Title: Properties of the Type B Histone Acetyltransferase Hat1
Abstract: 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 hat1Δ 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. 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 hat1Δ 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. During replication-coupled nucleosome assembly, newly synthesized histones H3 and H4 are deposited onto newly replicated DNA by the chromatin assembly factor CAF-1 (1Polo S.E. Almouzni G. Curr. Opin. Genet. Dev. 2006; 16: 104-111Crossref PubMed Scopus (124) Google Scholar). H3 and H4 form a heterodimer prior to deposition in both cytosolic and nuclear chromatin assembly complexes (2Tagami H. Ray-Gallet D. Almouzni G. Nakatani Y. Cell. 2004; 116: 51-61Abstract Full Text Full Text PDF PubMed Scopus (992) Google Scholar, 3Benson L.J. Gu Y.L. Yakovleva T. Tong K. Barrows C. Strack C.L. Cook R.G. Mizzen C.A. Annunziato A.T. J. Biol. Chem. 2006; 281: 9287-9296Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Notably, in species as divergent as humans, Drosophila, and Tetrahymena, newly synthesized H4 is acetylated in a conserved pattern at lysines 5 and 12 prior to its association with DNA (the sites are Lys-4 and -11 in Tetrahymena due to a deletion of the usual arginine residue at position 3) (4Chicoine L.G. Schulman I.G. Richman R. Cook R.G. Allis C.D. J. Biol. Chem. 1986; 261: 1071-1076Abstract Full Text PDF PubMed Google Scholar, 5Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (422) Google Scholar). Deacetylation of new H4 occurs gradually within 30–60 min following nucleosome assembly (6Jackson V. Shires A. Tanphaichitr N. Chalkley R. J. Mol. Biol. 1976; 104: 471-483Crossref PubMed Scopus (142) Google Scholar). The function of the acetylation of nascent H4 is currently unknown; however, when deacetylation is inhibited, newly replicated chromatin fails to mature properly (7Annunziato A.T. Seale R.L. J. Biol. Chem. 1983; 258: 12675-12684Abstract Full Text PDF PubMed Google Scholar). A likely candidate for the enzyme that acetylates newly synthesized H4 is Hat1, a type B histone acetyltransferase found in virtually all eukaryotic systems examined (8Annunziato A.T. Hansen J.C. Gene Expr. 2000; 9: 37-61Crossref PubMed Scopus (131) Google Scholar). Hat1 is recovered in cytosolic extracts, but there is evidence that it also resides in the nucleus (9Richman R. Chicoine L.G. Collini M.P. Cook R.G. Allis C.D. J. Cell Biol. 1988; 106: 1017-1026Crossref PubMed Scopus (40) Google Scholar, 10Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1997; 8: 96-108Abstract Full Text Full Text PDF Scopus (293) Google Scholar, 11Ruiz-Garcia A.B. Sendra R. Galiana M. Pamblanco M. Perezortin J.E. Tordera V. J. Biol. Chem. 1998; 273: 12599-12605Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Poveda A. Pamblanco M. Tafrov S. Tordera V. Sternglanz R. Sendra R. J. Biol. Chem. 2004; 279: 16033-16043Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 13Ai X. Parthun M.R. Mol. Cell. 2004; 14: 195-205Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 14Lusser A. Eberharter A. Loidl A. Goralik-Schramel M. Horngacher M. Haas H. Loidl P. Nucleic Acids Res. 1999; 27: 4427-4435Crossref PubMed Scopus (41) Google Scholar). Hat1 specifically acetylates free (non-nucleosomal) H4 in vitro, generating the Lys-5/Lys-12 acetylation pattern of newly synthesized H4 (4Chicoine L.G. Schulman I.G. Richman R. Cook R.G. Allis C.D. J. Biol. Chem. 1986; 261: 1071-1076Abstract Full Text PDF PubMed Google Scholar, 9Richman R. Chicoine L.G. Collini M.P. Cook R.G. Allis C.D. J. Cell Biol. 1988; 106: 1017-1026Crossref PubMed Scopus (40) Google Scholar, 10Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1997; 8: 96-108Abstract Full Text Full Text PDF Scopus (293) Google Scholar, 15Sobel R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; 269: 18576-18582Abstract Full Text PDF PubMed Google Scholar, 16Parthun M.R. Widom J. Gottschling D.E. Cell. 1996; 87: 85-94Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar, 18Kolle D. Sarg B. Lindner H. Loidl P. FEBS