Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and 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 mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, 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 evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

Histone Acetyltransferase 1 Links Metabolism and Transcription to Cell-Cycle Progression.

HAT1 is an essential gene in mammals that acetylates newly synthesized histone H4 and is involved in chromatin assembly and genome maintenance. In this issue of Molecular Cell, Gruber et al. (2019) show that HAT1 binds to and stimulates H4 gene expression in a growth-factor- and glucose-dependent manner.

Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is considered a gluconeogenic enzyme; however, its metabolic functions and regulatory mechanisms beyond gluconeogenesis are poorly understood. Here, we describe that dynamic acetylation of PCK1 interconverts the enzyme between gluconeogenic and anaplerotic activities. Under high glucose, p300-dependent hyperacetylation of PCK1 did not lead to protein degradation but instead increased the ability of PCK1 to perform the anaplerotic reaction, converting phosphoenolpyruvate to oxaloacetate. Lys91 acetylation destabilizes the active site of PCK1 and favors the reverse reaction. At low energy input, we demonstrate that SIRT1 deacetylates PCK1 and fully restores the gluconeogenic ability of PCK1. Additionally, we found that GSK3ß-mediated phosphorylation of PCK1 decreases acetylation and increases ubiquitination. Biochemical evidence suggests that serine phosphorylation adjacent to Lys91 stimulates SIRT1-dependent deacetylation of PCK1. This work reveals an unexpected capacity of hyperacetylated PCK1 to promote anaplerotic activity, and the intersection of post-translational control of PCK1 involving acetylation, phosphorylation, and ubiquitination.

An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality.

It has been well established that histone and DNA modifications are critical to maintaining the equilibrium between pluripotency and differentiation during early embryogenesis. Mutations in key regulators of DNA methylation have shown that the balance between gene regulation and function is critical during neural development in early years of life. However, there have been no identified cases linking epigenetic regulators to aberrant human development and fetal demise. Here, we demonstrate that a homozygous inactivating mutation in the histone deacetylase SIRT6 results in severe congenital anomalies and perinatal lethality in four affected fetuses. In vitro, the amino acid change at Asp63 to a histidine results in virtually complete loss of H3K9 deacetylase and demyristoylase functions. Functionally, SIRT6 D63H mouse embryonic stem cells (mESCs) fail to repress pluripotent gene expression, direct targets of SIRT6, and exhibit an even more severe phenotype than Sirt6-deficient ESCs when differentiated into embryoid bodies (EBs). When terminally differentiated toward cardiomyocyte lineage, D63H mutant mESCs maintain expression of pluripotent genes and fail to form functional cardiomyocyte foci. Last, human induced pluripotent stem cells (iPSCs) derived from D63H homozygous fetuses fail to differentiate into EBs, functional cardiomyocytes, and neural progenitor cells due to a failure to repress pluripotent genes. Altogether, our study described a germline mutation in SIRT6 as a cause for fetal demise, defining SIRT6 as a key factor in human development and identifying the first mutation in a chromatin factor behind a human syndrome of perinatal lethality.

Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction.

Caloric restriction (CR) extends the life span and health span of a variety of species and slows the progression of age-related hearing loss (AHL), a common age-related disorder associated with oxidative stress. Here, we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced-to-oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.

Sirt6 regulates lifespan in Drosophila melanogaster.

Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. Here, we characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD+-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. Our results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.

Metabolism and the Epigenome: A Dynamic Relationship.

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 that 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 epimetabolites is dynamically altered, highlighting known links to the epigenome and proposing other plausible connections.

Intrinsic catalytic properties of histone H3 lysine-9 methyltransferases preserve monomethylation levels under low S-adenosylmethionine.

