An N α-Acetyltransferase Responsible for Acetylation of the N-terminal Residues of Histones H4 and H2A

Abstract

A yeast gene has been identified that encodes a novel, evolutionarily conserved N α-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes. A yeast gene has been identified that encodes a novel, evolutionarily conserved N α-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes. Eukaryotic proteins are subject to two cotranslational modifications as the nascent polypeptides emerge from the ribosome, methionine cleavage by methionine aminopeptidase, and acetylation of the α-amino group of the N-terminal amino acid (1Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (355) Google Scholar). Removal of the methionine occurs only if the second amino acid is a small one such as serine, alanine, or glycine. Acetylation of the α-amino group on either methionine, or on the N-terminal residue resulting from methionine cleavage, occurs in the great majority of (but not all) eukaryotic proteins, and is accomplished by one of several N α-acetyltransferases (NATs) 1The abbreviations used are: NAT, N α-acetyltransferase; HAT, histone acetyltransferase; ACTH, adrenocorticotropin. (1Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (355) Google Scholar). A number of years ago we identified the genes for a yeast NAT, now called NatA, that consists of two subunits, Ard1 and Nat1 (2Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar). Subsequent work showed that NatA was responsible for the acetylation of many, but not all, yeast proteins beginning with small residues such as serine, alanine, or glycine (2Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar, 3Takakura H. Tsunasawa S. Miyagi M. Warner J.R. J. Biol. Chem. 1992; 267: 5442-5445Abstract Full Text PDF PubMed Google Scholar, 4Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar, 5Polevoda B. Sherman F. J. Biol. Chem. 2000; 275: 36479-36482Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Yeast ard1 and nat1 mutants are viable and the many proteins that are no longer N-terminally acetylated in the mutant strains are as stable as they are in a wild type strain (2Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar). Thus, there is no evidence that N-terminal acetylation serves to protect proteins from degradation in yeast. Ard1 protein has an acetyl-CoA binding motif found in members of the GNAT superfamily, enzymes that acetylate amino groups on proteins and other molecules (6Neuwald A.F. Landsman D. Trends Biochem. Sci. 1997; 22: 154-155Abstract Full Text PDF PubMed Scopus (387) Google Scholar). Thus, Ard1 is very likely to be the catalytic subunit of NatA. Two other NATs have been identified in yeast, called NatB and NatC, with catalytic subunits Nat3 and Mak3, respectively (4Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar, 7Tercero J.C. Wickner R.B. J. Biol. Chem. 1992; 267: 20277-20281Abstract Full Text PDF PubMed Google Scholar). NatB and NatC both acetylate proteins with N-terminal methionine residues, although they have different specificities dictated by the nature of the subsequent amino acids (4Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (175) Google Scholar). Histone H4 is a highly conserved protein with an N-terminal serine residue that is acetylated in both mammals and yeast (8Brandt W.F. Patterson K. von Holt C. Eur. J. Biochem. 1980; 110: 67-76Crossref PubMed Scopus (28) Google Scholar, 9DeLange R.J. Fambrough D.M. Smith E.L. Bonner J. J. Biol. Chem. 1969; 244: 319-334Abstract Full Text PDF PubMed Google Scholar). In our initial paper on NatA, we noted that H4 from ard1 or nat1 mutants had the same mobility on Triton-acid-urea gels as when isolated from wild type strains (2Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar). We therefore concluded that H4 was not acetylated by NatA. But, as noted above, NatB and NatC only acetylate proteins beginning with methionine, not serine, so the question arose as to whether another yeast NAT existed that was responsible for the N-terminal acetylation of H4. We therefore examined uncharacterized yeast genes that are members of the GNAT superfamily of acetyltransferases (6Neuwald A.F. Landsman D. Trends Biochem. Sci. 1997; 22: 154-155Abstract Full Text PDF PubMed Scopus (387) Google Scholar) to see if one of them might be the NAT that acetylates H4. Here we describe an evolutionarily conserved yeast protein, encoded by the gene we call NAT4, that N-terminally acetylates not only histone H4, but also H2A. Nat4 Expression Plasmid and nat4 Mutant—The NAT4 coding sequence was amplified from yeast genomic DNA by PCR and cloned into the expression vector, pET28a, as an EcoRI-XhoI fragment. This led to the expression