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PERSPECTIVE

Sir2 links chromatin silencing,metabolism, and aging1Leonard Guarente

Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139 USA

Aging is manifested by a progressive decline in vitalityover time leading to death. Studies in budding yeast al-low aging to be followed in individual pedigrees of cells,that is, those of mother cells, consequent to manyrounds of cell division (Mortimer and Johnston 1959).These studies have led to the general conclusion that thesilencing protein Sir2 is a limiting component of longev-ity; deletions of SIR2 shorten life span and an extra copyof this gene increases life span (Kaeberlein et al. 1999).Recent studies have spurred interest in Sir2 as a candi-date longevity factor in a broad spectrum of eukaryoticorganisms. SIR2 gene homologs have been found in avery wide range of organisms ranging from bacteria tohumans (Brachmann et al. 1995). Moreover, a biochemi-cal activity of Sir2 likely responsible for chromatin si-lencing, nicotinamide–adenine dinucleotide (NAD)-de-pendent histone deacetylase, has recently been discov-ered and shown to be broadly conserved (Imai et al.2000). In this review, I will briefly discuss silencing as itpertains to SIR2 and its relationship to aging. I will thentrace the studies that led to the discovery of the NAD-dependent histone deacetylase. I will next speculate howthe regulation of Sir2 by NAD could represent the linkbetween caloric intake and the pace of aging, which iswidely observed in many organisms (Weindruch et al.1986). Finally, I will present a speculative model of howa gradual disruption in chromatin silencing may occurand how such a change may cause aging.

Maintenance of chromatin silencing and genomestability by Sir2

Silencing of genomic DNA was first observed by repres-sion of genes near certain translocation breakpoints inDrosophila (for review, see Wakimoto 1998). Studies inDrosophila and yeast have led to the identification offactors that act in trans to mediate silencing. Amongthese are the proteins encoded by the yeast SIR genes,which are responsible for silencing at repeated DNA se-quences in yeast: mating type loci, telomeres, and therDNA. SIR2, SIR3, and SIR4 are all required for silencing

at mating type loci (Rine and Herskowitz 1987) and telo-meres (Gottschling et al. 1990), and SIR2, but not SIR3 orSIR4, is required for silencing in the rDNA (Bryk et al.1997; Smith and Boeke 1997). Silencing causes a moreclosed, inaccessible regional chromatin structure, as as-sayed by various probes of DNA accessibility (Loo andRine 1994; Bi and Broach 1997). Even though expressionof marker genes inserted into the rDNA is repressed,silencing of rDNA transcription itself may be more mod-est, as continued ribosome synthesis is essential forgrowth. The Sir proteins may also function in DNA re-pair by nonhomologous end-joining (NHEJ) (Tsukamotoet al. 1997; Boulton and Jackson 1998). In this regard, theSir2/3/4 proteins and Ku relocalize from telomeres tosites of DNA breaks to aid in their repair by NHEJ (Fig.1A) (Martin et al. 1999; Mills et al. 1999). A primary roleof the Sir complex at telomeres therefore may be to pro-vide a reservoir of factors that can be mobilized for theimmediate repair of DNA damage.

The function of Sir2 in promoting longevity in yeastmother cells appears to relate to silencing in the rDNA.The stability of the 100–200 tandem copies of rDNA onchromosome XII requires SIR2, as the frequency of re-combination at that locus increases about 10-fold in sir2mutants (Gottlieb and Esposito 1989). One of the prod-ucts of rDNA recombination is extrachromosomalrDNA circles (ERCs) (Fig. 1B), which, once formed, rep-licate and segregate preferentially to mother cell nuclei(Sinclair and Guarente 1997). ERCs thus accumulate inmother cells as they grow older and ultimately triggersenescence. At least one function of Sir2 in yeast longev-ity, therefore, is to forestall the appearance of the firstrDNA circle in mother cells by creating a silenced chro-matin structure.

