Wilson et al., 1983). The loss of global DNA methylation
during ageing is probably mainly the result of the passive
demethylation of
heterochromatic
DNA as a
consequence of a progressive loss of DNMT1 efficacy
and/or erroneous targeting of the enzyme by other
cofactors (Casillas et al., 2003). Several specific regions
of the genomic DNA become hypermethylated during
ageing. New findings demonstrated a widespread and
tissue specific age-related DNA methylation changes in
mice. A surprisingly high rate of hyper- and
hypomethylation as a function of age in normal mouse
small intestine tissues and a strong tissue-specificity to
the process has also been demonstrated. It has been
concluded that epigenetic deregulation is a common
feature of ageing in mammals (Maegawa et al., 2010).
Normal ageing cells and tissues show a progressive
loss of 5-methylcytosine content, primarily within DNA
repeated sequences, as well as in potential gene
regulatory areas . In addition, selected genes show
706 J. Med. Med. Sci.
progressive
age-related
increases
in
promoter
methylation, which, once a critical methylation density is
reached, have the potential to permanently silence gene
expression. These changes are highly mosaic within a
given tissue and introduce a high degree of epigenetic
variability in ageing cells (Calvanese et al., 2009). The
ageing associated epigenetic changes are shown in
Table 3.
DNA methylation in cancer
DNA methylation was the first epigenetic alteration to be
observed in cancer cells. Hypermethylation of CpG
islands at tumour suppressor genes switches off these
genes, whereas global hypomethylation leads to genome
instability and inappropriate activation of oncogenes and
transposable elements (Feinberg et al., 2004). It appears
that genomic DNA methylation levels, which are
maintained by DNMT enzymes, are delicately balanced
within cells. The over-expression of DNMTs is linked to
cancer in humans, and their deletion from animals is
lethal (Rodenhiser et al., 2006). Furthermore,
methylcytosine is capable of spontaneously mutating in
vivo by deamination to give thymine. Indeed, 37% of
somatic p53 gene mutations (and 58% of germ-line
mutations) occur at methyl CpGs, and these mutations
are strongly implicated in the cause of cancer (Rideout et
al., 1990). The importance of epigenetic alterations in
cancer progression was shown years ago when
methylation of the 50 CpG-island of the p16/CDKN2A
gene was proved to be responsible for its transcriptional
silencing in 20–40% of most common cancers (Herman
et al., 1995 ; Merlo et al., 1995). Several cancer
susceptibility genes, including BRCA1 and VHL, which
cause familial forms of breast and kidney cancer,
respectively, are silenced by methylation in a significant
percentage of sporadic forms of the respective tumor
types. Fifteen percent of sporadic breast cancers harbor
methylated BRCA1 genes and their gene-expression
profiles are identical to those of tumors from inherited
families in which BRCA1 is mutated; both are completely
distinct from those of other breast-cancer types (Jones et
al., 2002).
Several groups have been developing array-based
methods for genome-wide detection of methylation or
other epigenetic alterations such as histone modifications
(Callinan et al., 2006 ; Wu et al., 2006).
Interplay between DNA methylation and histone
modifications
It is important to note that there is a direct link between
DNA methylation and histone modifications. A number of
proteins involved in DNA methylation (e.g. DNMTs and
MBDs) directly interact with histone modifying enzymes
such as histone methyltransferases (HMTs) and histone
deacetylases (HDACs). The growing evidence for
dynamic inter/intra-regulation of these modifications,
position and modification-specific protein interactions,
and
biochemical/biophysical
interaction
between
modifications has strengthened the ‘histone code’
hypothesis, in which histone modifications are integral to
regulating the expression of the genome (Strahl et al.,
2000).
There are now several examples of modification
patterns and sequences that relate to gene activation,
some of which occur on the same histone tail or on the
same amino acid. Thus, if ubiquitination/sumoylation of
histones function to activate/repress, respectively, it will
be interesting to determine whether they occur on the
same lysine residues and whether , in a simple reciprocal
fashion, oppose one another's activity. In fact, it is now
believed that DNA methylation and histone methylation
are tied together in a loop where one modification is
dependent on the other. Altering this relationship will
almost certainly have severe consequences on the
epigenome and chromatin organization. Thus most, if not
all, factors that affect DNA methylation levels also affect
histone modifications. For instance, it appears that H3K9
methylation and DNA methylation are linked (Fuks,
2005). In mammals, DNA methyltransferases interact
with Suv39h H3K9 methyltransferases and loss of H3K9
methylation inSuv39h-knockout embryonic stem cells
decreases Dnmt3b-dependent CpG methylation at major
centromeric satellites (Lehnertz et al., 2003).
