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Epigenetic mechanisms in development, inheritance and disease
Nikki Minnebo, Aleyde Van Eynde, Mathieu Bollen*
Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, University of
Leuven, Belgium
*Corresponding author: Prof. Dr. M. Bollen, Laboratory of Biosignaling & Therapeutics, Department of
Cellular and Molecular Medicine, KULeuven, Campus Gasthuisberg, O&N1, Herestraat 49 Box 901, 3000
Leuven, Belgium. E-mail: Mathieu.bollen@med.kuleuven.be
Received: 02.12.2011 Accepted: 23.02.2012 Published: 05.03.2012
Abstract
Epigenetics is a relatively new and exciting field of the (bio)medical sciences. It confers an additional layer of
information that controls gene expression by mechanisms involving DNA methylation, histone modifications,
chromatin compaction and non-coding RNAs. As most epigenetic marks are mitotically transferable and
sometimes even stable during meiosis, these modifications can potentially be passed on to future generations.
Proper epigenetic signaling is essential for normal proliferation and differentiation, and epigenetic misregulation
is a key feature of many common diseases including cancer, diabetes and cardiovascular disease. As most
epigenetic marks are reversible, understanding the underlying disease mechanisms reveals the therapeutic
potential for interference with aberrant epigenetic signatures. These therapies hold great promises for a wide
range of applications. However, the creation of both potent and specific epigenetic therapies requires a detailed
insight into the patients’ epigenomic landscape. Here, we provide an overview of the major epigenetic signaling
pathways and their contribution to development and disease.
Keywords: epigenetics, chromatin, environment, inheritance, disease, therapy.
INTRODUCTION
We all originate from a single omnipotent
stem cell, the fertilized egg. Many cycles
of cell division, combined with a stepwise
differentiation process, produces the
thousands of billions of cells that constitute
our body. With each round of cell division
the genetic material or DNA is doubled
and passed on to the daughter cells,
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implying that each person is basically a
clone of cells. Eukaryotes are faced with
the problem to organize their large genome
(human cells contain about 3 billion
basepairs (bp), the equivalent of 2 meters
of DNA) into the micrometer dimensions
of a nucleus and at the same time keep the
DNA sufficiently accessible to regulatory
factors. Nature’s answer to this conundrum
is the nucleosome. Each nucleosome is
comprised of 146 bp of double-stranded
DNA, wrapped around an octamer of 2
copies of each of the histone proteins H2A,
H2B, H3 and H4. Successive nucleosomes
are linked by a short fragment of DNA
giving the appearance of a string of beads
(Fig. 1). This string is further coiled and
looped around scaffold proteins, giving
rise to the protein-DNA complex called
chromatin (1-5).
The human body contains about
210 different cell types that arise by
changes in the gene expression profile
during the stepwise differentiation of
pluripotent stem cells into fully committed
and terminally differentiated cells (Fig. 1).
This process is driven by epigenetic
mechanisms that determine which genes
become repressed or activated during
development (1;3;6;7). Although
epigenetics is a rather new scientific
discipline, the name epigenetics was
already coined in 1938 by Conrad H.
Waddington, an embryologist studying
Drosophila, who used it to describe the
link between a given phenotype and its
associated genotype (now termed
developmental biology) (8;9). He
demonstrated that a temperature shock
before puparium formation produced flies
with cross veinless wings, a phenotype that
could still be detected after 16 generations
(10). He believed that the morphological
and functional properties of an organism
were dictated by a program controlled by
the genome and influenced by the
environment. Since then, epigenetics has
been defined in many ways but the
common theme is that it describes an
additional, heritable layer of information,
on top of the DNA nucleotide sequence,
that influences the expression level of
subsets of genes (11).
Figure 1. The epigenome determines cell fate. All cells of an organism contain an identical genome, comprised
of an individual DNA sequence. Inside a cell, the DNA is condensed by its wrapping around a core of histone
proteins, forming the nucleosomes (red ellipses) and giving rise to a DNA-protein complex called chromatin.
Chromatin can be epigenetically modified to switch genes on or off. Examples of such epigenetic modifications
are DNA methylation (green triangle) and histone modifications (orange sphere). In addition, the local chromatin
structure can be changed by chromatin remodeling complexes (blue ellipse). ncRNAs (blue lines) have also been
implicated in gene regulation. Although an organism has only one single genome, it contains many different
epigenomes, which determine cell fate, function and phenotype. Cartoon cell types at the bottom from left to
right: red blood cell, neuron, osteocyt, muscle cell and epithelium.
DIFFERENT MECHANISMS OF
EPIGENETIC REGULATION
In mammalian cells, the major part of
chromatin is present in a highly condensed,
heterochromatic state, which is largely
devoid of transcription factors and thus
transcriptionally inactive. Euchromatin is
less condensed and mainly consists of
actively transcribed genes (12). Both
histones and DNA can be chemically
modified with various epigenetic markers
that change the affinity for chromatin-
binding proteins and/or influence the
electrostatic interaction between DNA and
histones (13;14).
