Sciadv 1600584

REVIEW
EPIGENETICS 2016 © The Authors, some rights reserved;

exclusive licensee American Association for
Epigenetics and aging the Advancement of Science. Distributed

under a Creative Commons Attribution
NonCommercial License 4.0 (CC BY-NC).
Sangita Pal1,2 and Jessica K. Tyler1* 10.1126/sciadv.1600584
Over the past decade, a growing number of studies have revealed that progressive changes to epigenetic information
accompany aging in both dividing and nondividing cells. Functional studies in model organisms and humans indicate
that epigenetic changes have a huge influence on the aging process. These epigenetic changes occur at various levels,
including reduced bulk levels of the core histones, altered patterns of histone posttranslational modifications and DNA
methylation, replacement of canonical histones with histone variants, and altered noncoding RNA expression, during
both organismal aging and replicative senescence. The end result of epigenetic changes during aging is altered local
accessibility to the genetic material, leading to aberrant gene expression, reactivation of transposable elements, and
genomic instability. Strikingly, certain types of epigenetic information can function in a transgenerational manner to
influence the life span of the offspring. Several important conclusions emerge from these studies: rather than being
genetically predetermined, our life span is largely epigenetically determined; diet and other environmental influences
can influence our life span by changing the epigenetic information; and inhibitors of epigenetic enzymes can influence
life span of model organisms. These new findings provide better understanding of the mechanisms involved in aging.
Given the reversible nature of epigenetic information, these studies highlight exciting avenues for therapeutic inter-
vention in aging and age-associated diseases, including cancer.
INTRODUCTION
Aging is a complex multifactorial biological process shared by all living as queen bees and worker bees (1, 12, 13). Although longevity studies on
organisms. It is manifested by a gradual decline of normal physiological the human population have shown that genetic factors could explain a
functions in a time-dependent manner. Organismal aging holds signif- fraction (20 to 30%) of the differences observed in life spans of mono-
icant importance for human health because it increases susceptibility to zygotic twins, the majority of the remainder of variation is thought to
many diseases, including cancer, metabolic disorders, such as diabetes, have arisen through epigenetic drift during their lifetime (9, 10, 12, 14, 15).
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cardiovascular disorders, and neurodegenerative diseases (1–4). On the Similarly, different environmental stimuli, including diet, cause differen-
other hand, cellular senescence, also called replicative senescence, is a tial alterations of stored epigenetic information to create a striking con-
specialized process, considered to be a potential endogenous anticancer trast in physical appearance, reproductive behavior, and life span of queen
mechanism, during which there is irreversible growth arrest in response and worker honeybees, despite their identical DNA content (16). In turn,
to potentially oncogenic stimuli (5). It is also considered a potential the resulting variability in the pattern of epigenetic information within
trigger to cause tissue remodeling during embryonic development individual cells in the population during aging leads to transcriptional
and following tissue damage, which also requires a proliferation arrest drift and genomic instability (Fig. 1). Being established by enzymes, epi-
(6). Cellular senescence bears many similarities to the aging process but genetic information is reversible. Hence, epigenetics holds great prospects
also shows distinct features. Although the causes of aging are poorly for targeting by therapeutic interventions, as opposed to genetic changes,
understood, there are continued efforts to delineate longevity pathways which are currently technically irreversible in humans. Accordingly, deli-
conserved among all eukaryotes. In recent years, great strides have been neating and understanding the epigenetic changes that happen during
made by numerous studies that efficiently categorized the cellular and aging is a major ongoing area of study, which may potentially lead the
molecular hallmarks of aging (7). Among these hallmarks, epigenetic way to the development of novel therapeutic approaches to delay aging
alterations represent one crucial mechanism behind the deteriorated and age-related diseases.
cellular functions observed during aging and in age-related disorders. There are different types of epigenetic information encoded within
By definition, epigenetics represents the reversible heritable mechanisms our epigenome, including but not limited to the presence or absence of
that occur without any alteration of the underlying DNA sequence. Al- histones on any particular DNA sequence, DNA methylation, chroma-
though the chromosomes in our genome carry the genetic information, tin remodeling, posttranslational modifications of the histone proteins,
the epigenome is responsible for the functional use and stability of that structural and functional variants of histones, and transcription of
valuable information; that is, it connects the genotype with the phenotype noncoding RNAs (ncRNAs) (1, 2, 10). Together, these different types
(8–11). These epigenetic changes can either be spontaneous or driven by of epigenetic information comprise our epigenome and are important
external or internal influences. Epigenetics potentially serves as the determining factors behind the function and fate of all cells and tissues,
missing link to explain why the pattern of aging is different between in both unicellular and multicellular organisms. Invariably, each of these
two genetically identical individuals, such as identical twins, or, in the different types of epigenetic information is functionally significant for
animal kingdom, between animals with identical genetic makeup, such the process of aging (Fig. 1). Growing evidence in recent years also clear-
ly implicates chromatin structure, which carries much of the epigenetic
1
Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, 1300 York information, as a major player during the aging process. The basic unit
Avenue, New York, NY 10065, USA. 2Genes and Development Graduate Program, Uni- of chromatin structure is the nucleosome, which consists of 147 base
versity of Texas Graduate School of the Biomedical Sciences at Houston, University of
Texas MD Anderson Cancer Center, Houston, TX 77030, USA. pairs of DNA wrapped around a histone octamer that comprises two
*Corresponding author. Email: [email protected] copies of each core histone protein, H2A, H2B, H3, and H4 (17, 18).
Pal and Tyler Sci. Adv. 2016; 2 : e1600584 29 July 2016 1 of 19

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The addition of linker histones, such as histone H1, and other non- complexity of the process of aging and the influence of overlapping
histone proteins, such as heterochromatin protein 1 (HP1), facilitates pathways on the outcome (22, 23).
the formation of higher-order repressive chromatin structures, such Hutchinson-Gilford progeria syndrome (HGPS) and Werner syn-
as heterochromatin (19). Packaging of the genomic DNA into the high- drome are rare human genetic disorders characterized by premature
ly organized chromatin structure regulates all genomic processes in the aging phenotypes with a shortened life span. This group of diseases re-
nucleus, including DNA replication, transcription, recombination, and sembles physiological aging to a certain extent, serving as excellent
DNA repair, by controlling access to the DNA (2). models to gain insight into the biology of aging in humans (24, 25). These
Studies on the aging of mammals are rather limited by the long life diseases are due to either a mutation in genes encoding the DNA repair
span of the commonly used model organisms. Thus, both nonverte- machinery or the A-type lamin, leading to disorganized chromatin
brate and invertebrate organisms, with their shorter life span and ease structures. The causative mutations behind these progeria syndromes in-
of genetic and environmental manipulations, gained popularity among dicate that genomic instability and chromatin deterioration are causes of
researchers in the aging field as experimental models for aging studies. human aging. Furthermore, the knowledge we gain from understanding
Among them, budding yeast or Saccharomyces cerevisiae is a highly in- the molecular pathology of these human premature aging diseases pro-
formative organismal model for aging studies with its genetic tools, vides us with useful information to understand the complex aging pro-
short life span, and fully sequenced genome (20, 21). Despite being uni- cess. Individuals with HGPS do not recapitulate all aging phenotypes
cellular, yeast has been an excellent model to identify and characterize because they usually show segmental progeria affecting multiple tis-
conserved basic biological processes, including aging. Yeast has been sues. By recapitulating some molecular and cellular changes that are
extensively used to identify genes and interventions responsible for life characteristics of the natural aging process, these models provide us
span extension and to gain insights into the aging processes of all eu- with a unique opportunity to understand the aging process in a human
karyotic organisms. In parallel, over the years, studies on invertebrate model (24, 25).
organisms, such as Drosophila melanogaster (flies) and Caenorhabditis Studies in humans and these powerful models of aging reveal that,
elegans (worms), and certain vertebrate models, such as mice, zebrafish, similar to all other biological structures inside the cells, the epigenome
naked mole rats, and, most recently, African turquoise killifish, have suffers from a progressive loss in its configuration during aging. This
also provided invaluable information to help us understand the results in a profound change in the chromosomal architecture, genomic
Chromosome mRNA
Transcriptional drift

Aging
DNA mutation
New transposable
element insertions
DNA methylation
Me Histone variant
Me Ac Me
Me Ac
Me Me
Ac Me Me Altered patterns of
Chromatin DNA mutation
histone modifications
modifiers
Normal chromatin state Abnormal chromatin state
Fig. 1. Overview of epigenetic changes during aging. In young individuals, the cells within each cell type have a similar pattern of gene expression,
determined in large part by each cell having similar epigenetic information. During aging, the epigenetic information changes sporadically in response to
exogenous and endogenous factors. The resulting abnormal chromatin state is characterized by different histone variants being incorporated, altered DNA
methylation patterns, and altered histone modification patterns, resulting in the recruitment of different chromatin modifiers. The abnormal chromatin state
in old cells includes altered transcription patterns and transcriptional drift within the population. The abnormal chromatin state in old cells also leads to new
transposable elements being inserted into the genome and genomic instability, including DNA mutations.

