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.     

Site specificity of yeast histone acetyltransferase B complex in vivo

Saccharomyces cerevisiae Hat1, together with Hat2 and Hif1, forms the histone acetyltransferase B (HAT-B) complex. Previous studies performed with synthetic N-terminal histone H4 peptides found that whereas the HAT-B complex acetylates only Lys12, recombinant Hat1 is able to modify Lys12 and Lys5. Here we demonstrate that both Lys12 and Lys5 of soluble, non-chromatin-bound histone H4 are in vivo targets of acetylation for the yeast HAT-B enzyme. Moreover, coimmunoprecipitation assays revealed that Lys12/Lys5-acetylated histone H4 is bound to the HAT-B complex in the soluble cell fraction. Both Hat1 and Hat2, but not Hif1, are required for the Lys12/Lys5-specific acetylation and for histone H4 binding. HAT-B-dependent acetylation of histone H4 was detected in the soluble fraction of cells at distinct cell cycle stages, and increased when cells accumulated excess histones. Strikingly, histone H3 was not found in any of the immunoprecipitates obtained with the different components of the HAT-B enzyme, indicating the possibility that histone H3 is not together with histone H4 in this complex. Finally, the exchange of Lys for Arg at position 12 of histone H4 did not interfere with histone H4 association with the complex, but prevented acetylation on Lys5 by the HAT-B enzyme, in vivo as well as in vitro.     

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.     

Hif1 is a component of yeast histone acetyltransferase B, a complex mainly localized in the nucleus

Hat1 is the catalytic subunit of the only type B histone acetyltransferase known (HAT-B). The enzyme specifically acetylates lysine 12, and to a lesser extent lysine 5, of free, non-chromatin-bound histone H4. The complex is usually isolated with cytosolic fractions and is thought to be involved in chromatin assembly. The Saccharomyces cerevisiae HAT-B complex also contains Hat2, a protein stimulating Hat1 catalytic activity. We have now identified by two-hybrid experiments Hif1 as both a Hat1- and a histone H4-interacting protein. These interactions were dependent on HAT2, indicating a mediating role for Hat2. Biochemical fractionation and co-immunoprecipitation assays demonstrated that Hif1 is a component of a yeast heterotrimeric HAT-B complex, in which Hat2 bridges Hat1 and Hif1 proteins. In contrast to Hat2, this novel subunit does not appear to regulate Hat1 enzymatic activity. Nevertheless, similarly to Hat1, Hif1 influences telomeric silencing. In a localization analysis by immunofluorescence microscopy on yeast strains expressing tagged versions of Hat1, Hat2, and Hif1, we have found that all three HAT-B proteins are mainly localized in the nucleus. Thus, we propose that the distinction between A- and B-type enzymes should henceforth be based on their capacity to acetylate histones bound to nucleosomes and not on their location within the cell. Finally, by Western blotting assays, we have not detected differences in the in vivo acetylation of H4 lysine 12 (acK12H4) between wild-type and hat1Delta, hat2Delta, or hif1Delta mutant strains, suggesting that the level of HAT-B-dependent acK12H4 may be very low under normal growth conditions.     

Histone H4 from cuttlefish testis is sequentially acetylated. Comparison with acetylation of calf thymus histone H4

The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.     

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.     

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.     

Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3.

Heterochromatin at yeast telomeres and silent mating (HM) loci represses adjacent genes and is formed by the binding and spreading of silencing information regulators (SIR proteins) along histones. This involves the interaction between the C terminus of SIR3 and the N terminus of histone H4. Since H4 is hypoacetylated in heterochromatin we wished to determine whether acetylation is involved in regulating the contacts between SIR3 and H4. Binding of H4 peptide (residues 1-34) acetylated at lysines Lys-5, Lys-8, Lys-12, and Lys-16 to an immobilized SIR3 protein fragment (residues 510-970) was investigated using surface plasmon resonance. We find that acetylation of H4 lysines reduces binding (K(a)) of H4 to SIR3 in a cumulative manner so that the fully acetylated peptide binding is decreased approximately 50-fold relative to unacetylated peptide. Thus, by affecting SIR3-H4 binding, acetylation may regulate the formation of heterochromatin. These data help explain the hypoacetylated state of histone H4 in heterochromatin of eukaryotes.     

