The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance.

Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.

The sirtuin SIRT6 regulates lifespan in male mice.

The significant increase in human lifespan during the past century confronts us with great medical challenges. To meet these challenges, the mechanisms that determine healthy ageing must be understood and controlled. Sirtuins are highly conserved deacetylases that have been shown to regulate lifespan in yeast, nematodes and fruitflies. However, the role of sirtuins in regulating worm and fly lifespan has recently become controversial. Moreover, the role of the seven mammalian sirtuins, SIRT1 to SIRT7 (homologues of the yeast sirtuin Sir2), in regulating lifespan is unclear. Here we show that male, but not female, transgenic mice overexpressing Sirt6 (ref. 4) have a significantly longer lifespan than wild-type mice. Gene expression analysis revealed significant differences between male Sirt6-transgenic mice and male wild-type mice: transgenic males displayed lower serum levels of insulin-like growth factor 1 (IGF1), higher levels of IGF-binding protein 1 and altered phosphorylation levels of major components of IGF1 signalling, a key pathway in the regulation of lifespan. This study shows the regulation of mammalian lifespan by a sirtuin family member and has important therapeutic implications for age-related diseases.

ModuleBlast: identifying activated sub-networks within and across species.

Identifying conserved and divergent response patterns in gene networks is becoming increasingly important. A common approach is integrating expression information with gene association networks in order to find groups of connected genes that are activated or repressed. In many cases, researchers are also interested in comparisons across species (or conditions). Finding an active sub-network is a hard problem and applying it across species requires further considerations (e.g. orthology information, expression data and networks from different sources). To address these challenges we devised ModuleBlast, which uses both expression and network topology to search for highly relevant sub-networks. We have applied ModuleBlast to expression and interaction data from mouse, macaque and human to study immune response and aging. The immune response analysis identified several relevant modules, consistent with recent findings on apoptosis and NFκB activation following infection. Temporal analysis of these data revealed cascades of modules that are dynamically activated within and across species. We have experimentally validated some of the novel hypotheses resulting from the analysis of the ModuleBlast results leading to new insights into the mechanisms used by a key mammalian aging protein.

The complex role of SIRT6 in carcinogenesis.

SIRT6, a member of the mammalian sirtuins family, functions as a mono-ADP-ribosyl transferase and NAD(+)-dependent deacylase of both acetyl groups and long-chain fatty acyl groups. SIRT6 regulates diverse cellular functions such as transcription, genome stability, telomere integrity, DNA repair, inflammation and metabolic related diseases such as diabetes, obesity and cancer. In this review, we will discuss the implication of SIRT6 in the biology of cancer and the relevance to organism homeostasis and lifespan.

Self-Assembled Cyclic d,l-α-Peptides as Generic Conformational Inhibitors of the α-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies.

Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-ß component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Sirtuin 6 protects the heart from hypoxic damage.

Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whether life extension is associated with cardioprotection against hypoxia. The proposed study is to develop approaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate the mechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG) with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyocytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewer damage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactate dehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI) binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechanism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein level of B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level of phospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, which might explain life extension.

Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide.

Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

SIRT6 exhibits nucleosome-dependent deacetylase activity.

The SIRT6 deacetylase is a key regulator of mammalian genome stability, metabolism and lifespan. Previous studies indicated that SIRT6 exhibits poor deacetylase activity in vitro. Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase. We show that SIRT6 associates with the nucleosome and deacetylates histones H3 and H4 when they are packaged as nucleosomes, but not as free histones. In contrast, SIRT1 shows the opposite characteristics. Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.

SIRT6, a protein with many faces.

Sirtuins are NAD(+) dependent deacylases enzymes. There are seven mammalian sirtuins, SIRT1-SIRT7, which are localized to different cellular compartments and are capable of diverse catalytic activities. SIRT6 is a key regulator of healthy ageing. In the past decade our understanding of SIRT6 significantly increased in many different aspects. We know its cellular localization, catalytic activities, substrates and the pathways it is involved in. This review discusses the recent discoveries regarding the SIRT6 enzyme.

Copy number variation as a tool for implementing pregnancy as an aging model.

Copy number variations (CNV) are a major contributor to genome variability and have been linked to aging and other degradable phenotypes such as pregnancy physiology. To demonstrate how pregnancy can be used as a model of aging, we used CNVs from pregnant mice. Candidate CNVs were selected by applying case-control analysis in human centenarians compared with control groups. These CNVs were aligned with the mouse genome and their copy variation was assessed using qRT-PCR in liver and blood tissue samples from pregnant mice throughout pregnancy (baseline; first, second, and third trimester; post-partum). Eight of the ten selected CNVs demonstrated a significant decline/increase trend throughout the pregnancy followed by opposite direction soon after delivery in the liver and blood of the mouse tissues. Furthermore, significant differential expression was detected among the candidate CNVs' close vicinity genes (APA2A, LSS, RBDHF1, PLAAT1, and SCL17A2), but not in the WSCD2 gene. Establishing a genetic link between longevity and pregnancy is a significant step toward implementing the pregnancy process as a model for aging. These results in pregnant mice highlight the mechanism and similarities between pregnancy and aging. Investigating the mechanisms that cause such rejuvenation after labor could change our aging treatment paradigm.

Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation.

