Pflugers Arch - Eur J Physiol (2010) 459:247258
DOI 10.1007/s00424-009-0730-7
MOLECULAR AND GENOMIC PHYSIOLOGY
Epigenetic factors in aging and longevity
Silvia Gravina & Jan Vijg
Received: 4 August 2009 / Accepted: 26 August 2009 / Published online: 19 September 2009
# Springer-Verlag 2009
Abstract Epigenetics refers to phenotypic changes caused
by mechanisms that are unrelated to changes in the
underlying DNA sequence, most notably chromatin remod-
eling driven by histone modifications, and DNA methyla-
tion. Such variation is transmitted by cell division, but
generally not passed on through the germ line. An
increasing body of evidence supports a role for epigenetic
changes in the etiology of aging and its associated disease
sequelae. Here, we review the role of epigenetics in aging
and longevity with a focus on DNA methylation. Increased
understanding of those aging-related processes that are
driven by epigenetic mechanisms will allow for the
development of novel epigenetic-based diagnostic, preven-
tive, and therapeutic strategies for age-related diseases.
Keywords Aging . Epigenetics . DNA methylation .
Lifespan
Introduction
Epigenetic changes involve transmissible alterations in
gene expression caused by mechanisms other than changes
in DNA sequence. While, in the past, human biology was
considered the interplay between the DNA sequence of
one's genome and environmental variation, evidence has
been accumulating that such epigenetic modifications as
DNA methylation and histone modification can have a
primary role in phenotypic outcomes, including human
S. Gravina (*) : J. Vijg (*)
Department of Genetics, Albert Einstein College of Medicine,
1301 Morris Park Ave, Bronx,
New York, NY 10461, USA
e-mail: silvia.gravina@einstein.yu.edu
e-mail: jan.vijg@einstein.yu.edu
disease and aging. During various stages of mammalian
development, epigenetic patterns, most notably DNA
methylation, are reprogrammed genome-wide, generating
cells with a broad potential of differentiating into various
lineages [82, 88]. While epigenetic changes are essential for
development and differentiation, they can also arise later in
life either by non-random mechanisms, such as responses to
environmental change, or through stochastic errors in main-
taining fixed patterns of DNA or histone modification.
Epigenetic modifications, such as changes in DNA methyla-
tion or histone modification, can impact on chromatin folding
or non-coding RNAs. Probably the best-documented example
of epigenetic modification and the main topic of this review is
DNA methylation, which consists of a covalent addition of a
methyl group to the cytosine.
DNA methylation is carried out by DNA methyltrans-
ferases (DNMTs); at least three functional DNMTs have
been identified in eukaryotic systems. DNMT1 is the
enzyme primarily involved in the maintenance of DNA
methylation patterns during replication, and it preferentially
methylates hemimethylated DNA [83]. Other known
functional methyltransferases are DNMT3a and DNMT3b,
with higher de novo methylation activity than DNMT1.
DNMT3a and DNMT3b contribute to de novo methylation
during embryogenesis [77].
Clusters of unmethylated CpG pairs, termed CpG
islands, are located in approximately 40% of promoters of
mammalian genes, most notably housekeeping genes, and
are targets of transcription factors binding the unmethylated
DNA and initiating transcription. By contrast, methylated
CpGs are usually linked to silent DNA. DNA methylation
represses transcription directly by preventing the binding of
transcription factors to promoters [108] or indirectly by
recruiting methyl-CpG binding proteins [73]. While both
mechanisms may contribute to methylation-mediated gene
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silencing, it has been shown that many fully methylated
genes can be transcribed at nearly normal rates under
conditions where methyl-CpG binding proteins are effec-
tively absent [9].
DNA methylation patterns are intricately related to
changes in histone modification. Indeed, these two epige-
netic control systems are two sides of the same coin:
chromatin remodeling. The amino-terminal region of
histones is a hot spot for posttranslational modifications
that affect their interactions with DNA, other histones, or
chromatin remodeling proteins. The most studied of the
posttranslational modifications of histones are methylation
and acetylation of lysine residues in the amino-terminal
tails of histones H3 and H4 [31]. Histone methylation is
catalyzed by histone lysine methyltransferase (HLMTase)
[96], while histone acetyltransferase (HAT) and histone
deacetylases (HDACs) regulate, respectively, the acetyla-
tion and deacetylation of lysine residues [19]. In general,
increased acetylation of the histones in gene control regions
correlates with transcriptional activity, while methylation of
histones is primarily found in transcriptionally silenced
heterochromatin regions. Methylation of histone H3 at
lysine 9 (H3K9) usually leads to transcriptional repression
and can inhibit acetylation of the H3 tail at several lysines
[104]. The processes of histone modification and DNA
methylation are interdependent and both contribute to the
overall state of chromatin and its epigenetic control of gene
expression. For example, DNA methylation acts synergis-
tically with histone deacetylation to repress transcription.
