Archaeal Sir2

The conserved Sir2 family of proteins has protein deacetylase activity that is dependent on NAD (the oxidized form of nicotinamide adenine dinucleotide). Although histones are one likely target for the enzymatic activity of eukaryotic Sir2 proteins, little is known about the substrates and roles of prokaryotic Sir2 homologs. An archaeal Sir2 homolog interacts specifically with the major archaeal chromatin protein, Alba. Alba exists in acetylated and nonacetylated forms. Furthermore, Sir2 can deacetylate Alba and mediate transcriptional repression in a reconstituted in vitro transcription system. These data provide a paradigm for how Sir2 family proteins influence transcription and suggest that modulation of chromatin structure by acetylation arose before the divergence of the archaeal and eukaryotic lineages (Bell, 2002).

Genetic characterization of yeast Sir2

Mating type interconversion in Saccharomyces cerevisiae occurs by transposition of copies of the a or alpha mating type cassettes from inactive loci, HML and HMR, to an active locus, MAT. The lack of expression of the a and alpha genes at the silent loci results from repression by trans-acting regulators encoded by SIR (Silent Information Regulator) genes. Evidence is presented for the existence of four SIR genes. Inactivation of any of these genes leads to expression of cassettes at both HML and HMR. Unusual complementation properties are observed for a number of sir mutations. Specifically, some recessive mutations in different genes fail to complement. The correspondence between SIR1, SIR2, SIR3, SIR4 and other genes with similar roles (MAR, CMT, STE8 and STE9) is presented (Rine, 1987).

The yeast SIR2 gene is involved in regulating nucleosome phasing and transcription in the mating type system. SIR2 also plays another important role in the cell. Specifically, in wild-type SIR2 strains recombination between the tandemly repeated ribosomal RNA genes is depressed. In sir2 mutants, both mitotic and meiotic intrachromosomal recombination increase 10- to 15-fold. In striking contrast to its effect on rDNA, the SIR2 gene does not affect intrachromosomal recombination between non-rDNA gene duplications. Furthermore, in the absence of the SIR2 gene product, rDNA acquires a partial dependency on recombination gene functions (RAD50 and RAD52) that are normally dispensable for exchange in the rDNA array. Thus, SIR2 may function in excluding the rDNA region from the general recombination system. Thus SIR2's effect is not restricted to controlling mating type expression, but rather that SIR2 functions in a more general way in the genome (Gottlieb, 1989).

Genes placed near telomeres in S. cerevisiae succumb to position-effect variegation. SIR2, SIR3, SIR4, NAT1, ARD1, and HHF2 (histone H4) were identified as modifiers of the position effect at telomeres, since transcriptional repression near telomeres is no longer observed when any of the modifier genes are mutated. These genes, in addition to SIR1, repress transcription at the silent mating loci, HML and HMR. However, there were differences between transcriptional silencing at telomeres and the HM loci, as demonstrated by suppressor analysis and the lack of involvement of SIR1 in telomeric silencing. These findings provide insights into telomeric structure and function that are likely to apply to many eukaryotes. In addition, the distinctions between telomeres and the HM loci suggest a hierarchy of chromosomal silencing in S. cerevisiae (Aparicio, 1991).

