Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity

In budding yeast, acetylation of histone H3 lysine 56 (H3-K56) is catalyzed by the Rtt109-Vps75 histone acetyltransferase (HAT) complex, with Rtt109 being the catalytic subunit, and histone chaperone Asf1 is required for this modification. Cells lacking Rtt109 are susceptible to perturbations in DNA replication. However, how Asf1 regulates acetylation of H3-K56 and how loss of H3-K56 acetylation affects DNA replication are unclear. We show that at low concentrations the Rtt109-Vps75 HAT complex acetylates H3-K56 in vitro when H3/H4 is complexed with Asf1, but not H3/H4 tetramers, recapitulating the in vivo requirement of Asf1 for H3-K56 acetylation using recombinant proteins. Moreover, the Rtt109-Vps75 complex interacts with Asf1-H3/H4 but not Asf1. In vivo, the Rtt109-Asf1 interaction is also dependent on the ability of Asf1 to bind H3/H4. Furthermore, the Rtt109 homolog in Schizosaccharomyces pombe (SpRtt109) also displayed an Asf1-dependent H3-K56 HAT activity in vitro. These results indicate that Asf1 regulates H3-K56 acetylation by presenting histones H3 and H4 to Rtt109-Vps575 for acetylation, and this mechanism is likely to be conserved. Finally, we have shown that cells lacking Rtt109 or expressing H3-K56 mutants exhibited significant reduction in the association of three proteins with stalled DNA replication forks and hyper-recombination of replication forks stalled at replication fork barriers of the ribosomal DNA locus compared with wild-type cells. Taken together, these studies provide novel insight into the role of Asf1 in the regulation of H3-K56 acetylation and the function of this modification in DNA replication.     

Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes

Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors, but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109/Asf1 complex, but not the Rtt109/Vps75 complex, being critical for resistance to genotoxic agents.     

Structure of the Rtt109-AcCoA/Vps75 complex and implications for chaperone-mediated histone acetylation

Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27, and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ～12?? diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in?vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in?vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3-H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones before deposition.     

Utilizing Targeted Mass Spectrometry to Demonstrate Asf1-Dependent Increases in Residue Specificity for Rtt109-Vps75 Mediated Histone Acetylation

In Saccharomyces cerevisiae, Rtt109, a lysine acetyltransferase (KAT), associates with a histone chaperone, either Vps75 or Asf1. It has been proposed that these chaperones alter the selectivity of Rtt109 or which residues it preferentially acetylates. In the present study, we utilized a label-free quantitative mass spectrometry-based method to determine the steady-state kinetic parameters of acetylation catalyzed by Rtt109-Vps75 on H3 monomer, H3/H4 tetramer, and H3/H4-Asf1 complex. These results show that among these histone conformations, only H3K9 and H3K23 are significantly acetylated under steady-state conditions and that Asf1 promotes H3/H4 acetylation by Rtt109-Vps75. Asf1 equally increases the Rtt109-Vps75 specificity for both of these residues with a maximum stoichiometry of 1:1 (Asf1 to H3/H4), but does not alter the selectivity between these two residues. These data suggest that the H3/H4-Asf1 complex is a substrate for Rtt109-Vps75 without altering selectivity between residues. The deletion of either Rtt109 or Asf1 in vivo results in the same reduction of H3K9 acetylation, suggesting that Asf1 is required for efficient H3K9 acetylation both in vitro and in vivo. Furthermore, we found that the acetylation preference of Rtt109-Vps75 could be directed to H3K56 when those histones already possess modifications, such as those found on histones purified from chicken erythrocytes. Taken together, Vps75 and Asf1 both enhance Rtt109 acetylation for H3/H4, although via different mechanisms, but have little impact on the residue selectivity. Importantly, these results provide evidence that histone chaperones can work together via interactions with either the enzyme or the substrate to more efficiently acetylate histones.     

Interaction with the histone chaperone Vps75 promotes nuclear localization and HAT activity of Rtt109 in vivo

Modification of histones is critical for the regulation of all chromatin-templated processes. Yeast Rtt109 is a histone acetyltransferase (HAT) that acetylates H3 lysines 9, 27 and 56. Rtt109 associates with and is stabilized by Nap1 family histone chaperone Vps75. Our data suggest Vps75 and Nap1 have some overlapping functions despite their different cellular localization and histone binding specificity. We determined that Vps75 contains a classical nuclear localization signal and is imported by Kap60-Kap95. Rtt109 nuclear localization depends on Vps75, and nuclear localization of the Vps75-Rtt109 complex is not critical for Rtt109-dependent functions, suggesting Rtt109 may be able to acetylate nascent histones before nuclear import. To date, the effects of VPS75 deletion on Rtt109 function had not been separated from the resulting Rtt109 degradation; thus, we used an Rtt109 mutant lacking the Vps75-interaction domain that is stable without Vps75. Our data show that in addition to promoting Rtt109 stability, Vps75 binding is necessary for Rtt109 acetylation of the H3 tail. Direct interaction of Vps75 with H3 likely allows Rtt109 access to the histone tail. Furthermore, our genetic interaction data support the idea of Rtt109-independent functions of Vps75. In summary, our data suggest that Vps75 influences chromatin structure by regulating histone modification and through its histone chaperone functions.     

