Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The process of coordinated DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3–H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three his-tone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3–H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3–H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.Key words: Gen5, Rtt109, chromatin, nucleosome assembly, genome integrity     

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.     

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.     

Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.     

CAF-1 and Rtt101p function within the replication-coupled chromatin assembly network to promote H4 K16ac,preventing ectopic silencing

Replication-coupled chromatin assembly is achieved by a network of alternate pathways containing different chromatin assembly factors and histone-modifying enzymes that coordinate deposition of nucleosomes at the replication fork. Here we describe the organization of a CAF-1-dependent pathway in Saccharomyces cerevisiae that regulates acetylation of histone H4 K16. We demonstrate factors that function in this CAF-1-dependent pathway are important for preventing establishment of silenced states at inappropriate genomic sites using a crippled HMR locus as a model, while factors specific to other assembly pathways do not. This CAF-1-dependent pathway required the cullin Rtt101p, but was functionally distinct from an alternate pathway involving Rtt101p-dependent ubiquitination of histone H3 and the chromatin assembly factor Rtt106p. A major implication from this work is that cells have the inherent ability to create different chromatin modification patterns during DNA replication via differential processing and deposition of histones by distinct chromatin assembly pathways within the network.     

O2 sensing: only skin deep?

In this issue, two papers implicate histone H3 lysine 56 acetylation in histone deposition in chromatin. Li et al. (2008) show that acetylation of H3K56 promotes S phase chromatin assembly that is mediated by the histone chaperones CAF-1 and Rtt106. Chen et al. (2008) establish that the acetylation mark promotes chromatin reassembly following DNA double-strand break repair.     

A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance

In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109Δ mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with the production of H3 K56ac and restores histone binding to chromatin assembly proteins CAF-1 and Rtt106. Therefore, we demonstrate that enhanced association with chromatin assembly factors can be accomplished not only by acetylation-mediated charge neutralization of H3K56 but also by the replacement of the positively charged lysine with an acidic residue. These data suggest that removal of the positive charge on lysine 56 is the functionally important consequence of H3K56 acetylation. Additionally, the suppressive function of K56E requires the presence of a second H3 allele, because K56E impairs growth when it is the sole source of histones, even more so than does constitutive H3K56 acetylation. Our studies therefore emphasize how H3 K56ac not only promotes chromatin assembly but also leads to chromosomal malfunction if not removed following histone deposition.     

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.     

Mms1 and Mms22 stabilize the replisome during replication stress

Mms1 and Mms22 form a Cul4(Ddb1)-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101(Mms1/Mms22) ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1-Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.     

Regulation of rtt107 recruitment to stalled DNA replication forks by the cullin rtt101 and the rtt109 acetyltransferase

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.     

Proliferating cell nuclear antigen and ASF1 modulate silent chromatin in Saccharomyces cerevisiae via lysine 56 on histone H3

The formation and stability of epigenetically regulated chromatin is influenced by DNA replication and factors that modulate post-translational modifications on histones. Here we describe evidence that PCNA can affect silencing in Saccharomyces cerevisiae by facilitating deposition of H3 K56ac onto chromosomes. We propose that PCNA participates in this process through a pathway that includes replication factor C, the chromatin assembly factor Asf1p, and the K56-specific acetyltransferase Rtt109p. We show that mutation of POL30 or loss of K56-acetylation in rtt109 and histone H3 mutants enhances silencing at the crippled HMR locus HMRae via restoring Sir binding and that pol30 mutants with silencing phenotypes have reduced levels of H3 K56ac. Although loss of acetylation on H3 K56 was generally compatible with silencing, mutations at this residue also led to defects in silencing an ADE2 reporter at HMR and abolished silencing when combined with cac1 or pol30-8. These silencing phenotypes are analogous to those in asf1 mutants or pol30-6 and pol30-79 mutants with defects in ASF1-dependent pathways. On the basis of these findings, we propose that mutations in DNA replication factors alter acetylation of H3 K56. We show that this defect, in turn, contributes to misregulation of epigenetic processes as well as of cellular responses to DNA damage.     

A Reversible Histone H3 Acetylation Cooperates with Mismatch Repair and Replicative Polymerases in Maintaining Genome Stability

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.     

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between Δmms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a Δmms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.     

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

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.