Interplay between Metabolism and Epigenetics: A Nuclear Adaptation to Environmental Changes.

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Mol Cell. Author manuscript; available in PMC 2017 June 02. Published in final edited form as: Mol Cell. 2016 June 2; 62(5): 695–711. doi:10.1016/j.molcel.2016.05.029.

Interplay between metabolism and epigenetics: a nuclear adaptation to environmental changes Jean-Pierre Etchegaray1,2 and Raul Mostoslavsky1,2,3,* 1The

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts 02114, USA

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2The

MGH Center for Regenerative Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA

3The

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA

Abstract

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The physiological identity of every cell is maintained by highly specific transcriptional networks that establish a coherent molecular program that is in-tune with nutritional conditions. The regulation of cell-specific transcriptional networks is accomplished by an epigenetic program via chromatin modifying enzymes, whose activity is directly dependent on metabolites such as acetylCoA, S-adenosylmethionine and NAD+ among others. Therefore, these nuclear activities are directly influenced by the nutritional status of the cell. In addition to nutritional availability, this highly collaborative program between epigenetic dynamics and metabolism is further interconnected with other environmental cues provided by the day-night cycles imposed by circadian rhythms. Herein, we review molecular pathways and their metabolites associated with epigenetic adaptations modulated by histone- and DNA-modifying enzymes and their responsiveness to the environment in the context of health and disease.

INTRODUCTION

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Epigenetics is defined by the heritable traits that do not involve alterations in DNA sequence, but rather chemical changes within the chromatin. These chemical changes entail posttranslational modifications of histones proteins, DNA methylation and DNA oxidation events. The inheritance aspect of epigenetics is mostly understood with regard to DNA methylation. However, epigenetics mediated by histone modifications remain an open question and an active area of research (reviewed in Zhu and Reinberg, 2011). Nevertheless, these histone modifications directly modulate chromatin structure and consequently influence gene expression. More specifically, the establishment of epigenetic signatures is accomplished by specific sets of enzymes that add or remove different types of posttranslational modifications including acetyl or methyl groups to distinct histone *

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Etchegaray and Mostoslavsky

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residues, thereby having a direct influence on transcription (reviewed in Bannister and Kouzarides, 2011; reviewed in Verdin and Ott, 2015). In most cases, acetylation of histones

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is associated with transcription activation, while histone methylation can promote either activation or repression depending on the targeted residue within a particular histone. This concept was originally described as the ‘histone code’, which is increasingly evolving into a complex web of posttranslational modification networks impacting multiple nuclear processes, including transcription, DNA replication and DNA repair (Jenuwein and Allis, 2001; reviewed in Wang et al., 2004). Additionally, specific epigenetic modifying enzymes can also alter DNA by introducing methyl groups into cytosine bases mostly at CpG dinucleotide-rich genomic loci. Removal of these methyl groups is accomplished by successive oxidations of methylated cytosines by specific DNA hydroxylase enzymes in conjunction with the DNA base excision repair machinery. Importantly, the activity of all these epigenetic modifying enzymes relies on the availability of specific metabolites, therefore chromatin modifications are directly responsive to particular cellular metabolic states. Metabolism is defined as the set of life-sustaining chemical modifications within all cells of an organism. These chemical modifications are catalyzed by specific enzymatic reactions to maintain overall cellular and tissue homeostasis in respond to environmental conditions such as availability of nutrients. In this review, we focus on the molecular mechanisms by which intermediate metabolites like α-ketoglutarate (α-KG), and cofactors including acetyl-coenzyme A (acetyl-CoA), S-adenosylmethionine (SAM) and nicotinamide adenine dinucleotide (NAD+), influence the activity of various epigenetic modifying enzymes, which ultimately affect gene expression in response to nutrient availability. We will provide examples indicating that cells evolved “sensing” mechanisms that detect nutrient changes and adapt their transcriptional programs through epigenetic means, which feeds back into metabolic homeostasis. Additionally, we included the influence of circadian rhythms on metabolites that could impact the activity of chromatin regulators.

Chromatin dynamics driven by metabolites The activity of most enzymes involved in dynamic chromatin modifications is dependent on intermediary metabolites, including acetyl-CoA, S-adenosylmethionine (SAM), ATP, nicotinamide adenine dinucleotide (NAD+), flavin adenine dinucleotide (FAD), alphaketoglutarate (α-KG, also know as 2-Oxaglutarate), and uridine diphosphate (UDP). These metabolites function as substrates and/or cofactors influencing the enzymatic activity of chromatin modifiers, thereby coupling chromatin-dependent gene regulation with the metabolic state of the cell. Here, we describe the molecular interconnections of this coupling system and the consequences of its deregulation.

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Acetyl-CoA and Histone Acetylation The acetylation of lysine residues in histones represents one of the best-characterized posttranslational modifications, historically implicated as part of the histone-code hypothesis initially proposed by Thomas Jenuwein and David Allis (Jenuwein and Allis, 2001). The positively charged lysine residues are neutralized by the acetyl groups, which decreases the ionic interaction between DNA and histones resulting in an open chromatin structure and thereby, an epigenetic mark associated with positive transcriptional regulation. This histone

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acetylation-dependent open chromatin acts as a docking site for the recruitment of bromodomain-containing proteins functioning as epigenetic readers or effectors, which play critical roles in gene regulation (reviewed in Verdin and Ott, 2015).

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Acetyl-CoA is a key intermediary metabolite produced during catabolism and anabolism and it is the universal substrate for the acetylation of histones (Lee et al., 2007; reviewed in Shahbazian and Grunstein, 2007). The activity of histone acetyltransferases (HATs) relies on intracellular levels of acetyl-CoA, which stands as a prominent example of the interplay between metabolism and chromatin dynamics. From a metabolic point of view, Acetyl-CoA serves as a feeding supply molecule in the tricarboxylic acid (TCA) cycle within the mitochondria and it also participates as a building block in the production of macromolecules such as lipids, cholesterol and amino acids (Figure 1A). Acetyl-CoA is generated via activation of acetate (via the ACSS2 enzyme), the thiolytic cleavage of βketoacyl CoA along with β-hydroxy acids, and the oxidative decarboxylation of pyruvate. In general terms, the generation of acetyl-CoA is associated with the breakdown of carbohydrates and fats, via glycolysis and β-oxidation, respectively (reviewed in Pietrocola et al, 2015). Under conditions of limited glucose availability, such as fasting, fatty acid betaoxidation becomes the major cellular metabolic pathway associated with the generation of acetyl-CoA promoting the TCA cycle to maintain mitochondrial-dependent ATP biosynthesis. The levels of acetyl-CoA are quite dynamic and are directly depending on nutrient availability. Several studies support the direct interconnection between histone acetylation and availability of acetyl-CoA. Indeed, genome-wide analyses from yeast to mammals demonstrate that the interplay between histone acetyltransferase activity and gene expression depends on the availability of acetyl-CoA. For example, yeast grown in conditions of high acetyl-CoA precursors such as glucose, galactose or ethanol exhibit histone hyperacetylation at various growth related genes including the ones encoding ribosomal RNA translation-associated proteins. In this way, yeast cells tune epigeneticdependent gene expression to modulate their growth based on nutrient availability (Cai et al., 2011a, Cai et al., 2011b). Studies in mammalian cells also support a role for acetyl-CoA in transcriptional activation via histone acetylation. More specifically, histone acetylation increases under conditions that favor the expression of the acetyl-CoA synthetase enzyme, adenosine triphosphate (ATP)-citrate lyase (ACL), which converts glucose-derived citrate into acetyl-CoA in the nucleus in response to high glucose availability or growth-factor stimulation (Wellen et al., 2009). Importantly, deletion of ACL decreases histone acetylation, which consequently reduces the expression of genes involved in glycolysis (Wellen et al., 2009). Additionally, dynamic translocation of the pyruvate dehydrogenase complex (PDC), consisting of three enzymes that convert pyruvate into acetyl-CoA, from the mitochondria into the nucleus is essential for histone acetylation (Sutendra et al., 2014). Overall, the data strongly favors the idea that high levels of acetyl-CoA result in a more permissive chromatin configuration by increasing the acetylation of histones, which in-turn promotes gene expression that modulates cell growth under these “nutrient favorable” conditions. In this context, acetyl-CoA acts as a biosensor of the metabolic state that triggers the regulation of specific genes involved in growth and proliferation in response to nutrient availability through an epigenetic mechanism involving histone acetylation (Figure 1A). These results



