The Epigenetics of Aging in Invertebrates

Aging is an unstoppable process coupled to the loss of physiological function and increased susceptibility to diseases. Epigenetic alteration is one of the hallmarks of aging, which involves changes in DNA methylation patterns, post-translational modification of histones, chromatin remodeling and non-coding RNA interference. Invertebrate model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, have been used to investigate the biological mechanisms of aging because they show, evolutionarily, the conservation of many aspects of aging. In this review, we focus on recent advances in the epigenetic changes of aging with invertebrate models, providing insight into the relationship between epigenetic dynamics and aging.


Introduction
Aging is an inevitable, time-dependent process in most living organisms, which involves functional decline, a steady increase in a plethora of chronic diseases, and ultimately death [1]. According to different biological scales, aging can be divided into "four layers": (I) the organism's decline in physical function and increased susceptibility to diseases; (II) systemic immune, metabolic and endocrine dysfunction; (III) cellular malfunction; and (IV) failure of biomolecular maintenance [2]. Epigenetics mainly acts in layers three and four, but also impacts other levels. Much research on aging has focused on genetic manipulation, and changing the activity of numerous genetic pathways can lead to lifespan extension in model organisms, for example, the insulin/Insulin-like growth factor-1) pathway (IIS), Target of rapamycin (TOR) signaling, Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and sirtuins [3]. However, recently more attention has focused on epigenetic changes, which have come to be considered one of the hallmarks of aging [4]. Chromatin structure is altered because of the loss of histone protein during aging [5]. There is global DNA hypomethylation during ontogenesis [6]. Most brain functions, including synaptic plasticity, learning and memory, decline with age when epigenetic changes occur, including changes in microRNA (miRNA) levels [7,8]. Studies have showed that aging in humans is also associated with epigenetic drift [9]. Model animals, such as Caenorhabditis elegans and Drosophila melanogaster, have been long used for the study of aging. Worms and fruit flies have natural advantages because of their short life cycles, being easy to house and feed, the power of available genetic manipulations, and the conservation of many mammalian aging signaling pathways. The average lifespan is 2 to 3 weeks for C. elegans at 20 • C, and 70 days for Drosophila at 25 • C [10]. Simple models have provided valuable insights into the aging process; for example, the IIS signaling pathway was first discovered in C. elegans and was later found to be Figure 1. DNA methylation and demethylation. DNA cytosine methylation is catalyzed by DNA methyltransferases (DNMTs) to form 5-methylcytosine (5mC). Then, it can be oxidized iteratively by ten-eleven translocation (TET) protein to develop to 5-hydromethylcytosine (5hmC), 5formylcytosine (5fC), and 5-carboxylcytosine (5caC), respectively. Finally, 5-carboxylcytosine (5caC) can be demethylated into the cytosine through the catalysis of a series of enzymes. Figure modified from Kohli and Zhang (2013) [17].

Histone Modifications in Invertebrate Aging
Nucleosomes, the basic structures of eukaryotic chromatin, are made up of dimers of each core histone (H2A, H2B, H3 and H4). Histone modifications provide another layer of regulation beyond the DNA sequence itself. During the aging of organisms, the level of histone gradually decreases [5,13,27] but histone modifications show different changes. Histone modifications comprise several types, such as methylation, acetylation, phosphorylation and ubiquitylation [28]. Methylation and acetylation are thought to be most well-characterized modifying methods associated with aging [29]. Here, we will discuss those two types of modifications ( Figure 2). DNA methylation and demethylation. DNA cytosine methylation is catalyzed by DNA methyltransferases (DNMTs) to form 5-methylcytosine (5mC). Then, it can be oxidized iteratively by ten-eleven translocation (TET) protein to develop to 5-hydromethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), respectively. Finally, 5-carboxylcytosine (5caC) can be demethylated into the cytosine through the catalysis of a series of enzymes. Figure modified from Kohli and Zhang (2013) [17].

