Precise Characterization of Human Histones in the H2A Gene Family by Top Down Mass Spectrometry Michael T. Boyne II,†,⊥ James J. Pesavento,‡ Craig A. Mizzen,§,| and Neil L. Kelleher*,†,‡,|,⊥ Department of Chemistry, Biophysics, and Computational Biology, and Department of Cell and Developmental Biology, Institute for Genomic Biology, The Center for Top Down Proteomics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received August 16, 2005
Top Down analysis revealed that at least fourteen genes encoding histone H2A are coexpressed in HeLa cells. Characterization of these species1 revealed that all except H2A.Z and H2A.F/Z were R-Nacetylated, H2A.O and H2A.C,D,I,N,P were the most abundant, and those exceeding ∼10% abundance lacked post-translational modifications. This unequivocal identification of H2A forms illustrates the advantages of Top Down Mass Spectrometry and provides a global perspective of H2A regulation through the cell cycle. Keywords: histone H2A • Fourier transform mass spectrometry (FTMS) • Top Down • post-translational modification (PTM) • SILAC • cell cycle • ubiquitination • electron capture dissociation (ECD) • histone code • chromatin
Introduction The central dogma of molecular biology describes the transcription of DNA into RNA followed by the translation of RNA into protein as the fundamental tenet of life. This conceptually simple idea of cellular function fails to address biological regulation through post-translational modification (PTM) of proteins. In addition, the evolution of structurally similar but functionally distinct variants adds to proteome complexity in higher eukaryotes. DNA is highly structured and condensed into nuclear chromatin by its intimate association with a family of proteins known as histones. About 150 base pairs of DNA are wrapped around an octamer of core histones (two copies of each H2B, H2A, H4, and H3) to form a nucleosome,2 the fundamental subunit of chromatin. Increasingly, histones are believed to play multiple roles in eukaryotic biology beyond the compaction of DNA to permit packaging into nuclei. Histones and their numerous post-translationally modified forms are believed to recruit other DNA-associated proteins,3 mark genes for silencing,3,4 and may transmit information in an epigenetic fashion to progeny cells during development to act as a cellular memory bank.5 They are also implicated in DNA repair and replication.6 It is apparent in chromatin biology that understanding the modification states of histones is important for elucidating their regulatory and structural roles.7 Compared to H4, histones H2A, H2B, H3, and linker histone H1 have diverged more rapidly, * To whom correspondence should be addressed. 600 S. Mathews, 53 Roger Adams, Lab, Urbana, IL 61801. Tel: (217) 244-3927. E-mail: Kelleher@ scs.uiuc.edu. † Department of Chemistry. ‡ Biophysics and Computational Biology. § Department of Cell and Developmental Biology. | Institute for Genomic Biology. ⊥ The Center for Top Down Proteomics.
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having multiple gene family members and variants, all of which are thought to potentially have specific functions.8 This complexity suggests the possibility that differential modifications and epigenetic regulation may occur among variants. To gain a complete biological picture, gene family members must be distinguished from one another and precisely characterized at the biochemical level. The use of Top Down Mass Spectrometry (MS)9 and the concept of Shotgun Annotation10 now allow gene specific identification11 and PTM characterization11-14 for human proteins. Utilizing Fourier Transform Mass Spectrometry (FTMS), accurate data on intact proteins and their fragment ions formed by Electron Capture Dissociation15 during tandem MS (MS/ MS) facilitate the characterization of cSNPs, alternative splice events, and PTMs by providing information on 100% of the primary protein structure.11 A logical extension of this technique is the targeted analysis of closely related protein species while concurrently characterizing any PTMs they harbor at the protein level.16 Here, we report a broad survey of products of the H2A gene family, with a total of 12 gene family members and two variants17 observed from asynchronously growing cell. Upon sodium butyrate treatment, H2A.Z was the most responsive, showing up to three acetylations versus only one for the other H2A gene products. We further report no significant changes in the relative expression ratios of the H2A family members throughout the cell cycle, with an initial application of SILAC18 showing incorporation of labeled amino acids primarily during S-phase and little variation in synthesis between family members.