The N-terminal domain of DNMT1 can bind to the
HDACs, thereby promoting histone deacetylating and
suppressing gene transcription [87]. Fuks et al. have shown
that DNMT3a can also recruit HDACs, thus leading to
transcriptional silencing [35].
One example of the power of epigenetics in regulating
gene expression is the phenomenon of position effect
variegation (PEV) [40]. PEV refers to the mosaic expres-
sion of a gene in otherwise homogeneous cell populations.
PEV was initially described in X-ray-irradiated fruit flies
undergoing a chromosomal inversion, which places the
white gene, normally located on the distal tip of the X
chromosome, close to the pericentric heterochromatin.
Normally, white is expressed in the adult eye resulting in
a red eye phenotype, but due to the inversion, the eye
contains patches of red and white tissue because the
expression of white is variegating [72]. PEV has also been
documented in other model organisms [23].
As it now appears, the epigenome is much more
dynamic than its sequence-based counterpart, undergoing
extensive alterations during development, differentiation,
and a variety of cellular responses that are essential for life.
The large volume of epigenomic transactions, including
maintenance, suggests a fairly high chance of errors.
Pflugers Arch - Eur J Physiol (2010) 459:247258
Indeed, epigenetic errors can cause disease by chromatin
remodelling, leading to aberrant gene expression. This is
especially important during development, where mistakes
in the series of carefully orchestrated changes in epigenetic
patterns can lead to congenital disorders and multisystem
pediatric syndromes [101]. However, epigenetic abnormal-
ities have also been found to be causative factors in adult-
onset diseases, including neurologic disorders, cancer, and
autoimmune disease [84, 92]. Interestingly, there is evidence
that epigenetic defects can also contribute to segmental
progeroid syndromes and, possibly, to aging itself [49]. Here,
we review the epigenetics of aging and lifespan, with a
particular focus on DNA methylation, the best understood
example of epigenetics. We discuss the emerging role of
epigenetics in susceptibility to age-related disease and
consider the possibility that aging in part is driven by an
epigenetic-mediated loss of phenotypic plasticity (Fig. 1).
Chromatin properties and systematic vs stochastic
change
It is increasingly being appreciated that chromatin is a
highly dynamic entity. Chromatin remodeling is most
dramatic during development, when genome-wide changes
in epigenetic marks orchestrate chromatin in a way that
allows it to generate different organs and tissues providing
the broad array of functions needed by the organism to
maintain itself and reproduce. Such changes are systematic
and are clearly purposeful. As will be discussed below,
similar changes may occur during normal aging for reasons
that may include signals from the environment, resulting in
a stress response. However, like the genome, the epigenome
is also subject to stochastic changes, called epimutations by
Holliday [48]. As we will see later, such epimutations have
been demonstrated to be critically important as causal
factors in the age-related increase in cancer, but could also
play a key role in driving other aging processes.
Stochastic changes in the epigenome could be due to
errors in maintaining established patterns of DNA methyl-
ation or histone modification. How frequent are such
mistakes? For DNA methylation, direct analysis is difficult
since methylation patterns need to be derived from the two
complementary strands of an individual DNA molecule.
Using hairpin-bisulfite PCR, specifically developed for this
purpose, Laird et al. analyzed the fidelity of methylation
transmission in the CpG island of the X-linked FMR1 gene
in human lymphocytes [61]. Their results indicate that the
fidelity of inheritance of the methylated state of each
cytosine in the hypermethylated allele is 0.96 per cell
division. When looking at the active, unmethylated X
chromosomal locus, they found that the fidelity of
inheritance of the unmethylated state was as high as 0.99.
Pflugers Arch - Eur J Physiol (2010) 459:247258
Fig. 1 Epigenetics of aging.
DNA hypomethylation can
initiate chromosome instability
while DNA hypermethylation in
promoter regions suppresses
expression of normal genes
(e.g., tumor suppressor genes).
Age-related hypermethylation or
hypomethylation could
theoretically impair or enhance
normal gene responsiveness to
environmental signals, thus in
turn contributing to a
generalized functional decline
However, for the hypermethylated locus on the inactive X
chromosome, the fidelity of inheritance of the unmethylated
state was only 0.83 (a de novo methylation efficiency of
0.17). These results clearly show that errors in maintaining
DNA methylation occur. Thus far, similar information for
histone modification patterns is lacking.
The stochastic nature of epigenetic changes is consistent
with new findings demonstrating a stochastic component of
gene expression regulation. In the past few years, through
work carried out in our laboratory [2, 12], it was shown that
aging is associated with increased stochastic deregulation of
cellular gene expression, giving rise to cellular mosaics of
diverging transcriptomes. This could reflect epigenetic drift,
affecting function and cellular responses to environmental
challenges (Fig. 2).