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integrity, and gene expression patterns (1, 10). Where examined, these A rather important unresolved paradox for the heterochromatin loss
effects are mostly conserved all the way from single-celled organisms, model of aging was the observation of loss of heterochromatin during
such as budding yeast, to complex multicellular eukaryotes. These con- aging at the same time as the formation of senescence-associated het-
served mechanisms help us gain a clearer picture of the aging process. erochromatin foci (SAHF), which are domains of heterochromatin over
Here, we will discuss the epigenetic changes that have emerged in recent proliferation-promoting genes in senescent cells (42). For a long time,
years to significantly influence the aging process, from studies on single- SAHF have been perceived as another difference between the aging pro-
celled model organisms to human models of aging. We will synthesize cess and replicative senescence (43). However, very recently, global re-
the recent developments in this area and highlight potential future organization of genome architecture has been reported in senescent
directions. cells using Hi-C (43) and FAIRE (44) analyses, leading to a proposed
two-step mechanism for the formation of SAHF. This two-step mech-
anism consists of heterochromatin decondensation in senescent cells, as
THE HETEROCHROMATIN LOSS MODEL OF AGING seen during organismal aging, followed by the heterochromatinization
of formerly euchromatin regions (Fig. 2) that spatially cluster to form
One of the earlier proposed models of aging was the “heterochromatin the compact SAHF structures. This process has been referred to as het-
loss model of aging” (26, 27). This model suggests that the loss of heterochromatin redistribution (45) and provides a unifying theme to re-
erochromatin that accompanies aging leads to changes in global nuclear solve the paradox.
architecture and the expression of genes residing in those regions, di-
rectly or indirectly causing aging and cellular senescence. As with any
other model of aging, the heterochromatin loss model is supported by GLOBAL HISTONE PROTEIN REDUCTION DURING AGING
experimental data, but there are also confounding observations. Loss of
transcriptional silencing due to decay of the heterochromatin occurs Recently, the aging research field has experienced a leap from the earlier
during aging in all eukaryotes examined from yeast to humans (26–30), paradigm of the heterochromatin loss model. Not only is the hetero-
and there is evidence that accelerating or reversing this process can chromatin reorganized during aging, but a global loss of core histone
either shorten or lengthen life span, respectively (Fig. 2). Gene silencing proteins from the genome during aging has also been observed in
requires the absence of histone acetylation within heterochromatin re- multiple scenarios, and this has been shown to be a cause of aging
gions. Accordingly, treatment with histone deacetylase (HDAC) inhi- in yeast.
bitors or deletion of genes encoding HDACs, such as yeast SIR2 or its In budding yeast, replicative aging is accompanied by loss of approx-
sirtuin counterparts in metazoan species, shortens life span, whereas imately half of the core histone proteins (46, 47). The extensive nucleo-

chemical activation or overexpression of SIR2 or sirtuins extends life some loss from the entire yeast genome during replicative aging (Fig. 2)
span (31, 32). Yeast Sir2 was first recognized as an H4 K16Ac deace- results in global increased transcription in aged cells (48). These global
tylase, whereas mammalian SIRT1 is an H3 K9Ac or H4 K16Ac deace- changes in transcription and chromatin structure were only detectable
tylase (32). However, it is now appreciated that sirtuins deacetylate not because of the development of novel normalization approaches for ge-
only histones but also many other transcriptional regulators, and in- nomic data sets (49). Notably, during yeast replicative aging, not only
deed, their roles in aging extend far beyond heterochromatin, including transcriptionally silent regions showed marked derepression but also all
genome maintenance (33). It has been previously observed in budding genomic regions showed transcriptional up-regulation, presumably be-
yeast that redistribution of Sir proteins from the silent mating–type loci cause of increased access of the transcription machinery to the DNA
to sites of increased genomic instability during aging causes loss of si- sequences (48).
lencing from heterochromatic mating–type loci to cause sterility and The drastic decline of the core histone proteins in S. cerevisiae is due
promote aging (34). Similarly, the mammalian homolog of Sir2, SIRT1, to the reduced protein synthesis of the histones (Fig. 2) (47). Clearly, the
has been shown to repress repetitive elements and other regions across fact that the levels of histone transcripts, like all other transcripts, actu-
the mouse genome. However, in response to DNA damage, SIRT1 is ally increase during replicative aging in yeast (46, 47, 50) has no func-
redistributed from these loci to DNA breaks, resulting in alterations tional consequence because of the reduced histone protein synthesis in
in gene expression that is comparable to those in the aging mice brain old yeast. Other cells may indeed use transcriptional regulation to re-
(33). Therefore, repression of heterochromatic repeat elements seems to duce histone levels during aging because quiescent muscle stem cell
be evolutionarily conserved and extremely critical in life span mainte- aging is accompanied by reduced histone transcript levels (51). Supply
nance (33, 35). Another example where it is clear that loss of hetero- of extra H3 and H4 histone proteins, either from an inducible promoter,
chromatin promotes aging is at the ribosomal DNA (rDNA) locus, by deleting the genes encoding the Hir repressor of histone gene
where loss of silencing of the rDNA promotes genomic instability and transcription, or by deleting the gene encoding the protein Tom1 that
aging in budding yeast (36, 37). Further evidence supporting the hetero- is involved in the degradation of excess histone proteins, results in life
chromatin loss theory comes from the analysis of chromatin structures span extension of yeast, identifying loss of histone proteins as a cause of
from HGPS patients and, most recently, from a Werner syndrome stem aging (47, 52). The pathway of life span extension used by supplying
cell model of premature aging (10, 38–41). These disorders exemplify extra histones appears to be independent from the pathway of calorie
how deregulation of heterochromatin, illustrated by global loss of het- restriction (CR) in budding yeast (47), which is a widely accepted way to
erochromatin marks (discussed more below) and altered heterochro- extend life span of seemingly all species (53). The life span extension
matin structure, accelerates aging. Although some of the defects observed that results from lithium exposure in worms is accompanied by in-
in premature aging patients have also been observed in aged individuals creased histone gene expression (54), although it is not known whether
(39), it is still debatable whether the findings are equally applicable to phys- the increased histone gene expression played a causative role in life
iological aging. span extension. The reduced bulk core histone protein levels observed

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during replicative aging is not restricted to budding yeast and has also rejuvenated population (48). Most of the nucleosomal positions are
been observed during aging in worms (55), during replicative aging of maintained in aged cells, whereas nucleosome occupancy is reduced
human diploid primary fibroblasts (56), and in senescent human cells to half the normal proportion and nucleosome locations on particular
(57). Notably, global histone loss has not yet been reported in mitotically DNA sequences become less stringent or fuzzier. Additionally, partial
dividing mammalian tissues in vivo. In human primary fibroblasts, the overexpression of histone proteins H3 and H4 partially reversed the
reduced synthesis of new histones during replicative senescence was a transcriptional defects observed during aging. It will be intriguing to in-
consequence of the shortened telomeres that activate the DNA damage vestigate in the future whether the longevity effect conferred by supply-
response (56), potentially explaining the mechanism by which telomere ing extra histones is equally pertinent in multicellular organisms.
shortening limits the number of cell divisions. Hence, loss of core histones
may be a more generalized phenomenon observed with aging in many
organisms. GENOMIC INSTABILITY RESULTING FROM CHROMATIN
Decreased levels of core histone proteins in aged cells would likely RELAXATION DURING AGING AND THE “AGING BY
cause a marked effect on the chromatin landscape by providing in- TRANSPOSITION” MODEL
appropriate access to the genetic material. The consequences of having
limiting amounts of histone proteins in the DNA during age progres- A more relaxed chromatin structure, due to heterochromatin loss or
sion have been elaborately examined in a recent global analysis of chro- histone loss, is predictive of not only transcriptional deregulation (as
matin from aged cells in budding yeast, in comparison to the young discussed above) but also genome instability. Indeed, the reduced
Extra histones
Integration
Yeast replicative aging cDNA
Reverse transcription
X (organismal aging) RNA
Retrotransposable element Retrotransposable element

(Ty ) (Ty )

Knockdown of Alu RNAs
Integration
Human cell cDNA
Reverse transcription
X replicative senescence RNA

(LINEs, SINEs) (LINEs, SINEs)
Calorie restriction
Integration
Mouse organismal aging cDNA
X Reverse transcription
RNA

(LTR RTE, LINEs, SINEs) (LTR RTE, LINEs, SINEs)
Fig. 2. Alterations in chromatin structure during aging leads to activation of retrotransposons. The schematic at the top depicts the reduction in the
level of bulk histone proteins during aging (for example, as seen in budding yeast), leading to a more open chromatin structure and subsequent transcrip-
tional activation of the normally silenced Ty elements. The resulting transcripts from the retrotransposable elements are reverse-transcribed into cDNAs that
reinsert elsewhere into the genome of old cells. Overexpression of histones reverses the loss of histones, reduces Ty retrotransposition, and extends repli-
cative life span. The schematic in the middle depicts the heterochromatin reorganization that occurs during aging, as seen, for example, in tissue culture cells
during replicative senescence. The normally heterochromatinized retrotransposable elements become euchromatinized, leading to their transcription and
transposition elsewhere into the genome. Knockdown of the Alu element transcripts in human adult stem cells leads to escape from senescence and reduced
genomic instability. The schematic at the bottom depicts the heterochromatin reorganization that occurs in mouse tissues during aging and is accompanied
by activation of retrotransposable elements (RTEs). Calorie restriction reduces the level of retrotransposition in the aged mice genome.