Silencing requires particular lysines in the extendedamino-terminal tail of histones H3 and H4 (Thompson etal. 1994; Hecht et al. 1995; Braunstein et al. 1996). Theseand other lysines of the tail are acetylated in active chro-matin but deacetylated in silenced chromatin (Braun-stein et al. 1993, 1996). The deacetylated histones evi-dently can fold into a more compact, closed nucleosomalstructure (Luger et al. 1997). These considerations led tothe suggestion that Sir2 could be a histone deacetylase.Further evidence for this claim arose from the globaldeacetylation of yeast histones observed when Sir2 was1E-MAIL [email protected]; FAX (617) 253-8699.

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overexpressed (Braunstein et al. 1993). However, at-tempts to demonstrate a histone deacetylase activity bySir2 in vitro initially met with failure.

Sir2 is a conserved NAD-dependent histone deacetylase

Unlike SIR3 and SIR4, the SIR2 gene is broadly con-served in organisms ranging from bacteria to humans(Brachmann et al. 1995). Studies on the bacterial homo-log, cobB, led to the conclusion that this gene could sub-stitute for another bacterial gene, cobT, in the pathwayof cobalamin synthesis (Tsang and Escalante-Semerena1998). cobT was known to encode an enzyme that trans-ferred ribose–phosphate from nicotinic acid mono-nucleotide to dimethyl benzimidazole. Thus, it seemedpossible that Sir2 proteins might be equipped to catalyzea related reaction at the nicotinamide–ribose bond inNMN and perhaps nicotinamide-adenine dinucleotide(NAD), in the latter case resulting in transfer of ADP–ribose. Indeed, it was shown by Frye (1999) that Sir2proteins from bacteria, yeast, or mammals were able totransfer 32P from NAD to a protein carrier, suggestingthat they were ADP–ribosyl transferases. Subsequentwork proved that Sir2 could, in fact, transfer ADP–ri-bose, albeit in a reaction that proceeds only weakly invitro (Tanny et al. 1999). This latter study led to theproposal that the ADP–ribosyltransferase activity of Sir2was essential to the in vivo function of silencing.

In studying this ADP–ribosyl transferase reaction, wenoticed that peptides of the amino-terminal tails of his-tone H3 or H4 could accept 32P from NAD, but only ifthe peptides were acetylated. Using acetylated H3, weseparated the Sir2-modified product by chromatographyand found by mass spectrometry that the molecularweight of the product was actually smaller by 42, indi-cating that the major modification catalyzed by Sir2 wasdeacetylation and not ADP ribosylation (Imai et al.2000). When NAD was omitted, no deacetylation by Sir2occurred. NADH, NADP, or NADPH could not substi-tute for NAD in this reaction. The weak ADP–ribosyl-transferase reaction did not generate sufficient levels ofproduct to allow detection by this physical method.

Because the deacetylase activity of Sir2 occurs prefer-entially on histone residues that are essential for silenc-ing, we infer that it is this activity, rather than the ADP–ribosyltransferase, that triggers silencing in vivo. Con-sistent with this claim, a mutation of Gly-270 of Sir2 toAla reduces the ADP–ribosyltransferase by 93%, but re-duces the deacetylase activity by only 20% and still canfunction in silencing, repression of rDNA recombina-tion, and extension of life span (Imai et al. 2000). Thus,Sir2 is an NAD-dependent histone deacetylase that maylink metabolism and silencing in vivo (Fig. 2). The role ofthe ADP–ribosyltransferase in vivo is still not clear, butthis activity is evidently separable from the deacetylase,as a known inhibitor of mono-ADP–ribosyltransferasesselectively inhibits the one activity of Sir2 and not theother (Imai et al. 2000). The ADP–ribosyltransferase mayturn out to be important to the function of Sir2 in DNArepair, as nuclear mono- and poly-ADP–ribosyltransfer-ases have been associated with DNA repair in mamma-

Figure 1. Functions of Sir2 in yeast. Sir2mediates silencing at telomeres, alongwith Sir3, Sir4, and Ku, (A) and at the re-peated rDNA (B) without these other fac-tors. Telomeric proteins respond to DNAdouble-strand breaks (DSBs) by moving tosites of damage in S-phase in a pathwayrequiring MEC1, RAD9, RAD53. In a sir2mutant, homologous recombination in therDNA increases leading to more ERCs.

