DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, acetylation of histones H3 and H4 plays an important role in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in the relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATM (ATAXIA TELANGIECTASIA MUTATED) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.

INTRODUCTION

Repair of DNA DSBs (double-strand breaks), one of the most cytotoxic forms of DNA damage, takes place in the context of chromatin. Histones form the protein component of chromatin, and play critical roles in all aspects of DNA metabolism and genome maintenance. A total of eight histone proteins form the core nucleosome particle, around which 147 bp of DNA wraps in a left-handed superhelix to form the lowest-order structure of chromatin [1]. Each nucleosome is composed of two each of H2A, H2B, H3 and H4, 11–22 kDa proteins, in a tripartite assembly with a central (H3/H4)2 tetramer flanked by two H2A/H2B dimers. Localized to the outer surface of the assembled nucleosome octamer, the N-terminal tails of H3 and H4 are rich in basic amino acids which confer a positive charge and high-affinity binding with negatively charged DNA [2].

Histones are subject to post-translational modification which residue-specific modification determines chromatin structure or function, including acetylation, phosphorylation, methylation, ubiquitination, SUMOylation and ADP-ribosylation. Post-translational histone modifications contribute to the varied and dynamic configuration of chromatin [3]. Post-translational modifications also orchestrate interaction of the histone tails with other proteins, as combinations of multiple modifications impart a histone ‘code’ that signals specifically to interacting protein partners. Chromatin configuration is intrinsically linked to gene expression and is tightly regulated during growth and development, displaying dynamic responses to changes in the environment [4].

Chromatin remodelling activities have been shown to be important in both the signalling and repair of DSBs in mammalian and yeast systems [5]. However, our understanding of chromatin modifications and their importance in plant DNA repair is limited to the phosphorylation of S139 (single-letter code has been used for amino acids) of the histone variant H2AX by the DSB signalling kinase ATM (ATAXIA TELANGIECTASIA MUTATED) [6]. This is a well-characterized and highly conserved response to induction of DNA DSBs, dependent on upstream MRN [MRE11 (meiotic recombination 11)–RAD50 (radiation sensitive 50)–NBS1 (Nijmegen breakage syndrome 1)] complex signalling in plants, yeast and animals [7,8]. In animals, H2AX phosphorylation has been shown to function in establishing stable repair foci at the site of DNA DSBs [9].

Chromatin remodelling during DNA repair in yeast and mammals involves hyperacetylation of H3 and H4 as early epigenetic changes to chromatin following DNA damage [5,10]. In addition to altering localized chromatin structure, these modifications function in DNA repair signalling pathways; histone H3 acetylation on K14 (H3K14 acetylation) stimulates autophosphorylation of ATM [11]. In contrast, there are no data concerning histone acetylation in the plant response to DSBs, although acetylation has been reported after UV-treatment of plants, which predominantly induces formation of cyclobutane pyrimidine dimers and 6-4 photoproducts. UVB-tolerant maize lines showed high levels of covalent changes to histones compared with the UVB-sensitive lines. Hyperacetylation of H3 (diacetyl) and H4 (tetracetyl) N-termini in UVB-tolerant lines were enriched in the promoter and transcribed regions of genes associated with UVB response, implicating acetylation in the transcriptional response to UV-induced DNA damage [12].

Previous studies utilized MS analysis of plant histones in Arabidopsis [13,14], alfalfa [15] and soybean [16] to identify methylated and acetylated histone isoforms. In the present study we have used a LC (liquid chromatography)–MS/MS (tandem MS) approach to analyse post-translational modifications that are present on H3 and H4 following DNA-damage induction by IR (ionizing radiation), which results in a range of damage products including SSBs (single-strand breaks) and DSBs. We identify several novel forms of acetylated H3 and H4 and demonstrate dynamic changes in the relative abundance of specific histone modifications upon induction of DNA damage in Arabidopsis.

EXPERIMENTAL

Plant growth

Wild-type Columbia-0 seeds were sterilized in 70% ethanol for 5 min, and resuspended in sterile 0.1% agar. Seeds were plated individually in Murashige and Skoog (Sigma) medium (20 g/l sucrose, 0.5 g/l Mes and 8 g of phytoagar; Duchefa). After a cold treatment of 2 days at 4°C, plates were transferred to a growth chamber (16 h of light and 8 h of dark cycle) and grown for 2 weeks before harvesting. IR treatment was delivered using a 320 kV X-ray irradiation system (NDT Equipment Services).