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provided one of the first evidences that, despite serving epigenetic functions, chromatin marks are much more dynamic than previously considered. Sirtuins, NAD+ and histone deacetylation


In metabolism, NAD functions as an electron transfer molecule in redox reactions, and it is found in the form of an oxidizing (NAD+) or a reducing agent (NADH). Thus, NAD+ accepts, while NADH donates electrons in various enzymatic reactions. NAD+ is produced de novo from the amino acid tryptophan or via the salvage pathway upon recycling of its precursors nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR) (Figure 1B). A critical enzyme of the salvage pathway, nicotinamide phosphorybosyltransferase (NAMPT) catalyzes the biosynthesis of NAD (reviewed in Imai and Guarente, 2014). NAD+ participates in various oxidative pathways such as glycolysis, the TCA cycle and fatty acid beta-oxidation. In addition, NAD+ functions as an obligated cofactor for the Class III histone deacetylase (HDAC) enzymes known as sirtuins (reviewed in Imai and Guarente, 2014). Sirtuins represent a unique enzyme family, conserved from yeast to mammals, with a gamut of roles including metabolic fitness, DNA repair, and mitochondrial functions. All together, these functions set apart sirtuins as enzyme promoting healthspan in multiple organisms (reviewed in Finkel et al., 2009, reviewed in Choi and Mostoslavsky, 2014). Their NAD dependency determines that the activity of sirtuins is dependent on nutrient availability; in turn, sirtuins directly affect the metabolic status of the cell. Consequently, NAD+ functions as a biosensor of nutrient availability, thereby coupling the metabolic state with sirtuin-dependent catalysis. Such “sensing/effector” loop establishes a highly regulated mechanism to sustain metabolic homeostasis. Although NAD+ is an obligatory cofactor for the activity of all seven mammalian sirtuins, the biological role of these enzymes differs from one another based on their differences in subcellular localization, tissue-specific expression and susbtrate specificity (reviewed in Finkel et al., 2009). SIRT1 -2, -6 and -7 are localized in the nucleus and target the deacetylation of specific histone residues. For instance, acetylated histone H3 at lysine 9 (H3K9ac) and -14 (H3K14ac) within specific gene loci is targeted by SIRT1, whereas global genomic deacetylation of acetylated histone H4 at lysine 16 (H4K16ac) during mitosis is catalyzed by SIRT2. SIRT6 exhibit specificity for deacetylating H3K9ac and H3K56ac, while SIRT7 targets H3K18ac within various gene loci (reviewed in Etchegaray et al., 2013; Nakahata et al., 2009; Vaquero et al., 2006; Michishita et al., 2008; Kawahara et al., 2009; Zhong et al., 2010; Barber et al., 2012). In recent studies, SIRT6 was shown to also deacetylate H3K18, specifically on pericentric heterochromatin (Taselli et al., 2016), suggesting that at least for some of these enzymes, their specific targets may be context dependent.

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The idea that fluctuating levels of NAD+, as a function of nutrient availability, could influence sirtuin catalysis is supported mostly by the work on SIRT1. More specifically, a significant decrease in NAD+ levels, due to impaired NAD+ salvage observed in certain conditions such as aging and diabetes has negative repercussions on SIRT1 activity. Deficient SIRT1 activity due to NAD+ depletion, in particular in high-fat diet treated mice, can be rescued upon supplementation with NMN, which is a natural precursor of NAD+, as mentioned above (Yoshino et al., 2011; Gomes et al., 2013; Yoon et al., 2015). Certain dietary conditions such as caloric restriction (CR) stimulates AMPK signaling pathway



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causing an upregulation of NAMPT, the rate-limiting enzyme in NAD+ biosynthesis, thereby leading to an increased SIRT1 activity (Canto and Auwerx, 2011; Ho et al., 2009). This increase in SIRT1 activity improves metabolic homeostasis through multiple means. Particularly, SIRT1 deacetylates PGC1α, enhancing gluconeogenesis and hepatic glucose output in the liver, and mitochondrial function in skeletal muscle (Rodgers et al., 2005; Gerhart-Hines et al, 2007) (Figure 1B). Levels of NAD+ and SIRT1 function are also modulated by circadian rhythms, as described below.


Another intriguing sirtuin that appears to link epigenetics to metabolism is SIRT6 (Figure 1B). This member of the sirtuin family evolved to be almost exclusively on chromatin, acting as an H3K9 and H3K56 deacetylase (reviewed in Etchegaray et al., 2013; reviewed in Kugel and Mostoslavsky, 2014). Although its high affinity binding for NAD indicates that its activity is less dependent on NAD levels (Pan et al., 2011), recent studies found, that chromatin context (Gil et al., 2013) and, intriguingly, the presence of free fatty acids (FFA) significantly enhance in vitro SIRT6 activity. Specifically, conformational changes induced by free-fatty acids such as myristic, oleic and linoleic acids were shown to stimulate the deacetylase activity of SIRT6 up to 35-fold in vitro (Feldman et al., 2013). Mechanistically, we have learned most of SIRT6 functions through the analysis of SIRT6 deficient mice. SIRT6 deficiency causes genomic instability along with a severe and fatal hypoglycemia (Mostoslavsky et al., 2006; Toiber et al., 2013). Follow up studies demonstrated that, mechanistically, SIRT6 deacetylates H3K9ac and H3K56ac to repress the transcription of HIF1α-driven glycolytic genes (Zhong et al., 2010). In the absence of SIRT6, glycolysis is upregulated, and the uncontrolled uptake of glucose by specific tissues such as brown fat (due to constitutive expression of the glucose transporter GLUT-1) is responsible for the lethal phenotype in SIRT6 deficient mice. This “glycolytic switch” due to the absence of SIRT6 plays a crucial role in the metabolic adaptation of cancer cells, as discuss in detail below. Given the effect of FFA on SIRT6 activity, one could speculate that nutrient conditions where FFA are increased (for instance, under starving conditions where betaoxidation is induced) could stimulate SIRT6 activity to repress glycolytic genes, in turn maintaining this pathway in check when cells are rather using alternative fuels (Figure 1B). SIRT6 has also been shown to modulate PGC1α upon conditions of nutrient stress, by an interesting feedback mechanism where SIRT6 deacetylation of the acetyl transferase GCN5 causes increased PGC1α activity, in turn modulating gluconeogenesis in the liver (Dominy et al., 2012). Collectively, the sirtuin-mediated histone deacetylation and its dependency on NAD+ levels, establishes an intimate relationship between chromatin dynamics and metabolism.

