Epigenetics and aging

Epigenetics and aging

Maturitas 74 (2013) 130–136 Contents lists available at SciVerse ScienceDirect Maturitas journal homepage: www.elsevier.com/locate/maturitas Review...

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Maturitas 74 (2013) 130–136

Contents lists available at SciVerse ScienceDirect

Maturitas journal homepage: www.elsevier.com/locate/maturitas

Review

Epigenetics and aging Patrizia D’Aquila, Giuseppina Rose, Dina Bellizzi, Giuseppe Passarino ∗ Department of Cell Biology, University of Calabria, 87036 Rende, Italy

a r t i c l e

i n f o

Article history: Received 5 November 2012 Accepted 11 November 2012

Keywords: Epigenetics Aging Age-related diseases Mitochondrial DNA

a b s t r a c t Over the past two decades, a growing interest on the research of the biological basis of human longevity has emerged, in order to clarify the intricacy of biological and environmental factors affecting (together with stochastic factors) the quality and the rate of human aging. These researches have outlined a complex scenario in which epigenetic marks, such as DNA methylation and numerous histone modifications, are emerging as important factors of the overall variation in life expectancy. In fact, epigenetic marks, that are responsible of the establishment of specific expression programs and of genome stability, represent a “drawbridge” across genetic, environmental and stochastic factors. In this review we provide an overview on the current knowledge and the general features of the epigenetic modifications characterizing the aging process. © 2012 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

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4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA methylation and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. General features of DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. DNA methylation and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histone modifications and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. General features of histone modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Histone modifications and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non coding RNA and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria as modulators of aging epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Provenance and peer review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Aging is a process of slow and gradual deterioration of the functional capacities that makes the individual particularly susceptible to environmental challenges and more prone to a variety of illnesses, and leads to a dramatic reduction of the individual survival probability and, ultimately, to death [1,2]. Aging affects all organisms, but lifespan is species-specific; in addition, among and within populations, a noticeable inter-individual variability exists with respect to the rate and the quality of aging. This heterogeneity

∗ Corresponding author. Tel.: +39 0984492932; fax: +39 0984492911. E-mail address: [email protected] (G. Passarino). 0378-5122/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.maturitas.2012.11.005

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results from a complex interaction of genetic, environmental and stochastic factors [3]. The continuous changes that occur during the process of aging can be observed not only in the individual’s anatomy and physiology, but also at cellular and molecular levels. In fact, numerous studies revealed that an extensive remodeling of gene-expression profiles, driving physiological and/or pathological changes in different tissues, takes place with aging [4]. In disentangling the molecular basis of the above changes, exciting revelations have emerged by a branch of research known as “aging epigenetics”. Epigenetics refers to the study of mitotically and, in some cases, meiotically hereditable changes of a phenotype that tightly regulate the cell-type specific expression of genetic information without affecting the DNA sequence [5]. Epigenetic patterns are established

