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. 2015 Apr 3;24(8):1224–1231. doi: 10.1002/pro.2673

Charge-neutralization effect of the tail regions on the histone H2A/H2B dimer structure

Kazumi Saikusa 1,*, Singo Shimoyama 1,†, Yuuki Asano 1,‡, Aritaka Nagadoi 1, Mamoru Sato 1, Hitoshi Kurumizaka 2, Yoshifumi Nishimura 1, Satoko Akashi 1,*
PMCID: PMC4534173  PMID: 25752661

Abstract

It is well known that various modifications of histone tails play important roles in the regulation of transcription initiation. In this study, some lysine (Lys) and arginine (Arg) residues were acetylated and deiminated, respectively, in the histone H2A/H2B dimer, and charge-neutralization effects on the dimer structure were studied by native mass spectrometry. Given that both acetylation and deimination neutralize the positive charges of basic amino acid residues, it had been expected that these modifications would correspondingly affect the gas-phase behavior of the histone H2A/H2B dimer. Contrary to this expectation, it was found that Arg deimination led to greater difficulty of dissociation of the dimer by gas-phase collision, whereas acetylation of Lys residues did not cause such a drastic change in the dimer stability. In contrast, ion mobility-mass spectrometry (IM-MS) experiments showed that arrival times in the mobility cell both of acetylated and of deiminated dimer ions changed little from those of the unmodified dimer ions, indicating that the sizes of the dimer ions did not change by modification. Charge neutralization of Arg, basicity of which is higher than Lys, might have triggered some alteration of the dimer structure that cannot be found in IM-MS but can be detected by collision in the gas phase.

Keywords: histone H2A/H2B dimer, acetylation, deimination, electrospray ionization, ion mobility-mass spectrometry

Introduction

In eukaryotic cells, genetic information is written on chromosomes in nuclei. The minimum structural unit of chromosomes is a nucleosome core particle (NCP), composed of two copies each of the core histones, H2A, H2B, H3, and H4, and 146 bp of DNA.1 It is considered that various modifications of histone tail regions play important roles in regulation of transcription initiation.2–5 For example, acetylation of amino groups in lysine (Lys) residues generally activates transcription initiation and deacetylation triggers transcription repression. In addition to various kinds of histone modifications, many proteins, such as histone chaperones and chromatin remodeling factors, are involved in the regulation of transcription initiation.6–10 Consequently, such a system enables to the expression of necessary proteins at particular times and locations.

In our previous studies, the histone H2A/H2B dimer was prepared with recombinant histone H2A and H2B monomer proteins and its gas-phase behavior was investigated using ion mobility-mass spectrometry (IM-MS).11,12 The intact ions of the dimer were generated using nanoelectrospray ionization (nanoESI) in the native-like state, and subjected to IM-MS. It was found that the histone H2A/H2B dimers presented two groups of collision cross-section (CCS) values in the gas phase,11 and that the difference in the CCS values was due to varying behavior of the flexible tail regions.12 It was also found that CCS of the acetylated histone dimers was little changed from that of the unmodified dimers, suggesting that charge repulsion of the flexible tail regions was not the reason for the varied sizes of the dimer ions in the gas phase.12