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nucleosome occupancy in old yeast led to increased genomic perturba- Ty transcription and retrotransposition in old yeast by overexpressing
tions, including higher levels of DNA breaks, damaged foci formation, histones (Fig. 2) (48). Life span–extending interventions, such as CR,
inter- and intrachromosomal translocations, insertion of mitochondrial have also been shown to counteract the increased expression of retro-
DNA into the nuclear genome, and high levels of retrotransposition transposons in aged mice (Fig. 2) (66).
(48). Notably, partial overexpression of histone proteins H3 and H4 The increased mobility of retrotransposons, observed in the gen-
partially reversed the transcriptional defects observed during aging omes of aged cells and tissues from multiples species, provides strong
and reduced retrotransposition, indicating that the increased retrotrans- evidence in favor of a recently hypothesized model of aging: aging by
position in old yeast was a consequence of histone loss during aging transposition (73). According to this model, these mobile elements and
(48). Increased levels of DNA breaks or unrepaired DNA damage, as their transposases are the driving force to cause structural dysregulation
illustrated by the formation of g-H2AX foci (a hallmark of DNA dam- of the genome to manifest aging phenotypes. Indeed, in an adult stem
age), have been observed in aged cells from multiple species, including cell model of ex vivo aging, entry into senescence was accompanied by
aged mice, senescent human cells, and cells derived from patients with increased transcription from the SINE/Alu retrotransposable elements
premature aging disorders (58–61). This suggests that DNA damage and persistent DNA damage foci (70). Experimental suppression of the
may be a driving force behind organismal aging, although the full ex- transcripts from Alu elements reversed the arrested phenotype and
tent of the role of DNA damage during age progression still needs to eradicated the DNA damage foci, directly indicating that retrotranspo-
be explored. son transcription was driving the entry into senescence (Fig. 2). Given
It is a long-standing belief that accumulation of DNA mutations the many recent links between activation of retrotransposable elements
over a period of time affects organismal aging because mutation accu- and aging, it is interesting to note that transposition also becomes more
mulation happens as a function of age in different organisms, such as frequent during cancer development (74, 75), wherein aging is the high-
flies or mice and, in some cases, also correlates with life span (62, 63). est risk factor for most cancers. Neurodegeneration, another disease of
However, a recent study indicated that accumulation of somatic DNA aging, is also characterized by increased retrotransposition (76, 77). Fu-
mutations, at least in budding yeast, does not have a causal role in aging ture studies will hopefully illuminate the full influence of transposition
(64). Therefore, it is yet to be conclusively determined whether the per- on the process of aging and the contribution of the age-dependent in-
sistent DNA damage observed in aging cells is the result of the increased crease in transposition to these other human diseases.
susceptibility of the accessible genome to accrue DNA damage with time Notably, the processes described in this section are unlikely to
or the inability of aged cells to efficiently repair the damage, or a combi- function independently from each other, and one event may lead to an-
nation of both. Notably, another recent analysis of the DNA repair tran- other during the aging process. For example, heterochromatin decay
scriptome of liver in species with significant life span differences revealed and histone loss lead to retrotransposition and changes in gene expres-

that the longer-lived species express certain DNA repair genes at higher sion. But what causes the heterochromatin decay and block in histone
levels compared to species with shorter life span, suggesting a superior protein synthesis during aging in the first place? We are currently
genome maintenance mechanism in the longer-lived species, which could limited in our knowledge of the sequence of the causal events during
potentially be responsible for their longer life span (65). aging in healthy individuals. Attempts should be made in the near fu-
Another form of genomic instability observed during aging that ture to define the cascade of events during age progression to attain a
results from heterochromatin decay is increased mobility of endoge- comprehensive view of the aging process and to identify the initial caus-
nous genetic elements, called retrotransposable elements (Fig. 2). Retro- ative events.
transposable elements have been implicated in the aging process by
several lines of evidence across organisms, including budding yeast, flies,
worms, and mice, and even during cellular senescence in adult human HISTONE VARIANT CHANGES WITH AGING
stem cells (Fig. 2) (44, 48, 66–70). Retrotransposable elements are tightly
silenced by heterochromatin in young cells or organisms. However, the In addition to the canonical histone proteins H2A, H2B, H3, and H4,
loss of heterochromatin with aging leads to increased expression of oth- there are nonallelic variants of most of the histone proteins that are
erwise silent retrotransposons, essentially causing transposition of sometimes distinctly different in their primary sequence and are crucial
these elements, because the transcripts are reverse-transcribed to for regulating chromatin dynamics by performing specialized functions
make a genomic cDNA copy that gets integrated elsewhere into the ge- (78, 79). Unlike core histone proteins, the age-associated changes ob-
nome (Figs. 1 and 2). The end result is increased mobility of retrotran- served for histone variants are more diversified and somewhat dispar-
sposable elements within genomes, resulting in disruption of cellular ate, depending on which model organisms are studied. Histone H3.3 is a
homeostasis during aging (71). Molecular insight into this process histone H3 variant that is incorporated into the genome in a replication-
was recently provided in studies of SIRT6 in mouse cells. While SIRT6 independent manner, in contrast to the replication-dependent histone
is best known for being a deacetylase for H3 K9Ac and H3 K56Ac (32), variants H3.1 and H3.2 (80). H3.3 is expressed constitutively in meta-
it also exhibits adenosine 5′-diphosphate (ADP)–ribosylase activity. Ac- zoan cells, unlike the canonical H3.1 that is mainly expressed in the S
cordingly, SIRT6-mediated mono–ADP-ribosylation of KAP1 was shown phase. Therefore, it seemed rather predictable that H3.3 would play a
to promote its interaction with HP1 and the packaging of L1 LINE key role in chromatin maintenance during senescence, when cells are no
elements into repressive heterochromatin (72). During aging, SIRT6 longer dividing. Indeed, H3.3 is the major form of histone H3 in the
is sequestered away from the L1 elements to, presumably, DNA breaks chromatin of senescent human cells, along with an N-terminally cleaved
elsewhere in the genome, resulting in the activation of the retrotransposons form of the H3.3 variant, which is termed H3.3cs1 (81). It appears that
(72). Proof that the loss of chromatin-mediated silencing of retrotrans- excess incorporation of H3.3 can drive senescence because ectopic ex-
posable elements during the course of aging directly leads to their pression of either H3.3 or H3.3cs1, but not H3.1, was able to induce
increased transposition during aging came from the ability to repress senescence in the absence of any oncogenic stimuli (81). The induction

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of senescence was most robust for the cleaved variant H3.3cs1, revealing gene transcription, DNA repair, DNA replication, effective condensa-
the importance of proteolytic processing of H3.3 during senescence. tion of chromatin, and many others, which affect almost all biological
The deposition of H3.3cs1 onto DNA was mainly attributed to the his- processes in eukaryotes, including aging. The histone posttranslational
tone chaperones ASF1a and UBN1 and putatively to HIRA in senescent modifications are added and removed by enzymes, usually in a tightly
cells (81). Notably, deletion of the yeast counterpart of HIRA, the Hir regulated manner. However, during aging, the abundance, activity, and
complex, extended replicative life span of yeast, but this is more likely function of some of these enzymes change, leading to alterations in the
due to the resulting increase in histone gene expression that counters epigenome.
the loss of core histones during aging (47). A similar correlation of Among the histone modifications that are known to affect the lon-
increased H3.3 incorporation during aging has also been observed in gevity process, the most prominent ones are acetylation and methyla-
the aged mouse brain, where H3.1 levels gradually decline with age tion of lysine residues. In budding yeast, acetylation present on the
and H3.3 gradually accumulates (82). The incorporation of H3.3 into globular domain of histone H3 on lysine 56 (H3 K56Ac) and acetylation
actively transcribed DNA regions in neuronal cells and its efficient at the N-terminal tail of histone H4 on lysine 16 (H4 K16Ac) both in-
turnover in neuronal cells are crucial to maintain cell type–specific gene fluence replicative aging, although through distinct mechanisms. It
expression as well as neuronal plasticity and cognition (82). It appears that seems that either the dynamic nature or a balanced level of each acety-
the histone variant H3.3 and its cleavage should be considered as impor- lation mark is crucial for the cell because either inability to acetylate
tant new players in maintaining functional chromatin structure during these residues or the presence of mutations that mimic permanent acet-
senescence. ylation reduces replicative life span of budding yeast (46, 47). Levels of
There are multiple different variants of histone H2A, and one in par- H3 K56Ac decrease during yeast aging (46, 47). Meanwhile, deletion
ticular has been implicated in aging. The H2A variant macroH2A is a of the genes encoding the relevant HDACs that remove H3 K56Ac,
prominent feature of the SAHF (83, 84). MacroH2A’s function is gen- Hst3, and Hst4 leads to a shortened yeast life span (46, 93) and ge-
erally assumed to be transcriptional repression (85, 86). Although nomic instability (46), including a high frequency of loss of hetero-
macroH2A has not been presumed to be important for the formation zygosity (94). The notion that there is a precise level of H3 K56Ac for
of SAHFs, it was suggested that macroH2A is crucial to maintain the optimal life span is borne out by the fact that overexpression of Hst3
transcriptional silencing at those sites (83, 84). The level of macroH2A shortens life span, whereas addition of one extra gene copy of HST3 or
increases in an age-dependent manner during replicative senescence in HST4 extends life span (95). Similarly, deletion of the yeast histone acetyl-
cultured human fibroblast cells and also in several tissues of aged mice transferase (HAT) that acetylates H3 K56Ac, Rtt109, or the histone
and primates (87). More recently, enrichment of macroH2A1 at sites chaperone Asf1, which assists in the acetylation, causes a shortened life
encoding the senescence-associated secretory phenotype (SASP) genes span (47). Exactly how a slightly elevated level of H3 K56Ac promotes