Histone Modifications in Invertebrate Aging
Nucleosomes, the basic structures of eukaryotic chromatin, are made up of dimers of each core histone (H2A, H2B, H3 and H4). Histone modifications provide another layer of regulation beyond the DNA sequence itself. During the aging of organisms, the level of histone gradually decreases [5,13,27] but histone modifications show different changes. Histone modifications comprise several types, such as methylation, acetylation, phosphorylation and ubiquitylation [28]. Methylation and acetylation are thought to be most well-characterized modifying methods associated with aging [29]. Here, we will discuss those two types of modifications ( Figure 2). . DNA methylation and demethylation. DNA cytosine methylation is catalyzed by DNA methyltransferases (DNMTs) to form 5-methylcytosine (5mC). Then, it can be oxidized iteratively by ten-eleven translocation (TET) protein to develop to 5-hydromethylcytosine (5hmC), 5formylcytosine (5fC), and 5-carboxylcytosine (5caC), respectively. Finally, 5-carboxylcytosine (5caC) can be demethylated into the cytosine through the catalysis of a series of enzymes. Figure modified from Kohli and Zhang (2013) [17].

Histone Modifications in Invertebrate Aging
Nucleosomes, the basic structures of eukaryotic chromatin, are made up of dimers of each core histone (H2A, H2B, H3 and H4). Histone modifications provide another layer of regulation beyond the DNA sequence itself. During the aging of organisms, the level of histone gradually decreases [5,13,27] but histone modifications show different changes. Histone modifications comprise several types, such as methylation, acetylation, phosphorylation and ubiquitylation [28]. Methylation and acetylation are thought to be most well-characterized modifying methods associated with aging [29]. Here, we will discuss those two types of modifications ( Figure 2). A global loss of histone protein makes the chromatin structure looser. Histone methylation and acetylation show dynamic changes with age. These changes work together to contribute to the aging process.

Histone Methylation in Invertebrate Aging
Histone methylation often takes place on lysine residues and is associated with aging in C. elegans and D. melanogaster. Like DNA methylation, histone methylation also requires methyltransferases and demethylases. Histone methyltransferases and demethylases are called KMTs/HMTs and KDMs/HDMs, correspondingly. We will review the roles of H3K4me3, H3K9me3, H3K27me3 and H3K36me3 associated with aging, respectively.

H3K4me3
H3K4me3 is an epigenetic chemical modification involved in the regulation of gene expression. H3K4me3 showed diverse changes with age in worms. The canonical pattern surrounding the transcriptional start sites, which marks the 5 end of genes [30], is established at an early stage during development and then remains stable with age. Non-conventional H3K4me3 regions preferentially mark gene bodies and are acquired during adulthood, and they further show age-dependent changes, which increase with age [31]. There are three major complexes responsible for generating H3K4me3 in mammals: the COMPASS complex, the Trithorax complex and the Trithorax-related complex [32]. Deficiencies of ASH-2, WDR-5, and SET-2 (H3K4 methyltransferases in the COMPASS complex) reduce global H3K4me3 levels at the larval L3 stage and extend worm lifespan [33]. Knockdown or mutation of RBR-2 from H3K4me3 demethylase was able to increase the level of H3K4me3 and then decrease lifespan. This affects the lifespan of animals in a germline-dependent manner [33]. Histone methylation has been implicated in transgenerational epigenetic regulation in C. elegans [34]. The loss of WDR-5.1 or SET-2 in worms impairs the transmission of stress adaptation in the progeny [35].
It was reported that there are still links between chromatin modifiers and fat metabolism. In C. elegans, the COMPASS H3K4me3 methyltransferase deficiency extends lifespan and promotes fat accumulation with a specific enrichment of mono-unsaturated fatty acids (MUFAs) in the intestine, by upregulating delta-9 fatty acid desaturase. This process acts mostly in the germline to regulate intestinal fat accumulation and lifespan, implying a germline-to-intestinal communication [36]. Importantly, these complexes can target RSKS-1/S6K in the germline, which is a key conserved substrate of mTOR complex 1 [37]. This suggests that the histone modification can act on mTOR signaling pathways to extend lifespan ( Figure 3).
A connection between metabolism and epigenetics also exists in Drosophila. Reduced levels of some enzymes involved in methionine metabolism disrupt its metabolism, which directly affects histone methylation levels. For example, the reduction of little imaginal discs (LID), the H3K4me3 demethylase, can counter the effects on histone methylation due to reduction of SAM-S (S-adenosylmethionine synthetase) [38]. However, the effect of LID in Drosophila lifespan is sex-specific, as the male Drosophila is more sensitive to the loss of LID-dependent H3K4me3 demethylation than the female [39]; however, the molecular mechanism for this remains unclear. Hcf (the Drosophila homolog of Hos cell factor 1) associates with the histone H3K4 methyltransferase Trithorax-related (Trr) to maintain H3K4 monoand tri-methylation, regulating the Hippo pathway, which controls tissue and organ size through the regulation of cell proliferation and apoptosis [40].
Mutations in KDM5, another H3K4me3 demethylase, contribute to cognitive defects in flies and humans [41,42]. KDM5 can also regulate component genes of the immune deficiency (IMD) signaling pathway and maintain the host-commensal bacteria homeostasis in a demethylase-dependent manner. It was recently shown that a Drosophila mutant deficient in kdm5 displayed gut dysbiosis, abnormal social behavior, and aberrant immune activation, and these phenotypes can be improved by Lactobacillus plantarum administration. This suggested a link between the gut microbiome and intellectual disability patients [43]. From all these results, H3K4me3 might act as a pro-aging factor to regulate lifespan. elegans, the COMPASS H3K4me3 methyltransferase deficiency extends lifespan and promotes fat accumulation with a specific enrichment of mono-unsaturated fatty acids (MUFAs) in the intestine, by upregulating delta-9 fatty acid desaturase. This process acts mostly in the germline to regulate intestinal fat accumulation and lifespan, implying a germline-to-intestinal communication [36]. Importantly, these complexes can target RSKS-1/S6K in the germline, which is a key conserved substrate of mTOR complex 1 [37]. This suggests that the histone modification can act on mTOR signaling pathways to extend lifespan ( Figure 3).

