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Rtt109 Acetylates Histone H3 Lysine 56 and Functions in DNA Replication

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Science  02 Feb 2007:
Vol. 315, Issue 5812, pp. 653-655
DOI: 10.1126/science.1133234

Abstract

Acetylation of histone H3 lysine 56 (H3-K56) occurs in S phase, and cells lacking H3-K56 acetylation are sensitive to DNA-damaging agents. However, the histone acetyltransferase (HAT) that catalyzes global H3-K56 acetylation has not been found. Here we show that regulation of Ty1 transposition gene product 109 (Rtt109) is an H3-K56 HAT. Cells lacking Rtt109 or expressing rtt109 mutants with alterations at a conserved aspartate residue lose H3-K56 acetylation and exhibit increased sensitivity toward genotoxic agents, as well as elevated levels of spontaneous chromosome breaks. Thus, Rtt109, which shares no sequence homology with any other known HATs, is a unique HAT that acetylates H3-K56.

Nucleosomes are the basic repeat structure of eukaryotic chromatin, each consisting of ∼146 base pairs of DNA wrapped around a histone octamer (1). Many diverse cellular functions are regulated through the modulation of nucleosome structure, and posttranslational modifications of core histones are key to this modulation (24). Indeed, histone modifications are known to play an important role in transcriptional regulation. However, the role of histone modifications in DNA replication is not well studied (5). Acetylation of lysine 56 on histone H3 (H3-K56) has been implicated in regulating replication, because H3-K56 is transiently acetylated during S phase. In addition, cells with alterations in H3-K56 acetylation display increased sensitivity toward certain DNA-damaging agents (610). Histone deacetylases responsible for deacetylating H3-K56 have recently been discovered (Hst3 and Hst4) (11, 12), but the histone acetyltransferase (HAT) that globally acetylates H3-K56 has not been found (8).

To identify the H3-K56 HAT, we screened 4700 viable yeast deletion mutants for their effect on H3-K56 acetylation using antibodies that specifically recognize acetylated H3-K56 (10) (H3-K56Ac; Fig. 1A, top). From this screening procedure, two genes were identified that, when deleted, abolished H3-K56 acetylation: ASF1 and RTT109 (Fig. 1A). Western blot analysis of whole-cell extracts prepared from the asf1Δ and rtt109Δ mutant strains (table S1) confirmed that deletion of ASF1 or RTT109 abolishes H3-K56 acetylation. As a control, methylation of H3 lysine 79 was unaffected (Fig. 1B). Others have also recently shown that mutation of Asf1 abolishes acetylation of H3-K56 (11, 13). However, Asf1 is a histone chaperone and does not appear to have intrinsic HAT activity (14).

Fig. 1.

(A) Rtt109 and Asf1 are required for H3-K56 acetylation. The total amount of protein from yeast cell extracts was revealed by Ponceau S staining (Pon S, left panel) and H3-K56 acetylation was detected by Western blot with antibodies that recognize H3 acetylated at lysine 56 (H3-K56Ac, right panel). Membrane regions surrounding the spots corresponding to H3-K56A, rtt109Δ, asf1Δ (arrowheads), and wild-type (WT) cells are shown. (B) Acetylation of H3-K56 is not detected in H3-K56A, rtt109Δ, and asf1Δ mutant cells. Western blot was performed to examine whole-cell extracts from cells as shown, with antibodies against histone H3 (H3), H3-K56Ac, H3 methylated at lysine 79 (H3-K79me), and PCNA.

RTT109 was originally identified in a genetic screen for regulators of transposition of the yeast retrotransposon Ty1 (15), but its biochemical function remained unknown. Therefore, we tested whether recombinant Rtt109 had HAT activity toward H3-K56. Purified recombinant Rtt109 was incubated with H3/H4 tetramers in the presence of [3H]acetyl-coenzyme A (acetyl-CoA), and the incorporation of [3H]acetate into proteins was analyzed. Indeed, Rtt109 incorporated [3H]acetate into proteins (Fig. 2A) and did so in a concentration-dependent manner (fig. S1). Furthermore, Rtt109 acetylated itself and H3-K56, but not H4 (Fig. 2, B and C), in vitro. Similar to recombinant Rtt109, Rtt109 purified from yeast cells also acetylated H3-K56 (Fig. 2D and fig. S2). Thus, Rtt109, both in recombinant form and as a complex purified from yeast cells, exhibits HAT activity toward H3-K56, but not H4, in vitro.