has been reported in response to oncogenic stimuli, promoting SASP longevity, whereas too much or too little H3 K56Ac shortens life span, is
transcriptional activation and propagating senescence through a posi- not clear. However, H3 K56Ac has been implicated in driving chroma-
tive feedback loop in a paracrine manner (88). The SASP leads to en- tin assembly, transcriptional regulation of genes including the histones
doplasmic reticulum stress that triggers the production of reactive themselves, genomic stability, and DNA replication (96–98); therefore,
oxygen species. The subsequent DNA damage and activation of the any of these functions may be optimized with slightly more H3 K56Ac
ATM signaling pathway are important for the removal of macroH2A1 to promote longevity.
from the SASP genes, which results in their transcriptional repression. In contrast to the reduced levels of H3 K56Ac during aging, the levels
Hence, this study demonstrates a role for macroH2A1 in transcriptional of H4 K16Ac increase during yeast replicative aging (46). Similar
activation. Basically, a massive redistribution of macroH2A1 occurs changes in the levels of these two histone acetylation marks also occur
during senescence, with the macroH2A1 leaving the SASP genes and during replicative aging in cycling human fibroblasts, where there is a
potentially relocalizing to SAHFs, along with other heterochromatin substantial decrease in H3 K56Ac and an increase in H4 K16Ac in late-
marks (88, 89). Hence, macroH2A1 appears to be a critical regulator passage cells (56). The increased level of H4 K16Ac in old yeast cells is
of chromatin structure and chromatin dynamics during senescence. attributed to the progressive decline in levels of the relevant HDAC Sir2
during aging (46). Overexpression of Sir2 extends life span (99). In
agreement, deletion of SAS2, which encodes the HAT that acetylates
HISTONE MODIFICATION CHANGES DURING AGING H4 K16, extends life span (46). Hence, the increase in H4 K16Ac that
occurs during aging is driving aging, or pro-aging, whereas manipula-
The histone components of chromatin are subject to a wide variety of tions that reduce H4 K16Ac levels extend life span. The pro-aging in-
posttranslational modifications. Each modification, either alone or in fluence of H4 K16Ac accumulation has been suggested to manifest
combination with others, enables regulation of the use of the underlying through effects on the chromatin structure at the telomeres (46), which
DNA sequence. Functionally, histone modifications either disrupt the is reminiscent of the heterochromatin loss model of aging. Notably, this
chromatin organization or provide new binding surfaces for recruit- work (46) provided direct evidence for aging regulation through mod-
ment of other proteins to specific regions of the chromatin. The type ification of H4 K16Ac by Sir2, the founding member of the sirtuin pro-
of posttranslational modification, the amino acid that is modified, tein family, in addition to the already existing aging theories for this
and the other histone posttranslational modifications nearby determine protein. The other modes by which Sir2 or sirtuins have been proposed
their exact functional outcome. The functional complexity of the stead- to influence aging (responding to nutrient availability through inflam-
ily rising number (currently well over 1000) of these histone modifica- matory response, apoptosis, DNA repair, cell cycle, mitochondrial
tions is yet to be fully understood (90–92). The presence of this vast functions, etc.) will not be discussed here further because they are not
array of modifications on histones orchestrates the functional re- directly related to epigenetics and are covered in other recent reviews
sponses, which are highly diversified. These range from regulation of (100, 101).

REVIEW
Other HATs and HDACs have also been implicated in aging. Dele- modification levels from cell populations or tissues misses important
tion of a component of the NuA4 HAT extends yeast replicative life additional information on gene-specific effects that would hopefully
span (52). NuA4 acetylates histone H4 but also has several nonhistone be available soon from profiling histone modifications by ChIP-seq,
substrates that may be relevant to its role in limiting longevity, such as eventually in individual cells, during aging.
Pck1p, which encodes a phosphoenolpyruvate carboxykinase enzyme, Evidence of histone lysine methylation playing a causal role during
Tap42p, a putative downstream member in the mammalian target of aging comes from studies modulating the levels of histone methyltrans-
rapamycin (mTOR) pathway, and/or Nnt1p, a putative nicotinamide ferases and demethylases. RNA interference (RNAi) screens in C. elegans
N-methyltransferase (102). Deletion of genes expressing components identified several histone methyltransferases, including ASH-2, SET-2,
of the histone H3 deacetylase complex Rpd3 extends replicative life span and SET-9, and several demethylases, including UTX-1, as potential
of yeast (103) and organismal life span of flies (104). Rpd3-mediated factors influencing the outcome of aging (55, 115). Reduction in the lev-
deacetylation appears to limit longevity through multiple pathways el of H3 K4me3 methylation, which serves as a signature of active chro-
(105), highlighting the pleiotropic effects of altering levels of histone matin, by knockdown of the H3 K4 methyltransferase SET-2 or the
acetylation. Further studies are required to fully understand the molec- components of the regulatory complex of H3 K4me3, ASH-2 and
ular mechanisms used by these HATs and HDACs to influence life span. WDR-5, caused life span extension in fertile worms. In contrast,
Histone acetylation also appears to play an important role in the knockdown of the H3 K4me3 demethylase protein RBR-2 shortened life
aging brain. Global histone hypoacetylation occurs in the repeated span of worms. On the other hand, overexpression of RBR-2 has also been
DNA elements in aged mice brain, which suggests a loss of chromatin shown to be sufficient for extending life span of worms (116). It is unclear
integrity with aging (106). Aged mice brains also show memory impair- how reduced levels of H3 K4me3 increase longevity, but it is evident that a
ment that has been proposed to be due to failure to up-regulate acety- limiting amount of this modification is favorable for optimal C. elegans
lation at histone H4 K12, which is a histone mark promoting transcription life span (117). Consistent with the findings in C. elegans, flies deficient
elongation, and due to the resulting altered gene expression pattern. in Lid, the RBR-2 ortholog in D. melanogaster, have shortened life span
Restoration of H4 K12 acetylation recovered both gene expression (117, 118). However, this is only evident in male flies, implying that
and learning behavior in aged mice, signifying the critical role of this the effect of this demethylase may be sex-specific in some organisms.
histone acetylation in aged mice brains (107). Alteration in the level of H3 K27me3 also influences life span of both
The trends in changed levels of histone methylation during aging worms and flies. Contrary to H3 K4me3, H3 K27me3 is a prominent mark
have also been extensively examined recently. Significant changes dur- of repressed chromatin. Attenuation of the H3 K27me3 demethylase
ing aging have been seen for trimethylation marks on histone H3 lysines UTX-1 in C. elegans extends life span through an insulin-dependent
4, 9, 27, and 36 (H3 K4me3, H3 K9me3, H3 K27me3, and H3 K36me3, pathway (119, 120). The opposing effects of H3 K4me3 and H3 K27me3