Hence, epigenetic alterations during aging could be
systematic, as well as stochastic. The former can be
addressed using advanced platforms based on genome-
wide analysis. However, stochastic changes need to be
analyzed at the single-cell or single-molecule level, and
suitable, genome-wide technology for that purpose is still
lacking. Indeed, cell-to-cell heterogeneity is considered
one of the major stumbling blocks of biological inves-
tigations of aging [66]. Below, we will also discuss the
need for single-cell epigenomics and the various
approaches being taken to overcome current limitations.
The emerging role of epigenetics in aging and longevity
Epigenetic information is only partially stable and destined
to change during development and in driving essential
249
somatic functions. This makes it a more likely candidate for
errors than its more stable DNA sequence counterpart,
changes in which have been well-documented and increase
during aging [17, 25, 37, 67, 102]. Indeed, epigenomic
alterations are now increasingly recognized as part of aging
and its associated pathologic phenotypes. Most of the
available data on epigenetic changes in human or animal
cells and tissues involve systematic changes, i.e., affecting
the exact same site in a large enough fraction of the cell
population to be detectable through current assays. Atten-
tion has thus far been focused on changes in DNA
methylation at cytosines, primarily in the aforementioned
CpG islands, which are usually unmethylated in normal
cells. Early studies indicated a general demethylation in
most vertebrate tissues with aging [86, 111]. In mitotically
active tissue, it is possible that this reflects some deficiency
in maintenance remethylation, as this normally occurs after
DNA replication. Such a general loss of 5-methylcytosine
during aging may de-suppress silenced retrotransposons,
such as L1, to increase genome instability [3, 68].
There are numerous reports in the literature on changes
in methylation status of individual genes during aging. Tra
et al. [100] compared more than 2,000 loci in T
lymphocytes isolated from newborn, middle-aged, and elderly
people, and found that 29 loci (approximately 1%) changed
DNA methylation status with age: 23 cases of hypermethyla-
tion and six cases of hypomethylation. Most of these
methylation changes happened before middle age. Interestingly,
the same CpG islands affected in human T cells also changed
with age in other human tissues (esophagus, lung, and
pancreas), suggesting that methylation instability is locus-
specific. The study was carried out using restriction landmark
250
Errors in DNA
Diet
methylation
Environment maintenance
SYSTEMATIC STOCHASTIC
Homogeneous gene Transcriptional
expression profile noise
Fig. 2 Epigenetic changes and effects of stochasticity. Recent findings
support the idea that aging is characterized by transcriptional instability,
which may be due in part to the loss of normal epigenetic patterns. In the
past, we have shown that aging is associated with increased molecular
randomization of cellular genomes and transcriptomes, which is
consistent with one of the most attractive concepts in the field: the idea
that aging is a time-dependent process of dysdifferentiation
genome scanning (RLGS), a genome-wide method based on
two-dimensional separation of DNA fragments obtained by
NotI digestion, which is methylation-sensitive. In the same
study, the authors also evaluated methylation patterns in T cells
and various tissues from aging mice, but could not detect sig-
nificant changes in the murine tissues, thus revealing a possible
difference in stability between mice and humans. However,
other studies, as we will discuss later, in mice and rats, did find
evidence for epigenetic changes during aging [7, 106].
In a recent study, Siegmund et al. examined DNA
methylation changes during the course of development,
maturation, and aging of the human cerebral cortex. The
authors looked at DNA methylation status at 50 loci in the
temporal neocortex of 125 subjects ranging in age from
17 weeks of gestation to 104 years old, including two
psychiatric disease cohorts, i.e., Alzheimer's disease and
schizophrenia. Most of the loci analyzed represent promoter
CpG islands of genes expressed in the cerebral cortex; a
portion of these genes is also implicated in cancer biology.
The authors observed a robust and progressive rise in DNA
Pflugers Arch - Eur J Physiol (2010) 459:247258
methylation levels across the lifespan for eight out of 50 loci,
with another 18 loci defined by a sharp rise within the first
months or years after birth. The authors also observed that
ALU and other repetitive elements either showed a
significant decrease in DNA methylation during the first
decade of life, followed by relatively little change during
subsequent maturation and aging, or showed relatively little
change across the lifespan [95].
Genes that might be more vulnerable to epigenetic changes
could be those of which one allele is normally suppressed
while the other one is active. For example, imprinted genes, a
subset of mammalian genes that is monoallelically expressed
in a parent-of-origin manner due to epigenetic mechanisms
that confer a parent-specific memory to individual cells, could
suffer from derepression of the inactive allele [30]. A list of
all human imprinted genes can be found in the database
http://igc.otago.ac.nz/Search.html. Another special case of
allele-specific epigenetic suppression is X-chromosome
inactivation, a mechanism by which one of the two copies
of the X chromosome present in female mammals is silenced
by packaging into transcriptionally inactive heterochromatin
[76]. Hence, it is conceivable that, during aging, genes on
the silent chromosome are derepressed by demethylation or
incomplete maintenance of methylation patterns. Indeed,
multiple authors have reported evidence for a relaxation of
epigenetic control of X-linked and imprinted gene expres-
sion during aging [7, 106].