Lett. 1998; 421: 109-114Crossref PubMed Scopus (34) Google Scholar). In human cells, Hat1 functions as a member of the HAT-B complex, which also contains the small protein p46 (also known as Rbap46) (10Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1997; 8: 96-108Abstract Full Text Full Text PDF Scopus (293) Google Scholar). In budding yeast (Saccharomyces cerevisiae) the p46 orthologue is termed Hat2p (11Ruiz-Garcia A.B. Sendra R. Galiana M. Pamblanco M. Perezortin J.E. Tordera V. J. Biol. Chem. 1998; 273: 12599-12605Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 16Parthun M.R. Widom J. Gottschling D.E. Cell. 1996; 87: 85-94Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). The nuclear form of the yeast Hat1 complex contains an additional subunit, Hif1p (12Poveda A. Pamblanco M. Tafrov S. Tordera V. Sternglanz R. Sendra R. J. Biol. Chem. 2004; 279: 16033-16043Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 13Ai X. Parthun M.R. Mol. Cell. 2004; 14: 195-205Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In S. cerevisiae, Hat1p has been demonstrated to be important for the proper establishment of telomeric silencing and for the repair of damaged DNA; in combination with mutations of the acetylatable lysines of histone H3, deletion of HAT1 causes loss of telomeric silencing and increased sensitivity to the DNA-damaging agent MMS 3The abbreviations used are: MMS, methyl methanesulfonate; HAT, histone acetyltransferase. (19Kelly T.J. Qin S. Gottschling D.E. Parthun M.R. Mol. Cell. Biol. 2000; 20: 7051-7058Crossref PubMed Scopus (117) Google Scholar, 20Qin S. Parthun M.R. Mol. Cell. Biol. 2002; 22: 8353-8365Crossref PubMed Scopus (156) Google Scholar). Moreover, it has been shown that Hat1p is recruited to the sites of DNA double-stranded breaks, where it acetylates H4 at Lys-12 (21Qin S. Parthun M.R. Mol. Cell. Biol. 2006; 26: 3649-3658Crossref PubMed Scopus (58) Google Scholar). It is possible that Hat1p participates in gene silencing and DNA repair by acetylating H4 during localized chromatin assembly. The structure of Hat1p from S. cerevisiae has been solved, and a model has been proposed for the interaction of the H4 N-terminal domain with the catalytic site (there is as yet no Hat1p:H4 tail co-crystal) (22Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. Cell. 1998; 94: 427-438Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 23Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 501-507Crossref PubMed Scopus (18) Google Scholar) (see Fig. 1). The H4 tail is postulated to lie in a shallow channel on the concave face of the enzyme. When Lys-12 of H4, i.e. the lysine that is most readily acetylated by yeast Hat1p, is aligned adjacent to the acetyl-CoA moiety, the lysines at positions 8 and 16 come in close proximity to two acidic regions on the surface of the enzyme (Fig. 1, top). It has therefore been proposed that electrostatic interactions between Lys-8/Lys-16 and the negatively charged patches serve to hold the H4 tail in place during the acetylation of Lys-12 (22Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. Cell. 1998; 94: 427-438Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In a previous study, we tested this hypothesis by performing in vitro HAT assays using variously acetylated H4 tail peptides. In support of the model, our results demonstrated that prior acetylation of the H4 tail at Lys-8 and -16 dramatically reduced the ability of recombinant yeast Hat1p and native human HAT-B to acetylate Lys-12 in vitro (24Makowski A.M. Dutnall R.N. Annunziato A.T. J. Biol. Chem. 2001; 276: 43499-43502Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). To further explore the interaction of the H4 N-terminal domain with Hat1, we have extended our analysis to include amino acid substitutions of the H4 tail at lysines 8 and 16. We found that the presence of lysine at these sites is not absolutely required for enzyme activity, but that a positive charge at Lys-8/Lys-16 is critical. Our results also implicate the N-terminal serine of H4 in the enzyme-substrate interaction. It