Methods Cell Synchronization. HeLa S3 cells were grown to a density of (2-3) × 105/mL in Joklik’s modified MEM with 5% NCS and 10.1021/pr050269n CCC: $33.50
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Human Histones in the H2A Gene Family
synchronized on the G1/S-phase border by a double thymidine block procedure.19 Cells were washed in pre-warmed PBS and then released into regular media or media that contained 13 15 C, N labeled valine and arginine (Isotec, Miamisburg, OH) supplemented with extensively dialyzed serum. The degree of synchrony was assessed by flow cytometry every hour following the second release. The representative mid-S, late-S/G2, M/G1, and mid-G1 phases were determined to occur 4, 8, 11.5, and 14 h post-release, respectively. Cells harvested at these time points were collected by centrifugation, washed twice in ice cold PBS and then either snap frozen in liquid nitrogen or processed for nuclear isolation directly. Nuclei Isolation and Histone Extraction. Washed cell pellets (2 × 107 cells) were suspended in NIB-250: 15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose, 1 mM dithiothreitol, 10 mM sodium butyrate, protease inhibitor cocktail set III (Calbiochem, 100:1 v:v), and phosphatase inhibitor cocktail set II (Calbiochem, 100:1 v:v) plus 0.3% NP-40 at 10:1 v:v ratio. Cells were lysed by gentle mixing and incubation on ice for 5 min. Nuclei were pelleted at 600 × g for 5 min. at 4 °C and then washed twice with NIB250 without detergent. Bulk histone was promptly extracted from the pelleted nuclei using 0.4 N H2SO4 at a 5:1 v:v ratio. The nuclei were left in acid on ice for at least 30 min, mixing intermittently. The nuclear suspension was spun down at 3000 × g for 5 min., the supernatant transferred to another tube, and the histones precipitated by adding 20% (w:v, final) trichloroacetic acid (TCA) and incubating overnight at 4 °C. The histone precipitate was then washed once with ice cold 0.1% HCl:acetone, twice with acetone, and air-dried before HPLC separation. Chromatography. Histone extracts (5%
Figure 3. (A) Sequence Alignment of H2A forms identified. Highlighted amino acids indicate sequence variant positions. Black lines show hypothetical Glu-C digest fragments. (B) Percent Sequence Identity of the H2A species identified. In some cases, they differ by as little as a single amino acid change, for example H2A.A (Arg) and H2A.G (Lys) are 99% identical.
of the total expressed in asynchronously growing cells. H2A.Z and H2A.F/Z are expressed at the lowest levels with the initial methionine removed, but the majority of these variants were otherwise un-modified. All remaining H2A family members were characterized as initial methionine cleaved, R-N-acetylated. It is notable that H2A.O and H2A.Q, which coelute under the conditions used here, are only distinguishable by their C-terminal sequences. H2A.Q contains a histidine 123 deletion and a glycine to serine change at position 128 resulting in a net 107 Da mass loss (Figure 3). In contrast to Top Down MS, which readily distinguishes the two forms both by intact mass and by C-terminal fragment ions, a bottom-up approach would require detection of 4 specific peptides of the 8 expected in a Glu-C digest representing ∼70% of the sequence to discriminate between these closely related gene family members (Figure 3). Identification of the C-terminal peptide would allow for conclusive proof of H2A.Q in the sample, however, identification of the corresponding C-terminal peptide for H2A.O does not provide a definitive identification. H2A.A, H2A.G, H2A.C,D,I,N,P,
Table 1. Summary of Fragmentation Data and Expectation Values for Gene-specific Identification of H2A Proteins Accomplished in This Study
Histone
H2Aa
H2A.Oc H2A.Qc Histone H2Ad,e H2A.Ed H2A.C,D,I,N,Pd,f H2A.Ld H2A.Gd H2A.Ad,g H2A.Z h H2A.F/Z h,i
IRMPD
ECD
experimental (Da)
theoretical (Da)
PTMs
b ions
y ions
eValues
c ions
13997.9 13890.8 13808.7 13838.7 13993.9 14007.8 14009.9 14037.9 13413.4 13369.4
13955.8 13848.8 13766.8 13796.8 13951.9 13965.9 13967.9 13995.9 13413.5 13369.5
N-Ac 8 N-Ac N-Ac N-Ac N-Ac N-Ac N-Ac N-Ac none none
8 6 6 8 6 7
2 1 1 3 4 1
2.E-04 2.E-05 2.E-02 6.E-08 5.E-08 2.E-06
5 6 3
1 10 3
6.E-04 9.E-11 9.E-06
27 23 24 23 24 12 32 15 33 26
b
z•
ions
eValuesb
27 21 20 17 26 12 29 13 30 22
2.E-57 2.E-34 6.E-41 7.E-34 2.E-43 9.E-18 2.E-51 3.E-23 2.E-69 2.E-56
a UniProt Protein Names are listed unless otherwise noted. b Expectation Values are a measure of identification confidence, lower scores are better. c H2A.O and H2A.Q coelute in one peak (H2A-1). d Histone H2A, H2A.E, H2A.C,D,I,N,P, H2A.L, H2A.G, H2A.A coelute in one peak (H2A-2). e Unnamed Histone H2A Family Member. UniProt Protein Name OTTHUMP0000001617. f Five different H2A gene family members encode the same amino acid sequence. g H2A.M has a conflict sequence reported A37G and H38N, which is the same amino acid sequence as. g H2A.Z and H2A.F/Z coelute in one chromatographic peak (H2AZ). i Hypothetical protein H2AV; H2A histone family, member V, isoform 1.