respectively), mostly as changes in the methylation marks that indicate on longevity are not surprising, given their opposing effects on
loss of heterochromatic structure with aging. In parallel, reduction in transcription where they function as a toggle switch to determine devel-
HP1 levels was evident during aging in whole flies, with a decreased level opmental patterns of gene expression (121). However, high H3 K27me3
of H3 K9me2 and altered levels of H3 K4me3 (29). However, when the does not always extend life span. In D. melanogaster, evidence suggests
profiles of the repressive histone marks were analyzed in aging fly heads, that heterozygous mutations in components of Polycomb repressive
an increase in the level of H3 K9me3 was observed, signifying the im- complex 2 (PRC2), namely E(z) and Esc, decrease the global level of
portance of tissue-specific expression pattern changes during aging in H3 K27me3 and extend life span in male flies (117, 122). These results
multicellular animals (108). Histone methylation pattern analysis in reveal species- and sex-specific discrepancies and indicate that excess
aged SAMP8 (senescence-accelerated mouse prone 8) brain tissues re- chromatin-mediated repression may sometimes be detrimental to
vealed decreased H4 K20me1 and H3 K36me3 and an increase in H3 life span.
K27me3 (109). Fibroblast cells collected from individuals with HGPS Mechanistic insights into the role of histone modifications during
showed decreased H3 K9me3 and H3 K27me3 with the expected reduc- aging have come from studies of H3 K9me3. Cells and tissues from a
tion of HP1a (40), given that HP1 binds to H3 K9me3. There was also progeria mouse model had increased levels of H3 K9me3 and the
an accompanying increase in transcript levels from the pericentric methyltransferase SUV39H1, along with defective DNA repair within
satellite DNA (40). The same study also revealed an increase in the level heterochromatin (123). Following DNA damage, compact chromatin
of H4 K20me3, which serves as a signature epigenetic mark of con- structures undergo rapid chromatin remodeling to provide access to
stitutive heterochromatin, demonstrating a shift in the epigenetic land- DNA repair factors. However, increased H3 K9me3 levels may possibly
scape in HGPS patients (40). Although the global pattern of histone hinder the heterochromatin remodeling, leading to sustained DNA
methylation differs in different organisms, presumably due to differen- damage, early senescence, and a shortened life span (123). Indeed, de-
tial involvement of longevity pathways during the aging process, in gen- pletion of the major H3 K9me3 methyltransferase SUV39H1 rescued
eral, the trend mostly reveals the increase in appearance of activating the defect in DNA repair and premature aging, leading to an extended
histone methylation marks and disappearance of repressive histone life span (123).
methylation marks during aging, signifying the loss of compact chro- Most recently, H3 K36me3 has been found to promote longevity. A
matin architecture in aging cells, tissues, or organisms (110). In recent high-throughput histone mutation screen in S. cerevisiae for identifying
years, some efforts have been made in either certain stem cell popula- mutants with altered life span pinpointed this modification (124).
tions or tissue types to delineate the differences between young populations Whereas the methyltransferase mutant for H3 K36 (set2∆) had a
and the aged counterparts or to determine the regulatory mechanisms shortened replicative life span, the H3 K36 demethylase mutant
from high-throughput sequencing approaches, including chromatin (rph1∆) had an increased life span (124). Reduction of H3 K36me3 dur-
immunoprecipitation–sequencing (ChIP-seq) and micrococcal nuclease– ing replicative aging in yeast leads to a more open chromatin confor-
sequencing (MNase-seq) (51, 111–114). However, examination of bulk mation that exposes cryptic promoters, and the resulting cryptic

REVIEW
transcripts may limit life span. Deletion of the relevant demethylase ablation of ATP-dependent nucleosome remodeler function have been
generates a more compact chromatin conformation, resulting in life linked to several disease syndromes and cancers (130, 131). Recently,
span extension (124). Although this study also identified increased cryp- some members of the ATP-dependent nucleosome remodeler families
tic transcripts during C. elegans aging, it remains to be validated whether have also been linked to the aging process.
a similar mechanism also applies in mammals. An interesting correla- Both physiological aging and premature aging syndromes reveal
tion exists between the gene expression change during aging and the signs of extensive alteration of chromatin structure, followed by
level of H3 K36me3 in gene bodies in C. elegans and D. melanogaster increased susceptibility to persistent DNA damage (132). Although sev-
(125). The gene bodies of genes with a robust expression change during eral studies suggest that altered chromatin conformations occur with
aging are marked with almost undetectable levels of H3 K36me3 in both age (43, 44), almost no direct connections established the involvement
organisms, suggesting a conserved mechanism for this modification in of ATP-dependent nucleosome remodelers until recently. The C. elegans
promoting longevity by preventing age-dependent changes in mRNA homolog of the catalytic subunit of the NURD chromatin remodeling
expression. Inactivation or depletion of the methyltransferase met-1 re- complex, LET-418/Mi2, has recently been identified as a longevity-
sulted in a global reduction of H3 K36me3 level, increased changes in determining factor because let-418 deficiency leads to prolonged life span
gene expression during aging, and shortening of life span in C. elegans and increased environmental stress resistance. This phenotype is partially
(125). Together, these studies suggest that loss of histone H3 K36 meth- dependent on the transcription factor DAF-16/FOXO. Genetic interaction
ylation during aging leads to aberrant gene expression, reminiscent of a analysis further suggests that let-418 may serve as a life span determinant
transcriptional drift-like effect, which presumably limits life span. In this by acting through the germ cell loss pathway. This protein is highly evo-
light, it is of interest that a recent report showed that pharmaceutical lutionarily conserved, and indeed, it has been found that these increased
suppression of transcriptional drift extends C. elegans life span by longevity and enhanced stress resistance functions are also conserved
delaying the onset of mortality (126). It is tempting to speculate that in fruit flies and plants (133). One study that aims to identify the mo-
deterioration of the epigenome during aging is responsible for the tran- lecular basis for the age-related defect in chromatin structure used cells
scriptional drift that causes the onset of mortality (Fig. 1). from HGPS patients and noted that components of the NURD ATP-
Histone ubiquitination is another modification that has been impli- dependent nucleosome remodeler complex, including RBBP4 and
cated in yeast replicative life span. A genome-wide screen for long-lived RBBP7, were substantially reduced in HGPS cells (132). Conversely, ex-
strains in yeast identified components of the histone deubiquinase perimental depletion of RBBP4 and RBBP7 in cell culture caused an in-
module (DUBm) of the SAGA (Spt-Ada-Gcn5-acetyltransferase) crease in endogenous DNA damage similar to what has been observed in
complex (127), including SCF73, the yeast ortholog of human Ataxin- HGPS cells and cells from aged individuals. Furthermore, it appeared
7. This protein serves as an adapter protein in the complex, which links likely that the defect in chromatin structure precedes the increased en-

the core SAGA complex with DUBm. Although the complex contains dogenous DNA damage observed under the experimental conditions,
both histone acetyltransferase and DUB activities, life span analysis of suggesting that the altered chromatin structure due to loss of NURD
different mutants suggests that a specific reduction in DUB function function leads to DNA damage or a defect in DNA repair. Loss of NURD
may regulate life span in yeast because strains that lack components subunits was evident not only in HGPS cells but also in cells collected
of DUBm showed marked life span extension. Life span extension from aged individuals. In summary, loss of NURD subunits seems to
was not evident in strains lacking the acetylation-specific SAGA com- be an early event during both physiological and premature aging, which
ponents but was seen with yeast lacking Spt8 (52), which recruits the contributes to alterations in the chromatin structure, making the genome
SAGA complex to promoters. Further analysis revealed that the life more susceptible to DNA damage (132). Although the exact mechanism
span extension observed in DUBm mutant strains is due to their in- behind the declining level of NURD components is yet to be determined,
volvement in multiple longevity pathways, including Sir2-dependent this study implicates the functional decline of an ATP-dependent nucleo-
pathways (127). Histone ubiquitination regulates transcription and some remodeling complex in aging.
DNA damage response (128), and it will be interesting to determine Compelling evidence for involvement of an ATP-dependent nucleo-
which of these functions of histone ubiquitination is relevant to longev- some remodeling complex in limiting longevity comes from a large-
ity. In the future, it will be important to determine whether the role of scale study that measured the effect of single gene deletions on replicative
histone ubiquitination in aging observed in yeast also holds true for life span of budding yeast (134). The absence of the Isw2 ATP-dependent
multicellular organisms. nucleosome remodeler allowed yeast cells to live longer (124). Further
experimental evidence suggests that the extended life span was through
the same pathway as CR but was somewhat distinct from the well-studied
NUCLEOSOME REMODELING AND AGING suppression of the TOR signaling pathway during CR. Rather, in this case,
a novel pathway is used, which involves up-regulation of several stress
Although the nucleosome structure itself is rather rigid, the cell has response genes that are also up-regulated as a response to genotoxic
evolved complex machineries to enable specific nucleosomes at precise stresses during CR. In the absence of Isw2, a subset of these genes, includ-
genomic locations to become very dynamic, as required, to facilitate dif- ing Rad51, is activated. Rad51 is a crucial factor required for homologous
ferent genomic processes. This regulated alteration of nucleosome recombination (HR)–mediated DNA repair pathways (135). Transcrip-
architecture during different biological events is mediated mainly by tional activation of Rad51 may provide the cells with the potential advan-
adenosine 5′-triphosphate (ATP)–dependent nucleosome remodeling tage to efficiently repair DNA using HR, empowering the cells to live
complexes (129). Using the energy of ATP hydrolysis, ATP-dependent longer. In agreement with this prediction, yeast life span was extended
nucleosome remodelers work closely with other activities, including by addition of a single extra gene copy of RAD51 (134). An earlier study
histone-modifying enzymes and histone chaperones, to package, evict, that investigated the mechanism of life span regulation by the FOXO
or slide nucleosomes in a highly regulated manner (130). Mutations and transcription factor DAF-16 in worms identified the ATP-dependent

