560 VOLUME 12 NUMBER 7 JULY 2005 NATURE STRUCTURAL & MOLECULAR BIOLOGY

Getting a grip on O-acetyl-ADP-riboseKevin G Hoff & Cynthia Wolberger

Members of the sirtuin protein family link gene silencing and heterochromatin formation to NAD+-dependent histone deacetylation. Two papers now implicate O-acetyl-ADP-ribose, the metabolite produced by this reaction, as a small-molecule effector that binds to heterochromatic proteins.

Expression of eukaryotic genes and mainte-nance of chromosome stability are governed by post-translational modification of proteins that package DNA into chromatin. The conversion of actively transcribed, euchromatic regions into transcriptionally silent and more compact heterochromatin is important in many pro-cesses, including X-chromosome inactivation in mammals, suppression of DNA recombination and aging1,2. The histone proteins in hetero-chromatic and euchromatic regions contain different patterns of post-translational modi-fications suggesting that distinct modifications may have particular functional consequences. Histone deacetylation is required for tran-scriptional silencing in budding yeast3–5 and is catalyzed by the Sir2 protein. Sir2 belongs to a large and highly conserved family of pro-teins, known as sirtuins, that are found in virtually all organisms6,7. In contrast to pre-viously identified histone deacetylases, which catalyze the removal of acetyl groups by simple hydrolysis, sirtuins deacetylate lysine residues in a reaction that uses the high-energy cofactor nicotinamide adenine dinucleotide (NAD+). Sirtuins consume NAD+ in the course of the deacetylation reaction, cleaving the glycosidic bond with nicotinamide and producing the metabolites 2′- and 3′-O-acetyl-ADP-ribose (AADPR) along with the deacetylated peptide (Fig. 1a). Because deacetylation is involved in other forms of transcriptional repression, the main function of sirtuins has been presumed to be the deacetylation of lysine residues.

Why, then, is such a baroque and energetically expensive mechanism used rather than simple hydrolysis, and why has the sirtuin reaction

been so conserved throughout evolution? Part of the answer is that the use of NAD+ allows the regulation of heterochromatin formation

The authors are in the Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205-2185, USA.e-mail: cwolberg@jhmi.edu

Figure 1 Sirtuin activity and heterochromatin. (a) Sirtuins use NAD+ to deacetylate lysine residues, yielding a mixture of 2′- and 3′-O-acetyl-ADP-ribose (AADPR), nicotinamide and the deacetylated substrate. (b) AADPR and SIR complex assembly. In yeast, Sir2 is recruited to chromatin as part of a complex with Sir3 and Sir4, where it deacetylates Lys16 of histone H4. The AADPR generated by the reaction binds to the Sir2–Sir3–Sir4 complex, promoting incorporation of additional Sir3 and spreading of the Sir proteins along the chromatin, thereby establishing silenced chromatin. Deacetylation of Lys16 of H4 also increases the affinity of Sir3 for H4 tails. (c) AADPR and X-inactivation. In human cells, AADPR generated by the sirtuin SirT1 binds to variant histone macroH2A1.1, which is postulated to promote its incorporation into silenced, heterochromatin regions through an as yet unknown mechanism, thereby inactivating the X chromosome.

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NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 12 NUMBER 7 JULY 2005 561

by nicotinamide, a potent non-competitive inhibitor of sirtuins8, and by the presence of adequate nuclear concentrations of free NAD+. A lingering speculation has been whether AADPR is somehow used by the cell as a signal-ing molecule. AADPR has been shown to block maturation of frog oocytes9, but until now no precise function had been assigned to AADPR and no targets were known.

In an exciting development, two papers now identify a plausible role for AADPR in hetero-chromatin formation. A recent report by Liou et al.10 published in Cell examines the potential effect of AADPR on the ability of yeast silent information regulator (Sir) proteins to estab-lish silenced chromatin. Transcriptional silenc-ing at telomeres and at the silent mating-type loci is established by recruitment of a complex of proteins containing Sir2, Sir3 and Sir4. This Sir2–Sir3–Sir4 complex then polymerizes and spreads along the chromatin fiber, establish-ing a transcriptionally silent and function-ally heterochromatic state11. Spreading of the silencing proteins depends on the presence of enzymatically active Sir2 (ref. 12). Because Sir3 and Sir4 bind preferentially to hypoacetylated histone tails, the primary role of Sir2 in silenc-ing was thought to be the deacetylation of histone tails, in particular lysine 16 of histone H4 (ref. 13). In a tour de force of biochemical and biophysical methods, Liou et al.10 present a compelling case that the binding of AADPR to the Sir2–Sir3–Sir4 complex triggers morpho-logical changes that may cause the Sir complex to polymerize along chromatin. The authors provide two lines of evidence. Using solution assays, they found that addition of acetylated peptide and NAD+ increases the stoichiometry of Sir3 in the Sir2–Sir3–Sir4 complex, which is known to accompany Sir-mediated hetero-chromatin spreading in yeast. In an intricate series of electron micrographs, the authors found significant differences in the morpho-logy of Sir2–Sir3–Sir4 complexes that assemble in vitro depending on whether AADPR is pres-ent. In the absence of other components, the Sir complex is roughly spherical, whereas com-plexes that assemble in the presence of AADPR adopt a markedly different, cylindrical form. The elongated shape is promoted by addition of acetylated peptide and NAD+, or of purified AADPR on its own, suggesting that the confor-mational transition is triggered by AADPR and does not depend on binding of the Sir complex to peptide.

