Characterization of Post-Translational Modifications of Histone H2B

Characterization of Post-Translational Modifications of Histone H2B-Variants Isolated from Arabidopsis thaliana Eveline Bergmu1 ller,† Peter M. Gehrig,‡ and Wilhelm Gruissem*,†,‡ Institute of Plant Sciences, ETH Zurich, Universita¨tstrasse 2, 8092 Zurich, Switzerland, and Functional Genomics Center Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Received April 17, 2007

Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants. Keywords: Histone H2B • mass spectrometry • MALDI-TOF/TOF • FTICR • post-translational modifications • Arabidopsis

Introduction Eukaryotic nuclear DNA is compacted more than 7-fold by winding around histones in nucleosomes, the basic unit of chromatin. Nucleosomes can be further assembled into supramolecular structures, which provide chromatin with dynamics necessary for transcriptional activity and gene regulation. The nucleosome consists of 146 bp of DNA that is wrapped around a histone octamer, which is formed by dimers of the core histones H2A, H2B, H3, and H4. The N-termini of histones are enriched in lysine and arginine residues, which protrude out of the nucleosome and are therefore accessible to dynamic post-translational modifications. Lysine residues can be acetylated, mono-, di-, and trimethylated, while arginine is mono- and dimethylated (symmetric and asymmetric).1-4 Other modifications that are found in histones include phosphorylation of serine and threonine residues, and ubiquitination and sumoylation of lysine.5-7 Once DNA is packaged into chromatin, its function is controlled by the ordered recruitment of diverse enzymatic complexes that chemically modify nucleosomes, remodel their structure, or change their position * Corresponding author: Wilhelm Gruissem, Institute of Plant Sciences, LFW E56.1, ETH Zurich, Universita¨tstrasse 2, 8092 Zurich, Switzerland. Tel., +41-44-6320857, fax, +41-44-6321079; e-mail, [email protected]. † Institute of Plant Sciences. ‡ Functional Genomics Center Zurich. 10.1021/pr0702159 CCC: $37.00

 2007 American Chemical Society

relative to the DNA.8 The type and combination of histone modifications influence gene regulation, and current models suggest that they act synergistically to alter chromatin structure and regulate transcription.9,10 Thus, histone modifications may establish a code that is “read” by other proteins and that directs the function of chromatin-remodeling machines and transcription factors.11-13 Chromatin remodeling is important for genome integrity, epigenetic inheritance, and in genome defense,14 as well as during development.15 Although the existence of a histone code is still controversial, there is increasing evidence to support this concept. For example, recruitment of HP1 (heterochromatin protein-1) by trimethylation of H3 at Lys-9 can be reversed by phosphorylation of Ser-10 during M-phase of the cell cycle.16 These results suggest that a regulatory read-out exists for combinatorial post-translational histone modifications by regulatory proteins. At the same time, they illustrate that more information is needed to understand the complexity of multiple histone modifications.17 In most organisms, multiple genes encode several subtypes of each class of histones, which are very similar and can only be distinguished by a few differences in their amino acid sequences. Most of the histone genes are expressed during S-phase, and it is currently unknown if all subtypes can equally form complexes that are assembled into nucleosomes. Other histone variants differ in their function and/or in the way they Journal of Proteome Research 2007, 6, 3655-3668

