Sumoylation of Sir2 differentially regulates transcriptional silencing in yeast

Silent information regulator 2 (Sir2), the founding member of the conserved sirtuin family of NAD⁺-dependent histone deacetylase, regulates several physiological processes including genome stability, gene silencing, metabolism and life span in yeast. Within the nucleus, Sir2 is associated with telomere clusters in the nuclear periphery and rDNA in the nucleolus and regulates gene silencing at these genomic sites. How distribution of Sir2 between telomere and rDNA is regulated is not known. Here we show that Sir2 is sumoylated and this modification modulates the intra-nuclear distribution of Sir2. We identify Siz2 as the key SUMO ligase and show that multiple lysines in Sir2 are subject to this sumoylation activity. Mutating K215 alone counteracts the inhibitory effect of Siz2 on telomeric silencing. SUMO modification of Sir2 impairs interaction with Sir4 but not Net1 and, furthermore, SUMO modified Sir2 shows predominant nucleolar localization. Our findings demonstrate that sumoylation of Sir2 modulates distribution between telomeres and rDNA and this is likely to have implications for Sir2 function in other loci as well.     

Sir2基因与端粒沉默的研究进展

转录沉默是基因表达调控的重要方式,它对于保持细胞的不同分化状态和维持染色质的稳定性至关重要。沉默信息调节因子2（silent information regulator 2,Sir2）参与酵母交配型基因沉默（silent mating type）、端粒区基因沉默以及核糖体DNA（rDNA）沉默。端粒区的基因沉默可能是酵母衰老过程中的机制之一,处于沉默状态的染色质中的许多基因无转录活性,可能由此影响酵母生长。     

Enzymatic activities of Sir2 and chromatin silencing

Heritable domains of generalized repression are a common feature of eukaryotic chromosomes and involve the assembly of DNA into a silenced chromatin structure. Sir2, a conserved protein required for silencing in yeast, has recently been shown to couple histone deacetylation to cleavage of a high-energy bond in nicotinamide adenine dinucleotide (NAD) and the synthesis of a novel product, O-acetyl-ADP-ribose. The deacetylase activity provides a direct link between Sir2 and the hypoacetylated state of silent chromatin. However, the unusual coupling of deacetylation to cleavage and synthesis of other bonds raises the possibility that deacetylation is not the only crucial function of Sir2.     

Importance of the Sir3 N terminus and its acetylation for yeast transcriptional silencing

The N-terminal alanine residues of the silencing protein Sir3 and of Orc1 are acetylated by the NatA Nalpha-acetyltransferase. Mutations demonstrate that the N terminus of Sir3 is important for its function. Sir3 and, perhaps, also Orc1 are the NatA substrates whose lack of acetylation in ard1 and nat1 mutants explains the silencing defect of those mutants.     

Locus specificity determinants in the multifunctional yeast silencing protein Sir2

Yeast SIR2, the founding member of a conserved gene family, acts to modulate chromatin structure in three different contexts: silent (HM) mating-type loci, telomeres and rDNA. At HM loci and telomeres, Sir2p forms a complex with Sir3p and Sir4p. However, Sir2p's role in rDNA silencing is Sir3/4 independent, requiring instead an essential nucleolar protein, Net1p. We describe two novel classes of SIR2 mutations specific to either HM/telomere or rDNA silencing. Despite their opposite effects, both classes of mutations cluster in the same two regions of Sir2p, each of which borders on a conserved core domain. A surprising number of these mutations are dominant. Several rDNA silencing mutants display a Sir2p nucleolar localization defect that correlates with reduced Net1p binding. Although the molecular defect in HM/telomere-specific mutants is unclear, they mimic an age-related phenotype where Sir3p and Sir4p relocalize to the nucleolus. Artificial targeting can circumvent the silencing defect in a subset of mutants from both classes. These results define distinct functional domains of Sir2p and provide evidence for additional Sir2p-interacting factors with locus-specific silencing functions.     

Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.