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SAM and DNA/Histone methylation Methylation of both histone H3 and H4 occurs at lysine and arginine residues (reviewed in Di Lorenzo and Bedford, 2011). Unlike histone acetylation, methylation does not appear to affect the ionic charge of the chromatin, but rather functions as a docking site for the recruitment of specific chromodomain-containing proteins (reviewed in Gayatri and Bedford, 2014). Furthermore, histone methylation is an epigenetic mark associated with either repression or activation of transcription, depending on the type of residue and the number of methyl group(s) added, along with its location within the N-terminal regions of



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either histone H3 or H4 (reviewed in Greer and Shi, 2012). For instance, methylation of H3K4, H3K48 and H3K79 is associated with transcriptional activation, while methylation of H3K9 and H3K27 preclude transcriptional repression. Other examples include the mono and –tri methylation of histone H4 at lysine 20 (H4K20me1 or H4K20me3), which cause compaction of chromatin leading to gene inactivation. Regarding methylation of arginine residues, di-methylation of histone H3 at arginine 17 (H3R17me2) is an activation mark, while di-methylation of H3 at arginine 2 (H3R2me2) correlates with transcriptional repression. These chromatin dynamics rely on the interplay between a diverse population of enzymes adding (histone methyltransferases) and excising (histone demethylases) methyl groups. In addition to histone methylation, the DNA base cytosine can be methylated by the DNA methyltransferase enzymes DNMT1, DNMT3a and DNMT3b, which produces 5methylcytosine (5mC), a major transcriptional repressive mark in many eukaryotes (reviewed in Bergman and Cedar, 2013).


The universal donor of methyl groups to both DNA and histone methyltransferase enzymes is the intermediary metabolite S-adenosylmethionine (SAM) (Takusagawa et al., 1998) (Figure 1C). Changes in methionine metabolism altering levels of SAM can directly influence trimethylation of H3K4 and consequently modulate gene expression (Mentch et al., 2015). More specifically, cells grown under conditions of methionine restriction exhibit a decrease on H3K4me3 lowering the expression of genes associated with the one-carbon metabolism pathway, which is used for the conversion of homocysteine into methionine (Mentch et al., 2015). This crosstalk between metabolism and epigenetics suggest a feedback mechanism whereby decreased levels of SAM could serve to negatively regulate the activity of the histone demethylase, KDM5B/JARID1B in order to maintain the levels of H3K4me3 (Mentch et al., 2015). The biosynthesis of SAM is derived from the condensation of the aminoacid methionine and ATP, by the rate-limiting enzyme methionine adenosyltransferase (MAT) (Sakata et al., 1993; Reytor et al., 2009). The intermediary metabolite S-adenosylhomocysteine (SAH) is the byproduct of SAM during methyltransferase reactions. SAH is further hydrolyzed to homocysteine, which is recycled back to methionine by using L-methyltetrahydrofolate (5-MTHF) as a methyl group donor, which is derived from the essential vitamin B9 or folic acid (reviewed in Gut and Verdin 2013; reviewed in Kaelin and McKnight, 2013). A homeostatic ratio between SAM and SAH levels is maintained via recycling of homocysteine to methionine. Importantly, SAH is a potent inhibitor of both DNA and histone methyltransferases. Thereby, the SAM/SAH ratio serves as a biosensor of the cellular metabolic state influencing the activity of methyltransferase enzymes that culminate in chromatin changes in response to nutrient availability. The requirement of the amino acid threonine as an essential source of SAM was demonstrated in mouse embryonic stem cells (mESCs) as a critical mechanism to maintain the pluripotency state via regulation of H3K4 methylation (Shyh-Chang et al., 2013). Threonine provides the necessary amount of glycine and acetyl-CoA, which are required for the biosynthesis of SAM by the mitochondrial enzyme threonine dehydrogenase (TDH). Depletion of either threonine or TDH causes a significant decrease in methylation of H3K4, which consequently affects cell proliferation and promotes differentiation (Alexander et al., 2011; Shyh-Chang et al., 2013). More specifically, this threonine or TDH restriction selectively diminished H3K4me2 and H3K4me3, which are part of a ‘bivalent’ epigenetic



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signature that is critical for the maintenance of the pluripotency state by keeping the transcription of developmentally related genes in a poised state (Bernstein et al., 2006). Therefore, just like the metabolites described above, SAM, folate and threonine are also part of a bio-sensing metabolic mechanism coupling nutrition availability with chromatin modifications, which ultimately impact the expression of genes in response to the environment. FAD+ and α-KG involved in Histone/DNA demethylation


Similar to acetyltransferases, deacetylases and methyltransferases, enzymes removing methyl groups from DNA and histones, known as demethylases, also utilize metabolites as substrate and cofactors. There are two classes of evolutionary conserved family of histone demethylases, the LSD and the Jumonji C (JmjC)-domain containing proteins (reviewed in Dimitrova et al., 2015). LSD1, also known as KDM1A, was the first histone demethylase enzyme to be identified (Shi et al., 2004). LSD1 targets demethylation of H3K4me1 and H3K4me2 as well as H3K9me1 and H3K9me2 (Shi et al., 2004; Ciccone et al., 2009; Shi et al., 2007). Another member of the LSD family, LSD2/KDM1B, also catalyzes demethylation of H3K4me1 and H3K4me2 (Karytinos et al., 2009). Surprisingly, LSD2 has been recently shown to have an E3 ubiquitin ligase activity to promote the proteosomemediated degradation of O-GlcNAc transferase (OGT), an enzyme required to add the sugar moiety O-GlcNAc as a posttranslational modification to a variety of substrates (Yang et al., 2015). OGT-dependent O-GlcNAcylation is discussed in more detail below. Both LSD1 and LSD2 use flavin adenine dinucleotide (FAD) as cofactor (Forneris et al., 2005). FAD is produced in the mitochondria and cytoplasm from riboflavin, also know as vitamin B2. Enzymes involved in the synthesis (FAD synthase) and hydrolysis (FAD pyrophosphatase) of FAD also exist in the nucleus providing a dynamic pool of nuclear FAD by balancing the ratio between its oxidized (FAD+) and reduced (FADH2) forms, Thus, the ration of FAD+/ FADH2 modulates the levels of nuclear FAD to be used as cofactor for epigenetic regulation (Giancaspero et al., 2013). Thus, FAD acts as a biosensor of the metabolic state causing a direct impact in epigenetic regulation by modulating the activity of histone demethylases. In this context, in adipocytes, transcription repression triggered by LSD1-dependent demethylation of H3K4me at promoters of genes involved in energy expenditure, such as PGC-1α and the pyruvate dehydrogenase kinase 4 (PDK4), is dependent on the availability of cellular FAD (Hino et al., 2012). In contrast to the LSD family, JmjC-domain containing enzymes function in an iron (Fe2+) and α-KG-dependent demethylation reaction (reviewed in Dimitrova et al., 2015). Alpha-ketoglutarate (α-KG), also known as 2-oxoglutarate, is produced from isocitrate by the mitochondrial enzymes isocitrate dehydrogenase-2 (IDH2) and -3 (IDH3) as an intermediate of the TCA cycle (Figure 1C). α-KG is also produced in the cytoplasm via IDH1-mediated catalysis. In addition to isocitrate, α-KG is also synthesized from amino acids such as arginine, glutamine, histidine and proline (Kaelin and McKnight, 2013). Based on homology conservation, the JmjC-domain containing proteins belong to a large family of enzymes targeting demethylation of various histone substrates including methylated H3K4, H3K9, H3K27, H3K36 and H4K20 (reviewed in Black et al., 2012). The role of α-KG levels in influencing the activity of JmjC-domain containing enzymes remains to be determined. Nevertheless, as discussed in detail below, cancerassociated mutations in isocitrate dehydrogenase (IDH1 and IDH2) enzymes converting α-



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KG into 2-hydroxyglutarate (2-HG) (Ward et al., 2010), as observed in gliomas, can alter KDM4C/JHDM3C-dependent demethylation of H3K9me3 in genes associated with differentiation of neural progenitor cells (Lu et al., 2012).