DNA methylation on CpG dinucleotides
DNA can be methylated by the chemical
addition of a methyl (CH3-) group to the
C5 position of cytosine bases (15). In
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22
mammals, mainly cytosines followed by a
guanine (CpG) are highly methylated (Box
1). Whereas CpGs are statistically
underrepresented and dispersed throughout
the genome, there are regions of high CpG
clustering called CpG islands, mostly in
the promoter region of coding sequences.
In fact, approximately 70% of all annotated
gene promoters contain a CpG island
which makes this one of the most common
promoter elements in the genome.
Virtually all housekeeping genes as well as
some differentiation genes have an
associated CpG island. Within islands,
most cytosine bases are unmethylated to
allow transcription of the associated gene.
In contrast, CpGs that are dispersed
throughout the genome are usually
methylated (16;17).
In human cells, there are three main
enzymes that catalyze the cytosine
methylation reaction: DNA
methyltransferase (DNMT)1, DNMT3a
and DNTM3b. DNMT1 is a maintenance
enzyme that copies the methylation pattern
from mother to daughter strands after
replication. The two other enzymes are de
novo methyltransferases that establish the
methylation pattern during early
embryonic development (18). DNA
methylation can have profound effects on
the structure of large chromosome
fragments, but is also implicated in the
fine-tuning of the expression of single
genes. Dispersed CpG methylation
stabilizes the integrity of the genome by
silencing parasitic DNA sequences such as
transposons. Although not firmly
established for mammals, this process has
been well documented for plants and fungi,
where transposons are specific targets for
DNA methylation. Methylated cytosines
are prone to spontaneous deamination
resulting in their conversion to thymine.
Such point mutations can prevent
transposons from changing their position in
the genome (19;20). In addition, DNA
methylation in the transposon promoter
region can prevent its expression.
DNA methylation in promoter
regions of protein coding genes represses
their transcription. Two distinct underlying
mechanisms have been proposed: [1] the
methyl mark prevents the recruitment of
DNA-binding transcription factors and
RNA polymerase to target genes (21), and
[2] some proteins specifically recognize
methylated CpGs and themselves recruit
histone modifying complexes that establish
a transcriptionally inactive chromatin
environment (18). Recently, it has been
proposed that DNA methylation also
directs nucleosome positioning in the
promoter region of inactive genes (22;23).
For imprinted genes, the DNA
methylation pattern differs between the
maternal and paternal genome, allowing
expression from only one of the two
alleles. One of the first discovered
imprinted genes was insulin-like growth
factor 2 (IGF2). A loss of imprinting (LOI)
at this region results in a doubling of IGF2
expression levels and is often associated
with an increased frequency of intestinal
tumors (24).
Finally, DNA methylation also
plays a crucial role in X-chromosome
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inactivation. In mammals, one of the two
female X-chromosomes becomes silenced
during early embryonic development to
compensate for the single copy in male
cells. In this way, X-linked genes will be
expressed at an equal level in both male
and female cells. X-inactivation is initiated
at the X-inactivating centre from which a
17-kb long non-coding (nc)RNA, called
Xist, is transcribed (25). This ncRNA then
coats the future inactivated X-chromosome
in cis, resulting in the recruitment of
Polycomb group (PcG) proteins (see
below) and DNMTs that silence most X-
linked genes (26;27). Moreover, the key
PcG protein EZH2 interacts directly with
all three DNMT’s, providing a link
between DNA methylation and histone
modifications (28).
The histone code
Histones are globular proteins and their N-terminal tails protrude from the nucleosome core
structure. These tails can carry different posttranslational covalent modifications (Box 1)
including acetylation, methylation, phosphorylation, ubiquitylation and sumoylation. Histone
modifications do not provide a simple on-off switch to regulate gene expression but rather act
combinatorially as reflected by the term “histone code” (29-32).
Histone modifications can function as docking sites for other DNA-regulatory
Box 1: Techniques for studying epigenetic modifications
The development of several research tools has been crucial in the mapping of epigenetic
modifications. The most thoroughly studied modification is DNA methylation. Initially,
differences in methylation of a particular sequence could only be detected with methylation-
specific restriction enzymes. This technique severely limited the range of sequences that could
be investigated, a drawback that was solved by the discovery of bisulphite treatment
(19).
This technique reproducibly converts unmethylated cytosines into uracil bases but leaves
methylated cytosines unchanged. Differences in DNA methylation at any given sequence can
subsequently be investigated by PCR with methylation-specific primers.