H3K9me3
H3K9me3 is a further covalent methylation of histone protein. One of the HMTs that has been identified in C. elegans is histone-modifying enzyme MET-2, which targets H3K9. It was reported that MET-2 associates with a conversed DNA repair protein SMRC-1, which limits DNA damage and promotes DNA replication and repair and genome stability in the germline in worms [44]. Recently, JMJD-1.2 was found to have a demethylase activity towards several lysine residues on Histone 3 (H3) in C. elegans. Jmjd-1.2 is expressed abundantly in the germline, where it controls the level of H3K9/K23/K27me2 both in mitotic and meiotic cells. However, jmjd-1.2 mutants are more sensitive to replication stress, and the progeny of mutant animals exposed to hydroxyurea show increased embryonic lethality and mutational rate, which suggests a role for jmjd-1.2 in the maintenance of genome integrity after replication stress [45]. Since genomic instability is one of the hallmarks of aging and is influenced by histone modification, we hypothesize that these hallmarks interact with each other during aging, making the mechanisms of aging more complicated. Associated with silenced heterochromatin regions, H3K9me3 serves as the binding site for heterochromatin protein 1 (HP1), which plays roles in heterochromatin formation, stabilization, and propagation [46]. In aged flies, the enrichment of H3K9me3 and HP1 was strikingly reduced. There is also an age-related change in the nuclear organization of H3K9me3 and HP1. Nuclei of fat body cells from young animals show a characteristic intensely concentrated staining for H3K9me3, while older animals show a more diffuse staining pattern [47]. Meanwhile, in the adult Drosophila mid-gut, the loss and dispersion of H3K9me3 and HP1 leads to the loss of chromatin stability in intestinal cells with age. Knockdown of su(var)3-9, methyltransferase for H3K9me3 or HP1α leads to intestinal stem cell (ISC) aging through genomic stress, JNK signaling, and apoptotic death in ECs [48]. The KDM4 family is highly conserved across species and reverses di-and tri-methylation of histone H3 lysine 9 (H3K9) and lysine 36 (H3K36). In Drosophila, Kdm4 is necessary for development because loss-of-function mutants do not survive past the early second instar larvae stage [49]. Apoptosis and DNA damage were involved in lethality [49]. Shown by the above evidence, H3K9me3 may contribute to DNA damage and genomic instability to promote the aging process.