Methyl-CpG-binding proteins may recruit histone
deacetylase complexes to deacetylate histone tails so
that the tails become suitable for serving as substrates
for methylation. In contrast with this sequential process,
MBD-containing HMTs may bind directly to methylated
DNA to methylate histone tails. Alternatively, it is also
possible that chromodomain-containing proteins bind to
methylated
histone
tails
and
recruit
DNA
methyltransferase (DNMT) to methylate adjacent CpG
sequences. Irrespective of the sequence of events, it is
likely that a concerted action of HMT and HDAC
complexes may play an important role in methylated DNA
silencing (Zhang et al., 2001).
In contrast to the above predictons it has been also
observed that
transcription of mouse DNA
methyltransferase 1 (DNMT 1) is regulated by both E2FRb-HDAC dependent and -independent pathways. It has
been identified that the promoter region and major
transcription start sites of mouse Dnmt1 and found two
important cis-elements within the core promoter region.
One is an E2F binding site, and the other is a binding site
for an as yet unidentifed factor. Point mutations in the two
cis-elements decreased promoter activity in both nontransformed and transformed cells. Thus, both sites play
a critical role in regulation of DNMT 1 transcription in
proliferating cells (Hiromichi et al., 2003).
As DNA methylation is found to be linked to histone
Jha et al. 707
Table 2. Chromatin-Remodeling Complexes (taken and modified after obtaining permission from the author and publisher).
Complex
Organism
ATPase
Mass (MDa)
No.
of
Subunits
S. cerevisiae
S. cerevisie
D. melanogaster
H. sapiens
H. sapiens
H. sapiens
Swi2/Snf2
Sth1
Brahma
Hbrm
CHD4
BRG1
2
1
2
2
1.5
2
11
15
ND
10
18
10
S. cerevisiae
S. cerevisiae
D. melanogaster
D. melanogaster
D. melanogaster
H. sapiens
ISWI1
ISWI2
ISWI
ISWI
ISWI
hISWI
0.4
0.3
0.5
0.7
0.2
0.5
4
2
4
5
4
2
Xenopus
H.sapiens
Mi-2
Mi-2
------
6
7
SWI/SNF family
SWI/SNF
RSC
Brahma
h SWI/SNF
NRD
h SWI/SNF
ISWI family
I SWI1
I SWI2
NURF
CHRAC
ACF
RSF
Mi-2/CHD family
Mi-2
NuRD
Table 3. Ageing associated epigenetic changes in different tissues of different species [taken after obtaining permission
from the author and publisher (Calvanese et al., 2009)].
708 J. Med. Med. Sci.
deacetylation in the same manner, methylation of
histone H4 by arginine methyltransferase PRMT1 is
essential in vivo for many subsequent histone
modifications knocking out of PRMT1 gene leads to a
domain-wide loss of histone acetylation on both histones
H3 and H4, as well as an increase in H3 Lys9 and Lys27
methylation, both marks associated with inactive
chromatin (Huang et al., 2005).