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As described above, methyl groups added to the fifth carbon of cytosine (5mC) by DNA methyltransferases, comprises DNA methylation, which is enriched at CpG containing genomic loci. Even though a DNA demethylase has not been discovered, DNA demethylation could be achieved through stepwise oxidation reactions driven by the teneleven translocation enzymes TET1, TET2 and TET3, which use α-KG and Fe2+ as cosubstrate and cofactor, respectively (reviewed by Pastor et al., 2013). TET enzymes catalyze the successive oxidations of 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by hydrolyzing α-KG in a Fe2+-dependent reaction (Tahiliani et al., 2009; reviewed in Kohli and Zhang 2013) (Figure 1C). Importantly, the activity of both TET enzymes and jumonji domain-containing histone demethylases (JmjKDMs) can be inhibited by intermediates of the TCA cycle such as fumarate and succinate, which are downstream of α-KG (Xiao et al., 2012). This supports an interconnection between metabolic rate and demethylase activity, which is reflected by changes in chromatin dynamics, thereby representing another example of a nutrient sensing mechanism influencing chromatin outputs.


Thus, DNA methylation can be enzymatically reversed upon TET-mediated oxidations of 5mC (Weber et al., 2016). However, accumulating evidence shows these TET-dependent oxidized products can also function as epigenetic determinants involved in gene regulation (Koh et al., 2011; Ficz et al., 2011; Hon et al., 2014; Etchegaray et al., 2015). In this context, several chromatin regulators along with transcription factors can specifically bind in vitro with high affinity to DNA fragments containing 5hmC, 5fC or 5caC (Spruijt et al., 2013). Moreover, a crystal structure was recently solved showing the elongating RNA polymerase II forming a complex with a 5caC-containing DNA fragment (Wang et al., 2015), further supporting the idea of TET-mediated oxidations to be more than just intermediates of DNA demethylation but also epigenetic marks with specific roles in transcription regulation. Ascorbate (vitamin C) functions as a cofactor that enhances TET activity by interacting with the catalytic domain to promote recycling of Fe2+ and/or to assist functional protein folding to stimulate catalytic activity (Minor et al., 2013; Yin et al., 2013; Blaschke et al., 2013; Dickson et al., 2013; Chen et al., 2013). Vitamin C is an essential water-soluble micronutrient that exists as ascorbate anion under physiological pH conditions. De-novo synthesis of ascorbate occurs in the liver via glucose biosynthetic pathway in rodents. However, in humans, ascorbate cannot be synthesized due to a mutated and nonfunctional Lgulonolactone oxidase, which is the enzyme that catalyzes the last step of ascorbate biosynthesis (Linster et al., 2007). Therefore, in humans ascorbate needs to be supplemented through dietary sources. Thus, vitamin C is another mediator in the interface between the epigenome and metabolic environments by linking the activity of TET enzymes and histone demethylases to nutrient sources. Supporting this notion is the fact that vitamin C can globally modify the status of DNA methylation in mammals. For instance, human embryonic stem cells (hESCs) exhibit a widespread DNA demethylation of a large number of genes in response to ascorbate (Chung et al., 2010). Ascorbate-dependent DNA demethylation was also shown to enhance the generation of induced pluripotent stem cells Mol Cell. Author manuscript; available in PMC 2017 June 02.


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(iPSCs) during somatic cell reprogramming known to be accompanied by genome-wide DNA demethylation and the enrichment of TET-dependent production of 5hmC (Esteban et al., 2010; Stadtfeld et al., 2012; Blaschke et al., 2013; reviewed in Young et al., 2015). The molecular mechanisms underlying metabolic adaptations through epigenetic regulatory programs established by ascorbate-dependent TET activity remain to be fully determined. Other metabolites involved in chromatin modifications


Apart from the post-translational modifications described above, histone modifications include the addition of moieties such as phosphorylation (depending on ATP availability), O-linked N-acetylglucosamine (O-GlycNAcylation), sumoylation, ubiquitination, and a variety of acyl-moieties including butyrylation, proprionylation, malonylation, succinylation and crotonylation (Crotonyl-CoA). These post-translational modifications are directly depending on the availability of specific metabolites, whose biosynthesis relies on nutritional levels. For instance, O-GlcNAc, a product of the hexosamine biosynthetic pathway, which is subsidiary to the glycolytic pathway, is incorporated at histone’s serine and threonine residues by the N-acetylglucosamine transferase (OGT) enzyme. O-GlcNAc is a monosacaride sugar moiety found in both serine and threonine residues of glycosylated targeted proteins within the nucleus and cytoplasmic (reviewed in Gambetta and Muller 2015). O-GlcNAcylation is catalyzed by OGT using uridine diphosphate (UDP)-GlcNAc as donor of the GlcNAc moiety, which is removed by the O-GlcNAcase (OGA) enzyme (Kreppel et al., 1997; Gao et al., 2001). Thus, O-GlcNAcylation depends on the balanced activities of OGT and OGA enzymes (Figure 2). O-GlcNAcylation is abolished in the absence of OGT, whose expression is essential for viability of mouse embryonic stem cells (mESCs) and hence mouse development (Shafi et al., 2000). Concordantly, mice lacking OGA enzyme exhibit constitutively high levels of O-GlcNAcylation resulting in a developmental delay followed by death shortly after birth (Yang et al., 2012). OGlcNAcylation is directly dependent on the metabolic state of the cell, since intracellular UDP-GlcNAc is synthesized through the hexosamine biosynthesis pathway, which is dependent on glucose availability as well as fatty acids, amino acids and nucleotides. From an epigenetic perspective, O-GlcNAcylation is found as a post-translational modification in histones as well as several chromatin-bound proteins including transcriptional regulators involved in DNA methylation and chromatin dynamics (reviewed in Lewis and Hanover 2014). Mass spectrometry analysis show dynamic changes of O-GlcNAcylation patterns in histones H2A, H2B and H4 during mitosis and in response to heat-shock (Sakabe et al., 2010). Histone H2B is O-GlcNAcylated at serine 112, both in vitro and in cell culture, and promotes monoubiquitination of H2B at lysine 120, which is implicated in transcriptional activation (Fujiki et al., 2011; Nardini, et al., 2013) (Figure 2). Both O-GlcNAcylation and phosphorylation target serine and threonine residues and are therefore capable of mutually inhibiting one another. Indeed, mass spectrometry analysis showed an inverse correlation between O-GlcNAcylation and phosphorylation of histone H3 at serines’ 10 and 28 along with threonine 32 during mitosis (Fong et al., 2012). Moreover, inhibition of OGA, the enzyme responsible for removing O-GlcNAc, impairs the transition from G2 to M phase during cell cycle, similarly to the phenotype observed upon blocking mitosis-specific phosphorylation of histone H3 (Fong et al., 2012). Thus, a switch between O-GlcNAcylation and phosphorylation of histone H3 was hypothesized to regulate G2-M transition during cell



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cycle progression (Fong et al., 2012). In addition to phosphorylation by various cyclindependent kinases (CDK7 of the TFIIH complex, CDK8, CDK9, CDK12 and CDK13) (reviewed in Buratowsky 2009 Egloff et al., 2012), the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) is also O-GlcNAcylated (Figure 2). More specifically, serine 5 of CTD is targeted by a dual competition between phosphorylation and O-GlcNAcylation (Kelly et al., 1993; Comer et al., 2001; Ranuncolo et al., 2012; Lu et al., 2016). Because phosphorylated CTD at serine 5 is a prerequisite for Pol II transcriptional pausing, it is tempting to speculate that O-GlcNAcylation of this serine residue might regulate the formation of the paused Pol II transcriptional complex. Therefore, by targeting histones and Pol II, O-GlcNAcylation acts as a bridge between the metabolic state and transcription by functioning as a nutrient-responsive glycosylation sensor to modulate gene expression. Nevertheless, the physiological consequences of OGT-mediated O-GlcNAcylation of Pol II CTD at serine 5 during transcription remain to be determined. Notably, this dynamic interplay between phosphorylation and O-GlcNAcylation also occurs in all TET enzymes (TET1, TET2 and TET3) to potentially regulate their activity and/or genomic targets in response to environmental conditions (Bauer et al., 2015). Additionally, TET enzymes can directly interact with OGT and facilitate the O-GlcNAcylation of histone H2B at serine 112 to coordinate transcriptional regulation (Chen et al., 2013). In mouse ESCs, association of OGT regulates TET1 activity at CpG-rich sequences near gene promoters (Vella et al., 2013). Furthermore, TET-OGT interactions promote O-GlcNAcylation of Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, to stimulate tri-methylation of H3K4 at gene promoters (Deplus et al., 2013). Thus, gene expression can be regulated through TET-OGT-mediated epigenetic changes in response to nutrient availability. However, it remains to be determined whether genes targeted by OGTTET complex exert a metabolic adaptation. Interestingly, the activity of the core pluripotent factors OCT4 and SOX2 is increased upon O-GlcNAcylation, which is essential for ESC self-renewal and somatic cell reprogramming (Jang et al., 2012) (Figure 2).

