Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase
Seongmin JangChanshin KangHan-Sol YangTae‐Yang JungHans HebertKa Young ChungSeung Joong KimSungchul HohngJi‐Joon Song
85
Citation
27
Reference
10
Related Paper
Citation Trend
Abstract:
DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.Keywords:
Histone octamer
Histone H2B
Histone Methylation
Histone code
The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.
Histone octamer
Histone code
Histone H4
Histone Methylation
Cite
Citations (0)
Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.
Histone octamer
Histone code
Histone Methylation
Chaperone (clinical)
Histone H4
Histone-modifying enzymes
Cite
Citations (3)
Histone octamer
Histone code
Histone Methylation
Histone-modifying enzymes
Cite
Citations (54)
The topography of the interaction between histone H1 and the histone octamer has been investigated. Bovine thymus nuclei or enzymatically fragmented chromatin were treated 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, which catalyzes the formation of covalent bonds between residues of proteins in electrostatic contact. Histone H1-core histone dimers were identified and the segments of molecules participating in crosslinking were elucidated. The results demonstrate that the major histone H1-core histone dimer generated upon carbodiimide crosslinking of intact nuclei, chromatin, or mononucleosomes consists of the segment of histone H1 containing amino acids 74-106 crosslinked to the segment of histone H2A containing amino acids 58-129. Thus, the central globular region of histone H1 intimately contacts the histone octamer. Besides histone H1-H2 dimers, two other histone H1-containing crosslinked products were detected. In these instances, the segments of histone H1 molecules containing amino acids 1-72 were shown to participate in crosslinking. The histone H1 contact points defined here all occur within mononucleosomes and not between nucleosomes. These results permit the formulation of a testable model for the arrangement of histone H1 along polynucleosome chains.
Histone octamer
Histone code
Histone Methylation
Cite
Citations (145)
In vitro studies on nucleosome core particles (NCPs) and nucleosomes have generally been limited to the use of histone proteins isolated from chromatin. Numerous reliable and well-established methods have been described of obtaining single histone proteins in significant quantity (e.g., refs. 1 and 2, and references therein). Briefly, the histone complexes (histone octamer, or histone tetramer and histone dimer) are isolated from "long chromatin," which is extracted from nuclei. The histone complexes can be further fractionated into individual histone proteins. This approach suffers from several disadvantages. First, the procedure is time-consuming and depends on the availability of fresh tissue or blood from the organism of choice. Second, histone proteins isolated from natural sources are often degraded by contaminating proteases (3). Third, histone isotypes and posttranslational modifications of histone proteins give rise to heterogeneity. The extent of heterogeneity and modification strongly depend on the type and developmental state of the tissue from which chromatin is isolated and can vary significantly between different batches. Fourth, and most important, only naturally occurring histone proteins can be obtained by this method.
Histone octamer
Histone code
Histone Methylation
Cite
Citations (453)
Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.