For studying the reactivation of inactive X-linked genes,
mice are often used harboring an X-autosomal translocation.
In such mice, it is always the normal, non-translocated X
chromosome that is inactivated. Using various approaches for
detecting allele-specific transcripts, this makes it possible to
verify whether, during aging, a suppressed gene is re-
activated. For example, in the mouse, an approximately 50-
fold age-related increase in liver cells that had re-activated the
gene ornithine transcarbamoylase on the inactive X chromo-
some has been observed. This was ascribed to an age-related
reduction in DNA methylation [106]. Age-associated loss of
epigenetic repression of X-linked genes has been analyzed
quantitatively for the gene Atp7a in various tissues of the
same X-autosomal translocation mouse model [7]. The
results indicated a mean organ-specific relaxation of gene
repression in the oldest group of mice of up to 2.2% (this is
the fraction of transcripts from the inactive X relative to its
active sister chromosome). Using the same quantitative
methodology, Fu et al. observed a similar relaxation of gene
repression for the imprinted Igf2 gene, the expression of
which increases in aging mouse liver up to 6.7% [34].
Another model ideally suited for studying age-related
variation in the epigenome is the monozygotic twin. In a
recent study, global and locus-specific epigenetic differ-
ences between identical twins were reported to arise with
age [32]. In this study, involving 80 twins varying in age
Pflugers Arch - Eur J Physiol (2010) 459:247258
from 3 to 74 years, a variety of epigenetic markers,
including histone modification, X-chromosome inactiva-
tion, total genomic 5-methylcytosine content, sequence-
specific DNA methylation and global DNA methylation
status (by RLGS), were assessed in blood in comparison
with measures of gene expression (using microarrays and
quantitative real-time PCR). The results indicate that, while
these monozygotic twins are epigenetically indistinguish-
able early in life, at older age, they start to exhibit
remarkable differences in overall content and genomic
distribution of 5-methylcytosine DNA and histone acetyla-
tion, affecting their gene-expression profiles. This diver-
gence of genetically identical individuals over time could
be stochastic (due, for example, to early, random epimuta-
tions) or systematic when driven by different cellular
responses to environmental changes. However, the study
did not examine the same individuals serially over time, so
we cannot know from these data whether DNA methylation
changed or was different historically.
In a recent longitudinal study, Bjornsson et al. [8] looked
at epigenomic changes in blood collected from the same
individuals in two separate populations (one from Iceland
and one from Utah, USA) over time. The Iceland cohort was
composed of unrelated samples while the Utah samples were
family-based. From measuring global DNA methylation by a
luminometric methylation assay, a quantitative measurement
of genome-wide methylation, they observed time-dependent
changes in global methylation within the same individuals in
the two separate populations, showing decreases, as well as
increases, in methylation greater than 20% over an 11- to 16-
year span. Interestingly, in the Utah population, the observed
changes in DNA methylation showed a familial clustering.
This clustering occurred for both decreased and increased
methylation, although it was most striking for decreased
methylation. While shared family environment could explain
this clustering, most family members did not share house-
holds during the majority of the time between samplings.
Therefore, this familiality suggests that methylation stability
is genetically determined. DNA sequence variation at loci
involved in DNA methylation maintenance may contribute
to this familial clustering.
In conclusion, most or all studies on DNA methylation
during aging support the hypothesis that aging is associated
with a relaxation of epigenetic control. However, while this
may contribute to some of the well-documented age-related
phenotypes, such as functional decline and the development
of age-related diseases, the functional relevance of age-related
epigenetic changes remains largely unknown, possibly with
the exception of cancer. As we will discuss later in the review,
epigenetic deregulation has been clearly linked to the etiology
of cancer; for example, hypermethylation has been associated
with the silencing of genes involved in cell cycle regulation,
tumor-cell invasion, DNA repair, apoptosis, and cell signaling.
251
This epigenetic deregulation could provide the tumor cell with
a growth advantage, allowing it to metastasize and further
increase its genetic instability, which in turn will lead to
further changes advantageous to the tumor.
Interestingly, Wang et al. [105] provided evidence for a
link between epigenomic relaxation and Alzheimer's disease.
Using MALDI-TOF mass spectrometry (see further below for
a description of this method), these investigators analyzed the
promoter methylation of 12 potential susceptibility genes for
Alzheimer's disease in post-mortem late-onset Alzheimer's
disease (LOAD) brain samples and lymphocytes, as com-
pared to normal, healthy controls, of an age range of between
65 and 85 years. The results indicated only minor epigenetic
changes in a subset of genes. However, deviations for
individuals from the norm (obtained from the average in the
control samples) were significantly higher in the LOAD
patients, suggesting an age-specific epigenetic drift that may
influence age-of-onset and disease progression.
Hence, it may be difficult to find epigenetic changes that
can be associated with defined phenotypic consequences.