3655

Published on Web 08/11/2007

research articles are deposited on DNA (replication-coupled or replicationindependent).18,19 For example, histone H3 can be grouped into the canonical H3-variants, the replacement H3.3-variants, and the centromeric CenH3-variant.20,21 H3.3-variants appear to be incorporated into DNA independent of the cell cycle, and they are associated mainly with actively transcribed gene loci. Therefore, not only histone modifications, but also the types of histones that are incorporated into the nucleosome influence chromatin structure and gene activity.21,22 Histone modifications have been studied intensively during the past few years since their significance in gene regulation became apparent. However, the information on histone modifications is not complete, especially concerning the possible combinations of modifications and their function. Immunoblotting using antibodies that detect specific modified histone amino acids have now provided valuable insights into the association of histone modifications with gene activity. But this approach has limitations, since it does not allow detection of new or multiple modification sites and it cannot distinguish between different histone variants. Mass spectrometry provides an excellent alternative approach to identify post-translational modifications, and some histones have already been characterized by MS/MS techniques.23-34 Post-translational modifications of plant histones have only been studied in a few cases.35-37 While some information is available on H3 modifications, no post-translational modifications of H2B-variants have been reported for Arabidopsis. In humans, several acetylation sites of H2B histones were determined by mass spectrometry on Lys-5, Lys-11, Lys-12, Lys-15, Lys-16, and Lys-20.33,34 Recently Medzihradszky et al.28 reported post-translational modifications of Tetrahymena H2B, which were studied by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) using electron capture dissociation (ECD). They identified methylation and acetylation sites in two H2B histones that differ by only three amino acids. Since H2B histones from plants have an N-terminal extension, which is distinct from that of humans and other organisms, it is difficult to compare the modification sites with each other. The Arabidopsis genome has 11 H2B genes, which encode proteins that differ mainly in the amino acid sequence of their N-terminal tails. Here, we report the isolation of Arabidopsis H2B histones and identification of their post-translational modifications using LC-MALDI-TOF/TOF and LTQ-FTICR mass spectrometry analysis. Our work represents the first detailed characterization of H2B histones from higher plants and will be the basis to investigate the role of Arabidopsis H2Bvariants and their modifications in chromatin structure and gene expression.

Experimental Procedures Nuclei Isolation and Extraction of Histones. Arabidopsis thaliana Col-0 cells were cultured in MS medium containing 3% sucrose, 1× Murashagi & Scoog medium, 500 µg/L NAA, and 50 µg/L kinetin. One-fifth of the cell volume was transferred to fresh medium every 7 days. For nuclei preparation, cells were harvested after 7 days. The pellet was suspended in nuclear extraction buffer (0.32 M sucrose, 25 mM Tris/HCl, pH 8, 10 mM MgCl2, 5% dextran, 2.5% Ficoll, 45 µM spermine, 0.2% Triton X-100, 2.5 mM DTT, and 5 mM Na-butyrate) and centrifuged at 900g for 10 min. The crude nuclei pellet was washed with NEB buffer without spermine, loaded on a Percoll gradient (40%, 60%, 80%), and centrifuged for 30 min at 1000g in a swing-out rotor. Purified nuclei were treated with nuclei 3656

Journal of Proteome Research • Vol. 6, No. 9, 2007

Bergmu1 ller et al.

lysis buffer (50 mM Tris/HCl, pH 8, 100 mM NaCl, 3 mM EDTA, 1% CHAPS, and 1× Roche protease inhibitor) and centrifuged at 10000g. The pellet was suspended in 0.3 M H2SO4 and stirred twice for 16 h at 4 °C. The supernatant containing the histones was dialyzed against 100 mM acetic acid. Isolation of Euchromatin-Enriched Fractions. Euchromatin-enriched fractions were isolated by a method modified for Arabidopsis.38-40 Briefly, intact nuclei were suspended in buffer N (15 mM Tris-HCl, pH 7.8, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 1 mM DTT, 320 mM sucrose, and 1× protease inhibitor cocktail (Roche) and normalized by estimating the DNA concentration and adjusting it to 400 µg/mL. The OD260/280 ratio was typically around 1.1-1.3 due to the immense amount of proteins in the extract. A total of 1.2 mg of DNAnormalized nuclei was treated with 10 U MNase for 20 min at room temperature and 10 min on ice. The suspension was centrifuged at 10 000g for 10 min at 4 °C, and the supernatant was called S1 fraction and should contain euchromatin. To obtain heterochromatin, the pellet was resuspended in 2 mM EDTA containing protease inhibitors, incubated 10 min on ice, and centrifuged. The S2 supernatant is enriched in heterochromatin, and the remaining pellet is mostly matrix-associated chromatin. Extraction of DNA. DNA was isolated from chromatin by chloroform/phenol extraction as described in van Bokland et al.39 Briefly, 20% SDS was added to a final concentration of 2% and incubated for 15 min at 50 °C. Afterward, potassium acetate was added to a concentation of 1 M and centrifuged at 12 000 rpm for 15 min. The supernantant was extracted with phenol/ chloroform, and the DNA was precipitated with ethanol. The DNA was resuspended in TE-buffer and loaded onto a 1% agrose gel. The DNA was stained with ethidiumbromide. Separation of Histones by Reversed-Phase High-Performance Liquid Chromatography (HPLC). Histones were separated by reversed-phase HPLC using a Vydac C4 column (250 mm × 4.6 mm, 5 µM, 300 Å). Individual histones were eluted with the following gradient: 0-10% B from 0 to 10 min; 1033% B, from 10-20 min; 33-46% B, from 20 to 30 min; 4664% B, from 30 to 85 min; 64-100% B, from 85 to 90 min; 100% B, from 90 to 95 min; 100-0% B, from 95 to 97 min; 0% B, from 97 to 115 min. Solvent A was 5% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA); solvent B was 80% ACN and 0.1%TFA. The flow rate was 0.7 mL/min, and 0.5 mL fractions were collected with an automated fraction collector between 20 and 84 min. SDS-PAGE and Western-Blot Analysis. For separation, histones were loaded on a 15% SDS-PAGE mini gel and either visualized by Coomassie R250 or immunoblotted using antidimethylated-Lys-9 histone H3 (H3K9diMe) from Upstate (rabbit polyclonal lgG, Cat 07-441, Lot 29698) in a 1:2000 dilution or anti-dimethylated-Lys-4 histone H3 (H3K4diMe) from Upstate (rabbit antiserum, Cat 07-030, Lot 26335) in a 1:5000 dilution. Tryptic Digestion of H2B. Preparative RP-HPLC fractions of H2B were collected, dried in a speed-vac and dissolved in 50 mM NH4HCO3. Prior to digestion, the samples were heated for 15 min at 60 °C. For digestion, samples were incubated 1:200 with trypsin (Promega) for 2 h at 37 °C. Adding TFA to a final concentration of 0.1% stopped the reaction. Histones extracted from gels were subjected to in-gel tryptic digest as described previously41 using trypsin in a ratio of one part trypsin to 10 parts of protein. Proteins were digested overnight at 26 °C. After

