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Sirtuins comprise a family of NAD+-dependent protein deacetylases and ADP-ribosyltransferases. Mammalian SIRT1 - a homolog of Sir2, the prototypical member of the sirtuin family - is an important regulator of metabolism, cell differentiation and senescence, stress response, and cancer. As a NAD+-dependent enzyme, SIRT1 regulates gene expression programs in response to cellular metabolic status, thereby coordinating metabolic adaptation of the whole organism. Several important mechanisms have emerged for SIRT1-dependent regulation of transcription. First, SIRT1 can modulate chromatin function through direct deacetylation of histones, as well as by promoting alterations in the methylation of histones and DNA, leading to the repression of transcription. The latter is accomplished through the recruitment of other nuclear enzymes to chromatin for histone methylation and DNA CpG methylation, suggesting a broader role of SIRT1 in epigenetic regulation. Second, SIRT1 can interact and deacetylate a broad range of transcription factors and coregulators, thereby regulating target gene expression both positively and negatively. Cellular energy state, specifically NAD+ metabolism, plays a major role in the regulation of SIRT1 activity. Recent studies on the NAD+ biosynthetic enzymes in the salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1), have revealed important functions for these enzymes in SIRT1-dependent transcription regulation. The collective molecular actions of SIRT1 control specific patterns of gene expression that modulate a wide variety of physiological outcomes.
The regulation of gene expression in response to internal and external signals allows cells to control a wide variety of cellular processes in a coordinated manner to direct physiological, as well as pathophysiological, outcomes. Signal-regulated transcription by RNA polymerase II (Pol II), which controls the expression of protein-coding genes in the chromatin environment of the genome, is a complex process that requires a host of protein factors and multipolypeptide complexes [1-3]. These include sequence-specific DNA binding transcription factors, as well as a variety of enzymes that (1) covalently modify or de-modify histones and other transcription-related proteins, (2) mobilize or structurally alter nucleosomes, or (3) enhance the loading and activity of the Pol II machinery at gene promoters. The concerted actions of these enzymes allow an exquisite level of control of gene expression.
Members of the sirtuin family of NAD+-dependent protein deacetylases, which include the nuclear proteins Sir2 (yeast) and SIRT1 (mammals) (Fig. 1), represent an evolutionarily conserved class of regulatory enzymes [4, 5]. Sirtuins play key roles in a variety of cellular processes from yeast to mammals, including signal-regulated transcription by Pol II . Sir2, the prototypical sirtuin family member, regulates gene silencing at the mating type loci (HML and HMR), the ribosomal DNA (rDNA) locus, and telomeres [4, 7]. These and other functions of Sir2 are dependent on its ability to deacetylate nucleosomal histones . Among mammalian sirtuins, SIRT1 is the family member most closely related to Sir2. SIRT1 controls cell differentiation, metabolism, circadian rhythms, stress responses, and cellular survival [4, 8, 9], and the levels of SIRT1 fluctuate during various cellular processes . As with yeast Sir2, many of the functions of SIRT1 are dependent on its deacetylase activity, which is directed towards nucleosomal histones (e.g., histone H4 lysine 16 or “H4K16”) [11, 12], as well as a wide range of transcription factors and other nuclear regulatory proteins [4, 5]. The unique requirement for the metabolic co-enzyme NAD+ as a substrate for both Sir2 and SIRT1 catalytic activity provides a crucial link between cellular metabolic status and gene regulation. In addition, the deacetylation reaction by sirtuins generates a unique NAD+ metabolite, O-acetyl-ADP-ribose (OAADPR, Fig. 1) [4, 5]. OAADPR plays an essential role in yeast SIR (silent information regulator) complex assembly . It also binds to macrodomains and may modulate the function of macrodomain-containing proteins [14, 15]. Multiple enzymatic activities, including Nudix hydrolases, contribute to OAADPR hydrolysis [16-19], suggesting a role for these enzymes in regulating the cellular functions of Sir2 and SIRT1.