Instead, aging could be associated not with all or nothing
DNA methylation changes, but with a generalized loss of
normal epigenetic patterns. This deregulation may contribute
to the aging phenotype and perhaps once a critical threshold of
epimutations is reached, the tissue or organ, may start to
malfunction. Such decreased epigenetic control during aging
would be in keeping with the so-called dysdifferentiation
theory of aging proposed by Richard Cutler [57]. According
to this hypothesis, the primary process of aging is the time-
dependent drifting away of cells from their proper state of
differentiation [78]. In order to test this hypothesis, Cutler
and co-workers assessed whether, during aging, genes that
are normally not expressed in a tissue are gradually
derepressed. Indeed, they found an age-dependent increase
in the expression of the globin gene in non-erythroid tissues
such as liver and brain, thus suggesting a relaxation of
normal gene repression [78].
One of the best examples of the possible clinical
consequences of an accumulation of epigenetic errors during
aging is cancer. Aging is one of the highest risk factors for
cancer: a hallmark of cancer is that the accumulation of
mutations and epimutations provides the tumor cells with the
attributes to escape growth control, evade the immune system,
and invade tissues to finally lead to metastatic disease [103].
In his epigenetic progenitor model, Feinberg et al. [29]
proposed that cancer originates from stem cells that have
been primed to become neoplastic through a series of global
epigenetic disruptions. This is then followed by stochastic,
genetic, and epigenetic changes that drive tumor progression
[29].
From an epigenetic perspective, cancer is commonly
characterized as showing global DNA hypomethylation and
site-specific promoter hypermethylation. DNA hypomethy-
252
lation initiates chromosome instability and increased tumor
frequency, as has been shown in mouse models [26, 36,
91]. It is possible that one of the early epigenetic events, as
proposed by Feinberg et al. [29], consists of DNA hyper-
methylation of tumor suppressor genes, which has been
described for RB, p16, APC, and MLH1 [5, 6, 54]. For
example, promoter hypermethylation of p16 has been
documented in 15 cancer types, ranging from 9% to 49%
hypermethylation, while the BRCA1 promoter was found
hypermethylated in 10%15% of women with no familial
breast cancer [53]. Other gene targets are some of the
aforementioned imprinted genes, which are expressed only
from one parental allele, representing 1% of autosomal
genes. Deregulation of imprinted genes has been linked to
cancer risk, which is due to the haploid state of the
imprinted genes that eliminates any protection against
deleterious effects of recessive mutations [29].
Another interesting example of how increased genomic
instability and epigenetic defects may contribute to the
aging phenotype is represented by segmental progeroid
syndromes, a group of diseases in which patients develop
features of aging early in life. One of the best-known
progeroid syndromes is Hutchinson-Gilford Progeroid
Syndrome (HGPS, so called childhood progeria) [46].
HGPS is a rare syndrome characterized by some traits
typical of the aging phenotype, such as alopecia, reduced
bone density, poor muscle development, and premature
death (at age 1215), usually by myocardial infarction or
stroke [21]. HGPS is most often caused by a splicing defect
in exon 11 of the LMNA gene, which encodes lamins A
and C, major structural elements of the nucleus [90]. Cells
from HGPS patients show dramatic defects in nuclear
envelope structure [60], increased genomic instability,
DNA damage, and defects in the DNA repair machinery
[63, 94]. In particular, it appears that, in the cells of
individuals with HGPS, the repair machinery is not as
efficiently recruited to sites of damage and, consequently,
damage such as DNA breaks is not as efficiently repaired
[63]. HGPS cells exhibit an increased amount of nuclei
with foci of phorphorylated histone H2AX (-H2AX),
which is usually considered a marker for DNA double-
strand breaks and also for senescence [15, 63]. HGPS cells
also show epigenetics defects, including a decrease in the
heterochromatic markers histone H3 lysine methylation
(H3K9Me) and HP1 protein [89, 90, 94]. Interestingly,
Scaffidi et al., through a quantitative single-cell analysis,
showed that the percentage of cells with reduced H3K9Me
in cells from old donors (>80 years) was between 40% and
90%, comparable to that found in HGPS patient cells and
approximately more than two times higher than the
percentage found in young donors. In the same study, they
showed that the percentage of -H2AX foci is about 3.5
times higher in cells from old individuals compared with
Pflugers Arch - Eur J Physiol (2010) 459:247258
young ones. These results with cells from HGPS patients,
as well as normal aged individuals, suggest a causal
connection between disrupted nuclear architecture, reduced
heterochromatin, and human aging.
Another interesting example of a possible relationship
between aging and epigenetic changes comes from studies
in the yeast Saccharomyces cerevisiae (Brewer's yeast). In
this species, a progressive accumulation of extrachromo-
somal rDNA circles (ERCs) has been demonstrated to be
the cause of replicative aging [97]. Sir2, a NAD-dependent
HDAC necessary for transcriptional silencing near telo-
meres and mating type loci [42], has emerged as a key
regulator of health and lifespan [55]. It has been shown
that, in yeast, deletion of Sir2 shortens while overexpres-
sion increases replicative lifespan [55]. The anti-aging
effects of Sir2 in yeast are, at least in part, due to
translocation of the Sir2 from telomeres and mating type
loci to rDNA. These repeats are prone to recombination to
generate ERCs; the resulting ERCs are segregated to the
mother cell, accumulate, and ultimately cause senescence,
thus shortening the lifespan. Sir2 prevents recombination
and the formation of ERCs, thereby extending lifespan.