In the present study, to investigate in more detail the charge-neutralization effects of the basic amino acids in flexible tail regions on the gas-phase characteristics of the histone H2A/H2B dimer, we performed two types of modifications of basic amino acids: acetylation of Lys and deimination of arginine (Arg), and compared the stability and mobility of the intact modified dimer ions in the gas phase. To prepare the samples, the histone H2A/H2B dimer was modified with the enzymes histone acetyltransferase (HAT) or peptidylarginine deiminase 4 (PAD4). These enzymes specifically modify the side chains of the basic amino acids Lys and Arg in histone proteins and neutralize the positive charges on the side chains (Supporting Information Scheme 1). HAT acetylates ε-amino groups of specific Lys residues of histone proteins.13 By acetylation of specific Lys residues in the NCP histone proteins, transcription initiation is generally activated.13 PAD4 deiminates guanidine groups of Arg residues in the unstructured regions of the histone proteins,14 and Arg residues are converted into citrulline residues. In addition, PAD4 specifically deiminates monomethylated Arg residues, which are derivatized by cofactor-associated arginine methyltransferase 1 (CARM1)15 or protein arginine methyltransferase 1 (PRMT1).16 Mono- and di-methylation of Arg residues generally triggers transcription activation, and it has been found that deimination of unmodified or monomethylated Arg residues consequently results in repression of transcription activation.17,18 Biological functions of these modifications of the histone proteins in the NCP have been characterized, as described above, but modification effects on the biophysical characteristics of the NCP and histone multimers have not been investigated in detail. It is still neither clear how the structures of the NCP and histone multimers are altered by acetylation or deimination nor has it been determined how their structural changes are reflected in their functions. Accordingly, in the present study, acetylated and deiminated H2A/H2B dimers were subjected to native mass spectrometry (MS) under different measurement conditions, and the dimer stability in the gas phase was evaluated. In addition, arrival times in the mobility cell of the dimers before and after the modifications were compared by IM-MS experiments. Using these analyses, we investigated the charge-neutralization effects of basic amino acids on the gas-phase characteristics of the histone H2A/H2B dimer, and discuss here the differences in these two modifications.

Results and Discussion

Mass determination of the modified H2A/H2B dimer

In the present study, amino groups of some Lys residues were acetylated, whereas guanidine groups of some Arg residues were deiminated. By acetylation, the mass of each Lys residue increases by 42 Da, and such a mass increase of proteins is easy to identify using matrix-assisted later desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). As previously reported, the most abundant ions of the H2A and H2B monomers correspond to 6- and 7-acetylated H2A and H2B, respectively [Fig. 1(A)].12 In contrast, deimination leads to only a 1 Da mass increase for each Arg residue, and such a small mass difference for a histone monomer protein (14 kDa) cannot accurately be identified by MALDI-TOFMS. Accordingly, to determine the extent of deimination, we utilized electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) under denaturing conditions, preparing the sample in acidic solution with acetonitrile [Fig. 1(B)], as previously reported.19 The mass increases for the dimer constituents, H2A and H2B, by deimination were approximately 2.7 and 2.0 Da (average, n = 3), respectively, suggesting that the numbers of deiminated Arg residues were approximately 2.7 and 2.0 on average for the H2A and H2B monomers, respectively. This finding shows that the extent of acetylation of Lys residues was much larger than that of Arg deimination under the present experimental conditions.

Figure 1.

Figure 1

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Mass spectra of the H2A/H2B dimer with and without modification under denaturing conditions. (A) MALDI-TOF mass spectra of the unmodified (upper) and acetylated (lower) H2A/H2B dimer. The numbers of acetyl groups introduced into the histone proteins are indicated in blue for H2A and red for H2B. (B) ESI mass spectra of the unmodified (upper) and deiminated (lower) H2A/H2B dimer under denaturing conditions. Blue and red arrows indicate peaks of multiply charged ions of the dissociated histone H2A and H2B monomers, respectively.

The modified sites were analyzed by peptide mass mapping. For acetylation, as we have already reported, MALDI-TOF peptide mass mapping with V8 protease revealed that Lys residues in the N-terminus regions in both H2A and H2B were extensively modified but that those on the core α-helices were not modified (Supporting Information Table 1).12 In the case of deimination, V8 protease, arginyl endopeptidase, and lysyl endopeptidase were complementarily used to identify the deiminated sites. To accurately analyze the mass of deiminated peptides, primarily ESI-TOFMS was used. It revealed that Arg residues in the N-terminus regions of H2A and H2B were somewhat modified, whereas no modified Arg residue was identified in the region extending from Arg42 to the C-terminus of H2A. As for H2B, no modification was suggested for the regions extending from Ala58 to Lys85 and from Glu93 to the C-terminus, but Arg86, which is located on the flexible loop between two helices, was slightly deiminated, as summarized in Figure 2 and Supporting Information Table 2. These results suggest that some positive charges on the N-terminus regions of both H2A and H2B were neutralized by acetylation or deimination but that basic residues in the helical regions remained unmodified.