Many of the functions of Sir2, including the locus-specific gene silencing effects noted above [4, 7], are dependent on its dynamic association with chromatin in response to different environmental or physiological states. For example, as yeast cells age, the Sir proteins relocate from the silent mating type loci to the nucleolus. The concomitant loss of silencing leads to sterility and aging [20-22]. In addition, Sir proteins relocate to DNA breaks in a DNA damage checkpoint-dependent manner [23-25]. Interestingly, a recent study has revealed parallel functions of SIRT1 in mammalian cells. Specifically, Oberdoerffer et al. have shown that DNA damage causes a global redistribution of SIRT1 away from silent repeat sequences and gene promoters to sites of DNA damage . The redistribution of SIRT1 (1) is required for efficient DNA double strand break repair and genome stability, (2) protects mice from irradiation-induced cancer, and (3) causes deregulation of SIRT1 bound genes .
In this review, we will examine the available literature describing how the localization and enzymatic activity of Sir2 and SIRT1 at specific sites in the genome regulate gene expression (Fig. 2). The focus will be on SIRT1, but Sir2 will also be discussed when involved in parallel processes.
Many of the functions of Sir2 and SIRT1 are dependent on their ability to deacetylate nucleosomal histones at specific residues [4, 5]. For example, Sir2 is essential for establishing and maintaining silent chromatin in yeast, which requires deacetylation of H4K16 [4, 5]. In this regard, age-associated decreases in Sir2 protein are accompanied by an increase in H4K16 acetylation and a loss of histones at specific subtelomeric regions, which results in impaired transcriptional silencing .
SIRT1 has been shown to have parallel functions in mammalian cells, promoting chromatin silencing and transcription repression. SIRT1 is recruited to chromatin through interactions with a variety of chromatin-associated proteins, such as transcription factors and coregulators (see the text and Table 1, below). Upon recruitment to chromatin, SIRT1 can deacetylate H4K16, H3 lysine 9 (“H3K9”) and H3 lysine 14 (“H3K14”) [11, 12]. In addition, SIRT1 interacts directly with histone H1 and deacetylates H1 lysine 26 (“H1K26”) . Beyond direct deacetylation of histones, SIRT1 also interacts with histone-modifying enzymes and regulates their activity. For example, SIRT1 binds and deacetylates the histone acetyltransferase p300, which inhibits p300 enzymatic activity . This has the potential to promote hypoacetylation of nucleosomal histones and affect gene expression outcomes.
Histone acetylation and histone methylation are often coordinately regulated . In this regard, the binding of SIRT1 to chromatin and the concomitant deacetylation of nucleosomal histones can promote alterations in histone methylation. For example, in an integrated reporter gene assay, recruitment and activity of SIRT1 at the promoter induced H4 lysine 20 monomethylation (H4K20me) and H3 lysine 9 trimethylation (H3K9me3), and decreased H3 lysine 79 dimethylation (H3K79me2) . Although in most cases an understanding of the detailed mechanisms is still lacking for SIRT1-induced histone methylation, recent reports suggest that SIRT1 can recruit histone methyltransferases (e.g., suppressor of variegation 3–9 homolog 1 or SUV39H1) to target sites, thereby regulating both histone acetylation and methylation at these sites. SIRT1 interacts with and deacetylates the catalytic domain of SUV39H1, the principle enzyme responsible for catalyzing H3K9me3 . These interactions recruit SUV39H1 to chromatin and increase the methyltransferase activity of SUV39H1, leading to increased levels of H3K9me3 at SIRT1 target sites. Consistent with this observation, loss of SIRT1 greatly inhibits SUV39H1-dependent H3K9me3 and impairs the localization of heterochromatin protein 1 (HP-1), an H3K9me3-binding protein .
