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R. Sendra, Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, C/Dr Moliner 50, 46100-Burjassot, València, Spain Fax: +34 96 354 4635 Tel: +34 96 354 3015 E-mail: firstname.lastname@example.org
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.
Histone acetylation is a highly dynamic post-translational modification involved in the regulation of chromatin activity in eukaryotic organisms [1,2]. Although the mechanism is not completely understood, the long-known link between histone acetylation and gene expression was definitively settled by the identification of a number of transcriptional regulators as histone acetyltransferases (HATs) and histone deacetylases. Acetylation influences transcription by facilitating the access of the transcriptional machinery to the DNA sequence and by creating specific recognition sites for regulatory proteins that promote transcription .
Histone acetylation has also been proposed to be involved in chromatin assembly during replication [1,3]. This notion emerged from the finding in different eukaryotic organisms that newly synthesized histones are acetylated [4,5], and deacetylated shortly after their incorporation into chromatin . In many eukaryotes, newly synthesized histone H4 assembled onto nascent DNA is diacetylated on Lys5 and Lys12 [5,7]. The N-terminus of newly synthesized histone H3 is also acetylated, but in a more heterogeneous and less conserved manner [5,8,9]. It is considered that the acetylation of histones may somehow favor their deposition onto DNA mediated through specific interactions with histone chaperones .
The enzyme that is assumed to catalyze the specific acetylation of newly synthesized histone H4 on its N-terminal tail is the type B HAT, HAT-B complex. Enzymes operationally classified as type B, in contrast to type A, only acetylate histones not associated with DNA, and are not involved in transcriptional regulation. HAT-B enzymes were originally isolated from cytosolic extracts [10–15], but several immunolocalization analyses have indicated a mainly nuclear localization [16–20]. In vitro, native HAT-B enzymes from a wide variety of species establish the specific Lys5/Lys12 acetylation pattern characteristic of newly synthesized histone H4 [10,13,15–17,21–23]. In the yeast Saccharomyces cerevisiae, the HAT-B complex consists of at least three protein subunits [19,20]: the catalytic subunit, Hat1; the enzymatic activity stimulatory protein, Hat2 ; and Hif1, which, in vitro, has histone chaperone and chromatin assembly activity . Recently, Hat1 and Hat2 have been found to interact with the origin recognition complex, suggesting a novel role for the Hat1–Hat2 subcomplex at the replication fork . It has been reported that although recombinant Hat1 is able to modify Lys5 and Lys12 [13,25], the isolated HAT-B complex exclusively acetylates Lys12 of histone H4 [13,22]. Deletions of HAT1, HAT2 or HIF1 produce no apparent phenotype [13,19,20,22], but combined with specific mutations in the N-terminus of histone H3, cause defects in both telomeric gene silencing [19,20,26] and resistance to DNA-damaging agents [20,27]. Such defects are reproduced by the substitution of Lys for Arg at position 12 of histone H4, but not at position 5 [26,27]. Moreover, Hat1 is recruited to the sites of DNA double-strand breaks, where it is specifically required for the histone H4 acetylation on Lys12, but apparently not on Lys5 .
Despite many correlations linking the acetylation of histone H4 with chromatin assembly, direct evidence actually indicates that the specific histone H4 Lys5/Lys12 diacetylation pattern, and also the HAT-B enzymes that generate it, are dispensable for this process. In yeast, the substitution mutation of Lys5 and Lys12 of histone H4, in combination with deletion of the histone H3 N-terminus, does not result in defective chromatin assembly, either in vitro or in vivo , although the acetylation state of newly synthesized yeast histone H4 is not known. Likewise, in chicken DT40 cells, it has been shown that HAT1 is not necessary for replication-coupled chromatin assembly . Thus, the biological role of the conserved Lys5/Lys12 acetylation of histone H4 and hence the function of the HAT-B enzymes found in all eukaryotes are elusive.
Many reports have described the characterization and the site specificity of type B enzymes from different species in vitro [10,13,15–17,21–23,31], but analyses of their in vivo specificity are few and not at all conclusive . Only recently has it been demonstrated in chicken DT40 cells that the homozygous HAT1 deletion results in a reduced diacetylation level on Lys5 and Lys12 of histone H4 in a cytosolic extract . S. cerevisiae Hat1 was the first HAT to be identified , and moreover its biochemical properties, both as an isolated subunit and as part of the HAT-B complex [13,19,20,25,31–33], have been studied. Despite all these studies, its in vivo site specificity has not been directly ascertained.
In this article, we demonstrate that both Lys12 and Lys5 of non-chromatin-bound histone H4 are authentic targets of acetylation for the S. cerevisiae HAT-B complex in vivo. Moreover, these positions are acetylated in histone H4 associated with the HAT-B enzyme from the yeast soluble fraction. The requirements for the distinct components of the complex for the acetylation and the association of histone H4 have also been analyzed.
