Epigenetic mechanisms of dietary restriction induced aging in Drosophila

    Epigenetic mechanisms of dietary restriction induced aging in Drosophila Ting Lian, Uma Gaur, Deying Yang, Diyan Li, Ying Li, Mingyao Yang PII: DOI: Reference:

S0531-5565(15)30039-5 doi: 10.1016/j.exger.2015.08.015 EXG 9685

To appear in:

Experimental Gerontology

Received date: Revised date: Accepted date:

4 April 2015 4 August 2015 25 August 2015

Please cite this article as: Lian, Ting, Gaur, Uma, Yang, Deying, Li, Diyan, Li, Ying, Yang, Mingyao, Epigenetic mechanisms of dietary restriction induced aging in Drosophila, Experimental Gerontology (2015), doi: 10.1016/j.exger.2015.08.015

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ACCEPTED MANUSCRIPT Epigenetic mechanisms of dietary restriction induced aging

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in Drosophila

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Ting Lian, Uma Gaur, Deying Yang, Diyan Li, Ying Li, Mingyao Yang*

Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan

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Agricultural University, Chengdu 611130, P.R.China

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Abstract

Aging is a long-standing problem that people are always interested in. Thus, it is critical to understand the underlying molecular mechanisms in aging and explore the

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most efficient method to extend life expectancy. To achieve this goal, a wide range of

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systems including cells, rodent models, budding yeast, C. elegans and flies have been employed for decades. In recent years, the effect of dietary restriction (DR) on

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lifespan is in the prime focus. Although we have confirmed that reduced insulin and/or insulin-like growth factor (IGF) and target of Rapamycin (TOR) signaling can increase Drosophila lifespan, the precise molecular mechanisms and nutritional

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response landscape of diet-mediated aging is ambiguous. Epigenetic events have been considered as the major contributors to lifespan extension with response to DR. The role of DNA methylation in aging is well acknowledged in mammals and rodents where it has been shown to impact aging by regulating the transcription, though the mechanism of regulation is not limited to only transcription. In Drosophila, the contribution of methylation during DR in aging is definitely less explored. In this review, we will update the advances in mechanisms of DR, with a particular focus on methylation as an upcoming target for aging studies and discuss Drosophila as a powerful model to understand mechanisms of aging with response to diet. Key words: Drosophila；Aging；Methylation；Signaling pathways * Corresponding author: Mingyao Yang: [email protected]

ACCEPTED MANUSCRIPT 1. Introduction Aging is one of our greatest social concerns since it is most often accompanied by a range of detrimental physiological events such as the progressive impairment of

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organs systems and immune functions resulting in a decreased ability to resist diseases. Consequently, several ailments increase with age, such as sarcopenia, most

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types of cancer, Alzheimer’s disease, Parkinson’s disease, stroke, type2 diabetes mellitus (López-Otín et al., 2013). The molecular mechanisms underlying aging and these age-related symptoms remain largely unknown.

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Kinds of factors contribute to aging including genetic composition and stochastic events such as environment. Particularly, the stochastic events play the major role

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(Herskind et al., 1996). Over the last 20 years, many studies have demonstrated that dietary and genetic alterations can also increase the healthy lifespan of laboratory

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model organisms (Fontana and Partridge, 2015), sparking an active surge among

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scientists to identify the factors that cause/accelerate aging with the goal of ultimately slowing the process and alleviating its detrimental symptoms (López-Otín et al.,

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2013). In the recent past, the major focus has shifted towards exploring the role of diet and its effect on aging phenotype. Calorie restriction (CR) is a dietary regimen that is based on low calorie intake by

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20-40% without causing malnutrition. At the same time, dietary restriction (DR) is a common intervention whereby a reasonable reduction of total nutrient intake is practiced without causing health problems. Strictly, DR also includes CR. Therefore, we will use term DR for the rest of our text. A substantially growing number of reports have identified the effectiveness of DR on improving health, delaying or preventing cancer or other diet/age-related diseases thereby significantly extending the health lifespan, from yeast to invertebrates even mammals (Cypser et al., 2013; Emran et al., 2014; Lee et al., 2014; Levine et al., 2014; Fontana and Partridge, 2015). Based on the previous evolutionary conservation studies, Drosophila has been used as a model organism for aging studies. Many research findings have suggested that diet can not only influence the physiological changes but also affect the signaling pathways such as the insulin/IGF signaling, the TOR pathway and the less explored