Nuclei isolation

Seedlings were ground in liquid nitrogen to a fine powder and further homogenized in homogenization buffer [25 mM Pipes (pH 7.0), 10 mM NaCl, 5 mM EDTA (pH 8.0), 250 mM sucrose, 0.5% Triton X-100, 20 mM 2-mercaptoethanol, 0.2 mM PMSF and 1/50 ml protease inhibitor tablet (Roche catalogue number 04693132001)] for approximately 1 min in a Waring blender. The homogenate was filtered through 300 μm, 100 μm and 56 μm Sefar Nitex membrane layers and the filtrate was centrifuged at 4600 g for 15 min at 4°C and the supernatant removed. The pellet was then washed in homogenization buffer and centrifuged at 1000 g for 10 min at 4°C a further two times. The nuclear pellet was then resuspended in nuclei storage buffer [50 mM Hepes (pH 7.6), 110 mM KCl, 5 mM MgC12, 50% (v/v) glycerol and 1 mM DTT (dithiothreitol)]. For protein samples used in immunoblotting, the nuclear pellet after the first centrifugation was suspended in nuclei electrophoresis buffer [5% SDS, 0.5 M Tris (pH 6.8) and 1 mM DTT].

Acid extraction of histones

Nuclei were centrifuged at 1000 g for 10 min at 4°C and the supernatant removed. The remaining pellet was resuspended in prechilled extraction buffer (0.5 M HCl and 0.2 mM PMSF) and vortexed. Samples were placed on a rotator at 4°C for between 30 min and overnight. Samples were then centrifuged at 16000 g for 10 min at 4°C, and the supernatant removed and mixed 2:1 with prechilled 100% TCA (trichloracetic acid)/water (final TCA concentration was 33%). Samples were mixed well and placed on ice for between 30 min and overnight. Samples were centrifuged at 16000 g for 10 min at 4°C and the supernatant poured off. The pellet was washed twice with 100% prechilled acetone and centrifuged at 16000 g for 5 min and then left to dry. Samples were stored at −80°C.

Tricine-SDS/PAGE gel electrophoresis

Protein samples were separated using 10% acrylamide/bisacrylamide (19:1) Tricine gels containing ethylene glycol (30%, v/v) according to [17] using the Protean II xi Cell electrophoresis system (catalogue number 165-1834, Bio-Rad Laboratories). Protein samples were dissolved in Laemmli buffer (5% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.1 M Tris (pH 6.8) and 0.1% Bromophenol Blue) and denatured at 90°C for 10 min, and loaded on to the gel alongside Precision Xtra Plus protein marker (161-0377, Bio-Rad Laboratories) and calf thymus histones (catalogue number 10223565001, Roche). Proteins were electrophoresed at 100 V for 24 h using Tricine electrophoresis buffer (catalogue number B48, Fermentas). Proteins were stained with Instant Coomassie (Expedeon ISB01L) for between 30 min and 1 h. The protein bands were excised and overlaid with 20% acetic acid and 5% methanol in 1.5 ml microfuge tubes. For immunoblotting, protein samples were separated on 16% acrylamide/bisacrylamide (19:1) minigels and electrophoresed for 2.5 h at 150 V.

Western blotting

Protein concentrations in resuspended nuclear pellets were quantified using the Lowry protein assay (Bio-Rad Laboratories) with BSA as a standard. After electrophoresis, proteins were transferred on to a nitrocellulose membrane (catalogue number 10402495, Whatman) for 1 h at 100 V. The blots were probed with the following rabbit polyclonal primary antisera: anti-H3 (1:10000 dilution; catalogue number H0164, Sigma); anti-H3K18acK23ac (1:10000 dilution; catalogue number 17-615, Millipore); anti-H4K5acK8acK12acK16ac (1:5000 dilution catalogue number 06-598, Millipore); anti-H4K8ac (1:10000 dilution; catalogue number 06-760-MN, Millipore); anti-H4K12ac (1:5000 dilution; catalogue number 06-761-MN, Millipore); or anti-H4K16ac (1:10000 dilution; catalogue number 06-762-MN, Millipore). Immune complexes were detected by alkaline-phosphatase-conjugated anti-(rabbit IgG) (1:30000 dilution; catalogue number A2556, Sigma–Aldrich) and developed in BCIP (5-bromo-4-chloroindol-3-yl phosphate)/NBT (Nitro Blue Tetrazolium) solution (Invitrogen).

Arg-C digestion

In-gel Arg-C digestion was performed after reduction with DTE (dithioerythritol) and S-carbamidomethylation with iodoacetamide. Gel pieces were washed twice with 50% (v/v) aqueous acetonitrile containing 25 mM ammonium bicarbonate, then once with acetonitrile and dried in a vacuum concentrator for 20 min. Gel pieces were rehydrated by adding 10 μl of 0.02 g/l Arg-C solution in 25 mM ammonium bicarbonate, and after 10 min sufficient 25 mM ammonium bicarbonate solution was added to cover the gel pieces. Digests were incubated overnight at 37°C.

Propionylation

The digest supernatant was removed and adjusted to pH 10 by the addition of aqueous ammonium hydroxide, followed by the addition of 10 μl of propionic anhydride and incubation at 60°C for 1 h. Post-propionylation the samples were acidified with TFA (trifluoroacetic acid), dried down and reconstituted in 0.1% aqueous TFA.