Figure 2.

Figure 2

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Summary of peptide mass mapping of deiminated histone proteins. Peptides observed in the mass spectra are mapped on the primary and secondary structures of H2A and H2B. The secondary structures of the dimer are extracted from the crystal structure of the human nucleosome core particle (PDB: 3AFA). Peptides including deiminated sites were determined mainly using ESI-TOFMS of arginyl endopeptidase, lysyl endopeptidase, and V8 protease digests of the deiminated H2A/H2B dimer. Green, blue, and red arrows indicate the observed peptides for the arginyl endopeptidase, lysyl endopeptidase, and V8 protease digests. The number of the modified groups in each peptide is indicated on the corresponding arrow. The numbers in parentheses show the dominant number of modification for the corresponding peptide observed in the mass spectra.

Stability of the modified H2A/H2B dimer ion

For observation of the intact histone H2A/H2B dimer ions using native MS, the sample should be prepared in a high concentration of ammonium acetate to avoid dissociation of the dimer.19 This step is necessary, owing to the high hydrophobicity of the interface region between the histone H2A and H2B monomers. Accordingly, the samples were prepared in relatively concentrated ammonium acetate solutions and subjected to native MS to characterize the behavior of the histone H2A/H2B dimer.

The gas-phase stability of the H2A/H2B dimers with and without modification was investigated for samples in 0.5M ammonium acetate using native MS at varied cone and collision voltages on Q-tof2 (Fig. 3). For the unmodified dimer, primarily intact dimer peaks were observed associated with some peaks of dissociated monomers [Fig. 3(A)]. On increasing the cone voltage from 50 V to 60 V with keeping the collision voltage at 20 V, intensities of the dissociated monomer peaks were little changed. Additional elevation of the cone voltage up to 70 V, monomer intensities improved. Furthermore, a change in the collision voltage from 20 V to 30 V with keeping the cone voltage at 70 V, dissociation was a little facilitated [Fig. 3(A)]. Similar changes in the mass spectra were observed for the acetylated H2A/H2B dimer: mainly intact dimer peaks were observed under a relatively mild condition. When the cone and collision voltages were set at 70 V and 30 V, respectively, intensities of the dissociated monomer peaks significantly improved [Fig. 3(B)]. In contrast, the deiminated H2A/H2B dimer showed different behavior from those of the unmodified and acetylated dimers; intensities of the dissociated monomer peaks did not improve extensively under severer conditions [Fig. 3(C)].

Figure 3.

Figure 3

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NanoESI-TOF mass spectra of the unmodified (A), acetylated (B), and deiminated (C) H2A/H2B dimer in 0.5M ammonium acetate. Mass spectra obtained under two conditions, cone 50 V and collision 20 V (upper panel) and cone 70 V and collision 30 V (lower panel), are indicated. Blue and red arrows indicate the peaks of dissociated H2A and H2B monomers. Arrowheads show multiply charged ions of the intact H2A/H2B dimers. Charge states of the dimer ion and monomer ions are indicated in black regular letters and in cyan italic letters, respectively, on the peak tops.

Figure 4 summarizes the relative ratios of ion intensities of the dissociated monomer ions to the sum of ion intensities of the dimer and monomer ions. It was found that an increase in cone voltage from 50 V to 60 V did not alter the relative intensities of the dissociated monomers. An increase in collision voltage from 20 V to 30 V, with keeping 70 V of cone voltage, did not show a large increase in the monomer ratio. In contrast, an increase in cone voltage from 60 V to 70 V facilitated the dissociation of the H2A/H2B dimer ions regardless of modification. This suggests that collision induced dissociation in the first vacuum chamber by increasing the cone voltage up to 70 V is effective to dissociate the unmodified and modified H2A/H2B dimer when the sample is prepared in 0.5M ammonium acetate. It was also found that the unmodified dimer showed the highest monomer ratio under every measurement condition, and that the deiminated dimer showed a considerably lower monomer ratio than the acetylated dimer throughout experiments. These findings suggest that the deiminated H2A/H2B dimer is more stable in the gas phase than the unmodified or acetylated dimers. This issue is discussed also in the last section.