ACCEPTED MANUSCRIPT epigenetic molecular mechanisms (Bjedov et al., 2010; Ford et al., 2011; Alic et al., 2014; McCauley and Dang, 2014; Yan et al., 2015). In this review, we will summarize DR induced pathways and the downstream

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epigenetic molecular mechanisms of aging with a prime focus on DNA methylation. Alongside we compiled the experimental evidences in different model organisms

events leading to healthy lifespan and longevity. 2. DR in Drosophila aging

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supporting the use of Drosophila as a tool to understand the underlying molecular

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Recent studies indicate that DR mediates longevity in Drosophila which is influenced not only by protein intake but also by the interplay between carbohydrate

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and protein ingestion. It is worthwhile mentioning here that the individual nutrients and particularly the levels of proteins/AAs rather than calories alone are key

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mediators of lifespan in model organisms including yeast, worms, flies, rodents, and

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also associated with the risk of aging-related diseases such as cancer, diabetes in humans (Mirzaei et al., 2014). Also, one report have denied the role of DR in

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increasing lifespan achieved by high-protein diet, yet, lifespan could be extended by controlling the ratio of macronutrients in ad libitum-fed mice (Solon-Biet et al., 2014), suggesting the overlapping role of balanced dietary macronutrients and DR in

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longevity (Piper et al., 2011). DR protocols in Drosophila have been practiced by changing the concentration of live yeast (Chippindale et al., 1993) or diluting the nutrient coordinately on the basis of normal food medium (Chapman and Partridge, 1996). Median lifespans of female flies are extended much more than males by DR (60%, 40% concentration of the standard laboratory diet, respectively) (Magwere et al., 2004) and female fecundity is reduced accordingly, even though the specific nutrients role in extending lifespan have been identified recently. In addition, alteration of dietary amino acid balance can mimic the beneficial DR effects in flies—lifespan extension (Grandison et al., 2009).. Taken together, the DR protocol needs to be optimized for the adequate nutritional intake necessary for flies growth and development.

ACCEPTED MANUSCRIPT 3. The genetic pathways involved in Drosophila aging with response to DR Extensive studies have suggested following nutrient sensing and signaling pathways which play key modulators of the longevity with response to DR;

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insulin/IGF signaling (IIS) pathway, Target of Rapamycin (TOR), AMP-dependent protein kinase (AMPK) and SIRT (sirtuin) (Speakman and Mitchell, 2011).

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3.1 IIS pathway

IIS pathway participates in the development and growth of Drosophila and is considered as an important candidate for endocrine control of DR induced longevity

components are evolutionarily conserved.

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in worms, flies and mice (Fontana and Partridge, 2015; Nassel et al., 2013), whose

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In Drosophila, IIS pathway includes one insulin/IGF-like tyrosine kinase receptor (INR), eight insulin-like peptide loci (dilps 1-8) and one forkhead transcription factor

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FOXO encoded by dfoxo (Kannan and Fridell, 2013). IIS was always reduced in

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response to DR, showing the fact that DR-induced longevity is fostered by repression of Insulin/IGF signaling (Clancy et al., 2001). Mutations that extend lifespan/ slow

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aging also pave the way for illuminating the role of IIS pathway in control of DR-mediated longevity. Firstly, as one of the main intracellular components, insulin/IGF-like tyrosine kinase receptor (INR), combined with its substrate chico, the

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phosphatidyl-inositol 3-kinase (PI3K) Dp 110/p60 and the PI3K target protein kinase B (PKB, also known as DAktl) form a signaling pathway that regulates growth, size and lifespan (Tatar et al., 2001). Secondly, loss of chico can extend fly’s median-lifespan in homozygotes and heterozygotes up to 48% and 36%, respectively (Clancy et al., 2001). While the homozygote females lifespan reached the maximum at the higher food dilution concentration than the control females, and they lived a shorter lifespan in response to other food concentrations (Clancy et al., 2002), suggesting that chico females are likely to intake less food and dietary restricted, or IIS pathway acts in a positive role in extending fly’s lifespan with response to DR. Furthermore, the mutants of dfoxo, the downstream effector of the IIS pathway, which is negatively regulated by IIS, can also highlight the role for IIS in diet-induced Drosophila longevity (Kannan and Fridell, 2013; Tatar et al., 2014). Although Min