Direct identification of Lys12 and Lys5 of soluble, non-chromatin-bound histone H4 as in vivo targets of acetylation by yeast Hat1
Previous work in our laboratory failed to detect any defect in the in vivo steady-state level of acetylation on Lys12 of histone H4 in hat1, hat2 or hif1 null mutant strains as compared to the wild-type under normal growth conditions . The apparent independence of histone H4 Lys12 acetylation from the HAT-B enzyme in vivo was actually interpreted as a consequence of the very short half-life of this modification, which would make detection difficult.
We persisted in investigating the in vivo specificity of the yeast HAT-B complex, and found that incubation of cells with hydroxyurea (HU) resulted in an increase of the histone H4 isoform with acetylated Lys12 (H4K12ac) in a HAT1-dependent manner (Fig. 1A). HU is a ribonucleotide reductase inhibitor that causes a depletion of deoxynucleotides, and thereby slows down DNA synthesis. The acetylation analysis was carried out by immunoblotting with a specific antibody to H4K12ac. Cells were incubated in the presence of 200 mm HU (a concentration commonly used to synchronize yeast cultures) for the indicated time periods. In wild-type cells, HU promoted acetylation of histone H4 Lys12, which is reflected by an increase in the H4K12ac level 2 h after HU addition. In contrast, the H4K12ac amount did not significantly change in hat1Δ mutant cells, even after 12 h of incubation (Fig. 1A). Importantly, an antibody against the C-terminus of histone H3 (anti-H3Ct), used as a control for histone loading, did not detect differences in the amount of histone H3 between the two strains, indicating that cells lacking Hat1 display normal levels of histones during the course of HU treatment.
To investigate whether HU induces an increase of the Hat1 protein, we used a yeast strain expressing a hemagglutinin (HA)-tagged version of Hat1. The Hat1–HA protein level did not increase with HU incubation time, but actually slightly diminished (Fig. 1B). Apparently, HU treatment does not alters the enzymatic activity of the HAT-B complex, as the chromatographic HAT profiles and activity levels, in particular that corresponding to the HAT-B peaks, were very similar in HU-treated and untreated cells (supplementary Fig. S1).
We investigated whether HAT1-dependent histone H4 Lys12 acetylation was also increased in response to other genotoxic agents, such as methylmethanesulfonate, phleomycin, and 4-nitroquinoline n-oxide (4NQO). Like HU, these other agents increased the amount of H4K12ac in wild-type but not in hat1Δ cells (supplementary Fig. S2). Fluorescence-activated cell sorting (FACS) analysis (supplementary Fig. S2) revealed a certain degree of qualitative correlation between the H4K12ac level and the enrichment of the culture in S-phase cells. The most potent effect on both was generated by HU.
In order to further examine the effect of HAT1 deletion on acetylation of histone H4 Lys12, we purified histones from wild-type and hat1Δ mutant yeast chromatin, before and after incubation with 200 mm HU for 3 h. In agreement with our previous results , immunoblotting analysis revealed no difference in histone H4 Lys12 acetylation between purified histones from wild-type and mutant cells left without HU treatment. However, in striking contrast to the results obtained with whole cell extract (WCE), we did not observe a significant difference in histone H4 Lys12 acetylation between the two strains after HU incubation (Fig. 2A). As histones were obtained from isolated chromatin, these results show that HU-induced, Hat1-dependent histone H4 acetylation (Fig. 1A) is restricted to non-chromatin-bound, soluble, ‘free’ histone H4. To investigate this further in yeast, spheroplasts of wild-type and hat1Δ cells (HU-treated and untreated) were lysed and fractionated by centrifugation into soluble and chromatin pellet fractions, as shown in Fig. 2B. A significant amount of H4K12ac was found in the soluble fraction of wild-type cells after incubation with HU, but not in hat1Δ mutant cells (Fig. 2C). H4K12ac was even detected in the soluble fraction of untreated wild-type cells, although its level increased substantially after treatment with HU. In addition, antibodies against the recombinant yeast full-length histone H4 (anti-ryH4) and the C-terminus of histone H3 (anti-H3Ct), which recognize the corresponding histones independently of the modification state, revealed that HU treatment increased the amount of soluble histone H4 and histone H3, as had been previously described [4,15,34]. Such an accumulation of histones was identical in wild-type and hat1Δ mutant cells. With respect to the chromatin fractions, histone H4 Lys12 acetylation was not significantly different between wild-type and hat1Δ cells, supporting the results obtained with purified histones.
We investigated the requirement for Hat1 for acetylation of other acetylatable positions on histone H4 and histone H3 in the soluble fraction (Fig. 3). The results clearly indicate that histone H4 Lys5 is an authentic target for Hat1 in vivo. As shown in the immunoblot in Fig. 3, like H4K12ac, the histone H4 isoform with acetylated Lys5 (H4K5ac) was detected in the soluble fraction of wild-type cells, but not of hat1Δ mutant cells. In addition, HU treatment also increased the amount of Hat1-dependent H4K5ac. In contrast, acetylation at the other potentially acetylatable sites within the histone H4 N-terminus, Lys8 and Lys16, was hardly visible on soluble histone H4, although strong bands on histone H4 in purified control histones were observed. In any case, their acetylation levels were independent of Hat1.
