The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1

Abstract

It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ~450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing.

Figure 1

Detection of a 450-kD Sas2p-containing complex. Yeast extracts were separated by Superose 6 size exclusion chromatography, and fractions were analyzed for Sas2p by Western blot analysis (anti-Myc or anti-HA monoclonal antibodies). (Top panel), Superose 6-separated whole cell extracts prepared from strain YJW265 expressing Sas2p–Myc. (Middle panel) Whole cell extracts prepared from strain YJW214, expressing Sas2p–HA. The Sas2p–HA complexes were enriched by Mono Q anion-exchange chromatography before separation by Superose 6. (Bottom panel) Superose 6–separated whole cell extracts prepared from strain YJW213 expressing the GAL1-regulated HA–Sas2p. Arrows mark peak fractions for molecular weight standard proteins.

Figure 2

Sas4p and Sas5p are components of the Sas2 complex. (A) Extracts were prepared from strains YJW265, YJW228, and YJW229 expressing the C-terminal Myc-tagged Sas2, Sas4, and Sas5 proteins, respectively. The sizes of the Sas2, Sas4, and Sas5 complexes were determined by analysis of elution profiles from Superose 6. (B) Comparison of the Sas2p–Myc elution profiles from Superdex 200 size exclusion chromatography following fractionation of whole cell extracts prepared from wild type (YJW265) and sas4Δ (YJW269), sas5Δ (YJW270), and sas4Δ sas5Δ (YJW271) mutant strains. Shown are Western blots of column fractions probed with Myc-antibodies.

Figure 3

Sas4p and Sas5p coimmunoprecipitate with Sas2p. Coimmunoprecipitation experiments were performed with both whole cell extracts (WCE) and Superose 6 fraction 28 prepared from double-tagged strains HA–Sas2p, Sas4p–Myc (YJW230) and HA–Sas2p, Sas5–Myc (YJW231; indicated at the bottom of the panel). Protein fractions were incubated with HA-antibodies, and 10% of input (in), 10% of supernatant (sup), and bead (be) fractions from the immunoprecipitates were probed for Myc-tagged proteins.

Figure 4

Purification of the SAS complex. (A) Schematic representation of the chromatographic steps applied for purification of the SAS complex. (B) The SAS complex was purified from YJW276. (Top) A silver-stained gel of fractions following Superdex 200 chromatography. (Bottom) Western blot of Superdex 200 fractions. Specific bands in the smaller Sas2 complex are indicated by their molecular weight. Sas4p–Myc and His–Sas2p–Flag were detected by Western blot analysis with anti-Myc and anti-Flag antibodies. (C) Peptide sequences obtained from mass spectrometry analysis are shown. Numbers at the left and right of the sequence indicate the position in each protein, respectively.

Figure 4

Purification of the SAS complex. (A) Schematic representation of the chromatographic steps applied for purification of the SAS complex. (B) The SAS complex was purified from YJW276. (Top) A silver-stained gel of fractions following Superdex 200 chromatography. (Bottom) Western blot of Superdex 200 fractions. Specific bands in the smaller Sas2 complex are indicated by their molecular weight. Sas4p–Myc and His–Sas2p–Flag were detected by Western blot analysis with anti-Myc and anti-Flag antibodies. (C) Peptide sequences obtained from mass spectrometry analysis are shown. Numbers at the left and right of the sequence indicate the position in each protein, respectively.

Figure 5

The conserved acetyl-CoA binding motif of Sas2p is required for HML and telomeric silencing. (A) Sequence comparison of acetyl-CoA binding motifs of Sas2p, Sas3p, and Esa1p. The positions of the mutated amino acid residues are shown. The residues of Esa1p involved in acetyl-CoA binding are indicated in gray, and underlined amino acids represent buried residues (Yan et al. 2000). (B) The effect of the SAS2 acetyl-CoA binding site mutations on silencing at HML was assayed by measuring mating efficiencies. Strains expressing wild-type and mutant alleles of SAS2 from CEN-based plasmids containing the SAS2 promoter (YJW348, YJW350, YJW352, YJW353, YJW354, and YJW355; ordered from left to right), 2μ-based plasmids also containing the SAS2 promoter (YJW349, YJW351, YJW356, YJW357, YJW358, and YJW359), and 2μ-based plasmids regulated by the GAL10 promoter (YJW281, YJW282, YJW283, YJW284, YJW285, and YJW286) were patched onto plates lacking uracil and replica plated onto minimal medium spread with a lawn of MATα cells. Quantitative mating analyses of the same strains are expressed as a percentage of diploids formed per viable cells. (C) To evaluate the expression of a telomere-proximal URA3 gene, serial dilutions of saturated cultures were inoculated onto plates lacking leucine in the presence and absence of 5-FOA. Silencing was compared for the following strains (top to bottom): YJW324, YJW328, YJW330, YJW369, YJW370, YJW371, YJW326, YJW332, YJW334, YJW375, YJW376, and YJW377.