Understanding histone acetyltransferase Rtt109 structure and function: how many chaperones does it take?

Rtt109 (Regulator of Ty1 Transposition 109) is a fungal-specific histone acetyltransferase required for modification of histone H3 K9, K27 and K56. These acetylations are associated with nascent histone H3 and play an integral role in replication-coupled and repair-coupled nucleosome assembly. Rtt109 is unique among acetyltransferases as it is activated by a histone chaperone; either Vps75 (Vacuolar Protein Sorting 75) or Asf1 (Anti-silencing Function 1). Recent biochemical, structural and genetic studies have shed light on the intricacies of this activation. It is now clear that Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. This reinforces that Asf1 and Vps75 activate Rtt109 via distinct molecular mechanisms. Structures of Rtt109-Vps75 further imply that Vps75 positions histone H3 in the Rtt109 active site. These structures however raise questions regarding the stoichiometry of the Rtt109-Vps75 complex. This has ramifications for determining the physiological Rtt109 substrate.     

Vps75, a new yeast member of the NAP histone chaperone family

Homologues of nucleosome assembly protein 1 (NAP1) are found throughout eukaryotes. Here we identify and characterize a new NAP family histone chaperone from budding yeast, named Vps75. Purified Vps75 preferentially binds histone H3/H4 tetramers and is capable of assembling nucleosomes in vitro. In vivo, Vps75 is associated with the chromatin of both active and inactive genes and telomeres. Others have previously reported that Vps75 forms a complex with Rtt109, required for acetylation of histone H3 lysine 56 (H3 Lys-56). Cells lacking RTT109 are sensitive to hydroxyurea, pointing to a role in replication. We show that VPS75 is not required for H3 Lys-56 acetylation and that vps75Delta cells are insensitive to hydroxyurea, suggesting that although Rtt109 and Vps75 associate and are likely to be functionally connected, they also have separate roles.     

Autoacetylation of the histone acetyltransferase Rtt109

Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear. Here, we examined the mechanism of Lys-290 acetylation and found that Rtt109 catalyzes intramolecular autoacetylation of Lys-290 ～200-times slower than H3 acetylation. Deacetylated Rtt109 was prepared by reacting with a sirtuin protein deacetylase, producing an enzyme with negligible HAT activity. Autoacetylation of Rtt109 restored full HAT activity, indicating that autoacetylation is necessary for HAT activity and is a fully reversible process. To dissect the mechanism of activation, biochemical, and kinetic analyses were performed with Lys-290 variants of the Rtt109-Vps75 complex. We found that autoacetylation of Lys-290 increases the binding affinity for acetyl-CoA and enhances the rate of acetyl-transfer onto histone substrates. This study represents the first detailed investigation of a HAT enzyme regulated by single-site intramolecular autoacetylation.     

A small molecule inhibitor of fungal histone acetyltransferase Rtt109

The histone acetyltransferase Rtt109 is the sole enzyme responsible for acetylation of histone H3 lysine 56 (H3K56) in fungal organisms. Loss of Rtt109 renders fungal cells extremely sensitive to genotoxic agents, and prevents pathogenesis in several clinically important species. Here, via a high throughput chemical screen of >300,000 compounds, we discovered a chemical inhibitor of Rtt109 that does not inhibit other acetyltransferase enzymes. This compound inhibits Rtt109 regardless of which histone chaperone cofactor protein (Asf1 or Vps75) is present, and appears to inhibit Rtt109 via a tight-binding, uncompetitive mechanism.     

Structure and histone binding properties of the Vps75-Rtt109 chaperone-lysine acetyltransferase complex

The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4)(2) tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4)(2). Investigation of Vps75-Rtt109-(H3-H4)(2) and Vps75-(H3-H4)(2) complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.     

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z'' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.     