Histone octamer
Chromatosome
Linker DNA
Histone Methylation
Histone code
Cite
Citations (43)
Histone octamer
Histone code
Histone-modifying enzymes
Histone Methylation
Cite
Citations (0)
The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures. The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures. Within the eukaryotic cell nucleus the genetic information is organized in a highly conserved structural polymer, termed chromatin, that supports and controls crucial functions of the genome. The fundamental unit of eukaryotic chromatin, the nucleosome, consists of 146 base pairs of genomic DNA wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4. The amino-terminal tails of each of the four core histones are subject to several types of covalent modifications, including acetylation, methylation, and phosphorylation. These modifications affect lysines (acetylation, mono-, di-, and trimethylation), serines and threonines (phosphorylation), and arginines (mono- and two types of dimethylation). These particular modifications can alter the global dynamics of chromatin structure and function (1Fischle W. Wang Y.M. Allis C.D. Histone and chromatin cross-talk.Curr. Opin. Cell Biol. 2003; 15: 172-183Crossref PubMed Scopus (983) Google Scholar, 2Strahl B.D. Allis C.D. The language of covalent histone modifications.Nature. 2000; 403: 41-45Crossref PubMed Scopus (6585) Google Scholar, 3Vaquero A. Loyola A. Reinberg D. The constantly changing face of chromatin.Sci. Aging Knowledge Environ. 2003; 2003: RE4Crossref PubMed Scopus (176) Google Scholar). Alternatively incorporation of histone variants into the nucleosome can also introduce variation in chromatin (4Ausio J. Abbott D.W. Wang X. Moore S.C. Histone variants and histone modifications: a structural perspective.Biochem. Cell Biol. 2001; 79: 693-708Crossref PubMed Scopus (59) Google Scholar). Each class of histone proteins consists of several subtypes that are encoded by different genes except for the H4 histones where all the different genes encode identical amino acid sequences. These genes are highly similar in sequence, are synthesized primarily during the S phase of the cell cycle, and code for the bulk of the cellular histones. There are also nonallelic variants of the major histones or replacement histones expressed at low but constant levels throughout the cell cycle that have significant differences in primary sequence (5Alvelo-Ceron D. Niu L. Collart D.G. Growth regulation of human variant histone genes and acetylation of the encoded proteins.Mol. Biol. Rep. 2000; 27: 61-71Crossref PubMed Scopus (14) Google Scholar, 6Kamakaka R.T. Biggins S. Histone variants: deviants?.Genes Dev. 2005; 19: 295-310Crossref PubMed Scopus (291) Google Scholar). The histone H3 family can be divided into three subtypes: H3.1, H3.2, and the replacement subtype H3.3 (7Franklin S.G. Zweidler A. Non-allelic variants of histones 2a, 2b and 3 in mammals.Nature. 1977; 266: 273-275Crossref PubMed Scopus (236) Google Scholar, 8Frank D. Doenecke D. Albig W. Differential expression of human replacement and cell cycle dependent H3 histone genes.Gene (Amst.). 2003; 312: 135-143Crossref PubMed Scopus (51) Google Scholar). A comparison of the translated amino acid sequences of the known human H2A and H2B variants with their respective molecular masses is shown in Table I (9Albig W. Trappe R. Kardalinou E. Eick S. Doenecke D. The human H2A and H2B histone gene complement.Biol. Chem. 1999; 380: 7-18Crossref PubMed Scopus (33) Google Scholar, 10Marzluff W.F. Gongidi P. Woods K.R. Jin J. Maltais L.J. The human and mouse replication-dependent histone genes.Genomics. 