Histone Acetylation and Deacetylation

The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C4 reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4. The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C4 reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4. The basic structural unit of eukaryotic chromosomes is a DNA·protein complex called the nucleosome. The nucleosome consists of a DNA molecule associated with a histone octamer comprised of pairs of the core histones H2A, H2B, H3 and H4. The nucleosomes are joined by linker DNA and histone H1 to form chromatin. Each core histone has a globular region, the histone fold domain, which is involved in histone-histone interactions (1.Arents G. Burlingame R.W. Wang B.C. Love W.E. The nucleosomal core histone octamer at 3.1 Å resolution: a tripartate protein assembly and a left-handed superhelix.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10148-10152Google Scholar), and the wrapping of DNA around the nucleosome core (2.Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 Å resolution.Nature (London). 1997; 389: 251-260Google Scholar). The N-terminal “tail regions” extend outside of the nucleosome particle where they can interact with DNA and with other regulatory proteins or transcription factors (2.Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 Å resolution.Nature (London). 1997; 389: 251-260Google Scholar, 3.Bradbury E.M. Reversible histone modifications and the chromosome cell cycle.Bioessays. 1992; 14: 9-16Google Scholar, 4.Banéras J.L. Martin A. Parello J. The N tails of histones H3 and H4 adopt a highly structured conformation in the nucleosome.J. Mol. Biol. 1997; 273: 503-508Google Scholar). These tails are essential but appear partially redundant. At least one of the two tails for both the H2A-H2B and H3-H4 pairs must be intact to maintain viability of yeast cells (5.Ling X. Harkness T.A. Schultz M.C. Ficher-Adams G. Grunstein M. Yeast histone H3 and H4 amino termini are important for nucleosome assembly in vivo and in vitro: redundant and position independent functions in assembly but not in gene expression.Genes Dev. 1996; 10: 686-699Google Scholar). The tails account for 28% of the core histone sequences and are extremely basic because of a high proportion of lysine and arginine. In the 2.8-Å nucleosome crystal structure, the electron densities for these tails are largely not observed (6.Lugar K. Richmond T.J. The histone tails of the nucleosome.Curr. Opin. Genet. Dev. 1998; 6: 140-146Google Scholar). Presumably their binding sites lie outside of the core particle. Several studies indicate that these tails do not contribute significantly to the primary wrapping of DNA in the nucleosome. Trypsinized nucleosome core particles are just as stable as intact particles with respect to perturbations in temperature, high salt concentrations, and accessibility to DNase I (7.Ausio J. Dong F. van Hold K.E. Use of selectively trypsinized nucleosome core particles to analyze the role of the histone tails in the stabilization of the nucleosome.J. Mol. Biol. 1989; 206: 451-463Google Scholar, 8.Hays J.J. Clark D.J. Wolffe A.P. Histone contribution to the structure of DNA in a nucleosome.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6829-6833Google Scholar). Neutron scattering studies have shown that hyperacetylation of histones has little or no effect on core particle structure in solution (9.Imai B.S. Yau P.M. Baldwin J.P. Ibel K. May R.P. Bradbury E.M. Hyperacetylation of core histones does not cause unfolding of nucleosomes. Neutron scatter accords with disc structure of nucleosomes.J. Biol. Chem. 1986; 201: 8784-8792Google Scholar). However, the histone tail sequences are highly conserved, and the reversible acetylation of the ε-amino groups of specific lysine residues has been implicated in key regulatory events (3.Bradbury E.M. Reversible histone modifications and the chromosome cell cycle.Bioessays. 1992; 14: 9-16Google Scholar, 10.Allfrey V.G. Li H.J. Eckhardt R.A. Chromatin and Chromatin Structure. Academic Press, New York1977: 167-191Google Scholar, 11.Grunstein M. Histone acetylation in chromatin structure and transcription.Nature (London). 1997; 389: 349-352Google Scholar, 12.Brownell J.E. Allis C.D. Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.Curr. Opin. Genet. Dev. 1996; 6: 176-184Google Scholar, 13.Turner M.T. O’Neill L.P. Histone acetylation in chromatin and chromosomes.Semin. Cell Biol. 1995; 6: 229-236Google Scholar, 14.Wade P.A. Pruss D. Wolffe A.P. Histone acetylation: chromatin in action.Trends Biochem. Sci. 1997; 22: 128-132Google Scholar, 15.Kornberg R.D. Lorch Y. Chromatin-modifying and remodeling complexes.Curr. Opin. Genet. Dev. 1999; 9: 148-151Google Scholar). The acetylation of histone H4 is restricted to lysine 5, 8, 12, and 16 (16.Clarke D.J. O’Neill L.P. Turner B.M. Selective use of H4 acetylation sites in the yeast S. cerevisiae..Biochemistry. 1993; 294: 557-561Google Scholar). Another known post-translational modification is methylation of lysine 20 (17.Borun T.W. Pearson D. Paik W.K. Studies of histone methylation during the HeLa S-3 cell cycle.J. Biol. Chem. 1972; 247: 4288-4298Google Scholar), which precludes acetylation at this site (18.Annunziato A.T. Eason M.B. Perry C.A. Relationship between methylation and acetylation of arginine rich histones in cycling and arrested HeLa cells.Biochemistry. 1995; 34: 2916-2924Google Scholar). Random histone acetylation would yield four mono-acetylated isoforms, six di-acetylated isoforms (Lys-5/Lys-8, Lys-5/Lys-12, Lys-5/Lys-16, Lys-8/Lys-12, Lys-8/Lys-16, and Lys-12/Lys-16), four tri-acetylated isoforms (Lys-5/Lys-8/Lys-12, Lys-5/Lys-12/Lys-16, Lys-8/Lys-12/Lys-16, and Lys-5/Lys-8/Lys-16), and one tetra-acetylated isoform. Table I shows the N-terminal sequence of H4, which contains all four acetylation sites (Lys-5, −8, −12, and −16) and the single methylation site at lysine 20. There are clearly functional differences among lysine residues. However, the specific role of each of these forms will remain unclear until measurable differences in the chromatographic, electrophoretic, and mass spectrometric properties of these forms can be correlated with cellular events (19.Rice J.C. Allis C.D. Histone methylation versus histone acetylation: new insights into epigenetic regulation.Curr. Opin. Cell Biol. 2001; 13: 263-273Google Scholar). This study is directed toward that goal.Table IPeptide fragments of histone H4 observed by MALDI-TOF mass spectrometry Open table in a new tab Histone acetylation is a very specific phenomenon with various isoforms playing distinct roles (13.Turner M.T. O’Neill L.P. Histone acetylation in chromatin and chromosomes.Semin. Cell Biol. 1995; 6: 229-236Google Scholar). Acetylation is a dynamic phenomenon with the steady state mediated by the opposing activities of histone acetyltransferases (HATs) 1The abbreviations used are: HAT, histone acetyltransferase; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; PSD, post-source decay; ESI, electrospray; MS/MS, tandem mass spectrometry; CID, collision-induced dissociation. and deacetylases. These activities involve large regulatory complexes that are capable of responding to specific DNA sequences and can contain transcription factors, regulatory ligands, and signal transduction and cell cycle proteins. Acetylation plays a role in nucleosome assembly. Newly synthesized histone H4 is di-acetylated in the cytoplasm at Lys-5 and Lys-12 by B-type histone acetyltransferase (HAT B) (20.Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Google Scholar). CAF-1 (chromatin assembly factor 1) is a complex of proteins that deposits Lys-5, Lys-12 di-acetylated H4 into chromatin. It has also been found that human CAF-1 readily assembles newly synthesized H3 and H4 onto replicating DNA in vitro. Presumably the specific acetylation patterns of nascent histones are required for their assembly in vitro (21.Kaufman P.D. Kobayashi R. Kessler N. Stillman B. The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.Cell. 1995; 81: 1105-1114Google Scholar). The acetylation pattern found in mature nucleosomes differs from that of newly incorporated histones. Increased acetylation is generally correlated with transcriptionally active or poised genes. Histone acetylation within nuclei is effected by A-type histone acetyltransferases (HAT A) that are likely to participate in gene activation. An in-gel HAT assay was used to purify and clone the cDNA for the major macronuclear HAT A, p55, from Tetrahymena (22.Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Roth S.Y. Allis C.D. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation.Cell. 1996; 84: 843-851Google Scholar). The gene encoding this enzyme is homologous to the yeast transcriptional co-activator gene Gcn5. The yeast protein Gcn5p, with ADA2 and ADA3, interacts with enhancer binding factors thereby establishing a direct mechanistic relationship between acetylation and gene activation (23.Guarente L. Transcriptional coactivators in yeast and beyond.Trends Biochem. Sci. 1995; 20: 516-521Google Scholar). The yeast protein has also been shown to possess HAT activity. Gcn5p and p55 both contain a bromodomain (12.Brownell J.E. Allis C.D. Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.Curr. Opin. Genet. Dev. 1996; 6: 176-184Google Scholar), which has been shown to be important for the assembly and activity of multisubunit transcriptional activation complexes (12.Brownell J.E. Allis C.D. Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.Curr. Opin. Genet. Dev. 1996; 6: 176-184Google Scholar). The bromodomain may tether HAT A to specific chromosomal sites, linking histone acetylation and gene activation (12.Brownell J.E. Allis C.D. Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.Curr. Opin. Genet. Dev. 1996; 6: 176-184Google Scholar, 22.Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Roth S.Y. Allis C.D. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation.Cell. 1996; 84: 843-851Google Scholar, 24.Hays S.R. Dollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. The bromodomain, a conserved sequence found in human, Drosophila and yeast proteins.Nucleic Acids Res. 1992; 20: 2603Google Scholar). The Gcn5p·ADA complex interacts functionally with the SWI·SNF complex, which is part of the RNA polymerase II holoenzyme (12.Brownell J.E. Allis C.D. Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation.Curr. Opin. Genet. Dev. 1996; 6: 176-184Google Scholar, 25.Peterson C.L Tamkun J.W. The SWI/SNF complex: a chromatin remodeling machine?.Trends Biochem. Sci. 1995; 20: 143-146Google Scholar). Further evidence for the connection between acetylation and transcription activation is the finding that H4 shows increased Lys-16 acetylation in the chromosome of Drosophila J. R. Turner B.M. histone H4 on the chromosome is associated with in Dev. Scholar). In the chromosome of H4 is Turner B.M. The chromosome in is by a of histone H4 acetylation. A for gene 1993; Scholar). that H4 in transcriptionally is in H4 histones from the of Drosophila B.M. J. Histone H4 isoforms acetylated at specific lysine residues chromosomes and chromatin in Drosophila 1992; and yeast M. Allis C.D. transcriptional in acetylation Biol. 1996; are acetylated only at that this modification is important in gene in these Histone acetylation and deacetylation may roles in cell cycle (3.Bradbury E.M. Reversible histone modifications and the chromosome cell cycle.Bioessays. 1992; 14: 9-16Google Scholar). The of which has no and Press, to between acetylation specific for and H4 in cells was found to be acetylated at Lys-5, and In the was at in was at the will be by that is to the of acetylation isoforms as a of cellular events and gene Allis and C.D. R. histone acetylation in of Natl. Acad. Sci. U. S. A. Scholar, R.E. Cook R.G. Allis C.D. acetylation of histone H4 by a histone acetyltransferase as by Biol. Chem. used and to determine the acetylation sites of newly synthesized H4 in and have been used by M. A. Histone H4 from is Biol. Chem. Scholar), and Press, Scholar), A.W. D. K. C. of histone Biochem. Scholar), and of sites of acetylation in histone 1992; Scholar). M. A. Histone H4 from is Biol. Chem. observed that the tri-acetylated form of H4 is and that the di-acetylated and mono-acetylated forms are and Lys-12 that Lys-5 is acetylated Lys-12 M. A. Histone H4 from is Biol. Chem. Scholar). In H4 B.M. the 1993; Scholar), Lys-16 is the only mono-acetylated and all di-acetylated forms also involve that Lys-16 is acetylated Turner used specific for acetylated forms of H4 to that Lys-5 and Lys-12 are in mono-acetylated H4 from a of