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at pre-conceptional and gestational level. In fact, both paternal and maternal exposure to environmental factors during gametogenesis or gestation has been demonstrated to be responsible of the offspring’s epigenome [6]. On the other hand, during the early stages of life, the inherited epigenetic status undergoes several changes to ensure an appropriate process of cell development and differentiation. Non-random mechanisms such as environmental stimuli or stochastic errors are able to induce changes in epigenetic profiles at both early and late life stages, being in most cases responsible for many processes occurring during the lifespan, including development, differentiation, stress response, and pathological conditions [7]. Although the epigenetic role in the above processes has been extensively investigated, little is yet known about the relationship between epigenetics and aging. In this review we will focus on the correlation with aging of the best known epigenetic marks, such as DNA methylation and post-translational modifications of histones, that occur overtime in higher organisms and cooperate in chromatin remodeling, leading to a dynamic regulation of gene expression. We will also highlight the role of mitochondria in modulating the above modifications. 2. DNA methylation and aging 2.1. General features of DNA methylation DNA methylation is a covalent biochemical modification consisting in the addition of a methyl group to the aromatic ring of cytosine (5-mC) predominantly located 5 to a guanosine (CpG) [8]. This process prevalently involves CpGs located into intergenic and intronic CpG-poor regions as well as in repetitive sequences, most of which derived from transposable elements, thus hindering the event of amplification and new insertion in the genome [9]. Unmethylated CpG dinucleotides are, instead, concentrated in CpGrich regions, termed CpG islands (CGIs), sequences of about 1 kb in length and with a CG content greater than 55%, that are mostly associated to the promoter regions and to the first exons of almost 60% of genes, including most housekeeping genes and half of all tissue-specific genes [10,11]. Conversely, Maunakea et al. revealed also the presence of methylated CGIs within intra- and intergenic regions, that are likely to be involved in a fine regulation of alternative transcripts, as emerged by the cell type specific expression of SHANK3 [12]. Different biological functions, such as development and differentiation, genomic imprinting, X chromosome inactivation and parasitic DNA suppression are mediated by DNA methylation, as it influences chromatin structure and, thus, inducing gene silencing. This process takes place either inhibiting the transcription factor binding to DNA target sequences or facilitating the recruitment of methyl-binding proteins [13,14]. DNA methylation has also been rarely reported to occur asymmetrically outside the CpG context (non-CpG methylation). This methylation, so far reported in plants, has been described more recently occurring in mammal embrional stem cells and in promoter regions of different genes, although its biological significance is unknown [15,16]. DNA methylation takes places after DNA replication and is mediated by a family of DNA methyltransferases (DNMTs) that includes DNMT1, DNMT3A, DNMT3B and DNMT3L [17,18]. These enzymes transfer a methyl group from S-adenosyl-lmethionine (SAM) to deoxycytosine, producing 5-methylcytosine and S-adenosylhomocysteine. DNA methylation patterns are also determined by DNA demethylases, that operate: (i) by preventing DNMT1 accessibility to newly replicated DNA strands; (ii) by recruiting enzymes belonging to the mismatch-repair pathways; (iii) through processes initiated by DNMT3A and DNMT3B themselves [19]. In this context, recent evidence demonstrated the

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capacity of the Ten-11 translocation family proteins (TET1-3) to oxidize 5-mC to 5-hydroxymethyl-cytosine (5-hmC) [20,21]. Despite 5-hmC is commonly considered an intermediate of the demethylation process of 5mC residues, both for its lowest levels in the genome and its short half-life, this species is emerging to be another epigenetic marker (the “sixth base”), having important roles in epigenetic reprogramming and regulation of tissue-specific gene expression [22,23]. 2.2. DNA methylation and aging DNA methylation patterns are not fixed; during various stages of mammalian development they are reprogrammed to ensure the normal mammalian embryogenesis and cell differentiation. Therefore, the above patterns can change during lifetimes in response to several stimuli from the external and internal environment. These stimuli are able to induce loss or gain of DNA methylation that can be propagated during cell division and sometimes transmitted across generations, resulting in permanent maintenance of the acquired phenotype [24]. Starting from the pioneering studies of Berdyshev and Vanyushin, several epigenetic alterations are now increasingly recognized as part of aging and aging-related diseases [25–28]. For instance, a complex relation between epigenetic control and Xlinked or imprinted genes occurs in aging. In particular, it was demonstrated an age-reduction in DNA methylation of the inactive X chromosome, particularly in the myeloid cell lineage of peripheral blood cells [29]. As a result, a positive correlation between age and degree of somatic X chromosome inactivation (XCI) skewing was observed as well as in cancer, autoimmune disorders and other diseases [30,31]. In disentangling the role of epigenetics in human aging, a significant contribution has been provided by studies in which global and gene-specific methylation levels were assessed in mono(MZ) and dizygotic (DZ) twins, mainly in order to elucidate the phenotypic divergence of MZ over time with respect to their susceptibility to diseases or other phenotypes [32,33]. Two independent works reported that lower epigenetic differences occur between MZ than DZ twins. In addition, although MZ twins are epigenetically indistinguishable during the early years of life, older individuals exhibited significant tissue-specific differences in their overall content and genomic distribution of 5-methylcytosines, affecting global gene-expression [34,35]. Moreover, Wong et al., through a gene-specific longitudinal study, found that epigenetic differences between MZ twins already occur during the course of childhood development, thus suggesting that environmental factors in the early stage of life can establish long-lasting epigenetic changes [36]. The relationship between the epigenetic changes and aging was also confirmed by observation of time-dependent changes in global DNA methylation levels in Icelandic unrelated individuals and in three-generation families from Utah [37]. The finding of familial clustering of methylation supports the idea that the DNA methylation stability is genetically determined. Taken together, the above evidence demonstrate that in lifetime the epigenome can be regulated by genetic, stochastic (due to random epimutations) or systematic (in response to environmental changes) factors. A gradual loss of total methylcytosine content with age occurs in most vertebrate tissues including humans [38]. This hypomethylation predominantly affects non island-CpGs and interspersed repetitive sequences (IRSs), such as Alu and human endogenous retrovirus K (HERV-K), through different age-dependent mechanisms [39–41]. More recently, Heyn et al. corroborated and extended the above findings demonstrating that the age-associated hypomethylation is present in all genomic compartments,