Figure 4.

Figure 4

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Plots of the relative ratio of observed ion intensities of the dissociated H2A and H2B monomers to the sum of the ion intensities of the dimer and dissociated monomers. Relative ratio of the monomer, R, is calculated by the equation R = A/(A + B), where A is that of the 4+, 5+, 6+, 7+, 8+, and 9+ charged ions of H2A and H2B monomers and B is the sum of the peak intensities of the 10+, 11+, and 12+ charged ions of the intact dimer. Mass spectra were obtained under four conditions: (1) cone 50 V and collision 20 V, (2) cone 60 V and collision 20 V, (3) cone 70 V and collision 20 V, and (4) cone 70 V and collision 30 V.

Size of the modified H2A/H2B

NanoESI-IM-MS was then performed to determine whether acetylation and deimination led to alteration in the gas-phase conformation of the H2A/H2B dimer. Figure 5 shows arrival time distributions of 10+, 11+, and 12+ charged ions of the H2A/H2B dimers with and without modifications. Given that the arrival time of each dimer ion did not change according to the concentration of ammonium acetate (data not shown), the samples were prepared in 4 M ammonium acetate to minimize dissociation and enhance the intensity of the intact dimer ions. In our previous IM-MS study of the H2A/H2B dimer, it was found that flexible tail regions led to the generation of two populations in the arrival-time distributions.11,12 Moreover, in this study, two populations were clearly observed for the 11+ charged ion, the most abundant ion, in the mass spectra. This finding was distinctly confirmed by the two-dimensional contour plots of m/z and arrival times (Supporting Information Figs. 1 and 2). Neither acetylation nor deimination strongly affected the behavior of the flexible tail regions in the gas phase.

Figure 5.

Figure 5

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Arrival-time distributions of the 10+, 11+, and 12+ charged ions of the acetylated (A–C) and deiminated (D–F) H2A/H2B dimer obtained by ESI-IM-MS for the samples prepared in 4 M ammonium acetate. Dotted lines indicate arrival-time distributions of the unmodified dimer ions and solid lines correspond to those of the acetylated or deiminated dimer ions.

With respect to the sizes of the acetylated or deiminated H2A/H2B dimer ions, no large change in arrival times was observed. The arrival-time distributions of the 10+, 11+, and 12+ ions of the acetylated dimer showed a slight delay from the corresponding ions of the unmodified dimer [Fig. 5(A–C)], as observed in our previous study.12 Given that acetylation causes a mass increase of each amino group of a Lys residue by 42 Da, it is possible that a mass increase due to acetylation led to a slight increase in size and a small delay of mobility, a result that was confirmed by calculation of the theoretical CCS values of acetylated dimers generated by in silico acetylation of the MD-simulated dimers.12 For the deiminated dimer, arrival-time distribution plots of the 10+, 11+, and 12+ charged ions were almost coincided with those of the unmodified dimer [Fig. 5(D–F)], implying that the dimer structure was not altered by deimination. These results show that the sizes of the dimer ions in the gas phase were unaffected by charge neutralization of some basic residues in the flexible tail regions.

