Introduction
In mammals, aging is
a complex phenomenon likely to result from a myriad of molecular changes.
Studying mammalian aging experimentally represents an obvious challenge that
has hampered our understanding of the molecular mechanisms underlying this
process. In culture, cells have a limited proliferative lifespan in which they
undergo an irreversible cell cycle arrest known as replicative senescence [1]. Cellular senescence corresponds to an
irreversible exit from the cell cycle and can be triggered in primary cells by
different stimuli, including expression of activated oncogenes (oncogene-induced
senescence), or serial passaging (replicative senescence). Specifically,
cellular senescence is believed to occur when cellular growth is promoted while
cell cycle progression is inhibited, as
observed for example upon strong mitogenic
stimuli [2,3]. Irrespective of the stimulus
used to drive senescence, senescent cells become flat and enlarged, and are
positive for β-galactosidase activity
in acidic conditions [4]. Senescence
is believed to reflect the aging process at a cellular level, and therefore
represent an ideal model system to study the molecular basis of aging. While
there has been some question as to whether cellular senescence contributes to
organismal aging, it is believed that senescence can lead to the reduction of
the regenerative potential of the stem cell pool. Additionally, accumulation of
senescent cells in tissues could impair tissue function [5].
Importantly, recent studies using skin biopsies from mammals have shown that
senescent cells accumulate, in vivo, during aging and can end up representing
more than 15% of total cells in aged animals [6]. This study
suggests that cellular senescence is a hallmark of organismal aging.
Importantly,
chromatin modifications have been shown to play a role in the determination of
the senescent phenotype. In eukaryotic cells, DNA is tightly associa-ted with
histones as well as non-histones proteins to form the chromatin fiber.
Specifically, histones are assembled as octamers composed of two copies of each
core histone, H2A, H2B, H3 and H4 to form the nucleosome. Through
post-translational modifications and protein recruitment, the nucleosome is an
essential component of dynamic chromatin regulation. Histones are subjected to
specific modifications including acetylation, methylation, phosphorylation,
ubiquitylation, ADP-ribosylation and sumoylation, while DNA itself can be
methylated [7,8]. These specific modifications result in higher order
chromatin structure and regulate DNA accessibility. Importantly, the
combination of different modifications results in specific transcriptional or
structural outcomes, underlining the existence of a "histone code" [9,10]. Such a code tremendously enhances the degree of
information coded solely by the DNA molecule. Therefore chromatin modifications
impact all biological processes related to DNA, including gene expression and
genomic stability, both of which are at the nexus of the senescent phenotype.
Here we discuss the evidence suggesting that chromatin modifications can be the
driving force behind the senescent phenotype and their implications in
organismal aging.
Age
related DNA methylation changes
Early experiments in mammalian cells have
demonstrated the occurrence of a global decline in DNA methylation in cultured
primary fibroblasts from mice, hamsters, and humans compared to their
immortalized counterparts. These observations suggested for the first time that
replicative senescence may correlate with changes in chromatin structure [11].
Importantly, these results have also been confirmed in vivo [12], as the
5-methyldeoxycytidine content in DNA isolated from mouse livers, brains, and
small intestinal mucosa significantly decreased as the animals age. The overall
decline of methylation results mostly from the loss of DNA methylation at
repetitive regions. Repetitive sequences, including satellite repeats at
pericentric and centromeric loci, and interspersed repeat sequences, represent
about 45% of the mouse genome [13] and are
normally highly methylated. It is widely accepted that DNA methylation
correlates in most cases with the generation of a repressive chromatin
structure, thus preventing potential deleterious recombination within
repetitive DNA sequences. Interestingly, these deleterious recombinations
accumulate with age, leading to increased incidence of disease including cancer
[14], suggesting
that the progressive loss of DNA methylation at repeat sequences may account,
at least in part, for the accumulation of genomic aberration in aging
organisms. In addition, since DNA methylation is normally associated with gene
repression, it is conceivable that hypomethylation of these regions results in
gene re-expression at these loci.