MS

HPLC was performed using a nanoAcquity UPLC system (Waters) equipped with a nanoAcquity Symmetry C18, 5 μm trap (180 μm×20 mm; Waters) and a nanoAcquity BEH130 1.7 μm C18 capillary column (75 m×250 mm; Waters). The trap wash solvent was 0.1% aqueous formic acid and the trapping flow rate was 10 μl/min. The trap was washed for 5 min after sample loading before switching flow to the capillary column. The separation used a gradient elution of two solvents: solvent A (0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate for the capillary column was 300 nl/min, column temperature was 60°C and the gradient profile was as follows: initial conditions 5% solvent B (2 min), followed by a linear gradient to 35% solvent B over 20 min and then a wash with 95% solvent B for 2.5 min. The column was returned to initial conditions and re-equilibrated for 25 min before subsequent injections.

The nanoLC system was interfaced to a maXis UHR-QTOF (ultrahigh-resolution quadrupole-time-of-flight) mass spectrometer (Bruker Daltonics) with a nano-electrospray source fitted with a steel emitter needle [180 μm outside diameter ×30 μm inside diameter; Proxeon). Positive-ion MS and CID (collision-induced dissociation)–MS/MS spectra were acquired using AutoMSMS mode. Instrument control, data acquisition and processing were performed using Compass 1.3 SR3 software (micrOTOF control, Hystar and DataAnalysis, Bruker Daltonics). Instrument settings were: ion spray voltage, 1500 V; dry gas, 6 l/min; dry gas temperature, 160°C; and ion acquisition range m/z, 50–2200. AutoMSMS settings were for MS 0.5 s (acquisition of survey spectrum) and for MS/MS (CID with N2 as collision gas) ion acquisition range m/z, 350–1400; 0.1 s acquisition for precursor intensities above 100000 counts; for signals of lower intensities down to 1000 counts acquisition time increased linearly to 1.5 s; the collision energy and isolation width settings were automatically calculated using the AutoMSMS fragmentation table; three precursor ions; absolute threshold 1000 counts; preferred charge states; and 24 singly charged ions excluded. Two MS/MS spectra were acquired for each precursor and previously selected target ions were excluded for 60 s.

Database searching

MS/MS data were submitted to database searching using a locally running copy of the Mascot program (version 2.3, Matrix Science), through the Bruker ProteinScape interface (version 2.1). Spectra were searched against the IPI (International Protein Index) Arabidopsis database (37150 sequences and 14876683 residues). The search criteria specified were: enzyme, Arg-C; fixed modifications, carbamidomethyl (C); variable modifications, acetylation (K), oxidation (M), propionylation (K, N-term); peptide tolerance, 10 ppm; MS/MS tolerance, 0.1 Da. Peptides with an expect value of 0.05 or lower were considered significant.

RESULTS

Isolation of Arabidopsis histones and preparation for LC–MS/MS

Histones isolated from Arabidopsis seedling tissue and separated by electrophoresis were identified by immunoblotting with H3 antisera (Figures 1A and 1B). Discreet bands of H4 and H3 were excised from the gel and samples were reduced, alkylated and digested in-gel with Arg-C protease rather than trypsin which produces suboptimal histone peptides for MS due to their high lysine and arginine content. Endoproteinase Arg-C cleaves at arginine residues only, producing histone peptides of 3–14 residues in length (Figure 1C). Propionylation of H3 and H4 peptides was carried out to modify the ϵ-amino groups of lysine residues and the N-termini to increase the hydrophobicity and retention of peptides on the C18 column prior to MS/MS. Following LC–MS/MS, the spectra were searched against the IPI Arabidopsis database, and the expected histone was the top-scoring match in each instance. We obtained sequence coverage of 45% and 54% of H3 and H4 respectively, with the N-termini of both H3 and H4 being well covered (Figures 1D and 1E).

Isolation of histones and peptides for identification by MS

Figure 1
Isolation of histones and peptides for identification by MS

Histones were isolated from Arabidopsis seedlings and analysed by peptide digestion and MS/MS. (A) Coomassie Blue-stained Tricine gel. (B) Schematic overview of preparation of H3 and H4 N-terminal peptides for LC–MS/MS. (C) Immunoblot with H3 antisera. Lane 1 and 2, plant histones; lane 3, calf thymus. (D) H3 amino acid sequence with identified peptides in bold and underlined. (E) H4 amino acid sequence with identified peptides indicated as bold underlined text.

Figure 1
Isolation of histones and peptides for identification by MS

Histones were isolated from Arabidopsis seedlings and analysed by peptide digestion and MS/MS. (A) Coomassie Blue-stained Tricine gel. (B) Schematic overview of preparation of H3 and H4 N-terminal peptides for LC–MS/MS. (C) Immunoblot with H3 antisera. Lane 1 and 2, plant histones; lane 3, calf thymus. (D) H3 amino acid sequence with identified peptides in bold and underlined. (E) H4 amino acid sequence with identified peptides indicated as bold underlined text.