is further demonstrated that Lys-12 of H4 is the preferred substrate for the human cytosolic HAT-B complex and that Lys-12 is typically acetylated prior to Lys-5 in generating diacetylated H4. Finally, in contrast to results obtained with S. cerevisiae,we have shown that hat1Δ Schizosaccharomyces pombe cells exhibit heightened sensitivity to DNA damage in the absence of concurrent mutations of H3 N-terminal domain. Peptide Synthesis, in Vitro HAT Assays—Custom 20-mer peptide synthesis, preparation of HeLa cytosolic extracts, and in vitro HAT filter-binding assays were performed as previously described (17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar, 24Makowski A.M. Dutnall R.N. Annunziato A.T. J. Biol. Chem. 2001; 276: 43499-43502Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 25Benson L.J. Annunziato A.T. Methods. 2004; 33: 45-52Crossref PubMed Scopus (8) Google Scholar) using 4–16 μCi/ml [3H]acetyl-CoA (1–3 Ci/mmol; PerkinElmer Life Sciences). Reaction times are given in the figure legends (see Figs. 2 and 3). In vitro peptide assays using recombinant yeast Hat1p (yHat1p) from S. cerevisiae were performed following a modified procedure of Kleff et al. (26Kleff S. Andrulis E.D. Anderson C.W. Sternglanz R. J. Biol. Chem. 1995; 270: 24674-24677Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) as described previously (yHat1p was the generous gift of Robert Dutnall) (24Makowski A.M. Dutnall R.N. Annunziato A.T. J. Biol. Chem. 2001; 276: 43499-43502Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 25Benson L.J. Annunziato A.T. Methods. 2004; 33: 45-52Crossref PubMed Scopus (8) Google Scholar). In some cases, HAT assays were performed in the presence of 0.5 mm okadaic acid (e.g. to prevent the dephosphorylation of Ser-1 in phosphorylated H4 tail peptides). Okadaic acid did not interfere with HAT activity. Peptide dephosphorylation reactions were performed at 37 °C using 2–6 μg of bacterial alkaline phosphatase (Sigma) per μg of peptide in 20 mm Tris-HCl, pH 8.0. Protocols for performing HAT assays on immobilized peptide substrates are given in detail elsewhere (25Benson L.J. Annunziato A.T. Methods. 2004; 33: 45-52Crossref PubMed Scopus (8) Google Scholar).FIGURE 3Effects of phosphorylation of Ser-1 and of combined phosphorylation and acetylation on the acetylation of H4 N-terminal peptides by HeLa HAT-B and yeast Hat1p. A, unacetylated (UN) and Ser-1-phosphorylated (S1P) H4 N-terminal peptides were incubated for 10 min with a HAT-B extract and [3H]acetyl-CoA. In some cases, peptides were treated with alkaline phosphatase (+AP) prior to the acetylation reaction. B, unacetylated, monoacetylated at lysines 5 or 12 (K5Ac, K12Ac), phospho-acetyl dimodified (S1P-K5Ac, S1P-K12Ac), and Lys-5/Lys-12 diacetylated (K5-K12Ac) H4 N-terminal peptides were incubated for 10 min with a HAT-B extract and [3H]acetyl-CoA. C, unacetylated, monoacetylated at lysines 5 or 12 (K5Ac, K12Ac), phosphoacetyl dimodified (S1P-K5Ac, S1P-K12Ac), and Lys-5/Lys-12 diacetylated (K5-K12Ac) H4 N-terminal peptides were incubated for 20 min with yeast Hat1p and [3H]acetyl-CoA; all reactions were performed in triplicate, except for the Lys-5/Lys-12 diacetylated peptide, which is the average of two trials. For A–C, results are expressed as a percentage of incorporation into the unacetylated H4 peptide. D, selected results from C were replotted by normalizing the K5Ac and K12Ac peptides to 100% incorporation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Acetylation of Recombinant H4, Triton X-100/Acid/Urea Gel Electrophoresis—S100 cytosolic extracts prepared from HeLa cells were immunodepleted of native H4 using antibodies that recognize H4 acetylated at lysines 5 and 12 (the pattern of acetylation found in cytosolic H4) as described previously (17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar). Treatment was overnight at 4 °C; immunodepletion was monitored by Western blotting. HAT reactions used 100 μlof immunodepleted S100 extract, 1.5 μg of recombinant H4 (Upstate Biotechnology), and 10 μm unlabeled acetyl-CoA. We have previously shown that Hat1 is the only HAT activity detected in HeLa S100 extracts (17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar). The reaction proceeded for 2.5 min at 37 °C and was terminated by placing the tube for 5 min into a slurry of dry ice in 95% ethanol. H4 was recovered by acid extraction and precipitated using 25% trichloroacetic acid (final concentration); pellets were washed in acidified acetone (0.05 m HCl) and then 100% acetone and dried (17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar, 25Benson L.J. Annunziato A.T. Methods. 