ACCEPTED MANUSCRIPT and his colleagues suggested the independent role of DR-induced longevity with IIS in Drosophila (Min et al., 2008), that is, knockdown of fly’s dfoxo shorten lifespan

while the flies continue to respond normally to DR. Over-expression of dfoxo in the

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fat body, which induces the reduced IIS activity, resulted in longer lifespans than the controls under increased food intake especially the maximum lifespan is achieved at

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the higher food concentrations. Thus, reduced IIS activity and over-expression of dfoxo play a role in fly’s lifespan response to DR, while other mechanisms such as the TOR pathway can compensate for the phenotype observed by loss of fly’s dfoxo

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(Fontana and Partridge, 2015). Yet, here, we could not overlook the interaction of gene (dfoxo) and diet nutrition space that is an untested region. As Piper et al. (Piper

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et al., 2011) discussed, in order to test robustly whether the mutation of dfoxo is required in DR, the Geometric Framework (GF) could be used as a tool to handle and

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explore the mechanisms of different genotypes under comprehensive nutritional

3.2 TOR pathway

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landscapes.

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In Drosophila, another pathway has also emerged as a nutrient-sensing channel, that is, the target of Rapamycin (TOR) (Colombani et al., 2003). TOR was identified from yeast to all eukaryotes and functions with two distinct complexes, TOR

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complex1 (TORC1) and TOR complex2 (TORC2). TORC1 is considered as the “signaling core” because of integrating intra and extracellular environmental cues (Kapahi et al., 2010). The role of TOR pathway is parallel to, but also interact with, IIS pathway in regulating fly’s growth (Chen et al., 2013). Furthermore, inhibition of TOR signaling pathway can extend health lifespan in yeast, nematodes and Drosophila by genetic or pharmacological interventions (Kapahi et al., 2010). In TOR signaling network, TORC1 is the central element containing the TOR kinase which modulates cellular responses to amino acids by regulating translation initiation, ribosomes, autophagy and endocytosis (Grewal, 2009; Kannan and Fridell, 2013). TOR kinase can inactivate the 4eBP and then inhibit the animal growth, and it also activates kinase S6K, which involves in translation initiation and elongation in case of abundant amino acids, indicating that TOR is directly regulated by amino

ACCEPTED MANUSCRIPT acids flux (Ma and Blenis, 2009). The role of TOR is both autonomous and systemic with response to DR. In addition, over-expression of dTsc2 (Drosophila tuberous sclerosis complex 2) can extend the lifespan of Drosophila, and this effect is stronger

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with the lower concentration of yeast extract, suggesting that up-regulation of TOR

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between TOR and DR (Kapahi et al., 2004, 2010).

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pathway can affect fly’s feeding behavior and revealing a possible relationship

Several reports support the role of TOR in regulating the DR-induced longevity, for example, the loss of 4eBP have a meaningful effect on Drosophila lifespan

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undergoing a limited range of protein-restricted diets lacking essential sterols and lipids (Zid et al., 2009). But 4eBP mutants showed an identical response with wild

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type when yeast was diluted from 16% to 0%, pin-pointing the fact that DR-induced longevity is likely to be independent with 4eBP and perhaps TOR (Tatar et al., 2014).

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This could be the effect of only one slice of the protein-carbohydrate response

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landscape rather than the comprehensive nutritional effects. As argued in the IIS section, if we would like to resolve whether TOR is required in aging response to DR,

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estimation of the longevity reaction norms across the protein:carbohydrate response landscape will be needed. 3.3 AMPK

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AMPK, AMP-activated protein kinase, has been considered as the principal energy sensor (Hardie et al., 2012). Several studies have confirmed the conservation of AMPK at both DR and aging in C. elegans and Drosophila, which is becoming the link between energy status, DR effect and longevity (Schulz et al., 2007; Fukuyama et al., 2012; Stenesen et al., 2013). AMPK can modulate the aging-related pathways including TOR/S6K, FOXOs, Sirtuins, CRTCs (cAMP-responsive element-binding protein (CREB)-regulated transcriptional coactivators (CRTCs), NF-κB signaling and other mediators (Burkewitz et al., 2014). Since there are not many reports about the role of AMPK in Drosophila DR effect and longevity, here we discuss some evidences in C. elegans which is a parallel model organism. When C. elegans undergoes a DR stress by diluting E. coli food source, AMPK is activated, which can accelerate the FOXO/DAF-16 dependent transcription and phosphorylation at some