Fig. 2.

Rtt109 is a HAT that acetylates itself and H3-K56. (A) GST-Rtt109 displays HAT activity. Purified recombinant Rtt109 and REGα, a proteosome-binding protein used as a control, were incubated with recombinant H3/H4 tetramers in the presence of [3H]acetyl-CoA, and the incorporated [3H]acetate was detected by scintillation counting. The mean and standard deviation from three independent experiments are shown. (B) Recombinant Rtt109 acetylates itself and H3, but not H4. Samples from HAT assays as described in (A) were resolved using SDS polyacrylamide electrophoresis (SDS-PAGE), followed by Coomassie brilliant blue staining (CBB), to detect total proteins and autoradiography to detect 3H acetylated labeled proteins. (C) H3-K56 is acetylated by recombinant Rtt109. HAT assays were performed using unlabeled acetyl-CoA, and the presence of acetylated H3-K56 was determined by Western blot analysis. (D) Rtt109 purified from yeast cells acetylates H3-K56. Experiments were performed as described in (C), except for the source of Rtt109. REGα in (C) and the No-tag sample in (D) were incubated for 6 hours; the rest of the samples were incubated for the times indicated. (E) Mutation of three conserved aspartate residues affects H3-K56 acetylation. Acetylation of H3-K56 was analyzed in yeast cells expressing rtt109 mutants of the indicated conserved aspartate residues. (F and G) In vitro assays using Rtt109 mutant proteins purified from yeast cells show a loss of HAT activity by selected aspartate mutants. (H) Rtt109 mutant proteins interact with Vps75. Vps75-TAP (tandem affinity purification) was purified from wild-type or rtt109 mutant cells, and copurified Rtt109 or mutants were detected by Western blot.

Analysis of the amino acid sequence of Rtt109 did not reveal homology to the catalytic domain of any known HATs. Canonical HATs such as Gcn5 and Esa1 use a glutamate residue conserved in each HAT family to deprotonate lysines before acetylation (16). We reasoned that Rtt109 may use a negatively charged residue for catalysis in a similar manner. Thus, we made 11 site-specific rtt109 mutants by replacing aspartate (D) and glutamate (E) residues with alanines (A) (fig. S3) and tested their effects on H3-K56 acetylation in yeast cells. Nine of these mutants had little effect on H3-K56 acetylation (fig. S4), but two, D89A and DD287 288AA, resulted in the loss of H3-K56 acetylation in yeast cells (Fig. 2E). Each of these three aspartate residues was then mutated to asparagine (N). The D89N mutant cells lost H3-K56 acetylation, and the DD287 288NN mutant cells exhibited a significant reduction in H3-K56 acetylation, whereas the D287N and D288N mutants had little effect (Fig. 2E). Consistent with the effect of these mutants on H3-K56 acetylation in yeast cells, in vitro HAT assays revealed that the Rtt109 mutant proteins D89A, D89N, and DD287 288AA lost the ability to acetylate H3, whereas the Rtt109 mutant DD287 288NN exhibited a reduced level of HAT activity, and the single mutants D287N and D288N had similar levels of activity compared with wild-type enzyme (Fig. 2, F and G). The inability of Rtt109 mutants to acetylate H3 was not likely due to disruption of the overall structure of the mutant proteins, because these Rtt109 mutants still bound Vps75, a known Rtt109-interacting protein (17) (Fig. 2H). These results demonstrate that D89 is essential for the HAT activity, whereas D287 and D288 are not essential, but contribute to this activity. D89 may serve as the deprotonation residue, and D287 and D288 may serve other functions such as binding to histones and/or acetyl-CoA. All of these aspartate residues are conserved among the Rtt109 family members (fig. S3).

