Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109

Abstract

Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Delta suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.

FIG. 1.

Involvement of Rtt109 chaperones Asf1 and Vps75 in DNA damage response and Rtt109 stability. (A) Lack of H3-K56ac is correlated with elevated γH2A. WCE from the indicated strains were analyzed by immunoblotting after 15% SDS-PAGE with the antibodies shown to the right of each panel (α-γH2A, -H3-K56ac, and -H3, anti-γH2A, -H3-K56ac, and -H3 antibodies, respectively). (B) Activation of the Rad53 checkpoint kinase (*), as monitored by in situ autophosphorylation, observed in rtt109Δ and asf1Δ cells but not in vps75Δ cells. By comparison, the level of Rad53 activation in wild-type (WT) cells treated with MMS (0.1%, 2 h) is shown. The upper autophosphorylating species (†) is independent of DNA damage and serves as a loading control. (C) Inability to acetylate H3-K56 correlates with genotoxin sensitivity. Indicated strains were grown to an OD at 600 nm (OD₆₀₀) of ≅0.5 before being plated at fivefold serial dilutions on YPD medium with or without MMS. All strains are isogenic with MSY421 (36). (D) Members of the Rtt109 epistasis group (10) all show checkpoint activation in cycling cells, but only the rtt109Δ strain shows elevated γH2A. (E) The Vps75 histone chaperone stabilizes Rtt109. TAP was performed on WCE from the indicated strains and purified complexes visualized by silver staining. Immunoblotting of the WCE used for TAP shows that Rtt109 is expressed and soluble in the vps75Δ background (see panel F). (F) Rtt109-TAP is unstable in the absence of Vps75. WCE were collected at the indicated times after addition of the translation inhibitor cycloheximide (5). Growth curve analysis (OD₆₀₀) indicates that each strain arrested with similar kinetics (not shown). WCE were analyzed by immunoblotting with the antibodies to the right of the panel.

FIG. 2.

Rtt109 acetylates both H3-K56 and H3-K9 in vitro. (A) Rtt109 is an H3-K9 HAT in vitro. HAT assays contained the indicated recombinant factors in the presence of [¹⁴C]acetyl-coA using chicken core histones as a substrate. Reactions were separated by 15% SDS-PAGE and either Coomassie or silver stained, treated for fluorography, and then imaged or were immunoblotted with the indicated antibodies (α-H3K56ac and -H3K9ac [anti-H3-K56ac and -H3-K9ac antibodies, respectively]). (B) HAT assays performed as described for panel A, with the exception that core histones (chicken) or recombinant H3 (Xenopus) was used as a substrate and that several dilutions of rRtt109 were used, as indicated. (C) HAT assays performed as described for panel A, with the exception that several dilutions of the respective histone chaperones were used, as indicated. (D) Asf1 stimulates the HAT activity of Rtt109-Vps75 toward both H3-K9 and H3-K56. Factors were affinity purified from the indicated yeast strains, and HAT assays were performed as for panel A. α-TAP, anti-TAP antibody.

FIG. 3.

Rtt109 acetylates H3-K9 in vivo. In panels A through E, immunoblotting on WCE from the indicated strains was performed with the indicated antibodies after 15% SDS-PAGE. (A) Individual deletion of RTT109, VPS75, or ASF1 results in reduced levels of H3-K9ac. WT, wild type; α-H3-K9ac, -H3-K18ac, and -H3-K4me2, anti-H3-K9ac, -H3-K18ac, and -H3-K4me2 antibodies. (B) VPS75 and ASF1 do not control each other's expression. α-TAP, -H3-K56ac, and -H3, anti-TAP, -H3-K56ac, and -H3 antibodies, respectively. (C) Deletion of RTT109 reduces the peak of S-phase-specific accumulation of H3-K9ac. Strains were synchronized in G₁/S with α-factor before release into the cell cycle. Passage through a single cycle takes ∼90 min under optimal conditions. Synchronized progress was monitored by expression of the G₂/M cyclin Clb2. Immunostaining of H3 was used as a loading control. α-CLB2, anti-CLB2 antibody. (D) The Hst3/Hst4 HDACs deacetylate H3-K56ac but not H3-K9ac. The hst3Δ hst4Δ double mutant is isogenic with the WT (7). (E) H3-K9ac is independent of H3-K56ac. H3-K9ac levels are indistinguishable from WT levels in an unacetylatable H3-K56R mutant, as are H3-K56ac levels in an H3-K9R mutant. Strains are isogenic with MSY421 (36). (F) Inability to acetylate H3-K9 has no effect on DNA repair/genome stability. Genotoxin sensitivity assays are performed as described for Fig. 1C. Strains are isogenic with MSY421 (36).

FIG. 4.

Chaperone involvement in H3-K9 acetylation by the Gcn5 and Rtt109 HATs in vivo. (A) Deletion of either GCN5 or RTT109 reduces cellular levels of H3-K9ac. The gcn5Δ (JF81) and rtt109Δ (SC218) strains are isogenic with the wild-type (WT) strain BY4741. The second set of rtt109Δ (AT398) and sas3Δ (AT411) strains are isogenic with WT-Y3656. α-H3, -H3-K18ac, -H3-K9ac, and -H3-K56ac, anti-H3, -H3-K18ac, -H3-K9ac, and -H3-K56ac antibodies, respectively. (B) H3-K9ac is absent in the gcn5Δ rtt109Δ double mutant. (C) Double deletion of the GCN5 and RTT109 genes leads to synthetic sickness. Growth analysis of strains in the BY4741 background was performed as described for Fig. 1C. α-H4-K12ac, anti-H4-K12ac antibody. (D) Absence of H3-K9ac in gcn5Δ asf1Δ but not rtt109Δ asf1Δ double mutants. (E) Absence of H3-K9ac in gcn5Δ vps75Δ but not asf1Δ vps75Δ double mutants.

FIG. 5.

Asf1 interacts with the nuclear Hat1 complex. (A) Asf1 copurifies with H4-specific HAT activity that is independent of its ability to promote H3-K56ac. Core histone acetylation assays and their analyses are as described in the legend for Fig. 3. α-TAP, anti-TAP antibody. (B) Asf1 copurifies with Hat1-dependent H4-K5- and -K12-specific HAT activity. In vitro HAT assays are as described for Fig. 3. α-H3, -H4K5ac, and -H4K12ac, anti-H3, -H4-K5ac, and -H4-K12ac antibodies, respectively. (C) Asf1 interacts with the nuclear Hat1 complex. Immunoblotting was carried out with anti-TAP or anti-Myc (α-MYC) after tandem affinity purification of Asf1-TAP and 10% SDS-PAGE. (D) Association of the Asf1 histone chaperone with the nuclear Hat1 complex (Hat1-Hat2-Hif1) is dependent on the Hat2 subunit. Silver staining or immunoblotting was carried out after tandem affinity purification of Asf1-TAP followed by 10% SDS-PAGE. (E) Acetylation of H4-K5 and H3-K56 may perform parallel roles in DNA repair. The unacetylatable H4-K5R/H3-K56R double mutant is synthetically sensitive to the S-phase inhibitor HU relative to the corresponding single mutants. Growth analysis of strains in an MSY421 background (36) was performed as described for Fig. 1C. WT, wild type.

FIG. 6.

Model illustrating the roles of histone chaperones and HATs during chromatin assembly.