Identification of acetylated lysines in histones H3 and H4 following DNA damage

Acetylation of lysine residues has been shown to be both involved in the DDR (DNA damage response) in mammals and yeast [18,19] and in the transcriptional response to UV-irradiation in maize [12]. To investigate global acetylation patterns after DNA damage in Arabidopsis, we investigated the acetylation of H3 and H4 N-termini following a 160 Gy dose of X-rays. Good coverage of the N-terminal regions of both H3 and H4 was achieved by LC–MS/MS and Mascot searching to identify peptides (Figures 1D and 1E). Several criteria were used to confirm the assignment of sites of acetylation within peptides. Acetylation could readily be distinguished from trimethylation, which gives a mass increase that is 36 mDa higher than acetylation, using the high mass accuracy of the Bruker maXis. Low mDa differences between measured and calculated peptide masses were obtained (Table 1), which were consistent with acetylation and ruled out trimethylation. Mascot assignments of acetylation positions along with the spectra were submitted to the Prophossi spectrum annotation tool (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl) [20], which labels spectra with product ions, and also matches all low-abundance and doubly charged product ions. Finally, manual inspection was performed to ensure the presence of crucial b- and y-type product ions in the MS/MS spectra (Figures 2 and 3).

Table 1
Acetylated peptides from H3 and H4 N-termini identified by LC-CID–MS/MS

Acetylated lysines are indicated by Kac. Ion scores and expect values are from Mascot. Measured peptide masses are monoisotopic values calculated from masses of doubly charged precursor ions that can include propionyl groups (see Figure 3). Mass errors are differences between measured and theoretical masses.

Histone Peptide Acetylation sites Ion score Expect value Measured mass (Da) Mass error (mDa) 
H3 K9STGGKacAPR17 K14 80 2.0×10−7 998.551 
H3 K9acSTGGKacAPR17 K9 K14 51 1.6×10−4 984.535 
H3 K18QLATKacAAR26 K23 58 3.3×10−5 1083.640 
H3 K18acQLATKacAAR26 K18 K23 63 9.8×10−6 1125.651 
H4 G4KGGKacGLGKGGAKR17 K8 55 5.0×10−5 1535.879 
H4 G4KGGKGLGKGGAKacR17 K16 80 1.5×10−7 1423.832 
H4 G4KacGGKGLGKGGAKacR17 K5 K16 60 1.2×10−5 1521.864 
H4 G4KGGKacGLGKGGAKacR17 K8 K16 70 1.3×10−6 1465.843 
H4 G4KGGKGLGKacGGAKacR17 K12 K16 63 6.4×10−6 1465.841 
H4 G4KacGGKGLGKacGGAKacR17 K5 K12 K16 84 5.2×10−8 1451.820 <1 
H4 G4KGGKacGLGKacGGAKacR17 K8 K12 K16 46 3.0×10−4 1507.849 
H4 G4KacGGKacGLGKacGGAKacR17 K5 K8 K12 K16 67 2.7×10−6 1493.829 
Histone Peptide Acetylation sites Ion score Expect value Measured mass (Da) Mass error (mDa) 
H3 K9STGGKacAPR17 K14 80 2.0×10−7 998.551 
H3 K9acSTGGKacAPR17 K9 K14 51 1.6×10−4 984.535 
H3 K18QLATKacAAR26 K23 58 3.3×10−5 1083.640 
H3 K18acQLATKacAAR26 K18 K23 63 9.8×10−6 1125.651 
H4 G4KGGKacGLGKGGAKR17 K8 55 5.0×10−5 1535.879 
H4 G4KGGKGLGKGGAKacR17 K16 80 1.5×10−7 1423.832 
H4 G4KacGGKGLGKGGAKacR17 K5 K16 60 1.2×10−5 1521.864 
H4 G4KGGKacGLGKGGAKacR17 K8 K16 70 1.3×10−6 1465.843 
H4 G4KGGKGLGKacGGAKacR17 K12 K16 63 6.4×10−6 1465.841 
H4 G4KacGGKGLGKacGGAKacR17 K5 K12 K16 84 5.2×10−8 1451.820 <1 
H4 G4KGGKacGLGKacGGAKacR17 K8 K12 K16 46 3.0×10−4 1507.849 
H4 G4KacGGKacGLGKacGGAKacR17 K5 K8 K12 K16 67 2.7×10−6 1493.829 

Identification of mono- and di-acetylated peptides from histone H3

Figure 2
Identification of mono- and di-acetylated peptides from histone H3

CID–MS/MS spectra were redrawn and annotated using ProPhoSI (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl). Precursor ion m/z values and measured masses are indicated; all precursor ions were doubly charged. A bold black line indicates that a b- (purple) or y-ion (green) is observable in the spectrum. Acetylated lysine residues are in red; propionylated lysine residues are in blue; and N-terminal proprionylation is labelled Pr.

Figure 2
Identification of mono- and di-acetylated peptides from histone H3

CID–MS/MS spectra were redrawn and annotated using ProPhoSI (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl). Precursor ion m/z values and measured masses are indicated; all precursor ions were doubly charged. A bold black line indicates that a b- (purple) or y-ion (green) is observable in the spectrum. Acetylated lysine residues are in red; propionylated lysine residues are in blue; and N-terminal proprionylation is labelled Pr.