of full-length Nat4 protein with a His-tag at the N terminus. The protein was expressed well in Escherichia coli and purified by Ni2+ affinity chromatography. Yeast Δnat4 mutants were constructed by replacing the entire NAT4 open reading frame either with the kanMX6 gene (strain OS1) or with the S. pombe his5 + gene (strain OS4), as described previously (10Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar). Acetylation Reactions—Histones or peptides were acetylated in 40-μl reactions with 75 mm Tris·Cl (pH 8.8), 135 mm NaCl, 0.2 mg/ml chicken erythrocyte histones or 50 μm peptide, 1 μCi of [3H]acetyl-CoA (Amersham Biosciences, 4.2 Ci/mmol), and Nat4 or Hat1 enzymes. Reactions were incubated at 37 °C for 30 min. After incubation, the reactions were spotted to Whatman P81 cation-exchange paper and air dried. The dried papers were washed for 5 min three times with 1 m sodium carbonate (pH 9.0), followed by a wash in acetone. Papers were air-dried, and radioactivity was quantitated in a liquid scintillation counter. Histone Purification—Yeast histones were purified for sequencing as described previously (11Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Scopus (407) Google Scholar). The histones were separated by electrophoresis on 15% SDS-polyacrylamide gels. After electrophoresis, proteins were blotted onto polyvinylidene difluoride membranes (0.2 μm; Bio-Rad). The membranes were washed with water five times for 5 min and then stained with 0.2% Ponceau S for 1 min to visualize the proteins, followed by a wash in water. Sections of the membrane with specific histone bands were excised, soaked briefly in 50% methanol, and washed with water five times. The membrane fragments were air dried and subjected to protein sequencing. For the acid-urea gels, histones were purified as described previously (12Waterborg J.H. J. Biol. Chem. 2000; 275: 13007-13011Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In Vivo [35S]Methionine Labeling and Protein Extraction—Yeast cells were grown in synthetic complete medium lacking methionine to 2 × 107/ml. A 1-ml culture was labeled for 15 min with 100 μCi of trans-[35S]methionine (ICN) and harvested by centrifugation. The cells were washed with 1 ml of ice-cold water and resuspended in 100 μl of water containing protease inhibitors (Roche Applied Science). Cells were lysed by vortexing with the same volume of glass beads. A 0.1-ml solution with 0.6% SDS, 2% mercaptoethanol, 0.1 m Tris·Cl (pH 8.0), which has been kept at 100 °C, was immediately added to the disrupted cell mixture and vortexed for 1 min. After a brief centrifugation 100 μl of the supernatant was mixed with 10 μl of a solution containing 0.5 mg/ml RNase, 1 mg/ml DNase, 500 mm Tris·Cl (pH 7.0), and 50 mm MgCl2. Protein extracts were frozen in dry ice/ethanol and later analyzed by two-dimensional gel electrophoresis at Cold Spring Harbor Laboratory. The sequence of the protein encoded by yeast ORF, YMR069w, has the acetyl-CoA binding motifs characteristic of members of the GNAT superfamily (6Neuwald A.F. Landsman D. Trends Biochem. Sci. 1997; 22: 154-155Abstract Full Text PDF PubMed Scopus (387) Google Scholar). Furthermore, the protein has sequence similarity to the catalytic subunits of the three known NATs, Ard1, Nat3, and Mak3, and is of similar size (Fig. 1). We expressed and purified the protein encoded by YMR069w in E. coli. The recombinant protein (that we call Nat4 based on the results to be presented below) was able to acetylate a peptide with a sequence corresponding to the N-terminal 28 amino acids of H4, but not a corresponding H3 peptide or an unrelated peptide, adrenocorticotropin (ACTH; Fig. 2A). We chose ACTH because NatA is able to acetylate this peptide in vitro (13Park E.C. Szostak J.W. EMBO J. 1992; 11: 2087-2093Crossref PubMed Scopus (140) Google Scholar). Notably, Nat4 protein could not acetylate chicken histones even though a known histone acetyltransferase (HAT), Hat1, could acetylate both the H4 peptide and histones (Fig. 2A). The observation that Nat4 could acetylate a synthetic H4 peptide but not chicken histones suggested that the protein might be a NAT for H4, because chicken H4 is already fully acetylated on its N-terminal serine. Further support for this idea came from studies comparing in vitro acetylation of a completely unacetylated H4 peptide with one internally acetylated on all four acetylatable lysines, namely residues 5, 8, 12, and 16. As can be seen in Fig. 2B, Nat4 could acetylate the internally tetraacetylated peptide almost as well as the unacetylated peptide, whereas Hat1 could only acetylate the peptide with internal unacetylated lysines.Fig. 2In vitro assays with Nat4 and Hat1. Assays were done in duplicate, and