Both SIRT1 and SUV39H1 are also found in a complex with nucleomethylin, a nuclear protein that binds to H3K9me2 at the rDNA locus . This complex, eNoSC (energy-dependent nucleolar silencing complex), senses cellular energy status and regulates silent chromatin formation at the rDNA locus through H3 deacetylation and H3K9 methylation. These observations suggest that the NAD+-dependent deacetylase activity of SIRT1 in the eNoSC complex provides a critical link between energy status and rRNA transcription . Interestingly, DBC1 (deleted in breast cancer 1) disrupts the SIRT1-SUV39H1 complex. DBC1 directly binds to both SIRT1 and SUV39H1 and inhibits their enzymatic activity [32-34]. This activity of DBC1 may play an important role in the regulation of heterochromatin formation and genomic stability. In addition to its interaction with SUV39H1, SIRT1 has also been found in the PRC4 (Polycomb repressive complex 4) complex with Ezh2 (Enhancer of zeste homolog 2), a histone methyltransferase, and may therefore indirectly regulate H3K27 and H1K26 methylation .
Recent advances in mass spectrometry technology, including linear trap quadrupole (LTQ) orbitrap and Fourier transform mass spectrometry (FTMS), have made it possible to directly examine combinatorial post-translational modifications (PTMs) on histone tails [36-39]. One interesting use of these technologies would be to further decipher the role of SIRT1 in coordinating histone modifications, such as acetylation and methylation, and to understand how SIRT1-dependent modification of histones regulates target gene expression.
SIRT1 has been shown to associate with the promoters of aberrantly silenced tumor suppressor genes (i.e., genes whose promoter CpG islands are hypermethylated) in cancer cells . Inhibition of SIRT1 leads to re-expression of these genes . Interestingly, in cell lines where these promoters are not hypermethylated and the genes are expressed, no SIRT1 binding is detected . One of the tumor suppressor genes repressed by the actions of SIRT1 is CDH1, the gene encoding E-cadherin. Using an experimental model in which a DNA double strand break was induced in a reporter construct containing the CDH1 promoter, O'Hagan et al. examined the relationship between DNA damage and SIRT1-dependent gene silencing . In their assays, SIRT1 bound to the region surrounding the DNA break in the reporter construct, consistent with the results of genomic localization (i.e., the ChIP-chip) studies showing global redistribution of SIRT1 upon DNA damage . Importantly, SIRT1 binding is required for recruitment of DNA methyltransferase 3B (DNMT3B) and the subsequent heritable methylation of CpG islands at the reporter gene promoter . These observations reveal a critical role for SIRT1 in DNA methylation and epigenetic silencing, although details of the mechanism remain to be elucidated.
SIRT1 interacts with a wide variety of DNA-bound or chromatin-associated factors (i.e., sequence-specific DNA binding transcription factors and coregulators, respectively) and is recruited to their target binding sites in the genome . These include p53 [42, 43], the FOXO transcription factors [44, 45], the circadian clock protein BMAL1 , the nuclear receptors LXR, estrogen receptor α, and androgen receptor [47-50], the hypoxia-inducible factor 2α , the coregulator PGC1α [52-57], and the DNA repair and anti-apoptotic factor Ku70  (Table 1). Deacetylation of these non-histone targets by SIRT1 is largely responsible for the complexity of transcriptional regulation by SIRT1 since it contributes to both negative and positive regulation of target gene expression . The activity of SIRT1 with these target proteins underlies its function in cell differentiation, metabolism, circadian regulation, stress response, and survival (Table 1). Several excellent reviews of this subject have recently been published [5, 6, 59-62] and the reader is directed to these reviews for a detailed discussion of this subject. In the following section, we describe one example of SIRT1 regulation of a non-histone target as an example.
Here we use peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 alpha (PGC-1α) as an example to discuss in more detail how SIRT1 regulates target gene expression through interactions with specific transcription factors and coregulators. PGC-1α is a transcriptional coactivator that controls mitochondrial biogenesis and respiration, and whole-body energy expenditure [63, 64]. To achieve these functions, PGC-1α serves as a coactivator for a variety of transcription factors, including the PPARs, thyroid hormone receptors, glucocorticoid receptors, estrogen receptors, estrogen-related receptors (ERRs), myocyte enhancer factor-2 (MEF-2) and the FOXO transcription factors [65-71].