Despite
a global decrease in total DNA methylation, specific sites in the genome become
hypermethylated as mammals age [15], suggesting
that the enzymatic activity associated with DNA methylation is not entirely
impaired during the aging process. This increase in DNA methylation occurs
mainly at CpG islands, which are typically unmethylated in normal tissues. However,
methylation at discrete CpG islands increases during the aging process [15].
Specifically, the locus encoding the estrogen receptor gene has been shown to
become increasingly methylated at CpG islands in human colon in an age
dependent fashion [16]. Similarly,
genes encoding ribosomal RNA become progressively methylated in rat liver [17]. Finally, multiple
tumor suppressor or tumor related genes such as APC, E-cadherin, GSTP1, and p16INK4A
have shown different levels of methylation in an age and tissue specific manner
[15,18,19].
While
the significance of these changes remains unclear, these observations, pointing
to the differential modulation of DNA methylation at gene-specific loci with
age, are at first glance in agreement with the "redistribution of chromatin
modifiers" (or RCM) hypothesis [20]. According
to the RCM hypothesis in aging, chromatin modifiers leave their normal target
loci as the cell ages, and are relocalized to other sites, thus altering the
chromatin structure in two ways. However, the substrate specificity of
methyl-transferases makes this unlikely in the case of DNA methylation. Various
enzymes account for the methylation events that occur on genomic DNA in
mammals. Dnmt1 is responsible for methylating newly replicated DNA and,
accordingly, uses hemimethylated DNA as a substrate; it is therefore
responsible for maintaining methylation patterns [21]. In
contrast to Dnmt1, the related Dnmt3a and Dnmt3b enzymes contribute to de
novo methylation, in that they can methylate previously unmethylated DNA [22]. Enzymatic
assays have revealed a decrease in maintenance methylation concomitant to an
increase in de novo methylation in senescent human fibroblasts compared
to their early passage counterparts [23]. Accordingly,
the amount of transcripts corresponding to Dnmt1 decrease significantly as
fibroblasts senesce, while Dnmt3b transcription is up-regulated, suggesting the
loss of methylation results at least in part from changes in Dnmts
transcriptional regulation [24].
Collectively, these observations suggest that during the aging process, a
change in the balance between de novo methyl-transferases and maintenance
methyl-transferases is likely to contribute to the changes observed in
chromatin structure. Whether these changes in DNA methylation directly
contribute to cellular aging or represent a mere consequence of this phenotype
remains an unresolved issue.
Histone
modifications, histone modifiers, and senescence
As
presented above, histones are modified at many residues within the tails or
even the globular domains, where different covalent modifications can be found [10,25].
Specific histone modifications have yet to be fully studied and understood in
organismal aging. Deciphering the contribution of specific histone modifiers to
the aging phenotype is impaired by the usually pleiotropic effects of these
enzymes, whose inactivation leads in many cases to cell lethality.
In spite of these impediments, specific
histone modifications have been linked to the aging process. For example, trimethylation
of histone H4 at lysine 20 (H4K20me3), a hallmark of constitutive heterochromatin,
increases in rats livers with age, and is found upregulated in a cellular model
of progeria [26,27]. Since
genetic inactivation of Suv4-20, a family of enzymes responsible for this
modification, results in proliferation defects due to increased sensitivity to
DNA damage, the specific function of these enzymes in cellular senescence
remains unclear [28]. In
addition, the modulation of Suv4-20 methyl-transferase activity or expression
level in young versus old cells or tissues has yet to be investigated. By
contrast, a direct and well-established function for a histone modifier in
senescence is that of the histone methyl-transferase EZH2. As reviewed in [29], the INK4A
locus is a major player in the induction of senescence in mammalian cells. Upon
replicative or oncogenic stress, the products of this locus, the p19ARF
and p16INK4A proteins, accumulate, leading to growth arrest. Recent
studies have implicated an EZH2-containing complex (known as PRC2) in the
transcriptional repression of the INK4A locus in proliferating cells. Upon
signals triggering senescence, EZH2 levels decrease, concomitant with the loss
of H3K27me3 mark at the INK4A locus [30]. While
these observations definitely implicate a histone methyl-transferase on the
induction of a senescence transcriptional program, whether active demethylation
on H3K27 is also involved in this process remains unclear [31]. Finally,
the histone demethylases KDM2a and KDM2b, which target methylated H3K36,
prevent senescence by modulating the p53 and Rb pathway [32,33].
