Sir3-nucleosome interactions in spreading of silent chromatin in Saccharomyces cerevisiae

Abstract

Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH domain, while one, L738P, localized to the AAA ATPase-like domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding domains in Sir3.

FIG. 1.

Isolation of dominant negative SIR3 alleles. (A) Two silent mutations were introduced into the SIR3 ORF (denoted by asterisks) to allow excision of specific fragments. The resulting SIR3-containing plasmid was digested with two adjacent restriction endonucleases (shown above the Sir3 diagram) and cotransformed with the corresponding PCR fragment (I to IV) into a TELVR::ADE2 reporter strain. Red or red/white sectored colonies are indicative of wild-type silencing, while white colonies indicate loss of silencing. These white transformants were isolated as positive hits. (B) Sample plate with a positive hit circled (left) and sample restreaks (right). (C) Locations of Sir3 mutations, with the primary sequence of the BAH domain shown in the box below. Mutations are indicated by red tick marks and red letters. L738P is the single mutation in the AAA+ domain. (D) Locations of the mutated residues in the BAH domain mapped onto the structure of the domain (PDB-ID 2FL7; see reference16). Ala181, Val196, and Ser204 are not shown as they lie internally in the structure. Lys219 and Leu738 are not part of the structure.

FIG. 2.

Dominant negative silencing defects for telomeric URA3 and mating-type TRP1 (TELVIIL::URA3 hmrΔe::TRP1) reporters, respectively. The indicated Sir3 alleles are expressed from a CEN vector (pDM832 and its mutant derivatives). Growth on medium with 5-fluoroorotic acid (+FOA) and lack of growth on medium lacking tryptophan (−Trp) indicate silencing. Tenfold serial dilutions of wild-type and mutant strains were plated on the indicated growth medium. Most BAH mutants strongly disrupt silencing. L738P has a weaker defect (row 25). Replacing Leu738 with alanine does not disrupt silencing (row 26).

FIG. 3.

Recessive silencing defects of Sir3 mutants. The indicated plasmids, producing wild-type or mutant Sir3, were introduced into hmrΔe::TRP1 TELVIIL::URA3 sir3Δ::Kan^R cells and assayed for silencing as described in the Fig. 2 legend. All mutants identified in the screen exhibit a complete loss of silencing (rows 3 to 24). The reduced growth in K202E (row 18) on SC −Leu −Trp medium is attributable to slower growth of this strain rather than partial rescue of silencing. L738A has a partial silencing defect (row 25). +FOA, with fluoroorotic acid; −Trp, without tryptophan.

FIG. 4.

Sir3 proteins lacking the BAH domain disrupt silencing in a dominant negative manner. (A) Sir3-BAHΔ dominantly disrupts silencing (row 2). Mutations expressed in the context of an isolated BAH domain (BAH only) do not disrupt silencing dominantly (rows 4 to 6). +FOA, with fluoroorotic acid; −Trp, without tryptophan. Silencing assays were performed as described in the Fig. 2 legend. (B) Western blot for expression of BAH only shows that the mutant BAH proteins are expressed at the same levels as wild-type BAH. Full-length Sir3-FLAG runs at ∼120 kDa. (C) Dominant negative mating defects of Sir3-BAHΔ and Sir3-L738P at HML. BAHΔ mating efficiency is 0.39 ± 0.11 (mean ± standard deviation) (n = 3) relative to that of wild-type SIR3; L738P mutant has no dominant defect, with a relative efficiency of 1.05 ± 0.112 (n = 2). (D) Recessive mating defect at HML relative to efficiency of vector-only control. Wild-type Sir3 has a mating efficiency of 9.7 × 10⁴ ± 1.2 × 10⁴ (mean ± standard deviation) (n = 3). Sir3-BAHΔ does not rescue the mating defect of sir3Δ mutant (0.3 ± 0.08; n = 3). Sir3-L738P partially rescues the mating defect of sir3Δ mutant at 1.9 × 10³ ± 1.5 × 10³ (n = 3). wt, wild type; rel., relative.

FIG. 5.

Mutant Sir3 proteins are recruited to chromatin and interfere with total Sir3 spreading. (A) Dominant negative mutants are recruited to chromatin in cells that express wild-type SIR3. FLAG-tagged wild-type or mutant Sir3 was expressed from a CEN plasmid (pSir3) in a strain that also expressed endogenous SIR3 (pSir3-FLAG + SIR3⁺). The FLAG-tagged Sir3 was immunoprecipitated from lysates and the level of enrichment at TELVIR (TEL 0.07) was determined. sir3Δ represents a sir3Δ control strain with empty vector. Averages and standard errors are shown for the results of two experiments. (B) ChIP for total Sir3 in cells that express both wild-type SIR3 and the indicated construct on a CEN plasmid (pSir3-FLAG + SIR3⁺). sir3Δ is a control strain which had endogenous SIR3 deleted and was transformed with empty vector. The level of enrichment was measured at 0.07, 0.7, and 2.4 kb from the end of chromosome VIR and is normalized to wild-type Sir3 at TEL0.07. Averages are shown for the results of three to five experiments, with the exception of E95K, for which are the averages of the results of two experiments are shown. (C) Sir3 point mutants are unable to spread along chromatin in the absence of wild-type Sir3. FLAG-tagged Sir3 was expressed from a CEN plasmid which was the only copy of SIR3 (pSir3 + sir3Δ). sir3Δ (3Δ) carries an empty vector. sir2Δ (2Δ) has SIR2 deleted but expresses endogenous SIR3. Averages and standard errors are shown for the results of two (TEL0.7, TEL2.4) or three (TEL0.07) experiments. wt, wild type; rel., relative.