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including promoters, exonic, intronic and intergenic regions [42]. Besides global DNA hypomethylation, a robust and progressive rise in DNA methylation levels for promoters of specific loci has been described across lifespan. The first gene showing an association between aging and promoter DNA methylation was the one encoding for estrogen receptor (ER). Successively, significant hypermethylation was found in genes encoding for ribosomal DNA clusters as well as in those involved in DNA binding and regulation of transcription, thus suggesting that deregulation of these genes could affect a large spectrum of intracellular pathways [43,44]. Genes for tumor suppression (COX7A1, LOX, RUNX3, TIG1, p16INK4A, RASSF1, DUSP22), development and growth (IGF2, cFos), cell–cell adhesion (CDH1), metabolism (ELOVL2, SLC38A4, SLC22A18, MGC3207, ECRG4, ATP13A4, AGPAT2, LEP), DNA repair (MLH1) and control of signal transmission (FZD1, FZD7) exhibited altered DNA methylation patterns in aging, displaying sometimes tissue- and cell type-specific features with consequent different functional outcomes [41,45,46]. Taken together, these studies demonstrate a loss of the epigenetic control in aging, suggesting its correlation with age-related pathological phenotypes, such as cancer, neurodegenerative and cardiovascular disorders, and with physiological processes of aging itself including psychophysical and immune decline, sarcopenia and frailty [47–49]. Age-dependent alterations in expression and/or functions of DNA methyltransferases as well as environmental factors contribute to changes in DNA methylation above described. An under-expression of DNMT1 and DNMT3A in human lymphocytes T from elderly individuals and in senescent fibroblasts and an increase of DNMT3B in fibroblast was described. These findings support previous observations about decrease and increase, respectively, in global and specific promoter gene methylation [50,51]. What is more, insufficient DNA methylation in Dnmt1+/− mice has been reported to cause immunosenescence, autoimmunity thus affecting healthy aging [52]. Lastly, dietary deficiencies in nutrients, including folate, choline and methionine, elements such as zinc, selenium or arsenic and UV light have been demonstrated to affect DNA methylation status in a tissue- and age-dependent manner. An example is provided by the age-related hypomethylation in rat of the ER promoter following exposure in utero to a high-fat diet [53]. What is more, calorie restriction, the sole dietary intervention increasing longevity in different species, leads to aberrant DNA methylation patterns likely by modulating DNMT activities [54,55]. 3. Histone modifications and aging 3.1. General features of histone modifications In eukaryotes the fundamental subunit of chromatin is the nucleosome, which consists of approximately 147 base pairs of double-stranded DNA wrapped around an octamer of small, basic histone proteins (H2A, H2B, H3 and H4). Histone H1 is involved in linking the nucleosomes together, facilitating the formation of higher-order structure of chromatin [56]. Histones undergo several epigenetics modifications, including acetylation, methylation, ubiquitylation, phosphorylation and ADP ribosylation, occurring within the core, the amino- and carboxyterminal tails and, rarely, within the globular domains. These modifications, carried out mainly by multiprotein enzymes, induce changes in nucleosome structure and function, directly contributing to the genome compartmentalization into domains, such as heterochromatin and euchromatin, transcriptionally silent and active, respectively. Additionally, modified histones are involved in gene activity regulation by their interactions with specific effectors [57]. Besides DNA methylation, acetylation and methylation of histones are the most well-characterized epigenetic marks.