Epigenetic Therapy
Epigenetic therapy, the use of drugs to correct epigenetic
defects, is a new and rapidly developing area of
pharmacology. Because so many diseases, such as
cancer, involve epigenetic changes, it seems reasonable
to try to counteract these modifications with epigenetic
treatments. These changes seem an ideal target because
they are by nature reversible, unlike DNA sequence
mutations. The most popular of these treatments aim to
alter either DNA methylation or histone acetylation. The
emerging
use of drugs that modulate epigenetic
alterations, including the hypomethylating agents and
histone deacetylase inhibitors, is an exciting advance for
cancer treatment. These agents have shown great
promise in the treatment of several hematologic
malignancies, especially myelodysplastic syndromes,
acute myeloid leukemia, and
cutaneous T-cell
lymphoma. The potential reversibility of epigenetic states
offers an exciting opportunity for novel cancer drugs that
can reactivate epigenetically silenced tumor-suppressor
genes (Esteller et al., 2005; Jha et al., 2010). Blocking
either DNA methyltransferase or histone deacetylase
activity could potentially inhibit or reverse the process of
epigenetic silencing. DNA methyltransferases and
histone deacetylases are the two major drug targets for
epigenetic inhibition to date, for instance
histone
deacetylase inhibitors induce apoptosis in peripheral
blood lymphocytes along with histone H4 acetylation and
the expression of the linker histone variant, H1. Histone
deacetylase inhibitors induce apoptosis in peripheral
blood lymphocytes along with histone H4 acetylation and
the expression of the linker histone variant, H18
(Sourlingas et al., 2001).
Vorinostat/SAHA, has been approved by the FDA for
use as second-line therapy in patients with cutaneous Tcell lymphoma (CTCL). The other agents include (1)
sodium phenylbutyrate; (2) MS-275; (3) valproic acid; (4)
depsipeptide (FK228); (5) LBH-589; and (6) CI-994
(Mann et al., 2007 ; Duvic et al., 2007).
The histone deacetylase inhibitor valproic acid inhibits
cancer cell proliferation via down-regulation of the
alzheimer amyloid precursor protein. Based on these
observations, the data suggest that APP down-regulation
via HDAC inhibition provides a novel mechanism for
pancreatic and colon cancer therapy (Venkataramani et
al., 2010). Histone deacetylase inhibitors suppress
inflammatory activation of rheumatoid arthritis patient’s
synovial macrophages and tissues (Chen et al., 2001). It
has been proposed that HDAC inhibition promotes
neuronal outgrowth and counteracts growth cone
collapse through CBP/p300 and P/CAF-dependent p53
acetylation (Gaub et al., 2010).
The most broadly used DNA methyltransferase
inhibitor, 5-aza-2’-deoxycytidine (5-aza-CdR), clinically
referred to as decitabine, has been shown to be have
toxic effects aside from its demethylating properties and
has been found to be mutagenic in vivo (Jackson et al.,
1997). Moreover, 5-aza-CdR has been shown to be
capable of transcriptionally activating genes with
unmethylated promoters (Soengas et al., 2001) which
leads to increased acetylation and H3 lysine 4
methylation (Nguyen et al., 2002) suggesting this drug
can induce chromatin remodeling independently of its
effects on cytosine methylation. The commonly used
drugs targeting methylation are azacytidine (5azacytidine),
decitabine
(5-aza-2’-deoxycytidine),
fazarabine (1-β-D-arabinofurasonyl-5-azacytosine), and
dihydro-5-azacytidine (Goffin et al., 2002). These are all
derivatives of deoxycytidine with some modification at the
fifth position in the pyrimidine ring. Other drugs include
zebularine and antisense oligodeoxynucleotides. Dietary
phytochemicals
particularly
catechol-containing
polyphenols were shown to inhibit DNMT and reactivate
epigenetically silenced genes (Fang et al., 2003). Certain
dietary polyphenols, such as (–)-epigallocatechin 3gallate (EGCG) from green tea and genistein from
soybean, have recently been demonstrated to inhibit DNA
methyltransferases (DNMT) in vitro. This inhibitory activity
is associated with the demethylation of the CpG islands in
the promoters and the reactivation of methylationsilenced genes such as p16INK4a, retinoic acid receptor ß,
O6-methylguanine methyltransferase, human mutL
homolog 1, and glutathione S-transferase- , (Millar et al.,
1999 ; Jarrard et al.,1998; Izbicka et al., 1999; Nakayama
et. al., 2000 ; Kinoshita et al., 2000 Sasak et al., 2002;
Jarrard et al., 1997 et al., Chi et al., 1997). These
activities have been observed in human esophageal,
colon, prostate, and mammary cancer cell lines, and the
activity can be enhanced by the presence of histone
deacetylase inhibitors or by a longer-term treatment
(Fang et al., 2007). The combined inhibition of DNA
methylation and histone acetylation enhances gene reexpression and drug sensitivity in vivo (Steele et al.,
2009). Curcumin and one of its major metabolites,
tetrahydrocurcumin can inhibit M. SssI, a DNMT1 analog,
activity (Liu et al., 2009).