The activity of PGC-1α is regulated at both transcriptional and posttranslational levels. Importantly, acetylation by GCN5 and SRC3 negatively regulates PGC-1α activity [72, 73]. Lysine to arginine mutations in PGC-1α, which block acetylation of lysine residues and mimic the deacetylated state, markedly increase the activity of PGC-1α [52, 56]. In vivo, SIRT1 directly interacts with and deacetylates PGC-1α at multiple lysine residues , thereby enhancing the activity of PGC-1α. Recent studies indicate that SIRT1-dependent activation of PGC-1α plays a major role in regulating metabolic adaptations in different tissues [52, 54-56]. For example, during fasting, gluconeogenesis in the liver is upregulated to maintain normal blood glucose levels. Both SIRT1 expression and activity are induced in the fasted liver, leading to increased activity of PGC-1α .
Once PGC-1α is induced, it regulates gluconeogenic, glycolytic, and fatty acid oxidation genes through co-activation of transcription factors, such as FOXO1 [65, 74, 75]. Additionally, SIRT1 can also directly interact with and potentiate FOXO1 activity . Therefore, increased hepatic SIRT1 activity enhances gluconeogenesis and inhibits glycolysis through activation of both PGC-1α and FOXO1. Similar interactions between SIRT1 and PGC-1α have been reported in skeletal muscle cells under fasting and low glucose conditions, where activated PGC-1α induces expression of genes involved in mitochondrial fatty acid oxidation 
Interestingly, in vivo treatment with the SIRT1 activator resveratrol in mice leads to deacetylation and activation of PGC-1α and enhanced mitochondrial function in several tissues [53, 54]. Increased insulin sensitivity, exercise performance and thermogenic activity are observed in these mice, suggesting that SIRT1 is a potential target for improvement of general health and treatment of metabolic diseases [53, 54]. In fact, SIRT1 activators are in clinical trials for Type 2 Diabetes (http://www.sirtrispharma.com/pipeline.html). Other promising therapeutic areas for SIRT1 include mitochondrial, cardiovascular, neurodegenerative, and inflammatory diseases, as well as various forms of cancer .
The cellular functions of Sir2 and SIRT1 are dependent upon its deacetylase activity, which requires the metabolic coenzyme NAD+. In addition, Sir2 and SIRT1 activity are inhibited by nicotinamide (NAM), which is (1) a by-product of NAD+-dependent protein modifications by sirtuins and poly(ADP-ribose) polymerases (PARPs) and (2) a precursor for NAD+ biosynthesis through a salvage pathway [78-80]. The regulation of Sir2 and SIRT1 activity by NAD+ and its metabolites provides a crucial link between cell metabolism and transcriptional regulation. This was first characterized with respect to calorie restriction (CR)-induced lifespan extension in yeast [81, 82], but has since been expanded to mammalian systems [83-86].
Studies in yeast have shown that the effect of CR on lifespan requires an intact NAD+ salvage pathway, and overexpression of the enzymes in this pathway extends yeast lifespan [81, 82, 87]. Because no overall change in NAD+ levels is observed in the long-lived yeast strains , other metabolic parameters regulated by the NAD+ salvage pathway, including NAD+:NADH ratio [88, 89] and NAM levels [80, 82, 90], have been proposed as the actual signal that regulate Sir2 activity during CR. Studies in mammalian cells have focused on the role of two enzymes in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1), in controlling SIRT1 function in the nucleus. The salvage pathway comprised by these enzymes uses NAM as the precursor for NAD+ synthesis in the nucleus (Fig. 3) and, therefore, can regulate SIRT1 activity through both NAM removal and NAD+ production.