Identification of mono-, di-, tri- and tetra-acetylated peptides from histone H4

Figure 3
Identification of mono-, di-, tri- and tetra-acetylated peptides from histone H4

CID–MS/MS spectra were redrawn and annotated using ProPhoSI (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl). Precursor ion m/z values and measured masses are indicated; all precursor ions were doubly charged. A bold black line indicates that a b- (purple) or y-ion (green) is observable in the spectrum. Acetylated lysine residues are in red; propionylated lysine residues are in blue; and N-terminal proprionylation is labelled Pr.

Figure 3
Identification of mono-, di-, tri- and tetra-acetylated peptides from histone H4

CID–MS/MS spectra were redrawn and annotated using ProPhoSI (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl). Precursor ion m/z values and measured masses are indicated; all precursor ions were doubly charged. A bold black line indicates that a b- (purple) or y-ion (green) is observable in the spectrum. Acetylated lysine residues are in red; propionylated lysine residues are in blue; and N-terminal proprionylation is labelled Pr.

Acetylated peptide isoforms for two N-terminal H3 peptides, K9STGGKAPR17 and K18QLATKAAR26, and one N-terminal H4 peptide, 4GKGGKGLGKGGAKR17, were identified in DNA-damage-treated plants (Table 1). Both mono- and di-acetylated forms of the two H3 peptides were present in addition to the non-acetylated forms. Their presence indicates that each of the lysines K9, K14, K18 and K18K23 were incompletely acetylated in histone H3. In this analysis, acetylation of K9 is only observed in combination with K14 acetylation, and likewise, acetylation of K18 is only identified where K23 is acetylated. In the case of the single N-terminal H4 peptide, G4KGGKGLGKGGAKR17, there were eight acetylated isoforms identified (two monoacetylated, three diacetylated, two triacetylated and one tetra-acetylated), which demonstrated acetylation of lysines K5, K8, K12 and K16 in histone H4 in different combinations and all four simultaneously. Of the eight different acetylated forms of the H4 N-terminal peptide isolated from irradiated Arabidopsis, CID–MS/MS evidence for only the tetra-acetylated isoform in higher plants has been published [12,21].

The MS/MS evidence for monoacetylated K9STGGKacAPR17 (where ac indicates an acetylated residue) from histone H3 is very strong, with both b- and y-ion series observing that they lack only a single ion each (Figure 2A). The spectrum of K9acSTGGKacAPR17 also contains a y-ion series that lacks only one ion (the y1), plus a series of four b-ions (Figure 2B). The evidence for K18QLATKacAAR26 and K18acQLATKacAAR26 is also strong, with both y-ion series missing only the y1 ion (Figures 2C and 2D).

For the two monoacetylated G4KGGKGLGKGGAKR17 H4 peptides, convincing evidence for the assigned sites of acetylation was contained in the extensive b- and y-ion series observed (Figures 3A and 3B). Likewise, spectra of all three of the diacetylated peptides contain y-ion series that are complete except for the y1 ion (Figures 3D and 3E), and two also contain extensive b-ion series (Figures 3D and 3E). Assignment of the sites of acetylation in G4KGKacGLGKGGAKacR17 is supported by the prominent y5, y7 and y12 ions at m/z 530, 771 and 1409 respectively (Figure 3C). Two triacetylated histone H4 peptides, G4KGKacGLGKacGGAKacR17 and G4KacGKGLGKacGGAKacR17, were also identified by Mascot with expect values of 5.2×10−8 and 3.0×10−4 respectively (Table 1). The y-ion series for these two peptides are nearly complete, and the N-terminal b-ion series that are also present confirm the absence of acetylation of K8 or K5 in the two isoforms (Figures 3F and 3G respectively). The hyperacetylated form with acetyl modifications present at each of the four lysine residues (G4KacGKacGLGKacGGAKacR17), has previously been identified in UV-irradiated maize by Casati et al. [12]. Their published CID–MS/MS spectrum is very similar to that of the N-terminally propionylated version we obtained, which contains a nearly complete series of y-ions, plus a series of five b-ions from the N-terminus (Figure 3H).

Identification of methylated lysines in histone H3 following DNA damage

Methylation of lysine residues can be associated with gene silencing, for example, H3K9 methylation is necessary for silencing and the dimethylated form is associated with regions of heterochromatin, whereas trimethylated H3K27 and H3K9 are both found in euchromatin [3,14,22]. The highest levels of methylation were identified in the histone H3 isoform H3.1, including monomethylation of lysine residues K9 and K27, consistent with the role of H3.1 in packaging heterochromatin [13]. Analysis of methylated lysine residues in a peptide sequence was manually validated by confirming the presence of the diagnostic b- and y-type product ions in the MS/MS spectrum. The mass difference between b- and y-type product ions indicating trimethylation will result in addition of 42.047 to the mass of lysine, whereas acetylation will only add 42.011 Da. The accuracy of the mass measurements in the present study was sufficient to discriminate the 36 mDa mass difference between these two modifications (Table 2). We identified two unique methylated peptides of H3 from irradiated plants. One isoform was dimethylated at K9, which is enriched in heterochromatic regions [22], was concomitantly acetylated at K14 (K9me2STGGKacAPR17; where me indicates a methylated residue); the assignment is supported by a series of y-ions that lacks only the y1 ion, plus intense b4 and b7 ions (Figure 4A). The other was monomethylated at K9 (K9meSTGGKAPR17); the assignment is supported by a series of y-ions that lacks only the y1 ion (Figure 4B).