Several
phytochemicals
inhibit
the
DNA
methyltransferase activity with betanin being the weakest
while rosmarinic and ellagic acids were the most potent
modulators (up to 88% inhibition) (Paluszczak et al.,
2010). Curcumin
and genistein cause reversal of
hypermethylation and reactivation of RARβ2 gene in
SiHa cell line (a squamous cervical cancer cell line) (Jha
Jha et al. 709
et al., 2010). Histone deacetylase (HDAC) inhibitors like
trichostatin A, SAHA (Suberoylanilide hydroxamic acid)
etc. are also being tried as potential chemotherapeutic
agents.
However, epigenetic therapy has its limitations, such as
the fact that both DNMT as well as HDAC inhibitors may
activate oncogenes due to lack of specificity, resulting in
accelerated tumour progression. Moreover, epigenetic
states, once corrected, may revert back to the original
state because of the reversible nature of DNA
methylation patterns. Indeed, combinations of DNA
methyltransferase and histone deacetylase inhibitors
appear to synergize effectively in the reactivation of
epigenetically silenced genes (Shi et al., 2003 ;
Thiagalingam et al., 2003). Reports suggest that reduced
histone acetylation or H3K4me2 methylation and
increased dimethyl-H3-K9 methylation play a critical role
in the maintenance of promoter DNA methylation
associated RASSF1A gene silencing in prostate cancer
(Kawamoto et al., 2007). Using biological and statistical
criteria, four hypermethylated genes CDKN2B, MLF-1,
PCDH8, HOXD8 and four hypomethylated genes CD37,
HDAC1, NOTCH1 and CDK5 were identified, where
aberrant methylation was associated with inverse
changes in mRNA levels. Prominent and aberrant
promoter methylation in Mantle Cell Lymphoma (MCL)
suggests that differentially methylated genes can be
targeted for therapeutic benefit in MCL (Leshchenko et
al., 2010). Combination trials are underway to test this
concept in the clinic. Caution in using epigenetic therapy
is necessary because epigenetic processes and changes
are so widespread. To be successful, epigenetic
treatments must be selective to irregular cells; otherwise,
activating gene transcription in normal cells could make
them cancerous, so the treatments could cause the very
disorders they are trying to counteract.
CONCLUSIONS
Epigenetic changes like histone modifications and DNA
methylation play an important role in several of the
biological processes like cancer, ageing and
development. Epigenetics has reached a new level of
maturity over the past few years, with many findings
highlighting the intimate link between DNA methylation
and histone modifications. As a result of these
discoveries, we have begun to unlock the long-standing
mystery of how CpG methylation patterns are
established. Histone deacetylation and H3K9 methylation
appear to pave the way for CpG methylation. In addition,
evidence suggests that the DNA methylation associates
with histone deacetylation and H3K9 methylation to
generate a selfpropagating cycle that promotes
transcriptional repression. Yet our understanding of the
interplay between these epigenetic modifications is still
incomplete. A major challenge is to uncover the mutual
reinforcements of repression and the different states of
covalent histone and DNA modification required to
silence specific genomic regions in specific cases of
epigenetic regulation.
An important question is whether DNA methylation
interacts
with other histone modifications besides
deacetylation and H3K9 methylation. The findings
described in this review point to a possible connection
with H3K27 and H4K20me3 methylation. Further studies
are going on to confirm, if the links between CpG
methylation and H3K27 in addition to H4K20me3
methylation are strong enough to support the proposed
hypotheses.
There is tremendous potential in epigenetics. We
might have just begun to uncover the link between
histone and DNA modifications. Epigenetic therapy is a
new and rapidly developing area in pharmacology. To
date, most trials of epigenetic drugs have been
conducted to evaluate their effects on cancers, many of
which have shown promising results. The past few years
have seen many exciting discoveries and undoubtedly,
many more are yet to come. The future of this field is
having a vast potential and it is expected that it would
solve many of the biological mysteries.
ACKNOWLEDGEMENTS
The authors acknowledge the financial assistance
provided by CSIR (Council of Scientific and Industrial
Research), India to AKJ.
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