NAMPT, which catalyzes the conversion of NAM to nicotinamide mononucleotide (NMN) (Fig. 3) , localizes to both the cytoplasm and nucleus [92, 93]. In addition, an extracellular form of the protein has been reported to act as an NAD+ biosynthetic enzyme (eNAMPT) [94, 95], a cytokine named PBEF (pre-B cell colony-enhancing factor) , and a adipocytokine with insulin-mimetic function (visfatin) , although the later study has been retracted Biochemical analyses suggest that NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway , while in vivo studies demonstrate that NAMPT expression levels closely correlate with cellular NAD+ production [83, 85, 86, 99-102]. The enzymatic activity of NAMPT also directly reduces cellular NAM levels . Both of these actions of NAMPT have the potential to stimulate SIRT1 activity.
The role of NAMPT in the regulation of SIRT1 activity has been explored in a variety of cell types, including skeletal myoblasts, vascular smooth muscle cells (SMCs), and chondrocytes. Restriction of nutrients in skeletal myoblasts stimulates NAMPT expression, leading to the modulation of cellular NAD+/NADH ratio, as well as NAM levels . These changes activate SIRT1, which promotes a SIRT1-dependent impairment of myoblast differentiation . Similarly, in human vascular smooth muscle cells (SMCs), NAMPT is upregulated during cell maturation  and declines during cell aging . These changes in NAMPT expression are accompanied by similar changes in cellular NAD+ levels, which regulate SMC function in a SIRT1-dependent pathway [84, 101]. Intriguingly, ectopic expression of NAMPT delays SMC senescence through activation of SIRT1 . Together, these studies illustrate how NAMPT-dependent regulation of SIRT1 plays a critical role in muscle cell differentiation, maturation, and senescence. In human chondrocytes, SIRT1 is recruited to the promoter of a cartilage-specific gene through interactions with SOX9 . At this gene, NAMPT stimulates SIRT1 activity and target gene expression through NAD+ production . These studies illustrate how the gene regulatory activity of SIRT1 is controlled by cellular NAD+ production.
Recent studies have connected the actions of NAMPT and SIRT1 in the regulation of the circadian rhythm. The circadian rhythm in mammals is controlled by internal “clocks” that temporally regulate biological functions in response to environmental and physiological cues in a process that involves negative feedback regulation of gene expression . Many of the core clock proteins are transcription factors, including CLOCK (a protein acetyltransferase [104, 105]) and BMAL1, which form a DNA-binding heterodimer. CLOCK:BMAL1 heterodimers promote the expression of the genes encoding Period (PER) and Cryptochrome 1 (CRY1) through the CLOCK-dependent acetylation of histones and transcription factors . Once synthesized, the PER and CRY1 proteins form heterodimers that inhibit CLOCK:BMAL1-dependent transcription (Fig. 4). A key aspect of this regulatory pathway is the acetyltransferase activity of CLOCK, which can acetylate histones H3 and H4 , as well as non-histone substrates including its binding partner BMAL1  and potentially other clock proteins . BMAL1 acetylation facilitates the association of the repressor CRY1 with CLOCK:BMAL1, thereby forming a crucial link in the feedback regulation of circadian rhythm  (Fig. 4).
Exciting new developments in this field have revealed the involvement of SIRT1 and NAMPT in circadian regulation. SIRT1 binds to CLOCK and is recruited to the CLOCK:BMAL1 complex at circadian gene promoters [46, 106]. At these sites, SIRT1 deacetylates H3K9 and H3K14 , as well as non-histone proteins BMAL1  and Per2 . Deletion or inhibition of SIRT1 leads to alterations in the circadian cycle, as revealed by changes in circadian gene expression profiles [46, 106]. Interestingly, SIRT1 activity is regulated in a circadian manner [46, 106], suggesting that SIRT1 may be a crucial link between cellular metabolism and the circadian clock. Two new reports have provided evidence to support this view. These studies revealed that the gene encoding NAMPT (i.e., NAMPT) is a direct target for regulation by CLOCK:BMAL1 heterodimers [85, 86]. Consequently, NAMPT expression and cellular NAD+ biosynthesis are subject to circadian regulation and exhibit circadian oscillations. The increased NAMPT-dependent NAD+ production leads to higher SIRT1 activity. The activated SIRT1, in turn, acts to inhibit the transcription of NAMPT through interactions with the CLOCK:BMAL1 complex at the NAMPT promoter. This completes a feedback loop involving NAMPT/NAD+/SIRT1 and CLOCK:BMAL1 [85, 86, 107] (Fig. 4).