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Figure 4. (A,B) Comparison of asynchronously grown H2A (A) to Sodium Butyrate treated H2A (B). C-Fragmentation map localizing an additional acetylation at lysine 5. D-Intact protein profile of multi-acetylated H2A.Z; asterisk, singly charged contaminant ion.
and H2A.L contain the same C-terminal peptide and would require 3 specific peptides in a Glu-C digest to be measured in order to identify H2A.O. This highlights the efficiency of the Top Down approach for both distinguishing gene family members and providing semiquantitative information on relative expression changes in a gene-specific fashion. Treatment with 10 mM sodium butyrate, a well-known histone deacetylase inhibitor, showed a minor increase in global acetylation after 54 h (Figure 4B vs the Figure 4A control). Using ECD, the additional acetylation was localized to lysine 5 on both H2A.O and H2A.Q (Figure 4C), which correlates well with previously published data of modification sites of H2A.31 Broadband data for intact H2A.E, H2A.C,D,I,N,P, H2A.G, H2A.A, and the unnamed family member show a similar result as Figure 4B, a single additional acetylation (data not shown). Surprisingly, the only H2A species that exhibits a marked increase in acetylation is H2A.Z which shows three 42 Da shifts in a broadband intact spectrum (Figure 4D). These mass shifts have not been localized precisely due to low expression of H2A.Z, but are due to multiple acetylations within the Nterminal tail (IRMPD data not shown). To further understand H2A and its dynamics, a HeLa S3 culture was synchronized using a double thymidine block and then released in normal media. Culture samples (about 106 cells) were collected at 2, 6, 10, and 15 h post-release, and H2A was isolated as described. FT mass spectra of intact H2As were taken to monitor for changes in the relative expression ratios of different H2A family members as well as detect any modification dynamics that may occur in a cell cycle regulated fashion (Supporting Information Figure 2). Intact mass spectra were compared and the sum-weighted average of the intensity of the H2A forms present in each chromatographic peak was calculated for each charge state and normalized (Supporting Information Figure 3). No major modification dynamics of H2A were observed during the cell cycle time course. Increased phosphorylation or decreased acetylation during mitosis,32,33 as reported previously, were not detected. Moreover, there was no apparent change in the expression ratios (>15%) of the different gene family members during the course of the cell cycle (Supporting
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Figure 5. (A) Intact mass spectrum of ubiquitinated H2A.O, 22539.5 Da (+28 charge state shown). (B) Compiled sequence tag and search output are shown with possible mass discrepancies. N-terminally acetylated H2A.O shows an 8541.6 Da mass shift indicating ubiquitination. (C,D) Graphical fragment map of H2A.O and ubiquitin supporting the identification of ubiquitinated H2A.O.
Information Figure 3). However, the 2 h and 10 h time points contained a 22 539.5 Da species at very low levels that was not apparent in any of the asynchronous samples or any other time points (Figure 5a). This species was isolated in the mass spectrometer and fragmented by IRMPD. With an initial search of ProSight PTM in absolute mass mode not yielding any results, a sequence tag was complied from the data (P V Y M A A V [I|L] E), and it was searched against the human database in ProSight PTM. Five proteins in the database contained this sequence of amino acids: histones H2A.O, H2A.Q, H2A.W, H2A.Y, and H2A.FY (Figure 5b). Hypothetical mass shifts for each of these proteins were calculated with and without R-Nacetylation. The R-N-acetylated form of H2A.O showed a mass discrepancy of 8541.6 Da, suggesting it was ubiquitinated (Figure 5b,c). The fragment ion list was searched against the ubiquitin amino acid sequence and 5 b ions matched supporting ubiquitination assignment for H2A.O (Figure 5d). The timing of this modification correlated with previously published reports of ubiquitinated H2A’s cell cycle dependence, disappearing during G2-mitosis and re-appearing somewhere near the mitosis-G1 transition.34 The application of SILAC can distinguish between newly synthesized and old histones. We expected that the ratio of labeled H2A to unlabeled H2A would approach 50% due to the duplication of chromatin during S-phase. However, the data of Figure 6 show that only an approximate 1:3 ratio or ∼25% incorporation was observed. These results presumably were attributable to the incorporation of natural amino acids during labeling under the conditions employed, resulting in proteins containing both labeled and unlabeled L-valine and L-arginine. Since the isotopic distribution was still centered at the expected mass, this could not have affected all newly synthesized molecules, but peak broadening on the low mass side probably contributed to the lower than expected ratios. In general, the SILAC data show a uniform incorporation of labeled amino acids into newly formed H2A family members during S-phase. As early as 4 h post-release (i.e., mid-S phase), new H2A.O protein is detectable (Figure 6A). The relative ratio of labeled H2A.O associated with chromatin to unlabeled H2A.O increases Journal of Proteome Research • Vol. 5, No. 2, 2006 251
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discussions and technical assistance. M.T.B. was supported on an NIH Institutional NRSA in Cellular and Molecular Biology (5T32 GM 07283). The laboratory of N.L.K. was supported by the National Institutes of Health (GM 067193-03). The laboratory of C.A.M. was supported by the University of Illinois and the Roy J. Carver Charitable Trust (Grant No. 04-76).