2002; 80: 487-498Crossref PubMed Google Scholar). Additional potential histone variants retrieved by querying the translated EMBL database are also listed (Table I). There are five replacement H2A histones, including H2AZ, macroH2A, H2A-Bbd, and H2AX, that differ considerably in sequence from the bulk H2A sequences (4Ausio J. Abbott D.W. Wang X. Moore S.C. Histone variants and histone modifications: a structural perspective.Biochem. Cell Biol. 2001; 79: 693-708Crossref PubMed Scopus (59) Google Scholar, 11Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Histone H2A variants H2AX and H2AZ.Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (628) Google Scholar, 12Bao Y. Konesky K. Park Y.J. Rosu S. Dyer P.N. Rangasamy D. Tremethick D.J. Laybourn P.J. Luger K. Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA.EMBO J. 2004; 23: 3314-3324Crossref PubMed Scopus (157) Google Scholar). These proteins are present in smaller amounts and have been known as minor variants because of their rarity.Table IComparison of protein sequences and molecular masses of histone H2A and H2B subtypesa Name of the variants derived from Swiss-Prot.b The amino acids in bold indicate a variation in amino acid composition compared with the first amino acid sequence listed.c Average molecular masses of the proteins with acetylated amino termini.d Histone variants retrieved by searching the Swiss-Prot/Uni-Prot databases (www.expasy.org).e Amino acids in conflict.f Histone H2AZ subtypes vary extensively from the sequence of the other H2A variants, and varied amino acids were not included in the table. Also macroH2A1, macroH2A2, and H2A-Bbd were not mentioned in the table.g Additional potential histone variants retrieved by querying the translated EMBL database (TrEMBL). Open table in a new tab a Name of the variants derived from Swiss-Prot. b The amino acids in bold indicate a variation in amino acid composition compared with the first amino acid sequence listed. c Average molecular masses of the proteins with acetylated amino termini. d Histone variants retrieved by searching the Swiss-Prot/Uni-Prot databases (www.expasy.org). e Amino acids in conflict. f Histone H2AZ subtypes vary extensively from the sequence of the other H2A variants, and varied amino acids were not included in the table. Also macroH2A1, macroH2A2, and H2A-Bbd were not mentioned in the table. g Additional potential histone variants retrieved by querying the translated EMBL database (TrEMBL). Relative to core histones H3 and H4, considerably less information on variants and post-translational modifications is available for histones H2A and H2B. Recently a mass spectrometric analysis of human core histone identified the variants H2A1 (H2AA, P02261), H2B1 (H2BR, P06899), and H2BF (H2BF, P33778) albeit with no MS/MS analysis confirmation (13Galasinski S.C. Louie D.F. Gloor K.K. Resing K.A. Ahn N.G. Global regulation of post-translational modifications on core histones.J. Biol. Chem. 2002; 277: 2579-2588Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To date, human histone modifications reported for histone H2A include acetylation on Lys-5 and -9 and phosphorylation on Ser-1 (14Goll M.G. Bestor T.H. Histone modification and replacement in chromatin activation.Genes Dev. 2002; 16: 1739-1742Crossref PubMed Scopus (108) Google Scholar, 15Turner B.M. Cellular memory and the histone code.Cell. 2002; 111: 285-291Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar, 16Matthews H.R. Waterborg J.H. Freedman K.B. Hawkins H.C. The Enzymology of Post-translational Modifications of Proteins. 2. Academic Press, London1985: 125-185Google Scholar). For histone H2B, multiple acetylation sites were identified previously on Lys-5, -12, -15, and -20 (14Goll M.G. Bestor T.H. Histone modification and replacement in chromatin activation.Genes Dev. 2002; 16: 1739-1742Crossref PubMed Scopus (108) Google Scholar, 15Turner B.M. Cellular memory and the histone code.Cell. 2002; 111: 285-291Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar, 16Matthews H.R. Waterborg J.H. Freedman K.B. Hawkins H.C. The Enzymology of Post-translational Modifications of Proteins. 