Locus specificity determinants in the multifunctional yeast silencing protein Sir2

Abstract

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.

Fig. 1. PCR mutagenesis of SIR2 and screen for locus-specific mutants. (A) SIR2 plasmid GLC16 (CEN/LEU2/KanMX4 backbone) used both as a template for mutagenic PCR and as a backbone for transformation and ‘gap repair’. (B) Strategy for PCR-based mutagenesis of SIR2. Unique restriction sites (with nucleotide/amino acid positions given in parentheses) used to prepare three different linear plasmid molecules for ‘gap repair’ are indicated. The SIR2 open reading frame is indicated by the open arrow, and the location of the three different PCR products used for mutagenesis are shown by lines below. (C) Two strains (GCY23 and GCY40) used in the screen, their relevant reporter loci, and expected phenotypes for wild-type SIR2, class I and class II mutants.

Fig. 2. Phenotypes of class I and class II mutants. Reporter strain GCY23 was transformed with plasmids (GLC16 or derivatives) containing the SIR2 mutant alleles indicated, SIR2 wild type or no SIR2 insert (vector). Ten-fold serial dilutions of cells were plated on SC-Leu+FOA (to measure TPE), SC-Leu-Trp (HMR silencing), Pb²⁺/G418 (rDNA silencing) and SC-Leu (total number of plasmid-containing cells).

Fig. 3. Mutations responsible for class I and class II phenotypes. (A) Graphical representation of Sir2p, depicting location of class I and class II mutations. The gray box indicates the highly conserved core domain of Sir2p (amino acids 254–498) and the mutated residues conferring class I and class II phenotypes are represented by black bars. (B) A list of mutations, divided according to class. Mutations in bold letters have been identified more than once, whereas those in parentheses do not contribute to the silencing phenotype of the mutant and have no effect on silencing when recloned by themselves. These changes are shown because some subsequent assays were performed with alleles containing these mutations. We observed an average of 2.8 mutations per clone for class I mutants, and 2 mutations per clone for class II mutants, including silent mutations. Because of the relative abundance of class II mutants, only those containing a minimum number of mutations were selected for subsequent analysis. The following pairs of mutants behaved similarly in all the subsequent assays, and thus only one mutant per group is shown in subsequent figures: sir2-12/sir2-423, sir2-146/sir2-472, sir2-213/sir2-404 and sir2-366/sir2-408/sir2-424. A summary of mutant phenotypes is shown in Supplementary Tables I and II, available at The EMBO Journal Online.

Fig. 4. Dominance test of class I and class II mutations. The SIR2 wild-type strain GCY66 was transformed with plasmids (GLC16 or derivatives) containing either class I or class II mutations, SIR2 wild type or no insert (vector). (A) Six different class I mutations (as indicated) assayed for telomeric silencing (adh4::URA3-TelVIIL). (B) Class II mutations assayed for rDNA silencing using the colony-color marker MET15. Overnight cultures of cells transformed with the plasmids indicated were spotted onto Pb²⁺/G418 plates.

Fig. 5. Sub-nuclear localization of Sir2p and class II mutant proteins. A sir2Δ strain (GCY16) was transformed with a centromeric plasmid containing SIR2 or class II mutations (as indicated), in which the proteins are fused at their C-termini to GFP. Cultures were prepared and viewed by fluorescence microscopy as described in Materials and methods.

Fig. 6. Determination of the integrity of the Sir complex at telomeres by Sir3p–GFP localization. A sir2Δ SIR3–GFP strain (GCY150) was transformed with LEU2 integrative plasmids coding for SIR2 or class I mutations, as indicated. (A) Sir3p–GFP delocalizes from telomeres in the absence of Sir2p. Comparison of GFP fluorescence in SIR2 and sir2Δ strains. DAPI (4′,6-diamidine-2-phenylindole) staining of DNA is shown on the right. (B) Sir3p–GFP shows a novel localization pattern in the presence of all class I mutations.

Fig. 7. Co-localization of Sir4p and Nop1p by indirect immunofluorescence in sir2 class I mutants. Cells (SIR2 and the five sir2 class I mutants indicated) were stained with both anti-Sir4p (detected by a DTAF (5-([4,6-dichlorotriazin-2-yl] amino)-fluorescein) conjugated antibody, green) and anti-Nop1p (detected by a Cy5-conjugated antibody, red). Independent signals from Sir4p and Nop1p are shown in the first two columns. The merge of these two images for each strain is shown on the right, where the coincidence of Sir4p and Nop1p signals is yellow.

Fig. 8. In vitro interaction of wild-type and class II mutant proteins with Net1p. Binding of Myc epitope-tagged Sir2 proteins from whole-cell yeast extracts (wild type and class II mutants, as indicated) to GST–Net1(566–1189) protein bound to glutathione beads. Bound protein was detected by Western blotting using a monoclonal antibody against the Myc tag.

Fig. 9. Targeted silencing at telomeres, HMR and the rDNA locus. (A) Schematic representation of targeting silencing loci: HMR (YG451), adh4::URA3 on Chr. VII-L (YG581) or downstream of a 5S rRNA gene (GCY62). All of the targeting strains are also sir2Δ::kanMX4. (B) Effect of Gbd–Sir2p class II mutants (as indicated) targeted to the rDNA, with Gbd–Sir2p and Gbd alone for comparison. (C) Effect of Gbd–Sir2p class I mutants (as indicated) targeted to either a telomere or HMR. Gbd–Sir2p and Gbd alone serve as controls.