REVIEW
nucleosome remodeler SWI/SNF as a crucial cofactor in transcriptional methylation becomes more and more divergent because of epigenetic
activation of DAF-16 target genes. The well-known prolongevity effect drift caused by environmental factors or spontaneous stochastic errors
of DAF-16/FOXO is dependent on SWI/SNF because RNAi inactivation in the process of transmission of DNA methylation (12). Epigenetic
of core subunits of the SWI/SNF complex abolished DAF-16/FOXO– drift leads to unpredictable differences in the methylome among aging
mediated life span extension (136). Deletion of the catalytic subunit of individuals (Fig. 3). However, some of the methylation changes that oc-
the ATP-dependent nucleosome remodeler Chd1 has also been shown cur with age are directional and involve specific regions of the genome.
to extend yeast replicative life span in a comprehensive analysis of repli- This fact indicates that at least part of the DNA methylation changes
cation life span in almost 4700 deletion mutants (52). Given the con- during aging are not stochastic but could be associated with biological
served roles of the ATP-dependent nucleosome remodelers from yeast mechanisms involved in the aging process. DNA methylation, as well as
to humans, it will be intriguing to explore the potential roles of these re- life span, also differs between organisms of identical genetic makeup,
modelers to modulate life span in mammals. such as queen and worker honeybees, when subjected to different en-
vironmental stimuli, such as diet, which results in an altered gene expres-
sion pattern. Strikingly, similar methylome profiles were experimentally
DNA METHYLATION CHANGES DURING AGING found following RNAi-mediated silencing of a DNA methyltransferase
enzyme, Dnmt3, in laboratory-bred bees (16). Although it is yet to be
DNA methylation is one of the most extensively studied and best- determined whether the diet-induced DNA methylation changes are
characterized epigenetic modifications during aging (Fig. 3) (137). In indeed responsible for causing the transcriptional shift observed in these
young cells, the majority of CpGs within the genome have cytosine cases, it provides great promise for unraveling a key concept of epige-
methylation. CpG methylation within promoters leads to transcription- netic reprogramming.
al repression, through the formation of compact chromatin structures, With a few exceptions, mammalian aging is more commonly associated
such as heterochromatin. Conversely, promoters of genes that are high- with CpG hypomethylation, especially at repetitive DNA sequences
ly expressed are devoid of DNA methylation, hence their name—CpG (Fig. 3) (138–144). This is likely to be at least partly responsible for
islands. DNA methylation is particularly important during develop- the loss of heterochromatin during aging. Loss of CpG methylation
ment, when it is used to silence genes in tissues in which their expression at repetitive sequences will heighten the risk of retrotransposition
is never going to be needed, or at developmental stages in which they no events and, hence, genomic instability during aging, given that the retro-
longer need to be expressed. As identical twins age, the pattern of DNA transposons comprise much of the repetitive DNA. The global decrease
LINE Non–CpG island gene CpG island gene SINE CpG island gene LTR

CpG poor
DNA repeats promoter CpG island DNA repeats CpG island DNA repeats
LINE Non–CpG island gene CpG island gene SINE CpG island gene LTR
CpG poor
DNA repeats promoter CpG island DNA repeats CpG island DNA repeats
Key:
Unmethylated CpG Methylated CpG Transposable elements
Fig. 3. Summary of DNA methylation changes during aging. Young mammalian cells are characterized by DNA hypermethylation over the genome, with
the exception of CpG islands within the promoters of expressed genes. In particular, DNA repeats, such as LINE, SINE, and long terminal repeat (LTR) trans-
posable elements, are heavily DNA-methylated, helping to maintain them in a constitutive heterochromatin state. During aging, there is general DNA hy-
pomethylation over the genome, which mostly occurs in a stochastic manner within the cell population. Loss of DNA methylation leads to activation of
normally silenced DNA sequences like the transposable elements. However, DNA methylation also increases in a nonstochastic manner over the CpG islands
of certain genes, correlating with their heterochromatinization and silencing.

REVIEW
in DNA methylation upon aging may be attributed to the progressive de- mechanistic details of this phenomenon are unclear (162). One of the
cline in levels of the DNA methyltransferase DNMT1 (137). The contri- remaining challenges in the analyses of DNA methylation during aging
bution of DNA methylation to aging is further suggested by a Dnmt1+/− is to identify the causal pathways that contribute to the functional de-
mouse model that showed reduced age-dependent impaired learning cline of the DNA methylome during aging.
and memory function (145). In addition to the general DNA hypo- The age-dependent changes in the DNA methylome are reminiscent
methylation that occurs during aging, progressive age-dependent loss of those occurring in cancer. Global hypomethylation at the repetitive
of DNA methylation also occurs at specific gene promoters, including regions and site-specific hypermethylation at certain promoters have
ITGAL and IL17RC, leading to their transcriptional induction and sub- also been reported during cancer, suggesting a potential connection be-
sequent autoimmune responses (146, 147). Simultaneous with the general tween age-dependent DNA methylation changes and increased cancer
and localized DNA hypomethylation during aging, hypermethylation risk observed in the elderly population (163, 164). Notably, local hyper-
occurs at specific CpG sites of the genome, presumably to repress expres- methylation events during aging sometimes occur at the promoters of
sion of specific genes (Fig. 3) (137, 142, 148, 149). A recent analysis also tumor-suppressor genes, potentially preceding cell transformation
observed loss of DNA methylation from cis regulatory elements, such as events. The analogy between cancer and aging also extends to the
enhancer regions in pancreatic b cells, and suggested the possibility that PcG target genes because both aging and cancer are accompanied by
transcription factor binding may hinder the ability of DNA methyltrans- DNA hypermethylation of these genes. Hence, a better understanding
ferases to eventually recognize those sites, resulting in aberrant gene ex- of the reasons behind the changes in the DNA methylome during aging
pression in aged cells (150). The hypothesis seems promising but is yet to may help us to also understand the causes of cancer.
be addressed during aging in different cells, tissues, or organisms. How-
ever, our current knowledge about the limited tissue types tested so far did
not show a striking global correlation between DNA methylation and al- ncRNAs AND AGING
terations in gene expression profile during hematopoietic stem cell aging
or in whole blood during aging (151, 152). ncRNAs are the most recent players in the epigenetics field, influencing
The development of novel next-generation sequencing technologies seemingly all biological processes in virtually all organisms. The advent
for genome-wide assessment of DNA methylation levels has permitted and widespread use of deep sequencing has provided unbiased insights
the confirmation and extension of earlier studies. A comparison be- into the eukaryotic genome, including the existence and functional roles
tween the methylome of CD4+ T cells from newborn and centenarian of ncRNAs. In contrast to earlier beliefs, it is now widely accepted that
individuals further solidifies the notion of global decreases in DNA approximately 60 to 90% of the human genome is transcribed, with
methylation with aging, accompanied by heterogeneous DNA methyl- some variability observed in other organisms (165, 166), giving rise to

ation in the centenarian genome (153). Generally, age-related DNA an enormous array of ncRNAs (165–169). Until recently, most of the
methylation changes are more prominent in CpG islands (Fig. 3), but studies focused on the short ncRNAs, but the functional importance
tissue-specific biases are observed more frequently outside those sites of long ncRNAs (lncRNAs) is now becoming apparent. Although it is
(45, 149). DNA methylation changes occur at several specific sites dur- likely that the majority of these functions are epigenetic, with the
ing aging in a highly reproducible way, but the complete functional ncRNAs having significant influence on modulating gene expression
relevance of these changes is yet to be explored and understood. How- and chromatin packaging, the complete array of biological functions
ever, the reproducibility of DNA methylation changes during aging at of ncRNAs is yet to be understood (170, 171). Disruption of ncRNA
some sites is such that age prediction can be made fairly accurately by function has been implicated in numerous disease conditions, such as
analyzing DNA methylation patterns in only three specific CpG sites, at cancer, neurodegenerative disorders, cardiovascular disorders, and
least for blood DNA (154). aging (172, 173).
Not surprisingly, given their functional interdependence, DNA The occurrence of ncRNAs has been highly conserved through evo-
methylation and histone modifications are intertwined to exert the lution; even budding yeast is now appreciated to have many ncRNAs.
changes observed during aging. Numerous studies have reported age- ncRNA transcription from the rDNA locus, which is silent under
induced DNA hypermethylation of specific loci that contain known normal conditions, serves as a life span determinant of this unicellular
Polycomb group protein (PcG) target genes in both mice and humans organism through its function to regulate rDNA stability (174). Muta-
(142, 149, 154–157). PcGs repress gene expression by H3 K27 trimethy- tions that prevent expression of the ncRNAs from the rDNA locus lead
lation in a dynamic manner that is reversible by H3 K4 methylation, as to life span extension in budding yeast (174). As discussed above, ex-
needed, during development. Whereas hypermethylation of DNA upon pression of the ncRNAs from Alu elements during adult human stem
aging is enriched at the genomic regions carrying bivalent histone cell aging ex vivo promotes entry into senescence, and knockdown of
marks (that is, both H3 K4me3 and H3 K27me3) at poised promoters, the Alu transcripts reverses senescence (70). During aging of both
DNA hypomethylation co-occurs with the histone modification marks worms and mice, a decline in either mRNA or protein level has been
H3 K9Ac, H3 K27Ac, H3 K4me1, H3 K4me2, and H3 K4me3 that are observed for Dicer (175), suggesting likely defective small ncRNA
found mostly at enhancer regions (158, 159). During replicative senes- (sncRNA) processing in these organisms during aging. Indeed, the ma-
cence, there are alterations in DNA methylation patterns globally and at jority of microRNAs (miRNAs), a class of sncRNAs, have been shown
specific sites, as observed during organismal aging. Notably, cell passage to be down-regulated with age (175–177). Notably, this phenotype can
numbers and the population doublings can be accurately predicted be reversed following CR in mice (175). In the absence of Dicer in mice,
from the methylation pattern at specific CpG sites (160). This altered there are signs of early senescence, signifying its role in longevity (175).
DNA methylation pattern during replicative senescence correlated with A decrease in Dicer levels has also been observed in adipocytes collected
the change of expression of SIRT1 (161). This might result from the ef- from elderly humans, suggesting the possibility of a conserved mecha-
fect of SIRT1 on DNA methylation of PcG target genes, although the nism of sncRNA dysregulation during aging in mammals (175).