Yeast cells expressing mutants lacking H3-K56 acetylation (K56A, K56R) display increased sensitivity toward DNA-damaging agents (6, 8), including camptothecin (CPT), hydroxyurea (HU), and methyl-methanesulfonate (MMS) (16). Therefore, the sensitivity of rtt109Δ cells and rtt109 site-specific mutant cells to these genotoxic agents was determined. The rtt109Δ cells and cells expressing rtt109 aspartate mutants (D89A, D89N, and DD287 288AA) that lacked H3-K56 acetylation exhibited sensitivity toward CPT, HU, and MMS to a degree similar to cells expressing the H3-K56 mutants. Furthermore, the DD287 288NN cells, where H3-K56 acetylation was reduced but not abolished, were not as sensitive as rtt109Δ cells (Fig. 3, A and B). Moreover, rtt109Δ H3-K56R and rtt109Δ H3-K56A double-mutant cells displayed similar sensitivities toward these DNA-damaging agents as either single mutant alone (Fig. 3A and fig. S5). In contrast, cells expressing rtt109 site-specific mutants where H3-K56 acetylation was not affected were resistant to these DNA-damaging agents (Fig. 3B and fig. S6). These results suggest that the ability of Rtt109 to suppress sensitivity toward DNA-damaging agents is mainly mediated by its HAT activity toward H3-K56.

Fig. 3.

Cells lacking Rtt109 and H3-K56 acetylation are sensitive to DNA-damaging agents and exhibit elevated levels of spontaneous Rad52 foci. (A) Cells lacking Rtt109 or containing mutations at H3-K56 are more sensitive to DNA-damaging agents than wild-type cells. Tenfold serial dilutions of yeast cells of the genotype indicated at the left were spotted onto YPD medium alone or YPD medium containing CPT, HU, or MMS. Note that the rtt109Δ H3-K56R mutant cells grew slightly better than rtt109Δ single mutant cells when H3 and H4 were expressed on a plasmid. (B) Cells expressing rtt109 mutants unable to acetylate H3-K56 display a similar sensitivity toward DNA-damaging agents as the rtt109Δ mutant cells. (C and D) Asynchronous rtt109Δ and H3-K56R mutant cells exhibit an increase in the percentage of cells containing Rad52-YFP foci compared with WT cells. Rad52-YFP and differential interference contrast images from a representative field of WT and various mutants are shown (C), and the percentage of cells containing Rad52 foci are calculated (D). Cells were separated into two categories on the basis of whether a cell displayed a bud (S/G2/M) or not (G1).

In budding yeast, Rad52 forms spontaneous foci, predominantly during S and G2-M phases of the cell cycle, and these foci are thought to be sites of repair of DNA lesions (18, 19). Cells with mutations in proteins involved in DNA metabolism, such as Top3 exhibit elevated levels of Rad52 foci, possibly due to an increase in spontaneous chromosome breaks (20). The rtt109Δ and H3-K56R single- and double-mutant cells showed a substantial increase in Rad52 fused with yellow fluorescent protein (Rad52-YFP) foci (Fig. 3, C and D). Moreover, the rtt109Δ H3-K56R double-mutant cells did not exhibit more Rad52 foci than either rtt109Δ or H3-K56R mutant alone (Fig. 3D). Thus, the increase in Rad52-YFP foci observed in rtt109Δ mutant cells appears mainly to be due to loss of H3-K56 acetylation. Supporting this idea, acetylation of four other H3 lysine residues (K9, K14, K18, and K23) was not altered in the rtt109Δ mutant cells (fig. S7). Taken together, these data indicate that Rtt109-mediated acetylation of H3-K56 during S phase protects DNA from damage.

Here we have shown that Rtt109 is a member of a novel HAT family that acetylates H3-K56. The rtt109Δ mutant exhibited a synthetic lethal or slow-growth phenotype with a mutant allele of PCNA (proliferating cell nuclear antigen), pol30-79, which is defective in DNA replication and repair (21), but not with the PCNA mutant allele, pol30-8, which is defective in epigenetic silencing (22) (fig. S8A). The rtt109Δ mutant also exhibited a synthetic lethal/slow growth phenotype with a mutation in DNA polymerase α (fig. S8B) and was previously found to genetically interact with Orc2 and Cdc45 mutations (23, 24). All of these proteins are involved in DNA replication. The genetic interactions between Rtt109 and the proteins involved in DNA replication suggest that the rtt109Δ mutant cells are defective in certain aspects of DNA replication. In support of this idea, the rtt109Δ mutant exhibits synthetic lethal or slow-growth phenotypes with mutations in genes such as RAD52, which are involved in homologous recombination (25), a process that is needed to resolve stalled replication forks (26). Thus, H3-K56 acetylation by Rtt109 is closely linked to DNA replication.

Supporting Online Material

www.sciencemag.org/cgi/content/full/315/5812/653/DC1

Materials and Methods

Figs. S1 to S8

Tables S1 and S2

References

References and Notes

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