Purification of Sir3 and Sir2/Sir4 from yeast extracts. Silver stained SDS-polyacrylamide gels showing the purification of TAP-tagged Sir3 (A) and Sir4 (B) from strains DMY1737 and DMY1704, respectively. The left panel in A shows a control purification from the parental strain lacking the TAP tag (SF10). Proteins were identified by mass spectrometry. The band marked with an asterisk (∗) in B was identified as Ssb1, a yeast Hsp70 protein, which is a common contaminant of TAP purifications. Lanes 1 to 4 show successive elution fractions from calmodulin-Sepharose, the second column used in the purifications. (C) Western blots showing the migration of Sir3 in a Superose 6 gel filtration column in crude yeast extracts (Sir3-TAP, top panel) and after purification on IgG-Sepharose and calmodulin-Sepharose (Sir3-CBP, top panel, same material as in A, lane 3). Molecular size markers used for calibration of the sizing column were thyroglobulin (670 kDa) and aldolase (158 kDa).

Requirement for SIR2, SIR3, and SIR4 for assembly of silent chromatin. (A) Schematic diagram showing the location of PCR primers corresponding to the HML, HMR, and MATa loci on chromosome III, the subtelomeric region on the right arm of chromosome VI (TEL and VI-R), and the rDNA repeats on chromosome XII used in chromatin immunoprecipitation experiments. The locations of PCR primers are indicated under each region as thick bars. The telomeric primers amplify DNA fragments from 0.35, 0.6, 1.4, 2.8, and 4.7 kb from the telomeric repeats. The HML and HMR primers flank the E silencers (HML-E and HMR-E) or are located within HMLα or HMRa, respectively. The rDNA primers amplify fragments within the nontranscribed spacer regions (NTS1 and NTS2) and the 35S rRNA coding region. Strains used were the wild type (W303-1a) and sir2Δ (SF3), sir3Δ (SF4), and sir4Δ (SF5) mutants. (B) Phosphorimager data of chromatin immunoprecipitation experiments showing the association of the Sir2, Sir3, and Sir4 proteins with silencers (HML-E and HMR-E) and DNA immediately adjacent to telomere VI-R in wild-type (lanes +), sir2Δ (2Δ), sir3Δ (3Δ), and sir4Δ (4Δ) strains. PCR amplifications of anti-Sir2, anti-Sir3, and anti-Sir4 chromatin immunoprecipitations and input DNA from WCL are shown. (C) Association of Sir2, Sir3, and Sir4 in the same genetic backgrounds as in B with silent chromatin regions that are more distal from initiation sites and with a control nonsilenced locus (GAL1). (D) Association of Sir2, Sir3, and Sir4 with rDNA.

Enzymatic activity of Sir2 is required for association of Sir2, Sir3, and Sir4 proteins with telomeric DNA regions and the HML mating type locus. (A and B) Chromatin immunoprecipitation was carried out from a sir2 deletion strain (sir2Δ), a SIR2 wild-type strain (SIR2⁺), and strains containing sir2 alleles that encode enzymatically inactive Sir2 proteins (sir2-H364Y and sir2-G262A) with anti-Sir2, anti-Sir3, and anti-Sir4 antibodies. Panels show phosphorimager data of PCR amplifications corresponding to input (WCL) and immunoprecipitated chromatin for the indicated regions of the VI-R telomere and the HML locus. The following strains were used: DMY1865 (sir2Δ), DMY1866 (SIR2⁺), DMY1865 (sir2-H364Y), and DMY1867 (sir2-G262A). See Fig. 2A for locations of primers.

Requirement for the enzymatic activity of Sir2 in localization of Sir2 and Sir3 to rDNA and the role of histones H3 and H4 in rDNA silencing. (A) Chromatin immunoprecipitation was carried out from sir2Δ, SIR2⁺, and sir2-H364Y strains using an anti-Sir2 antibody. See Fig. 3 legend for strain names. (B) Chromatin immunoprecipitation experiments showing that the association of Sir3 with rDNA requires the enzymatic activity of Sir2. Association of Sir3 with rDNA fragments in sir2Δ, SIR2⁺, and sir2-H364Y strains carrying either wild-type SIR4 or sir4Δ is shown. (C) Loss of rDNA silencing in histone H3 and H4 mutants. Tenfold serial dilutions of wild-type or histone mutant strains were plated on complete medium or medium lacking uracil (SD−Ura), and plates were photographed after 2 to 3 days of growth at 30°C.

Enzymatic activity of Sir2 is required for localization of Sir2, Sir3, and Sir4 proteins to telomeric foci but not for localization of Sir2 to the nucleolus. The localization of Sir2-GFP, Sir3-GFP, and Sir4-GFP in cells containing either wild-type SIR2 (A to C, DMY1247, DMY2165, and DMY2167, respectively) or the enzymatically inactive sir2-H364Y (D to F, DMY1249, DMY2166, and DMY2168, respectively) is shown. Panels A and D show the localization of Sir2-GFP and sir2-H364Y-GFP, respectively. Colocalization of Sir2-GFP (G to J, DMY1247) and sir2-H363Y-GFP (K to N, DMY2164) with the nucleolar marker Nop1 confirms that Sir2-H364Y-GFP is localized to the nucleolus. See Table 1 for strains.

Hypoacetylation of histone H4 associated with HMR-E, HML-E, telomeric DNA regions, and rDNA requires Sir2. Chromatin immunoprecipitations were carried out using an anti-acetylated H4 antibody from SIR2⁺ (W303-1a), sir2Δ (SF3 or DMY1865), sir2-H364Y (DMY1866), sir2G262A (DMY1867), sir3Δ (SF4), and sir4Δ (SF5) strains. PCR amplifications of immunoprecipitated DNA (lanes 1 to 8) and WCL (lanes 9 to 12) for the HM silencers (A), telomeric DNA regions (B), rDNA (C), and nonsilenced MATa and ACT1 loci (D) are shown.

Model for assembly of silent chromatin domains in yeast. (A) Assembly of the Sir complex at silencers and telomeres is proposed to occur in a stepwise fashion involving at least three steps: transient binding, stable association, and spreading of the Sir proteins. In step 1, the Sir2/4 heterodimer transiently binds to the silencer or the telomere via interactions with Rap1/Sir1 and Rap1/yKu70, respectively. This binding does not require the enzymatic activity of Sir2 and is independent of Sir3. In fact, Sir4 can bind to silencers in sir2Δ and sir3Δ cells (see Fig. 2). The association of Sir3 with the silencer can also occur independently of Sir2 but requires Sir4. The Sir4 interactions with the silencer- and telomere-bound factors are based on two-hybrid assays (42, 63, 64). Stable association of the complex with chromatin (step 2) and spreading (binding to regions distal from nucleation sites, step 3) requires all three Sir proteins and the NAD-dependent deacetylation activity of Sir2 (23; this study). (B) Stable association of Sir2 with rDNA requires Net1 but does not require the NAD-dependent deacetylase activity of Sir2 and probably occurs in the absence of deacetylation (step 1) (57; this study). An unknown protein(s) is likely responsible for targeting Net1/Sir2/Cdc14 to rDNA and plays a role analogous to that of silencer and telomere binding proteins. Sir2 activity is required for rDNA silencing and hypoacetylation of histone H4 associated with rDNA (step 2). ADPRAc, O-acetyl-ADP-ribose.