Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1

Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2Δ mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2Δ shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2Δ strains. By strong SIRT1 overexpression in sir2Δ cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2Δ mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.沉默信息调节因子家族（Sirtuins）是一类与酵母Sir2p蛋白（yeast protein Sir2p）同源的III类组蛋白去乙酰化酶（class III histone deacetylases）。已有研究证实，哺乳动物Sirt1可通过其去乙酰化酶活性，靶向组蛋白与非组蛋白底物，参与能量代谢、脑功能调控、炎症反应及衰老进程。为验证Sirt1能否在酵母细胞中替代Sir2p发挥功能，我们在酿酒酵母sir2Δ突变菌株（S.cerevisiae sir2Δ mutant strain）中表达了全长人源Sirt1蛋白。从酵母到人类，染色质（chromatin）的结构具有高度保守性，因此酵母染色质是在体内底物环境中评估、抑制或激活异位组蛋白去乙酰化酶活性（ectopic histone deacetylase activity）的理想模型体系。sir2Δ突变株会呈现一系列依赖于Sir2p去乙酰化酶活性的异常表型。我们分析了Sir2p正常作用的三类沉默位点：核糖体DNA（ribosomal DNA）、端粒（telomeres）及交配型位点（mating type loci）。此外，我们还验证了sir2Δ菌株的典型特征指标：染色体外核糖体DNA环（extrachromosomal ribosomal DNA circles）的生成水平与组蛋白高度乙酰化（histone hyperacetylation）程度。通过在sir2Δ细胞中强力过表达SIRT1基因，我们发现该突变株的部分分子表型几乎恢复至野生型状态。具体而言，rDNA区域的转录沉默（transcriptional silencing）得以修复，染色体外rDNA环的形成受到显著抑制，且组蛋白H3K9与H4K16位点的乙酰化水平显著降低。而在其余研究位点——HM位点（HM loci）、端粒及亚端粒区域（sub-telomere），并未观察到功能互补现象。综上，本研究结果表明：① SIRT1基因可在rDNA位点上互补sir2Δ突变株的多种分子表型；② 利用酵母体系对Sirt1的活性开展体内筛选具备可行性。