In budding yeast, there is evidence that the N-terminal tail of newly synthesized histone H3 is monoacetylated preferentially on Lys9, but also on Lys14, Lys23, or Lys27 . Except for Gcn5, which is responsible for histone H3 Lys9 acetylation , the acetylation enzymes for the other positions are unknown. We detected histone H3 acetylated at these positions in the soluble fraction, although with varying degrees of intensity. Except for the histone H3 isoform with acetylated Lys14, which apparently did not change, the other three histone H3 isoforms increased after HU treatment. In neither case did loss of Hat1 have any effect on acetylation at these Lys residues (Fig. 3).
Recent evidence also indicates acetylation in the globular domains of histone H3 and histone H4. In yeast, acetylation of histone H3 Lys56 and histone H4 Lys91 has been described, and both seem to be linked to nucleosome assembly [35,36]. As shown in Fig. 3, the histone H3 isoform with acetylated Lys56 and the histone H4 isoform with acetylated Lys91 were detected in the soluble fraction, and the levels of both were significantly increased by HU treatment, but the amount of neither of them was dependent on the presence of Hat1.
Although some caution must accompany the interpretation of the immunoblotting assays, due to a possible lack of reactivity or specificity of the antibodies, our results indicate that Hat1 is apparently not involved in the acetylation of any site on soluble histone H4 and histone H3 except for Lys12 and Lys5 of histone H4.
Involvement of different components of the yeast HAT-B complex in the acetylation of soluble histone H4
We examined the presence of histone H4 acetylation on Lys12 and Lys5 in soluble fractions obtained from wild-type and hat1Δ, hat2Δ and hif1Δ deletion strains. Deletion of the HAT2 gene caused a considerable reduction in the acetylation of Lys12 and Lys5 of soluble histone H4 (Fig. 4). Consistently, very low immunosignals were also obtained in soluble fractions of HU-treated hat1Δ and hat2Δ cells. In contrast, the levels of Lys12 and Lys5 acetylation were equal in wild-type and hif1Δ soluble fractions. On the other hand, the amount of total soluble histone H4 was similar in all four strains, and was equally increased by HU treatment (as revealed with anti-ryH4). These data indicate that Hat2, but not Hif1, participates in the catalytic function of the HAT-B complex in vivo.
Hat1-dependent acetylation of soluble histone H4 throughout the cell cycle and in Rad53-deficient cells
We previously observed fairly constant levels of yeast Hat1 protein throughout the cell cycle . We therefore checked the presence of soluble H4K12ac at distinct cell cycle stages. Wild-type and hat1Δ mutant cells growing asynchronously were left without treatment or incubated with either α-factor, which arrests cells in G1 phase, or hydroxyurea or nocodazole, which prevent the G2/M transition, or transferred to minimal medium without a nitrogen source, which arrests cells in G0 phase. The cell cycle phases of the arrested cells were confirmed by DNA flow cytometry. Soluble histone H4 Hat1-dependently acetylated on Lys12 was present in cells arrested at all cell cycle stages, G1, S, G2/M and also G0 (Fig. 5A). Similar results were obtained with cells at different cell cycle stages from synchronized cultures by release from a α-factor block  (results not shown).
In S. cerevisiae, the checkpoint protein kinase Rad53 regulates histone protein levels, and thus Rad53-deficient yeast cells exhibit abnormally high amounts of soluble histones . We therefore investigated whether such an excess of soluble histone H4 is also acetylated by Hat1. For this purpose, we deleted the HAT1 gene in wild-type and rad53Δ mutant strains, and examined the levels of H4K12ac in the corresponding soluble fractions (Fig. 5B). As expected, asynchronously growing rad53Δ mutant cells displayed a higher amount of soluble histone H4 than wild-type cells, but only the excess soluble histone H4 from HAT1 cells was acetylated on Lys12. The accumulation of HAT-B-dependent acetylation of Lys12 in Rad53-deficient cells was further confirmed on yeast strains harboring the chromosomal RAD53 gene under the glucose-switched off GAL1 promoter in wild-type, hat1Δ, hat2Δ or hif1Δ strains (supplementary Fig. S3). Results showed that Lys12 acetylation of excess soluble histone H4 present in Rad53-deficient cells was absolutely dependent on Hat1 and Hat2, but not on Hif1.
