The Rtt109 histone acetyltransferase promotes nucleosome assembly and genome stability by acetylating K9, K27 and K56 on new non-nucleosomal histone H3 molecules through its interactions with either of two distinct histone chaperones, Vps75 or Asf1. H3K9 acetylation, in particular, is evolutionarily conserved from yeast to human cells and is the most prominent site of acetylation in the N-terminal tail of new H3 molecules in S. cerevisiae
(Adkins et al., 2007
; Kuo et al., 1996
). Despite the importance of the acetylation of new histones, the mechanism and the molecular basis of chaperone-mediated histone lysine acetylation specificity have gone largely unexplored.
To explore the mechanism by which histone chaperones promote Rtt109 HAT activity and lysine specificity, we determined the X-ray crystal structure of an Rtt109-AcCoA/Vps75 complex that revealed a 2-fold symmetrical heterotetrameric ring containing an interior cavity of ~12 Å diameter. Biochemical, enzymatic and in vivo studies further demonstrated that Rtt109-Vps75 contacts observed in the crystals are important for optimal H3K9/K27 but not H3K56 acetylation both in vitro and in yeast cells. A comparison of the Rtt109-AcCoA/Vps75 complex with the free Rtt109 and Vps75 proteins also showed that Vps75 binding to Rtt109 does not alter the Rtt109 active site, suggesting that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting histone H3 for acetylation.
We also used information derived from the structure of the Rtt109-AcCoA/Vps75 complex to demonstrate that Asf1 and Vps75 stimulate the HAT activity of Rtt109 via different mechanisms. Along with other groups, we previously reported the identification of residues generally important for Rtt109-mediated H3 acetylation, including Rtt109 residues Asp89, Tyr199, Trp222 and Asp287 (Han et al., 2007b
; Tang et al., 2008a
; Tsubota et al., 2007
). In this study, we extended these findings to residues that play more dedicated roles in Vps75-mediated activation of Rtt109 for H3K9 acetylation. We showed that the 130–179 segment of Rtt109 and other residues that contribute to interaction surfaces between Rtt109 and Vps75 play a particularly important role in Vps75-dependent histone H3K9 acetylation both in vitro
and in yeast cells. We also showed that Rtt109-R292 plays a more important role in Asf1-mediated histone acetylation.
The structure of the Rtt109-AcCoA/Vps75 complex, together with results from in vitro
enzymological assays and analysis of acetylation in yeast cells, also indicate that histone substrate binding occurs within the interior surface of the ring-shaped Rtt109/Vps75 complex. Three lines of evidence support this model. First, the acetyl groups of the two Rtt109-bound AcCoA cofactors in the complex point into the interior of the ring. Second, the internal surface of the ring shows a much higher degree of sequence conservation than the exterior. Third, we have identified several Rtt109 and Vps75 substitution mutations within the interior of the ring that reduce histone H3 acetylation in vitro
and H3K9/K27 but not H3K56 acetylation in vivo
. The 2:2 stoichiometry and 2-fold symmetry of the complex are also consistent with the observation that Vps75 preferentially binds (H3–H4)2
heterotetramers (Selth and Svejstrup, 2007
) and strongly suggests that both H3 molecules of the heterotetramer are acetylated for histone deposition. In this way, the Vps75 histone chaperone serves as a cofactor for Rtt109-mediated acetylation by both appropriately positioning histone H3K9 for acetylation in the Rtt109 active site and ensuring homogenous histone H3 acetylation before H3–H4 deposition into nascent chromatin.
The structure of the Rtt109-AcCoA/Vps75 complex also enabled us to design Rtt109 and Vps75 separation-of-function mutants. These mutations leave H3K56 acetylation unaffected, but completely abolish the H3K9/K27 acetylation that remains in gcn5Δ cells ( and ). Significantly, mutations that selectively perturb the Rtt109/Vps75 enzyme do not exacerbate the phenotypes of gcn5Δ cells nor do they exhibit phenotypes that are commonly observed in mutants where replication-coupled nucleosome assembly is defective. This is likely because of the overlapping substrate specificity of Gcn5, Rtt109/Asf1 and Rtt109/Vps75 for H3K9/K27 and suggests that the Rtt109/Vps75 complex contributes to, but is not essential for replication-coupled nucleosome assembly.
It is currently unclear why several distinct HATs (Hat1, Gcn5, Rtt109/Vps75, Rtt109/Asf1 and possibly others) contribute to the acetylation of new H3/H4 molecules. By analogy with isoenzymes, some of these HATs may not be functionally redundant under certain growth or cellular stress conditions. However, new tools will be necessary to determine whether this is the case. Thus far, studies aimed at assigning functions to the acetylation of newly synthesized histones have been mainly performed by creating yeast strains where H3 and H4 carry several lysine-to-arginine mutations in the N-terminal tails. This experimental strategy has been helpful, but suffers from two limitations. First, these mutations cripple the acetylation of both newly synthesized and pre-existing histones and global disruption of histone acetylation interferes with transcription. Second, phenotypes that result from histone lysine-to-arginine substitutions might be due to mutations of the lysines, rather than the absence of acetylation. Rtt109/Vps75 is an enzyme that exclusively acetylates non-nucleosomal histones at two specific residues in the N-terminal tails of new H3 molecules. Therefore, the structure-based Rtt109 mutants described here, which are specifically defective in H3K9/H3K27, but not H3K56 acetylation, may provide invaluable tools to investigate the elusive function of the acetylation of the N-terminal tails of newly synthesized histones.