2. Academic Press, London1985: 125-185Google Scholar, 17Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Patterns of histone acetylation.Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar). Modifications outside the NH2-terminal tails of histone H2A and H2B include ubiquitination in the carboxyl-terminal region on Lys-119 and -120, respectively (14Goll M.G. Bestor T.H. Histone modification and replacement in chromatin activation.Genes Dev. 2002; 16: 1739-1742Crossref PubMed Scopus (108) Google Scholar). In the past, microsequencing was widely used to identify modification sites of histone proteins (17Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Patterns of histone acetylation.Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar) and to obtain sequence information for the different histone subtypes (7Franklin S.G. Zweidler A. Non-allelic variants of histones 2a, 2b and 3 in mammals.Nature. 1977; 266: 273-275Crossref PubMed Scopus (236) Google Scholar, 18Doenecke D. Albig W. Bode C. Drabent B. Franke K. Gavenis K. Witt O. Histones: genetic diversity and tissue-specific gene expression.Histochem. Cell Biol. 1997; 107: 1-10Crossref PubMed Scopus (96) Google Scholar). Immunoblotting with specific antibodies is a convenient method for detecting known modifications as it requires only small amounts of samples. However, few antibodies against modified H2A and H2B (19Myers F.A. Chong W. Evans D.R. Thorne A.W. Crane-Robinson C. Acetylation of histone H2B mirrors that of H4 and H3 at the chicken beta-globin locus but not at housekeeping genes.J. Biol. Chem. 2003; 278: 36315-36322Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20White D.A. Belyaev N.D. Turner B.M. Preparation of site-specific antibodies to acetylated histones.Methods. 1999; 19: 417-424Crossref PubMed Scopus (61) Google Scholar) and specific histone subtypes (11Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Histone H2A variants H2AX and H2AZ.Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (628) Google Scholar, 18Doenecke D. Albig W. Bode C. Drabent B. Franke K. Gavenis K. Witt O. Histones: genetic diversity and tissue-specific gene expression.Histochem. Cell Biol. 1997; 107: 1-10Crossref PubMed Scopus (96) Google Scholar) are available. MS provides an ideal tool to overcome these limitations (13Galasinski S.C. Louie D.F. Gloor K.K. Resing K.A. Ahn N.G. Global regulation of post-translational modifications on core histones.J. Biol. Chem. 2002; 277: 2579-2588Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 20White D.A. Belyaev N.D. Turner B.M. Preparation of site-specific antibodies to acetylated histones.Methods. 1999; 19: 417-424Crossref PubMed Scopus (61) Google Scholar, 21Medzihradszky K.F. Zhang X. Chalkley R.J. Guan S. McFarland M.A. Chalmers M.J. Marshall A.G. Diaz R.L. Allis C.D. Burlingame A.L. Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS).Mol. Cell. Proteomics. 2004; 3: 872-886Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Zhang K.L. Tang H. Huang L. Blankenship J.W. Jones P.R. Xiang F. Yau P.M. Burlingame A.L. Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 306: 259-269Crossref PubMed Scopus (126) Google Scholar, 23Bonaldi T. Imhof A. Regula J.T. A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications.Proteomics. 2004; 4: 1382-1396Crossref PubMed Scopus (94) Google Scholar, 24Zhang K.L. Tang H. Analysis of core histones by liquid chromatography-mass spectrometry and peptide mapping.J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003; 783: 173-179Crossref PubMed Scopus (34) Google Scholar, 25Zhang L.W. Eugeni E.E. Parthun M.R. Freitas M.A. Identification of novel histone post-translational modifications by peptide mass fingerprinting.Chromosoma. 2003; 112: 77-86Crossref PubMed Scopus (220) Google Scholar, 26Galasinski S.C. Resing K.A. Ahn N.G. Protein mass analysis of histones.Methods. 