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.     

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.     

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     

Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei

Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G₂/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G₂/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.     

HST3/HST4-dependent deacetylation of lysine 56 of histone H3 in silent chromatin

The composition of posttranslational modifications on newly synthesized histones must be altered upon their incorporation into chromatin. These changes are necessary to maintain the same gene expression state at individual chromosomal loci before and after DNA replication. We have examined how one modification that occurs on newly synthesized histone H3, acetylation of K56, influences gene expression at epigenetically regulated loci in Saccharomyces cerevisiae. H3 K56 is acetylated by Rtt109p before its incorporation into chromatin during S phase, and this modification is then removed by the NAD⁺-dependent deacetylases Hst3p and Hst4p during G2/M phase. We found silenced loci maintain H3 K56 in a hypoacetylated state, and the absence of this modification in rtt109 mutants was compatible with HM and telomeric silencing. In contrast, loss of HST3 and HST4 resulted in hyperacetylation of H3 K56 within silent loci and telomeric silencing defects, despite the continued presence of Sir2p throughout these loci. These silencing defects in hst3Δ hst4Δ mutants could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p could not restore silencing in hst3Δ hst4Δ mutants. Together, our findings argue that HST3 HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.     

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.     

Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.     

The histone chaperone ASF1 regulates the activation of ATM and DNA-PKcs in response to DNA double-strand breaks

The Ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are activated by DNA double-strand breaks (DSBs). These DSBs occur in the context of chromatin but how chromatin influences the activation of these kinases is not known. Here we show that loss of the replication-dependent chromatin assembly factors ASF1A/B or CAF-1 compromises ATM activation, while augmenting DNA-PKcs activation, in response to DNA DSBs. Cells deficient in ASF1A/B or CAF-1 exhibit reduced histone H4 lysine 16 acetylation (H4K16ac), a histone mark known to promote ATM activation. ASF1A interacts with the histone acetyl transferase, hMOF that mediates H4K16ac. ASF1A depletion leads to increased recruitment of DNA-PKcs to DSBs. We propose normal chromatin assembly and H4K16ac during DNA replication is required to regulate ATM and DNA-PKcs activity in response to the subsequent induction of DNA DSBs.     

Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly

In mammals, heterochromatin is characterized by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone H3. It is currently unclear whether there is a coordinated transmission of these two epigenetic modifications through DNA replication. Here we show that the methyl-CpG binding protein MBD1 forms a stable complex with histone H3-K9 methylase SETDB1. Moreover, during DNA replication, MBD1 recruits SETDB1 to the large subunit of chromatin assembly factor CAF-1 to form an S phase-specific CAF-1/MBD1/SETDB1 complex that facilitates methylation of H3-K9 during replication-coupled chromatin assembly. In the absence of MBD1, H3-K9 methylation is lost at multiple genomic loci and results in activation of p53BP2 gene, normally repressed by MBD1 in HeLa cells. Our data suggest a model in which H3-K9 methylation by SETDB1 is dependent on MBD1 and is heritably maintained through DNA replication to support the formation of stable heterochromatin at methylated DNA.     

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.     

In vivo study of the nucleosome assembly functions of ASF1 histone chaperones in human cells

Histone chaperones have been implicated in nucleosome assembly and disassembly as well as histone modification. ASF1 is a highly conserved histone H3/H4 chaperone that synergizes in vitro with two other histone chaperones, chromatin assembly factor 1 (CAF-1) and histone repression A factor (HIRA), in DNA synthesis-coupled and DNA synthesis-independent nucleosome assembly. Here, we identify mutants of histones H3.1 and H3.3 that are unable to interact with human ASF1A and ASF1B isoforms but that are still competent to bind CAF-1 and HIRA, respectively. We show that these mutant histones are inefficiently deposited into chromatin in vivo. Furthermore, we found that both ASF1A and ASF1B participate in the DNA synthesis-independent deposition of H3.3 in HeLa cells, thus highlighting an unexpected role for ASF1B in this pathway. This pathway does not require interaction of ASF1 with HIRA. We provide the first direct determination that ASF1A and ASF1B play a role in the efficiency of nucleosome assembly in vivo in human cells.     

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.     

The histone chaperone Asf1p mediates global chromatin disassembly in vivo

The packaging of the eukaryotic genome into chromatin is likely to be mediated by chromatin assembly factors, including histone chaperones. We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1) in vivo. Analysis of chromatin structure by accessibility to micrococcal nuclease and DNase I digestion demonstrated that the chromatin from CAF-1 mutant yeast has increased accessibility to these enzymes. In agreement, the supercoiling of the endogenous 2mu plasmid is reduced in yeast lacking CAF-1. These results indicate that CAF-1 mutant yeast globally under-assemble their genome into chromatin, consistent with a role for CAF-1 in chromatin assembly in vivo. By contrast, asf1 mutants globally over-assemble their genome into chromatin, as suggested by decreased accessibility of their chromatin to micrococcal nuclease and DNase I digestion and increased supercoiling of the endogenous 2mu plasmid. Deletion of ASF1 causes a striking loss of acetylation on histone H3 lysine 9, but this is not responsible for the altered chromatin structure in asf1 mutants. These data indicate that Asf1p may have a global role in chromatin disassembly and an unexpected role in histone acetylation in vivo.     

A novel role for histone chaperones CAF-1 and Rtt106p in heterochromatin silencing

The histone chaperones CAF-1 and Rtt106p are required for heterochromatin silencing in the yeast Saccharomyces cerevisiae. Although it has been suggested that CAF-1 is involved in the maintenance of heterochromatin silencing, their exact functions during this process are not well understood. Here, we show that CAF-1 and Rtt106p are involved in the early stages of heterochromatin formation. The binding of Sir proteins to telomeric heterochromatin is significantly reduced and, additionally, Sir proteins are mislocalized in cells lacking CAF-1 and Rtt106p. At the HMR locus, CAF-1 and Rtt106p are required for the initial recruitment of Sir2p and Sir3p, but not Sir4p, to the HMR-E silencer, where silencing initiates, as well as the efficient spreading of all of these Sir proteins to the distal a1 gene. Moreover, silencing at the HMR locus is dramatically reduced in cells lacking CAF-1, Rtt106p, and Sir1p. Thus, these studies reveal a novel role for CAF-1 and Rtt106p in epigenetic silencing and indicate that the spreading of heterochromatin, a poorly understood process, requires histone chaperones.     

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.     

The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents

The removal of histones from DNA and their subsequent replacement is likely to be necessary for all processes that require access to the DNA sequence in eukaryotic cells. The histone chaperone chromatin assembly factor 1 (CAF-1) mediates histone H3-H4 assembly during DNA replication and nucleotide excision repair in vitro. We have found that budding yeast deleted for the genes encoding CAF-1 are highly sensitive to double-strand DNA-damaging agents. Our genetic analyses indicate that CAF-1 plays a role in both homologous recombination and nonhomologous end-joining pathways and that the function of CAF-1 during double-strand repair is distinct from that of another histone H3-H4 chaperone, anti-silencing function 1 (ASF1). CAF-1 does not protect the genome by assembling it into a damage-resistant chromatin structure, because induction of CAF-1 after DNA damage is sufficient to restore viability. Furthermore, CAF-1 is not required for repair of the DNA per se or for DNA damage checkpoint function. CAF-1-mediated resistance to DNA damage is dependent on the ability of CAF-1 to bind PCNA, indicating that PCNA may recruit CAF-1 to sites of double-strand DNA repair. We propose that CAF-1 has an essential role in assembling chromatin during double-strand-DNA repair.     

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.