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In addition to acetylation, the histone acetyltransferase p300 also catalyzes the crotonylation of histone lysine residues. Recent genome-wide studies show that histone crotonylation is located within regulatory elements of actively transcribed genes and functions as a more potent activator of transcription compared to histone acetylation. More specifically, these studies demonstrated differential levels of histone acetylation versus crotonylation by modulating intracellular levels of acetyl-CoA versus crotonyl-CoA (Sabari et al., 2015). Thus, these findings demonstrate the direct interconnections between the availability of metabolites such as acetyl-CoA and crotonyl-CoA, and how they impact p300-mediated histone-tail modifications, which ultimately influence gene expression in response to nutritional availability. However, apart from tissue culture-based approaches, the physiological relevance of how p300-dependent crotonylation of histones is modulated by the availability of crotonyl-CoA and its influence in gene expression remains to be demonstrated in vivo. Importantly, since histone acetylation is reversed upon HDAC activity, and the fact that p300 catalyzes both acetylation and crotonylation of histones, one might expect the co-removal of these modifications by HDAC enzymes. In this context, the sirtuin family of enzymes, particularly SIRT5 and SIRT6 were shown to remove a diversity of acylgroups other than acetate. Specifically, SIRT5 can act as a mitochondrial desuccinylase and



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demalonylase, while SIRT6 can remove long-fatty acyl groups from TNFα (Du et al., 2011; Jiang et al., 2013). In addition, all sirtuins can directly hydrolyze 13 different acyl groups from histone H3 in vitro, and both SIRT1 and SIRT2 function as efficient de-crotonylases (Feldman et al., 2013). Because of their nuclear localization, SIRT1, SIRT2, SIRT6 and SIRT7 are all appealing candidate enzymes for de-crotonylation of histones in vivo, leading to further speculations of how availability of metabolites such as acetyl-CoA and crotonylCoA will promote an enzymatic competition between p300 and sirtuins to regulate epigenetic dynamics and ultimately gene transcription under different nutritional conditions.

Interplay between metabolism, chromatin and disease


Growing amount of research support the notion that dysfunctional chromatin dynamics are strongly correlated with various diseases including cancer. For instance, large-scale genomewide analysis of various cancer model systems revealed a strong correlation between aberrant epigenetic modifications and tumor progression, thereby highlighting the importance of chromatin modifying enzymes in cancer. Consequently, chromatin modifiers are viewed as promising biomarkers to diagnose various diseases and also as targets for therapeutic intervention in medicine. The general idea is that cancer cells manipulate their epigenome to achieve fast growth, even under suboptimal nutrient conditions. A mounting body of research discoveries supports the idea that changes affecting metabolic enzymes can influence epigenetic dynamics and consequently the expression of genes (reviewed in Janke et al., 2015). Remarkably, changes in the concentration of various metabolites can alter the properties of tumors through epigenetic modifications. Metabolites with either oncogenic or tumor suppressive functions have been described. In the following sections, we focus on how metabolites and metabolic enzymes including α-KG, IDH, OGT, O-GlcNAcylation and L-ascorbic acid (vitamin C), influence the activity of chromatin enzymes, and their impact on disease. TET and IDH enzymes

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As mentioned above, α-KG is a critical intermediary metabolite of the TCA cycle and is produced upon oxidative decarboxylation of isocitrate, which is catalyzed by isocitrate dehydrogenase enzymes, IDH1 and IDH2. Mutations in these enzymes result in the production of the oncometabolite 2-hydroxyglutarate (2-HG) (Dang et al., 2009; Ward et al., 2010), which acts as a competitive inhibitor of α-KG, thereby altering the activity of α-KGdependent enzymes such as TETs (Figure 3). Consequently, accumulation of 2-HG leads to a diminished TET-dependent oxidation of 5mC into 5hmC, that is associated with several cancers including gliomas and hematological malignancies (Xu et al., 2011; reviewed in Huang and Rao, 2014). In fact, IDH mutant tumors exhibit DNA hypermethylation phenotypes, consistent with the loss of TET activity (Figueroa et al., 2010). Concomitantly, mutually exclusive TET2 and IDH mutations have been found in hematological cancers, suggesting that the TET-dependent epigenomic changes in IDH mutant tumors may drive these tumors (Ko et al., 2015). The description of a mutant TET1 enzyme was the first demonstration that low production of 5hmC is associated with acute myeloid leukemia (AML) (Ono et al., 2002; Lorsbach et



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al., 2003; Tahiliani et al., 2009). TET2 mutations within the catalytic domain were also shown to impair the oxidation of 5mC into 5hmC in hematological cancers (Ko et al., 2015).

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The loss of 5hmC in cancer can be viewed as the loss of an epigenetic mark required to establish transcriptional signatures for proper tissue function and/or as the loss of DNA demethylation. Indeed, studies support the loss of 5hmC as an epigenetic hallmark in various types of human cancers (Chen et al., 2013; Jin et al., 2011; Ko et al., 2010; Kudo et al., 2012; Mason et al., 2013; Muller et al., 2012; Orr et al., 2012; Perez et al., 2012; Xu et al., 2011; Yang et al., 2013). Additionally, altering TET-mediated oxidations also result in aberrant DNA methylation in the genome that consequently leads to cancer (Ko et al., 2010; Pronier et al., 2011; Chang et al., 2014). Thus, the α-KG dependency of TET activity suggests a crosstalk between metabolism and epigenetic dynamics. However, direct evidence showing that changes in α-KG levels can affect TET-dependent DNA oxidations is limited. However, in IDH mutations, limiting the availability of α-KG due to the production of 2-HG supports the idea of a metabolic interplay with TET-dependent epigenetic dynamics. Importantly, as mentioned above, fumarate and succinate, which are metabolites downstream of αKG, act as potent inhibitors of (JmjC)-domain containing histone demethylases and TET enzymes, thereby altering gene regulation (Figure 3). Indeed, ectopic expression of mutant versions of the fumarate and succinate oxidizing enzymes FH and SDH, found in various human cancers (Baysal et al., 2000; Astuti et al., 2001; Hao et al., 2009; Kaelin 2009; Bayley et al., 2010; Oermann et al., 2012) cause genome-wide inhibition of H3K4 and H3K9 demethylation and TET-dependent production of 5hmC (Xiao et al., 2012). Overall, these studies provide strong evidence that dysregulation in metabolic intermediates could directly influence epigenetically-driven cancers. Jmj-KDMs