This study is a genomewide analysis of histone deacetylase function in yeast. The trichostatin A (TSA)-sensitive histone deacetylase (HDAC) Rpd3p exists in a complex with Sin3p and Sap30p in yeast that is recruited to target promoters by transcription factors including Ume6p. Sir2p is a TSA-resistant HDAC that mediates yeast silencing. The transcription profile of rpd3 is similar to the profiles of sin3, sap30, ume6, and TSA-treated wild-type yeast. A Ume6p-binding site was identified in the promoters of genes up-regulated in the sin3 strain. Two genes appear to participate in feedback loops that modulate HDAC activity: ZRT1 encodes a zinc transporter and is repressed by RPD3 (Rpd3p is zinc-dependent); BNA1 encodes a nicotinamide adenine dinucleotide (NAD)-biosynthesis enzyme and is repressed by SIR2 (Sir2p is NAD-dependent). Although HDACs are transcriptional repressors, deletion of RPD3 down-regulates certain genes. Many of these are down-regulated rapidly by TSA, indicating that Rpd3p may also activate transcription. Deletion of RPD3 represses ('silences') reporter genes inserted near telomeres. The profiles demonstrate that 40% of endogenous genes located within 20 kb of telomeres are down-regulated by RPD3 deletion. Rpd3p appears to indirectly activate telomeric genes sensitive to histone depletion by repressing transcription of histone genes. Rpd3p also appears to activate telomeric genes repressed by the silent information regulator (SIR) proteins directly, possibly by deacetylating lysine 12 of histone H4. Bioinformatic analyses indicate that the yeast HDACs RPD3, SIR2, and HDA1 play distinct roles in regulating genes involved in cell cycle progression, amino acid biosynthesis, and carbohydrate transport and utilization, respectively (Bernstein, 2000).

Silent information regulator (Sir) 2 is a limiting component of the Sir2/3/4 complex, which represses transcription at subtelomeric and HM loci. Sir2p also acts independently of Sir3p and Sir4p to influence chromatin organization in the rDNA locus. Deleted and mutated forms of Sir2p have been tested for their ability to complement and/or to disrupt silencing. The highly conserved C-terminal domain of Sir2p (aa 199-562) is insufficient to restore repression at either telomeric or rDNA reporters in a sir2Delta background and fails to nucleate silencing when targeted to an appropriate reporter gene. However, its expression in an otherwise wild-type strain disrupts telomeric repression. Similarly, a point mutation (P394L) within this conserved core inactivates the full-length protein but renders it dominant negative for all types of silencing. Deletion of aa 1-198 from Sir2(394L) eliminates its dominant negative effect. Thus two distinct functional domains in Sir2p, both essential for telomeric and rDNA repression have been defined: the conserved core domain found within aa 199-562 and a second domain that encompasses aa 94-198. Immunolocalization and two-hybrid studies show that aa 94-198 are required for the binding of Sir2p to Sir4p and for the targeting of Sir2p to the nucleolus through another ligand. The globular core domain provides an essential silencing function distinct from that of targeting or Sir complex formation that may reflect its reported mono-ADP-ribosyl transferase activity (Cockell, 2000).

The yeast SIR2 gene and many of its homologs have been identified as NAD(+)-dependent histone deacetylases. To get a broader view of the relationship between the histone deacetylase activity of Sir2p and its in vivo functions, eight highly conserved residues in the core domain of SIR2 have been mutated. These mutations have a range of effects on the ability of Sir2p to deacetylate histones in vitro and to silence genes at the telomeres and HM loci. Interestingly, there is not a direct correlation between the in vitro and in vivo effects in some of these mutations. The histone deacetylase activity of Sir2p is necessary for the proper localization of the SIR complex to the telomeres (Armstrong, 2002).

Enzymatic characterization of yeast Sir2

Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes (Imai, 2000).

Members of the SIR2 family catalyze a NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assembly-linked and transcription-related acetyltransferases. No evidence is found that these proteins ADP-ribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes (Landry, 2000).

Yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD(+) and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. The production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres generates O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, it has been demonstrated that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect is mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose has been demonstrated (Borra, 2002).