Table 2
Methylated peptide isoforms from H3 and H4 N-termini identified by LC-CID–MS/MS

Acetylated lysine is indicated by Kac and dimethylated lysine by Kme2. Ion scores and expect values are from Mascot. Measured peptide masses are monoisotopic values calculated from masses of doubly charged precursor ions that include propionyl groups (see Figure 4). Mass errors are differences between measured and theoretical masses.

Histone Peptide Modification sites Ion score Expect value Measured mass (Da) Mass error (mDa) 
H3 K9meSTGGKAPR17 K9 41 2.8×10−4 1026.587 
H3 K9me2STGGKacAPR17 K9 K14 35 7.8×10−3 1026.581 
Histone Peptide Modification sites Ion score Expect value Measured mass (Da) Mass error (mDa) 
H3 K9meSTGGKAPR17 K9 41 2.8×10−4 1026.587 
H3 K9me2STGGKacAPR17 K9 K14 35 7.8×10−3 1026.581 

Identification of methylated and acetylated peptides from histone H3

Figure 4
Identification of methylated and acetylated peptides from histone H3

CID–MS/MS spectra were redrawn and annotated using ProPhoSI (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl). Precursor ion m/z values and measured masses are indicated; all precursor ions were doubly charged. A bold black line indicates that a b- (purple) or y-(green) ion is observable in the spectrum. Acetylated lysine residues are in red; propionylated lysine residues are in blue; dimethylated lysine residues are in orange; and N-terminal proprionylation is labelled Pr.

Figure 4
Identification of methylated and acetylated peptides from histone H3

CID–MS/MS spectra were redrawn and annotated using ProPhoSI (http://www.compbio.dundee.ac.uk/prophossi/bin/prophossi-cgi.pl). Precursor ion m/z values and measured masses are indicated; all precursor ions were doubly charged. A bold black line indicates that a b- (purple) or y-(green) ion is observable in the spectrum. Acetylated lysine residues are in red; propionylated lysine residues are in blue; dimethylated lysine residues are in orange; and N-terminal proprionylation is labelled Pr.

Increase in acetylation of the H3 N-terminus following DNA damage

Previous reports indicate that the diacetylated forms for histone H3 K9acK14ac and K18acK23ac constitute some of the lowest abundance detectable modifications in plants [13]. Monoacetylated H3 peptides varied in relative abundance, K14ac being most abundant, then K18K23, whereas K9ac was not detected [13]. In the present study, four acetylated peptides on the H3 N-terminus were identified. The Arg-C digest after propionylation generated two peptides, K9STGGKAPR17 and K18QLATKAAR26, which display acetylation at each of the four lysine residues (K9, K14, K18 and K23). However, our analysis does not reveal whether residues K14 and K18 are both acetylated within a single molecule of histone H3. The hyperacetylation of the H3 N-terminus was observed in response to DNA damage in mammals by the use of antisera specific to each lysine modification [23], but this analysis also did not reveal whether concurrent acetylation of multiple residues occurs in the same molecule. To further characterize hyperacetylation following X-ray treatment in Arabidopsis, a commercially available antibody to K14acK18ac was used to investigate whether K14 and K18, the residues spanning the two peptide fragments generated by the Arg-C digest, were both acetylated in irradiated tissue by immunoblotting. The antibody was used to detect differences in relative abundance of lysine modifications in protein samples from irradiated and unirradiated tissues (Figure 5A). Diacetylation of K14 and K18 was detected in all tissues, and was shown to increased abundance following IR in Col-0 (Figure 5B). Acetylation of K14 and K18 is therefore detectable as an epigenetic mark present on histones in the absence of genotoxic stress that increases in response to DSB induction. A similar response was shown in the atm-null mutant background; however, a band of greater intensity is present following IR treatment, indicating that acetylation persists in the absence of a fully functioning DDR, and suggests that diacetylation of K14 and K18 occurs independently of ATM-mediated signalling.

Relative abundance of H3K14acK18ac and acetylated forms of H4 N-terminus increases following IR

Figure 5
Relative abundance of H3K14acK18ac and acetylated forms of H4 N-terminus increases following IR

(A) Coomassie Blue-stained Tricine-SDS/PAGE gel. (B) Western blot analysis using H3K14acK18ac. (C) Coomassie Blue-stained Tricine-SDS/PAGE gel. (D) Immunoblot for tetra-acetylated H4K5acK8acK12acK16ac. (E) Coomassie Blue-stained gel (F). Western blots with anti-H4K5ac (G), anti-H4K12ac (H), anti-H4K16ac antibodies. Lane 1, Col-0 unirradiated; lane 2, Col-0 160 Gy IR; lane 3, atm-3 unirradiated; and lane 4, atm-3 160 Gy IR.