ACCEPTED MANUSCRIPT unidentified sites and then increase the worm’s stress resistance and extends lifespan (Greer et al., 2007). According to a report in flies (which is not DR specific), the lifespan can be extended by over-expression of AMPK through increasing the ratio of

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AMP:ATP and ADP:ATP (Stenesen et al., 2013). AMPK-SIRT1-NF-κB plays a critical role in suppressing the immune responses resulting from DR, and suggests a

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role in mediating lifespan (Salminen and Kaarniranta, 2012). AMPK can also impact aging by involving in a variety of processes such as autophagy, mitochondria biogenesis, lipid metabolism, central control of energy homeostasis, stem cells, tissue

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rejuvenation and protein synthesis (Burkewitz et al., 2014). 3.4 SIRT

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Sirtuins (SIRT) are nicotinamide adenine dinucleotide-dependent protein deacetylases and consist of seven highly conserved members (SIRT1-7) in mammals.

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SIRT1, SIRT6 and SIRT7 are located in the cell nucleus, SITR3, SIRT4, SIRT5 are

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located in mitochondria, only SIRT2 is located in cytoplasm (Frye, 2000) and the ortholog of Sirtuins is Sir2 (silent information regulator 2, now termed sirtuins) in

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flies and yeast (Guarente, 2000). In Drosophila, Sir2 (Drosophila Sir2, dSir2) has five members, Sir2, Sirt2, Sirt4, Sirt6 and Sirt7 (Hoffmann et al., 2013). Sirtuins family mediate various biological processes such as protein acetylation,

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metabolism, aging and age-related diseases including neuro-degenerative diseases (AD) in worms, flies, rodents and humans (Kim et al., 2007; Guarente, 2011; Nakagawa and Guarente, 2011). The direct role of Sir2 in DR-mediated alteration of lifespan in Drosophila has been revealed (Rogina and Helfand, 2004; Wood et al., 2004; Bauer et al., 2009). Although dSir2-mediated extension of the Drosophila life span is controversy (Burnett et al., 2011), new studies have showed that the over-expression of dSir2 can mimic DR effect. Over-expression of fat body Sir2 can extend fly’s lifespan in normal lab diet and knockdown of fat body dSir2 abrogates the extension of fly’s lifespan especially the median lifespan with response to DR, demonstrating that the role of dSir2 in regulating fly’s lifespan is diet-dependent (Banerjee et al., 2012). However, Hoffmann et al. (Hoffmann et al., 2013) found that over-expression of dSir2 in adult fly’s fat body can extend both female and male

ACCEPTED MANUSCRIPT lifespan and the effect of dSir2 in mediating the longevity is independent of diet based on the mismatching of transcriptional profile on fat bodies after DR and dSir2 over-expression. Furthermore, the increased dSir2 expression induced a longer

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lifespan in dSir2 dose-dependent manner (Whitaker et al., 2013). Combined together, the first conclusion can be drawn which is that the fly’s lifespan can be extended at an

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increased Sir2 expression in specific tissue with proper experimental systems. But the conflict still exists whether Sir2 can mediate DR-dependent lifespan extension, the most likely reason is the overlook of the expression level of transgenes (Sinclair and

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Verdin, 2012), perhaps the ideal expression level of Sir2 will link to DR effect. 4. The potential epigenetic mechanisms underlying Drosophila aging with

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response to DR

Aging process is affected by stochastic events, among which, environmental factors

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attribute the most. Till now, DR is most widely applied in various animal model

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especially Drosophila. A number of reports have confirmed that DR- related lifespan extension is likely to be involved in age-related epigenetic architectural changes, such

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as DNA methylation and histone modifications including histone acetylation, methylation, phosphorylation and biotinylation (Milagro et al., 2011; Hou et al., 2014; Jacobsen et al., 2014; Zhu et al., 2014). Out of these, DNA methylation has been the

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epigenetic research center by virtue of its more stability than other epigenetic modifications (Kondo, 2009). In addition, scientists are more interested in studying histone methylation because of its role in extending lifespan in invertebrates and even mammals (Greer et al., 2010; Jin et al., 2011; Larson et al., 2012; Takahashi et al., 2012; McCauley and Dang, 2014). In more recent studies, DR has been shown to increase active and healthy lifespan among multiple species despite of the protocols differences. As one of the most important epigenetic modifications, DNA methylation shows aberrant pattern with aging, that is, DNA methylation is decreased globally but increased locally, while DR can reverse the aberrant DNA methylation pattern by controlling at specific loci (Muñoz-Najar and Sedivy, 2011). Since there is no exact report on epigenetic influence of DR in Drosophila, we gathered some evidences in other model species.

