Calorie restriction (CR) promotes healthy aging in diverse species. Recently, it has been shown that fasting for a portion of each day has metabolic benefits and promotes lifespan. These findings complicate the interpretation of rodent CR studies, in which animals typically eat only once per day and rapidly consume their food, which collaterally imposes fasting. Here, we show that a prolonged fast is necessary for key metabolic, molecular and geroprotective effects of a CR diet. Using a series of feeding regimens, we dissect the effects of calories and fasting, and proceed to demonstrate that fasting alone recapitulates many of the physiological and molecular effects of CR. Our results shed new light on how both when and how much we eat regulate metabolic health and longevity, and demonstrate that daily prolonged fasting, and not solely reduced calorie intake, is likely responsible for the metabolic and geroprotective benefits of a CR diet.

Many chromatin modifying enzymes require metabolic cofactors to support their catalytic activities, providing a direct path for fluctuations in metabolite availability to regulate the epigenome. Over the past decade, our knowledge of this link has grown significantly. What began with studies showing cofactor availability drives global abundances of chromatin modifications has transitioned to discoveries highlighting metabolic enzymes as loci-specific regulators of gene expression. Here, we cover our current understanding of mechanisms that facilitate the dynamic and complex relationship between metabolism and the epigenome, focusing on the roles of essential metabolic and chromatin associated enzymes. We discuss physiological conditions where availability of these 'epi-metabolites' are dynamically altered, highlighting known links to the epigenome and proposing other plausible connections.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate cellular epigenetic states. This metabolism-epigenome link enables the sensitization of chromatin methylation to altered SAM abundance. However, a chromatin-wide understanding of the adaptive/responsive mechanisms that allow cells to actively protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear de novo mono-methylation of H3 Lys 9 (H3K9) at the expense of global losses in histone di-and tri-methylation. Under SAM-depleted conditions, de novo H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide the first evidence for active epigenetic adaptation and persistence to metabolic stress.

Deleterious changes in energy metabolism have been linked to aging and disease vulnerability, while activation of mitochondrial pathways has been linked to delayed aging by caloric restriction (CR). The basis for these associations is poorly understood, and the scope of impact of mitochondrial activation on cellular function has yet to be defined. Here, we show that mitochondrial regulator PGC‐1a is induced by CR in multiple tissues, and at the cellular level, CR‐like activation of PGC‐1a impacts a network that integrates mitochondrial status with metabolism and growth parameters. Transcriptional profiling reveals that diverse functions, including immune pathways, growth, structure, and macromolecule homeostasis, are responsive to PGC‐1a. Mechanistically, these changes in gene expression were linked to chromatin remodeling and RNA processing. Metabolic changes implicated in the transcriptional data were confirmed functionally including shifts in NAD metabolism, lipid metabolism, and membrane lipid composition. Delayed cellular proliferation, altered cytoskeleton, and attenuated growth signaling through post‐transcriptional and post‐translational mechanisms were also identified as outcomes of PGC‐1a‐directed mitochondrial activation. Furthermore, in vivo in tissues from a genetically heterogeneous mouse population, endogenous PGC‐1a expression was correlated with this same metabolism and growth network. These data show that small changes in metabolism have broad consequences that arguably would profoundly alter cell function. We suggest that this PGC‐1a sensitive network may be the basis for the association between mitochondrial function and aging where small deficiencies precipitate loss of function across a spectrum of cellular activities.

Biochemical, proteomic, and epigenetic studies of chromatin rely on the ability to efficiently isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast , which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state and is applicable for both parallel multisample spin-column purification and large-scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes and features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy. The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies and serve as a mild, acid- and detergent-free sample preparation method for MS analysis.

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Summary Maintaining zinc homeostasis is an important property of all organisms. In the yeast Saccharomyces cerevisiae, the Zap1 transcriptional activator is a central player in this process. In response to zinc deficiency, Zap1 activates transcription of many genes and consequently increases accumulation of their encoded proteins. In this report, we describe a new mechanism of Zap1-mediated regulation whereby increased transcription of certain target genes results in reduced protein expression. Transcription of the Zap1-responsive genes RTC4 and RAD27 increases markedly in zinc-deficient cells but, surprisingly, their protein levels decrease. We examined the underlying mechanism further for RTC4 and found that this unusual regulation results from altered transcription start site selection. In zinc-replete cells, RTC4 transcription begins near the protein-coding region and the resulting short transcript leader allows for efficient translation. In zinc-deficient cells, RTC4 RNA with longer transcript leaders are expressed that are not efficiently translated due to the presence of multiple small open reading frames upstream of the coding region. This regulation requires a potential Zap1 binding site located farther upstream of the promoter. Thus, we present evidence for a new mechanism of Zap1-mediated gene regulation and another way that this activator protein can repress protein expression.

29S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone 30 methyltransferases that regulate cellular epigenetic states. This metabolism-epigenome link 31 enables the sensitization of chromatin methylation to altered SAM abundance. However, a 32 chromatin-wide understanding of the adaptive/responsive mechanisms that allow cells to 33 actively protect epigenetic information during life-experienced fluctuations in SAM availability 34 are unknown. We identified a robust response to SAM depletion that is highlighted by 35 preferential cytoplasmic and nuclear de novo mono-methylation of H3 Lys 9 (H3K9) at the 36 expense of global losses in histone di-and tri-methylation. Under SAM-depleted conditions, de 37 novo H3K9 mono-methylation preserves heterochromatin stability and supports global 38 epigenetic persistence upon metabolic recovery. This unique chromatin response was robust 39 across the mouse lifespan and correlated with improved metabolic health, supporting a 40 significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies 41 provide the first evidence for active epigenetic adaptation and persistence to metabolic stress. 42 43 44 persistence 109 responses to Met-restriction in both HCT116 cells and C57BL/6J liver (Figure 1D-1E). In 110 HCT116 cells, Met-restriction stimulated distinct biphasic changes in global PTM profiles (Figure 111 1D). Phase I (0 hr-45 min) was rapid and marked by a trending upregulation of H3K4me2/3, 112PTMs known to mark transcriptionally active promoters. Phase II (90 min-24 hrs) was 113 characterized by global decreases in di-and tri-histone methylation. Decreased levels of di-114 and tri-methylated peptides were accompanied by increases in acetylated and unmodified 115 peptide species. Similarly in C57BL/6J mice, histone PTM responses were marked by 116 significant decreases in histone di-and tri-methylation that essentially matched the patterns 117 found in HCT116 cells during the prolonged Phase II response ( Figure 1E and Figure S1E-S1J). 118Decreased higher state (di-and tri-) histone methylation both in vitro and in vivo highlights a 119 decreased methylation capacity resulting from prolonged Met and/or SAM depletion. Together, 120 these observations suggest perturbed methyl-donor metabolism is capable of stimulating 121 significant changes in histone, but not global 5mC, methylation abundance. Furthermore, 122 similarities between the metabolic and epigenetic responses to Met-restriction across both 123 systems support the use of in vitro Met-restriction as a model for mechanistic follow-up studies. 124 125 Decreased SAM Availability Drives Robust Histone Methylation Response 126 127 Dramatic reduction of intracellular Met and SAM correlated with onset of the in vitro 128 Phase I and II histone PTM changes, respectively (Figure 1D and Figure S1A-S1B). This 129 implies depletion of individual methyl-metabolites may be capable of stimulating distinct histone 130 modifying pathways. To determine if the global losses in di-and tr...