Epigenetic regulation of caloric restriction in aging

DNA methylation is one of the most important epigenetic modifications. It provides a stable and heritable component of epigenetic regulation. DNA methylation primarily occurs on cytosine residues of CpG dinucleotides, which are frequently clustered into CpG islands at the regulatory sites of gene promoter regions. The amount of DNA methylation in a gene control region generally inversely correlates with gene activation [37, 38]. The methyl groups on CpG dinucleotides can recruit multiple transcriptional complex proteins, including methylation-sensitive transcription factors and methyl-binding proteins that are often associated with gene silencing [39]. Therefore, DNA methylation plays an important role in the regulation of gene expression, maintenance of DNA integrity and stability in many biological processes, such as genomic imprinting, normal development, cell proliferation and aging [40‐42]. The patterns of DNA methylation are dynamically mediated by at least three independent DNA methyltransferases (DNMTs): DNMT1, DNMT3a and DNMT3b. DNMT1 performs a maintenance function during cell division, while DNMT3a and DNMT3b act as de novo methyltransferases after DNA replication by adding a methyl moiety to the cytosine of CpG dinucleotides that have not previously been methylated [43‐47].

During aging processes, there is a progressively reduced capability for homeostasis and loss of chromatin integrity, predominantly due to aberrant gene expression [48]. DNA methylation regulation plays a crucial role during aging processes. Age causes a dramatic change in the distribution of 5-methylcytosine (the product of DNA methylation) across the genome. This leads to a decrease in global DNA methylation [49‐54]. Although genome-wide levels of methylation decrease with aging, the promoter regions of many specific genes tend to switch from unmethylated to methylated status, resulting in gene silencing, which may include promoters of several tumor- and/or aging-related genes, such as RUNX3 and TIG1 [53, 55] (Table 2). These findings suggest an essential role of aging-associated DNA methylation changes in the regulation of aging-related diseases such as cancer.

The evidence suggests that the biological effects of CR are closely related to chromatin function [56]. In fact, acting as an important environmental intervention, CR is speculated to exert its aging-delaying effect through its capacity to increase genomic stability. Reversal of aberrant DNA methylation during aging is believed to be the most effective mechanism for CR to maintain chromatin function and subsequently influence aging processes.

As discussed previously, two major changes in DNA methylation occur during aging progression. These changes involve globally decreased but locally increased DNA methylation status. Interestingly, CR is likely to recover these aging-induced aberrant DNA methylation patterns, but by specific loci control rather than globally [57] (Figure 1). Studies of the comparison of DNA methylation levels in pancreatic acinar cells between CR-fed rats and control rats fed ad libitum suggest that CR increased the methylation level of proto-oncogenes such as Ras [30] (Table 2). A hypermethylated gene promoter will often be recognized by transcriptional repressor complexes, leading to silencing the expression of these oncogenes, which contributes to the effects of CR on cancer prevention. Although the majority of CR research has been based on experimental animal studies, we have established an in vitro mammalian cellular system to mimic CR-controlled longevity by the reduction of glucose, the main caloric resource in cell culture medium [31]. In our current studies of human cells, DNA hypermethylation of an E2F-1 binding site was found in the promoter of the p16 ^INK4a gene, an important tumor suppressor and aging-associated gene. This DNA hypermethylation of the E2F-1 binding site blocks access of E2F-1 (an active transcription factor of p16 ^INK4a ) to the p16 ^INK4a promoter, resulting in p16 ^INK4a downregulation, which contributes to CR-induced lifespan extension (Table 2 and Figure 1). In this regard, there is a strong tendency for the DNA methylation pathway to predominately control key cancer-related genes during CR, suggesting a close connection between aging and cancer.

On the basis of the preceding discussion, we confirm that DNMTs play a crucial role in maintaining or rewriting DNA methylation profiles. Consistently, DNMT1 activity is significantly elevated in response to CR to correct the decreased methylation level during aging [31]. Further studies have also indicated that CR-caused Dnmt3a level changes in the mouse hippocampus may benefit mouse brain function during aging [58]. Both DNMT1 and DNMT3b play a critical role in regulating cellular senescence in human stem cells [59]. Therefore, it is highly possible that CR modulates DNA methylation, depending on expression levels and/or enzymatic activities of individual DNMTs (Figure 1).

Because of the critical roles of DNMTs in the control of aging and aging-associated diseases such as cancer and DNMT inhibitors such as azacitidine (5-azacytidine) and decitabine (5-aza-2'-deoxycytidine) have been widely used for cancer treatment in both experimental studies and clinical trials [60] (Table 3). Moreover, some bioactive food components with DNMT inhibition properties, such as green tea polyphenols and soybean genistein, have shown cancer prevention and inhibition activities by reducing DNA hypermethylation of key cancer-causing genes [61‐63] (Table 3). These are important and encouraging findings that imply the potential translation of these bioactive dietary compounds into intervention targets and strategies for the prevention and treatment of human cancer.

Since restricted caloric intake induces a series of metabolic responses to nutrition deficiency, effective regulation of metabolic processes to adapt to this change could be another important mechanism underlying the effect of CR on longevity. One approach to interpreting CR in the regulation of metabolic pathways is through interventions to treat human obesity, which has become an important public health issue in recent years. Obesity is a common metabolic disorder characterized by excessively accumulated body fat and is closely related to a series of human diseases, including diabetes, hypertension, dyslipidemia, cardiovascular complications and even cancer, which are recognized causes of accelerated aging [64]. Therefore, obesity prevention could be a key underlying factor in the anti-aging effects of CR. Because of its substantial and promising effects in promoting weight loss, CR is widely used in clinical weight control intervention [65]. Current studies focusing on short-term CR interventions in obese human subjects revealed that hypocaloric diets cause DNA methylation changes in specific loci, such as ATP10A, WT1 and TNF-α, which could be used as early indicators of a response to the metabolic effects and as predictors of outcomes in weight-loss programs [66‐68]. Although further CR studies have identified a pool of DNA methylation-controlled candidate genes that may be closely correlated with metabolic pathways, widespread methylation changes on numerous gene loci that facilitate CR in reprogramming the DNA methylation profile may also explain a powerful and universal effect of CR in influencing different aspects of human health. Thus, better understanding the functions of these DNA methylation-sensitive genes may contribute not only to optimizing personal weight-loss plans, but more important, to developing a novel application in slowing down of aging processes and the prevention of aging-related diseases.