cell and that Lys-16 are the to be acetylated B.M. the 1993; Scholar). of cells with histone as the in the direction of acetylation of histones G. Bradbury V.G. of histone deacetylation to of forms of histone H3 and H4 and increased DNase I of associated DNA Natl. Acad. Sci. U. S. A. Scholar). A.W. D. K. C. of histone Biochem. have that for H4 from and from butyrate-treated HeLa cells Lys-16 is the but of groups is specific and sites and Lys-5 in N-terminal direction. from butyrate-treated HeLa the isoform was found to be as as the and isoforms. In all the the were by the of and the for histone and which involved of by mass spectrometric of the separated shows for R.D. of histone sequence and modifications by mass spectrometry and tandem mass 1993; the use of ionization mass spectrometry and tandem mass spectrometry to the sequence and modifications of histone the use of mass spectrometric to the acetylation sites of histone H4 to the and the of H4 acetylation isoforms. Our mass spectrometric shows and with high that the acetylation of H4 in HeLa cell nuclei proceeds from lysine 16 to lysine These results the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Our results also revealed that lysine 20 is in all the acetylated isoforms, as well as the non-acetylated isoform of H4. HeLa from were in with as Yau Bradbury E.M. and of acetylated histone H3 and Biol. Chem. or from Cell histones were by the with for to were in and histones were from nuclei with N and separated reverse-phase C4 C4 on a with a A of with a of and was H4 with were by 1) and Histone H4 bands were excised from the gel and digested with K. Burlingame The role of mass in protein mass spectrometry and Chem. 1999; Scholar). of histones were in 20 of and at with of of solution digested peptides were a mass of all peptides were by a with in the A of to at was within the mass of to and were for each and peptides were observed as in the were for of were with of a in were by peptide mass of peptides were on a time-of-flight mass with a cell with nanoelectrospray was in the of to both mass spectrometric and of and a mass of at least with was of was into a nanoelectrospray mass were to mass of peptides and to their from of stable of were for sequence analysis by tandem mass Histone H4 were by and The single histone H4 all acetylated isoforms was excised and digested with trypsin. The peptides were by MALDI-TOF mass spectrometry to determine their mass The specific peptide sequences of histone H4 were to the mass in by the with mass The are in Table in which the acetylation sites in each specific peptide sequence are lysine residues are to by acetylated lysine residues are The mass for the peptide sequences to the various acetylated isoforms are in Table the mass of the peptide sequences that contain acetylation sites and Table with the mass of all of N-terminal of histone H4 Table that only four of the 15 acetylated peptide sequences were observed in the MALDI-TOF of HeLa histone H4. The mass of these four were and These to the tetra-acetylated peptide sequence the tri-acetylated peptide sequence the di-acetylated peptide sequence and the mono-acetylated peptide sequence The at mass to peptide sequence from the tri-acetylated isoform of histone H4 in which the lysine was not during the of this the at the of increased The at mass is to peptide sequence where lysine 20 is modified by two The sequence was by of N-terminal Histone H4 Open table in a new tab The modified peptides were to collision-induced to the acetylation The peptide sequence to the at mass was by to be the 5, 8, 12, of peptide sequence of histone shown in the are and and and a of as in the A and one at were also observed. The of and are to acetylated lysine This the to peptide which is with the tetra-acetylated peptide sequence a tandem mass spectrometry the of mass at was as peptide sequence where lysine residues 8, 12, and 16 are all acetylated The other of molecular mass to the peptide sequence mass is is by this The of of this peptide be because of at to two peptides and The mass at can be as peptide sequence mass where lysine and are or peptide sequence mass where lysine 12 and 16 are The results from the tandem mass spectrometry