A Sir2/3/4 complex with Rap1 silences heterochromatin in yeast

The maintenance of transcriptional silencing at HM mating-type loci and telomeres in yeast requires the SIR2, SIR3, and SIR4 proteins, none of which appear to be DNA-binding proteins. SIR3 and SIR4 interact with a carboxy-terminal domain of the silencer, telomere, and UAS-binding protein RAP1. SIR3 and SIR4 were identified in a two-hybrid screen for RAP1-interacting factors and was shown that SIR3 interacts both with itself and with SIR4. The interaction between RAP1 and SIR3 can be observed in vitro in the absence of other yeast proteins. Consistent with the notion that native SIR proteins interact with the RAP1 carboxyl terminus, it has been shown that mutation of the endogenous SIR3 and SIR4 genes increases transcriptional activation by LexA/RAP1 hybrids. To test the importance of the RAP1-SIR3 interaction for silencing, mutations were identified in the RAP1 carboxyl terminus that either diminish or abolish this interaction. When introduced into the native RAP1 protein, these mutations cause corresponding defects in silencing at both HMR and telomeres. It is proposed that RAP1 acts in the initiation of transcriptional silencing by recruiting a complex of SIR proteins to the chromosome via protein-protein interactions. These data are consistent with a model in which SIR3 and SIR4 play a structural role in the maintenance of silent chromatin and indicate that their action is initiated at the silencer itself (Moretti, 1994).

The distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 were examined in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions (Lieb, 2001).

Initiation of transcriptional silencing at mating type loci and telomeres in Saccharomyces cerevisiae requires the recruitment of a Sir2/3/4 (silent information regulator) protein complex to the chromosome, which occurs at least in part through its association with the silencer- and telomere-binding protein Rap1p. Sir3p and Sir4p are structural components of silent chromatin that can self-associate, interact with one another, and bind to the amino-terminal tails of histones H3 and H4. A small region of Sir3p between amino acids 455 and 481 has been identified that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir complex formation or histone binding. SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres. However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-terminal truncations that are defective in Sir3p binding. This difference may be explained by the ability of the Rap1p carboxyl terminus to interact independently along with Sir4p. Significantly, the Rap1p-Sir4p two-hybrid interaction does not require Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p. It is proposed that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggested that Rap1p-mediated recruitment of Sir proteins operates through multiple cooperative interactions, at least some of which are redundant. The physical separation of the Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through the stabilization of Sir complex-nucleosome interactions (Moretti, 2001).

In Saccharomyces cerevisiae, heterochromatin-like regions are found near telomeres and at the silent mating-type loci, where they can repress genes in an epigenetic manner. Several proteins are involved in telomeric heterochromatin structure, including Rap1, Sir2, Sir3, Sir4, yKu70 (Hdf1), yKu80 (Hdf2), and the N termini of histones H3 and H4. By recognizing cis-acting DNA-binding sites, Rap1 is believed to recruit Sir and other silencing proteins and determine where heterochromatin forms. The integrity of heterochromatin also requires the binding of Sir proteins to histones that may form a scaffold for Sir protein interactions with chromatin. This study describes how the heterochromatin complex may form initially and how it differs from the complex that spreads along the chromosome. Close to the telomere end, Sir4 can bind Rap1 independently of Sir2, Sir3, yKu70/yKu80, and the intact H4 N terminus. In contrast, Sir4 binding requires all of the silencing factors further along telomeric heterochromatin. These data indicate that Sir4 binding to Rap1 initiates the sequential association of Sir and other proteins, allowing the subsequent spreading of the heterochromatin proteins along the chromosome (Luo, 2002).

Transcriptional silencing at the budding yeast silent mating type (HM) loci and telomeric DNA regions requires Sir2, a conserved NAD-dependent histone deacetylase, Sir3, Sir4, histones H3 and H4, and several DNA-binding proteins. Silencing at the yeast ribosomal DNA (rDNA) repeats requires a complex containing Sir2, Net1, and Cdc14. The native Sir2/Sir4 complex is composed solely of Sir2 and Sir4 and native Sir3 is not associated with other proteins. The initial binding of the Sir2/Sir4 complex to DNA sites that nucleate silencing, accompanied by partial Sir2-dependent histone deacetylation, occurs independently of Sir3 and is likely to be the first step in assembly of silent chromatin at the HM loci and telomeres. The enzymatic activity of Sir2 is not required for this initial binding, but is required for the association of silencing proteins with regions distal from nucleation sites. At the rDNA repeats, histone H3 and H4 tails are required for silencing and rDNA-associated H4 is hypoacetylated in a Sir2-dependent manner. However, the binding of Sir2 to rDNA is independent of its histone deacetylase activity. Together, these results support a stepwise model for the assembly of silent chromatin domains in Saccharomyces cerevisiae (Hoppe, 2002).

Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones. This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4. Since H4 is hypoacetylated in heterochromatin, this study investigates whether acetylation is involved in regulating the contacts between SIR3 and H4. Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance. Acetylation of H4 lysines was found to reduce binding of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide. Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin. These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes (Carmen, 2002).

Sir2 and life span in yeast and C. elegans

The SIR genes are determinants of life span in yeast mother cells. Life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed (Kaeberlein, 1999).

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. A model is proposed in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span (Anderson, 2002).

In C. elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favor entry into the dauer state during larval development and extend lifespan in adults. Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults. In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan. Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor. A survey was performed of the lifespan of C. elegans strains containing duplications of chromosomal regions. A duplication containing sir-2.1-the C. elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50%. Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway. These findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms (Tissenbaum, 2001).

Inhibitors of Sir2

In a cell-based screen for inhibitors of Sir2p, a compound, splitomicin, was identified that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe, it has been demonstrated that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells (Bedalov, 2001).

The yeast transcriptional repressor Sir2p silences gene expression from the telomeric, rDNA, and silent mating-type loci and may play a role in higher order processes such as aging. Sir2p is the founding member of a large family of NAD-dependent deacetylase enzymes, named the sirtuins. These proteins are conserved from prokaryotes to eukaryotes, but most remain uncharacterized, including all seven human sirtuins. A reverse chemical genetic approach would be useful in identifying the biological function of sirtuins in a wide variety of experimental systems, but no cell-permeable small molecule inhibitors of sirtuins have been reported previously. A high throughput, phenotypic screen in cells has led to the discovery of a class of sirtuin inhibitors. All three compounds inhibit yeast Sir2p transcriptional silencing activity in vivo, and yeast Sir2p and human SIRT2 deacetylase activity in vitro. Such specific results demonstrate the utility and robustness of this screening methodology. Structure-activity relationship analysis of the compounds identified a key hydroxy-napthaldehyde moiety that is necessary and sufficient for inhibitory activity. Preliminary studies using one of these compounds suggest that inhibition of sirtuins interferes with body axis formation in Arabidopsis (Grozinger, 2001).

Mammalian Sir2

The complete sequence of a human gene, SIR2L, with homology to the yeast SIR2 gene is presented. Comparison of the predicted sequence of the protein encoded by the SIR2L gene (SIR2Lp) with Sir2p or other proteins with homology to Sir2p reveals 20%-33% overall identity and four highly conserved regions; the significance of these regions is currently unknown. SIR2L codes for a 2.1kb transcript which is expressed in various human tissues. The expression level of the transcript is found to be relatively high in the heart, brain and skeletal muscle tissues and low in lung and placenta. The intracellular location of SIR2Lp was visualized by fusion to the Green Fluorescent Protein or with a FLAG-tag. The results indicate that unlike Sir2p in yeast, SIR2Lp in human cells is found primarily in the cytoplasm. Using a mammalian inducible expression system, it has been observed that unlike SIR2 in yeast, overexpression of SIR2L in human cancer cells has no effect on cell growth (Afshar, 1999).

The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Mammalian Sir2alpha physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits a NAD-dependent p53 deacetylation induced by Sir2alpha, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2alpha represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2alpha point mutant increases the sensitivity of cells in the stress response. Thus, these findings implicate a p53 regulatory pathway mediated by mammalian Sir2alpha. These results have significant implications regarding an important role for Sir2alpha in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy (Luo, 2001).

DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis. The protein product of the gene hSIR2(SIRT1), the human homolog of the S. cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor. Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53. In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity. It is proposed that hSir2 is involved in the regulation of p53 function via deacetylation (Vaziri, 2001).