ACCEPTED MANUSCRIPT 4.1 Histone methylation in model species It has been reported that maternal diet composition such as the alteration of choline, protein and fat can change the level of histone methylation in rat offspring influencing

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the phenotype such as coat color of pups, the character of tails, and the hepato-carcinogenesis (Waterland and Jirtle, 2003; Waterland et al., 2006; Davison et

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al., 2009; Mehedint et al., 2010). Based on these observations, it is suggested that the deficiency of methyl-donor will have a direct effect on histone marks, leading to the breakage or remodeling of chromatin, which is then involved in neoplasia, and

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consequently alters the lifespan. The histone methylation modification that has been fully investigated so far is H3K9me (histone methylation on position 9 of histone H3;

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if it is trimethylated, it will be called as H3K9me3) (Zhang et al., 2003; Zhang et al., 2004; Margueron et al., 2005). So far, H3K4, H3K9, H3K27, H3K36, H3K79 and

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H4K20 have been found to be related with chromatin state, transcriptional repression,

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gene expression and DNA damage responses (Margueron et al., 2005; Martin and Zhang, 2005; Shilatifard, 2006; Berger, 2007).

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In Drosophila, HP1 (Heterochromatin Protein 1), is an integral component of heterochromatin located at the heterochromatin sites and controls heterochromatin levels. HP1-related heterochromatin is necessary to slow aging phenotypes in

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Drosophila, because heterochromatin level plays an indispensable role in maintaining adult muscle integrity, nuclear stability and function thereby having an effect on the animal mobility and lifespan. As one of the binding sites of HP1, in aging flies, H3K9me3 levels are increased, while dimethylation levels are reduced, and HP1 can also bind to H3K9me2 (Bannister et al., 2001; Lachner et al., 2001; Wood et al., 2010; Larson et al., 2012). The reduced levels of H3K9me2 are associated with the loss of HP1, which can induce the breakage of the gut muscle fibers and increase the instability of rDNA locus in Drosophila. When HP1 levels are reduced, it will induce the increase of rRNA transcription and decrease the capacity of protein synthesis and ribosome biogenesis. Consequently, it will shorten fly’s health lifespan. Conversely, over-expression of HP1 could extend the median and maximum lifespan (Larson et al., 2012). In addition, Siebold and his colleagues also confirmed that changes in

ACCEPTED MANUSCRIPT H3K27me3 levels have an effect on Drosophila lifespan. If the polycomb repressive complex 2 (PRC2) components E(z) and Ese are mutated, total level of H3K27me3 would be decreased and fly’s lifespan would be extended (Siebold et al., 2010).

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Furthermore, choline deprivation of diet will change the level of H3 methylation, among which, H3K9me1 is decreased by 25% in the ventricular and subventricular

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zones whereas H3K9me2 is decreased by 37% in the pyramidal layer in brain. Consequently, neurogenesis and apoptosis in fetal brain will be altered (Mehedint et al., 2010). On the basis of the interaction between histone methylation level, DR and

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aging, our understanding of vertebrates especially the humans will be enhanced. 4.2. DNA methylation in Drosophila and other species

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The cytosine methylation (5-mc), generally, called CpG island in vertebrates, is widely detected on the genomes of many prokaryotes and eukaryotes such as bacteria,

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plants, and mammalian cells (Law and Jacobsen, 2010; Zemach et al., 2010). Yet,

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DNA methylation is not universal given that some species including C. elegans, N. crassa, D. discoidium, S. mansoni and D. melanogaster, were long thought to lack or

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own the low amounts of DNA methylation (Simpson et al., 1986; Gowher et al., 2000; Tamaru and Selker, 2001; Katoh et al., 2006; Geyer et al., 2011; Raddatz et al., 2013; Capuano et al., 2014; Takayama et al., 2014). Nevertheless, various methylated

