Combinatorial Modification of Human Histone H4 Quantitated by Two-dimensional Liquid Chromatography Coupled with Top Down Mass Spectrometry (2008)

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >104 using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date. Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >104 using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date. Histones are a class of proteins around which DNA is wrapped and packaged inside a eukaryotic nucleus. Two molecules of each core histone H2A, H2B, H3, and H4 together with ∼146 bp of DNA form the fundamental unit of chromatin called the nucleosome. These proteins are heavily modified, with combinations of these enzymatic modifications thought to form a “histone code” orchestrating epigenetic processes such as long-term gene silencing and gene activation (1Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2153) Google Scholar), higher level chromatin packaging (2Felsenfeld G. Groudine M. Nature. 2003; 421: 448-453Crossref PubMed Scopus (811) Google Scholar), and DNA repair mechanisms (3Downs J.A. Nussenzweig M.C. Nussenzweig A. Nature. 2007; 447: 951-958Crossref PubMed Scopus (246) Google Scholar). All of these activities change with relation to the cell cycle, a sequence of events during which a cell commits to DNA replication (G1), replicates its DNA (S), prepares for mitosis (G2), and undergoes cell division (M) (4Schafer K.A. Vet. Pathol. 1998; 35: 461-478Crossref PubMed Scopus (384) Google Scholar). Histone synthesis and deposition are largely coupled to DNA replication during S phase (5Kaufman P.D. Curr. Opin. Cell Biol. 1996; 8: 369-373Crossref PubMed Scopus (43) Google Scholar). As a cell doubles its nuclear DNA, there is a concomitant doubling of the content of histones and nucleosomes. Even though antibodies have been used to track single modifications, the fate of preexisting histone modifications and the acquisition of new histone modifications during the cell cycle is not well understood because this approach is unable to distinguish previously modified forms from newly modified ones (6Rice J.C. Nishioka K. Sarma K. Steward R. Reinberg D. Allis C.D. Genes Dev. 2002; 16: 2225-2230Crossref PubMed Scopus (201) Google Scholar, 7Fang J. Feng Q. Ketel C.S. Wang H. Cao R. Xia L. Erdjument-Bromage H. Tempst P. Simon J.A. Zhang Y. Curr. Biol. 2002; 12: 1086-1099Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 8Spada F. Chioda M. Thompson E.M. J. Cell Biochem. 2005; 95: 885-901Crossref PubMed Scopus (12) Google Scholar). However, an epigenetic mechanism presumably exists to faithfully transmit patterns of histone modification and chromatin structure to ensure normal cellular function over successive generations (9Turner B.M. Cell. 2002; 111: 285-291Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar). Dynamic changes in the PTMs 4The abbreviations used are: PTM, post-translational modification; TDMS, Top Down mass spectrometry; MS, mass spectroscopy; HILIC, hydrophilic interaction chromatography; RP-HPLC, reverse phase hydrophilic interaction liquid chromatography; FTMS, Fourier transform MS; ECD, electron capture dissociation; PRMT, protein arginine methyltransferase; SWIFT, stored waveform inverse Fourier transform. affecting the N-terminal tails of the core histones, which comprise ∼25–30% of their individual masses, are thought to modulate their interactions with both DNA and other proteins (10Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6623) Google Scholar). Great efforts have been made to catalogue the types and sites of modifications and ascertain their biological contexts. For example, it has been asserted that methylation of lysine 9 in human H3 is a mark for heterochromatin by specifically interacting with heterochromatin-associated protein 1 (11Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (698) Google Scholar, 12Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2184) Google Scholar). Nevertheless, the true biological significance of such observations remains to be defined in the absence of quantitative data regarding modification type, co-occurrence with other PTMs on the same histone, and abundance in bulk chromatin. Antisera have been used to provide qualitative descriptions of the genomic distributions of specific histone PTMs in various organisms (e.g. Refs. 13Kurdistani S.K. Tavazoie S. Grunstein M. Cell. 2004; 117: 721-733Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar and 14Fischer J.J. 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PubMed Scopus (201) Google by the relative This the intact mass on the relative of each intact mass and not on the absolute in the intact mass be isobaric (e.g. a mass to H4 be a of acetylation of a single intact mass of As previously in our the of these called be used to the with an of for to J.J. PubMed Scopus (201) Google Scholar). is the relative of each protein form be for that a form over the from and and a for for that comprehensive of all histone H4 modifications and their from 1 is in of histone H4 by of modified from HeLa cells on a as previously W. H. J. 