Figure 5
Relative abundance of H3K14acK18ac and acetylated forms of H4 N-terminus increases following IR

(A) Coomassie Blue-stained Tricine-SDS/PAGE gel. (B) Western blot analysis using H3K14acK18ac. (C) Coomassie Blue-stained Tricine-SDS/PAGE gel. (D) Immunoblot for tetra-acetylated H4K5acK8acK12acK16ac. (E) Coomassie Blue-stained gel (F). Western blots with anti-H4K5ac (G), anti-H4K12ac (H), anti-H4K16ac antibodies. Lane 1, Col-0 unirradiated; lane 2, Col-0 160 Gy IR; lane 3, atm-3 unirradiated; and lane 4, atm-3 160 Gy IR.

Loss of tetra-acetylated H4 tail following DNA damage: K16 and K12 most likely targets for deacetylation

Hyperacetylation of the H4 tail has been shown to localize to transcription start sites of loci implicated in the DDR in maize [12], and be an epigenetic modification at DSB sites in studies with mammalian cells undergoing DDR [24]. Our MS analysis of Arabidopsis histones isolated from tissues undergoing repair of DSBs following X-ray treatment revealed the tetra-acetylated form was present, along with seven other isoforms. The tetra-acetylated peptide was found to decrease in abundance following X-ray treatment in Col-0, but increase in abundance in the atm-null mutant background, again illustrating that acetylation persists in the absence of a fully functioning DDR (Figures 5C and 5D). To identify deacetylation sites in Col-0 histones following IR, we used antibodies against each individual acetylated lysine residue; histones displayed consistent levels of acetylation on K5 (Figure 5F), whereas K12 and K16 show a decrease in relative abundance after irradiation and therefore represent candidate sites for deacetylation during the DDR (Figures 5G and 5H).

DISCUSSION

We used an LC–MS/MS approach to investigate acetyl and methyl modifications of Arabidopsis histones following DNA damage induction by X-ray treatment and investigated the DNA-damage specific modification of individual peptides through Western blot analysis.

Our MS analysis provided good sequence coverage of both H3 and H4 (around 50% coverage) through the inclusion of Arg-C digestion and propionylation steps. The four H3 acetylated isoforms peptides described in the present study were also identified in a nucleosome-wide characterization of Arabidopsis histones, which additionally identified the monoacetylated peptide K18acQLATK23 [14]. In addition, a study of the relative abundance of peptide isoforms of histones isolated from Arabidopsis inflorescence tissue showed that the unmodified H3 peptide K18QLATKAAR26 was the most abundant isoform, followed by the K18ac, K18acK23ac and then K23ac isoforms [13]. The MS analysis the present study was not quantitative, however we identified the two least abundant forms, K23ac and K18acK23ac, demonstrating the sensitivity of our analysis and confirming the presence of these isoforms following genotoxic stress. Studies of Arabidopsis using quantitative MS analysis in addition to an immunological detection report that K9ac accounts for a few per cent of the total histone population [13], and in human cells, H3K9ac levels are reduced in response to DNA damage [18]. This is consistent with the failure to detect this species in X-ray treated plant tissues, whereas the mutually exclusive methylated peptide was identified in the present study. The present study also detected the dimethylated and acetylated H3 peptide K9me2K14ac, previously shown to be low in abundance in both Arabidopsis [13] and alfalfa [15].

To investigate whether any hyperacetylated forms are associated with the plant response to genotoxic stress, immunoblotting was performed using antiserum specific against acetylated histone peptides. Acetylation of H3K14acK18ac increased in response to X-ray induction relative to untreated controls, indicating potential roles for chromatin modification in the plant DDR. Similar increases in H3K14ac were observed in yeast cells following UV treatment [25]. Although acetylation confers structural changes to chromatin, the relationship of these covalent modifications in the DDR is complex, as the combination of covalent histone marks also initiates ATP-dependent remodelling activities [24], with acetylation of the H3 tail dramatically increasing nucleosome repositioning by the RSC complex [26]. Although the present study demonstrates hyperacetylation of Arabidopsis H3 in response to DNA damage, this acetylation occurs in the absence of ATM indicating that this modification is independent of (or upstream of) the well-characterized ATM-kinase mediated DDR in plants.

Extensive acetylation of Arabidopsis histone H4 was detected in the present study, including nine forms of H4 G4KGGKGLGKGGAKR17 and unmodified, mono-, di-, tri- and tetra-acetylated peptide isoforms represented (Figure 6, residues 1–9). Zhang et al. [14] identified acetylation sites by LC–MS/MS on six smaller tryptic peptides that are contained within the sequence GKGGKGLGKGGAKR (Figure 6, peptides a–f). Their analysis identified modified peptides in common with the present study, which additionally demonstrates the acetylation of K5 without concomitant acetylation of K8 (Figure 6, peptides 8 and 9). This result contrasts with previous models of acetylation in plants [21], in which acetylation of K5, K8, K12 and K16 in Brassica oleracea was proposed to occur sequentially beginning with K16 and proceeding towards the N-terminal K5. In addition, peptides 2 and 7 (Figure 6) are also inconsistent with the proposed model, but suggest that acetylation of K16 is common.

