Lysine Succinylation and Lysine Malonylation in Histones

Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism. Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism. Histones and p53 are among the proteins that are found to be most frequently modified (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8034) Google Scholar, 2Dai C. Gu W. p53 post-translational modification: Deregulated in tumorigenesis.Trends Mol. Med. 2010; 16: 528-536Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Collective efforts from the research community identified at least 12 types of protein post-translational modifications (PTMs), 1The abbreviation used is:PTMpost-translational modification. 1The abbreviation used is:PTMpost-translational modification. most of which were identified by mass spectrometry (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8034) Google Scholar, 3Garcia B.A. Shabanowitz J. Hunt D.F. Characterization of histones and their post-translational modifications by mass spectrometry.Curr. Opin. Chem. Biol. 2007; 11: 66-73Crossref PubMed Scopus (118) Google Scholar, 4Sakabe K. Wang Z. Hart G.W. β-N-acetylglucosamine (O-GlcNAc) is part of the histone code.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 19915-19920Crossref PubMed Scopus (280) Google Scholar, 5Kruse J.P. Gu W. SnapShot: p53 posttranslational modifications.Cell. 2008; 133: 930-30.e1Abstract Full Text PDF PubMed Scopus (124) Google Scholar). In addition, the search for histone PTMs has not been exhausted and, not only novel sites, but also novel types of modifications continue to be discovered. For example, Hart and co-workers (4Sakabe K. Wang Z. Hart G.W. β-N-acetylglucosamine (O-GlcNAc) is part of the histone code.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 19915-19920Crossref PubMed Scopus (280) Google Scholar) recently identified O-GlcNAc modification as a new type of histone PTM. They demonstrated that O-GlcNAcylation is dynamically changed during mitosis and in response to heat shock. post-translational modification. post-translational modification. Mounting evidence suggests that histone PTMs play a crucial regulatory role in diverse biological processes, such as cell differentiation and organismal development, and that aberrant modification of histones contributes to diseases, including cancer (6Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (816) Google Scholar, 7Martin C. Zhang Y. Mechanisms of epigenetic inheritance.Curr. Opin. Cell Biol. 2007; 19: 266-272Crossref PubMed Scopus (179) Google Scholar). Thus, understanding this epigenetic process and its roles in cellular physiology and diseases demands a comprehensive understanding of all possible histone modifications. At least two major mechanisms are thought to be associated with contributions of histone PTMs to dynamic chromatin-templated processes (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8034) Google Scholar, 6Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (816) Google Scholar). First, histone PTMs can directly modulate the packaging of chromatin by altering chemical structures of histones or internucleosomal interactions, through a change of the net charge, hydrogen bonding, size, or hydrophobicity in substrate PTM residues. A modified chromatin therefore in turn regulates the access of DNA-binding proteins, such as transcription factors. For example, neutralization of positive charges of lysine residues has been shown to disrupt interactions between positively charged lysine side chain and negatively charged DNA. Second, histone PTMs regulate chromatin structure and function by recruiting PTM-specific binding proteins (also called “readers”), which recognize modified histones via specialized structural folds, such as bromo, chromo, and plant homeo domain (PHD) domains (8Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling.Nature. 2006; 442: 86-90Crossref PubMed Scopus (875) Google Scholar, 9Wysocka J. Swigut T. Milne T.A. Dou Y. Zhang X. Burlingame A.L. Roeder R.G. Brivanlou A.H. Allis C.D. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.Cell. 2005; 121: 859-872Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Zeng L. Zhou M.M. Bromodomain: An acetyl-lysine binding domain.FEBS Lett. 2002; 513: 124-128Crossref PubMed Scopus (552) Google Scholar). Conversely, histone PTMs can also function by inhibiting the interaction of specific binders with chromatin. The remarkable regulatory potential of histone marks has been well illustrated in histone lysine acetylation and lysine methylation. Modifications at different locations (in the residues of histones) are involved in either activation or repression of gene expression. Acetylation versus methylation at the same histone site can be associated with very different transcriptional programs (11Jenuwein T. Allis C.D. Translating the Histone Code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google Scholar). Interestingly, lysine methylation exists in three forms: mono-, di-, and tri-methylation. Subtle chemical differences in these modifications may lead to very different outcomes. Different forms of lysine methylation can be enriched in different parts of chromatin (heterochromatin or euchromatin) (12Martin C. Zhang Y. The diverse functions of histone lysine methylation.Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1591) Google Scholar). They can also be associated with different transcriptional regulatory elements of human genome. For example, histone H3K4 monomethylation specifically marks gene promoters, whereas H3K4 trimethylation is primarily associated with enhancers (13Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. Wang W. Weng Z. Green R.D. Crawford G.E. Ren B. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (2409) Google Scholar). We recently discovered two types of novel PTMs called lysine succinylation and lysine malonylation in non-histone proteins (see Fig. 1A) (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar, 15Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M111.012658)Abstract Full Text Full Text PDF Scopus (511) Google Scholar). In this study, we report that lysine succinylation and lysine malonylation are new types of histone PTMs. Our preliminary studies in Saccharomyces cerevisiae suggest lysine succinylation and malonylation in histones might have functional consequences. All chemicals, unless otherwise indicated, were of the highest purity available or analytical grade purchased from Sigma-Aldrich. 