2003; 31: 3-11Crossref PubMed Scopus (33) Google Scholar, 27Zhang K. Williams K.E. Huang L. Yau P. Siino J.S. Bradbury E.M. Jones P.R. Minch M.J. Burlingame A.L. Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry.Mol. Cell. Proteomics. 2002; 1: 500-508Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Several groups have combined LC-MS analysis and peptide mapping to identify histone proteins and to characterize their post-translational modifications. However, little and incomplete data have been reported for histones H2A and H2B (13Galasinski S.C. Louie D.F. Gloor K.K. Resing K.A. Ahn N.G. Global regulation of post-translational modifications on core histones.J. Biol. Chem. 2002; 277: 2579-2588Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 23Bonaldi T. Imhof A. Regula J.T. A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications.Proteomics. 2004; 4: 1382-1396Crossref PubMed Scopus (94) Google Scholar, 25Zhang L.W. Eugeni E.E. Parthun M.R. Freitas M.A. Identification of novel histone post-translational modifications by peptide mass fingerprinting.Chromosoma. 2003; 112: 77-86Crossref PubMed Scopus (220) Google Scholar). On the other hand, direct structural characterization of intact histone H2B from Tetrahymena has recently been achieved by FT ion cyclotron MS using electron capture dissociation. Implemented on a home-built instrument, this technology was used to characterize the two major histone H2B variants differing by only three amino acids and their post-translational modifications (21Medzihradszky K.F. Zhang X. Chalkley R.J. Guan S. McFarland M.A. Chalmers M.J. Marshall A.G. Diaz R.L. Allis C.D. Burlingame A.L. Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS).Mol. Cell. Proteomics. 2004; 3: 872-886Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In this study, we combined LC-MS analysis on intact proteins and peptide mapping (MS and MS/MS) to provide a detailed description of histones H2A and H2B subtypes of a human cell line as a basis for the investigation of their potential roles in chromatin structure and function. Jurkat cells were cultured in RPMI 1640 medium containing 10% FCS, 100 IU of penicillin/streptomycin, and 10 μg/ml gentamycin. Cells were washed twice with PBS, and the cell pellet was suspended in lysis buffer (250 mm sucrose, 50 mm Tris-HCl, pH 7.5, 25 mm KCl, 5 mm MgCl2, 0.2 mm phenylmethylsulfonyl fluoride, 50 mm NaHSO3, 45 mm sodium butyrate, 10 mm 2-mercaptoethanol, 0.2% Triton X-100) and centrifuged at 800 × g for 15 min to obtain nuclei. For preparing histones (28Ryan C.A. Annunziato A.T. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1999: 21.2Google Scholar), nuclei were extracted in 6 volumes 0.2 m H2SO4 (16 h at 4 °C). After centrifugation at 16,100 × g, supernatants were precipitated by trichloroacetic acid (25%, final concentration). The pellet was washed with 50 mm HCl in acetone and then with acetone and subsequently dissolved in β-mercaptoethanol (0.1%) in water. The histone aliquots were acidified with TFA to a final concentration of 0.1% and centrifuged for 2 min at 16,100 × g, and an aliquot of the supernatant corresponding to 11 μg of histones was subjected to micro-LC-ESI-MS analysis. After a desalting step of 3 min with 0.1% TFA in 2% ACN on a Vydac C18 precolumn (1-mm inner diameter × 5 mm, 5-μm particle size), the separation of the histone proteins was achieved by reversed-phase HPLC using a Vydac C18 column (150 mm × 1 mm, 5-μm particle size). Individual histones were eluted from the column by applying a multistep gradient of acetonitrile (0–44% B in 5 min, 44–49% B in 17 min, isocratic gradient 5 min at 49%, 49–62% B in 57 min; solvent A: 0.1% TFA in 2% ACN; solvent B: 0.1% TFA in 80% ACN, 50 μl/min). The flow was split after UV detection (214 nm), 42 μl/min were directed to the automated fraction collector and collected into 96-well plates, and 8 μl/min were directed into the mass spectrometer