The three main categories of epigenetic marks, DNA methylation (DNAm), post-translational histone modifications (PTHMs) and small non-coding RNAs, are considered as central regulators of the aging process [ 4 12 ]. Epigenetics patterns are greatly remodeled during aging, and this phenomenon has been implicated in the development of multiple age-related diseases [ 13 14 ]. One important characteristic of epigenetic factors is their plasticity: epigenetic changes are reversible and they can be modulated by a wide range of environmental factors, including lifestyle habits and dietary interventions [ 15 ].

Caloric restriction (CR), i.e., the reduction of caloric intake (by 10% to 40%) without causing malnutrition, has proven to be by far the most effective intervention that can extend the maximum lifespan in a wide range of organisms including yeast, nematodes, flies, and rodents [ 6 ]. Interestingly, observations also demonstrated an effect on healthspan (i.e., time spent being healthy), coincident with a significant decrease in age-related diseases such as cardiovascular events, diabetes, neurodegenerative diseases, and cancers [ 7 10 ]. The beneficial effects of CR occur through an extreme wide range of molecular mechanisms, largely overlapping with aging hallmarks, among which epigenetic factors have recently gained interest.

Aging can be defined as a time-dependent and progressive decline in functional status which ultimately results in death. This complex and multifaceted phenomenon, which is a major risk factor for a wide range of non-communicable and chronic diseases, is the result of a complex interplay between genetic, environmental, and stochastic variables. While some researchers have suggested that aging is genetically programmed, others sustain that it is the result of the progressive accumulation of damage coupled with a decline in maintenance [ 1 3 ]. While this topic is still controversial, the scientific community agrees that aging is characterized by specific hallmarks, which rely on well-defined biological pathways [ 4 5 ]. The identification of the mechanistic processes underlying aging has one important consequence: it provides new potentially modifiable targets and justifies anti-aging interventions.

2. DNA Methylation

14,16,21,22,25,26, DNA methylation consists in the addition of a methyl group to a cytosine residue, preferentially in a CpG dinucleotide. During aging, there is a marked remodeling of genomic DNA methylation patterns, which has been extensively investigated and implicated in multiple common age-related diseases [ 13 17 ]. Different types of DNA methylation changes coexist during aging. Firstly, the global genomic DNA methylation level decreases with age, mainly reflecting a decrease in DNA methylation of repetitive genomic regions and interspersed elements [ 18 19 ]. Secondly, differential methylation of specific genomic loci occurs, especially with an age-related hypermethylation in gene promoters [ 20 23 ]. Finally, there is an increase in the inter-individual divergence between patterns of DNA methylation (named DNA methylation drift) [ 24 27 ], and an increase in the rate of epimutations [ 28 ].

c-myc [ Ras [33,34,35,36, The two first studies reporting the impact of CR on DNA methylation were published in 1993: CR was shown to induce an increase in the methylation of the proto-oncogenes 29 ] and 30 ] in mice livers and pancreatic acinar rat cells, respectively, repressing the trend towards age-associated hypomethylation [ 29 30 ]. Twenty years later, Ions et al. analyzed the impact of CR on a genome-wide scale and correlated epigenetic data with transcriptomic ones [ 31 ]. Indeed, it has been described that CR impacts gene expression across numerous organisms, preventing changes observed in the aging transcriptomes [ 32 37 ]. Analyzing publicly available datasets, Ions et al. reported a significant overlap between the genes that showed altered expression in response to CR and those whose methylation varies during aging [ 31 ].