H3K27me3
Like H3K9me3, H3K27me3 is another typical epigenetic mark, which usually denotes transcriptional silencing. H3K27me3 levels increased in aged flies [50]. Polycomb repressive complex 2 (PRC2) is a multiprotein complex that catalyzes the methylation of H3K27. In Drosophila, heterozygous mutations in E(z) and ESC, both being core subunits of PRC2, increase longevity and reduce adult levels of H3K27me3 [51]. Mutations in trithorax (trx), which is an antagonist of polycomb silencing and a methyltransferase for H3K4me3, elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. The mutants in E(z) and esc exhibit increased resistance to oxidative stress and starvation, and these phenotypes are suppressed by trx mutations too [51]. These results suggest that the H3K4me3 Trx complex and the H3K27me3 PRC2 complex may work together to regulate animal lifespan. Metabolic homeostasis is intimately connected with aging and lifespan regulation [52]. A reduction of H3K27me3 by PRC-deficiency promotes healthy lifespan in a glycolysis-dependent manner, as perturbing glycolysis diminishes the pro-lifespan benefits mediated by PRC-deficiency [50]. The C. elegans SET domain protein MES-2, an ortholog of E(z) (a subunit of PRC2), provides H3K27 methylation activity [53]. RNAi against mes-2 extends lifespan significantly in wild type C. elegans. This also shows that the mechanism of life extension is independent of the germline [54].
The protein complex UTX-1 is a type of histone demethylase specific for H3K27me3, and it is a marker linked to chromatin repression. Recent work has shown that KDM6A/UTX is the target of metformin in prolonging lifespan through altering global H3K27me3 levels in mice [55]. The mutant of utx-1 in flies raises the H3K27me3 level, limiting lifespan by downregulating glycolytic genes [50]. However, global somatic H3K27me3 levels decrease with age in germline-deficient worms [56]. RNAi of the utx-1 gene extends the mean lifespan of C. elegans by 30% [57]. Meanwhile, both knockdown and heterozygous mutations of utx-1 extend lifespan and increase the global levels of the H3K27me3 mark in worms [56]. Unlike H3K4me3, which extends lifespan mostly in a germline-dependent manner, H3K27me3 demethylase UTX-1 regulates lifespan independently of the presence of the germline, but in a manner that depends on the insulin-FOXO signaling pathway [56]. The catalytic enzyme utx-1 has an opposite effect on lifespan in flies and worms because it targets different genes in regulating lifespans. Most importantly, this phenomenon is mainly accomplished by different change patterns of H3K27me3 in aged flies and worms, increasing in aged flies but decreasing in aged worms. This suggests that only the optimal levels of H3K27me3 can maintain a healthy lifespan.

H3K36me3
H3K36me3 is a histone modification associated with active transcription, which plays crucial roles in a wide range of biological processes. A number of enzymes catalyze H3K36me. Loss of Rph1, a K36me2/3 demethylase, increases H3K36me3 level and extends lifespan in yeast [58]. Deficiency in the methyltransferase met-1 results in globally decreased H3K36me3, an increase in global mRNA expression change with age, and a shortened lifespan in C. elegans [59]. This indicates that global mRNA change level is negatively correlated with H3K36me3. H3K36me3 facilitates genomic stability via the promotion of DNA damage repair, both in DNA mismatch repair and double strand break pathways [60]. The suppression of spurious transcriptional initiation within the gene bodies involving a Set2/SETD2 (methyltransferase of H3K36) mechanism can ensure the fidelity of gene transcription [61]. While SET-18 is the H3K36 dimethyltransferase, a set-18 worm mutant extends lifespan and increases oxidative stress resistance in a daf-16-dependent manner. The level of muscle-specific set-18 is activated in aged worms (day 7 and day 11), attributable to the promotion of H3K36me2 and the inhibition of daf-16a expression; subsequently, longevity is shortened [62]. These results indicate that H3K36me3 and H3K36me2 have different roles in aging. H3K4me3 extends lifespan via maintaining transcription fidelity and genomic stability, while H3K36me2 causes limited lifespan through the IIS pathway.