Acetylation reaction, in which acetyl groups are transferred from acetyl-CoA to lysine residues of histones H3 and H4, is performed by histone acetyl transferases (HATs), which include the GNAT (Gcn5related N-acetyltransferase) superfamily, the MYST, p300/CBP and TFIIIC families, as well as the nuclear receptor co-activators (ACTR, SRC-1, TFII). This modification, found predominantly at promoters of transcribed units, results in chromatin decondensation and, thus, in transcription activation [58]. Conversely, histone deacetylases (HDACs) are able to remove acetyl group from histones, with consequent heterochromatin formation and transcriptional repression. Four different subfamilies of HDACs are known to date: class I, II and IV HDACs comprise eleven enzymes that share sequence homology and require Zn2+ for their deacetylase activity, whereas the class III HDACs comprise the members of the Sirtuin family (SIRTs), a group of NAD+ -dependent deacetylases [59]. The interplay between HDACs and HATs results in dynamic transitions of chromatin structure and, hence, in a switch between repressive and permissive chromatin. Moreover, it has been reported that all three DNA methyltransferases are able to suppress gene transcription being component of histone deacetylase repressory complexes [60]. Histone methylation occurs on lysine residues and is mediated by histone methyltransferases (HMTs) that can transfer up to three methyl groups for each site. Furthermore, protein arginine methyltransferases (PRMTs) can also mono- or dimethylate arginine residues on their guanidine nitrogen, with activation or inhibition of gene transcription [61]. In this context it is worth noting that H3 and H4 arginine residues can be deiminated to citrulline, antagonizing the possible activatory effect of arginine methylation. Through the different grade of modification of lysine and arginine residues, histone methylation can result in activation of inhibition of transcriptional processes, thus increasing the complexity of the epigenetic control on chromatin structure. An example of this complexity emerges from the H3 histone tail methylation: three methylation sites (H3K4, H3K36 and H3K79) are implicated in transcriptional activation, whereas methylation of H3K9, H3K27, and H4K20 has been associated to gene repression [62,63]. Histone demethylases have also been identified. LSD1 demethylates H3K4 and H3K9, inhibiting and activating transcription, respectively [64]. Moreover, it was observed that H3K9 and H3K36 methylation can be reversed by JHDM family members [65]. Considering the variety and the complexity of the effect that each histone modification has on each other, as well as on chromatin remodeling and in the transcription of the associated genes, it is clear as the dynamic integration of epigenetic signaling at histone level considerably extends the information potential of the genetic code, providing high flexibility to the epigenetic control of gene expression. 3.2. Histone modifications and aging The epigenetic remodeling during the lifetime is also characterized by the occurrence of different type or combination of histone modifications, accompanied by the gradual reduction of the histone levels that dramatically affect chromatin structure. In general, in vivo and in vitro studies have revealed a global increase of the trimethylation of H4K20 and of the phosphorylation of H3S10 as well as a decrease of the trimethylation of H3K9 and H3K27 and of the acetylation of tH3K9 with aging [66]. More detailed summary of the histone modifications occurring during aging has been reported (see Table 1 in [66]). More specifically, Saccharomyces cerevisiae cells undergo changes of both histone levels and their post-translational modifications during their replicative age. In fact, a reduction in the total protein levels of H3, H4 and H2A and the expression of acetylated H3K56 have been observed in old cells [67]. Moreover, studies on Caenorhabditis elegans demonstrated as