2004; 33: 45-52Crossref PubMed Scopus (8) Google Scholar). Triton X-100/acid/urea-PAGE and Western blotting were performed as described previously (17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar, 27Ryan C.A. Annunziato A.T. Curr. Protocols Mol. Biol. 1999; 21.2: 1-10Google Scholar). Antibodies used were as follows: anti-histone H4 acetylated on Lys-5 (1:1000 dilution; Serotec); anti-total H4 (1:500 dilution; Cell Signaling Technology); and anti-histone H4 acetylated on Lys-12 (1:1000 dilution; Upstate Biotechnology). Antibody specificity was verified by Western blotting of acetylated histones in the presence or absence of competing, variously acetylated, H4 N-terminal peptides; both of the anti-acetylated H4 antibodies strongly recognized Lys-5/Lys-12-diacetylated H4, as determined by Western blotting of histones resolved in Triton X-100/acid/urea gels. Deletion of the hat1 Gene from S. pombe—S. pombe yeast were routinely cultured on YEA medium (28Gutz H. J. Bacteriol. 1966; 92: 1567-1568Crossref PubMed Google Scholar). The genotypes of all yeast strains used in this study are given in Table 1. The gene encoding the S. pombe Hat1 protein (SPAC139.06; Uniprot accession number Q9UTM7) was deleted using the PCR-based one-step gene replacement technique described by Bahler et al. (29Bahler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie III, A. Steever A.B. Wach A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 943-951Crossref PubMed Scopus (1771) Google Scholar). PCR primers HAT-forward (5′-ATGAGTGCTGTTGATGAATGGGTACATAATGCCAATGAATGCATAGAAATTGTACAAGTAAACGAAAAGCGGATCCCCGGGTTAATTAA-3′) and HAT-reverse (5′-AAGATTGAGCAAGTTTTTGGCGTTTTCGAGGCGAATCTTCCTTAAGCTTGGGTAGCTTTTTGACAGAATTCGAGCTCGTTTAAAC-3′) were used to amplify the kanMX6 selectable marker from plasmid pFA6a-3HA-kanMX6. This PCR product, containing ∼60 bp of sequence homology flanking the hat1+ gene, was used to transform a diploid strain (constructed by mating FWP2 and FWP39 and selecting for Ade+ diploid cells due to intragenic complementation of the ade6-M210 and ade6-M216 alleles) to G418 resistance. Deletion of the hat1+ gene was confirmed by PCR. Tetrad dissection of the diploid transformant resulted in a 2:2 pattern of G418 resistant to G418-sensitive progeny, indicating that the hat1Δ deletion progeny are viable. Diagnostic PCR of the progeny revealed that the PCR product associated with the hat1Δ deletion co-segregates with G418 resistance. The hat1+ gene was subsequently deleted from strain 975 (h+) by homologous recombination using a PCR product containing the hat1Δ::kan locus amplified from one of the G418-resistant progeny to create strains LBP6 and LBP7. This gene deletion was also confirmed by PCR analysis.TABLE 1S. pombe strains used in this studyStrainGenotypeReference or Source975h+48Leupold U. Compt. Rend. Trav. Lab. Carlsberg Ser. Physiol. 1950; 24: 381-480Google ScholarFWP2h+ leul—32 ade6-M210C. HoffmanFWP6h- leul—32C. HoffmanFWP39h- ade6-M216C. HoffmanLBP6h+ hat1Δ::kanThis studyLBP7h+ hat1Δ::kanThis studyLBP8h- leul—32 hat1Δ::kanThis study Open table in a new tab The analysis of the MMS sensitivity of wild-type and mutant yeast was performed as previously described (20Qin S. Parthun M.R. Mol. Cell. Biol. 2002; 22: 8353-8365Crossref PubMed Scopus (156) Google Scholar). Sensitivity to UV light was tested at 80 and 120 joules, using a UVC 500 UV cross-linker (GE Healthcare). Acetylation by Hat1 Requires Positively Charged Amino Acids at Positions 8 and 16 of the H4 Tail—In a previous