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The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae - PubMed

  • ️Mon Jan 01 2001

The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae

S H Meijsing et al. Genes Dev. 2001.

Abstract

The acetylation state of histones plays a central role in determining gene expression in chromatin. The reestablishment of the acetylation state of nucleosomes after DNA replication and chromatin assembly requires both deacetylation and acetylation of specific lysine residues on newly incorporated histones. In this study, the MYST family acetyltransferase Sas2 was found to interact with Cac1, the largest subunit of Saccharomyces cerevisiae chromatin assembly factor-I (CAF-I), and with the nucleosome assembly factor Asf1. The deletions of CAC1 (cac1Delta), ASF1 (asf1Delta), and SAS2 (sas2Delta) had similar effects on gene silencing and were partially overlapping. Furthermore, Sas2 was found in a nuclear protein complex that included Sas4 and Sas5, a homolog of TAF(II)30. This complex, termed SAS-I, was also found to contribute to rDNA silencing. Furthermore, the observation that a mutation of H4 lysine 16 to arginine displayed the identical silencing phenotypes as sas2Delta suggested that it was the in vivo target of Sas2 acetylation. In summary, our data present a novel model for the reestablishment of acetylation patterns after DNA replication, by which SAS-I is recruited to freshly replicated DNA by its association with chromatin assembly complexes to acetylate lysine 16 of H4.

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Figures

Figure 1
Figure 1

(A) Interactions between Sas2, Sas4, and Sas5. (Left) Sas2 coimmunoprecipitated with myc–Sas4 and HA–Sas5. Coimmunoprecipitations were with strain AEY1558 transformed with pAE240 (Sas2) and with pAE612 (myc–Sas4) or pAE625 (HA–Sas5). Immunoprecipitations were probed for Sas2 with an anti-Sas2 antibody. (Center) myc–Sas4 coimmunoprecipitated with Sas2 and HA coimmunoprecipitated Sas5. For coimmunoprecipitation of myc–Sas4 with Sas2, extracts from strain AEY1559 transformed with pRS315 (sas2Δ) or pAE240 (Sas2) and pAE612 (myc–Sas4) were immunoprecipitated with anti-Sas2 antibody (α-Sas2). For coimmunoprecipitation of myc–Sas4 with HA–Sas5, AEY1559 transformed with pAE613 (myc–Sas4) and pAE625 (HA–Sas5) was used. Precipitates were analyzed by immunoblotting with an α-myc antibody. (Right) Sas5 coimmunoprecipitated with Sas2 and myc–Sas4. Immunoprecipitation between Sas2 and HA–Sas5 was performed in extracts from strain AEY1559 transformed with pRS315 (sas2Δ) or pAE240 (Sas2) and pAE625 (HA–Sas5). Immunoprecipitation between myc–Sas4 and HA–Sas5 was performed in strain AEY1558 transformed with pAE613 and pAE625. Immunoblotting of immunoprecipitations with α-HA was used to detect HA–Sas5. (B) Sas2, Sas4, and Sas5 coeluted from a gel filtration column. Elution profile of low-copy myc–Sas2, myc–Sas4, and Sas5–HA on a Sephacryl S300 column. The strain used was AEY2465 transformed with pAE778 (myc–Sas2) and pAE779 (myc–Sas4). Fractions were analyzed by immunoblotting with an α-myc (top) and an α-HA antibody (bottom). The elution peaks of marker proteins are indicated above. (C) Deletion of Sas4 altered the elution profile of the SAS-I complex. Strain AEY2424, transformed with pAE778 (myc–Sas2) and pAE779 (myc–Sas4; WT, top) or with pAE778 (myc–Sas2) only (sas4Δ, bottom) was used for gel-filtration experiments.

Figure 2
Figure 2

SAS-I was involved in rDNA silencing. (A) The deletion of SAS2, SAS4, or SAS5 increased repression of URA3 inserted in the rDNA locus. Silencing of URA3 was measured as growth on 5-FOA-containing medium. The strains used were AEY1778, AEY2279, AEY2360, and AEY2361 (from top to bottom). (B) Increased silencing of MET15 by sas2Δ in rDNA required SIR2. Strains were assayed for MET15 activity by streaking them on lead indicator medium. The strains used were AEY1201 (WT), AEY1202 (sir2Δ), AEY1195 (sas2Δ), AEY1978 (sas2Δ sir2Δ), AEY1755 (cac1Δ), and AEY2487 (sas2Δ cac1Δ). (C) Sas2 was physically associated with rDNA sequences. Chromatin immunoprecipitations (CHIPs) were performed with extracts from a wild type (AEY1), a sas2Δ strain (AEY269), and a cac1Δ asf11Δ (AEY2451) strain using α-Sas2 or no antibody (−). PCR was performed with increasing amounts of precipitate (black triangle) using primers specific to the nontranscribed spacer of RDN1 (NTS) and ACT1. The inverse image of PCR products resolved on ethidium bromide-stained 1% agarose gels is shown. (D) GFP-tagged Sas2 localized to the nucleus. The sas2Δ strain AEY474 was transformed with pAE94. Bar, 5 μm.