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PubMed Scopus Google Scholar). using to forms (e.g. 1 and from the most forms (e.g. an of a H4 form of mass with a and this mass to of two in a of the dynamic range the of the level forms the dynamic range of the analysis reported HILIC, MS, and to be This to a to detect an H4 form present in of million nucleosomes in the human nucleus. Two and the dynamic range we H4 from cells the of the cell cycle the in the many forms two forms with and the other with dimethylation Lys-20 with N-terminal acetylation and to be to for this which of histone H4 the H4 by to be and of the H4 and and However, the the cell cycle, the this form of H4 during the Cell analysis of each revealed many H4 present <1% in bulk chromatin. these the of a of Lys-20 methylation and acetylation the of these forms change during the cell cycle, as other have reported a the two events (6Rice J.C. Nishioka K. Sarma K. Steward R. Reinberg D. Allis C.D. 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Because of the for each HILIC, MS, and we our analysis to the three cell cycle and cell the same to assess the of our quantitative our for quantitation to the of the we have forms of histone H4 These forms N-terminal and from newly histone H4 by the of H4 of and As the cell from to the form by it as of the histone H4 present in the there a in the of monomethylated H4 and it dimethylated H4 from to during the from the to and there a of to the level in These an in methylation of monomethylated Lys-20 dimethylated Lys-20 during the cell cycle J.J. H. Cell. Biol. PubMed Scopus Google Scholar). and each of the histone H4 present in the nucleus. These present their the and during There in the level of Lys-20 Arg-3 mono- and dimethylation from to the robustness of as an all cell cycle 8, and to for the in characterization and quantitation of histone H4 and data from each biological for the H4 forms in with 42 to the of the three All analysis in assess a this analysis of to the the of the abundance of the modification the by protein form interaction the of the cell cycle on the of all forms of to be for the For the of from to in the to a statistically with 1 of This the of our and be understood by the of the form of histone H4 the during S phase and to the methylation of Lys-20 as the cell of H4 during the Cell of with Top Down mass enabled a dynamic range in of which and quantitation of histone histone H4 the most protein form of nuclear histone histone H4 and This in abundance not to a in as many as patterns of methylation on specifically lysine 20 and and arginine the combinations of in this on molecules with and dimethylated Lys-20 the cell acetylation site a the abundance of as Lys-20 and the as the site of the acetylation site each of the three cell cycle this remains during the cell cycle abundances, that H4 acetylation chromatin with other Allis C.D. S. A. PubMed Scopus Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar). However, other to the of each Lys-20 methylation such as the that is enhanced through by of Lys-20 is simultaneously to analysis of histone H4 forms during the cell cycle from the of analysis. the from not the form not is present the of this aLys-8, aLys-12, aLys-8, aLys-12, analysis. the from not the form not is present the of this in a new the data of are the dynamics of both histone H4 and methylation as the cell acetylation, a of observations be made to a in molecules and monomethylated molecules However, from to the molecules and a for the monomethylated dimethylated molecules by from to These data that methylation Lys-20 on and molecules is as the cell of Lys-20 methylation on and H4 during the three cell cycle phases 5, and This that acetylation not have an on Lys-20 methylation in to in data the two modifications K. J.C. Sarma K. Erdjument-Bromage H. J. Wang Y. S. P. Tempst P. Steward R. Allis C.D. Reinberg D. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). there has been histone to be for H4 Nevertheless, there is a change of Lys-20 during the cell to that the of Lys-20 methylation on molecules the same as on as have reported that acetylation Lys-20 methylation K. J.C. Sarma K. Erdjument-Bromage H. J. Wang Y. S. P. Tempst P. Steward R. Allis C.D. Reinberg D. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Lys-20 we cells H4 by H4 as molecules As the cells the abundance of the and molecules to Lys-20 the of the and dimethylated forms of molecules all histone H4 These that of H4 has on Lys-20 methylation in This new the of 42 H4 forms and their dynamic changes for three cell cycle work the biological significance of the dynamic methylation Lys-20 through the cell cycle and specific combinations of acetylation from the quantitative of H4 PTMs of the types and of modified forms of histone H4 in chromatin for the of an data with