The NMN produced by NAMPT is further converted into NAD+ by NMNAT (Fig. 3). Three NMNAT isoforms are found in mammals (NMNAT-1, -2, and -3) . Among them, NMNAT-1 localizes exclusively to the nucleus  and is likely the key NMNAT family member responsible for regulating SIRT1 activity. Studies using the Wallerian degeneration slow (WldS) mouse model, which contains a gene fusion encoding the N-terminal fragment of ubiquitination factor E4B (Ube4B) linked to full-length NMNAT-1 [110, 111], have revealed a critical function for NMNAT-1 in neuronal protection that may be SIRT1 dependent. Wallerian degeneration is the process by which the distal part of an injured nerve axon undergoes degeneration; this process is slowed in the WldS mouse. Overexpression of the chimeric WldS protein, which retains NMNAT-1 enzymatic activity, significantly delays the axonal degeneration process [110, 111].
Controversy exists over the mechanism of WldS action. In some experimental systems, NMNAT-1 enzymatic activity is required for the neuronal protection phenotype through a SIRT1-dependent pathway [112, 113], while in others, NMNAT-1 enzymatic activity is dispensible[114-117]. In one study using primary dorsal root ganglion neuron explants, the axon protective effect of WldS was mimicked by NMNAT-1 overexpression or addition of exogenous NAD+, and the protective effect of NAD+ required SIRT1 enzymatic activity . Interestingly, the cellular levels of NAD+ were not altered in the NMNAT-1 overexpressing cells [110, 112], suggesting that localized production of NAD+ in the nucleus, rather than total level of cellular NAD+, regulates SIRT1 function. Further studies are required to fully understand if and how NAD+-dependent interplay between NMNAT-1 and SIRT1 contributes to the WldS phenotype.
In a recent study, we uncovered a new aspect of NMNAT-1-dependent regulation of SIRT1 function during gene regulation . Using global gene expression analyses, we identified a group of genes that are (1) commonly regulated by NMNAT-1 and SIRT1 and (2) show SIRT1 binding to their promoters. These genes also show SIRT1-dependent recruitment of NMNAT-1 to their promoters. Knockdown of NMNAT-1 in this system reduced total cellular NAD+ levels and inhibited the deacetylation of H4K16 by SIRT1 at target gene promoters. These results demonstrate that (1) NMNAT-1 regulates SIRT1-dependent transcription through NAD+ production and (2) an NAD+ biosynthetic enzyme can be recruited to target gene promoters as a coregulator to modulate transcriptional outcomes (Fig. 5).
Colocalization of NMNAT-1 and SIRT1 at target gene promoters may regulate SIRT1 activity in several ways. For example, close proximity of NMNAT-1 and SIRT1 may facilitate more efficient NAD+ utilization by SIRT1, perhaps through a substrate channeling mechanism . Indeed, such a mechanism has been proposed previously for SIRT1 regulation by NAD+ biosynthetic enzymes . The interaction between NMNAT-1 and SIRT1 at target gene promoters may also promote allosteric regulation that enhances the enzymatic activity of either or both enzymes, as has been shown for NMNAT-1 and poly(ADP-ribose) polymerase 1 (PARP-1) . Finally, cellular signaling inputs may regulate interactions between NMNAT-1 and SIRT1, providing an additional level of regulatory control. Similar regulation has been reported for the interaction between NMNAT-1 and PARP-1 . Overall, colocalization of NMNAT-1 and SIRT1 at target gene promoters suggests that NMNAT-1 plays an important role in modulating SIRT1 activity during transcription regulation.