2,2,3,3-D4-succinic acid was purchased from Cambridge Isotope Laboratories (Andover, MA). Dulbecco's modified Eagle's medium and YPD medium were purchased from Fisher. Schneider's Drosophila medium was purchased from Invitrogen. HeLa and mouse embryonic fibroblast cells were obtained from the ATCC (Manassas, VA). All of the synthetic peptides used in this study were synthesized through customer synthesis using N-(9-fluorenyl)methoxycarbonyl-Lys (mono-tert-butyl succinate)-OH. HeLa and mouse embryonic fibroblast cells were grown to 95% confluence in high glucose (4.5 g/liter) Dulbecco's modified Eagle's medium (with glutamine and sodium pyruvate) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with 95% air and 5% CO2. Drosophila S2 cells were grown in Schneider's Drosophila medium containing 10% heat-inactivated fetal bovine serum at 26 °C until the cell density reached 1 × 107 cells/ml. Yeast (BY4741) cells were grown in YPD medium at 30 °C for 16 h with shaking at 230–270 rpm until A600 reached 2.4. For isotopic labeling, HeLa cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% penicillin-streptomycin, and 50 mm of sodium D4-succinate for 24 h until 95% confluence. Extraction of the histones followed the acid extraction method described previously (16Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). Chemical propionylation of histone extracts was performed using a procedure previously reported with slight modifications (17Garcia B.A. Mollah S. Ueberheide B.M. Busby S.A. Muratore T.L. Shabanowitz J. Hunt D.F. Chemical derivatization of histones for facilitated analysis by mass spectrometry.Nat. Protoc. 2007; 2: 933-938Crossref PubMed Scopus (279) Google Scholar). Briefly, 3 mg of histone extracts were dissolved in 50 μl of 100 mm ammonium bicarbonate buffer (pH 8.0) and added with 300 μl of propionic anhydride in 300 μl of methanol. Ammonium hydroxide was added to adjust the solution pH to ∼8.0. After incubation at 51 °C for 20 min, the mixture was dried in a SpeedVac. In-solution histone digestion was carried out as previously reported (18Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). Enrichment of lysine succinylated and malonylated peptides from tryptic digest of histones, with or without in vitro propionylation, by peptide immunoprecipitation with pan anti-succinyllysine and anti-malonyllysine antibodies (PTM Biolabs Inc., Chicago, IL), was carried out as described previously (18Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). Peptide samples were analyzed by a NanoLC-1D plus HPLC system (Eksigent Technologies, Dublin, CA) coupled to an LTQ Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA) as described previously (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). The peptides were eluted from a home-made capillary Jupiter C12 column (10-cm length × 75-μm inner diameter, 4-μm particle size, 90 Å pore diameter; Phenomenex, St. Torrance, CA) with a 2-h gradient of 2% to 80% HPLC solvent B (0.1% formic acid in acetonitrile, v/v) in solvent A at a flow rate of 200 nl/min. High resolution full scan MS spectra (from m/z 350 to 1800) acquired in the Orbitrap with resolution r = 60,000 at m/z 400 was followed by MS/MS fragmentation of the 20 most intense ions in the linear ion trap analyzer with collisionally activated dissociation energy of 35%. Verification of lysine succinylated peptides by HPLC/MS/MS analysis of synthetic peptides was used the same method as described previously (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). Briefly, the affinity-enriched histone its synthetic and their mixture were analyzed by respectively. The mass analysis was performed by search were by For protein the from and were human protein mouse protein Drosophila protein and Saccharomyces protein respectively. and were used for protein The identified proteins from were a new for PTM The identified protein were in The search mass for ion mass was as and for ion as was as with lysine lysine malonylation and lysine succinylation were as modifications. For the histone lysine propionylation was also as a modification. All of the peptide with ion 20 were verified their MS and MS/MS The used to for histone and is to and a of All were in to and a of The histone and histone were by gene synthesis and at The assays were described previously J. H. A of synthetic histone H3 and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). for the for all histone H3 and were from a study J. H. A of synthetic histone H3 and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). We the of histone lysine succinylation in eukaryotic cells the that all the major PTMs are present in histones, including but not to and O-GlcNAc modification. for the of lysine succinylation in histones, we carried out analysis of histones from eukaryotic using an anti-succinyllysine The lysine succinylation from the histones of all The can be by a peptide a at the but not its peptide The of the was demonstrated by using a peptide and (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). results that lysine succinylation is found in histones and an histone in eukaryotic sites, we histones from HeLa cells using a procedure described previously (16Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). The histones were in with or without chemical propionylation, and to using anti-succinyllysine as reported (18Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). succinylated peptides were analyzed by HPLC/MS/MS analysis and protein to sites in The residues can be identified a mass of 100 at the lysine The to the identification of sites in HeLa histones the same we also identified 7, 10, and 7 succinylation sites in histones from cells of and respectively. The MS/MS spectra were verified to the high of the peptide identification and are in the and for Fig. 4 and and mass of histone succinylation at high resolution MS/MS spectra of succinylation peptide from affinity-enriched HeLa histone using anti-succinyllysine pan the synthetic and the mixture of The the