The DNA end-joining protein Ku70 is one of several proteins that inhibit apoptosis by sequestering the proapoptotic factor Bax from the mitochondria. However, the molecular mechanism underlying Ku70-dependent inhibition of Bax is not fully understood. Here, we show that the absence of Ku70 results in the accumulation of ubiquitylated Bax. Under normal growth conditions, Bax ubiquitylation promotes its degradation. Upon induction of apoptosis in wild-type cells, a significant reduction in the levels of ubiquitylated Bax was observed, whereas in Ku70(-/-) cells, the ubiquitylated Bax was robustly accumulated. Addition of recombinant Ku70 into a protein extract of Ku70(-/-) cells resulted in a decrease in the levels of ubiquitylated Bax, even in the presence of proteasome inhibitors. Moreover, an in vitro deubiquitylation assay demonstrated that recombinant Ku70 hydrolyzed polyubiquitin chains into monoubiquitin units. Thus, Ku70 regulates apoptosis by sequestering Bax from the mitochondria and mediating Bax deubiquitylation. These results shed light on the role of proteasome inhibitors as tumor suppressors.

Dynamic regulation of N6,2'-O-dimethyladenosine (m6Am) in obesity.

The prevalent m6Am mRNA cap modification was recently identified as a valid target for removal by the human obesity gene FTO along with the previously established m6A mRNA modification. However, the deposition and dynamics of m6Am in regulating obesity are unknown. Here, we investigate the liver m6A/m methylomes in mice fed on a high fat Western-diet and in ob/ob mice. We find that FTO levels are elevated in fat mice, and that genes which lost m6Am marking under obesity are overly downregulated, including the two fatty-acid-binding proteins FABP2, and FABP5. Furthermore, the cellular perturbation of FTO correspondingly affect protein levels of its targets. Notably, generally m6Am- but not m6A-methylated genes, are found to be highly enriched in metabolic processes. Finally, we deplete all m6A background via Mettl3 knockout, and unequivocally uncover the association of m6Am methylation with increased mRNA stability, translation efficiency, and higher protein expression. Together, these results strongly implicate a dynamic role for m6Am in obesity-related translation regulation.

Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan.

In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases. Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor. Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.

The effects of environmental stressors on candidate aging associated genes.

BACKGROUND: Aging is defined as a biological and physical complex process that is characterized by the increase in susceptibility to diseases and eventually death. Aging may occur at different rates between and within species, especially or (it varies) among the long-lived ones. Here, we ask whether this diversity (e.g. aging phenotype) stems from genetic or environmental factors or as a combination between the two (epigenetics). Epigenetics play a central role in controlling changes in gene expression during aging. DNA methylation is the most abundant epigenetic modification among vertebrates and is essential to mammalian development. MATERIALS AND METHODS: In this study, we utilized the HELPtag assay to identify five candidate genes that were significantly hyper- or hypo-methylated across four different age groups in mice. The candidate genes were annotated using ensemble and their expression was further tested in vitro using the murine RAW 264.7 cell line to examine the effect of three environmental stressors (UV radiation, Hypoxia and fasting) on their expression. RNA was extracted at different time points followed by cDNA synthesis. Changes in gene expression were evaluated using qRT-PCR. RESULTS: We show that fasting and UV radiation reduced the viability of RAW264.7 cells. We also found a significant change in three candidate genes' expression levels during fasting (TOP2B, RNF13 and MRPL4). Furthermore, we found a significant change in the four candidate genes' expression levels following UVC treatment (TOP2B, RNF13, PKNOX1 and CREB5) and yet no changes were recorded in hypoxic conditions. CONCLUSION: Our results suggest that the model we used was a fitting model for the assessment of environmental stressors on candidate gene expression. In addition, we established a cellular response to the environment via changes in gene expression.

Age-related loss of gene-to-gene transcriptional coordination among single cells.

A long-standing model holds that stochastic aberrations of transcriptional regulation play a key role in the process of ageing. While transcriptional dysregulation is observed in many cell types in the form of increased cell-to-cell variability, its generality to all cell types remains doubted. Here, we propose a new approach for analysing transcriptional regulation in single-cell RNA sequencing data by focusing on the global coordination between the genes rather than the variability of individual genes or correlations between pairs of genes. Consistently, across very different organisms and cell types, we find a decrease in the gene-to-gene transcriptional coordination in ageing cells. In addition, we find that loss of gene-to-gene transcriptional coordination is associated with high mutational load of a specific, age-related signature and with radiation-induced DNA damage. These observations suggest a general, potentially universal, stochastic attribute of transcriptional dysregulation in ageing.

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation.

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.

SIRT6 Promotes Hepatic Beta-Oxidation via Activation of PPARα.

The pro-longevity enzyme SIRT6 regulates various metabolic pathways. Gene expression analyses in SIRT6 heterozygotic mice identify significant decreases in PPARα signaling, known to regulate multiple metabolic pathways. SIRT6 binds PPARα and its response element within promoter regions and activates gene transcription. Sirt6+/- results in significantly reduced PPARα-induced ß-oxidation and its metabolites and reduced alanine and lactate levels, while inducing pyruvate oxidation. Reciprocally, starved SIRT6 transgenic mice show increased pyruvate, acetylcarnitine, and glycerol levels and significantly induce ß-oxidation genes in a PPARα-dependent manner. Furthermore, SIRT6 mediates PPARα inhibition of SREBP-dependent cholesterol and triglyceride synthesis. Mechanistically, SIRT6 binds PPARα coactivator NCOA2 and decreases liver NCOA2 K780 acetylation, which stimulates its activation of PPARα in a SIRT6-dependent manner. These coordinated SIRT6 activities lead to regulation of whole-body respiratory exchange ratio and liver fat content, revealing the interactions whereby SIRT6 synchronizes various metabolic pathways, and suggest a mechanism by which SIRT6 maintains healthy liver.

Regulation of SIRT1 protein levels by nutrient availability.

The mammalian NAD+ dependent deacetylase, SIRT1, was shown to be a key protein in regulating glucose homeostasis, and was implicated in the response to calorie restriction. We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions. These results have important implications on the relationship between sirtuins, nutrient availability and aging.