REVIEW
miRNAs are sncRNAs that negatively control their target gene ex- network in mice (190). Loss of imprinting from the IGF2/H19 locus
pression posttranscriptionally and have been implicated in aging. Al- has been shown during aging in both mice and human prostate as-
though miRNAs do not alter the chromatin structure, they are sociated with reexpression of certain inactive genes and loss of binding
considered mediators of epigenetics because they lead to heritable of chromatin-associated protein CTCF (191). This may serve as a po-
changes in gene expression that do not involve a change in DNA tential reason for increased prostate cancer occurrence in aging men,
sequence. The best-characterized examples of roles of miRNAs during further supporting the connection between aging and cancer develop-
aging come from studies in C. elegans. Several miRNAs have been ment. Other lncRNAs that are implicated in major senescence-associated
shown to be involved in modulating life span and in controlling tissue pathways, such as p53/p21 pathways, are differentially expressed in pro-
aging (178, 179). One of the most prominent examples includes the reg- liferating early passage compared to senescent late-passage fibroblast
ulation of aging by the miRNA lin-4 and its pro-aging target miRNA cells (192, 193). Similarly, differential expression patterns of various
lin-14 (178). Loss of function of lin-4 shortens life span, whereas over- lncRNAs have been implicated in the pathophysiology of another
expression of lin-4 extends life span. In contrast, knocking down lin-14, age-onset neurological disorder, Huntington’s disease, wherein some
even in adult animals, extends life span (178). The key question is what of these lncRNAs have been postulated to modulate chromatin archi-
are the target genes of lin-14 that are regulating aging? Of all the miRNAs tecture and/or transcription (194, 195). Other ncRNAs, mostly products
reported in C. elegans, more than a quarter are differentially expressed of the RNAi pathway, are involved in heterochromatin assembly in re-
during aging, including some of the miRNA families conserved with petitive DNA elements in diverse organisms (196). Here, they function
humans (176, 177, 179). Whereas some miRNAs have an antiaging effect to target histone-modifying activities in DNA repeats through reader
by promoting longevity in C. elegans, others show a pro-aging effect by proteins. Other ncRNAs act to organize the three-dimensional orga-
antagonizing longevity (177, 179, 180). Even the expression pattern of nization of chromosomes or as boundaries or insulators to prevent the
some miRNAs serves as a predictor of longevity. However, the evolution- spreading of heterochromatin (197). Whether the changes in chromatin
arily conserved nature of some of these miRNAs indicates that their role structure that occur during aging are due to changes in these particular
in life span regulation likely extends beyond this organism to larger eu- ncRNAs is yet to be examined.
karyotes, including humans. Differential expression of miRNAs is also
evident in mice and humans during aging, although the differences are
not always present in all organs (181–184). TRANSGENERATIONAL EPIGENETIC CHANGES THAT
One of the most notable features of human aging is neurodegenera- AFFECT AGING
tion and reduced brain function. Premature death of neurons is
considered to be a major feature of neurodegenerative diseases. Studies According to biological dogma, genetics governs all the inherited traits

of neurodegenerative processes implicate ncRNAs. Whereas some of across generations, and epigenetic modifications are reset upon passage
the miRNAs correlate with neuroprotection, others clearly contribute through the germ line. However, over the years, this notion was chal-
toward neurodegenerative diseases and/or aging (173). For example, lenged when evidence of epigenetic inheritance through meiosis became
the mir-34 family appears to be an important determinant for brain acknowledged in certain processes, such as flower symmetry and color
aging in flies and maybe also in worms (185). There are higher levels in plants, or coat color and size in mice (198, 199). Recently, longevity
of mir-34 in Alzheimer’s disease mouse model brains and samples mediated by histone methylation was shown to be epigenetically in-
collected from Alzheimer’s disease patients (186, 187). The prosurvival herited for several generations (198), implicating transgenerational ep-
factor BCL2 and the antiaging deacetylase SIRT1 both serve as targets of igenetic inheritance for the first time in the regulation of life span.
mir-34, and the expression of the latter correlates inversely with mir-34 Deficiencies in either of the three components of H3 K4me3 methylase
expression (181, 188), revealing a potential mechanism for mir-34 func- complex (ASH-2, WDR-5, or SET-2), in only the parental generation,
tion in the aged brain. Similarly, another miRNA, mir-144, seems to be resulted in life span extension in C. elegans in the three subsequent gen-
enriched in aged brains and may also contribute to age-associated neu- erations, in the absence of methylase deficiency in these offsprings.
rodegeneration through down-regulation of key protective factors However, only the parents with the deficiencies in the H3 K4me3 reg-
(173). Genetic studies have demonstrated that the longevity-modulating ulatory complex, and not their wild-type long-lived offspring, had re-
miRNAs lin-4 and lin-14 in C. elegans function in the same pathway as duced global H3 K4me3 levels. Hence, altered histone methylation per
DAF-2 and DAF-16 (178), modulating life span through the insulin/ se was not transgenerationally inherited. Instead, microarray analysis
insulin-like growth factor 1 (IGF-1) signaling pathway. There are reports revealed that there were persistent changes in gene expression through-
of multiple miRNAs involved in the mTOR pathway, although exper- out the generations upon manipulation of the H3 K4me3 regulatory
imental evidence of these miRNAs regulating life span is still limited complex in the parents (198), which could potentially be responsible
(181). Clearly, changes in miRNA levels during aging are a means to for the transgenerational inheritance of long life span. Further experi-
regulate target gene expression, in addition to chromatin changes. mentation is needed to identify the pathways responsible for the trans-
lncRNAs themselves also serve as important regulators of generational inheritance of longevity and to explore whether this
transcription through their interaction with chromatin or chromatin- epigenetic memory is generalizable to other species. A useful approach
associated factors, modulating aging and senescence directly or in- to study the inheritance of aging phenotypes would be to follow the lead
directly. One such example includes a specific lncRNA, Gas5, which of a recent study examining epigenetic germ line inheritance of diet-
is highly expressed in aged mice brain and has been associated with im- induced obesity and insulin resistance in mice (200). This study used
paired learning (189). Another bona fide example is H19 lncRNA, a dif- in vitro fertilization to ensure exclusive inheritance through the gametes
ferentially spliced product from the H19 gene located at the IGF2/H19 and showed that the parental high-fat diet renders the offspring more
imprinted locus, which interacts with methyl-CpG–binding domain susceptible to developing obesity and diabetes. It is tempting to specu-
protein 1 to form a complex to repress expression of an imprinted gene late that this novel mode of inheritance may illustrate how epigenetics

REVIEW
could have contributed to evolution, whereby the ancestors’ environmental cancer agents to treat age-related increases in carcinogenesis (201). Clear-
exposure determined the fate of the descendants. Given the intriguing ly, some of the antiaging effects of CR are through the down-regulation of
nature of the subject, more studies will undoubtedly further explore this the nutrient-sensing mTOR pathway (Fig. 4), and this has been discussed
exciting direction in the near future. elsewhere (204).
Because of the challenges for humans in adherence to a CR regimen,
significant research emphasis has been placed on finding effective “CR
LIFE SPAN–EXTENDING REGIMENS THAT FUNCTION, AT mimetics.” These are compounds or drugs that can essentially mimic
LEAST IN PART, THROUGH EPIGENETIC CHANGES the prolongevity effects of CR in humans by using the same pathways
as CR, without actual restriction of calorie intake. In this context, several
There is considerable interest in extending health span as well as life new, as well as already existing marketed, drugs are being assessed
span. Accordingly, improvements in our knowledge of the basic mech- (205, 206). Given that the sirtuins promote longevity in diverse species,
anisms that cause aging have led to the field that identifies different they have attracted considerable interest as drug targets. The first sirtuin-
ways to promote longevity. Experimental manipulations involving ge- activating compounds (STACs) to be identified included plant-derived
netic, epigenetic, environmental, or even pharmacological interventions metabolites, such as flavones, stilbenes, anthocyanidins, and chalcones
in different model organisms have been extremely informative for dem- (207). The best known of these is resveratrol. Resveratrol and synthetic
onstrating the potential of manipulating the processes under study, to STACs have been shown to induce physiological changes and gene
delay aging. Of all the hallmarks of aging, epigenetic alterations have expression changes that are similar to those that are induced by CR
attracted attention as a promising avenue for life span extension be- and to extend life span in many model organisms (207). The ability
cause, unlike most of other age-promoting processes, epigenetic altera- of resveratrol to extend life span in mice depends on them being fed a
tions can be modulated by inhibition or stimulation of the relevant high-fat diet because there was no extended life span when mice were
enzymes. Thus, interventions targeting epigenetic information hold im- given resveratrol on a standard diet (208). Although resveratrol stimu-
mense potential to extend life span and to counteract age-associated dis- lates Sir2/SIRT1 activity in vitro (209), its stimulatory interaction with
eases, including cancer and neurodegenerative disorders. In this section, SIRT1 depended on binding to the 7-amino-4-methylcoumarin (AMC)
we will discuss the relationship of well-known life span–extending in- fluorophore within the histone peptide substrate used to measure deace-
terventions with epigenetics. tylase activity (210). The fluorophore used in the in vitro HDAC assay
Some of the most effective life span extension regimens function at mimics bulky hydrophobic amino acids, and resveratrol only stimulates
least in part through epigenetic pathways (Fig. 4). Of all the experimen- SIRT1-mediated deacetylation of natural substrates that have large hy-
tal manipulations known to date, by far the most accessible means drophobic residues C-terminal to the acetyl lysine (211–214). The recent