Histone H4 acetylated on Lys12 and Lys5 is associated with the HAT-B complex in the yeast soluble fraction
To gain further insights into the organization and the molecular determinants of the yeast HAT-B complex, we attempted to determine: (a) whether HAT-B enzyme, present in the soluble fraction, contains associated histone H4; (b) the acetylated sites; and (c) the involvement of the different HAT-B components in histone H4 binding. To address these questions, we performed immunoprecipitation experiments with soluble extracts from yeast strains that express tagged forms of each of the three components of the HAT-B complex (Hat1–HA, Hat2–HA, and Hif1–Myc). Immunoprecipitates (bound fractions), input materials and unbound materials were examined by immunoblotting with antibodies against histone H4, histone H3, and acetylated isoforms. All three HAT-B components, Hat1, Hat2 (Fig. 6A, lanes 6 and 15, respectively) and Hif1 (Fig. 6B, lane 27) coimmunoprecipitated H4K12ac. Furthermore, histone H4 present in the soluble extracts from yeast cells lacking Hat1 or Hat2 was not coprecipitated with any of the other complex components (Fig. 6A, lanes 9 and 21; and Fig. 6B, lanes 30 and 33), indicating that both Hat1 and Hat2 are necessary for histone H4 binding. In contrast, both Hat1 and Hat2 were still able to coprecipitate H4K12ac in the absence of Hif1 (Fig. 6A, lanes 12 and 18), indicating that Hif1 is dispensable for the interaction of histone H4 with Hat1/Hat2. Results corresponding to Fig. 6A,B were entirely reproduced when blots were probed with the antibody to H4K5ac (results not shown).
When the blots were probed with anti-H3Ct, an immunosignal was not obtained in any of the immunoprecipitates of Hat1, Hat2, or Hif1 (Fig. 6A, lanes 6, 12, 15 and 18; and Fig. 6B, lane 27). These results are disturbing, because it is assumed that histone H3 and histone H4 form tetramers  or dimers , with an equal stoichiometry. It is well known that histone H3 is particularly susceptible to proteolytic degradation. We cannot completely rule out the possibility that histone H3 proteolysis is also occurring in yeast soluble extracts in our experiments, but its presence in input and unbound fractions argues against this possibility. Moreover, intact histone H3, and also H4K12ac, were detected in immunoprecipitates from soluble extracts of cells expressing Flag-tagged Cac1 or Asf1, two histone H3/H4 chaperones (Fig. 6C). These additional controls also indicate the absence of specific histone H3 degradation under our immunoprecipitation assay conditions. Likewise, none of the specific antibodies to acetylated histone H3 used in Fig. 3 generated immunosignals corresponding to histone H3 on the HAT-B complex immunoprecipitates (results not shown). Altogether, our data suggest that histone H3 is not part of the HAT-B complex in the soluble fraction of yeast cells.
In vivo, the HAT-B complex requires an acetylatable Lys at position 12 for acetylation on Lys5, but not for binding histone H4
Recombinant yeast Hat1, as well as native HAT-B enzymes from various species, modify histone H4 in vitro on Lys12 preferentially over Lys5 [13,17,22,23,25,31]. Thus, HAT-B complex acetyltransferase activity results in an ordered acetylation, with Lys12 being acetylated before Lys5 [23,31]. To investigate the in vivo requirement for histone H4 Lys12 on the acetylation of Lys5 and also on the histone H4–HAT-B complex association, we made use of yeast strains expressing wild-type or a K12R substitution mutant histone H4 (H4K12R) from a centromeric plasmid as the only source of histone H4. In addition, these strains contained Hat1 or Hif1 tagged with the HA epitope. First, we checked that K12R substitution does not interfere with the recognition of H4K5ac by the antibody to H4K5ac (supplementary Fig. S4). In agreement with this, antibody to H4K5ac yielded bands with similar intensity on WCEs prepared from cells containing wild-type histone H4 or H4K12R (Fig. 7A). However, when soluble fractions were analyzed with the same antibody, cells expressing wild-type histone H4 were characterized by a well-defined band, whereas in cells expressing H4K12R, only a very weak signal was detected (Fig. 7A). These results imply that an acetylatable Lys at position 12 is essential for the efficient acetylation of Lys5 of soluble histone H4.
Analyses of histone H4 association with the HAT-B complex were performed by coimmunoprecipitation and subsequent immunoblotting. As expected, the antibody to H4K12ac (control) generated a signal in immunoprecipitates from soluble extracts containing wild-type histone H4 (Fig. 7B, lanes 3 and 9) but not in those containing H4K12R (Fig. 7B, lanes 6 and 12). Remarkably, anti-ryH4 revealed that as much Hat1–HA as Hif1–HA coimmunoprecipitated both wild-type and K12R mutant histone H4 (Fig. 7B, lanes 3, 6, 9 and 12). This finding demonstrates that Lys12 and its acetylation are not involved in the binding of histone H4 to the HAT-B complex. In addition, the antibody to H4K5ac revealed that H4K12R coprecipitated with Hat1 was acetylated very weakly on Lys5 (Fig. 7B, lane 6).