Specifically, overexpression and loss-of-function experiments have shown that
this function is accomplished through their ability to demethylate H3K36me
histones at the p15INK4B locus, leading to its repression. Together,
these data demonstrate that histone modifiers are able to contribute in a
gene-specific manner to the induction or prevention of cellular senescence. By
contrast, the contribution of chromatin modifiers to cellular senescence and
aging as global regulators of chromatin structure remains elusive and awaits
genome-wide approaches to definitively ascertain these functions.
Similar
to what was reported for DNA methylation, the total levels of histone
acetylation are likely to change as the organism ages. Consistent with this
observation, the levels of the histone deacetylase HDAC-1 decrease upon serial
passaging of primary human fibroblasts [34].
Importantly, treatment of primary human fibroblasts with the HDAC inhibitors
Trichostatin A (TSA) or sodium butyrate induce a senescence-like state,
suggesting that modulation of histone acetylation through class I and II HDACs
is an essential step in the establishment of senescence [35]. Whether
this effect results from global changes in histone acetylation or from the
transcriptional activation of discrete loci remains unknown. Seemingly
contradictory, the recent observation that HDAC1 overexpression in melanoma
cells is sufficient to induce an irreversible senescence program demonstrates
that the function of class I HDACs in senescence is likely to be more complex
than initially anticipated [36]. The precise contribution of HDAC1 and other class I and
II HDACs to the establishment and maintenance of the senescent state and
organismal aging will undoubtedly become more clear in the near future as
genome wide approaches, including promoter location analysis, allow the
delineation of HDACs targets upon pro-senescence signals.
The
complex case of sirtuins
The
members of the evolutionary conserved Sirtuin family of proteins are nicotinamide
adenine dinucleotide (NAD)-dependent proteins with histone deacetylase
activity, whose founding member is the yeast Sir2 protein. In yeast, worms and
flies, extra copies of sirtuins extend replicative lifespan, and the
deacetylase enzymatic activity of sirtuins is required for this function [37-40].
Additionally, Resveratrol, a potent inducer of sirtuins enymatic activity,
extends life span in these species, in a sirtuin-dependent manner [41]. The
correlation between sirtuins and aging in metazoans has led to an interest in
the mammalian homologs of Sir2, the SIRT proteins. The mammalian SIRT family
consists of seven members, SIRT1-7. SIRT1, however, is reported to be the most
similar to yeast Sir2. The contribution of SirT1 to senescence and aging in
mammals is complex, and may involve non-chromatin substrates. For instance,
SIRT1 was shown to deacetylate p53 in vivo, leading to p53 inactivation [42-44]. In
addition, loss-of-function experiments in mouse fibroblast demonstrated that
SIRT1 is required for replicative senescence resulting from chronic genotoxic
stress [42]. In these
experiments, SIRT1-/- mouse embryonic fibroblasts (MEFs) fail to upregulate p19ARF
upon serial passaging in normal culture conditions, correlating with their
inability to undergo replicative senescence. Whether this effect of SIRT1 on
p19ARF regulation involves chromatin regulation or involves the
deacetylation of a non-histone protein is unknown. Recently, a direct
correlation between SIRT1 regulation of chromatin structure and aging has been
uncovered in mouse embryonic stem cells [45]. Using
genome-wide location analysis, it was demonstrated that, upon genotoxic stress,
SIRT1 is delocalized from repeat sequences and numerous gene promoters to bind
to sites of DNA damage, in a process consistent with the afore-mentioned "RCM"
hypothesis (for redistribution of chromatin modifiers). Such relocalization may
affect the aging process in a dual manner: First, it may suppress genomic
instability by contributing to DNA repair upon chronic genotoxic stress that
occurs in aging organisms. Second, the loss of SIRT1-driven transcriptional
repression at its natural targets correlates with the deregulation of gene
expression consistent with what is observed in aging tissues [45]. These
observations strongly suggest that forced expression of SIRT1 may represent a
strategy to alter aging-related chromatin changes in mammals.