S-adenosylmethionine (SAM) is the methyl donor for site-specific methylation reactions on histone proteins, imparting key epigenetic information. During SAM-depleted conditions that can arise from dietary methionine restriction, lysine di- and tri-methylation are reduced while sites such as Histone-3 lysine-9 (H3K9) are actively maintained, allowing cells to restore higher-state methylation upon metabolic recovery. Here, we investigated if the intrinsic catalytic properties of H3K9 histone methyltransferases (HMTs) contribute to this epigenetic persistence. We employed systematic kinetic analyses and substrate binding assays using four recombinant H3K9 HMTs (i.e., EHMT1, EHMT2, SUV39H1, and SUV39H2). At both high and low (i.e., sub-saturating) SAM, all HMTs displayed the highest catalytic efficiency (kcat/KM) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in kcat values, apart from SUV39H2 which displayed a similar kcat regardless of substrate methylation state. Using differentially methylated nucleosomes as substrates, kinetic analyses of EHMT1 and EHMT2 revealed similar catalytic preferences. Orthogonal binding assays revealed only small differences in substrate affinity across methylation states, suggesting that catalytic steps dictate the monomethylation preferences of EHMT1, EHMT2, and SUV39H1. To link in vitro catalytic rates with nuclear methylation dynamics, we built a mathematical model incorporating measured kinetic parameters and a time course of mass spectrometry-based H3K9 methylation measurements following cellular SAM depletion. The model revealed that the intrinsic kinetic constants of the catalytic domains could recapitulate in vivo observations. Together, these results suggest catalytic discrimination by H3K9 HMTs maintains nuclear H3K9me1, ensuring epigenetic persistence after metabolic stress.

Ure(k)a! Sirtuins Regulate Mitochondria.

Increasing evidence suggests that multiple metabolic pathways are regulated by sirtuin-dependent protein deacetylation in the mitochondria. In this issue, Nakagawa et al. (2009) show that the sirtuin SIRT5 deacetylates and activates a mitochondrial enzyme, carbamoyl phosphate synthetase 1, which mediates the first step in the urea cycle.

Diet-Microbiota Interactions Mediate Global Epigenetic Programming in Multiple Host Tissues.

Histone-modifying enzymes regulate transcription and are sensitive to availability of endogenous small-molecule metabolites, allowing chromatin to respond to changes in environment. The gut microbiota produces a myriad of metabolites that affect host physiology and susceptibility to disease; however, the underlying molecular events remain largely unknown. Here we demonstrate that microbial colonization regulates global histone acetylation and methylation in multiple host tissues in a diet-dependent manner: consumption of a "Western-type" diet prevents many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Finally, we demonstrate that supplementation of germ-free mice with short-chain fatty acids, major products of gut bacterial fermentation, is sufficient to recapitulate chromatin modification states and transcriptional responses associated with colonization. These findings have profound implications for understanding the complex functional interactions between diet, gut microbiota, and host health.

Increased expression of SLC25A1/CIC causes an autistic-like phenotype with altered neuron morphology.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

Acss2 Deletion Reveals Functional Versatility via Tissue-Specific Roles in Transcriptional Regulation.

The coordination of cellular biological processes is regulated in part via metabolic enzymes acting to match cellular metabolism to current conditions. The acetate activating enzyme, acyl-coenzyme A synthetase short-chain family member 2 (Acss2), has long been considered to have a predominantly lipogenic function. More recent evidence suggests that this enzyme has regulatory functions in addition to its role in providing acetyl-CoA for lipid synthesis. We used Acss2 knockout mice (Acss2-/-) to further investigate the roles this enzyme plays in three physiologically distinct organ systems that make extensive use of lipid synthesis and storage, including the liver, brain, and adipose tissue. We examined the resulting transcriptomic changes resulting from Acss2 deletion and assessed these changes in relation to fatty acid constitution. We find that loss of Acss2 leads to dysregulation of numerous canonical signaling pathways, upstream transcriptional regulatory molecules, cellular processes, and biological functions, which were distinct in the liver, brain, and mesenteric adipose tissues. The detected organ-specific transcriptional regulatory patterns reflect the complementary functional roles of these organ systems within the context of systemic physiology. While alterations in transcriptional states were evident, the loss of Acss2 resulted in few changes in fatty acid constitution in all three organ systems. Overall, we demonstrate that Acss2 loss institutes organ-specific transcriptional regulatory patterns reflecting the complementary functional roles of these organ systems. Collectively, these findings provide further confirmation that Acss2 regulates key transcription factors and pathways under well-fed, non-stressed conditions and acts as a transcriptional regulatory enzyme.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Discovery and Mechanism of Small Molecule Inhibitors Selective for the Chromatin-Binding Domains of Oncogenic UHRF1.

Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition.

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.

Biological and catalytic functions of sirtuin 6 as targets for small-molecule modulators.

