[26 SIR2 Family of NADDependent Protein Deacetylases

By Jeffrey S. Smith, Jose Avalos, Ivana Celic, Shabazz Muhammad, Cynthia Wolberger, and Jef D. Boeke

Introduction

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor in numerous metabolic pathways, including glycolysis and the citric acid cycle. The reduction of NAD+ in these reactions produces NADH, which subsequently acts as the key electron carrier in the oxidative phosphorylation process of ATP generation. Oxidative phosphorylation oxidizes NADH to regenerate NAD+. The NAD+: NADH and NADP+:NADPH concentration ratios largely define the redox state of a cell. Changes in the redox state can dramatically affect multiple cellular processes including stress response, apoptosis, and aging. Some of these responses are due to the production of reactive oxygen species (ROS), but others are directly related to NAD+ itself.

The link between NAD+ and chromatin structure actually goes back to the 1970s, when it was demonstrated that a large amount of NAD+ synthesis occurs in the nucleus of vertebrate cells.1 Furthermore, one of the enzymes involved in NAD+ synthesis was shown to be associated with chromatin.2,3 It turns out there are at least two chromatin-related enzymatic activities that utilize NAD+ as substrates in the nucleus. The first is poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30), which hydrolyzes NAD+ and transfers the ADP-ribose moiety to itself and probably some other proteins. This activity is triggered by double-stranded DNA breaks that are bound by PARP.4 In DNA-damaged cells PARP activity can quickly deplete the cellular NAD+ pool, leading to necrotic cell death. The second chromatin-related enzymatic activity, identified more recently, is the NAD+-dependent deacety-lase activity of Sir2p.57 The silent information regulator 2 (Sir2) family of proteins is highly conserved, from bacteria to humans, and plays critical roles in

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7 J. S. Smith, C. B. Brachmann, I. Celic, M. A. Kenna, S. Muhammad, V. J. Starai, J. L. Avalos, J. C.

Escalante-Semerena, C. Grubmeyer, C. Wolberger, and J. D. Boeke, Proc. Natl. Acad. Sci. U.S.A.

transcriptional silencing and cellular life span regulation.6,8'9 In this chapter we describe SIR2 function and the relevant experimental techniques used in its study.

Sir2p as General Regulator of Transcriptional Silencing in Yeast

SIR2 is the founding member of the SIR2 family of proteins. It was originally identified from the budding yeast, Saccharomyces cerevisiae, as a gene required for transcriptional silencing of the HML and HMR silent mating type loci.10'11 Mutations in SIR2 result in derepression of a and a mating-type information genes located in HML and HMR, respectively. The result is expression of a and a mating-type information in the same haploid cell, which results in formation of the al/a2 repressor complex, repression of haploid-specific genes, and a nonmating pheno-type. There are three other SIR genes (SIRI, SIR3, and SIR4) that are required for silencing at HMR and HML. SIR2, along with SIR3 and SIR4, are required for transcriptional silencing at yeast telomeres.12 In this case, mutations in SIR2 derepress silenced reporter genes that are integrated adjacent to a telomere. More recently, silencing in the ribosomal DNA locus was shown to require SIR2, but not SIRI, SIR3, or SIR4. In this case, sir2 mutations cause a loss of recombinational suppression between rDNA repeats,13 and also derepression of polymerase II (Pol II)-transcribed reporter genes that are artificially integrated in the rDNA.14,15 Silencing in the rDNA is significant because it demonstrates that Sir2p can function in silencing independently of the other Sir proteins, and therefore must have an important activity, which turns out to be NAD+-dependent histone deacetylase activity.

Identification and Comparisons of SIR2 Gene Family

Additional SIR2 family members have been identified through a combination of degenerate polymerase chain reaction (PCR), low-stringency hybridization, and database searches. In yeast alone, four additional homologs were identified: HST1, HST2, HST3, and HST4,8 9 All Sir2-like proteins share a common core domain of approximately 250 amino acids that contains several highly conserved blocks of amino acids. The two motifs that are most diagnostic of the Sir2p family are GAGISTS(L/A)GIPDFR and YTQNID. See Fig. lA15a for a sequence alignment of the Sir2 core domain from various eukaryotic species. For clarity, an alignment

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15 J. S. Smith and J. D. Boeke, Genes Dev. 11,241 (1997).

l5a R. A. Frye, Biochem. Biophys. Res. Commun. 273,793 (2000).

SceSÎ SceHl Klact CalS2 HsapB Dmela Cele2 HsapD HsapA CalHl SceH2 Lntajo Tbruc Ecoli Styph Aacti Hpylo Phori A&eol Afuil îîtube Aful2 Celel

Tmari Scoel scesb Bsubt Saure HsapF consensus

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ipdlis EDA1 1 TFDDVXSlgKK 1 S|AOAVELFKT

1 -DVAELIKARAC 1 -GVARYMQSE! 1 • • - PVA! 1 -KIAAHMKSNP] 1 -QIARYIR! 1 -GLARFIBRNNI 1 FRDKWPEAMEK

rkilvltGAGiS sGipdfRs

1 -|aevarv1asi 1 -1 mdekllklfflaei

1 -SrkaaeiMak:

X S§KKF1S1 3' 1 SKOTUS!. S' 1 ¡«KELQRESI': 1 nfspLVSi 1 daofisya 1 1

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1 eSetlkhi dss! 1 KiWELAMfKOSS.

rkilvltGAGiS sGipdfRs

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Gly Ici fâfïipfyfhv

SceS2 SceHl Klact CalS2 HsapB Dmela Cele2 HsapD HsapA CalH2 SceH2 Lmajo Tbruc Ecoli Styph Aacti Hpylo Phori Aaeol Afull Mtube Afuia Celel Cele3 HsapC

SceH4 Tmari Scoel SceH3 Bsubt Saure HsapF consensus

76-----PENMYSPXlSl

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