Another mammalian sirtuin family member,
the SIRT6 protein, has been involved in aging-related chromatin changes in
mice. SIRT6 deficient mice display a premature aging-phenotype and die rapidly
after birth, displaying an acute multi-organ degenerative syndrome [46]. While SIRT6
has been shown to prevent telomere dysfunction in human cells by deacetylating
H3K9 at telomeric loci (see below), this function is not conserved in mouse
cells and therefore cannot account for the aging-phenotype in SIRT6-/- mice [47]. A recent
report shed light on a potential molecular mechanism for SIRT6-mediated
prevention of aging in mice [48]: SIRT6 was
shown to be tethered by NF-κB target genes and to deacetylate
H3K9 on these promoters, thus attenuating this signaling pathway. Since NF-κB has been implicated in the induction of a senescence specific program
and genetic alteration of NF-κB signaling rescues the aging
phenotype elicited by SIRT6 deficiency [48,49], it is
tempting to conclude that SIRT6 prevents aging at least partly through the
modulation of the chromatin structure and transcription program driven by this
specific signaling pathway. Finally, reinforcing the connection between DNA
damage and cellular senescence, SIRT6 was recently shown to deacetylate H3K9 at
sites of double strand breaks (DSB), and to be required for the mobilization of
DNA-PK at these sites and the subsequent resolution of DSBs [50].
Senescence
Associated Heterochromatic Foci and the induction of senescence
As
presented above, senescence, along with stem cell depletion, is one cellular
manifestation of organismal aging. A direct involvement of chromatin
modifications in the establishment of the senescent phenotype was recently
revealed in that senescent cells display specialized and discrete subnuclear
structures called S
enescence A
ssociated H
eterochromatic F
oci
(SAHF) [51]. SAHF were
first described in human primary fibroblasts driven to senescence via serial
passaging or oncogenic stress [52].
Importantly, these heterochromatic foci are not present in quiescent cells. SAHF
are easily stained in senescent human cells upon incubation with
4'-6-Diamidino-2-phenylindole staining (DAPI), resulting in the visualization
of bright positive nuclear foci. These foci are resistant to digestion by
nucleases, consistent with the dense heterochromatic nature of the
corresponding genomic loci. Indeed, SAHF contain chromatin marks that are
reminiscent of those found in constitutive heterochromatin, including
hypoacetylated histones, methylation of lysine 9 of histone H3, and
Heterochromatin Protein 1 (HP1) [52]. However,
SAHF also contain specific marks, absent from constitutive heterochromatin such
as enrichment of macroH2A and HMGA proteins and depletion of linker histone H1 [53-55].
Consistent with a function in the permanent cell cycle exit upon induction of
senescence, SAHF embed genomic loci encoding pro-proliferative proteins into
heterochromatin structures, thus preventing their accessibility by the
transcription machinery [52]. In
addition, it appears that each chromosome condenses into one single SAHF, where
the loci to be repressed are found in the interior or immediate periphery of
the corresponding focus [55]. The
molecular events leading to the formation of SAHF are still under
investigation, but several proteins have been demonstrated to contribute to the
formation of SAHF: They include the histone chaperones HIRA and Asf1, HP1g, HMGAproteins, and the H3K9me3 methyl-transferases
Suv39h1/h2 [54,56,57].
Impor-tantly, genetic disruption of Suv39h1 in mouse lymphocytes promotes
tumorigenesis by preventing oncogene-induced senescence upon activated Ras
overexpression [56] This
important result suggests that the formation of SAHF is essential for
senescence to occur in vivo. However, it is important to note that primary
fibroblasts inactivated for Suv39h1 and h2 remain susceptible to replicative
senescence [52,58]. This
discrepancy suggests that either the formation of SAHF upon replicative or
oncogene signal utilize different pathways, or that the enzymatic machinery
required for SAHF formation differs in lymphocytes and in fibroblasts. More
experiments are needed to precisely define the contribution of SAHF to the
senescent phenotypes. In addition, many questions regarding the generation of
these specialized loci remain, including the identity of the complex required
for the deacetylation of the loci embedded in SAHF, as well as the molecular
basis for the targeting and coordination of the different chromatin modifying
activities affecting SAHF formation. Finally, since all chromatin modifications
identified in SAHF have now been shown to be reversible, the irreversible
nature of senescence is likely to rely on additional molecular mechanisms yet
to be identified.