Summary of peptides from histone H4 observed by LC–MS/MS

Figure 6
Summary of peptides from histone H4 observed by LC–MS/MS

The nine GKGGKGLGKGGAKR peptides observed in the present study, one unmodified and eight acetylated, are labelled 1–9. Smaller peptides included within the sequence GKGGKGLGKGGAKR that were observed by Zhang et al. [14] (labelled a–f) are also shown for comparison. Sites of acetylation at lysine residues 5, 8, 12 and 16 are indicated by open circles.

Figure 6
Summary of peptides from histone H4 observed by LC–MS/MS

The nine GKGGKGLGKGGAKR peptides observed in the present study, one unmodified and eight acetylated, are labelled 1–9. Smaller peptides included within the sequence GKGGKGLGKGGAKR that were observed by Zhang et al. [14] (labelled a–f) are also shown for comparison. Sites of acetylation at lysine residues 5, 8, 12 and 16 are indicated by open circles.

The tetra-acetylated H4 tail has been shown to increase in abundance following UV exposure in UV-resistant maize [12], and is present specifically at transcription start sites of UV-tolerance-associated genes. In mammals, hyperacetylation of H4 occurs at endonuclease-induced DSBs; H4K8ac is enriched at break sites downstream of γ-H2AX, and two subunits of acetyltransferase NuA4 complex were also present [27]. Substitution of lysine resides of H4 at K5, K8, K12 and K16 by glutamine rendered cells more sensitive to DNA damage and impaired NHEJ (non-homologous end-joining) repair of DSBs demonstrating the importance of these residues in the mammalian DSB response [19]. Furthermore, tetra-acetylation is required for accumulation of BRCA1 (breast cancer 1) and Rad51 at DSB foci, and can extend for several kb either side of the DSB [27,28]. In contrast, the present study demonstrated that plants display a slight global decrease in relative abundance of tetra-acetylated H4 following 160 Gy IR. Interestingly this is not observed in atm mutants, which display a pronounced increase in the levels of tetra-acetylated H4 after exposure to X-rays. Mutant atm plants are deficient in the detection and signalling response to DSBs [6,29], and observed hyperacetylation of H4 in these lines after irradiation may be indicative of the accumulation of DSBs in the atm mutant background. Furthermore, the increased acetylation of H4 in atm mutants clearly indicates that hyperacetylation is upstream of ATM signalling. The observed deacetylation in irradiated plants may require functional ATM, as mammalian ATM interacts with the deacetylase HDAC (histone deacetylase) 1 in vivo with increased association following IR [30], and HDAC1 and HDAC2 have been shown to regulate acetylation and promote DSB repair [31].

Summary

Using LC–MS/MS we have identified several different acetylated H3 and H4 N-terminal acetylation states following X-ray treatment, including four not previously described in Arabidopsis. Western blot analysis demonstrated hyperacetylation of histone H3 and a global decrease in H4 acetylation in response to X-rays. Acetylation levels were greater in an atm-null background, clearly showing that histone acetylation occurs in the absence of ATM, although deacetylation activity may be compromised. Future work investigating the hyper- and hypo-acetylation response of H3 and H4 will determine the significance of these epigenetic changes during genotoxic stress and repair.

Abbreviations

     
  • ac

    acetylated

  •  
  • ATM

    ATAXIA TELANGIECTASIA MUTATED

  •  
  • CID

    collision-induced dissociation

  •  
  • DDR

    DNA damage response

  •  
  • DSB

    double-strand break

  •  
  • DTT

    dithiothreitol

  •  
  • HDAC

    histone deacetylase

  •  
  • IPI

    International Protein Index

  •  
  • IR

    ionizing radiation

  •  
  • LC

    liquid chromatography

  •  
  • me

    methylated

  •  
  • MS/MS

    tandem MS

  •  
  • TCA

    trichloracetic acid

  •  
  • TFA

    trifluoroacetic acid

AUTHOR CONTRIBUTION

Georgina Drury, Wanda Waterworth and Chris West conceived the study; Georgina Drury, Wanda Waterworth and Chris West designed and performed the experiments, and evaluated the data; MS experiments and related data analysis were performed by Adam Dowle, David Ashford and Jerry Thomas. The paper was written by Georgina Drury, Wanda Waterworth and Chris West, with editorial assistance from Adam Dowle, David Ashford and Jerry Thomas.

We thank Dr Iain Manfield (The Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, U.K.) for help and advice and Rajni Bhardwaj (University of Leeds, Leeds, U.K.) for assistance with medium and plant growth. We thank Dr David Martin (University of Dundee, Dundee, U.K.) who incorporated propionylation into Prophossi.

FUNDING

This work was supported by the Biotechnology and Biological Science Research Council (BBSRC) [grant number BB/G001723/1]. MS work was performed using instruments of the University of York, Centre of Excellence in Mass Spectrometry.

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