An important function of heterochromatin, therefore,
seems to be the suppression of recombination. This would
suggest an age-related loss of heterochromatin as a causal
factor in aging, which is in keeping with reported increases
in nucleosome spacing with age (in rats) and a lower
sensitivity to nucleases [56]. Orthologs of Sir2 have anti-
aging functions in other species, such as nematodes and
flies [85]. The role of the human ortholog SIRT1, in
longevity is not yet known, but there is some evidence
suggesting that, in mammals, the sirtuins (as the Sir2
proteins are called) play key roles in many important
physiological functions, as well as in age-related diseases
[43]. Interestingly, Oberdoerffer et al. [75] have recently
shown that, in mouse embryonic stem (ES) cells treated
with hydrogen peroxide, the Sir2 ortholog SIRT1 relocates
from repeat sequences and gene promoters to sites of DNA
double-strand breaks (DSBs) promoting DNA repair and,
hence, genomic stability. Transgenic mice overexpressing a
truncated form of p63 (a homolog of p53) show signs of
premature aging and decreased levels of SIRT1 [98]. This is
in keeping with a study by Baur et al., which showed that
resveratrol, a Sirt1 activator, extends the healthy lifespan of
mice on a high-fat diet [4].
New methods for measuring epigenetic changes
Most of the work reviewed above is based on classical,
single-locus assays for analyzing epigenetic marks. More
recently, studies on epigenetics have been greatly facilitated
by the development of improved methods for the analysis
Pflugers Arch - Eur J Physiol (2010) 459:247258
of epigenetic modifications on a global basis. For instance,
it is now possible to profile the sites of DNA methylation or
various covalent modifications to the histone tails through
genome-wide analysis either by hybridization to tiling
microarrays (ChIP-chip) [10] or using next-generation
sequencing (ChIP-Seq) [79].
A substantial effort is underway within the epigenomics
community to identify DNA methylation patterns and
histone marks on a genome-wide scale using those methods
in order to provide new insights into the regulation and
dynamics of DNA methylation in genomes. Here, we will
focus on DNA methylation assays we have now begun to
use routinely in our studies of aging and age-related
diseases.
The assay we use for the analysis of global DNA
methylation is termed the Hpa II-tiny fragment Enrichment
by Ligation-mediated PCR (HELP) assay [58]. The
technique compares genome representations generated by
the restriction enzyme Hpa II, which will not cut methyl-
ated CCGG sites, with those generated by its methylation
insensitive isoschizomer MspI, followed by ligation-
mediated PCR. These fragment mixtures are used as probes
to hybridize tiling microarrays for assessing the presence or
absence of Hpa II fragments at specific loci, a measure of
hypo- or hypermethylation, respectively. Its built-in control
(Msp I fragments) and the possibility of using customized
chips make this technique reliable and versatile, i.e., it can
be adapted to examine DNA methylation in certain regions
or in entire genomes. An analytical pipeline for HELP data
processing written in the R programming language was
recently published [99]. John Greally, the inventor of the
assay, is presently developing next-generation sequencing
approaches to substitute for current tiling array read-outs
(personal communication).
Many techniques for DNA methylation analysis are
based on bisulfite treatment of DNA to allow PCR-based
detection of methylated or unmethylated cytosines either
globally or in specific sequences. Sodium bisulfite is used
to convert cytosine residues to uracil in single-stranded
DNA, under conditions whereby 5-methylcytosine remains
non-reactive. The converted DNA can be amplified with
specific primers targeting specific regions of interest (such
as promoter regions), followed by downstream detection
techniques (Sanger sequencing, MassArray, microarrays,
next-generation sequencing). For example, the MassArray
technology allows precise and quantitative DNA methyla-
tion detection in selected target regions (300500 bp) based
on base-specific cleavage of single-stranded nucleic acids
coupled with MALDI-TOF MS detection [27]. After the
bisulfite treatment of the DNA, a PCR amplification step is
carried out to yield an amplicon with a T7 promoter tag.
Next, in vitro RNA transcription is performed on the
reverse strand followed by RNase A cleavage at specific
253
bases (U or C). Cleavage products are spotted on a chip
containing a matrix compound to assist in laser deioniza-
tion. Within the cleavage products, methylation-dependent
C to T variation appears as G/A generated from the reverse
strand by base-specific cleavage. These G/A variations
result in a mass difference of 16 Da per CpG site, which is
easily detected by the MassArray analyzer, resulting in a
signal pair pattern from the methylated and non-methylated
template DNA (http://www.sequenom.com/). Each assay
can read several CpG dinucleotides so that, for example,
a whole promoter can be analyzed in one experiment
(Fig. 3).