To investigate histone modification signatures or DNA-binding profiles of a particular nuclear
factor, ChIP
or chromatin immunoprecipitation is the method of choice (2). After
crosslinking, genomic DNA is fragmented and an antibody is used to precipitate the protein of
interest, together with fragments of associated DNA. After reversing the crosslinks, the
precipitated proteins are digested and the associated DNA is purified. Any DNA of interest
can then be quantified by qPCR, using appropriate primer sets.
A genome wide mapping
of chromatin signatures uses the hybridization of fluorescently
labeled DNA to a microarray. More recently, next-generation sequencing
provides a
greater coverage, superior sequencing resolution and requires less input material, which is
often limited for ChIP experiments (2).
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The histone code
Histones are globular proteins and their N-
terminal tails protrude from the
nucleosome core structure. These tails can
carry different posttranslational covalent
modifications including acetylation,
methylation, phosphorylation,
ubiquitylation and sumoylation.
Histone modifications do not provide a
simple on-off switch to regulate gene
expression but rather act combinatorially
as reflected by the term “histone code”
(29-32).
Histone modifications can function
as docking sites for other DNA-regulatory
proteins, each associated with a specific
biological process. In addition, they can
have direct activating or repressing
functions depending on the type and
localization of the modification. Some
histone modifications change the charge of
the histone tail. For example, positively
charged lysine residues lose their charge
upon acetylation; this weakens the histone
interaction with the negatively charged
DNA, which makes the DNA more
accessible for transcription factors,
resulting in transcriptional activation. In
addition, acetylated histones serve as
docking sites for bromodomain-containing
chromatin modifying proteins (30). In
contrast, histone methylation on lysine or
arginine residues does not change their
charge. Rather, methylated residues are
binding sites for chromodomain-containing
transcriptional activators or repressors
(32;33). For example, active genes are
trimethylated on lysine 4 of histone H3
(H3K4me3) near the transcription start
site. During transcription such genes also
become trimethylated on H3K36 across the
gene body. These methylated lysines
subsequently attract chromodomain-
containing transcription factors and
chromatin remodelers. Methylation of
H3K9 and H3K27 on the other hand are
strongly associated with gene silencing and
the establishment of a less accessible,
facultative heterochromatic structure (32).
For example, the H3K27me3 mark is
recognized by the chromodomain-
containing Polycomb protein that resides
within a multiprotein complex named
Polycomb Repressive Complex (PRC)1
(34). Another PRC1 protein called RING1,
is an enzyme that monoubiquitilates
histone H2A on K119 leading to a more
condensed chromatin state. By a still
unresolved mechanism, PRC1 can also
impair the binding or progression of RNA
polymerase along these genes leading to
transcriptional silencing (35-37). Finally,
DNMTs can be recruited by the H3K27
methyltransferase EZH2 to methylate the
target DNA, providing a more stable and
silent chromatin environment (28).
H3K27me3 is particularly
important for the silencing of key
differentiation factors during
embryogenesis (38-40). This repressive
mark is deposited by PRC2 which contains
three core proteins: the methyltransferase
EZH2 and its two co-activators SUZ12 and
EED. PcG proteins were first discovered in
Drosophila by mutations in a group of
transcription factor genes, called homeotic
(Hox) genes, that are important for early
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fly development (1;41). The name
Polycomb itself refers to the phenotype of
male flies carrying mutations in the
Polycomb gene. Male flies normally have a
set of bristles or “sex combs” on their first
pair of legs to help them during mating.
Mutations in the Polycomb gene caused
them to develop multiple sex combs on all
their legs, hence the name Polycomb (42).
In general, mutations in any of the PcG
protein encoding genes cause deficient
methylation of H3K27 and are associated
with developmental defects (also called
homeotic transformations), owing to their
important role in development and
differentiation (38;43). However, the PcG
proteins also regulate the expression of
hundreds of genes in differentiated cells,
and therefore should be viewed as key
regulators of transcription in all cells.
The biological counterpart of the
PcG proteins are the Trithorax Group
complexes that activate genes. As
suppressors of PcG mutant phenotypes,
some Trithorax complexes contain histone
methyltransferases such as Ash1 that
methylate H3K4. In addition, these
complexes can also contain components of
the ATP-dependent chromatin remodeling
complexes NURF or SWI/SNF that
facilitate the recruitment of the
transcriptional machinery (44;45).
ATP-dependent chromatin remodeling
Nucleosomes are not evenly spaced along
the DNA and often the removal or sliding
of nucleosomes along promoter areas is
necessary for transcription factors and
RNA polymerase to gain access to the
DNA. Indeed, the small promoter region
surrounding the transcription start site of
active genes is usually depleted of
nucleosomes (46). Nucleosome remodeling
is also indispensible for DNA polymerase
to copy the DNA along the nucleosomes
during the S-phase of the cell cycle (47).