FIG. 6.

SIR complex formation is not disrupted by the Sir3 mutations. (A) Coimmunoprecipitation of the indicated Sir3 mutants with Sir2 and Sir4. (B) Liquid chromatography-MS-MS results for purified Sir3 proteins. The relative abundance of predominant binding partners is unchanged by point mutations or deletion of the BAH domain #, total number of peptides; %, percent coverage; s.c., spectral count; α, anti; wt, wild type.

FIG. 7.

Sir3 self-interactions are unaffected by point mutations or deletion of the BAH domain. (A) The indicated Sir3-3xFLAG proteins were expressed from a CEN plasmid in cells expressing SIR3-13MYC₁₃. Coimmunoprecipitation for the FLAG tag indicated that none of the mutants disrupted Sir3 dimerization. (B) Coimmunoprecipitation as described for panel A; Sir3-BAHΔ and Sir3-BAHΔ/L738P interact with wild-type Sir3. The asterisk on the left indicates a cross-reacting background band. (C) One of the two BAH-BAH interfaces in the crystal structure described by Connelly et al. (6), highlighting a potential salt bridge. Glu84 is shown in red and Lys99 in yellow. (D) Silencing assays as described in the Fig. 2 (SIR3) and Fig. 3 (sir3Δ) legends. Sir3-K99E does not display a dominant negative silencing defect and Sir3-E84K/K99E does not restore silencing. +FOA, with fluoroorotic acid; −Trp, without tryptophan. (E) The BAH domain does not interact with itself (lane 6) or with full-length Sir3 (lane 7) in coimmunoprecipitation assays. TAP, dual protein A-CBP tag. (F) BAH-3xFLAG does not interact with Sir3-BAHΔ-13xMyc. (G) Sir3 oligomerization is unaffected by point mutations or deletion of the BAH domain. Sir3-3xFLAG was expressed from a high-copy Gal1 vector and purified via the FLAG tag. Three micrograms protein was resolved by native PAGE and silver stained. The pattern of oligomers (identified by arrows) is unchanged by point mutations and is shifted down for the smaller Sir3-BAHΔ protein. (H) As described for panel G but using Sir3-BAHΔ/L738P. The double mutant is able to oligomerize as assayed by native PAGE. α, anti; +, present; −, absent.

FIG. 8.

Loss of nucleosome binding in dominant negative BAH mutants. (A) Results of coimmunoprecipitation assays showing that D17G, E84K, and K202E have decreased binding to nucleosomes. L738P does not interfere with nucleosome binding as shown by the results of this assay. (B) Loss of nucleosome binding in the BAH point mutants S67P, E95K, E137G, P179L, and S212F.

FIG. 9.

In vitro histone tail-binding assay. (A) Sir3-L738P binds the histone tail more tightly, both in full-length Sir3 and Sir3-BAHΔ. The BAH domain does not contribute to binding as shown by the results of this assay. GST-H4(15-34) was incubated with Sir3 purified from yeast, and binding was detected by Western blotting. (B) Sir3-L738P binding is sensitive to mutations of residue 16. H4K16Q completely disrupts the interaction, whereas H4K16R binds to Sir3-L738P weakly. wt, wild type.

FIG. 10.

Electrostatic maps of the BAH domain of Sir3 (PDB-ID 2FL7) (17) and the surface of the yeast nucleosome (PDB-ID 1ID3) (52). The locations of the dominant negative mutations are indicated by yellow dots on BAH. Most of the residues which were mutated lie in an acidic patch, with the exception of the residues mutated as K202E and K209R/K209RG, which constitute a “lysine knob.” The histone H4 tail and H3K79 regions on the surface of the nucleosome may constitute an interaction region for the acidic patch in the BAH domain. The lysine knob may interact with DNA or the acidic patch near the H3K79 region on the surface of the nucleosome. See text for details.

FIG. 11.

Model for the role of Sir3 in spreading of silent chromatin. (A) Diagram of Sir3 and its histone/nucleosome-interacting domains. (B) Wild-type Sir3 can be recruited to chromatin through Sir3-Sir3 interactions. Sir3 anchors the spreading SIR complex to chromatin by interactions of CHB and BAH domains with the nucleosome (blue). BAH point mutants or the BAH deletion are unable to bind to the nucleosome, thus resulting in a loss of the anchoring function and of stable recruitment of additional Sir3 molecules. Sir3-L738P may bind to the nucleosome in a conformation that does not allow the BAH domain to engage with the nucleosome on the opposite face.