Histone Acetylation in Invertebrate Aging
Histone acetylation on lysine residues is another common histone modification, which plays a very important role in longevity regulation due to the direct connection with transcription promotion. Similar to methylation, it also requires the involvement of many enzymes, such as histone acetyltransferases (HATs) and histone deacetylases (HDACs). It was found that levels of H4K12ac, H3K9ac and H3K23ac increased in the midlife of Drosophila, compared with younger animals [63]. Early exposure to some mild stresses can slow down the aging process and extend lifespan through epigenetic changes. Histone acetylation levels of worms are increased under mild heat stress and are maintained into old age [64]. The expression of immune and detoxification genes also increased after such heat stress [64]. Histone acetyltransferase CBP-1 and the chromatin remodeling SWI/SNF complex (switch/sucrose non-fermenting, also known as the BAF complex) act as epigenetic modulators of the long-lasting defense responses [64]. When there is a decrease in the histone H4K12-specific acetyltransferase chameau, aging-associated phenotypes (such as raised oxygen consumption and acetyl-CoA levels as well as associated transcriptome changes) are alleviated, and longevity is prolonged [63]. In the meantime, Acetyl-CoA is a key metabolite in the TCA cycle and a cofactor for the acetylation of lysine residues, lowering the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL), and also promoting longevity and retarding aging-associated changes [63]. Indeed, histone acetylation patterns are susceptible to alterations in key metabolites such as acetyl-CoA and NAD + , allowing chromatin to function as a sensor of cellular metabolism [65]. This implies that basical metabolism could be coupled with the aging process via lysine acetylation. Recent evidence indicates that histone lysine acetylation is tightly involved in the control of learning and memory [66]. Loss of dCBP, an acetyltransferase catalyze H3K23 in Drosophila, can decrease H3K23ac levels and impair neuronal gene activation, resulting in defective courtship learning [67]. These results imply that acetylation at different lysine residues may have opposite effects on aging.
Members of the sirtuin family of NAD-dependent protein deacetylases and ADP ribosyltransferases have been studied extensively as potential anti-aging factors. Overexpression of SIR2, a member of the sirtuin family, extended lifespan in budding yeast [68]. Sir-2.1 and dSir2 are the homologs of S. cerevisiae SIR2 in C. elegans and Drosophila, respectively. Increased expression levels of Sir-2.1 and dSir2 extend the lifespan of worms and flies. However, the effect of Sir2 is variable because of differences in genetic background and the mutagenic effects of transgene insertions [69]. Further research revealed that the effects of increased dSir2 expression on lifespan in Drosophila are dosage-dependent. Significant lifespan extension is observed when dSir2 expression is induced between two-and five-fold [70]. This effect is tissue-specific, as overexpression of dSir2 in the pan-neuronal cells or fat body extended lifespan, whereas induction in motoneuron or muscles did not [71].
Rpd3 is a zinc-dependent histone deacetylase in Drosophila and a homolog of mammal HDAC1. Reduction or inhibition of rpd3 extends longevity, increases energy storage and downregulates gene expressions of the IIS pathway [72]. There is an overlap between rpd3 and IIS longevity pathways, as mutations in rpd3 and dfoxo showed weakened stress resistance compared with rpd3 single mutant flies [72]. Lifespan extension in rpd3 mutant flies may overlap with the mechanism of extension seen in dietary restriction (DR). dSir2 has been implicated in mediating the response to DR in metazoans. Flies with double mutations in rpd3 and dSir2 had a median lifespan shorter than control flies, while rpd3 mutants lived longer [73]. Continued exploration has found a potential interaction between rpd3-mediated longevity and the protein synthesis regulator 4E-BP (a downstream of the TOR signaling pathway), based on the reduced longevity for both rpd3 and 4E-BP mutants compared to the single mutants of rpd3 [74]. Therefore, rpd3 is associated with the IIS, DR and TOR signaling pathways to promote longevity.