Changes in histone modifications during in vitro maturation of porcine oocytes

Nuclear core histone modifications influence chromosome structures and functions. Recently, the involvement of histone acetylations in the cell memory of gene expression has been suggested in mouse oocyte maturation. At present, there is little available data on histone modifications in mammalian oocyte maturation. In the present study, we examined changes in the acetylation of histone H3 lysines 9 (H3K9) and 14 (H3K14), and histone H4 lysines 5 (H4K5), 8 (H4K8) and 12 (H4K12), and trimethylation of H3K9 during in vitro maturation of porcine oocytes. Immunocytochemical analyses revealed that the all of the lysines examined were highly acetylated in the germinal vesicle stage, and this level of acetylation was maintained until the first prometaphase. In the first metaphase, the lysines near the N-terminal end, H3K9 and H4K5, were completely deacetylated. The acetylation of the lysines far from the N-terminal end, H3K14, H4K8, and H4K12, was markedly decreased but still present. The acetylations were increased transiently at the first anaphase and telophase, and then decreased again at the second metaphase to the same level as the first metaphase. Since effective concentrations of trichostatin A (TSA) to inhibit the deacetylation were different in various lysine residues, multiple histone deacetylases (HDACs) were suggested to function during meiotic maturation. The trimethylation of H3K9 was maintained in a high level throughout maturation. These results suggest that the histone acetylation during porcine oocyte maturation is precisely controlled by the cell cycle.     

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.     

Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo

The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74 % decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50 %, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.     

K8 and K12 are biotinylated in human histone H4.

Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, e.g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin-peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modifications. Substitution of a given lysine (positive charge) with glutamate (negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines.     

Processing mechanism and substrate selectivity of the core NuA4 histone acetyltransferase complex

Esa1, an essential MYST histone acetyltransferase found in the yeast piccolo NuA4 complex (picNuA4), is responsible for genome-wide histone H4 and histone H2A acetylation. picNuA4 uniquely catalyzes the rapid tetra-acetylation of nucleosomal H4, though the molecular determinants driving picNuA4 efficiency and specificity have not been defined. Here, we show through rapid substrate trapping experiments that picNuA4 utilizes a nonprocessive mechanism in which picNuA4 dissociates from the substrate after each acetylation event. Quantitative mass spectral analyses indicate that picNuA4 randomly acetylates free and nucleosomal H4, with a small preference for lysines 5, 8, and 12 over lysine 16. Using a series of 24 histone mutants of H4 and H2A, we investigated the parameters affecting catalytic efficiency. Most strikingly, removal of lysine residues did not substantially affect the ability of picNuA4 to acetylate remaining sites, and insertion of an additional lysine into the H4 tail led to rapid quintuple acetylation. Conversion of the native H2A tail to an H4-like sequence resulted in enhanced multisite acetylation. Collectively, the results suggest picNuA4's site selectivity is dictated by accessibility on the nucleosome surface, the relative proximity from the histone fold domain, and a preference for intervening glycine residues with a minimal (n + 2) spacing between lysines. Functionally distinct from other HAT families, the proposed model for picNuA4 represents a unique mechanism of substrate recognition and multisite acetylation.     

Identification of five sites of acetylation in alfalfa histone H4.

Radioactive acetylation in vivo of plant histone H4 of alfalfa, Arabidopsis, tobacco, and carrot revealed five distinct forms of radioactive, acetylated histone. In histone H4 of eukaryotes ranging from fungi to man, acetylation is restricted to four lysines (residues 5, 8, 12, and 16) possibly caused by a quantitative methylation of lysine-20. Chemical and proteolytic fragmentation of the amino terminally blocked alfalfa H4 protein, dynamically acetylated by radioactive acetate in vivo, allowed protein sequencing and identification of selected peptides. Peptide identification was facilitated by analyzing fully characterized calf histone H4 in parallel. Acetylation in vivo of alfalfa histone H4 was restricted to the lysines in the amino-terminal domain of the protein, residues 1-23. Lysine-20 was shown to be free of methylation, as in pea histone H4. This apparently makes lysine-20 accessible as a novel target for histone acetylation. The in vivo pattern of lysine acetylation (16 greater than 12 greater than 8 greater than or equal to 5 = 20) revealed a preference for lysines -16 and -12 without an apparent strict sequential specificity of acetylation.     

Analysis of the histone acetyltransferase B complex of maize embryos.

Purified histone acetyltransferase B (HAT-B) from maize consists of two subunits, p50 and p45. Cloning of the cDNA and genomic DNA encoding the catalytic subunit p50 revealed a consensus motif reminiscent of other acetyltransferases. Internal peptide sequences and immunological studies identified p45 as a protein related to the Retinoblastoma associated protein Rbap. Antibodies against recombinant p50 were able to immunoprecipitate the enzymatic activity of p50 as well as p45. Consistent with the idea that HAT-B is involved in acetylation of newly synthesized histone H4 during DNA replication, mRNA and protein levels are correlated with S-phases during embryo germination. Inhibition of histone deacetylases by HC toxin or Trichostatin A caused a decrease of the in vivo expression of HAT-B mRNA. Regardless of its predominant cytoplasmic localization, a significant proportion of HAT-B-p50 is present in nuclei, irrespective of the cell cycle stage, suggesting an additional nuclear function.