Genetic alterations causing deregulations in the expression of Jmj-KDMs are associated with various human cancers. For instance, upregulation of KDM5A/JARID1A is associated with lung and hematopoietic cancer, but downregulated in melanoma. KDM5B/JARID1B is upregulated in breast, prostate and bladder cancer, while KDM5C/JARID1C is upregulated in prostate cancer but downregulated in renal carcinoma (reviewed in Park et al., 2016). As discussed above, the activity of Jmj-KDM enzymes is dependent on α-KG. However, it remains speculative whether fluctuations in α-KG correlate with Jmj-KDM driven cancers. Apart from cancer, Jmj-KDM enzymes may also influence metabolic diseases. For instance, loss of KDM3A/JHDM2A function causes a disruption of β-adrenergic-stimulated glycerol release in brown fat tissue as well as a decrease in fat oxidation and glycerol release in skeletal muscles resulting in obesity and hyperlipidemia (Tateishi et al., 2009). Additionally, KDM1A/LSD1 forms a co-repressor complex with the (NAD+)-dependent histone deacetylase SIRT1, which triggers the deacetylation of H4K16ac and demethylation of H3K4me, resulting in the repression of genes associated with the Notch developmental signaling pathway (Mulligan et al., 2011). Apart from influencing TET activity, vitamin C also functions as a cofactor for Jmj-KDMs (Figure 3). KDM2A/JHDM1, which specifically demethylates H3K36me, and KDM4A/ JHDM3A, which demethylates tri-methylated H3K9 and H3K36, require vitamin C for optimal catalytic activity in vitro (Klose et al., 2006; Tsukada et al., 2006). As described



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above, the degradation and recycling of ascorbate is largely controlled by redox status including NAD/NADH and FADH2/FAD, which can consequently impact both TETdependent DNA oxidation and histone demethylation. Therefore, metabolism of ascorbate has significant implications in the integrity of the epigenome and its disruption leads to disease. For instance, ascorbate enhances differentiation of dopaminergic neurons, which correlates with TET-mediated production of 5hmC and also demethylation of H3K27me3 by KDM6B/JMJD3 (He et al., 2015). Thus, failure to maintain this ascorbate-dependent epigenetic signature of higher 5hmC and lower H3K27me3 in dopaminergic neurons due to age-related ascorbate decline might result in neurodegenerative disorders such as Parkinson’s disease, which is characterized by the progressive loss of dopaminergic neurons (Reeve et al., 2014). Additionally, genetic variations in ascorbate transporters are associated with the risk of colorectal adenoma, bladder cancer, gastric cancer and non-Hodgkin lymphoma (reviewed in Camarena and Wang, 2016). However, the precise molecular functions of ascorbate, in the context of cancer development, remain to be determined.

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As mentioned above, fumarate and succinate are potent inhibitors of TET activity in vitro and can also decrease global levels of 5hmC in neuroblastoma cells, which consequently affects the expression of genes targeted by the hypoxia-inducible factor (HIF) (Laukka et al., 2016). Additionally, knocking down fumarate hydrogenase (FH) and succinate dehydrogenase (SDH) leads to the accumulation of fumarate and succinate, respectively, which can broadly inhibit demethylation of H3K4, H3K9, H3K27 and H3K79 by JmjKDMs as well as TET-dependent production of 5hmC in mouse liver cells (Xiao et al., 2012) (Figure 3). In addition, suppression or ectopic expression of tumor-derived FH and SDH enzymes lead to an upregulation of the Hoxa genes associated with increased H3K79me2, an epigenetic signature found in mouse mixed-lineage leukemia (MLL) and human acute myeloid leukemia (AML) patients, due to fumarate- and succinate-dependent inhibition of JmJ-KDMs (Xiao et al., 2012; Krivtsov et al., 2008). Additionally, both adrenal gland tumors (pheochromocytomas) and smooth muscle tumors exhibit increased TETdependent production of 5hmC and H3K9me3 associated with accumulation of fumarate and succinate due to inactivation of FH and SDH enzymes (Hoekstra et al., 2015). Therefore, these αKG-dependent enzymes are sensors of energy metabolism where αKG functions as an activator of TETs and Jmj-KDMs while fumarate and succinate repress their activities. Disrupting this metabolic-epigenetic crosstalk leads to cancer. SIRT6 is a tumor suppressor that modulates metabolism

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Another example of the interplay between metabolism, epigenetics and disease comes from the discovery that the sirtuin SIRT6 acts as a potent tumor suppressor by regulating glycolysis (Mostoslavsky et al., 2006; Zhong et al., 2010; Sebastian et al., 2012). Cancer cells adopt unique metabolic features to their advantage so they can by-pass cell cycle regulatory mechanisms for faster proliferation and growth. A universal mechanism utilized by cancer cells is the increment of glucose uptake that tilts the balance in favor of glycolysis (conversion of glucose to lactate). This metabolic switch allows cancer cells to use glycolytic intermediates as building blocks to synthesize macromolecules to promote rapid cell division. Under normal conditions, glycolysis occurs in the absence of oxygen, however in cancer cells, glycolysis is increased in the presence of oxygen, which is known as aerobic



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glycolysis or the ‘Warburg effect’ (Warburg 1956). As mentioned above, SIRT6 was originally identified as an epigenetic modulator of metabolism, by its ability to repress glycolytic genes (Mostoslavsky et al., 2006; Zhong et al., 2010). In the context of colorectal cancer, SIRT6 exerts its tumor suppressive function by inhibiting the Warburg effect. Thus, the loss of SIRT6 leads to an augmentation in the levels of acetylated H3K9 that results in the up-regulation of glycolytic gene expression promoting cellular transformation and consequently tumor growth and aggressiveness (Sebastian et al., 2012). In recent studies, SIRT6 was found mutated in different human tumors, and these mutations either inactivated or destabilized SIRT6 (Kugel et al., 2015), suggesting that such a tumor suppressive function for SIRT6 may be quite broad. In this regards, SIRT6 was recently described as a potent tumor suppressor in pancreatic cancer, acting as a repressor of the oncofetal protein Lin28b (Kugel et al., 2016). These results indicate that SIRT6 may have evolved to protect against tumorigenesis by epigenetic mechanisms that extend beyond its roles in glycolytic metabolism. Butyrate and HDAC


Butyrate is a short-chain fatty acid highly produced in the colon through microbiotamediated catabolism of dietary fiber. Oxidation of butyrate into acetyl-CoA is the major source of energy for colon cells (colonocytes). In the absence of butyrate, colonocytes undergo cell death. Fiber-rich diets prevent colon cancer by promoting butyrate-mediated inhibition of HDAC activity, which results in the down-regulation of transcriptional networks involved in the hyper-proliferation and transformation of colonocytes (reviewed in Goncalves and Martel 2013). More specifically, while normal colonocytes consume fiberderived butyrate in the TCA cycle, cancerous colonocytes, by relying on the Warburg effect to produce energy from glycolysis, accumulate butyrate which functions as an HDAC inhibitor, in turn upregulating expression of pro-apoptotic genes. Thereby, a diet rich in fiber prevents tumorigenesis through this unique metabolic/epigenetic crosstalk (Donohoe et al., 2012). With structural similarities to butyrate, the ketone body β-hydroxybutyrate (β-OHB) also inhibits HDAC activity, and exerts a protective effect against oxidative stress. β-OHB is a major source of energy to maintain ATP production during extended periods of fasting, caloric restriction (CR), ketogenic-rich diets or strenuous exercise. Both CR and ketogenicrich diets have a protective effect against neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease (reviewed in Gut and Verdin, 2013). Mechanistically, high levels of β-OHB inhibit the activity of HDAC1-3-4 leading to an increase acetylation of H3K9 and H3K14 on genes involved in the oxidative damage response including Foxo3a and Mt2 (Shimazu et al., 2013). Thus, the production of β-OHB during nutritional conditions of stress is important for both, the production of ATP to maintain metabolic homeostasis and the regulation of transcription to reduce oxidative stress.