study, we determined that prior acetylation of H4 tail peptides at Lys-8 and/or Lys-16 significantly reduced the ability of both the human HAT-B complex and recombinant yHat1p to acetylate lysines 5 and 12 of H4 (24Makowski A.M. Dutnall R.N. Annunziato A.T. J. Biol. Chem. 2001; 276: 43499-43502Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This was interpreted as supporting the model of the Hat1-H4 tail interaction proposed by Dutnall et al. (22Dutnall R.N. Tafrov S.T. Sternglanz R. Ramakrishnan V. Cell. 1998; 94: 427-438Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar); however, questions still remained concerning the requirement for (unacetylated) lysine at positions 8 and 16. One possibility was that a positive charge was itself sufficient and not lysine per se. Alternatively, it could be that lysine was uniquely required at these sites for Hat1 to recognize and acetylate the H4 tail. To test these alternatives, H4 tail peptides, containing either glutamine (to mimic acetylation) or arginine (to provide a nonspecific positive charge), were synthesized and tested in HAT assays using recombinant yHat1p. (It is important to emphasize that Lys-5 and Lys-12, the substrate lysines of Hat1, are unaltered in these experiments and thus remain potentially acetylatable.) The results are presented in Fig. 2A. Consistent with our previous results, substituting glutamine for lysine at Lys-8 and Lys-16 dramatically reduced the ability of yHat1p to acetylate the H4 tail peptide (Fig. 2, A and B). This confirms the importance of lysine and/or a positive charge at Lys-8 and Lys-16 for acetylation to occur. Surprisingly, substituting arginine at positions 8 and 16 resulted in acetylation as great or greater than that observed when the normal lysine residues were present at these sites (Fig. 2, A and B). Similar results were obtained for the native HeLa HAT-B complex (Fig. 2C). These results provide additional support for the proposed model of H4 tail binding and help further refine our understanding of the Hat1-H4 tail interaction. Clearly lysine is not uniquely required at positions 8 and 16 for Hat1 to recognize and acetylate the H4 tail. Nevertheless, it is of considerable importance to retain positive charges at these sites. Involvement of Ser-1 in Acetylation of the H4 Tail by Hat1—In an early study of post-translational modifications of newly synthesized histones, it was suggested that new H4 may be phosphorylated at the N-terminal serine residue (30Ruiz-Carrillo A. Wangh L.J. Allfrey V.G. Science. 1975; 190: 117-128Crossref PubMed Scopus (322) Google Scholar). It has also been reported that the NuA4 histone acetyltransferase is negatively regulated by phosphorylation of H4 at Ser-1 (31Utley R.T. Lacoste N. Jobin-Robitaille O. Allard S. Cote J. Mol. Cell. Biol. 2005; 25: 8179-8190Crossref PubMed Scopus (130) Google Scholar). It was therefore of interest to examine the effect of phosphorylation of H4 at Ser-1 on HAT-B activity. HAT assays were performed on an H4 N-terminal peptide phosphorylated at Ser-1 (Fig. 3A, S1P) (see supplemental Fig. S1 for a time course curve). Consistent with the findings of Utley et al. (31Utley R.T. Lacoste N. Jobin-Robitaille O. Allard S. Cote J. Mol. Cell. Biol. 2005; 25: 8179-8190Crossref PubMed Scopus (130) Google Scholar) concerning NuA4, an H4 peptide phosphorylated at Ser-1 was a relatively poor substrate for human HAT-B. Dephosphorylation of the S1P peptide with alkaline phosphatase restored acetylation (Fig. 3A), whereas treatment with bovine serum albumin had no effect (data not shown). To determine whether phosphorylation of Ser-1 would affect the acetylation of Lys-5 or Lys-12 differently, additional HAT-B assays were performed on dimodified H4 peptides that were phosphorylated at Ser-1 and also acetylated at either Lys-5 or Lys-12 (Fig. 3B). As expected and in agreement with our previous results (24Makowski A.M. Dutnall R.N. Annunziato A.T. J. Biol. Chem. 2001; 276: 43499-43502Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), prior acetylation at Lys-5 or Lys-12 reduced the