As shown in Figure 3, it was found that neither acetylation nor deimination caused a change in the observed charge state. The arrival times in the mobility cell were little altered, suggesting that no recognizable alteration occurred in the dimer structure. These results are consistent with the conclusion of the previous study by Kaltashov and Mohimen that the average charge states of the protein observed in the native mass spectra reflect the solvent-accessible surface area (ASA) of the protein.20 The average charge state corresponding to the core part of the unmodified H2A/H2B dimer structure, which was obtained by MD simulation, was calculated as 11.4 using the previously proposed equation of charge-to-ASA.21 This value is roughly consistent with the average-charge states observed for the unmodified and modified dimer in Figure 3. In the consecutive MD simulation of the dimer first in solution then in vacuo, the core region remained intact throughout simulation but the tail regions showed a compaction upon introducing to the gas phase.11 Furthermore, no remarkable effect was found on the sizes of the simulated structures from random alteration of the charged sites.11 These results suggest that the tail regions in the gas phase lose their flexibility and shrink in size, and protonation of the gas phase proteins occurs not only in the tail region but also the helical core part of the dimer, resulting in the charge states corresponding to the ASA value. This is consistent with the results of IM-MS that the unmodified and modified dimers showed similar CCS values.

Modification effects on the dimer structure

In this study, to avoid the dissociation of the H2A/H2B dimer in the native MS experiments, the samples were prepared in a high concentration of ammonium acetate. In solution, high ionic strength reduced charge repulsion of the tail regions and stabilized hydrophobic interactions in the dimer interface. Upon ionization, solvents were removed off but the H2A/H2B dimer ions could survive in the gas phase. Although a relatively high concentration of ammonium acetate reduced the electrostatic interactions, charge repulsion within the unmodified dimer might not have completely been eliminated, resulting in the highest ratio of the dissociated monomers in the mass spectra of the unmodified dimer.

As described above, Lys and Arg residues, mainly in the N-terminus flexible tail regions, were specifically modified by acetylation and deimination, and it was found that the number of acetylated Lys residues was much larger than that of deiminated Arg residues. Given that a larger number of positive charges in the H2A/H2B dimer were neutralized by acetylation than by deimination, a larger change in the structural characteristics might be expected for the acetylated dimer. Contrary to this expectation, it was found that deimination made dimer dissociation in the gas phase much more difficult than acetylation. This finding implies that deimination of a few Arg residues in H2A or H2B influences the gas-phase stability of the dimer more than does acetylation of from six to eight Lys residues in each histone monomer. With respect to the basicity in solution, the pKa value of ε-amino group of Lys residue is 10.53, whereas that of the guanidine group of Arg is 12.48,22 meaning that the basicity of the side chain of an Arg residue is larger than that of a Lys by two orders of magnitude. Furthermore, the gas-phase basicity of a Lys residue is 951.0 kJ/mol, whereas that of an Arg is 1006.6 kJ/mol.23 Such a difference in basicity of Lys and Arg residues not only in solution but also in the gas phase may account for the disparate behavior of the acetylated and deiminated H2A/H2B dimers in the gas phase, and charge neutralization of a few Arg residues in H2A and H2B may have strongly influenced the structural characteristics of the H2A/H2B dimer. Furthermore, a change in basicity of the deiminated dimer might have triggered some structural alteration that cannot be detected by IM-MS.

When NCP is acetylated, transcription initiation is generally activated, whereas NCP deimination triggers inhibition of transcription activation. That is, acetylation and deimination of NCP exert opposite effects, although both modifications neutralize the positive charges on the histone tails. The activation and repression mechanisms of transcription initiation triggered by histone modification have not clearly been identified. Considering that many proteins, such as chromatin remodeling factors and histone chaperones, are also required for regulation of transcription initiation, it is understandable that the tail-modification effect on the structure alone is not directly reflected to the biological function. It is intriguing to speculate that the biophysical characteristics of the acetylated and deiminated NCPs are associated with their function, and the characterization of modified NCPs using mass spectrometry is accordingly under current investigation.