the error bars indicate the spread in the data. A, recombinant Nat4 acetylates an unacetylated H4 peptide, but not H3 or ACTH peptides or chicken histones. Recombinant Hat1 acetylates both the H4 peptide and chicken histones. B, recombinant Nat4 acetylates both completely unacetylated (un Ac) and internally tetraacetylated (tetra Ac) H4 peptides, whereas Hat1 acetylates only the unacetylated peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next we constructed a mutant with a complete deletion of the ORF for Nat4, YMR069w. The mutant was viable and grew at a normal rate. Histones were purified from the mutant, as well as from a wild type and an ard1 strain (Fig. 3). The proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and the bands at the positions of H4 and H3 excised and subjected to Edman degradation and protein sequencing. Histone H3, which is known to be unblocked (i.e. not acetylated) at its N terminus, gave the expected N-terminal sequence, Ala-Arg-Thr-Lys. Histone H4 gave no readable sequence from the wild type and the ard1 strains, as expected if the protein was blocked by acetylation. However, a sequence corresponding to the first four amino acids of H4, Ser-Gly-Arg-Gly, was obtained for H4 from the strain with a deletion of ORF YMR069w (Fig. 3 and Table I). This strongly suggested that this gene encodes a NAT that acetylates the N terminus of H4, and thus the gene was named NAT4.Table IN-terminal sequences of yeast histones H4, H2A, and H2A.ZGeneProteinNo. of amino acidsN-terminal sequenceHHF1H4102SGRGKGGKGHHF2H4102SGRGKGGKGHTA1H2A131SGGKGGKAGHTA2H2A131SGGKGGKAGHTZ1H2A.Z133SGKAHGGKG Open table in a new tab Next, yeast histones were purified to homogeneity by high pressure liquid chromatography from four strains, wild type, an ard1 mutant, a nat4 mutant, and an ard1-nat4 double mutant. The histones were analyzed on an acid-urea gel, which separates them by size and by charge. As shown in Fig. 4, H4 showed the same pattern of isoforms in wild type and in the ard1 strain, thus confirming our previous conclusion that NatA does not acetylate H4. On the other hand, the H4 isoforms from the nat4 and nat4-ard1 strains migrated more rapidly than from the NAT4 strains, consistent with one extra positive charge due to one less acetylated amino group. Taken together with the H4 sequencing data described above, this experiment convincingly demonstrates that the H4 N-terminal serine is acetylated by Nat4. H3 exhibited the same electrophoretic mobility in all four strains, as expected, given that H3 is not N-terminally acetylated (Fig. 4). In addition, the electrophoretogram confirmed our previous result that histone H2B is a substrate for Nat1-Ard1 (2Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar). Note that some, but not all, of the H2B isoforms migrate more rapidly in both ard1 mutant strains. This is discussed below. To our surprise, H2A isoforms also migrated more rapidly when isolated from both nat4 mutant strains as compared with the NAT4 strains (Fig. 4). This suggested that H2A, which has an N-terminal serine residue, just as H4 does, also is a substrate for Nat4. We therefore sequenced purified yeast H2A from wild type and the nat4 mutant. H2A from the wild type strain gave no readable sequence, while H2A from the mutant gave the sequence Ser-Gly-Gly-Lys, which corresponds exactly to the first four amino acids of H2A (Fig. 3 and Table I). Thus, H2A also is N-terminally acetylated by Nat4. To examine whether other proteins were substrates for Nat4, we labeled yeast proteins with [35S]methionine in both a wild type strain and a nat4 mutant. A protein extract from each strain was then analyzed by two-dimensional gel electrophoresis followed by autoradiography. We used this method previously to show that many yeast proteins gain an extra positive charge, and thus shift to the basic side, in ard1 mutants (2Mullen J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google Scholar). However, a careful comparison showed that the pattern of protein spots on the gels from the NAT4 and the Δnat4 strains were almost identical (data not shown). The one visible difference was that the Δnat4 mutant had a missing spot at a molecular weight consistent with the predicted size of Nat4 protein. Thus, we saw no evidence that other proteins were substrates for Nat4. Histones H4 and H2A do not contain methionine and thus would not have been seen in the autoradiogram. Also, highly basic proteins like histones are not well resolved by the usual two-dimensional gel methods (14O'Farrell P.Z. Goodman H.M. O'Farrell P.H. Cell. 1977; 12: 1133-1141Abstract Full Text PDF PubMed Scopus (2583) Google Scholar). As mentioned above, the Δnat4 mutant was viable. The