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, these data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence (Langley, 2002).

Sir2 is a NAD+-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD+]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, tests were made to see whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD+-dependent deacetylase activity. The [NAD+]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD+]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD+]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression (Fulco, 2003).

SirT1 regulates energy metabolism and response to caloric restriction in mice

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. Mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, were studied,and it was found found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, caloric restriction did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction (Boily, 2008).

SIRT1 regulates circadian clock gene expression through PER2 deacetylation

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. This study shows that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry (Asher, 2008).

Pharmacological modulation of circadian rhythms by synthetic activators of the deacetylase SIRT1

Circadian rhythms govern a wide variety of physiological and metabolic functions in many organisms, from prokaryotes to humans. It has been reported that silent information regulator 1 (SIRT1), a NAD(+)-dependent deacetylase, contributes to circadian control. In addition, SIRT1 activity is regulated in a cyclic manner in virtue of the circadian oscillation of the coenzyme NAD(+). This study used specific SIRT1 activator compounds both in vitro and in vivo. A variety of compounds was tested to show that the activation of SIRT1 alters CLOCK:BMAL1-driven transcription in different systems. Activation of SIRT1 induces repression of circadian gene expression and decreases H3 K9/K14 acetylation at corresponding promoters in a time-specific manner. Specific activation of SIRT1 was demonstrated in vivo using liver-specific SIRT1-deficient mice, where the effect of SIRT1 activator compounds was shown to be dependent on SIRT1. These findings demonstrate that SIRT1 can fine-tune circadian rhythm and pave the way to the development of pharmacological strategies to address a broad range of therapeutic indications (Bellet, 2013).

Partitioning circadian transcription by SIRT6 leads to segregated control of cellular metabolism

Circadian rhythms are intimately linked to cellular metabolism. Specifically, the NAD(+)-dependent deacetylase SIRT1, the founding member of the sirtuin family, contributes to clock function. Whereas SIRT1 exhibits diversity in deacetylation targets and subcellular localization, SIRT6 is the only constitutively chromatin-associated sirtuin and is prominently present at transcriptionally active genomic loci. Comparison of the hepatic circadian transcriptomes reveals that SIRT6 and SIRT1 separately control transcriptional specificity and therefore define distinctly partitioned classes of circadian genes. SIRT6 interacts with CLOCK:BMAL1 and, differently from SIRT1, governs their chromatin recruitment to circadian gene promoters. Moreover, SIRT6 controls circadian chromatin recruitment of SREBP-1, resulting in the cyclic regulation of genes implicated in fatty acid and cholesterol metabolism. This mechanism parallels a phenotypic disruption in fatty acid metabolism in SIRT6 null mice as revealed by circadian metabolome analyses. Thus, genomic partitioning by two independent sirtuins contributes to differential control of circadian metabolism (Masri, 2014).

SIRT1 mediates central circadian control in the SCN by a mechanism that decays with aging

SIRT1 is a NAD(+)-dependent protein deacetylase that governs many physiological pathways, including circadian rhythm in peripheral tissues. This study shows that SIRT1 in the brain governs central circadian control by activating the transcription of the two major circadian regulators, BMAL1 and CLOCK. This activation comprises an amplifying circadian loop involving SIRT1, PGC-1alpha, and Nampt. In aged wild-type mice, SIRT1 levels in the suprachiasmatic nucleus are decreased, as are those of BMAL1 and PER2, giving rise to a longer intrinsic period, a more disrupted activity pattern, and an inability to adapt to changes in the light entrainment schedule. Young mice lacking brain SIRT1 phenocopy these aging-dependent circadian changes, whereas mice that overexpress SIRT1 in the brain are protected from the effects of aging. These findings indicate that SIRT1 activates the central pacemaker to maintain robust circadian control in young animals, and a decay in this activity may play an important role in aging (Chang, 2014).

Sir2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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