incorporation of [3H]acetate by ≥50% (note that in these cases one of the Lys-5/Lys-12 substrate sites is already acetylated). Notably, phosphorylation of Ser-1 further reduced the acetylation of H4 peptides acetylated at Lys-5 or Lys-12 (Fig. 3B). Indeed, phosphorylation of a peptide already acetylated at Lys-12 reduced further acetylation by HeLa HAT-B to background levels, indicating that acetylation at Lys-5 was essentially eliminated under these conditions. Identical results were obtained with yeast Hat1p, although in this case acetylation of the Lys-12ac peptide was itself much reduced relative to acetylation by the human enzyme (a reflection for the strong preference of yHat1p for this site (Fig. 3C) (16Parthun M.R. Widom J. Gottschling D.E. Cell. 1996; 87: 85-94Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 26Kleff S. Andrulis E.D. Anderson C.W. Sternglanz R. J. Biol. Chem. 1995; 270: 24674-24677Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). To better visualize the effect of phosphorylation of the H4 tail on yHat1p activity, the results of Fig. 3C were replotted by setting acetate incorporation into the Lys-5ac and Lys-12ac peptides at 100% (Fig. 3D). From this it can be seen that phosphorylation of S1P dramatically reduced acetylation of the H4 tail by yHat1p at both substrate sites. Acetylation of Immobilized Peptide Substrates—It remained formally possible that the observed reduction in acetylation of the Lys→ Gln and S1P peptides was caused by a failure of these altered substrates to adhere properly to filters in preparation for scintillation counting. To eliminate this potential artifact, HAT assays were also performed on peptide substrates that were immobilized on agarose beads, as we have previously described (25Benson L.J. Annunziato A.T. Methods. 2004; 33: 45-52Crossref PubMed Scopus (8) Google Scholar). Immobilized peptides yielded results that were in full agreement with those obtained from conventional HAT assays (supplemental Fig. S2). It is therefore concluded that lysines 8 and 16 (and potentially Ser-1) of the H4 N-terminal domain can modulate the Hat1-H4 tail interaction. Order of Acetylation of H4 by Hat1—The human HAT-B complex acetylates lysines 5 and 12 of H4 exclusively (10Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1997; 8: 96-108Abstract Full Text Full Text PDF Scopus (293) Google Scholar, 17Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (146) Google Scholar). To determine the order of acetylation of these sites, recombinant H4 was briefly incubated with HeLa HAT-B in vitro in the presence of unlabeled acetyl-CoA, and the reaction was rapidly quenched in a dry ice bath. The products were then separated in a Triton X-100/acid/urea gel (to resolve acetylated isoforms) and analyzed by Western blotting using antibodies that recognize H4 acetylated at either Lys-5 or Lys-12 (Fig. 4). Lys-12 is the preferred substrate of human HAT-B. When approximately half of newly acetylated H4 was still monoacetylated at Lys-12 (Fig. 4, lane 7), acetylation at Lys-5 was observed predominantly in the diacetylated isoform (lane 3). Thus, Lys-12 is typically acetylated first by HeLa HAT-B followed by acetylation at Lys-5 to generate the diacetylated product. Scans of the Western blot indicated that there was an ∼3-fold preference by HAT-B for Lys-12 over Lys-5, in agreement with a previous analysis of maize HAT-B (18Kolle D. Sarg B. Lindner H. Loidl P. FEBS Lett. 1998; 421: 109-114Crossref PubMed Scopus (34) Google Scholar), and consistent with the strong preference for Lys-12 by the yeast enzyme (16Parthun M.R. Widom J. Gottschling D.E. Cell. 1996; 87: 85-94Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 26Kleff S. Andrulis E.D. Anderson C.W. Sternglanz R. J. Biol. Chem. 1995; 270: 24674-24677Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). MMS Sensitivity of hat1Δ S. pombe Yeast—Deletion of the HAT1 gene from S. cerevisiae has no effect on cell growth under normal conditions (16Parthun M.R. Widom J. Gottschling D.E. 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