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cytosines (including 5-methylcytosine, N6-methyladenine, N4- methylcytosine ) have been identified in genomic DNA from prokaryotes to eukaryotes including Drosophila (Ratel et al., 2006; Zhang et al., 2015). The identification of DNA methyltransferase 2 (Dnmt2) and methyl-CpG binding domain (MBD) 2/3 in Drosophila has demonstrated the presence of low amounts of DNA methylation and it is likely to be considered that non-CpG methylation (CpT, CpA, CpC) also existed alongside (Lyko et al., 2000a; Lyko et al., 2000b; Kunert et al., 2003). Recently, a high resolution method, the bisulfite sequencing has been conducted to detect the DNA methylation patterns in Drosophila. But the existence of DNA methylation could not be defined clearly, since the sequencing depth was not sufficient enough to reach the detection limit of bisulfite sequencing (Lyko et al., 2010; Li et al., 2012; Raddatz et al., 2013; Zykovich et al., 2014). However, Takayama’s group observed

ACCEPTED MANUSCRIPT the presence of DNA methylation in Drosophila embryo by combining MeDIP with Bs-seq strategy (Takayama et al., 2014). Furthermore, the Drosophila genome harbors a well conserved homolog of the TET protein family, whose substrate is the genomic

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5mC. Tet gene expression is actively limited in the central nervous system and brain during Drosophila embryo development. During the later stages, the expression level

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of Tet gene is still maintained high in the brain and central nervous system compared with the salivary gland and the digestive system, suggesting the important role of Tet in the development of brain and central nervous system (Brody et al., 2002; Graveley

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et al., 2011). In mouse embryo, Tet protein and 5hmC functionally participate in the brain development, and in Drosophila embryo, Dnmt2 and Tet gene display the

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similar expression pattern (Münzel et al., 2010; Hahn et al., 2013). From these data, the complete deficiency of 5mC in Drosophila is not expected, at least, the modified

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cytosines should exist in some tissues or certain stages, especially the central nervous

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system with the highest expression of Tet and Dnmt2. Furthermore, in male Drosophila germline stem cells, the non-random segregation of Y and X

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chromosomes has been confirmed which is convincingly heritable and is carried out depending on the wild-type Dnmt2 protein (Yadlapalli and Yamashita, 2013). It is likely to be left with some methylation marks. Based on the above, we can speculate

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that some modified cytosines such as 5mC or 5hmC are also present during the non-random chromosome segregation in Drosophila. Therefore, the expression of Tet and Dnmt2 in tissues and stages and the non-random chromosomal segregation can help us to understand the possible DNA-based epigenetic mechanisms such as DNA methylation (Dunwell et al., 2013). A range of studies have identified that DR can induce DNA methylation depending on the diet composition and the tissue. Feeding the pregnant mice with low-protein diet can decrease the DNA methylation at both the PPAR alpha and glucocorticoid receptor loci in F1 and F2 male liver (Burdge et al., 2007; Lillycrop et al., 2008). Hyper-methylation and hypo-methylation have also been observed after undergoing a period of DR diet and over-feeding, respectively in humans (Bouchard et al., 2010; Milagro et al., 2011; Jacobsen et al., 2014). In addition, the deficiency of choline in

ACCEPTED MANUSCRIPT diet can alter the DNA methylation state of specific genes, thereby influencing the expression levels, consequently, impacting the hepatocarcinogenesis (Okabe et al., 2011). In all, it can be stated that diet components can mediate health lifespan by

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influencing DNA methylation state and regulating the transcription and expression of specific genes.

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5. Drosophila as a perfect model to study DNA methylation involved in diet related aging

As explained earlier, aging is accompanied by several physiological changes and

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reduction of all kinds of cell functions, even inducing some common aging-related diseases, such as cancer, diabetes, obesity, heart disease, hypertension. Although we

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have summarized the relationship between diet and aging through major genetic pathways and epigenetic modifications (Fig. 1), the precise molecular mechanisms

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about the effect of diet on aging are not very clear, especially on epigenetic

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modifications. A range of cell lines, rodent animals have been used to study the potential molecular mechanisms of aging, but Drosophila is considered as one of the

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most popular genetic model to study the complexity of the aging process with response to diet.

