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.     

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.     

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.     

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.     

Effects of acetylation of histone H4 at lysines 8 and 16 on activity of the Hat1 histone acetyltransferase

During nucleosome assembly in vivo, newly synthesized histone H4 is specifically diacetylated on lysines 5 and 12 within the H4 NH(2)-terminal tail domain. The highly conserved "K5/K12" deposition pattern of acetylation is thought to be generated by the Hat1 histone acetyltransferase, which in vivo is found in the HAT-B complex. In the following report, the activity and substrate specificity of the human HAT-B complex and of recombinant yeast Hat1p have been examined, using synthetic H4 NH(2)-terminal peptides as substrates. As expected, the unacetylated H4 peptide was a good substrate for acetylation by yeast Hat1p and human HAT-B, while the K5/K12-diacetylated peptide was not significantly acetylated. Notably, an H4 peptide previously diacetylated on lysines 8 and 16 was a very poor substrate for acetylation by either yeast Hat1p or human HAT-B. Treating the K8/K16-diacetylated peptide with histone deacetylase prior to the HAT-B reaction raised acetylation at K5/K12 to 70-80% of control levels. These results present strong support for the model of H4-Hat1p interaction proposed by Dutnall et al. (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and provide evidence for the first time that site-specific acetylation of histones can regulate the acetylation of other substrate sites.     

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.     

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.     

The Yeast Histone Chaperone Hif1p Functions with RNA in Nucleosome Assembly

Background

Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex) in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.

Results

To identify the factor(s) that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.

Conclusions

These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.     

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.     

Human histone acetyltransferase 1 protein preferentially acetylates H4 histone molecules in H3.1-H4 over H3.3-H4

In mammalian cells, canonical histone H3 (H3.1) and H3 variant (H3.3) differ by five amino acids and are assembled, along with histone H4, into nucleosomes via distinct nucleosome assembly pathways. H3.1-H4 molecules are assembled by histone chaperone CAF-1 in a replication-coupled process, whereas H3.3-H4 are assembled via HIRA in a replication-independent pathway. Newly synthesized histone H4 is acetylated at lysine 5 and 12 (H4K5,12) by histone acetyltransferase 1 (HAT1). However, it remains unclear whether HAT1 and H4K5,12ac differentially regulate these two nucleosome assembly processes. Here, we show that HAT1 binds and acetylates H4 in H3.1-H4 molecules preferentially over H4 in H3.3-H4. Depletion of Hat1, the catalytic subunit of HAT1 complex, results in reduced H3.1 occupancy at H3.1-enriched genes and reduced association of Importin 4 with H3.1, but not H3.3. Finally, depletion of Hat1 or CAF-1p150 leads to changes in expression of a H3.1-enriched gene. These results indicate that HAT1 differentially impacts nucleosome assembly of H3.1-H4 and H3.3-H4.     

Yaf9, a novel NuA4 histone acetyltransferase subunit,is required for the cellular response to spindle stress in yeast

Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.     

Identification of histone demethylases in Saccharomyces cerevisiae

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.     

Crystal structure of yeast Esa1 suggests a unified mechanism for catalysis and substrate binding by histone acetyltransferases

Esa1 is the catalytic subunit of the NuA4 histone acetylase (HAT) complex that acetylates histone H4, and it is a member of the MYST family of HAT proteins that includes the MOZ oncoprotein and the HIV-1 Tat interacting protein Tip60. Here we report the X-ray crystal structure of the HAT domain of Esa1 bound to coenzyme A and investigate the protein's catalytic mechanism. Our data reveal that Esa1 contains a central core domain harboring a putative catalytic base, and flanking domains that are implicated in histone binding. Comparisons with the Gcn5/PCAF and Hat1 proteins suggest a unified mechanism of catalysis and histone binding by HAT proteins, whereby a structurally conserved core domain mediates catalysis, and sequence variability within a structurally related N- and C-terminal scaffold determines substrate specificity.