Nucleosome remodeling complexes use
ATP as an energy source to alter histone-
DNA interactions. By relocating
nucleosomes to available acceptor DNA in
cis or even trans, nucleosome remodeling
ATPases establish a dynamic chromatin
state in which the overall packaging of
DNA is maintained, but specific sequences
can be transiently exposed. In mammals,
many different remodeling ATPases have
been discovered and they all associate with
a wide range of other proteins giving rise
to large multiprotein complexes with
distinct functions (47;48). For example, the
SWI/SNF family of ATPases can slide
nucleosomes along the DNA axis and even
eject histones from the nucleosomes,
whereas the ISWI family is only capable of
sliding nucleosomes (46). The Mi-2/NuRD
complex is unique in that it combines a
nucleosome sliding function with a histone
deacetylase (HDAC) activity through
interaction with HDAC1 and 2 (49;50).
Another epigenetically related process can
be found in the ISWI-type WICH complex,
which binds to replication foci during S-
phase. By keeping nucleosomes mobile, it
provides access to the newly replicated
DNA and prevents it from turning into
heterochromatin (51). In doing so, this
complex safeguards the transmission of the
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epigenetic state from mother to daughter
cells.
Long non-coding RNAs
ncRNAs are functional transcripts that do
not encode proteins. They represent a
significant part of the mammalian
transcriptome and regulate gene expression
at multiple levels. Besides small (20-25 bp)
ncRNAs, such as miRNA or siRNA, that
mainly regulate translation by targeting
mRNA for degradation (52), some of these
ncRNAs are quite large, up to several
hundred kb. These long ncRNAs have been
shown to regulate epigenetic gene
silencing, both in cis and in trans, by
binding to complementary DNA and/or by
association with chromatin-modifying
complexes (53-55). The Xist RNA is only
one example of a cis-inactivating ncRNA
(26;27). Another well-known case is Air, a
non-coding transcript of 108 kb that is
transcribed from the second exon of the
paternal allele of the IGF2 receptor gene
(56). Air binds and silences a 490-kb
region in cis and interacts with the H3K9
methyltransferase G9a (57). Yet another
ncRNA is the 91-kb Kcnq1ot1, which is
transcribed in the antisense direction from
an intron of the Kcnq1 gene. The internal
promoter for Kcnq1ot1 is only methylated
on the maternal chromosome, accounting
for the specific expression of Kcnq1ot1
from the paternal allele. Paternally
expressed Kcnq1ot1 recruits the
methyltransferase G9a and the PRC2
complex, which locally deposit the
repressive H3K9 and H3K27 methylation
marks, respectively, silencing protein
encoding genes inside the 1-Mbp Kcnq1
domain. Intriguingly, paternal imprinting
of Kcnq1ot1 is seen in placenta but not in
liver and thereby contributes to lineage
specific transcriptional silencing (58;59).
An example of a ncRNA-based
epigenetic silencing mechanism in trans is
HOTAIR. This 2.2-kb antisense transcript
is generated from the HOXC cluster on
chromosome 12 but represses transcription
across a 40-kb region in the HOXD cluster
on chromosome 2. HOTAIR also interacts
with the PRC2 complex and is required for
the deposition of the H3K27me3 mark
across the HOXD region. Accordingly, a
knockdown of HOTAIR results in an
expression of genes in the HOXD locus
and this is associated with a local drop in
the level of H3K27me3 (60;61).
A recent genome-wide analysis
identified over a thousand highly
conserved mammalian ncRNAs (62).
About 20% of these are bound by PRC2 in
various cell types whereas some newly
identified ncRNAs are bound by other
chromatin-modifying complexes.
Consistent with these findings, the siRNA-
mediated depletion of PRC2-interacting
ncRNAs results in the derepression of
associated genes (63). Given these recent
developments, ncRNAs can be predicted to
play an important general role in directing
chromatin-modifying complexes to their
target genes.
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27
EPIGENETIC PROCESSES ARE
CRUCIAL FOR EMBRYONIC
DEVELOPMENT
Studies in mice and Drosophila have
shown that a depletion of epigenetic
regulators often results in severe
developmental defects or even early
embryonic lethality. In mice for example,
the lack of DNMT1 results in embryonic
lethality at day E8.5-9 and DNMT1
heterozygous embryos show rudiments of
the major organs but they are smaller than
their wild type littermates and develop
more slowly (64). Moreover, loss of any of
the core PRC2 subunits results in severe
defects and lethality around gastrulation
(65-67). These effects suggest an essential
role of these chromatin-modifying proteins
in normal development.