Lamins,
progeria and the aging phenotype
Lamins belong to a family of intermediate
filaments, which are the main structural components of the nuclear lamina. They
have been shown to play a role in chromosome organization and to interact with
chromatin and DNA through lamin binding proteins [59].
Importantly, lamins are mutated in multiple human diseases, known as
laminopathies. The connection between lamins and aging was first suggested
through the genetic analysis of a progeria syndrome: Hutchinson-Gilford
progeria syndrome (HGPS) is a rare genetic disorder characterized by premature
aging. Patients are born normal, but develop age-associated disorders within a
year after birth. Characteristic symptoms include hair loss, artheriosclerosis,
loss of subcutaneous fat, growth retardation, osteoperosis, and aged skin [60]. HGPS is
caused by a single nucleotide substitution in the Lamin A gene resulting in a
cryptic splice site in exon 11 causing a 150 nucleotide-deletion from the 3'
end of the lamin A gene, leading to the synthesis of an abnormal protein,
LA∆50 [61,62].
Fibroblasts from young HGPS patients have relatively normal nuclei and nuclear
lamina. However, as these cells are passaged in culture, several abnormalities
become detectable in the nucleus. These include lobulation of the nuclear
envelope, thickening of the nuclear lamina, clustering of nuclear pores, and
loss of peripheral heterochromatin [59]. The
severity of these nuclear abnormalities is highly correlated with an overall
accumulation of the LA∆50 protein upon passage in culture [63].
Importantly, ectopic expression of LAΔ50 protein in normal
cells is sufficient to recapitulate the nuclear defects observed in cells
derived from HGPS patients [63]. Further
strengthening the connection with cellular aging is the recent observation that
similar mechanisms and defects occur in healthy individuals. Indeed,
fibroblasts from older healthy individuals (81 to 96 years) express the
LA∆50 transcript at low levels and display nuclear defects similar to
those from HGPS patients [64].
At the chromatin level, HGPS fibroblasts exhibit a
loss of nuclear peripheral heterochromatin, and, in cells derived from older
HGPS patients, several heterochromatin marks, including mono-methylated H3K9
and tri-methylated H3K9, are globally almost undetectable. As female HGPS
fibroblasts are passaged in culture, a reduction of H3K27 trimethylation is
observed on the inactive X chromosome. In fact, loss of this mark was detected in
early passage cultures before any nuclear defects were observed suggesting
heterochromatin defects precede nuclear defects [27]. The loss
of the H3K27 tri-methyl mark was also correlated with a 9-10 fold decrease in
EZH2, the enzyme responsible for maintenance of this mark [27].
Furthermore, late-passage HGPS cells show an upregulation of H4K20 tri-methyl [27], which, as
presented above, is also observed in livers of older rats [26].
Importantly, similar defects in heterochromatinization accumulate, though to a
lesser extent, in aging wild-type cells. Specifically, skin fibroblasts from
healthy old individuals (81-96 years) show decreased staining for HP1 and
H3K9me3 compared to the same cells from young individuals (3-11 years) [64].
The molecular mechanisms linking lamin A to
heterochromatin remains unclear, but two hypothesis are currently being
explored. First, the function of specific transcription factors could be
affected by mutations in Lamin A. One candidate is the retinoblastoma protein,
Rb, which binds directly to type-A lamins and whose function is regulated by
this association [65,66]. Given
the role of Rb in the regulation of histone methylation [67], including
H3K27, H3K9 and H4K20, it is conceivable that alteration of Lamin A affects
histone methylation through impairment of the Rb pathway. A second,
non-exclusive, hypothesis is that defects in Lamin A protein induce changes in
nuclear localization of specific loci. For example, chromosome territories can
be displaced from the nuclear periphery to the nuclear interior upon expression
of mutant Lamin A. This would be consistent with the recent demonstration that
Lamins associated microenvironments are organized into transcriptionally
defined domains [68]. Given the
connection between nuclear tethering and transcription/chromatin modifications
[69],
alterations in nuclear envelope structure resulting from Lamin A mutations are
likely to affect chromatin globally. Although still correlative, these studies
strongly suggest that loss of heterochromatin in the HGPS model could
potentially drive the premature aging process and may contribute to the normal
aging process.