operated in MS mode. Preparative HPLC fractions containing individual histones according to UV and MS chromatograms were pooled, dried (SpeedVac SC 110, Savant Instruments, Farmingdale, NY), and dissolved in 5–10 μl of 25 mm NH4HCO3 or 100 mm Tris-HCl, pH 8. The histones were digested either with endoproteinase Glu-C (sequence grade, Roche Diagnostics) in 25 mm NH4HCO3 at an enzyme ratio of 1:20 at 25 °C for 1–2 h, with trypsin (Promega, Madison, WI) at an enzyme ratio of 1:250 at 37 °C for 5 min in 100 mm Tris-HCl, pH 8, or with endoproteinase Arg-C (sequence grade, Roche Diagnostics) at an enzyme ration of 1:250 at 37 °C for 1–2 h in 100 mm Tris-HCl, pH 8. The reaction was stopped by adding formic acid to a final concentration of 0.1%. For desalting, 5–10 μl of peptide mixtures were absorbed on a POROS R3 column, washed with 0.2% formic acid, and desorbed sequentially with 2 μl of 10, 20, 30, and 50% methanol in 0.2% formic acid. LC-MS of intact histones was conducted using an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA) coupled to an LCQ electrospray ion trap mass spectrometer (LCQ, Thermo Finnigan, San Jose, CA). For optimal ESI conditions, spray voltage was set to 4000 V, capillary temperature was 200 °C, capillary voltage was set to 8 V, and the tube lens was set to 16 V. MS spectra were acquired by scanning over m/z range 200–2000 in 350 ms. The mass accuracy of the instrument for intact proteins was estimated to be 100 ppm. The molecular masses of the histones were determined after deconvolution of the multiply charged ion series (Bioworks software, Thermo Finnigan). Interpretation of MS spectra was done manually by comparing measured masses to calculated masses of histone sequences derived from Swiss-Prot (see Table I). For peptide mass fingerprints, ESI mass spectra were recorded on a QStar Pulsar hybrid quadrupole time-of-flight mass spectrometer equipped with a nanospray ion source (Applied Biosystems, Foster City, CA). The needles (Protana, Odense, Denmark) were loaded with the peptide mixtures and adjusted in front of the orifice, and the spray voltage was set between 900 and 1300 V. The instrument was scanning between 200 and 2000 Da. Parent ions were selected for CID. We achieved a mass accuracy of at least 0.02 Da with external calibration, thus allowing distinction between acetylation and trimethylation (Supplemental Table I) as well as between arginine and dimethyllysine (Supplemental Table II). Interpretation of MS and MS/MS spectra was done manually by comparing peptide measured masses to calculated masses derived from histone sequences (see Table I). We chose a strategy where histones were isolated from nuclei of Jurkat cells by acid extraction and were separated by C18 reversed-phase HPLC (Fig. 1). During separation, 15% of the HPLC effluent was analyzed directly by mass spectrometry with an ion trap LCQ instrument, and the remainder was collected. The fractions were pooled according to the UV and MS chromatograms and digested for detailed analysis by nano-ESI-MS and MS/MS. The HPLC chromatogram showed nine well resolved peaks that after MS data analysis and deconvolution of the multiply charged ion series were identified as histone proteins H1, H2A, H2B, H3, and H4 (Fig. 1). The assignments were based on the masses predicted from known amino acid sequences and allowing for post-translational modifications. No chromatographic separation was observed for histone H2B isoforms, whereas the H2A variants eluted in two distinct HPLC peaks. Compared with the traditional method using a C4 column (22Zhang K.L. Tang H. Huang L. Blankenship J.W. Jones P.R. Xiang F. Yau P.M. Burlingame A.L. Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 306: 259-269Crossref PubMed Scopus (126) Google Scholar, 23Bonaldi T. Imhof A. Regula J.T. A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications.Proteomics. 