Circadian rhythms linked to chromatin, metabolism and disease The circadian clock is a cell autonomous time-measuring system controlling daily rhythms of gene expression required to maintain cellular and tissue homeostasis. This allows for metabolic and behavioral processes to be in-tune with 24-hour of day/night cycles (reviewed in Reppert and Weaver, 2002). Disruption of the circadian clock results in arrhythmic



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physiology and behavior leading to neurodegenerative disorders including Alzheimer’s and Parkinson disease (Videnovic et al., 2014), depression and bipolar disorders (Roybal et al., 2007), learning and memory impairments (Smarr et al., 2014), metabolic syndrome (Kettner et al., 2015), obesity and diabetes (Kooijman et al., 2015), immunodeficiency (Schelermann et al., 2013), accelerated aging (Bunger et al., 2000) and cancer (Altman et al., 2015). About 10% of the genome display circadian expression at the mRNA level in several tissues (Akhtar et al., 2002; Panda et al., 2002; Storch et al., 2002; Ueda et al., 2002; Koike et al., 2012). For instance, circadian-regulated genes specific to the liver include key regulators of glucose metabolism such as Glut2, Pck1 and Gys2, as well as genes relevant to drug and cholesterol metabolism such as Cyp2a4, Cyp4a14, Cyp7a1 and Cyp2c55 (Hatanaka et al., 2010). In this way, the expression of these metabolic genes is coordinated to obtain maximum benefits at times of feeding.

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Circadian rhythms, epigenetics and metabolism

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Chromatin dynamics has been shown to occur in a circadian manner. For instance, histone acetylation levels exhibit 24-hour oscillation, underlying circadian gene expression (Etchegaray et al., 2003; Curtis et al., 2004; Naruse et al., 2004; Ripperger and Schibler 2006; Feng et al., 2011). More specifically, the histone acetyltransferases p300 and the CREB-associated factor, PCAF stimulate circadian gene expression via acetylation of histone H3 in liver and vasculature tissues (Takahata et al., 2000; Etchegaray, et al., 2003; Curtis et al., 2004). Dynamic acetylation of H3K9ac and H3K14ac within circadian genes is driven by the (NAD+)-dependent histone deacetylases SIRT1 and SIRT6 (reviewed in Masri and Sassone-Corsi, 2014) (Figure 4). Moreover, the core circadian protein CLOCK is itself a HAT, targeting H3K9, H3K14 and its transcription partner BMAL1 (Doi et al., 2006). Circadian gene expression is also regulated upon dynamic methylation of H3K4 by the histone methyltransferase mixed lineage leukemia, MLL1 and MLL3 enzymes (Katada et al., 2010; Valekunja et al., 2013). In this context, SIRT1 is implicated in the maintenance of these rhythms by targeting H3K4ac and direct deacetylation of MLL1 in a circadian manner (Aguilar-Arnal et al., 2015). Further, SIRT1 has also been shown to target circadian components (Asher et al;, 2008; Nakahata et al., 2008). Thus, circadian-dependent epigenetic dynamics represents an additional layer of complexity in gene regulation.

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Nevertheless, one of the major links between metabolism and circadian gene regulation is the metabolite NAD+. Interestingly, the expression of NAMPT, which is the rate-limiting enzyme in the biosynthesis of NAD+, is under circadian control (Figure 4). Therefore, the abundance of NAD+ is regulated via circadian rhythms. Accordingly, one would expect the activity of (NAD+)-dependent enzymes such as Sirtuins to be regulated by daily oscillations. Indeed, the cyclic expression of Nampt and consequently SIRT1 activity are abolished in circadian-deficient mice (Ramsey et al., 2009; Nakahata et al., 2009). Additionally, pharmacological inhibition of NAMPT recapitulates depletion of NAD+ oscillations and thereby impairs SIRT1 activity (Nakahata et al., 2009). In this context, mice deficient in the NAD+ hydrolase CD38 expression exhibit abnormal circadian behavior and metabolism (Sahar et al., 2011). As mentioned above, the ratios between NAD+ and NADH are a direct measurement of cellular redox equilibrium. While NADH is associated with activation of circadian gene expression, NAD+ is involved in repression of circadian transcription (Rutter



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et al., 2001). Interestingly, genome-wide analyses in liver tissues revealed a unique set of

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targeted genes by SIRT1 versus SIRT6 that consequently influence specific metabolic pathways (Masri et al., 2014). Thereby, NAD-dependent circadian regulation of both SIRT1 and SIRT6 interconnects cellular metabolism with the circadian machinery to maintain daily epigenetic oscillations to control gene expression in response to NAD+ availability. Thus, cells seem to have epigenetic-driven bioenergetic pathways coupled to day-night cycles of systemically regulated nutrient availability. However, this brings a plethora of highly complex tissue-specific molecular networks functioning in-concert with daily environmental cycles that are yet to be fully characterized.

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The circadian transcriptional regulator Rev-erbα uses heme as ligand and functions as a transcriptional repressor by forming a complex with the co-repressor, NCoR and HDAC3 (reviewed in Fang and Lazar 2015). As a feedback mechanism, Rev-erbα controls its own activity by modulating the levels of heme via repression of PGC-1α, which activates the expression of delta-aminolevulinate synthase-1 (ALAS-1), the rate-limiting enzyme in the biosynthesis of heme (Zheng et al., 2008; Estall et al., 2009) (Figure 5). PGC-1α is a transcriptional co-activator that regulates energy metabolism (Puigserver et al., 1998; Yoon et al., 2001; Wu et al., 1999; Lin et al., 2004; reviewed in Kelly and Scrapulla 2004; Lin et al., 2005). Notably, Rev-erbα functions as a key modulator of the circadian molecular feedback loop, thereby functioning as an energy sensor regulating circadian rhythms (Raghuram et al., 2007; Yin et al., 2007). In this context, both ALAS-1 and PGC1α exhibit circadian gene expression patterns in the liver and skeletal muscle (Kaasik and Lee, 2004; Liu et al., 2007). Additionally, changes in AMP/ATP ratios are sensed by the circadian clock through circadian-dependent degradation of the AMP-activated protein kinase (AMPK) (Lamia et al., 2009).

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Circadian rhythms defects in disease

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The Rev-erbα-NCoR-HDAC3 complex acts as a repressor of a cohort of tissue-specific circadian genes to interlock their daily oscillatory expression with the specific physiological function of each tissue (reviewed in Gerhart-Hines and Lazar 2015). For instance, genetic variations in human Rev-erbα are associated with bipolar disorder (Kripke et al., 2009; Severino et al., 2009; Etain et al., 2011). Rev-erbα–deficient mice exhibit mania-like behavior due to hyperactivity of dopaminergic neurons (Chung et al., 2014; Jager, et al., 2014). In liver tissue, both Rev-erbα and Rev-erbβ are recruited to similar loci genome-wide, and are enriched at metabolic genes to coordinate the synthesis and storage of fatty acids in a circadian manner (Bugge et al., 2012; Cho et al., 2012). Accordingly, in addition to altered circadian behavior, mice deficient in both Rev-erbα and Rev-erbβ have severe hepatic steatosis, which is associated with fatty liver disease (Bugge et al., 2012; Cho et al., 2012). Overall, both Rev-erbα and Rev-erbβ contribute to the maintenance of circadian rhythms and interconnect daily oscillations with the biology of various tissue-specific processes upon direct regulation of circadian output genes, whose expression is entwined with the unique physiology of each tissue. Convincing evidence supporting the coupling between circadian rhythms and metabolism came from circadian tissue-specific genetic ablations in mouse model systems that lead to



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metabolic syndrome pathologies (reviewed in Perelis et al., 2015). Indeed, disruption of circadian rhythms impairs glucose tolerance, reduces insulin secretion and alters proliferation of pancreatic β-cells causing diabetes mellitus (Marcheva et al., 2010). More recently, it was found that 27% of the transcriptome displays circadian oscillations in insulin producing β-cells of the pancreatic islet (Perelis et al., 2015). Concordantly, insulin secretion in response to nutrients is abrogated in circadian-deficient mice (Perelis et al., 2015). Depending on its nature, circadian deficiencies can lead to impaired gluconeogenesis and glucose intolerance leading to hyperglycemia and hypoinsulinemia. Furthermore, the circadian transcriptome and glucose metabolism in the liver were recently found to be reprogrammed via distal inputs from lung adenocarcinoma (Masri et al., 2016), Thus, circadian oscillations are critical for maintaining systemic glucose homeostasis.