It has to be noted that, while the methods of epigenetic
analysis have been greatly improved in the past decade,
allowing the generation of single-base resolution maps of
the entire epigenome, thereby greatly facilitating the
detection of systematic changes, stochastic variation needs
to be analyzed at the single cell level. Suitable assays for
this purpose are still lacking.
Epigenetic changes during cell senescence
Cellular senescence, the irreversible cessation of mitotic
activity, was originally considered as an in vitro model
system for in vivo aging [45]. Currently, cellular senes-
cence is known as a stress response and a major tumor
suppressor mechanism [14, 15]. The senescence response is
essentially a DNA damage response and can be induced by
clastogenic agents, such as ionizing radiation, or activated
oncogenes, i.e., through replicative stress [13, 15, 16].
Importantly, cell senescence can be triggered by an
excessive number of cell divisions, thereby shortening
telomeres and generating double-strand breaks [44, 112].
This is called replicative senescence and explains why
human cells in culture have a finite lifespan.
Senescence has been linked to a variety of morpholog-
ical changes, such as an enlarged cell shape, decreased
protein synthesis and degradation, resistance to apoptosis,
and increased activity of some lysosomal enzymes includ-
ing senescence-associated -galactosidase (SA-gal) [44,
50]. Senescent cells can be identified by cytological
markers of senescence-associated DNA damage foci
(SDFs); SDFs contain proteins that are associated with
DNA damage (such as -H2AX and p53-binding protein-1
(53BP1)) [22, 47].
Cell senescence entails widespread changes in gene
expression [20, 74]. Narita et al. [74] have shown that the
senescence phenotype is also associated with changes in
chromatin structure. Senescent cells tend to form special-
ized domains of heterochromatin, called senescence-
associated heterochromatin foci (SAHF). SAHF have been
proposed to play a role in the silencing of proliferation-
254
Fig. 3 EpiGram tab for the gene MKRN3. The EpiGram is a graphical
representation of methylation ratios found in each sample for the
amplicon studied. Each sample's nucleotide sequence is displayed as a
series of individual CpGs, which are color-coded circles on the same line.
The color within the circle denotes the level of methylation found at this
particular site in the selected sample. The color spectrum ranges from
yellow (or 0% methylated) CpG units to blue (100% methylated) CpG
promoting genes, thus contributing to the senescence-
associated proliferation arrest.
Cellular senescence is an important barrier to the
development of cancer in mammals due to its ability to
arrest proliferation and prevent neoplastic progression of
cells harboring oncogenic lesions [14, 93]. At the same
time, there is increasing evidence that an accumulation of
dysfunctional senescent cells contributes to mammalian
aging [15]. Indeed, senescent stromal cells may actually
promote the proliferation and tumorigenesis of epithelial
cells [13, 16, 59]. This is, in part, due to the capability of
senescent cells to secrete growth factors, as well as extracel-
lular matrix components, matrix-degrading enzymes, and
inflammatory cytokines that can disrupt tissue integrity and/
or stimulate nearby cells to proliferate, thus creating a pro-
oncogenic tissue environment [13, 16]. Furthermore, the
senescent phenotype has been linked to some age-related
diseases such as osteoarthritis, atherosclerosis, and liver
cirrhosis [69, 110].
At the moment, very little is known about the
relationship between DNA methylation and cell senes-
cence. Overall, 5-methylcytosine content and DNMT1
activity diminish when cells senesce [39]. Young and
Smith have shown that, in response to the demethylating
agent 5-azacytidine (5AZA), human fibroblasts undergo
senescence. Interestingly, human fibroblasts lacking p21
fail to undergo senescence after 5AZA treatment,
indicating that p21 is an essential component of this
5AZA-induced arrest [113, 114]. Nothing is so far known
about the methylation status of genes involved in
processes found associated with senescence.
Pflugers Arch - Eur J Physiol (2010) 459:247258
units. The example above is an EpiGram obtained for the studied
imprinted gene MKRN3 in liver of wild-type mice and mice harboring a
defect in non-homologous DNA end joining, a DNA double-strand break
repair process (Ku80/). The first ten lines represent five wild-type
samples (in duplicates), while lines 11 to 20 represent the Ku80/
samples. The last two lines correspond to a universal unmethylated
DNA sample, used as a conversion control
Environmental effects on the epigenome:
impact on aging
Recent findings support the idea that the epigenome is a
key target of environmental modifications with long-term
effects on disease susceptibility and aging. The aforemen-
tioned study on monozygotic twins [32] suggests that the
emerging differences in the epigenome as twins get older
may reflect environmental factors and lifestyle. One aspect
of the lifestyle is the diet; for example, a diet poor in folate
has been linked to genomic instability [52] and hypome-
thylation [33]. Interestingly, a deficiency in folate and
methionine has been shown as the cause of aberrant IGF2
imprinting [107].