PcG proteins also maintain the
pluripotency and self-renewal capacity of
embryonic stem (ES) cells by controlling
key developmental regulatory genes. For
example, ES cells lacking SUZ12 and EED
show a more differentiated phenotype
(39;68), and ES cells devoid of EZH2 are
unable to reprogram B cells towards
pluripotency (69). In ES cells, many genes
that are important for development contain
both active (H3K4me3) and repressive
(H3K27me3) chromatin marks and are
therefore named “bivalent” genes. During
further development, specific signaling
processes determine cell fate decisions by
activation or repression of these bivalent
domains (70). A nice example of such
lineage commitment was described for skin
development (71;72). EZH2 is highly
expressed in basal progenitor cells and
promotes their proliferation by repressing
the Ink4A-Ink4B locus, which encodes
inhibitors of cell cycle progression. As skin
cells differentiate and move to the surface
of the skin, EZH2 expression levels
decrease resulting in the expression of the
Ink4A-Ink4B locus and a proliferation
arrest. This study reveals that the EZH2-
containing PRC2 complex controls
epigenetic modifications both temporally
and spatially in tissue-restricted stem cells.
In this way, the PRC2 complex ensures
that the proliferation potential is
maintained and that undesirable
differentiation processes are repressed
(73). Similar conclusions have been drawn
from studies on the role of EZH2 in
neurogenesis (74), myogenesis (75;76),
hematopoiesis (77) and adipogenesis (78).
Another appealing example of
developmental epigenetics comes from the
honeybee (Apis mellifera). If a new queen
is needed in a honeybee population, one
larva is chosen to be fed large quantities of
royal jelly as an exclusive food source
during the first four days of its growth.
This type of early feeding triggers the
development of queen morphology,
including fully developed ovaries that are
needed to propagate the species.
Intriguingly, the silencing of DNMT3 in
honeybees causes similar effects as the
feeding of royal jelly, suggesting that this
food source contains a DNA methylation
inhibitor (79).
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TRANSGENERATIONAL
EPIGENETIC INHERITANCE
The transmission of genetic information
from parent to progeny is well understood
and generally accepted. However, the
transfer of a non-genetic determination of
phenotype has also been documented and
is now known as “soft inheritance” or
transgenerational epigenetic inheritance
(TEI) (80).
A first clear example of TEI came
from Överkalix, a desolate small town in
northern Sweden where, during the 19th
century, its small population would starve
if the harvest was poor. Several decades
later, Bygren and colleagues found that
pregnant women, suffering from food
deprivation, delivered children with an
increased risk of cardiovascular disease
(81). Similarly, the children of women who
were pregnant during the Dutch hunger
winter of 1944, suffered from impaired
glucose tolerance as an adult which was
correlated with less DNA methylation of
the imprinted IGF2 gene (82).
Unfortunately, large scale studies of
epigenetic inheritance in humans are scarce
and most information on TEI comes from
studies on plants and animals (80). For
example, a study in rats showed that
maternal grooming caused offspring to be
more fearful and this was linked to
promoter hypermethylation of the
Glucocorticoid Receptor gene in the
hippocampus. This stress-induced
phenotype was epigenetic but not
transferred through germ cells as cross-
fostering pups from one mother to the
other during the first week caused pups to
adopt the stress-phenotype of their new
mother (80;83).
Mice have been engineered with a
uniquely regulated agouti viable yellow
gene (Avy) that can be used as an easy
epigenetic read-out system (84). The Avy
gene gives mice a yellow coat color and is
correlated with a propensity for obesity
and diabetes when expressed
constitutively. This model system was used
to show that ethanol consumption by
pregnant females led to an increase in
DNA methylation in developing embryos
at the Avy locus, correlating with a change
in coat color from yellow to pseudoagouti
(brown). A similar switch in coat color was
observed when feeding pregnant mice a
methyl-rich diet, changing the phenotype
of the pups towards the healthy, longer-
lived direction (85).
Only very recently, a study of the
roundworm Caenorhabditis elegans (86)
showed that methylation of H3K4 plays a
key role in longevity. Deficiencies in the
H3K4 methyltransferases ASH-2 or WDR-
5 in the parental generation extends their
lifespan with about 20%, an effect that is
observed up until the third generation. This
transgenerational transmission depends on
the H3K4 demethylase RBR-2 and is
associated with epigenetic changes that
influence the expression level of key genes
involved in longevity.
Although clear examples of TEI
exist, several issues concerning this
phenomenon remain unclear. For an
epigenetic mark to be transmitted to future
progeny, these modifications must be
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29
present in the germ cells. However, the
paternal DNA becomes demethylated prior
to the first cell division of the zygote and
the maternal genome is demethylated after
several cleavage divisions. De novo
methylation is subsequently established in
the inner cell mass of the blastocyst
(87;88). Additionally, in sperm cells, the
histones are replaced by smaller
protamines without modifiable tails that
would disable any histone-linked
epigenetic information to be passed
through the male germ line (89). However,
it was recently found that some histone-
containing nucleosomes are retained in
sperm and these appear to be specifically
present at loci important for embryonic
development (90). Moreover, parts of the
genome seem to escape the genome-wide
demethylation process during
embryogenesis, potentially mediating
epigenetic inheritance. TEI can also be
partially explained by the presence of
developing germ cells inside the embryo of
a pregnant mother. Therefore,
environmental conditions acting on the
pregnant mother can also induce epigenetic
changes in the developing germ cells of the
embryo, accounting for the transmission of
a phenotype from grandparent to
grandchild (80;89).