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As described above, metabolism is coupled with circadian rhythms through SIRT1-mediated controlled circadian expression of Nampt. Deficient levels of NAD+ in liver, adipose tissue and skeletal muscle contribute to metabolic disorders in aging mice models that can be rescued upon supplementation with NMN (Ramsey et al., 2008; Yoshino et al., 2011). Supporting the role of circadian rhythms and (NAD+)-coupling in maintaining proper tissue physiology is the fact that circadian mutant mice display skeletal myopathy, heart failure and consequently die upon prolonged fasting periods, which are characteristic symptoms of mitochondrial disease (reviewed in Perelis et al., 2015). Indeed, circadian mutant mice have abnormal acetylation of mitochondrial enzymes and thereby altered electron transport chain, superoxide dismutase pathways, lipid oxidation, amino acid catabolism and TCA cycle (reviewed in Perelis et al., 2015).

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Overall, circadian rhythms are interconnected with various aspects of gene regulation and metabolic dynamics. Although the circadian clockwork itself is directly controlled by epigenetic dynamics such as histone acetylation and methylation, a direct link between circadian-dependent regulation of metabolism and epigenetic dynamics remains an open question.

CONCLUDING REMARKS

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In this review, we discussed how metabolism influences the enzymatic activity of several epigenetic modulators and its impact on gene expression in response to environmental conditions, nutrition availability and circadian cycles. A common theme in this crosstalk is the transcriptional regulation of rate-limiting enzymes to adjust the biosynthesis of metabolites, which are directly associated with dynamic epigenetic modifications. For instance, the levels of metabolites including acetyl-CoA, SAM and NAD+, can be modulated in response to environmental conditions such as nutrition, exercise and stress. Inturn, these metabolites can directly alter the activity of chromatin-modifying enzymes including HATs, HMTs and sirtuins along with transcription factors of the circadian clockwork, which consequently regulate expression of specific metabolic and growth associated genes as an adaptive response to the environment. Importantly, NAD+ levels are continuously oscillating throughout the day because the NAMPT expression is control by the circadian clock. Furthermore, the circadian clock directly regulates the epigenome via rhythmic acetylation of histones at circadian gene promoters, which constitute about 10% of



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the genome in liver tissue. Collectively, there is a hard-wired interdependency between metabolites, chromatin modifying enzymes and circadian rhythms as an interlocked molecular adaptation program to environmental conditions. These hard-wired molecular mechanisms should not be viewed as isolated phenomena, but rather as a complex network of coordinated pathways acting in response to multiple environmental stimuli. As many of the examples mentioned throughout this review, alterations in any of these components can lead to complex and often serious diseases. Although much of this highly dynamic hardwired molecular program is yet to be determined, such crosstalk can no longer be ignored. Undoubtedly, drugs targeting any of the above-discussed nodes will have profound impact in multiple diseases including cancer, diabetes, obesity and neurological disorders.

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Work in the Mostoslavsky’s laboratory is supported in part by NIH grants CA175727-01, R21CA198109-01, 1R21ES025638-01 and The National Pancreas Foundation (to R.M.). R.M. is a Kristine and Bob Higgins MGH Research Scholar, a Warshaw Institute Fellow, and the recipient of the Glenn Award for Research in Biological Mechanisms of Aging.

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Figure 1. Interplay between intermediary metabolites and epigenetics

Author Manuscript

A. Acetyl-CoA and histone acetylation. Various metabolic pathways lead to the formation of acetyl-CoA, which is then utilized as an acetyl group donor during histone acetyltransferasedependent acetylation of nucleosomal histones. B. NAD can be de novo synthesized from amino acids such as tryptophan and also through the salvage pathway. NAD+ is an obligatory cofactor for the activity of SIRT1 and SIRT6, which decetylase histone H3K9/14 and H3K9/56, respectively. Decetylation of histone H3 by these sirtuins modulate the expression of metabolic genes, thereby altering metabolic pathways such as glycolysis, gluconeogenesis, mitochondrial respiration, fatty acid oxidation and lipogenesis. C. Sadenosylmethionine (SAM) is generated through methionine byosinthesis pathway and it is the universal donor of methyl groups to both histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs). Within this metabolic pathway, S-adenosyl homocysteine (SAH) functions as a repressor of both DNMTs and histone lysine demethylases (KDMs). Alpha-ketogluterate (a-KG) is generated through the TCA cycle and serves as an obligatory cofactor for the catalytic activity of KDMs and ten-eleven translocation (TETs) enzymes. TETs oxidized DNA by successive catalysis of methylated cytosines into 5hydroxymethylcytosine (5hmC), 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC).



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Figure 2. Metabolic influence on epigenetic and transcriptional regulatory pathways

Metabolic pathways including glucose, glutamine and glucosamine lead to the biosynthesis of UDP-GlcNAc, which serves as a donor for the O-GlcNAcylation of TET enzymes by OGlcNAcyltransferase (OGT). O-GlcNAcylated TETs promote the O-GlcNAcylation of histone H2B. O-GlcNAcylated OCT4 and SOX2 is required for embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). O-GlcNAcylation of he C-terminal repeat (CTD) of RNA polymerase II (Pol II) is postulated to serve as a transcriptional regulatory mechanism.



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Author Manuscript Author Manuscript Figure 3. Interplay between metabolism, epigenetic and disease

Author Manuscript

While a-KG functions as a positive metabolite required for TEt activity, succinate and fumarate are both inhibitors of TET- and KDM-mediated catalysis. Mutated cancer derived SDH and FH enzymes lead to the accumulation of succinate and fumarate, thereby inactivating TET-mediated production of 5hmC and KDM-dependent demethylation of methylated H3K4 and H3K9. Vitamin C, however, activates TET-dependent generation of 5hmC.



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Author Manuscript Author Manuscript Author Manuscript Figure 4. At the crossroad between NAD and the circadian clock

Author Manuscript

NAMPT, the rate-limiting enzyme for the biosynthesis of NAD is under circadian regulation. Therefore, levels of NAD, the essential cofactor for the activity of sirtuins, exhibit 24-hr oscillatory patterns. SIRT1 has a direct participation in the circadian regulation of NAMPT, by modulating CLOCK and BMAL1 heterodimeric-dependent trans-activation of Nampt gene. Along with NAD, the activity of SIRT6 also depends of free fatty acids. Both SIRT1 and SIRT6 regulate different sets of circadian genes and metabolic pathways referred to as circadian partition where SIRT1 regulates peptides and cofactors, while SIRT6 controls lipids and carbohydrate metabolism.



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Figure 5. Interplay between circadian clock and metabolism

The circadian clock is composed of interlocking feedback loops consisting of transcription activators (CLOCK, BMAL1, RORa/b) and repressors (CRYs, PERs and Rev-erba/b). Circadian activity of coactivator and corepressor complexes underlies epigenetic dynamics of histone acetylation and methylation, resulting in circadian gene expression. The metabolite heme participates in a circadian feedback loop by promoting transcriptional inhibition of genes associated with adipogenesis, lipid and glucose metabolism via the corepressor complex formed by Rev-erba/b, HDAC3 and NCoR. This corepressor complex regulates the expression of PGC-1a, which activates the expression of ALAS-1, the ratelimiting enzyme required for the biosynthesis of heme. Thus, levels of heme itself are under circadian regulation.


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