The or ions with ion of the in peptide its synthetic and their mixture MS and MS/MS of and mass of histone succinylation at high resolution MS/MS spectra of in succinylation peptide from affinity-enriched HeLa histone the synthetic and the mixture of ion of the in peptide its synthetic and their mixture MS and MS/MS of We are that the identified mass of 100 is by lysine the histone peptides were MS/MS lysine succinylation is a new is to the structure of the identified peptides to that the mass of 100 is by lysine succinylation. MS/MS and HPLC are for peptide this we synthesized three and We carried out MS/MS and between the synthetic peptides and their in respectively. Our that the high resolution MS/MS fragmentation of in the synthetic and their mixture were the mixture of the in peptide and the synthetic a in the HPLC that the mass was by a the same we also the peptide identification for 4 and Fig. the of lysine succinylation in histones, we carried out in isotopic followed by HPLC/MS/MS analysis of histone peptides as described previously (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). In this we HeLa cells with isotopic for 24 The histones were and analyzed using the described was identified in histone peptides and and Fig. that histone lysine succinylation can be in to histone lysine by using a A evidence for roles of in J. 2005; PubMed Scopus Google Scholar). the biological function of lysine succinylation in histones, we the modified to and to succinylation and to acid to succinylated the we found that the but not or histone cell sites cell After as described J. H. A of synthetic histone H3 and 2008; Full Text Full Text PDF PubMed Scopus Google we found significant for all histone and with In addition, all of the histone at and was among the and we found that histone but not or was to a of succinylation of this In addition, we identified unique of histone a of at and with a the is in a whereas the at but has J. H. A of synthetic histone H3 and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). the at 37 and which is not in the malonylation has been reported recently in and cells C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M111.012658)Abstract Full Text Full Text PDF Scopus (511) Google Scholar). is not lysine malonylation exists in histone using with pan anti-malonyllysine and mass we identified one and two sites in histones from HeLa and S. cerevisiae cells, The peptides can be identified the of in their MS/MS spectra C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M111.012658)Abstract Full Text Full Text PDF Scopus (511) Google Scholar) and their high resolution ion Fig. and the potential function of lysine malonylation in we of the sites and histone to acid to modification and analyzed the of these as described J. H. A of synthetic histone H3 and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). We not significant change of for the with the cell in as shown in and Fig. In this study, sites of in were of which two sites are histone two histone two histone and one histone respectively. Interestingly, of these sites are the a modified in In two of the succinylated sites and are at the of its the histones with DNA. the succinylated sites, all of are to the DNA. The of these succinylated residues and the of lysine succinylation suggest that may with the interaction between histones and the negatively charged DNA. of histone which is to the of a in cell In addition, is that of the sites are the potential access to and the the succinylated is also well to be is carried out by in S. cerevisiae and is for gene activation in by methylation of the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Identification of succinylation this the that its function may be by this modification. is to be and acetylation this is for histone Zhang K. M. Acetylation in histone H3 domain regulates gene expression in 2005; 121: Full Text Full Text PDF PubMed Scopus Google Scholar). We identified lysine malonylation in and lysine succinylation in and of with acid in Fig. whereas of the site with or not change cell J. H. A of synthetic histone H3 and 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. A comprehensive of histone residues for H3K4 methylation.Nat. Mol. Biol. 2008; PubMed Scopus Google Scholar). histone the site and is to the DNA the change of from positive to at the site by malonylation might with its interaction with the of cell we have not performed to the of histone lysine succinylation and lysine malonylation at specific sites, studies suggest that the of these two modifications are histone lysine methylation and lysine the two most histone and are in with lysine In cells, histone lysine succinylation sites can be by without the and are histone and sites not the that histones are in the cells, PTMs not the that have biological an example, we recently demonstrated that histone lysine a histone PTM with lysine is associated with specific functions in transcriptional not we the that lysine malonylation and lysine succinylation is by chemical directly from and a of evidence that lysine succinylation and lysine malonylation in First, in this study, we have identified unique sites that have not been reported to either have lysine acetylation and or PTMs Second, of the sites we identified are among most of sites are in the domain and the of the of histones, which are among the residues that access for chemical we that histone PTMs and chemical histone PTMs may have significant biological the of these modifications be a for cellular acetylation the positive side chain of histone lysine chromatin structure and function by interactions between histones and DNA and recruiting a the lysine residues have with and of the major K. DNA binding the Opin. Biol. 8: PubMed Scopus Google a role in their interactions with DNA through succinylation and malonylation significant structural lysine acetylation by a positively charged to a In of lysine succinylation and malonylation are of the same as a in the substrate residues. the crucial role of histone lysine PTMs lysine acetylation and in processes, such a structural is to have significant for chromatin structure and lysine succinylation and malonylation are to play roles in histone structure and with