In summary, environmental
conditions (food, stress, toxins,…) can
cause epigenetic changes, resulting in an
altered phenotype (Fig. 2) and in some
cases these effects can be transferred to the
next generation(s). However, this does not
imply that these changes are stable
throughout evolution. The notion of soft
inheritance is still rather controversial and
is often linked with the work of Jean-
Baptiste Lamarck (1744-1829), who
proposed an evolutionary theory based on
the inheritance of properties acquired
during the lifetime of an organism that can
be summarized in contemporary terms as
“the environment contributes to an
inherited phenotype”. This theory contrasts
with that of Charles Darwin (1809-1882),
which is based on the competition between
individuals that show a (natural) genetic
variation and can be translated as “the (epi-
)genotype determines the phenotype”.
Although the Darwinistic view of
evolution is now broadly accepted, the
notion of epigenetic inheritance has re-
opened the scientific debate on the
contribution of the Lamarckianistic view
(89;91). Hard evidence of true epigenetic
“inheritance” is still missing as most
observed transgenerational effects tend to
fade away after a few generations (92).
However, there is evidence that
environmentally induced epigenetic
changes contribute to the variability
between individuals in a population,
thereby increasing the speed of evolution
in a Darwinistic manner. Indeed, in
contrast to genetic mutations, epigenetic
signals can be easily modified and provide
a powerful mechanism for rapid
adaptations to sudden environmental
changes (93).
Figure 2. Environmental factors can induce both genetic and epigenetic alterations. An organisms’
phenotype is determined by the combination of genetic and epigenetic determinants. Environmental factors such
as radiation or smoking can influence a phenotype by introducing genetic alterations. The mutation of the DNA
sequence is irreversible and sometimes alters the amino acid sequence of the encoded protein which may be the
cause of specific “inherited” diseases. Epigenetic alterations (epimutations) can also contribute to disease
development and even influence an organisms’ lifespan, but in contrast to genetic mutations, they are reversible.
Diseases caused by genetic mutations are hard to cure and often require surgery or chemotherapy to remove the
affected cells. In contrast, diseases that originate from epigenetic alterations hold great promise for conventional
treatment, as the epimutation can be restored to normal by inhibiting specific chromatin-modifying enzymes.
ABERRANT EPIGENETIC
SIGNALING IN DISEASE
DEVELOPMENT
The last decade of research has
tremendously increased our understanding
of the mechanisms underlying epigenetic
phenomena, both in normal development
and disease. Currently, it is well known
that environmental factors and lifestyle
choices not only cause DNA sequence
alterations but also change the epigenetic
marks on our chromatin. Aberrant
epigenetic signals can contribute to disease
development and can even be passed on to
future generations. Importantly, epigenetic
marks, unlike genetic mutations, are in se
reversible and scientists are beginning to
understand how they can manipulate these
P Belg Roy Acad Med Vol. 1: 19-40 N. Minnebo, A. Van Eynde and M. Bollen
___________________________________________________________________________
31
epigenetic signals to revert them to their
original states (Fig. 2).
Cancer comprises over 200
different diseases of abnormal cell growth
induced by a series of mutations, but also
involving epigenetic changes. DNA
methylation in cancer has been extensively
studied and the onset of cancer is generally
accompanied by a global demethylation of
the genome. This leads to a destabilization
of the genome and thus increases the risk
of gaining additional mutations (94).
Hypomethylation is also observed locally,
for example at the promoter of oncogenes,
increasing their expression levels.
Conversely, hypermethylation is observed
in the promoter region of tumor suppressor
genes, as well as genes involved in cell-
cycle regulation, DNA repair, apoptosis
and angiogenesis (95;96). Importantly,
many PcG proteins are themselves
oncogenes and their expression levels are
often elevated in cancer samples. For
example, SUZ12 is found to be
overexpressed in colon, liver and breast
cancer, while EZH2 is overexpressed in 16
different types of cancer and is correlated
with increased angiogenesis, metastasis
and a poor patient outcome (97). Indeed,
EZH2 can be considered a true oncogene
as removal of EZH2 from cancer cells
results in growth arrest whereas ectopic
expression of EZH2 in normal cells
promotes invasion and cell proliferation in
vitro (98). Detection of aberrant DNA
methylation and overexpression of PcG
proteins therefore hold great promise as
biomarkers for the diagnosis and prognosis
of cancer (99).