Supporting Information Available: Supporting Information Figures 1-3 and Tables 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References
Figure 6. (A,B,C,D,E) Broadband intact mass spectra of the H2A-1 peak from various SILAC cell cycle time points. Highlighted peaks indicate labeled H2A isoforms created by SILAC. F-The intensity of each H2A.O and H2A.Q species was calculated as a weighted abundance across all charge states, and the total amount of a specific H2A gene product was normalized to one.
through the cell cycle, roughly doubling from mid-S to lateS/G2 and doubling again from late-S/G2 to M/G1 (Figure 6BC). Loss of synchrony in the cell population and potential preferential turnover of pre-existing H2A may have contributed to the latter increase. The relative ratios for each of the H2A species studied here were tabulated, and they all show a similar trend (Supporting Information Figure 3). The lack of modifications on the most abundant forms of histone H2A in bulk chromatin diminishes the role it may play in the administration of a modification-based histone code. It is not beyond reason that particular forms of H2A may be the most important components for control at the nucleosomal level rather than any modifications H2A may contain. It should be noted that dynamic modification states might lie below what is detectable with the limited dynamic range of this system (50:1 to 300:1). Regardless, the results for global levels of H2A clearly indicate that the bulk of histone H2A gene family members are expressed with their N-terminal methionine removed, R-Nacetylated.
Conclusions Top Down MS provides a clear, global view of H2A expression dynamics that occur within a population of cells by unequivocally distinguishing closely related species from one another while concurrently analyzing any post-translational modifications. This gene-specific characterization is accomplished by recombining fragment ions in a logical manner to match the measured intact mass. Bottom Up MS lacks this clarity. While it is relatively straightforward to identify a peptide from a gene family, it is much more difficult to identify the exact gene family member due to their high sequence identity.35 Thus, Top Down is distinctive in its ability to decipher that part of any code that is written in the language of differential expression of gene family members or variant forms in addition to its advantages for PTM analysis.
Acknowledgment. The authors would like to thank Mike Roth, Paul Thomas, and Phil Compton for their valuable 252
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Cyclotron Resonance Mass Spectrometry for Versatile MS/MS above 10 kDa. J. Am. Soc. Mass Spectrom. 2004, 15 (7), 10991108. Wang, T. C. L.; Ricca, T. L.; Marshall, A. G. Extension of Dynamic Range in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Via Stored Waveform Inverse Fourier Transform Excitation. Anal. Chem. 1986, 58 (14), 2935-2938. Little, D. P.; Speir, J. P.; Senko, M. W.; O’Conner, P. B.; McLafferty, F. W. Infrared Multiphoton Dissociation of Large Multiply Charged Ions for Biomolecule Sequencing. Anal. Chem. 1994, 66 (18), 2809-2815. Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Automated Reduction and Interpretation of High-Resolution Electrospray Mass Specra of Large Molecules. J. Am. Soc. Mass Spectrom. 2000, 11 (4), 320-332. Taylor, G. K.; Kim, Y. B.; Forbes, A. J.; Meng, F.; McCarthy, R.; Kelleher, N. L. Web and Database Software for Identification of Intact Proteins Using “Top Down” Mass Spectrometry. Anal. Chem. 2003, 75 (16), 4081-4086. Expectation values are modified Pscores where the size of the database searched is factored into the scaling. As with Pscores, lower scores are better. H2A.L and H2A.G were identified from different samples of HeLa S3. The original identification of H2A family members were from cells ordered from the National Cell Culture Center, which contained H2A.L. Follow-up work with cell culture synchronization, small molecule treatment, and SILAC were cultured internally. They contained H2A.G (Supporting Information Figure 1). Similar changes in gene family expression for HeLa cells cultivated in different laboratories have been observed for H1. Meergans, T.; Albig, W.; Doenecke, D. Varied Expression Patterns of Human H1 Histone Genes in Different Cell Lines. DNA Cell Biol. 1997, 16 (9), 1041-1049.
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