Figure 3
Figure 3

Mutations in the HAT domain and the putative zinc finger disrupted functions of Sas2 in silencing. (A) Schematic representation of Sas2. (Gray) Region of Sas2 homologous to other MYST family proteins; (cross-hatched) the CCHC zinc finger; (black) acetyl-CoA–binding site (HAT). The mutations in the zinc finger (Zn−) and the acetyl-CoA-binding domain (HAT−) are indicated. (B) Point mutations in the HAT and zinc finger domains disrupt the function of Sas2 in HM silencing. Silencing of AEY474, a MATα HMRa-e** sas2Δ strain and MATa sir1Δ sas2Δ (AEY346) transformed with integrating vectors carrying SAS2 (pAE227), sas2 (HAT−) (pAE230), sas2 (Zn−) (pAE389), or no insert (pRS303) was monitored by patch-mating assays using MATα his4 or MATa his4 as mating tester lawns, respectively. (C) Mutations in Sas2 disrupted its ability to function in telomeric silencing. Silencing of a URA3 gene inserted near the left telomere of chromosome VII was tested in serial dilution assays on 5-FOA-containing medium. The strains used were derivatives of AEY1190 transformed with plasmids as in B. (D) The CCHC zinc finger of Sas2 was essential for its interaction with Sas4. Cell extracts were prepared from AEY2424 expressing myc–Sas4 and wild type (pAE240) or mutant Sas2 (HAT−, pAE321 and Zn−, pAE491). Sas4 was precipitated with α-myc, and the precipitates were immunoblotted using α-Sas2.

Figure 4
Figure 4

A mutation of lysine 16 to arginine in histone H4 displayed the same silencing phenotypes as sas2Δ at HMR, HML, and the telomeres. (A) H4 K16R restored silencing at a defective HMR allele. The panel shows the α-mating ability of MATα HMRa-e** strains carrying various H4 alleles. The strains used were AEY 1976 (wt), AEY1974 (K5, 8R), AEY2197 (K5, 12R), and AEY1956 (K16R). (B) H4 K16R caused HML derepression in a sir1Δ strain. The a-mating ability of the MATa sir1Δ strains AEY2221 (WT), AEY2222 (K5, 8R), AEY2223 (K5, 12R), and AEY2224 (K16R) is shown. (C) H4 K16R caused derepression of telomeric URA3. Silencing of URA3 was measured in serial dilution assays on 5-FOA medium. The strains used were AEY2302 (H4 wt), AEY2304 (H4 K16R), AEY1017 (wt), and AEY1190 (sas2Δ). (D) Additional deletion of SAS2 did not alter the suppression of HMRa-e** by H4 K16R. The α-mating ability of MATα HMRa-e** strains AEY 1976 (wt), AEY1956 (K16R), and AEY2475 (K16R sas2Δ) is shown.

Figure 5
Figure 5

Sas2 and Sas4 interacted with Cac1, but not Cac2 or Cac3. (A) Sas2 and Sas4 coimmunoprecipitated with Cac1. Cell extracts were prepared from AEY1558 or AEY1559 expressing myc–Cac1 (pAE614) or HA–Cac1 (pAE544) and myc–Sas4 (pAE613) or Sas2 (pAE240), as appropriate; precipitated with antibodies as indicated; and analyzed by immunoblotting. (B) Cac2 and Cac3 coimmunoprecipitated Cac1, but not Sas2. Extracts from strain AEY1558 expressing HA–Cac1 (pAE544), myc–Cac2 (pAE820), and Sas2 (pAE240) or strain AEY2463 (Cac3–myc) expressing HA–Cac1 (pAE544) and Sas2 (pAE240) were immunoprecipitated with α-myc and immunoblotted with α-HA (top) or α-Sas2 (bottom).