Prenatal overgrowth, hypoglycemia,
abdominal wall defects and a high
frequency of tumors are all characteristic
of the Beckwith-Wiedemann Syndrome
(BWS). Being a rare familial disorder
caused by epigenetic changes in several
genes, it is a true paradigm for
understanding the epigenetics of cancer.
Large-scale screenings of affected families
identified that LOI at the IGF2 gene is one
of the main determinants of this disease.
Normally, the maternal IGF2 allele is
methylated, restricting expression to the
paternal allele, but in BWS it is expressed
from both paternal and maternal alleles,
leading to a doubling of IGF2 expression.
LOI at the maternal locus was also
observed to be 5-fold more frequent in
colorectal cancer patients showing that
LOI of IGF2 confers a general cancer risk,
even though this epigenetic change
accounted for only 15% of all BWS
patients (88;100).
Various pathologies have been
linked to a disruption of the epigenetic
state, often referred to as epimutations.
Two imprinted-gene disorders associated
with mental retardation, Prader-Willy
Syndrome and Angelman Syndrome,
involve two adjacent reciprocally
imprinted genes. Usually, these syndromes
are associated with mutations in this locus,
located on chromosome 15, but some
patients have been identified where an
aberrant DNA methylation at the same
locus causes the disease (100). This
epimutation is the result of an allele that
has passed through the male germline
without proper clearing of the silent
P Belg Roy Acad Med Vol. 1: 19-40 N. Minnebo, A. Van Eynde and M. Bollen
___________________________________________________________________________
32
epigenetic state that was previously
established in the patients’ grandmother,
indicative of TEI (101).
Intriguingly, a controversial
association has been found between the
occurrence of epigenetic disorders (eg.
BWS) and assisted reproduction
techniques, such as in vitro fertilization
(102;103). However, the number of
reported cases is too low to draw firm
conclusions and it has not yet been
established how this increased incidence of
imprinting disorders is caused. Aberrant
epigenetic signals may arise by in vitro
manipulation of embryos or the unnatural
harvesting of egg and sperm cells. For
example, embryo culture medium
composition has been shown to affect
DNA methylation (104) and strongly
modifies the placental expression profile
long after embryo manipulations, showing
that stress of an artificial environment is
memorized after implantation (105).
Alternatively, abnormalities could already
have been present in the parents, leading to
reduced fertility and subsequent birth
defects. In this regard, an association
between reduced sperm concentration and
abnormal imprinting in spermatozoa has
provided a link between imprinting defects
and impaired gametogenesis (106).
Therefore, it has been hypothesized that
spermatozoa from infertile males, used for
in vitro fertilization, contain a higher
number of gametes with chromosomal
abnormalities and that a similar mechanism
might account for infertility in women.
Maternal nutritional imbalance and
metabolic disturbances that take place
during early development can have
profound effects on the health of offspring
and may even be transmitted to the next
generation. This theory is called “fetal
programming” and many common diseases
such as obesity, diabetes, cardiovascular
disorders, cancer, asthma and even
schizophrenia, take root in nutrition and
environmental effects during early
embryonic development (107). The
epigenome is most vulnerable to
environmental factors during
embryogenesis, when DNA synthesis rates
are high and the de novo DNA methylation
pattern is shaped for normal tissue
development (89). Moreover, oocytes have
a more open chromatin structure and a
longer life time than male germ cells,
making oocytes more vulnerable to
epigenetic modifications by environmental
factors (87). In addition, sperm nuclei are
much more likely to be cleared of any
epigenetic errors because their histones are
largely replaced by protamines (108).
THE PROMISE OF EPIGENETIC
DRUGS
Epigenetic diseases can (in part) be
prevented by adopting a healthy lifestyle.
In addition, since epigenetic modifications
are reversible, they can potentially be
reversed by targeting chromatin-modifying
enzymes. Some epigenetic drugs have
already been approved by the FDA and are
being used clinically. Decitabine or 5-aza-
2'-deoxycytidine is an example of a DNMT
inhibitor that is currently used to treat
myelodysplastic syndrome (MDS), a bone
P Belg Roy Acad Med Vol. 1: 19-40 N. Minnebo, A. Van Eynde and M. Bollen
___________________________________________________________________________
33
disease that can lead to leukemia (109).
The drug is a nucleoside analogue with a
modified cytosine ring that is incorporated
in the DNA of replicating cells and
therefore preferentially targets rapidly
growing cancer cells. The drug
stoichiometrically binds to and thereby
inhibits DNMTs, counteracting
hypermethylation of eg. tumor suppressor
genes (110). Low drug doses are needed to
limit toxic side effects and have been
shown to increase overall survival in
patients with MDS and prolong the time of
conversion from MDS to leukemia.
However, being incorporated in the
genome, decitabine can cause mutations in
daughter cells if the parental cell does not
die. Another problem is that decitabine is
toxic to the bone marrow, where blood
cells are constantly being synthesized.