toward life span extension is dietary restriction (DR) or CR, which structure of the complex of SIRT1, resveratrol, and an AMC-peptide
has profound effects in multiple organisms, including yeast, flies, worms, (210) is likely to guide the computational search and/or chemical design
mice, and even primates (53). Although CR has been shown to act of better compounds to stimulate SIRT1 deacetylase activity against its
through multiple different pathways, the precise unified mechanism native substrates. Promisingly, the synthetic STACs SRT1720 and
by which CR exerts its life span–extending effects still needs to be defined SRT2104 extend the life span of obese mice and protect against age-
(201). However, evidence suggests that epigenetic alterations, including related changes in multiple tissues (215).
DNA methylation and histone modifications, may play crucial roles in The antidiabetic drug metformin also induces effects similar to CR
life span extension by CR (201, 202). For example, CR is thought to be (216). Diabetes is considered an age-associated disease, and disturbances
important for regulating DNA methylation patterns at individual loci in in insulin signaling and carbohydrate homeostasis may essentially lead to
the genome, but not the global DNA methylation pattern, and this may other age-related complications, including cancer, if untreated. Along
occur with CR increasing the expression level and/or activities of certain with its antidiabetic properties, metformin supplementation has been
DNMT enzymes, such as DNMT1 and DNMT3b (9, 201). In agreement, shown to increase life span and health span in C. elegans (217, 218) and
some DNMT inhibitors have displayed preventive potential against age- male mice (219), together with some of the benefits seen with CR. A re-
associated diseases, such as cancer, implying the importance of studying cent study of more than 180,000 people showed that diabetes patients
these compounds to promote a healthy life span. Similarly, the regulation being treated with metformin not only lived longer than other diabetic
of histone modifications, such as histone acetylation and deacetylation, patients but also lived longer than the healthy control sample (220). Until
may play roles in regulating life span during CR. This is solidified by recently, metformin was thought to primarily function by regulating the
numerous studies performed on class III HDAC enzymes (Sir2 in yeast stress response and metabolic pathways (216). However, a very recent
and its mammalian sirtuin orthologs) in multiple model organisms, report provided evidence for epigenetic regulation potentially contribut-
wherein activation of this enzyme class has frequently been associated ing to the mechanism of action of metformin. Increased levels of SIRT1
with CR and life span extension (Fig. 4), suggesting that there may be were observed in human subjects treated with metformin (221). Further
a protective mechanism conferred by deacetylation during aging. Com- exploration is needed to fully understand how metformin mechanistically
parative analysis of the transcriptome of the brains of rats maintained on extends life span in humans. However, given that metformin is approved
a normal diet or CR regime during aging reveals that CR largely prevents by the U.S. Food and Drug Administration for use in humans, commer-
the differential gene expression that is observed during aging (203). Further- cially available, and relatively cheap, it has the potential to be revolution-
more, CR induces specific gene expression patterns in the cerebral cortex ary for increasing human health span and life span.
that offer neuroprotection, potentially by inducing mir-98-3p, which it- Several compounds that reduce global histone acetylation also ex-
self alters the activity of HDACs and HATs (203). In relation to this, the tend life span. Spermidine, a naturally occurring polyamine, directly
ability of HDAC inhibitors to reactivate key tumor suppressor genes inhibits HATs, maintaining histone H3 in a hypoacetylated state (222).
has been extensively analyzed for their potential application as anti- Polyamines result in resistance to stress and reduced rates of cell necrosis

REVIEW
during chronological aging in yeast and human cells and extend the tophagy, contributing to the aging process and diseases of aging. Hence,
life span of flies, worms, and human cells. In agreement, administration dietary and pharmacological manipulations that decrease acetyl-CoA
of spermidine through diet in aging mice also reduced age-related ox- levels might extend life span by stimulating autophagy. Notably, SIRT1
idative stress (222). Another potential way to reduce histone acetylation also stimulates autophagy, wherein SIRT1 induces FOXO-mediated ex-
globally is to feed a diet that depletes acetyl–coenzyme A (CoA), the pression of autophagy pathway components and deacetylates major
donor for acetylation reactions. But how does reducing acetylation ex- players of the autophagy induction pathway (Fig. 4) (228). Indeed, the
tend life span? Depletion of acetyl-CoA by starvation of mammalian longevity-inducing role of CR appears to be through the SIRT1-dependent
cells in culture and fasting in mice has been recently shown to induce induction of autophagy (229). Hence, it is tempting to speculate that
autophagy (223). In agreement, spermidine also induced autophagy, multiple different manipulations to extend life span, such as CR, acetyl-
extending chronological life span in yeast (223, 224). Autophagy may CoA depletion, polyamines, and sirtuin activation, all function through
be regarded as a mechanism of cellular “cleansing,” and its up-regulation a common pathway to stimulate autophagy, potentially through an ef-
has clear cellular benefits that would promote longevity (225). According- fect on histone acetylation (Fig. 4).
ly, note that stimulating autophagy is a common theme among multiple An exciting new way to improve health span and, subsequently, life
interventions that extend life span (Fig. 4) (226). Recently, histone acet- span in mice is to specifically kill off senescent cells with senolytic agents
ylation has also been implicated in the mechanism whereby autophagy is (230). Given the epigenetic changes that are required for the maintenance
activated in metazoans. Fly life span is extended and autophagy is induced of senescence, another potential avenue to extend life span may be to de-
when acetyl-CoA levels were reduced in fly brains (224). The mechanism velop senolytic drugs that target specific epigenetic enzymes.
wherein high acetyl-CoA levels block autophagy is not totally clear, but
it likely involves histone acetylation. In one study, the HAT p300, whose
substrates include histone H3, was required for high levels of acetyl-CoA CONCLUSION AND FUTURE DIRECTIONS (RESETTING THE
to block autophagy (223). In another study, high levels of H4 K16Ac (the AGING CLOCK)
substrate of Sir2/SIRT1) down-regulated autophagy, and low levels of H4
K16Ac induced autophagy (227). The natural increase in H4 K16Ac that The aging process is unquestionably complex. During an organism’s
occurs during aging, because of Sir2 loss, could lead to the decline of au- lifetime, aging cells undergo a plethora of changes and accumulate
Longevity

Calorie restriction STACs Metformin
Nutrients Ac
SIRT1 Autophagy
Ins
uli
n
1
SIRT1
F-
IG
Rapamycin AMPK Ac STACs

Akt Active ATG
Metformin
proteins
mTOR SIRT1
Ac
FOXO
Me
Me
X Me
Me
FOXO
ATG RNA
Me ATG genes
Cy cleus
Nu
Heterochromatin
top
reorganization
Intervention
DNMTs
during aging
lasm
Spermidine
Ac
HATs
Ac SIRT1
Acetyl CoA STACs
depletion
Ac Ac Ac
RNA
Ac
X
Ac
ATG genes
Fig. 4. Schematic showing how some external interventions trigger longevity, often at least partly through stimulating autophagy. The pink
writing refers to dietary, chemical, or therapeutic interventions that can extend life span, in at least some organisms (described in the text). Arrows indicate
stimulating effects, and blocked lines indicate inhibitory effects. This schematic is not meant to be exhaustive but highlights the pathways that alter the
epigenetic information and autophagy.

REVIEW
macromolecular damage. The aging phenotypes are manifested by the interventions on the epigenome in an aging individual, given the in-
summation of alterations of many different signaling cascades. Here, we terconnectivity of the epigenetic mechanisms.
attempted to provide an overview of some of the major changes in aging
cells, tissues, and organisms that have come to light in recent years
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Epigenetics and aging
Sangita Pal and Jessica K. Tyler
Sci. Adv., 2 (7), e1600584.

DOI: 10.1126/sciadv.1600584
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Normal chromatin state Abnormal chromatin state

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X (organismal aging) RNA

Retrotransposable element Retrotransposable element

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Retrotransposable element Retrotransposable element

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Rapamycin AMPK Ac STACs

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