Recruitment of the type B histone acetyltransferase Hat1p to chromatin is linked to DNA double-strand breaks

Type B histone acetyltransferases are thought to catalyze the acetylation of the NH₂-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH₂-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.     

Nuclear Hat1p complex (NuB4) components participate in DNA repair-linked chromatin reassembly

Chromatin is disassembled and reassembled during DNA repair. To assay chromatin reassembly accompanying DNA double strand break repair, ChIP analysis can be used to monitor the presence of histone H3 near the lesion. The chromatin assembly factor Asf1p, as well as the acetylation of histone H3 lysine 56, have been shown to promote chromatin reassembly when DNA double strand break repair is complete. Using Gal-HO-mediated double strand break repair, we have tested each of the components of the nuclear Hat1p-containing type B histone acetyltransferase complex (NuB4) and have found that they can affect repair-linked chromatin reassembly but that their contributions are not equivalent. In particular, deletion of the catalytic subunit, Hat1p, caused a significant defect in chromatin reassembly. In addition, loss of the histone chaperone Hif1p, when combined with an allele of H3 that mutates lysines 14 and 23 to arginine, has a pronounced effect on chromatin reassembly that is similar to that observed in an asf1Δ. The role of Hat1p and Hif1p is at least partially redundant with the role of Asf1p. Consistent with a more prominent role for Hif1p in chromatin reassembly than either Hat1p or Hat2p, Hif1p exists in complex(es) independent of Hat1p and Hat2p and influences the activity of an H3-specific histone acetyltransferase activity. Our data directly demonstrate the role of the nuclear HAT1 complex (NuB4) components in DNA repair-linked chromatin reassembly.     

Histone Acetyl Transferase 1 Is Essential for Mammalian Development,Genome Stability,and the Processing of Newly Synthesized Histones H3 and H4

Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat1^−/− neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1^−/− mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1^−/− MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly.     

Early‐onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (Hat1)

Histone acetyltransferase 1 (Hat1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of Hat1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that Hat1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of Hat1 results in a significant decrease in lifespan. Defects observed in Hat1^+/? mice are consistent with an early‐onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of Hat1 is linked to the normal aging process as Hat1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from Hat1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. Hat1^+/? mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that Hat1^?/? MEFs exhibit mitochondrial defects suggesting a critical role for Hat1 in mitochondrial function. Taken together, these data show that loss of Hat1 induces multiple hallmarks of early‐onset aging.     

The nuclear Hat1p/Hat2p complex: a molecular link between type B histone acetyltransferases and chromatin assembly

The yeast Hat1p/Hat2p type B histone acetyltransferase complex is localized to both the cytoplasm and nucleus. We isolate the nuclear form of the Hat1p/Hat2p complex and find that it copurifies with the product of the uncharacterized open reading frame YLL022C (named Hif1p). The functional significance of the association of Hif1p with the Hat1p/Hat2p complex is confirmed by the observation that hif1Delta and hat1Delta strains display similar defects in telomeric silencing and DNA double-strand break repair. Hif1p is a histone chaperone that selectively interacts with histones H3 and H4. Hif1p is also a chromatin assembly factor, promoting the deposition of histones in the presence of a yeast cytosolic extract. In vivo, the nuclear Hat1p/Hat2p/Hif1p complex is bound to acetylated histone H4, as well as histone H3. The association of Hif1p with acetylated H4 requires Hat1p and Hat2p providing a link between type B histone acetyltransferases and chromatin assembly.     

Properties of the type B histone acetyltransferase Hat1: H4 tail interaction, site preference, and involvement in DNA repair

The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between Hat1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast Hat1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between Hat1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the hat1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae hat1Delta strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of Hat1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for Hat1 deletion appears to be more variable.     

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.     

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     

Alterations in histone acetylation following exposure to <Superscript>60</Superscript>Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0–2 Gy ⁶⁰Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic?+?r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0–1 Gy ⁶⁰Co γ-rays. SAHA treatment significantly decreased dic?+?r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy ⁶⁰Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by ⁶⁰Co γ-radiation.     

Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing

The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.     

Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair

The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly. The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing. As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents. Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate. The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation. While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks. Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly.     

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.     

p300-mediated Acetylation of Histone H3 Lysine 56 Functions in DNA Damage Response in Mammals

The packaging of newly replicated and repaired DNA into chromatin is crucial for the maintenance of genomic integrity. Acetylation of histone H3 core domain lysine 56 (H3K56ac) has been shown to play a crucial role in compaction of DNA into chromatin following replication and repair in Saccharomyces cerevisiae. However, the occurrence and function of such acetylation has not been reported in mammals. Here we show that H3K56 is acetylated and that this modification is regulated in a cell cycle-dependent manner in mammalian cells. We also demonstrate that the histone acetyltransferase p300 acetylates H3K56 in vitro and in vivo, whereas hSIRT2 and hSIRT3 deacetylate H3K56ac in vivo. Further we show that following DNA damage H3K56 acetylation levels increased, and acetylated H3K56, which is localized at the sites of DNA repair. It also colocalized with other proteins involved in DNA damage signaling pathways such as phospho-ATM, CHK2, and p53. Interestingly, analysis of occurrence of H3K56 acetylation using ChIP-on-chip revealed its genome-wide spread, affecting genes involved in several pathways that are implicated in tumorigenesis such as cell cycle, DNA damage response, DNA repair, and apoptosis.     

Pleiotropic effects of the histone deacetylase Hos2 linked to H4‐K16 deacetylation,H3‐K56 acetylation,and H2A‐S129 phosphorylation in Beauveria bassiana

Histone acetyltransferases and deacetylases maintain dynamics of lysine acetylation/deacetylation on histones and nonhistone substrates involved in gene regulation and cellular events. Hos2 is a Class I histone deacetylases that deacetylates unique histone H4‐K16 site in yeasts. Here, we report that orthologous Hos2 deacetylates H4‐K16 and is also involved in the acetylation of histone H3‐K56 and the phosphorylation of histone H2A‐S129 and cyclin‐dependent kinase 1 CDK1‐Y15 in Beauveria bassiana, a filamentous fungal insect pathogen. These site‐specific modifications are evidenced with hyperacetylated H4‐K16, hypoacetylated H3‐K56, and both hypophosphorylated H2A‐S129 and CDK1‐Y15 in absence of hos2. Consequently, the Δhos2 mutant suffered increased sensitivities to DNA‐damaging and oxidative stresses, disturbed cell cycle, impeded cytokinesis, increased cell size or length, reduced conidiation capacity, altered conidial properties, and attenuated virulence. These phenotypic changes correlated well with dramatic repression of many genes that are essential for DNA damage repair, G₁/S transition and DNA synthesis, hyphal septation, and asexual development. The uncovered ability for Hos2 to directly deacetylate H4‐K16 and to indirectly modify H3‐K56, H2A‐S129, and CDK1‐Y15 provides novel insight into more subtle regulatory role for Hos2 in genomic stability and diverse cellular events in the fungal insect pathogen than those revealed previously in nonentomophathogenic fungi.     

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.