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members of the ASH-2 trithorax complex, ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2 were unfavorable to longevity and that H4K5 acetylation, but not total H4 protein levels, declines with age [68]. In mice, alteration in H4K12 acetylation was also associated with the age-related cognitive decline [69]. Furthermore, the loss of SIRT6, a H3K9 deacetylase involved in telomere function, in expression of aging-associated genes and in recruitment of DNA-PK catalytic subunit to chromatin in response to DNA damage, is able to induce a premature aging-like phenotype [70]. Peculiar histone modifications features were observed also in human progeroid syndromes. Specifically, the decrease in H3K9 and HP1 methylation, associated with an increase of H4K20 trimethylation has been considered one of the main molecular alterations in the Hutchinson–Gilford Progeroid Syndrome (HGPS) and in common aged human tissues [71]. Furthermore, Narita et al. demonstrated that either H3K9 hypermethylation (with the recruitment at E2F-responsive promoters of the retinoblastoma (Rb) tumor suppressor and heterochromatin proteins), or H3K4 demethylation modulate chromatin structure thus inducing the transcriptional silencing of the retinoblastoma target genes in senescent cells [72]. Epigenetic changes, such as decreased H3K27 methylation of insulin/IGF1 genes have been previously shown to modulate lifespan through gene expression regulation. Specifically, upon signals triggering senescence, p16 expression can be modulated by H3K27 trimethylation, that acts as recruitment signal for the oncogene and polycomb repressive complexes (PRC1and PRC2); moreover, the histone demethylases KDM2a and KDM2b, which target methylated H3K36, prevent senescence by modulating the p53 and Rb pathway [73,74]. Furthermore, experiments in Drosophila with HDAC inhibitors demonstrated that the expression of two heat shock proteins (hsp22 and hsp70), that play a positive role in lifespan determination, was found to be strongly affected by histone modifications [75]. Several evidence have demonstrated that histone modifications affect also nucleosome assembly, and, then, genome integrity. For example, H3K56 acetylation regulates the interaction between H3–H4 and the histone chaperones CAF-1, Rtt106 and Asf1, in order to promote the deposition of newly synthesized histones and the gathering of nucleosome [76]. Noteworthy, the above processes are influenced also by age-associated decline of the levels of histone chaperones. In addition, several histone-modifying enzymes are playing an important role in aging. In this context, special attention should be paid to the Sirtuin family, a class of evolutionary conserved NADdependent histone deacetylases (HDACs) involved in a wide range of intracellular processes, including chromatin remodeling, apoptosis, transcriptional silencing and lifespan and responsible for the regulation of two critical histone post-translational modifications, namely the acetylation of H3 and H4 at lysine 9 and 16, respectively [77]. In particular, an age-associated decline in Sir2 protein levels associated to a rise in H4K16 acetylation and loss of histones at specific subtelomeric regions was observed in replicatively old yeast cells [78]. Moreover, DOT1L, an histone methyltransferase that methylates H3K79 regulating cell proliferation, is down-regulated in aged tissues [79]. 4. Non coding RNA and aging The majority of eukaryotic genomes transcribes non-coding RNAs (ncRNAs) that growing evidence are reporting to be involved in different intracellular functions and part of the epigenetic machinery. These molecules have been classified in infrastructural, constitutively expressed ncRNAs and regulatory ncRNAs. The former include ribosomal, transfer, small nuclear, and small nucleolar RNAs, the latter include microRNAs (miRNAs), Piwi-interacting

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RNAs (piRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), promoter-associated RNAs (PARs) and enhancer RNAs (eRNAs) [80]. By mediating transcriptional gene silencing, regulatory ncRNAs have emerged to critically control gene expression in cell physiology and development, chromatin architecture, epigenetic memory, cell cycle regulation, energy metabolism, transcription and RNA splicing. Therefore, ncRNAs dysregulation appear to have decisive relevance in both pathological (cancer, neurological and cardiovascular diseases or cancer) and non-pathological (senescence) phenomena [81,82]. Conflicting reports have also suggested a possible association between siRNA-mediated gene suppression and other epigenetic marks such as histone modification and DNA methylation [83]. miRNAs are the best characterized small ncRNA influencing aging and lifespan. Several studies in both murine and human aging models reported that multiple miRNAs, including lin-4, miR1, miR-145 and miR-140, modulated insulin/IGF-1, IGF-1R, the insulin/IGF-1 receptor substrate ISR-1, as well as lipid metabolism and insulin secretion. Moreover, p53-p21-pRb and p16-pRb pathways, class I HDAC and SIRT1 activity, widely considered as critical regulators for cell senescence, are modulated by different miRNAs, including miR-34a, member of miR-106b family and miR-449a. Recently, Liu et al. exhaustively summarized the recently identified miRNAs involved in cell senescence (see Table 1 in [82] and references therein). 5. Mitochondria as modulators of aging epigenetics Over the past two decades, the growing interest in searching the biological and the environmental factors affecting the quality and the rate of human aging has also highlighted the significant involvement of mitochondrial function (and dysfunction) in aging [84,85]. In fact, extensive evidence, such us age-related changes in mitochondrial content, structure and function, as well as in mitochondrial DNA, is indicating that these organelles are able to modulate numerous intracellular signaling pathways which appear to be critically important for the maintenance of cellular homeostasis and, in turn, aging. A series of studies has demonstrated a decline of the mitochondrial respiration efficiency with age attributable either to a progressive down-regulation of genes encoding for mitochondrial proteins such as several subunits of cytochrome-c oxidase, NADH dehydrogenase and ATP synthase, or to the decline of mitochondrial biogenesis with aging [86,87]. The decline in the oxidative phosphorylation (OXPHOS) activity correlates with a wide spectrum of mtDNA mutations, including point mutations, large scale deletions and duplications which progressively accumulate in post-mitotic tissues during human aging [88,89]. The large increase in mtDNA mutations and the deficit in mitochondrial respiratory function have been attributed to the progressive and irreversible accumulation of oxidative damage by reactive oxygen species (ROS), generated primarily as by-product of the mitochondrial respiratory chain, but also by cytochrome P450 and peroxisomes metabolism, during the immune-inflammatory response, in the detoxification of xenobiotics and in response to several environmental agents [90]. Turk et al. reported that the presence of 8-hydroxyl-2 deoxyguanosine (8-OH-dG) in CpG nucleotide, the most studied DNA base lesion induced by oxidative stress in aging, diminishes the ability of DNA methyltransferase to methylate the adjacent cytosine, and of restriction nucleases to cleave the DNA [91]. In addition, Valinluck et al. demonstrated that the oxidation of guanosine in 8-OH-dG and/or 5-methylcytosine in 5-hydroxymethylcytosine in a CpG dinucleotide significantly reduces the binding affinity of MBDs to their recognition sequences,