38,39,40,41,42,43,44, Drosophila [ Recently, nine independent studies comparing the effect of age on DNA methylation patterns in animals fed ad libitum (AL) or with a CR diet have been published [ 25 45 ]. While CR seemed to have no impact on DNA methylation levels in adult 44 ], eight other studies collectively reported that CR is protective against age-related DNA methylation changes in mammals in different tissues types (kidney [ 38 ], blood [ 25 40 ], liver [ 39 42 ], hippocampus [ 41 ], and cerebellum [ 45 ]). Genomic regions which tend to become differentially methylated with age (aDMRs) experience less changes in animals under a CR diet: for example, analyzing the liver from female mice on a CR diet from 4 to 22 months of age, Cole et al. observed that CR increased methylation in hypomethylated aDMRs, while it decreased it in the hypermethylated aDMRs. Interestingly, Sziraki et al. observed a two-stage response to CR [ 40 ]. They analyzed the blood methylomes of four groups of mice, aged from 10 to 27 months, which all started CR at the age of 4 months. They found that the DNA methylome was initially shifted by CR in the same direction as aging but, secondarily, the cumulative changes associated with CR shifted it toward a younger state compared to control animals [ 40 ]. The remodeling of DNA methylation patterns associated with CR can target genomic regions associated with the development of age-related diseases. For example, in the kidney of old rats, CR was able to attenuate age-dependent methylation alterations in the promoters of genes that are associated with inflammation, cancer, or diabetes [ 38 ], while in mouse liver, CR had a specific impact on genes involved in lipid metabolism-related pathways, resulting in the regulation of the lipid profile (with an attenuation of the age-associated increase in liver triglyceride content) [ 39 ].

While the previously mentioned studies evaluated the impact of CR on DNA methylation directional changes (hypo- or hypermethylation), Maegawa et al. investigated its influence on DNA methylation drift. This progressive divergence of the epigenomes between different subjects over time has been correlated with lifespan in three different mammalian species (mouse, rhesus monkey, and human) [ 25 46 ]. Maegawa et al. demonstrated that CR was able to protect against this DNA methylation drift, both in mice and rhesus macaques. The observed effects were possibly dose-dependent, regarding both CR severity and duration: indeed, in monkeys exposed to 30% CR since middle age, the attenuation of age-related methylation drift as compared to AL fed controls was less pronounced than the one seen in mice exposed to 40% CR since early adulthood [ 25 ].

48, Nts1 gene promoter, correlating with the increase in its mRNA expression. The hypomethylation persisted even when CR was discontinued [ Interestingly, an important characteristic of CR is its capacity to induce a cellular memory which can persist even when it is discontinued [ 47 49 ]. DNA methylation could play a role in these long-lasting effects. It was demonstrated that even a short-term CR (one or two months) can reverse changes in aDMRs in rodents [ 38 39 ]. In four-month-old male mice, a one-month CR was able to induce significant changes in the expression of several genes, and 20 to 50% of these changes persisted two months after CR was discontinued [ 50 ]. Interestingly, concomitant significant changes in DNA methylation of the promoter regions of those genes were observed. For example, CR induced a significant decrease in the DNA methylation ofgene promoter, correlating with the increase in its mRNA expression. The hypomethylation persisted even when CR was discontinued [ 50 ]. Long-lasting effects of severe dietary restriction on epigenetics have also been observed in humans exposed prenatally to famine during the Dutch Hunger Winter in 1944–1945. Remarkably, it was observed that differential methylation persisted even after six decades [ 51 ].

ATP10A and WT1 genes in Milagro et al. study) [53, Epigenetic data on the effects of pure CR in humans are limited, as it is an intervention likely to be difficult to implement in the long-run in humans. To our knowledge, only two studies have reported the results of epigenomic responses to a hypocaloric diet intervention in humans [ 52 53 ], but no study has specifically evaluated the impact of CR on DNA methylation signatures of aging in humans. A reduced energy intake intervention was shown to cause DNA methylation changes at specific loci in obese subjects (three chromosomal regions (chromosomes 1p36, 4q21, and 5q13) in Bouchard et al. study;andgenes in Milagro et al. study) [ 52 53 ]. According to these studies, DNA methylation levels could also be used as predictors of weight loss, as they were significantly different between high and low responders to the nutritional intervention [ 52 54 ].