PARP-1 is another chromatin and transcription modulating enzyme that, like SIRT1, requires a source of nuclear NAD+ [122, 123]. SIRT1 and PARP-1 regulate many common pathways, including cellular differentiation, stress response and survival, and the two enzymes share the same intranuclear NAD+ pool. Consequently, they may influence each other's activity through competition for NAD+ [124, 125]. This may have a significant impact on SIRT1 function because PARP-1 is the major NAD+ consumer in the cell during stress responses [125, 126]. In response to genotoxic stress, PARP-1 activation can deplete intracellular NAD+ stores and, at the same time, release high levels of NAM. Both of these actions of PARP-1 can inhibit SIRT1 enzymatic activity.
Interestingly, recent studies have revealed direct regulation of PARP-1 activity by SIRT1, providing a mechanism for feedback control of PARP-1 activation in response to genotoxic stress (Fig. 6). For example, SIRT1 activation can inhibit poly(ADP-ribose) (PAR) synthesis by PARP-1 following H2O2 treatment . On the other hand, deletion of SIRT1 leads to a dramatic increase in PAR synthesis and apoptosis inducing factor (AIF)-mediated cell death following genotoxic insult . Furthermore, studies in cardiomyocytes have shown that PARP-1 is acetylated in response to cellular stress, which leads to the activation of PARP-1 enzymatic activity. . SIRT1 directly binds to and deacetylates PARP-1, therefore inhibiting PARP-1 activity . SIRT1 also directly inhibits the expression of the PARP-1 gene, thereby reducing cellular PARP-1 levels and activity even further. Interestingly, the physical interactions between SIRT1 and PARP-1 are regulated by NAD+, with higher NAD+ levels inhibiting the interaction .
Based on these observations, a detailed mechanism has emerged for cross-talk between SIRT1 and PARP-1 in stress response  (Fig. 6). PARP-1 is acetylated and activated following enhanced cellular stress. Poly(ADP-ribosyl)ation by PARP-1 markedly reduces cellular NAD+ levels, leading to inhibition of SIRT1 activity. When NAD+ levels are low, SIRT1 interacts with and deacetylates PARP-1 and inhibits expression of the PARP-1 gene, thereby reducing total cellular PARP-1 activity. These actions of SIRT1 block NAD+ depletion by PARP-1, thus forming a feedback loop to control PARP-1 activity. The interplay between PARP-1 and SIRT1 has been proposed to play an important role in determining cell fate between life and death in response to cellular stress.
A host of historical, as well as recent, studies have demonstrated clearly a role for yeast Sir2 and mammalian SIRT1 in NAD+-dependent gene regulation. In this regard, these deacetylases can (1) reduce the acetylation levels of specific lysines in histones and transcription factors to alter their function, (2) modulate the activity of other histone and protein-modifying enzymes (e.g., lysine methyltransferases), (3) collaborate with DNA methyltransferases to regulate the levels of DNA methylation, (4) interact physically and functionally with enzymes in the NAD+ biosynthetic pathway, and (5) interact physically and functionally with PARP-1 to regulate its activity. Although the details of SIRT1-dependent gene regulation are becoming clearer, many unanswered questions still remain. Additional studies are required to resolve the specific roles of nuclear and extranuclear NAD+ production in regulating the activity of SIRT1. In addition, the factors that lend specificity to SIRT1-dependent gene regulation at specific promoters under specific physiological conditions still need to be identified. Finally, a full cataloging of all of the proteins that are deacetylated by SIRT1, which will require a proteomic approach, is needed to fully understand the spectrum of SIRT1 functions in the nucleus.
The authors would like to thank members of the Kraus lab for helpful suggestions regarding this manuscript. The authors' research related to the topic of this review is supported by grants from the NIH/NIDDK (R01 DK069710) and the Endocrine Society to W.L.K. and a postdoctoral fellowship from the New York State Health Research Science Board to T.Z.
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