Finally, we investigated the in vitro activity of yeast Hat1 and Hat1-dependent type B complex towards wild-type or K12R mutant histone H4 in purified yeast core histones. A HAT-B complex, partially purified by anion exchange chromatography of soluble extracts from wild-type cells, was used in the enzymatic assays (Fig. 8A). As a control, equivalent chromatographic fractions from a hat1Δ mutant strain were also assayed. A recombinant yeast Hat1 (ryHat1) was also included in the assays (Fig. 8C). Whereas wild-type histone H4 was efficiently acetylated by native HAT-B enzyme, H4K12R was modified very weakly, if at all (Fig. 8A). Thus, in vitro as well as in vivo, Lys5 in H4K12R represents only a very poor substrate for the yeast HAT-B complex. Importantly, this finding supports the in vivo results that an acetylatable Lys at position 12 of soluble histone H4 is required for further modification on Lys5. Moreover, immunoblotting with antibody to H4K5ac, after HAT assays, revealed that the yeast HAT-B complex indeed acetylates Lys5 on wild-type histone H4. Figure 8B shows that incubation of yeast or chicken histones with acetyl-CoA and chromatographic fractions containing the HAT-B enzyme increased the H4K5ac immunosignal, which was not the case when hat1Δ fractions (or buffer solution) were used. Thus, these results demonstrate that the yeast HAT-B complex acetylates Lys5 in the context of intact histone H4 in vitro.
In contrast to Hat1 as part of the HAT-B complex, recombinant Hat1 was able to acetylate H4K12R, although to a lesser extent than wild-type histone H4 (Fig. 8C).
The main result of this study has been the demonstration that the S. cerevisiae HAT-B complex is involved in the acetylation of both Lys12 and Lys5 of soluble histone H4 in vivo. We showed that both Hat1 and Hat2 are essential for this specific histone H4 Lys12/Lys5 acetylation, whereas the third component of the complex, Hif1, is not. These results are in agreement with in vitro data indicating that the absence of Hif1 alters neither the activity nor the specificity of the rest of the HAT-B enzyme [19,20], whereas Hat2 has the ability to enhance the catalytic potential of the Hat1 subunit . Therefore, the functional role of Hif1 in the HAT-B complex is downstream of the acetylation of histone H4.
It had been previously determined that, in vitro, the yeast HAT-B complex exclusively acetylates Lys12 on histone H4 N-terminal synthetic peptides [13,22], whereas recombinant Hat1 modifies Lys12 and Lys5 [13,25]. Moreover, in yeast cells, indirect evidence has shown the involvement of Hat1 in the modification of Lys12, although not of Lys5, of histone H4 [26–28]. However, we have demonstrated that Lys5 is a bona fide target for acetylation by the yeast HAT-B complex in vivo. Remarkably, the inability of the HAT-B complex to acetylate histone H4 containing the K12R substitution, both in vivo and in vitro, indicates a sequential order of acetylation, with Lys12 being modified before Lys5. As Arg mimics unacetylated Lys, this inability to use Lys5 as a target on H4K12R strongly suggests that acetylation of Lys12 is a prerequisite for the subsequent acetylation of Lys5. An identical sequential order of site usage has been found for HAT-B enzymes isolated from maize and rat liver , and also from human cells , in vitro. Yeast recombinant Hat1 is able to modify H4K12R, which complements previous findings showing less stringent site specificity for Hat1 alone [13,25], and suggests the involvement of the other complex components in the site selection mechanism. In contrast to previous studies [13,22], we have found that, even in vitro, the yeast HAT-B complex modifies Lys5 as well as Lys12, just like other type B enzymes from diverse species [10,15,16,23,31]. The reason for this discrepancy may be the different substrates used. Earlier experiments, indicating Lys12 as the only acetylation site, were carried out with histone H4 N-terminal peptides [13,22]. We have used whole histone H4 of yeast or chicken erythrocytes, as in numerous other studies [10,15,16,23,31]. We suggest that an interaction of histone H4, beyond its N-terminus, with the HAT-B complex is needed in order to establish a physiological acetylation pattern. Interestingly, in line with this idea, in vitro, the human HAT-B enzyme acetylates the histone H4(1–41) N-terminal fragment more efficiently than the shorter histone H4(1–34) fragment . It seems reasonable that at least part of the differential potential as substrates of the two peptides is due to different positions being modified by the enzyme. Our data indicate a site specificity of the yeast HAT-B complex that exactly matches the specificity of other type B HATs [10,15,16,23,31], thus pinpointing a much higher degree of conservation of these enzymes than previously assumed.
The levels of acetylation on Lys16 and Lys8 are extremely low in the soluble histone H4 of wild-type cells, and also nearly undetectable on Lys12 and Lys5 in hat1Δ cells. These observations strongly suggest that, in yeast, Lys12 and Lys5 are the only N-terminal positions that are acetylated in soluble histone H4, and that the HAT-B complex must be the only enzyme involved in this specific modification. However, in contrast with these results, histone H4 acetylated on Lys16 and, to a greater degree, on Lys8 has been detected in the cytoplasmic fraction of chicken DT40 cells. In addition, chicken cells lacking Hat1 retain a significant level of histone H4 Lys12 and Lys5 acetylation in the soluble fraction . Although the acetylation pattern of the soluble H4 histones of yeast and chicken could be different, contamination with chromatin could also explain the presence of Hat1-independent histone H4 Lys12 and Lys5 acetylations and other acetylated positions in the soluble fraction.