in revealed that this the peptide sequence that of the sequence Although the mass of that lysine 12 and 16 are the of this peptide not be It from the di-acetylated isoform of histone H4 12 and 16 are or the tri-acetylated isoform of histone H4 in which lysine 5, 12, and 16 are and lysine is not acetylated and by Table However, the peptide sequence acetylated Lys-5 with a mass of to peptide sequence was not observed that the tri-acetylated isoform of histone H4 at sites Lys-5, and Lys-16 is likely to be the that lysine 20 is modified by two groups in all forms of histone H4, histone H4 was digested with which peptide shown in of and to and tetra-acetylated peptide isoforms is the mass of on the that the mass are mass mass of the is to that lysine 20 must be modified by two of lysine 20 in all H4 isoforms with of acetylation. lysine residues 5, 8, 12, and 16 are acetylation sites has been by and by MALDI-TOF and tandem mass spectrometry and are as sites for This only lysine 20 as the methylation The at lysine 20 was by the of the peptide sequence by have shown that tandem mass spectrometry can be used to the specific sites of modification of histone acetylation isoforms from a and that the is to sequence from from This is a in and of It also the associated with N-terminal acetylation of H4 which These studies have that there is a pattern to the acetylation of H4. butyrate-treated HeLa cells the only acetylated forms are as the tetra-acetylated form is acetylated at lysine 5, 8, 12, and the tri-acetylated form is acetylated at lysine 12, and the di-acetylated form is acetylated at lysine 12 and and the only significant amount of mono-acetylated form is modified only at position 16. This evidence for the of M. A. Histone H4 from is Biol. Chem. Scholar), Turner B.M. J. Histone H4 isoforms acetylated at specific lysine residues chromosomes and chromatin in Drosophila 1992; Scholar), and A.W. D. K. C. of histone Biochem. Scholar), these results are for histone from HeLa with a and the acetylation pattern of histones. The and of acetylation of histones at may and are by both HATs and by histone The of these two must be coordinated at a non-random distribution of isoforms for the cell is observed. The spectrometry may be to a of cells and to specific chromosomes or chromatin and to cell cycle events to a hypothesis a model where histone acetylation at lysine 16 and until the four sites are acetylated histone deacetylation must in the reverse direction. Our mass spectrometric results revealed that lysine in the N of histone H4, is di-methylated and that methylation in all isoforms of histone H4 of the of acetylation. It would be to this site plays role in acetylation. as part of the HAT and binding there special the pattern of acetylation 12 The acetylation of histones results in the of the on lysine residues in the core histone N which would the between histone N-terminal and studies revealed that acetylation the binding of the H4 N to DNA by six of in L. Yau Bradbury E.M. Studies of the DNA binding properties of histone H4 amino Biol. Chem. 1993; Scholar). with a model of DNA as a and H4 with of acetylation as the that non-acetylated H4 and mono-acetylated H4 cause from and of histone H4 in of the DNA G. R. between N-terminal of H4 and DNA is by the acetylation 1998; Scholar). non-acetylated H4 and mono-acetylated H4 have a of of the DNA and tetra-acetylated H4 significantly studies are required to determine the site of acetylation is as important as the of It is to that the site of histone acetylation plays important role in the binding of histones with transcription factors, because in is not The non-acetylated histones are likely to adopt specific to DNA chromatin but binding transcription factors or other specific proteins acetylation has been shown to transcription factor binding J.J. Pruss D. Wolffe A.P. A role for histone acetylation in transcription factor to nucleosomal 1993; Scholar, and with histone deacetylation and 1997; Scholar, J.C. C. Wolffe A.P. and of core histone the 1998; Scholar, M. P.A. C. Allis C.D. J.L. Acetylation of histone H4 plays a primary role in transcription factor binding to nucleosomal DNA in J. 1996; Scholar). the Cell for the of HeLa