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thus leading alterations in transcription [92]. Furthermore, the intracellular redox state is also able to modulate the activity of methionine adenosyltransferase (MAT), enzyme responsible for the synthesis of S-adenosylmethyonine (SAM) from l-methionine and ATP. In particular, oxidative/nitrosative stress, associated to a low ratio of GSH/GSSH reduces MATs activity [93,94]. These evidence have demonstrated the direct role for oxidative damages and, thus for mitochondrial dysfunction, in determining aberrant DNA methylation patterns in cancer and aging. Epigenetic processes also depend on the cellular bioenergetic systems, regulated by the mitochondrial electron transport chain activity and, in turn, by mitochondrial DNA. ATP, acetyl CoA, NADH and NAD+ are the main substrate of chromatin phosphorylation, acetylation and deacetylation reactions, thus contributing to the epigenetic process of chromatin remodeling [95,96]. Moreover, when OXPHOS is inhibited or insufficient, the mitochondrial NADH/NAD+ ratio increases, inhibiting the mitochondrial production of methylene-tetrahydrofolate and serine. This reduces methionine and, consequently, SAM production. Lastly, mitochondria contributes to epigenetics modification through the folate metabolism and the regulation of the metabolic switch between S-adenosyl-methionine and nucleotide synthesis through the onecarbon cycle [97]. Besides, mtDNA-specific interactions between mitochondria and nucleus influence global DNA methylation patterns. Smiraglia et al. found that in response to the depletion and repletion of mtDNA, DNA methylation patterns of several nuclear genes were significantly influenced, providing the first direct evidence that mtDNA modulates the epigenetic modifications in the nucleus [98]. In addition, it was reported that mtDNA variants, able to regulate several intracellular function and to induce changes in nuclear gene expression and associated with human longevity, influence global DNA methylation levels [99–103]. Either way, the oxidative phosphorylation efficiency and/or the activation of signaling pathways between mitochondrial and nuclear genome where considered to be responsible of mtDNA sequence-specific regulation of the epigenetic landscape, probably through the well known cross talk between mitochondrial and nuclear genome [104–106]. 6. Conclusion The literature reviewed in this paper reveals compelling evidence about the role of epigenetic modifications in the aging process. It emerges an interaction of genetically determined and environmentally induced factors affecting epigenetic patterns which on turn are correlated with the differences in interindividual susceptibility to functional decline and vulnerability to diseases in the elderly people. The molecular mechanisms underlying the above patterns as well as their hereditability are not yet clearly understood, also because tissue- and cell-specificity make extremely intricate their comprehension. In fact, whether the aging phenotypes induce the epigenetic changes or vice versa remain an open question that needs further investigations. Finally, the reversibility of epigenetic modifications provides new possibility of therapeutic intervention in the biomedical aging research. In fact, different molecules, including HDAC inhibitors as well as small ncRNAs have been introduced as promising novel drugs in preclinical and clinical trials against various physiological and pathological age phenotypes. Contributors All the authors were involved in the drafting, editing and approval of this manuscript.

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