Chromatin
structure of telomeres and the aging phenotype
Telomeres
are repetitive DNA elements at the tips of chromosomes that protect DNA ends
from recom-bination and degradation [70]. As mammals
age, their telomeres become shorter during each round DNA replication due to
the inability of the transcription machinery to completely replicate the ends.
Eventually, human telomeres reach a critically short length that activates the
DNA damage pathway leading to replicative senescence. Telomere attrition is
considered to be a mechanism for organismal aging in which shortened telomeres
lead to stem cells depletion and eventual loss of tissue regeneration [71].
Telomeres
from normal mouse lab strains are much longer than those from humans and
therefore, do not erode enough to induce senescence. However, studies in mice
have allowed elucidation of the mechanisms behind epigenetic regulation of
telomeres, which are likely to be relevant in other mammalian species. These
studies demonstrate that telomeres are heterochromatic in nature, and that loss
of heterochromatic marks may regulate telomere length, thus potentially
affecting cellular aging. Specifically, subtelomeric regions comprise
methylated DNA, as well as H3K9 and H3K20 methylated nucleosomes [72,73]. Loss of
DNA methyltransferase activity in mouse embryonic stem cells results in
decreased methylation at subtelomeric regions hence increased telomere
recombination [74]. Moreover,
MEFs deficent in the Suv39h1 and Suv39h2, H3K9 histone methyltransferases, have
abnormally long telomeres, in agreement with the hypothesis that chromatin
structure contributes to the regulation of telomere length [72]. In the
telomerase deficient mouse, critically short telomeres are characterized by the
loss of heterochromatin marks at subtelomeric regions including DNA
methylation, H3K9 and H4K20 trimethylation and histone hypoacetylation [75]. Therefore,
the interplay between chromatin modifications and telomere length appears to be
complex and remains to be fully investigated.
Recent studies in human cells have
directly linked chromatin modifications to cellular senescence. Human primary
fibroblasts, in which levels of the H3K9 deacetylase SIRT6 are experimentally
decreased, undergo premature cellular senescence [47]. These
cells exhibit telomere dysfunction and chromosomal end-to-end to fusions,
specifically linked to alteration of SIRT6 enzymatic activity. SIRT6 knockdown
human fibroblasts and SIRT6 deficient mouse cells show H3K9 hyperacetylation at
telomeric loci, thereby, demonstrating SIRT6 H3K9 deacetylase targets
telomeres [47]. This study
demonstrates that telomere length and function require epigenetic modifications
and alterations to these regulatory mechanisms lead to telomeric dysfunction
and a subsequent senescent phenotype
Concluding
remarks
As
organisms age, changes occur at many levels, including transcriptional
regulation and nuclear architecture. While it becomes clear that modulation of
the chromatin structure influences molecular events at the nexus of cellular
aging, several questions remain: First, are specific chromatin modifications a
cause or a consequence of cellular senescence? As presented above,
loss-of-function experiments have begun to shed light on the direct
contribution of specific modifiers to cellular aging. Second, if chromatin
modifiers can directly contribute to the aging phenotype, what is the molecular
circuitry leading to the modulation of their activities during the aging
process, and may it be altered as a therapeutic means? Finally, one of the
biggest challenges in the understanding of the aging process is to decipher the
connections between the seemingly independent molecular events that have been
reported in different settings of senescence. The recent development of small
molecules that interfere with specific histone modifiers and their use in
clinical trials, should provide new opportunities for the therapeutic
modulation of the aging phenotypes in the future.
We
thank Drs. Lawrence Gardner, Brooke Grandinetti, Petar Jelinic and Isabelle
Marie for helpful comments on the manuscript. We apologize to any colleague
whose work could not be cited due to space limitations. TD is supported by a
predoctoral NIH training grant CA009161. Work in the David lab is supported by
the American Federation for Aging Research (AFAR), The American Cancer Society
(ACS) and the March of Dimes.
The authors of this manuscript have no conflict of interests to declare.