Materials and Methods

Preparation of histone proteins

Histone H2A and H2B proteins were prepared according to previous reports.11,12,19,24 Extra residues, GSM (for acetylation) or GPGM (for deimination), derived from the expression vector were attached at the N-terminus of the histone protein after the cleavage of the histidine-tag region. The histone H2A and H2B monomers were subjected to dimer formation by refolding in a manner similar to that described previously.11,12,19

Acetylation of the H2A/H2B dimer

The H2A/H2B dimer was acetylated using histone acetyltransferase (HAT) p300 (Enzo Life Sciences, Farmingdale, NY) at a 10:1 substrate: enzyme ratio (w/w) as previously reported.12 The histone H2A/H2B dimer was incubated with HAT p300 in 50 mM Tris-HCl (pH 8.0), 10% glycerol, 0.1 mM EDTA, and 1 mM DTT in the presence of 1 mM acetyl-CoA at 37°C. After 26 h of incubation, acetylation was terminated by dialysis of the reaction mixture against 500 mM ammonium acetate with Micro-dialyzer TOR-3K (Nippon Genetics, Japan). The extent of acetylation was determined for each histone protein by mass measurement using MALDI-TOFMS (Autoflex, Bruker, Billerica, MA). To identify the acetylated sites, the modified dimer was digested with Staphylococcus aureus V8 protease (Roche Diagnostics, Mannheim, Germany) in 50 mM ammonium acetate (pH 4.0) at 37°C, desalted with c-tip (Nikkyo Technos, Tokyo, Japan), and subjected to peptide mass mapping using MALDI-TOFMS.12

Deimination of the H2A/H2B dimer

The H2A/H2B dimer was deiminated using recombinant PAD4, as reported previously.19 Deimination was performed by incubation of the histone H2A/H2B dimer with PAD4 (5 mU) in 20 mM Tris-HCl (pH 8.0), 2 M NaCl, 10 mM CaCl2, and 1 mM DTT at 37°C for 15 min. To determine the extent of deimination, the sample was acidified with 0.1% formic acid and then desalted using C18 ZipTip (Millipore, Billerica, MA) and subjected to mass measurement with a Q-tof2 mass spectrometer (Waters, Milford, MA). The measurement of accurate mass of the deiminated histones were performed three times. The stability of the deiminated dimer in the gas phase was evaluated using nanoESI-MS under the native-like condition as indicated below. To identify the deiminated Arg residues, the modified dimer was thermally denatured and then digested with a protease at 37°C and subjected to peptide mass mapping using infusion nanoESI-MS or MALDI-TOFMS after desalting with C18 ZipTip. Digestion with endoproteinase Arg-C (Roche Diagnostics) and lysyl endopeptidase (Wako Pure Chemical Industries, Kyoto, Japan) was performed in 20 mM Tris-HCl (pH 8.0) with 10 mM CaCl2. Digestion with S. aureus V8 protease (Roche Diagnostics) was performed in 25 mM ammonium bicarbonate (pH 7.8).

ESI-MS of histone dimer

Gas-phase dissociation of the histone dimers was investigated using Q-tof2 with a nanoESI source.19,25 The sample solutions of the modified dimers were dialyzed against 2 M ammonium acetate solutions (pH 6.8), and then diluted with Milli-Q water (Millipore) to adjust the ammonium acetate concentration to 500 mM. To obtain native ESI mass spectra, the sample solutions were deposited in gold-coated borosilicate capillaries (Humanix, Japan), and placed in the nanoESI source. The dimer ions were generated by applying 0.9 to 1.0 kV of capillary voltage. By altering the cone and collision voltages, dissociation of the dimer with or without modification was analyzed: 50, 60, or 70 V (cone voltage) and 20 or 30 V (collision voltage).

ESI-IM-MS of histone dimer

Ion mobility mass spectra for the histone dimers were acquired with or without modifications using a TriWave Synapt G2 HDMS (Waters) with a nanoESI source.26–28 The dimers in 4 M ammonium acetate were introduced to the nanoESI source in a manner similar to that used for mass measurement on Q-tof2. To perform IM-MS experiments, the following parameters were used: 0.8 to 1.0 kV capillary voltage, 20 V cone voltage, 4 V trap collision energy, 40 V trap bias voltage, 40 V Tri-Wave height, and 800 m/s TriWave velocity.

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Supplementary Information

pro0024-1224-sd1.pdf (306.3KB, pdf)

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