mutant grew normally at all temperatures tested on both rich medium and synthetic medium. The strain grew normally on galactose or glycerol medium. A nat4/nat4 homozygous diploid underwent normal sporulation and meiosis, leading to four viable haploids per spore. Three experiments failed to uncover any transcription defects for nat4 mutants. First, the strain could grow on a medium lacking inositol. This is a sensitive test, because many viable mutants in genes for HATs or other transcriptional coactivators are defective in transcription of the INO1 gene, leading to an inositol auxotrophy (15Pollard K.J. Peterson C.L. Mol. Cell. Biol. 1997; 17: 6212-6222Crossref PubMed Scopus (190) Google Scholar). Second, a nat4 mutation was introduced into a strain with an HO-lacZ reporter gene. No defect in lacZ expression was seen. Many mutations affecting the Gcn5 HAT complexes or the Swi/Snf chromatin remodeling complex have a defect in transcription of the gene K. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Peterson C.L. Genes Dev. 1999; PubMed Scopus Google Scholar). the nat4 mutation was into a strain with a mutation to for an F. PubMed Google Scholar, F. M. Trends 1992; 8: Full Text PDF PubMed Scopus Google Scholar). The strain the of an Three experiments that Nat4 protein acetylates the N terminus of H4. First, the recombinant protein acetylated an H4 peptide in even one already acetylated on the four acetylatable (Fig. Second, and H4 isolated from a nat4 mutant could be sequenced by Edman degradation and the expected N-terminal sequence, whereas H4 from a wild type yeast strain was blocked and could not be sequenced (Fig. 3). H4 from the mutant had an mobility on acid-urea gels, consistent with an extra positive charge (Fig. 4). The that all the H4 in the nat4 mutant suggested that all the H4 was acetylated by Nat4 in wild type yeast and that no other NAT could acetylate of the H2A from the nat4 mutant also in acid-urea gels (Fig. 4). This to sequence H2A. As with H4, H2A gave the expected sequence when isolated from the nat4 mutant and gave no sequence from wild type (Fig. 3). H2A has an N-terminal sequence that is similar to that of H4 I). one other yeast protein is similar to H4 and H2A, comparing the first amino and that is the H2A I). is that is also a substrate for Nat4, like H4 and H2A, has no methionine residues and thus could not have been seen in the two-dimensional gel recombinant purified from E. coli an was an substrate for in vitro acetylation by Nat4, while H2B was not acetylated (data not shown). In to H4, H2A, and there are other yeast proteins predicted to with As noted above, we not any mobility comparing proteins from wild type and nat4 mutant strains by two-dimensional gel electrophoresis we that Nat4 is dedicated to the acetylation of histones H4 and H2A and does not on other all the H4 and H2A isoforms had an mobility on the acid-urea gel, we that all of the H4 and H2A in yeast are acetylated by Nat4 (Fig. 4). But, as some, but not all, of the H2B isoforms had an mobility on acid-urea gels when isolated from ard1 mutants J.R. Kayne P.S. Moerschell R.P. Tsunasawa S. Gribskov M. Colavito-Shepanski M. Grunstein M. Sherman F. Sternglanz R. EMBO J. 1989; 8: 2067-2075Crossref PubMed Scopus (246) Google and Fig. 4). for this result is that NatA the catalytic acetylates H2B of the that the two yeast H2B genes for different proteins, one with and the other with NatA can only acetylate one of two and the other one either is not acetylated or is acetylated by as Nat4 is well conserved from yeast to not just in the binding but the of the protein (Fig. We that from also acetylate the N terminus of H4 and H2A, and H4 and H2A are known to be blocked from N-terminal sequencing in all species in which they have been and in the of the proteins, N-terminal acetylation has been by R.J. Fambrough D.M. Smith E.L. Bonner J. J. Biol. Chem. 1969; 244: 319-334Abstract Full Text PDF PubMed Google Scholar). is that nat4 mutants not readily observable in the mutant cell have four extra positive one for each H4 and H2A in the the mutant grew normally all Furthermore, the mutant had no observable transcription defects that do for mutants affecting the Swi/Snf chromatin remodeling the Gcn5 HAT complexes and the proteins in To to uncover we constructed nat4-ard1 and double mutants. We also constructed a strain with the nat4 mutation only one of the genes or of strains had and that of the mutants (data not shown). acetylation of the N-terminal serine on H4 and H2A is the be studies or a synthetic may uncover the for N-terminal acetylation of H4 and H2A. We for that this gene might a NAT, of the for Analysis and of at for protein Grunstein for peptides, and for