Hat1-dependent acetylated histone H4 is present in the soluble fraction in different cell cycle stages, which shows that, in addition to DNA replication, it may also participate in other processes outside of S phase. A dynamic nucleosome disassembly/reassembly process is a well-established feature of sites undergoing transcription [39,40], but a global histone H4 exchange independent of replication and transcription has also been described in yeast . Reassembly makes use of histones from the soluble pool [40,41], in which histone H4, as our results indicate, must be acetylated by the HAT-B complex.
In addition, we have found that excess histone H4 accumulating in the soluble fraction in cells treated with HU or in cells deficient in the Rad53-dependent histone degradation pathway  is acetylated by Hat1. It therefore seems that all new histone H4 molecules appearing in the soluble fraction contain the specific Lys12/Lys5 acetylation pattern generated by the type B enzyme.
In contrast to studies on different species where newly synthesized histone H4 in a diacetylated form was obtained from chromatin [4,5,42,43], we did not detect HAT-B-dependent acetylation on chromatin histone H4. In yeast, the Hat1-dependent acetylation could be eliminated immediately upon the deposition of histone H4 into chromatin. It cannot be ruled out that this deacetylation occurs either during, or even prior to, histone H4 deposition. Mutational analysis has shown that specific Lys residues in the N-termini of histone H3 and histone H4 play critical roles in nuclear import, suggesting that acetylation could serve to release histones from nuclear transport factors . Formally, for such a role, the deacetylation would not necessarily have to be postdeposition.
Although, in vitro, Hat1 and Hat2  and also Hif1  bind H4/H3 histones, in vivo, both Hat1 and Hat2, together, are involved in the physical interaction with histone H4, whereas Hif1 is not. Furthermore, both targets of acetylation, Lys12 and Lys5, are found to be acetylated in the histone H4 bound to the HAT-B complex. Current models propose that the acetylated state at the histone H4 N-terminus is involved in the stable binding of histone H4 to the HAT-B complex [1,20,24]. However, this is not consistent with the ability of H4K12R, which also lacks acetylation at Lys5, to be bound by the HAT-B complex. Even Hif1, in the context of the HAT-B complex, is associated with histone H4 that lacks acetylation at the N-terminal tail. As Hif1 exhibits chromatin assembly activity in vitro , we must not rule out completely its participation in chromatin assembly independently of histone H4 acetylation. Our data also suggest that the N-terminus (at least segment 1–12) is not involved in the stable association of histone H4 with the complex. Our previous two-hybrid assays indicated an in vivo interaction between Hif1 and fragment 1–59 of histone H4 that was dependent on Hat2 ; thus, the portion of histone H4 involved in the interaction with the HAT-B complex must be located between residues 13 and 59. Verreault et al.  found that helix 1 (residues 31–40) of histone H4, situated in the histone-fold domain, is critical for binding to the Hat2 human homolog p46. Reasonably, the yeast HAT-B complex could use the same determinants to bind histone H4, although additional contacts with Hat1 seem to be necessary for efficient and stable binding of histone H4. It is possible that all or some of these interactions are also responsible for the acetylation specificity of the HAT complex discussed above.
Although histone H3 has always been found with histone H4 , and they are usually obtained from the cells in a 1 : 1 ratio, we have not detected histone H3 in the HAT-B complex from the yeast soluble fraction. The controls carried out argue against the specific proteolytic degradation of histone H3 in the yeast fractions obtained and processed by our experimental procedures. Although these results must be interpreted with caution, overall they suggest either that histone H3 does not form part of the yeast HAT-B complex in the soluble fraction, or that its binding to histone H4 in the complex is weaker than in the nucleosomal tetramer or in predeposition histone H3/H4–chaperone complexes [34,38]. The possibility that Hat1 takes part in alternative complexes with distinct histone contents deserves to be taken into account. Different complexes containing Hat1 as a catalytic subunit have been described in yeast [20,24,33], although the presence of histones in all of them has not been analyzed. In any case, the absence of histone H3 in the HAT-B complex is not a reason to change significantly the widely assumed perception that this enzyme plays a role in deposition of histones during nucleosome assembly [45,46]. The recruitment of histone H3 could occur in a subsequent step, resulting in a transient, and less abundant, new HAT-B complex containing histone H3, or simultaneously to its transfer, together with acetylated histone H4, to a distinct predeposition complex. In any case, the presence of the HAT-B complex lacking histone H3 in the soluble cell fraction would indicate that the interaction of histone H4 with the complex and its acetylation by Hat1 must be events that occur very early after the biosynthesis of the histone H4 molecule.