Sirtuin 6 (SIRT6) is a nuclear NAD+-dependent deacetylase of histone H3 that regulates genome stability and gene expression. However, nonhistone substrates and additional catalytic activities of SIRT6, including long-chain deacylation and mono-ADP-ribosylation of other proteins, have also been reported, but many of these noncanonical roles remain enigmatic. Genetic studies have revealed critical homeostatic cellular functions of SIRT6, underscoring the need to better understand which catalytic functions and molecular pathways are driving SIRT6-associated phenotypes. At the physiological level, SIRT6 activity promotes increased longevity by regulating metabolism and DNA repair. Recent work has identified natural products and synthetic small molecules capable of activating the inefficient in vitro deacetylase activity of SIRT6. Here, we discuss the cellular functions of SIRT6 with a focus on attributing its catalytic activity to its proposed biological functions. We cover the molecular architecture and catalytic mechanisms that distinguish SIRT6 from other NAD+-dependent deacylases. We propose that combining specific SIRT6 amino acid substitutions identified in enzymology studies and activity-selective compounds could help delineate SIRT6 functions in specific biological contexts and resolve the apparently conflicting roles of SIRT6 in processes such as tumor development. We further highlight the recent development of small-molecule modulators that provide additional biological insight into SIRT6 functions and offer therapeutic approaches to manage metabolic and age-associated diseases.

Mechanism of activation for the sirtuin 6 protein deacylase.

The histone deacetylase sirtuin 6 (SIRT6) regulates numerous biological functions, including transcriptional repression, DNA repair, and telomere maintenance. Recombinant SIRT6 displays catalytic efficiencies 2 orders of magnitude greater for long-chain deacylation than deacetylation against peptide substrates; however, deacetylation can be enhanced by allosteric small-molecule activators. Here, we investigated the mechanisms of activated lysine deacetylation and enhanced long-chain acyl-group removal by SIRT6. Activity-based screening identified compounds that activated histone peptide deacetylation 18-48-fold. Chemical optimization based on structure-activity relationships yielded an activator with improved potency and selectivity for SIRT6. Using this novel activator, we conducted biochemical and kinetic analyses revealing that SIRT6 is activated via acceleration of a catalytic step occurring after substrate binding but before NAD+ cleavage. We identified a SIRT6 variant, R65A, that maintains basal deacetylase activity but cannot be activated and failed to enhance long-chain deacylation. Additional biochemical studies revealed that Arg-65 is critical for activation by facilitating a conformational step that initiates chemical catalysis. This work suggests that SIRT6 activation of deacetylation involves a similar mechanism to improved catalysis as that of long-chain deacylation. The identification of novel SIRT6 activators and the molecular insights into activation and catalysis presented here provide a foundational understanding for physiological SIRT6 activation and for rational design of activating molecules.

A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls.

Advances in mass spectrometry (MS) have revolutionized the ability to measure global changes in histone post-translational modifications (PTMs). The method routinely quantifies over 60 modification states in a single sample, far exceeding the capabilities of traditional western blotting. Thus, MS-based histone analysis has become an increasingly popular tool for understanding how genetic and environmental factors influence epigenetic states. However, histone proteomics experiments exhibit unique challenges, such as batch-to-batch reproducibility, accurate peak integration, and noisy data. Here, we discuss the steps of histone PTM analysis, from sample preparation and peak integration to data analysis and validation. We outline a set of best practices for ensuring data quality, accurate normalization, and robust statistics. Using these practices, we quantify histone modifications in 5 human cell lines, revealing that each cell line exhibits a unique epigenetic signature. We also provide a reproducible workflow for histone PTM analysis in the form of an R script, which is freely available at https://github.com/DenuLab/HistoneAnalysisWorkflow.

Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome.

Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Mechanisms and Dynamics of Protein Acetylation in Mitochondria.

Reversible protein acetylation is a major regulatory mechanism for controlling protein function. Through genetic manipulations, dietary perturbations, and new proteomic technologies, the diverse functions of protein acetylation are coming into focus. Protein acetylation in mitochondria has taken center stage, revealing that 63% of mitochondrially localized proteins contain lysine acetylation sites. We summarize the field and discuss salient topics that cover spurious versus targeted acetylation, the role of SIRT3 deacetylation, nonenzymatic acetylation, and molecular models for regulatory acetylations that display high and low stoichiometry.