Figure 6
Figure 6

(A) cac1Δ and sas2Δ were epistatic in HML silencing in sir1Δ strains. Stains used in a patch mating assay were MATa HMLα and sir1Δ cac1Δ (AEY2204), sir1Δ sas2Δ (AEY346), sir1Δ hir1Δ (AEY2236), sir1Δ sas2Δ cac1Δ (AEY2205), sir1Δ sas2Δ hir1Δ (AEY2228), sir1Δ cac1Δ hir1Δ (AEY2234), sir1Δ sas2Δ cac1Δ hir1Δ (AEY2230), and sas2Δ cac1Δ (AEY2206). MATα his4 as a mating tester. (B) cac2Δ and cac3Δ did not cause additional derepression in sir1Δ sas2Δ strains. The a mating ability of MATa strains AEY1290 (sir1Δ sas2Δ), AEY2205 (sir1Δ sas2Δ cac1Δ), AEY2508 (sir1Δ sas2Δ cac2Δ), and AEY2479 (sir1Δ sas2Δ cac3Δ) is shown. (C) sas2Δ and cac1Δ had additive effects on HMR repression. A colony color assay was used to assay repression of the ADE2 gene inserted at HMR. Yeast cells were incubated on YM plates supplemented with 10% of the normal adenine concentration, WT (AEY1676), sas2Δ (AEY2481), cac1Δ (AEY2483), and sas2Δ cac1Δ (AEY2486). (D) The α-factor response of MATa HMLα strains was measured by spreading them on complete medium plates containing 40 μg/mL α-factor and incubating them at 23°C. Approximately 100 cells per strain were scored after 17 h. Schmoo indicates individual cells that formed mating projections and remained arrested; schmoo cluster, individual cells that formed multiple mating projections and eventually divided at least once; and colony, cells that formed colony of round cells and did not appear to respond to α-factor. WT (AEY2), sas2Δ (AEY266), sir1Δ (AEY345), cac1Δ (AEY1403), sas2Δ cac1Δ (AEY2206), asf1Δ (AEY2430), sas2Δ asf1Δ (AEY2429), asf1Δ cac1Δ (2452), and sas2Δ asf1Δ cac1Δ (2491). (E) CAF-I activity was not altered in sas2Δ strains. (Left) CAF-I activity was immunoprecipitated with myc–Cac2. Immunoprecipitates from AEY1558 or AEY1808 transformed with pAE716 (myc–Cac2) were used in SV40 in vitro replication assays to determine nucleosome assembly activity. The autoradiogram (top) of the ethidium bromide stained gel (EtBr, bottom) shows increased negative supercoiling of replicated plasmid DNA on addition of myc–Cac2 immunoprecipitate from a wild-type strain (WT). In the absence of Cac1 (cac1Δ), immunoprecipitated myc–Cac2 was unable to increase supercoiling. (right) CAF-I activity was unaffected by the absence of Sas2. Increasing amounts of myc–Cac2 immunoprecipitates from wild-type (WT) or sas2Δ∷TRP1 strains (sas2Δ) were added to in vitro SV40 replication reactions and assayed as above.

Figure 7
Figure 7

The deletion of ASF1 had the same silencing phenotype as sas2Δ at HMR and HML, and Asf1 physically interacted with SAS-I. (A) Asf1 coimmunoprecipitated with Sas2 and Sas4. Extracts from strain AEY2493 (Asf1–HA) expressing Sas2 (pAE240) or myc–Sas4 (pAE612) were immunoprecipitated with α-Sas2 or preimmune serum (PI), or with α-myc versus no antibody (−) and immunoblotted with α-HA. (B) Disruption of ASF1 caused HML, but not HMR, derepression in sir1Δ strains. The MATα strains AEY1 (WT), AEY2426 (asf1Δ), AEY2490 (sas2Δ asf1Δ), and AEY2428 (asf1Δ sir1Δ) were tested for their α-mating. Likewise, the a-mating ability of the MATa strains AEY2 (WT), AEY2430 (asf1Δ), AEY2429 (sas2Δ asf1Δ), and AEY2431 (asf1Δ sir1Δ) is shown. (C) asf1Δ restored repression at a mutated HMR allele. Shown is the α-mating ability of MATα HMRa-e** strains and WT (AEY403), sas2Δ (AEY474), or asf1Δ (AEY2363). (D) Effect of combinations of sir1Δ, cac1Δ, asf1Δ, and sas2Δ on HML silencing. The a-mating ability of the MATa strains AEY2 (WT), 1290 (sir1Δ sas2Δ), AEY2431 (sir1Δ asf1Δ), AEY2470 (sir1Δ sas2Δ asf1Δ), AEY2471 (sir1Δ cac1Δ asf1Δ), and AEY2473 (sir1Δ cac1Δ asf1Δ sas2Δ) was tested. Results of quantitative mating assays are shown relative to a value of 1.0 for AEY2.

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