Strains, media and culture conditions
The yeast strains used in this study are listed in Table 1. All of the chromosomal integrated-tagged or deleted strains were generated by a one-step PCR-based strategy [47,48] as described previously . Growth and manipulation of yeast cells, and preparation of media, were performed according to standard procedures. Strains BQS1391 to BQS1394 were generated by replacing, also through homologous recombination with the appropriate PCR fragment, the natural promoter of the essential RAD53 gene by the regulatable GAL1 promoter . These strains were grown in rich media containing 2% galactose (YPGAL). The RAD53 expression was repressed by switching to a 2% glucose medium (YPD).
As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4), hat1Δ::TRP1
As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4), hat2Δ::HIS3
As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4), hif1Δ::his5+
Arrest of yeast cells at different cell cycle stages was achieved by addition of α-factor (Sigma-Aldrich, Madrid, Spain) to 4.5 μg·mL−1, HU (Sigma-Aldrich) to 200 mm, and nocodazole (Calbiochem, San Diego, CA, USA) to 15 μg·mL−1, and a subsequent incubation for 3 h. For the arrest caused by nitrogen deprivation, a minimal medium was prepared with yeast nitrogen base without ammonium sulfate (Pronadisa, Madrid, Spain). The cell cycle stages were assessed by microscopic inspection and by FACS analysis of propidium iodide-stained cells with an Epics XL (Coulter, Fullerton, CA, USA) flow cytometer. For the analysis of the effect of DNA-damage-inducing agents, YPD liquid exponentially growing yeast cultures were treated with HU, methylmethanesulfonate, phleomycin, and 4NQO, all of them purchased from Sigma-Aldrich, at the final concentrations and for the time periods indicated in the figure legends.
Preparation of cell extracts and cellular fractionation
WCEs for SDS/PAGE were prepared using an alkali method .
For the separation of the cellular content into a soluble fraction and a precipitate containing the chromatin, yeast cells were harvested by centrifugation (500 g, 5 min), washed in distilled water, and resuspended in 10 mL·g−1 of cells (fresh weight) of pretreatment medium (50 mm Tris/HCl, pH 7.5, 5 mm MgCl2, 1 m sorbitol, 75 mm 2-mercaptoethanol). After incubation for 10 min, cells were collected by centrifugation (500 g, 5 min), and dispersed in 4 mL·g−1 of digestion buffer (pretreatment medium but only 5 mm 2-mercaptoethanol). Spheroplasts were produced by incubation with zymolyase (40 U·g−1; Seikagaku, Tokyo, Japan) at 30 °C for 20–30 min with gentle agitation. Upon incubation, cell suspensions were diluted with 10 volumes of cold wash buffer (50 mm Mes/NaOH, pH 6.0, 10 mm MgCl2, 1 m sorbitol, 1 mm phenylmethanesulfonyl fluoride, 2 μm E64, 1 mm 2-iodoacetamide), and the spheroplasts were collected by centrifugation at 1000 g for 5 min. This and all subsequent steps were performed at 4 °C. Sedimented spheroplasts were washed once with the same buffer. Spheroplasts were lysed by adding 4 mL·g−1 of fractionation buffer [50 mm Tris/HCl, pH 7.6, 75 mm NaCl, 0.5 mm CaCl2, 0.1% (v/v) Tween-20, and the protease inhibitors phenylmethanesulfonyl fluoride 1 mm, 3,4-dichloroisocoumarin 25 μm, and the complete inhibitor cocktail (Roche, Basel, Switzerland)], and incubating for 10 min at 4 °C with orbital agitation. Soluble and pellet fractions were obtained by centrifugation at 16 000 g for 5 min. The examination of the enzymatic activity of the HAT-B complex from the soluble fraction was achieved upon its partial purification by chromatography onto Q-Sepharose HP (GE Healthcare, Little Chalfont, UK) (see below).
Yeast histones were purified by acid extraction of isolated chromatin as described previously .
Immunoblotting and immunoprecipitation assays
For western blotting of histones, WCEs or pellet fractions, from 1 × 106 cells, or soluble fractions, from 1 × 107 cells, were resolved by 15% SDS/PAGE. For analysis of HA-tagged, Myc-tagged or Flag-tagged proteins, the extracts and cellular fractions were electrophoresed on 8% SDS polyacrylamide gels. All protein gels were transferred to 0.2 μm pore nitrocellulose membranes as previously described . Membranes were routinely stained with Ponceau S as a loading control, and processed as described by the manufacturer of the ECL Advance Western blotting detection system (GE Healthcare). Blots were probed overnight at 4 °C with specific antibodies against different isoforms of histone H4 and histone H3, and against the specific tags. The primary antibodies used in immunoblotting were as follows. From Upstate Biotechnology (Lake Placid, NY, USA): anti-H4 acetylated at position 5, 12, or 16; and anti-H3 acetylated at position 9, 14, 27, or 56. From Abcam (Cambridge, UK): anti-H4 acetylated at residue 8 or 91, and also α-ryH4, anti-H3 acetylated at position 18 or 23, and α-H3Ct. The anti-HA clone 12CA5, and the anti-Myc clone 9E10 mouse monoclonal sera were from Roche. All primary antibodies were utilized at a dilution approximately 10 times higher than suggested by the manufacturer. The secondary antibodies were horseradish peroxidase-linked anti-rabbit or anti-mouse IgG (GE Healthcare), and were employed at a dilution of 1 : 50 000.