Figure 6

The SAS complex interacts with Asf1p. (A) Individually expressed ³⁵S-labeled Sas proteins were incubated with GST or GST–Asf1 fusion proteins coupled to glutathione Sepharose beads. Proteins that remained bound to the beads fraction (be) were subjected to SDS-PAGE. The amount of input (in) and supernatant (sup) are equivalent to 25% of the reaction in the assay. (B) Coimmunoprecipitation experiments were performed with whole cell extracts prepared from the double-tagged strains expressing the Sas4p–Myc (top, YJW416 and YJW417), Sas2p–Myc (middle, YJW414 and YJW415), Sas5p–Myc (bottom, YJW418 and YJW419), and Asf1p or Asf1p–HA (indicated at the bottom of the panel). Whole cell extracts were incubated with anti-HA antibodies, and 0.5% of input (in), 0.5% of supernatant (sup), and bead (be) fractions from the immunoprecipitates were probed for the Myc-tagged proteins. (C) The HA-tagged Sas proteins coimmunoprecipitate Asf1p. The Myc-tagged ASF1p expression plasmid was transformed into the untagged strain and the chromosomally integrated HA-tagged SAS2, SAS4, and SAS5 strains (YJW457, YJW458, YJW459, and YJW460). Results are as described in B. (D) The interaction between the SAS complex and ASF1p is not mediated via DNA/chromatin in the extracts. Coimmunoprecipitation experiments were performed in untreated WCE (lanes 1–3), presence of ethidium bromide (EtBr) (lanes 4–6), or presence of MNase (lanes 7–9). Whole cell extracts were prepared from strains expressing the Sas4p–Myc (top, YJW417), Sas2p–Myc (middle, YJW415), Sas5p–Myc (bottom, YJW419). Results are as described in Figure 6B except that 1% of input and supernatant was loaded.

Figure 7

DNA damage sensitivity and mating efficiencies of asf1, sir1, and sas2 mutants. (A) Effects of disruption of ASF1 and SIR1 and/or SAS2 on sensitivity to mutagens. Serial dilutions of wild type and mutant strains were spotted and grown on complete media in the absence (−), or presence of ultraviolet radiation (100 J/m²), MMS (0.006%), or HU (25 mM). Strains analyzed were W303-1a (wild type), YJW433 (asf1Δ), YJW252 (sir1Δ), YJW435 (asf1Δ sir1Δ), YJW253 (sas2Δ), YJW436 (asf1Δ sas2Δ), YJW258 (sas2Δ sir1Δ), and YJW437 (asf1Δ sas2Δ sir1Δ). (B) ASF1 contributes to silencing at HML. Mating assays are shown with quantitative mating assay results given below. Strains shown are W303–1a (wild type), YJW252 (sir1Δ), YJW253 (sas2Δ), YJW258 (sas2Δ sir1Δ), YJW433 (asf1Δ), YJW435 (asf1Δ sir1Δ), YJW436 (asf1Δ sas2Δ), and YJW437 (asf1Δ sas2Δ sir1Δ). (C) Deletion of ASF1 restores silencing to strains with mutations in HMR-E. Quantitative mating assay results are expressed relative to a value of 1.0 for wild-type W303–1b. Strains shown are YAB53 (ΔAΔE), YAB197 (ΔBsir1Δ), YGM75 (ΔEΔB), SY557 (asf1ΔΔAΔE), SY558 (asf1ΔΔB sir1Δ), and SY560 (asf1ΔΔEΔB).