Chromatin Alterations in Aging
Changes in DNA and histone modifications are finally shown in chromatin changes. During aging, loss of histone and heterochromatin causes the chromatin structure to loosen, resulting in loss of transcriptional silencing. Retrotransposable elements are silenced by anchoring heterochromatin. The loss of heterochromatin with aging also leads to increased expression of otherwise silent retrotransposons [13]. The resulting transcripts from the retrotransposable elements are reverse-transcribed into cDNAs, which reinsert elsewhere into the genome of old cells, leading to genomic instability [75]. It was reported that HP1 (heterochromatin protein 1) destabilization was found in aged Drosophila, and overexpression of HP1 extended lifespan [76]. Deficiency of HP1α in enterocytes (ECs) leads to intestinal stem cell (ISC) aging, implying the loss of heterochromatin stability, which may be the crucial mechanism for ISC aging [48]. It seems that keeping heterochromatin stable could promote longevity.
It was known that SWI/SNF, NuRD (nucleosome remodeling and deacetylase) and the polycomb system make a difference in chromatin regulation [77]. Polycomb complexes are associated with chromatin containing repressive marks and silent or low transcriptional states [78]. Studies have pointed to a genetic antagonism between the SWI/SNF complex and polycomb repressive complexes 1 and 2 (PRC1/2) in Drosophila. Deletion of the BAF ATPases catalytic subunit Smarca4 in mouse embryonic stem cells can cause a genome-wide increase in the localization of PRC1 and PRC2 and the abundance of H3K27me3, resulting in chromatin regulation [78]. The deficiency in CHD3, a subunit of the NuRD complex [79], elevated p53-dependent germline apoptosis, mainly due to the failure in the timely repair of double-stranded breaks, eventually leading to an increase in chromatin defects and apoptosis in worms [80]. In short, SWI/SNF contributes to the open chromatin state and active transcription; NuRD promotes the production of the repression of chromatin; and the polycomb complex promotes the formation of chromatin compression environments.

Non-Coding RNAs in Invertebrates during Aging
Non-coding RNAs (ncRNAs) comprise various RNA species, including microRNA (miRNA), tRNA-derived small RNA (tsRNA), ribosomal RNA (rRNA), piwi-interacting RNA (piRNA), circular RNA (circRNA), and lncRNA [81]. ncRNAs have a remarkable impact on gene expression and chromatin remodeling by binding to their targets [82]. Mostly, total miRNA and piRNA are gradually decreased during aging in C. elegans, whereas in contrast, tsRNA, rRNA and circRNA levels generally display age-dependent increases [81]. The genetic modulation of specific ncRNAs affects longevity and aging rates by modulating established aging-regulating protein factors [81]. Generally, miRNA affects the aging process by acting on specific genes and altering their expression. miRNAs specifically target the 3 -UTR of mRNAs to exert transcriptional repression. A highly conserved miRNA, miR-124, was significantly upregulated in APS-induced longevity of C. elegans by regulating ATF-6 (an endoplasmic reticulum stress-regulated transmembrane transcription factor) [83]. The specific overexpression of the miRNA let-7 in the Drosophila nervous system increased female median fly lifespan by~22% [84]. RNA polymerase III can generate non-coding RNAs, including tRNAs. A reduction in RNA polymerase III can extend lifespan in worms and flies [85]. Therefore, this sheds light on tRNAs' possible involvement in the aging process.
In animals, piwi-interacting RNAs (piRNAs) of 21-35 nucleotides in length silence transposable elements; nearly all animals rely on piRNAs to defend the germline genome from transposon expression [86]. Piwi proteins combine with pi-RNA to silence targets post-transcriptionally. In flies, piwi promote H3K9 methylation, a repressive chromatin mark, through the recruitment of Eggless (also known as dSetdb1) by the piwi-interacting mediator proteins Asterix and Panoramix [87]. Piwi can repress heterochromatin loss, age-related dysfunction and apoptosis in ISCs, subsequently maintaining somatic stem cell genomic integrity [88].
CircRNAs are stable because the lack of free 5 -and 3 -ends protects them against nuclease attack [89]. These circRNAs can function as microRNA sponges to regulate mRNA, ultimately changing gene expression [90]. The expression of circ_0005230 was elevated in human tumors and cholangiocarcinoma (CCA) cells, and it significantly facilitated cell growth, clone-forming ability and metastatic properties and inhibited cell apoptosis in CCA cells. Further study implies that circ_0005230 could directly sponge miR-1238 and miR-1299 to exert its oncogenic function [91]. In humans, circRNA may also be involved in the regulation of ovarian function and aging by targeting certain miRNAs [92]. Therefore, specific circRNAs may function as anti-aging elements.
LncRNAs with a variable length spanning from 200 bp up to several kilobases appear to be important for proper neurological functioning, with aberrant expression of lncRNAs leading to neurological disorders in Drosophila [93]. One study found that there is a high expression of the lncRNA fer1l4 in human tumor tissues. Moreover, GO enrichment analysis also revealed that fer1l4 may be involved in processes associated with tumorigenesis [94]. Recent research shows that lncRNAs are associated with organismal aging. The lncRNA tts-1 in C. elegans extends lifespan, respectively, in daf-2 (insulin/IGF-1 receptor) and clk-1 (mitochondrial gene) mutations by reducing ribosome levels in a way that promotes life extension [95]. Therefore, some lncRNAs regulate lifespan in classic signaling pathways such as IIS pathway.