Figure 2. Sir2 is an NAD-dependent histone deacetylase. Thedeacetylation of lysines in the amino-terminal tails of histonesH3 and H4 in nucleosomes (NUC) is proposed to convert activeto silenced chromatin. Sir2 is stimulated to carry out this reac-tion by NAD, the available levels of which are likely coupled tothe metabolic rate of cells.

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lian cells (Kreimeyer et al. 1984; Pero et al. 1985; Meyerand Hilz 1986).

Is Sir2 a link between metabolic rate and aging?

One of the most consistent observations in aging is thelink between metabolic rate and the pace of aging (Wein-druch et al. 1986). Thus, if the metabolic rate of an or-ganism is slowed down, for example, by lowering caloricintake or by lowering ambient temperature for cold-blooded animals, life span is significantly extended(Finch 1990). Interestingly, this link breaks down incomparisons between organisms. For example, rodentsand bats have comparable metabolic rates, yet bats liveup to 10-fold longer. Thus, each species appears to havea predetermined rate of aging that is further regulated bythe rate of metabolism integrated over the life time.

Calorie restriction appears to be efficacious in a widerange of organisms, including rodents (Weindruch et al.1986), worms (Lakowski and Hekimi 1998), yeast (Mull-er et al. 1980), and probably primates (Roth 1999). Thereare no data yet pertaining to humans. In addition to pro-moting longevity, a nutritious yet calorie-restricted dietgives rise to robust health and a high level of motor ac-tivity in experimental animals. The billion dollar ques-tion is: What is the mechanism by which calorie restric-tion increases life span? One school of thought relates tothe possible link between oxidative damage by reactiveoxygen species (ROS) and aging (Harman 1981). By thisreckoning, lowering calories simply lowers the produc-tion of ROS in mitochondria and thus slows aging. Adifferent view is that calorie restriction provokes a radi-cal shift in the metabolic strategy in cells, which some-how favors longevity. Gene-array analysis in calorie-re-stricted mice shows altered expression of ∼2% of genes,many involved in some aspect of cellular metabolism(Lee et al. 1997), suggesting that a simple shift in cellularmetabolism may favor longevity.

In this regard, the fact that the histone deacetylaseactivity of Sir2 is driven by NAD and thus linked tocellular metabolism is quite provocative. Might Sir2 pro-teins offer an explanation of how calorie restriction regu-lates longevity? If calorie restriction were to increase thelevels of available NAD, then Sir2 activity would likelybe enhanced, resulting in greater silencing and poten-tially a longer life span. It is likely that Sir2 is in com-petition with other NAD-using enzymes in cells for thedinucleotide. When cells have high levels of calories, the

carbon flow through glycolysis would be high (Fig. 3A).The glycolytic enzyme glyceraldehyde-3-P-dehydroge-nase (GAPDH) uses NAD, and the resulting NADH isrecycled by the delivery of electrons to oxygen via theelectron transport chain or, if oxygen is scarce, to acetyl-CoA to generate fermentation products. Thus, a substan-tial portion of the NAD pool may be recruited by thishigh flow of carbon through gylcolysis. In contrast, whencalories are restricted, the flow of carbon through glycol-ysis is low (Fig. 3B). Under these conditions more carbonis fully oxidized to CO2 via the enzymes of the TCAcycle in mitochondria, which also use NAD with theresulting NADH recycled via the electron transportchain. However, because the carbon flow is much lowerwhen calories are restricted, less NAD may be siphonedfrom the common pool, leaving more for other NAD-binding proteins, including Sir2. In conclusion, I suggestthat Sir2 proteins may link metabolic rate to the pace ofaging by sensing NAD levels and generating the man-dated level of chromatin silencing.