Surprisingly few studies have investigated genome-wide alterations in DNA methylation profiles in CR-induced longevity using in vivo or in vitro models. Thus, the complete methylation-regulated pathways and target genes that may be responsible for CR-induced longevity remain unknown. Further investigations in this particular area show promising prospects in developing novel clinical preventative or therapeutic approaches to aging-related degenerative diseases.

Histone modifications affect the basic structure of the chromatin unit, the nucleosome. The nucleosome consists of 146 bp of DNA wrapped around an octamer of histones (two copies of H2A, H2B, H3 and H4 monomers) [69]. In most cases, histone remodeling occurs at the N-terminal group of lysine (K) residues in histones by diverse modification patterns such as acetylation, methylation, ubiquitination and ADP ribosylation, among which histone acetylation or deacetylation changes are considered to be the most prevalent mechanisms of histone modifications [27]. Histone modifications are associated with both gene activation and gene repression. The combination of modifications within histone tails directly changes nucleosome configuration and results in the status of chromatin switching to either a compacted status (tight-close) or a relaxed status (loose-open) [70]. Therefore, histone modifications determine the level of openness of chromatin and thus the degree of gene activity within a certain DNA region. For example, a deacetylated histone lysine residue has the positive charge, which attracts the negatively charged DNA strand producing a compact chromatin state that is associated with transcriptional repression. By contrast, the modification of histone acetylation removes the positive charge and results in an open chromatin structure, which leads to active transcription (Figure 2).

The promising impact of the chromatin regulators on aging interference provides an excellent opportunity to prevent for human aging-related diseases by applying potential epigenetic drugs. An example of this is resveratrol, a natural compound found in grapes and red wine which has been demonstrated to extend lifespan in Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila through remodeling chromatin structure via mediation of SIRT1 activity [111‐113]. It has been reported that resveratrol can activate SIRT1 mechanisms and mimic SIRT1-induced CR cascades, leading to increased longevity [114]. In addition to its effect on longevity, this compound is known to positively influence metabolism and reduce fat and glucose levels, resulting in increasing glucose tolerance and activation of several signaling pathways that are relevant to antistress, antioxidation and increased mitochondrial biogenesis [115, 116]. These effects were illustrated by a current finding showing that resveratrol opposes the effects of a high-fat diet in mice [117]. Due to the toxicity of the high-fat diet, control animals in this study had early mortality, whereas resveratrol improved the health and survival rate of these mice, suggesting the important role of resveratrol in the aging process. Clinically, a total of 31 human studies involving resveratrol have been reported in the US national database http://​clinicaltrials.​gov/​. These studies aimed at investigating the potential role of resveratrol in diabetes, obesity, Alzheimer's disease and cancer (Table 3). These studies have revealed promising and universal effects of resveratrol by favorably altering cell proliferation, increasing cellular detoxification, protecting DNA damage, modulating metabolic processes and inhibiting tumorigenesis, which significantly improve human health and lead to increased human lifespan [118, 119].

Epigenetic therapy has shown powerful clinical potential in delaying aging and preventing aging-related diseases, especially cancer. As we have discussed previously, DNMT inhibitors, inlcuding azacitidine and decitabine, as well as HDAC inhibitors, such as depsipeptide, phenylbutyrate, valproic acid and suberoylanilide hydroxamic acid, have been widely used for cancer treatment in both experimental studies and clinical trials (Table 3). Studies have also indicated that resveratrol is a potent cancer chemopreventative agent. These findings are extremely encouraging, and future studies focusing on development of novel epigenetic drugs are urgently needed to develop effective clinical strategies to treat human aging-related diseases [120].

The significant epigenetic impact of CR on delaying aging and preventing aging-related diseases has motivated efforts to identify natural or synthetic compounds that mimic the effects of CR. A broad range of diets have been identified that mediate epigenetic processes, the so called "epigenetic diets," providing potential to reduce aging-associated disease incidence and possibly extending the quality and length of the human lifespan by simple consumption of such diets or extracted bioactive dietary compounds [121] (Table 3). As described previously, resveratrol represents an excellent example of an "epigenetic diet" and acts as a SIRT1 mimic that leads to increased longevity in vivo and in vitro [111‐119]. Other important epigenetic diets have recently been identified, such as green tea, broccoli sprouts and soybeans, and the bioactive compounds extracted from these diets have received extensive attention due to their profound effects on cancer prevention by altering the aberrant epigenetic profile in cancer cells [62, 63, 80, 122‐125]. In particular, long-term consumption of these epigenetic diets is highly associated with a low incidence of various aging-related degenerative diseases such as cancer and cardiovascular disease, suggesting that these bioactive diets may affect aging processes by altering chromatin profiles that also occur in CR [2]. For instance, global gene expression profiling can be used to identify useful compounds correlated with biological age. Dhahbi et al. [126] developed gene expression profiling methods to discover potential pharmaceuticals capable of mimicking the effects of CR, which may open a new avenue in the discovery of promising candidates that mimic CR and delay aging.