Several studies suggest the mother's behavior or diet can
affect epigenetic patterns in her offspring. For example, a
study carried out on Agouti pregnant mice showed that
feeding the mice with a diet rich in methyl donors
influences coat color, body weight, and health of their
progeny [24]. Another study conducted in rats showed that,
in the absence of appropriate nurturing, there is a
demethylation of the glucocorticoid receptor (GR) in the
hippocampus of the offspring and, as a consequence,
increased expression of the receptor gene later in life,
thereby leading to an increased stress sensitivity of the
adolescent rats [109].
Those findings are in keeping with a new theory called
the fetal basis of origins of adult-onset disease [71]. This
theory posits that the effects of in utero exposures can cause
permanent functional changes that result in increased
susceptibility to disease/dysfunction later in the life span.
Pflugers Arch - Eur J Physiol (2010) 459:247258
An example of this is the so-called Second World War
Dutch Hunger Winter from 19441945; children of cause of diseases. We have provided an overview of the
pregnant women exposed to famine were more susceptible
to diabetes, obesity, and cardiovascular disease later in life
[64]. This finding suggests that transient environmental
conditions early in human gestation can be recorded as
persistent changes in epigenetic information and have life-
long phenotypic consequences. Indeed, as discussed previ-
ously, early embryonic development is of special interest in
this respect because this is a crucial period for establishing
and maintaining epigenetic marks [82].
Several studies in mammals [11, 62, 70, 80, 81] are now
supporting the hypothesis that epigenetic alterations might be
inherited transgenerationally and affect the health of future
generations. For example, embryonic rats exposed to the
antiandrogenic compound vinclozolin show decreased sper-
matogenesis, and this trait is passed to several subsequent
generations [1]. Transgenerational epigenetic inheritance has
now also been demonstrated in other eukaryotic organisms,
for example, plants and yeast [18, 41]. The fact that an
epigenetically induced adaptation to the environment can be
passed through generations has a distinctly Lamarckian
flavor, which is a topic of discussion [51].
Conclusions and future prospects
It is becoming evident that epigenetics plays a key role in
the development of diseases associated with aging. Epi-
genetics has been considered to be at the epicenter of
modern medicine [28] because it explains the relationship
between an individual's genetic background, the environ-
ment, aging, and disease. The concept of DNA as a static,
fixed entity has been replaced by a new vision where DNA
is highly dynamic, capable of large fluctuations, and able to
interact actively with the environment. According to the
new findings, DNA responds to the environmental stimuli
by modifying its properties in adapting to changes, in order
to maintain an internal balance and proper functionality.
We have reviewed the pivotal role of epigenetics in
conferring to DNA this plasticity; thanks to the epigenetic
marks, cells know their role in life. Without its epigenetic
memory, cells would lack identity and function. In addition
to that, genes can have a memory and can learn by
experience, and the (epigenetic) marks left by such
experiences can be passed to daughter cells and to future
generations. This gives rise to a fascinating concept: health
and general physiology can be affected not only by the
interplay of our own genes and conditions of life but also
by the inherited effects of the interplay of our own genes
and the environment of our ancestors.
Despite the fact that the epigenome is highly dynamic, it
is also strictly controlled. As discussed, the loss of this
255
control would be detrimental and its relaxation can be the
role of the relaxation of this control in the development of
age-related diseases, giving emphasis to the concept that
aging, at least in part, is a time-dependent, epigenetically
mediated loss of phenotypic plasticity.
One of the most fascinating aspects of possible therapeutic
intervention is that epimutations can be corrected because
epigenetic marks are, by definition, reversible and dynamic.
To date, DNMT inhibitors (such as decitabine) and histone
deacetylase inhibitors (SAHA) are in clinical trials in cancer.
Many companies are currently focusing on HDAC inhibitors.
The first one, Vorinostat, has been approved by the FDA in
October 2006 for second-line therapy of cutaneous T-cell
lymphoma [65], while the combination of classic anti-cancer
treatment has been promising in reversing acquired chemo-
therapy resistance [38].
Many questions regarding epigenetics and its role in age-
related diseases still remain open. Do epimutations accu-
mulate stochastically with aging in different tissue types
and what are, eventually, the consequences of this accumu-
lation? Which epigenetic marks confer identity to the
cell, which genes are responsible for enhanced disease
susceptibility when epigenetically deregulated, which envi-
ronmental factors are deleterious for the epigenome and,
finally, which epigenetic biomarkers could be used for
detection of early-stage diseases?
The elucidation of the interaction of the environment
with the epigenome and their role in aging will allow for
the development of novel epigenetic-based diagnostic,
preventive, and therapeutic strategies. Manipulating and
understanding the epigenome holds promises for preventing
and treating age-related diseases.
Acknowledgements We thank the SENS Foundation, the National
Institute on Aging, and the Ellison Medical Foundation for supporting
the research of the authors.
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