2004; 4: 1382-1396Crossref PubMed Scopus (94) Google Scholar, 24Zhang K.L. Tang H. Analysis of core histones by liquid chromatography-mass spectrometry and peptide mapping.J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003; 783: 173-179Crossref PubMed Scopus (34) Google Scholar), reversed-phase HPLC using a C18 column was more efficient in separating the individual histones. The elution pattern was consistent and reproducible with similar results obtained for other human cell lines such as THP1 or HeLa cells (data not shown). To confirm the identity of histone H2A and H2B variants and to characterize their modifications, the corresponding fractions containing histones were hydrolyzed either with endoproteinase Glu-C, endoproteinase Arg-C, or trypsin. The resulting peptide mixtures were analyzed by nano-ESI-MS, measured peptide masses were compared with calculated masses derived from the histone H2A and H2B variants, and MS/MS analyses were performed. In the following, a detailed account of the peaks containing H2A and H2B histones is presented. The molecular masses of histone H2A in the first peak (see Fig. 1) were determined after deconvolution of the multiply charged ion series (Fig. 2A) and compared with the theoretical masses of the different H2A subtypes described in Table I. Inspection of the mass spectrum of intact H2A histones allowed a first assignment of several potential protein subtypes. The molecular masses at 13,421.8, 13,900.2, 14,006.8, and 14,046.9 Da fitted well with the calculated masses of histone H2AZ (13,421.5 Da), H2AQ (13,899.2 Da), H2AO (14,006.3 Da), and H2AA (14,046.3 Da), respectively (Fig. 2A). The results are compiled in Table II. Final assignments were confirmed by peptide mapping using nano-ESI-MS of digests of pooled histone fractions.Table IIAssignments of the variants for histone H2A contained in the first and second HPLC peakMassΔmAssignmentbThe major protein variants were assigned. The other variants may be present at lower levels.DeconvolutedaThe molecular masses of the histones were determined after deconvolution of the multiply charged ion series using an automated program.CalculatedDaFirst HPLC peak 13,421.813,421.5+0.3H2AZ 13,900.213,899.2+1.0H2AQ 14,006.814,006.3+0.5H2AO 14,046.914,046.3+0.6H2AA 14,046.914,048.3−1.4H2AO + 1 AccAcetylation is represented by Ac. 14,086.914,086.2+0.7H2AO + 1PdPhosphorylation is represented by P.Second HPLC peak 13,817.113,817.1+0.0Q96KK5 13,847.113,847.1+0.0H2AE 13,928.013,927.1+1.0H2AE + 1 P 14,002.214,002.3−0.1H2AC 14,017.014,018.3−1.3H2AG 14,017.014,016.3+0.7H2AL 14,044.714,046.3−1.6H2AA 14,044.714,044.3+0.4H2AC + 1 Ac 14,081.514,082.3−0.7H2AC + 1 Pa The molecular masses of the histones were determined after deconvolution of the multiply charged ion series using an automated program.b The major protein variants were assigned. The other variants may be present at lower levels.c Acetylation is represented by Ac.d Phosphorylation is represented by P. Open table in a new tab Endoproteinase Glu-C and endoproteinase Arg-C were used as enzymes to achieve 100% sequence coverage of histone H2A. Analysis of Glu-C peptides by MS/MS identified the characteristic peptide 42–56 containing a Met in position 51; this confirmed the presence of histone H2AO and H2AQ in this HPLC fraction (Table III). In contrast, the Glu-C peptide 42–56 and the Arg-C peptide 43–71 containing a Leu in position 51 were never detected in the digests of this HPLC peak, whereas they were always identified in the digests of the second HPLC peak containing H2A (see below). These results indicated the absence of histone H2AA in this fraction. In addition, the presence of histones H2AO and H2AQ in the first HPLC peak was confirmed with the identification of several other peptides: Glu-C-unmodified peptide 1–41 (m/z 560.60, [M + 8H]8+) with a Ser in position 16 and an Ala in position 40 (Fig. 2B), Glu-C peptide 