Two HDAC inhibitors, Vorinostat
and Romidepsin, are remarkably efficient
for treating cutaneous T-cell lymphoma
(96). However, the precise molecular
mechanisms for patient response still need
to be determined. Moreover, the HDAC
inhibitor trichostatin-A impairs blood
vessel formation by indirectly repressing
vascular endothelial growth factor, a key
regulator of tumor vascularization (111).
Another possible use of HDAC inhibitors
lies in their potential to overcome
resistance of cancer to conventional
therapies. Indeed, multiple HDAC
inhibitors reverse therapeutic resistance in
a subpopulation of cancer stem cells in
culture. Also the combination of DNMTs
and HDAC inhibitors is being clinically
tested as treatment for many different types
of cancer. Epigenetic cancer therapy can
thus be seen as a promising approach
because of its synergy with conventional
chemotherapy, reversing tumor
chemoresistance and increasing the
efficiency of radiotherapy (112). Finally,
there is a growing interest in therapies
targeted against PcG proteins, such as
EZH2, that are overexpressed in many
cancer samples. Recently, 3-
deazaneplanocin-A was reported to rather
selectively inhibit EZH2 and thereby
decrease repressive methylation of H3K27
as well as reactivate genes that became
aberrantly silenced in cancer (113).
Other epigenetic drugs rather
downregulate the expression of specific
sets of genes. For example, patients with
Chronic Obstructive Pulmonary Disease
have increased histone acetylation levels
on genes involved in inflammation.
Treatment of this disease includes the use
of corticosteroids that are thought to act, in
part, by promoting the recruitment of
HDAC2 to the promoter of the active
inflammatory genes. Conversely, the
antidepressant Imipramine is known to
induce acetylation of histones and reverse
depression-induced repressive chromatin
signatures. In rats, Imipramine inhibits
HDAC5 in the hippocampus (114).
Another promising epigenetic
curative tool is the use of induced
pluripotent stem cells (iPSCs). Stem cells
are cells with the potential to differentiate
into any kind of tissue. Therefore, they are
very attractive for the generation of
replacement tissues for a wide range of
disease conditions. In theory, stem cells
P Belg Roy Acad Med Vol. 1: 19-40 N. Minnebo, A. Van Eynde and M. Bollen
___________________________________________________________________________
34
from the patient can then be used to
regenerate his/her own damaged tissue,
bypassing the problem of finding suitable
donors. However, natural sources of stem
cells are limited and so bioengineered
iPSCs are rapidly gaining interest as a new
tool for regenerative medicine. Patient-
specific iPSCs are differentiated somatic
cells that are experimentally
reprogrammed to resemble embryonic
stem cells, typically by introducing stem
cell transcription factors that will
reprogram the cell (114;115). iPSCs can
then give rise to any cell type given the
right mix of differentiation stimuli. So far,
iPSC technology has demonstrated
therapeutic benefits by generating patient-
specific replacement tissues for a wide-
spectrum of disease conditions. Upon
transplantation in model systems, it was
shown beneficial for the treatment of sickle
cell anemia, ischemic heart disease,
Parkinson’s disease and hemophilia.
However, more work is needed to fully
understand the molecular mechanisms
underlying iPSC formation and
differentiation. For example, failure to
completely reverse repressive epigenetic
modifications might limit the potential of
iPSC formation. Moreover, it was reported
that iPSCs contain epigenetic marks that
resemble those found in cancer cells.
Therefore, iPSC need to be better
characterized before they can be used
therapeutically (114;115).
CONCLUSIONS AND FUTURE
PERSPECTIVES
The importance of a correct epigenetic
regulation is highlighted by its key
functions in both embryonic development
and the etiology of many pathologies.
Chromatin modifying enzymes are vital to
establish a correct lineage commitment
during terminal differentiation of stem
cells in the embryo. Both lifestyle choices
and environmental factors can influence
the epigenome and deregulation of
epigenetic processes can be the cause of
many diseases. It has also become clear
that epigenetic changes that accumulate
during the lifespan of an organism can be
passed on to future generations. However,
our knowledge about the epigenome is still
in its infancy and a greater understanding
of the function and regulation of
epigenetics is needed to identify attractive
drug targets and design novel therapeutic
strategies. Deep-sequencing technologies
(Box 1) have been developed that are
capable of mapping the epigenomic
landscape in high resolution and have
already been applied to identify DNA
methylation markers for cancer patient
diagnosis. Using this technology to map
combinations of epigenetic markers in
patient samples will provide a greater
understanding of gene regulation in disease
epigenomics and identify the factors that
mediate changes to important loci. This
will subsequently translate into the
development of more successful disease
treatments (2;116;117).
ACKNOWLEDGEMENTS
N.M. is a research fellow of the FWO-
Flanders.
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