How might a loss of silencing cause aging?

Models relating loss of silencing to aging have been de-scribed in yeast (Kennedy et al. 1995) and in mammaliancells (Howard 1996; Villeponteau 1997; Imai and Kitano1998). In yeast, one effect of Sir2-mediated silencing is arepression of recombination in the rDNA (Fig. 4). Thisrepression of genome instability delays the formation ofERCs, which will ultimately lead to the demise of agingmother cells. However, there is no good evidence thatERCs, or for that matter any extra DNA, accumulates inaging metazoan cells. Does this mean that the yeastmodel for aging bears no relevance to aging in higherorganisms? Not necessarily. The generation and accu-mulation of ERCs may be viewed as the molecular read-out of a breach in genomic silencing that leads to agingin yeast. However, I imagine that in other organismsdifferent read-outs are possible. The most obvious ofthese is the inappropriate gene expression that wouldresult from a loss of silencing (Fig. 4).

Although we do not know the targets of Sir2 silencingin the genomes of multicellular organisms, Sir2 proteinsmay help demarcate active and inactive regions that de-termine cell type. This surmise is strengthened by thefinding that a murine Sir2 also can function as an NAD-dependent histone deacetylase (Imai et al. 2000). Anygradual loss in silencing would lead to an erosion of therequired chromatin landscape, perhaps resulting in an

Figure 3. Possible role of NAD as mediator of calo-rie restriction. In calorie excess (A), glucose is oxi-dized at a high rate by glycolytic enzymes, whichsequesters a portion of the available NAD from thecommon pool. Thus, Sir2 activity is relatively low.In calorie restriction (B) the flow of carbon throughglycolysis and the TCA cycle is low, thus increasingavailable NAD, elevating Sir2-promoted silencing,and promoting a longer life span.

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alteration of phenotype such as dedifferentiation (Onoand Cutler 1978) or even cell death. These changes mayplay a causative role in the progressive deterioration ofvitality during aging.

Is there any evidence that changes in chromatin struc-ture may lie at the heart of aging in mammals? The clon-ing of animals by the “reprogramming” of adult cell nu-clei in oocytes (Wilmut et al. 1997; Wakayama et al.1998) may be relevant in this regard. Studies in sheep andmice, although still rather preliminary, give no evidencethat the clones will show accelerated aging or a reducedlife span. Assuming that the cloning process does notstrongly select for “young” cells in the adult soma, anyaging-related changes that have occurred in adult cellsare reversed by the reprogramming in oocytes. This re-programming may well be the resetting of the chromatinstructure to a zygotic landscape, which, in turn, resetsthe aging clock. Any irreversible changes in the DNA ofadult soma, for example, deletions or other mutations,could not be reset by cloning and are therefore not likelycauses of aging. That said, it has been repeatedly ob-served that DNA mutations do occur over time in adultsoma (Melov et al. 1995; Vijg 2000). These mutationsmay not contribute to any aging phenotype, but it ispossible that they will cause other anomalies in clonedanimals, such as higher cancer rates.

How might silencing be lost over time?

If, in fact, a loss of silencing can cause aging, it begs thequestion of how this loss might occur. Here, I present aspeculative model that takes into account the secondfunction of Sir2 proteins—as mediators of DNA repair. Isuggest that Sir2 is chronically recruited from silencedchromatin to sites of DNA damage to aid their repair(Fig. 5). After the DNA has been repaired, Sir2 wouldreturn to its prior residence. However, if this mobiliza-tion and resetting of Sir2 proteins were

life span is imminent, a therapeutic that compresses theperiod of morbidity may be more than a flight of fancy.

Acknowledgments

I thank S. Imai, B. Jegalian, B. Johnson, and H. Tissenbaum forcomments on the manuscript. Work in my lab was supported bygrants from the NIH, The Ellison Medical Foundation, TheSeaver Foundation, and The Howard and Linda Stern Fund.

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Leonard Guarente Sir2 links chromatin silencing, metabolism, and aging

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