Targets for Pharmacological Manipulation
Epigenetic markers have become particularly interesting because, in addition to acting as markers for the genetic regulation of aging, epigenetic mechanisms may be targets for drugs in aging and age-related diseases (Figure 4). Many drug trials have confirmed these proposals. Resveratrol, as an activator of Sir2/SIRT1 and AMPK, extends the lifespan of yeast [96], worms and fruit flies [97]. However, this lifespan-extension effect of resveratrol is abrogated by the SIR2 mutation [97,98]. Natural compounds, such as curcumin or alkylresorcinols, enhanced SIRT1 activity and extended the lifespan of Drosophila [98,99]. NAD + is a necessary cofactor for many metabolic pathways, such as glycolysis, fatty acid b-oxidation, and the TCA cycle. Also, NAD + is also a substrate of sirtuin. NAD + levels decline during aging across species [100,101], and supplementation of NAD + extended the lifespan of worms, mice [102] and flies [103]. NAD + precursors include nicotinamide (NAM), nicotinic acid (NA), tryptophan (Trp), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN); changes in these substances also affect sirtuins and then lifespan [104]. Therefore, Sir2/SIRT1 could be a promising target for aging intervention. LncRNAs with a variable length spanning from 200 bp up to several kilobases appear to be important for proper neurological functioning, with aberrant expression of lncRNAs leading to neurological disorders in Drosophila [93]. One study found that there is a high expression of the lncRNA fer1l4 in human tumor tissues. Moreover, GO enrichment analysis also revealed that fer1l4 may be involved in processes associated with tumorigenesis [94]. Recent research shows that lncRNAs are associated with organismal aging. The lncRNA tts-1 in C. elegans extends lifespan, respectively, in daf-2 (insulin/IGF-1 receptor) and clk-1 (mitochondrial gene) mutations by reducing ribosome levels in a way that promotes life extension [95]. Therefore, some lncRNAs regulate lifespan in classic signaling pathways such as IIS pathway.

Targets for Pharmacological Manipulation
Epigenetic markers have become particularly interesting because, in addition to acting as markers for the genetic regulation of aging, epigenetic mechanisms may be targets for drugs in aging and age-related diseases ( Figure 4). Many drug trials have confirmed these proposals. Resveratrol, as an activator of Sir2/SIRT1 and AMPK, extends the lifespan of yeast [96], worms and fruit flies [97]. However, this lifespan-extension effect of resveratrol is abrogated by the SIR2 mutation [97,98]. Natural compounds, such as curcumin or alkylresorcinols, enhanced SIRT1 activity and extended the lifespan of Drosophila [98,99]. NAD + is a necessary cofactor for many metabolic pathways, such as glycolysis, fatty acid b-oxidation, and the TCA cycle. Also, NAD + is also a substrate of sirtuin. NAD + levels decline during aging across species [100,101], and supplementation of NAD + extended the lifespan of worms, mice [102] and flies [103]. NAD + precursors include nicotinamide (NAM), nicotinic acid (NA), tryptophan (Trp), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN); changes in these substances also affect sirtuins and then lifespan [104]. Therefore, Sir2/SIRT1 could be a promising target for aging intervention. With aging, transcription levels of genes involving biosynthetic, metabolic and immune functions decline [105]. Histone acetylation promotes transcription activation, and deacetylation inhibits transcription, suggesting that pharmacological intervention in the process could impact longevity. Thus, HDAC inhibitors can increase longevity by promoting gene transcription. Inhibitors such as sodium 4-phenylbutyrate (PBA), sodium butyrate (SB), trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA) affect several pathways involved in the regulation of these gene expression patterns associated with healthy aging [106]. Metformin, the first drug chosen to be With aging, transcription levels of genes involving biosynthetic, metabolic and immune functions decline [105]. Histone acetylation promotes transcription activation, and deacetylation inhibits transcription, suggesting that pharmacological intervention in the process could impact longevity. Thus, HDAC inhibitors can increase longevity by promoting gene transcription. Inhibitors such as sodium 4-phenylbutyrate (PBA), sodium butyrate (SB), trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA) affect several pathways involved in the regulation of these gene expression patterns associated with healthy aging [106]. Metformin, the first drug chosen to be tested in a clinical trial aimed at targeting the biology of aging per se, may exert its anti-aging effect by acting on H3K27me3 [55].
Spermidine, a naturally occurring polyamine, directly inhibits HATs, maintaining histone H3 in a hypoacetylated state in human cells. This altered acetylation status leads to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, and human cells, thereby enhancing longevity [107]. Spermidine can also block the age-related changes of cardiac cell composition and function, enhance diastolic function without affecting systemic blood pressure, and extend lifespan in an autophagy-dependent manner [108]. Spermidine or similar compounds related to HATs or autophagy could be good candidates to extend lifespan.
It is expected that drugs targeting epigenetic marks are the most promising for aging intervention.