The Rtt109-Vps75 histone acetyltransferase complex acetylates non-nucleosomal histone H3

Acetylation of lysine 56 of histone H3 (H3-Lys-56) occurs in S phase and disappears during G(2)/M phase of the cell cycle. However, it is not clear how this modification is regulated during the progression of the cell cycle. We and others have shown that the histone acetyltransferase (HAT) Rtt109 is the primary HAT responsible for acetylating H3-Lys-56 in budding yeast. Here we show that Rtt109 forms a complex with Vps75 and that both recombinant Rtt109-Vps75 complexes and native complexes purified from yeast cells acetylate H3 present in H3/H4/H2A/H2B core histones but not other histones. In addition, both recombinant and native Rtt109-Vps75 HAT complexes exhibited no detectable activity toward nucleosomal H3, suggesting that H3-Lys-56 acetylation is at least in part regulated by the inability of Rtt109-Vps75 complexes to acetylate nucleosomal H3 during G(2)/M phase of the cell cycle. Further, Rtt109 bound mutant H3/H4 tetramers composed of histones lacking their N-terminal tail domains less efficiently than wild-type H3/H4 tetramers, and Rtt109-Vps75 complexes displayed reduced HAT activity toward these mutant H3/H4 tetramers. Thus, the N termini of H3/H4 tetramers are required for efficient acetylation of H3 by the Rtt109-Vps75 complex. Taken together, these studies provide insights into how H3-Lys-56 acetylation is regulated during the cell cycle.     

Cell Cycle– and Chaperone-Mediated Regulation of H3K56ac Incorporation in Yeast

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.     

Structural insights into histone H3 lysine 56 acetylation by Rtt109

Histone acetylation plays important roles for the regulation of many fundamental cellular processes. Saccharomyces cerevisiae Rtt109 is an important class of histone acetyltransferases (HATs), which promote genome stability by directly acetylating newly synthesized histone H3 lysine 56 (H3-K56) through an unknown mechanism. Here, we report the crystal structures of Rtt109 at 2.2 A and Rtt109/Acetyl-CoA binary complex at 1.9 A. The structure displays a vise-like topology with mixed three-layered alpha/beta module forming the central module, whose core region resembles the structure of GCN5 HAT domain and P300/CBP HAT domain. Using structural and biochemical analyses, we have discovered the catalytic active site and have identified Asp288 as the deprotonation residue and Lys290 as the autoacetylation residue. We have further proposed the unique H3-K56 anchoring pocket and the potential H3alphaN binding groove. Our work has provided structural insights to understand the acetylation mechanism of H3-K56 by Rtt109.     

The Rtt109 histone acetyltransferase facilitates error-free replication to prevent CAG/CTG repeat contractions

Lysine 56 is acetylated on newly synthesized histone H3 in yeast, Drosophila and mammalian cells. All of the proteins involved in histone H3 lysine 56 (H3K56) acetylation are important for maintaining genome integrity. These include Rtt109, a histone acetyltransferase, responsible for acetylating H3K56, Asf1, a histone H3/H4 chaperone, and Hst3 and Hst4, histone deacetylases which remove the acetyl group from H3K56. Here we demonstrate a new role for Rtt109 and H3K56 acetylation in maintaining repetitive DNA sequences in Saccharomyces cerevisiae. We found that cells lacking RTT109 had a high level of CAG/CTG repeat contractions and a twofold increase in breakage at CAG/CTG repeats. In addition, repeat contractions were significantly increased in cells lacking ASF1 and in an hst3Δhst4Δ double mutant. Because the Rtt107/Rtt101 complex was previously shown to be recruited to stalled replication forks in an Rtt109-dependent manner, we tested whether this complex was involved. However, contractions in rtt109Δ cells were not due to an inability to recruit the Rtt107/Rtt101 complex to repeats, as absence of these proteins had no effect on repeat stability. On the other hand, Dnl4 and Rad51-dependent pathways did play a role in creating some of the repeat contractions in rtt109Δ cells. Our results show that H3K56 acetylation by Rtt109 is important for stabilizing DNA repeats, likely by facilitating proper nucleosome assembly at the replication fork to prevent DNA structure formation and subsequent slippage events or fork breakage.     

Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B)

The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.     

Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights

Asf1 is a histone chaperone that favors histone H3/H4 assembly and disassembly. We solved the structure of the conserved domain of human ASF1A in complex with the C-terminal helix of histone H3 using nuclear magnetic resonance spectroscopy. This structure is fully compatible with an association of ASF1 with the heterodimeric form of histones H3/H4. In our model, ASF1 substitutes for the second H3/H4 heterodimer that is normally found in heterotetrameric H3/H4 complexes. This result constitutes an essential step in the fundamental understanding of the mechanisms of nucleosome assembly by histone chaperones. Point mutations that perturb the Asf1/histone interface were designed from the structure. The decreased binding affinity of the Asf1-H3/H4 complex correlates with decreased levels of H3-K56 acetylation and phenotypic defects in vivo.     

Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. We find that Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.