A new model for the establishment of silenced chromatin in yeast has emerged from these studies (Fig. 1b). When the Sir complex is recruited to chromatin, NAD+-dependent deacetylation of the histone tails generates

AADPR and local hypoacetylated chromatin. Deacetylation of histone tails increases the affinity of the Sir complex for chromatin, while the presence of AADPR triggers recruitment of additional Sir3 and an overall conformational change of the complex to an elongated polymer that spreads along the chromatin. Although the actual site for AADPR binding is unknown, an intriguing candidate is Sir3, which contains an ATP-binding domain similar in sequence to those of AAA ATPases, but which itself lacks any known catalytic activity. Another candi-date is Sir2 itself, which in the case of the yeast homologue, Hst2, has been shown to bind AADPR14. Although many mechanistic details remain to be worked out, this work presents a compelling case for a direct role of AADPR in silencing.

Another putative role for AADPR in binding directly to chromatin proteins is outlined in a paper on page 624 of this issue. Kustatscher et al.15 identify AADPR binding to the histone variant macroH2A1.1 (mH2A1), which is localized to heterochromatic regions in higher eukaryotes and is linked to X-chromosome inactivation (Fig. 1c). Unlike typical histones that contain the core histone fold and unstruc-tured tails, mH2A1 has an additional 20-kDa domain at its C terminus known as a macro domain. Although other macro domain–containing proteins bind poly(ADP-ribose), the authors found that macroH2A1 does not. In their search for other nucleotides that might bind to mH2A1, the authors decided to try the sirtuin reaction product, AADPR. mH2A1 binds preferentially to AADPR and, with some-what lower affinity, to mono(ADP-ribose), while showing much weaker affinity for other nucleotides. The authors determined the crys-tal structure of the H2A1 macro domain bound to a molecule of MES buffer and, guided by previously determined structures of a thermo-philic macro domain bound to MES and to APP-ribose, inferred the position of AADPR when bound to macroH2A1. The position-ing of AADPR within the nucleotide-binding pocket was supported by mutagenesis studies that defined residues important for AADPR binding. How (or whether) binding of AADPR to macroH2A affects mammalian hetero-chromatin is unclear. AADPR may influence the incorporation of mH2A into nucleosomes at heterochromatic regions or alter the inter-actions of nucleosomes containing mH2A. Interestingly, a splicing variant of macroH2A1 called mH2A1.2 that contains three addi-tional residues near the AADPR binding site has greatly reduced affinity for AADPR. Thus, if AADPR binding is indeed important for heterochromatin formation or spreading in

mammals, splice-site variants of mH2A may be exploited by the cell to yield very differ-ent effects on chromatin structure. Although further investigation of AADPR binding to macroH2A.1, and its biological consequences, is needed, these studies provide an important first step in understanding the ligand specific-ity of this variant histone.

The identification of AADPR binding part-ners heralds the beginning of a search for the precise mechanism by which AADPR acts as an effector molecule in the establishment of heterochromatin. It also holds out the tantaliz-ing likelihood that additional AADPR-binding proteins will be identified. Sirtuins are uni-versally conserved in nature, with organisms such as humans and flies containing up to seven homologs localized in various cellular compartments. Although it is possible that the AADPR generated by these enzymes ulti-mately diffuses into the nucleus, there may well be other non-nuclear classes of proteins whose activity could be modulated by binding to this interesting metabolite. In addition, mecha-nisms to control the cellular concentrations of AADPR in response to appropriate conditions might well also exist. The work by Liou et al.10 and Kustatscher et al.15 reveals yet another level of complexity relating to heterochromatin for-mation and provides evidence that AADPR is a second messenger. These papers are likely to represent just the beginning of a new avenue of investigation into the targets and pathways affected by this intriguing small molecule.

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