Crosstalk between Epigenetic Marks
Epigenetic markers are involved in many physiological processes, and their interaction makes these processes more complex. It was shown that there was an inverse correlation between DNA methylation and histone H3K4 methylation in human cells [109]. Subsequently, genome-wide research found that DNA methylation could discriminate promoters from enhancers through H3K4me1-H3K4me3 in the seesaw mechanism [110], suggesting that the balance of seesaw might be used to determine whether the body is in a normal state.
In many organisms, small RNAs modify chromatin via RNA interference pathways [111]. RNAi can produce long-term heritable responses that affect progeny [112]. In C. elegans, both exogenous and endogenous siRNAs can direct histone H3K27 methylation at targeted loci through the Nrde (nuclear RNAi defective) pathway, resulting in H3K27me3 status inheritance by progeny for multiple generations [113]. The induction of RNAi in met-2 (H3K9 methylation enzyme) mutant worms resulted in the transgenerational barrier being broken, while RNAi was stably inherited transgenerationally; consequently, the progeny become progressively sterile due to the accumulation of small RNAs coupled by defective H3K9 methylation [114]. Therefore, ncRNA together with methylation could modulate transgenerational inheritance.
In the Drosophila brain, miR-34 becomes upregulated in an age-associated manner and is functionally required for lifespan extension, whereas mir-34 loss was shown to accelerate brain aging and degeneration and a decline in survival [115]. A more recent study revealed that miR-34 repressed the translation of Pcl and Su(z)12 (two components of PRC2) transcripts, resulting in a reduction of PRC2 activity and less H3K27me3, promoting healthy aging [116]. Epigenetic marks' interaction also exists in age-related diseases. In colon cancer cells, lncRNA CCAT2 acts as a negative regulator of miRNA-145 biogenesis, implying a novel mechanism of lncRNA-miRNA crosstalk [117].
There is also a crosstalk between histone deacetylase inhibitors and H3K4 methylation marks in prostate cancer cells [118]. It was reported that H3K9me3 and DNA methylations interact with each other, probably through HP1 [119]. In addition, enhance RNA (eRNA) is one of the non-coding RNA molecules [120], which can bind CBP at enhancers and stimulate histone acetylation and transcription of target genes [121]. The crosstalk between epigenetic marks is even more complex than that described above, thus making the aging mechanism more compelling.

Conclusions
Epigenetic alteration involves changes in DNA methylation patterns, post-translational modification of histone, chromatin remodeling and non-coding RNA interference. These processes are each associated with the aging of D. melanogaster and C. elegans. However, we believe that the epigenetic landscape may be further complicated beyond the description above due to the crosstalk between epigenetic mechanisms. Understanding the epigenetic changes in the aging process can advance our knowledge of the mechanisms of aging. Drug research in epigenetics will be a powerful intervention in aging. A longer and healthier lifespan for humans could be achieved by leveraging the powerful genetic tools available for simple invertebrate models, in order to aid our understanding of aging mechanisms in the less tractable human system.

Acknowledgments:
We thank all lab members for invaluable comments on the manuscript and Julian Dow for comments and proofreading.

Conflicts of Interest:
The authors declare no conflict of interest. Trimethylation of lysine 27 on histone H3 protein subunit H3k36me3