For immunoprecipitation experiments, soluble cell fractions were incubated with rat anti-HA (3F10; Roche) or mouse anti-Myc (9E10) monoclonal sera to a final concentration of approximately 2.5 μg·mL−1, and incubated for 4 h at 4 °C. Forty microliters of pre-equilibrated protein G–Sepharose FF (GE Healthcare) was then added and incubated for 4 h on a rotating wheel. For pulling down Flag-tagged proteins (Cac1 and Asf1) from soluble fractions, mouse anti-Flag M2-agarose beads were utilized. After centrifugation (500 g for 1 min), supernatants were saved, and the beads were washed five times with 0.5 mL of washing buffer B [15 mm Tris/HCl, pH 7.6, 150 mm NaCl, 0.5 mm EDTA, 0.1% (v/v) Tween-20, 10% (v/v) glycerol, plus the protease inhibitors indicated above]. Input materials, first supernatants (unbound materials) and immunoprecipitates (bound materials) were resolved by SDS/PAGE and probed for the presence of tagged proteins and for coimmunoprecipitated histone H4 and histone H3 by immunoblotting.
Extraction of HAT enzymes and anion exchange chromatography
Extracts for fractionation of HAT enzymes were obtained by lysis of yeast spheroplasts in a low-salt medium. Briefly, yeast cells growing exponentially, or after 3 h of incubation with 200 mm HU, were harvested by centrifugation, and washed in distilled water. Spheroplasts, prepared as described above, were lysed in buffer containing 75 mm Tris/HCl (pH 7.9), 0.25 mm EDTA, 5 mm MgCl2, 10 mm 2-mercaptoethanol, 0.1% (v/v) Tween-20, and the protease inhibitors phenylmethanesulfonyl fluoride 1 mm, 3,4-dichloroisocoumarin 25 μm, and the complete inhibitor cocktail (Roche), and stirred for 30 min. The homogenates were ultracentrifuged for 1 h at 100 000 g, and the resulting supernatants, containing those ‘free’ or soluble HATs, such as the HAT-B complex, and devoid of the chromatin-associated HAT enzymes, were saved and dialyzed against buffer B [15 mm Tris/HCl, pH 7.9, 0.25 mm EDTA, 5 mm 2-mercaptoethanol, 0.05% (v/v) Tween-20, 10% (v/v) glycerol, 10 mm NaCl]. The dialyzed extracts were loaded onto Q-Sepharose HP columns, and after washing, bound proteins were eluted with a linear 80–400 mm NaCl gradient in buffer B. Fractions were collected and assayed for protein content (A280 nm) and HAT activity. ryHat1 was generated in bacteria as previously described , purified partially by anion exchange chromatography on Sep-Pak Accell plus QMA cartridges (Waters, Milford, MA, USA), and employed for the in vitro HAT specificity assays.
For determination of enzymatic activity in chromatographic fractions, a new assay method was used. Briefly, 12 μL of chromatographic fractions was mixed with 4 μg of chicken erythrocyte core histones and 0.005 μCi of [1-14C]acetyl-CoA (50 mCi·mmol−1; Moravek, Brea, CA, USA) in a final volume of 16 μL, and incubated for 20 min at 30 °C. The reaction was terminated by addition of 8 μL of SDS/PAGE sample solution [3×; 0.187 m Tris/HCl, pH 6.8, 6% (w/v) SDS, 1.5 m 2-mercaptoethanol, 30% (v/v) glycerol, 0.005% (w/v) bromophenol blue]. The amount of [14C]acetate incorporated into the protein substrates was quantified with an FLA-3000 phosphoimager (Fujifilm, Tokyo, Japan) or an InstantImager (Packard Meriden, CT, USA), and the relative radioactivity values were expressed as arbitrary units (a.u.). Full details of this method for assaying HAT activity will be given elsewhere.
In vitro HAT specificity assay
The histone specificity of partially purified HAT-B complex from the soluble cellular fraction was determined by HAT assays as previously described  (see also above), using chicken erythrocyte or yeast core histones and [1-14C]acetyl-CoA as substrates. Acetylated histone products were resolved by 15% PAGE in the presence of SDS, and subsequently the gels were stained with Coomassie brilliant blue, destained, dried, and exposed on phosphor-record image plates. Screens were scanned in an FLA-3000 fluorescent imager analyzer. Immunoblotting of the resulting acetylated histone products was also carried out, in some case, to examine the site specificity on histone H4.
We wish to thank Dr M. R. Parthun and A. Verreault for their generous gift of several yeast strains and antibodies. We also acknowledge Drs P. Loidl, M. Pamblanco and E. Ralph for their useful comments on the work and critical reading of the manuscript. This work has been supported by Grant BFU2005-02603 from Ministerio de Educación y Ciencia, Spain.