93–121 with a Lys in position 99, and Arg-C peptides 82–128 and 82–129 (Table III). Furthermore inspection of the Glu-C and Arg-C digests identified peptide 60–67 with Val in position 65 as well as peptides 68–95, 96–127, and 82–127 that were only characteristic for histone H2AZ variant (Table III). In conclusion, MS and MS/MS analyses of Glu-C and Arg-C digests confirmed the presence of histone variants H2AO, H2AQ, and H2AZ in the first HPLC peak.Table IIIList of peptides identified by MS/MS for histone H2A contained in the first and second HPLC peaksProteasesResiduesMassSequencesaBold amino acids represent residues that vary in the sequence, acK indicates a monoacetylation on Lys, and pS indicates a phosphorylation on Ser.VariantsbList of histone H2A variants identified in the protein mixtures with the same corresponding sequences.CalculatedMeasuredDaFirst HPLC peak Glu-C1–414476.54476.77SGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAEH2AO/Q Glu-C1–414518.514518.73SGRGacKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAEH2AO/Q Glu-C1–414556.464556.65pSGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAEH2AO/Q Glu-C42–561502.791502.76RVGAGAPVYMAAVLEH2AO/Q Glu-C60–67936.47936.48YLTAEVLEH2AZ Glu-C57–64950.49950.5YLTAEILEH2AO/Q Glu-C93–1213054.853054.83LNKLLGKVTIAQGGVLPNIQAVLLPKKTEH2AO/Q Glu-C65–923211.763211.78LAGNAARDNKKTRIIPRHLQLAIRNDEEH2AO/Q Glu-C68–953127.753127.71LAGNASKDLKVKRITPRHLQLAIRGDEEH2AZ Glu-C96–1273306.943306.96LDSLIKATIAGGGVIPHIHKSLIGKKGQQKTVH2AZ Arg-C1–111142.621142.64SGRGKQGGKARH2AO/Q Arg-C1–111184.631184.64SGRGacKQGGKARH2AO/Q Arg-C1–191816.021816.08AGGKAGKDSGKAKTKAVSRH2AZ Arg-C1–191858.031858.08AGGacKAGKDSGKAKTKAVSRH2AZAGGKAGacKDSGKAKTKAVSR Arg-C1–191900.041900.08AGGacKAGacKDSGKAKTKAVSRH2AZ Arg-C82–1295247.015247.22HLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGKH2AO Arg-C82–1285139.965140.01HLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHKAKSKH2AQ Arg-C85–1274568.594568.49HLQLAIRGDEELDSLIKATIAGGGVIPHIHKSLIGKKGQQKTVH2AZSecond HPLC peak Glu-C1–414476.54476.89SGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAEH2AL Glu-C1–414490.514490.89SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAEH2AC/E/Q96KK5 Glu-C1–414506.514506.33SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYSEH2AA/G Glu-C1–414532.524532.81SGRGacKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAEH2AC/E/Q96KK5 Glu-C1–414570.474570.25pSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAEH2AC/E/Q96KK5 Glu-C42–561484.831484.83RVGAGAPVYLAAVLEH2AA/C/E/G/L/Q96KK5 Glu-C57–64950.49950.49YLTAEILEH2AA/C/E/G/L/Q96KK5 Glu-C65–923211.763212.08LAGNAARDNKKTRIIPRHLQLAIRNDEEH2AA/C/E/G/L/Q96KK5 Glu-C93–1213054.853054.85LNKLLGKVTIAQGGVLPNIQAVLLPKKTEH2AC/E/G/Q96KK5 Glu-C93–1213082.853082.97LNKLLGRVTIAQGGVLPNIQAVLLPKKTEH2AA/L Arg-C1–1111
Histone octamer
Histone code
Histone Methylation
Histone H2B
Cite
Citations (139)
The genetic information of eukaryotes is organised in a nucleoprotein complex
called chromatin. The fundamental repeating unit of chromatin is nucleosome in
which 146 bp of DNA is wrapped around octamer of core histone proteins H2A,
H2B, H3 and H4. Histone H1 (linker histone) associates with DNA between two
nucleosomes. Core histones are highly conserved proteins among all eukaryotes.
The N-terminus of the core histones extends outwards from the nucleosome and
is flexible in nature whereas the globular C-terminus forms the scaffold of the
nucleosome (Fischle et al., 2003). Nature has evolved mechanisms to dynamically
alter chromatin structure like chromatin remodelling by ATP dependent chromatin
remodellers, covalent histone modifications, and replacement of histone proteins by
their respective variants.
Histone octamer
Histone code
Histone Methylation
Chromatosome
Histone-modifying enzymes
Linker DNA
Nucleoprotein
Cite
Citations (0)
Histone octamer
Histone code
Histone H4
Histone Methylation
Cite
Citations (162)