Compared with other lab models, the Drosophila is tractable on the following

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aspects. At first, Drosophila harbors a relatively shorter rearing period, convenience in raising in lab, lifespan and DR protocol is easy to control. Secondly, female fly’s fecundity can be assessed by counting the egg number, egg size and mortality can be assessed and presented on the survival curve. Last but not the least, in consideration of the tissue-specific expression of DNA methylation, the dissection of Drosophila tissue is practical in labs. The relationships among diet, DNA methylation and corresponding diseases based on specific tissues including brain, gut, heart, muscle can be studied easily (Gargano et al., 2005; Nishimura et al., 2011; Bell and Thompson, 2014; Chen et al., 2014). Besides, Drosophila genome shares more than 60% human genes, which is having several orthologues with Drosophila (Bier, 2005; Hoskins et al., 2015). Therefore, Drosophila has been used in the research programs associated with genetic molecular mechanisms of human aging and age-related

ACCEPTED MANUSCRIPT diseases, such as Parkinson’s disease (Feany and Bender, 2000; Auluck et al., 2002; Chen and Feany, 2005), Alzheimer’s disease (AD) (Shulman et al., 2014) and insomnia (Koh et al., 2006). In addition, present genetic approaches can be easily and

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widely applied in Drosophila (Helfand and Rogina, 2003). By employing Drosophila as a model to study the mechanism of aging with response to diet, we can identify and

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characterize single-gene mutations extending lifespan efficiently. Although by loss of

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Dnmt2 by RNA interference, there are no detectable consequences for fly

Fig. 1. The relationship between diet and aging through major genetic pathways and epigenetic modifications. Diet composition and intervention can influence aging by several pathways, such as IIS, TOR, AMPK, SIRT pathways and epigenetic modifications including DNA methylation and histone modification. These modifications can alter the chromatin state and gene expression levels, consequently, impact aging. In some cases, these pathways can interact with each other. Abbreviations: INR, insulin/IGF-like tyrosine kinase receptor; IIS, insulin/IGF signaling; IGF1, Insulin-like growth factor1; IRS1/2, insulin receptor substrate1/2; Foxo, forkhead box protein O; PI3K, phosphatidyl-inositol 3-kinase; TSC1/2, tuberous sclerosis complex protein1/2; TOR, the target of rapamycin; 4eBP, the eukaryotic translation initiation factor 4E binding protein; S6K, S6 ribosomal kinase; SIRT, silent information regulator; NF-κB, nuclear factor-κB.

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embryonic development, yet minor phenotypic aberrations are observed during the later development. Over-expression of Dnmt2 could induce the significant genomic

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hypermethylation (Kunert et al., 2003). Furthermore, the molecular mechanisms and

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pathways of Drosophila are conserved in mammals. For instance, the insulin/IGF

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signaling pathway, dTOR signaling pathway and some epigenetic modification such as histone methylation and DNA methylation, are extremely helpful in addressing the underlying

mechanisms

such

as

the

interaction

tissue-tissue,

gene-gene,

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gene-cell-tissue and tissue-specific functional decline during aging (Lee and Luo, 2001; McGuire et al., 2004; Karpac and Jasper, 2009; Karpac et al., 2011). Both IIS

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and TOR are also found to be important sensing pathways linked with cell growth and mechanisms in honey bee (Apis mellifera) (Mutti et al., 2011), which is closely related to Drosophila. Therefore, after discussing the genetic and practical aspects, we

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6. Conclusion

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response to DR.

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propose Drosophila as a perfect model to study epigenetic mechanisms of aging with

There are several mechanisms for dietary restriction, one of which focuses on epigenetics. Changes in DNA methylation with response to diet, especially the

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composition of diet nutrient, is responsible for the correlated phenotypic changes and the risk of age-related diseases. It is essential to explore the relationship between DNA methylation and aging. Although kinds of cell lines or other models have been applied in this research, still as we discussed above, employing Drosophila to study the DNA methylation mechanism that affects aging and animal lifespan will be a big challenge. Acknowledgements We thank Dr Matt Piper for his valuable comments on the manuscript. This work was supported by the grants from the Natural Science Foundation of China (No. 31471998), the “Thousand Talents Program” in Sichuan and the Innovative Research Team in University of Sichuan Bureau of Education (14TD0002). The authors declare no conflict of interest.

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age in disease-free human skeletal muscle. Aging cell 13, 360-366.

ACCEPTED MANUSCRIPT Highlights Dietary restriction (DR) induces lifespan extension in Drosophila.

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Various signaling pathways are involved in DR-dependent longevity.

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Epigenetic events have been considered as the important contributors to

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DR-induced lifespan extension.

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Drosophila is a powerful model to resolve epigenetic mechanism of aging

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with response to diet.

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