Arabodopsis Histone H2B-Variants Post-Translational Modifications

elution, tryptic peptides were lyophilized to dryness and stored at -80 °C until analysis. Linear Ion Trap/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Digested histones were analyzed by a hybrid linear ion trap Fourier transform mass spectrometer (LTQ-FT, Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source. An HPLC system was used online with the mass spectrometer. The system (Agilent 1100 nanoflow) consists of a solvent degasser, a nanoflow pump, and a thermostated micro-autosampler. Solvent A consisted of 3% ACN and 0.2% formic acid, and solvent B consisted of 100% ACN and 0.2% formic acid. The samples were automatically loaded onto a laboratory-made silica capillary column (i.d. 75 µm, length of 9 cm; BGB Analytik AG, Bo¨ckten, Switzerland) packed with C18 reversed-phase material (Magic C18 resins; 5 µm, 200-Å pore; Michrom BioResources, Auburn, CA). Peptides were eluded at a flow rate of 0.2 µL/min from the column with a 80 min gradient (0-12% buffer B from 0 to 20 min; 12-80% B, from 20 to 67 min; 80% B from 67 to 72 min). MS analysis was performed using data-dependent acquisition mode in which the mass spectrometer automatically switches between a high-resolution survey scan acquired by FTICR (resolution ) 100 000, m/z range 300-2000) followed by the acquisition of lower resolution fragmentation spectra in the linear ion trap (collision-induced dissociation at a target value of 10 000) of the four most abundant peptides eluting at a given time. Former target ions selected for MS/MS were dynamically excluded for 60 s. Total cycle time was approximately 10 s. The general mass spectrometric conditions were spray voltage, 1.9 kV; ion transfer tube temperature, 250 °C; normalized collision energy, 35% for MS2. Ion selection thresholds were 500 counts for MS2. An MS2 activation q ) 0.25 and activation time of 30 ms were applied. NanoLC Separation and MALDI Target Spotting of Tryptic Peptides. Peptide separation was performed on an Ultimate chromatography system (Dionex - LC Packings, Sunnyvale, CA) equipped with a Probot MALDI spotting device. A total of 10 µL of the samples was injected by using a Famos autosampler (Dionex - LC Packings) and loaded directly onto a 75 µm × 150 mm reversed-phase column (PepMap 100, 3 µm; Dionex LC Packings). Peptides were eluted at a flow rate of 300 nL/ min by using the following gradient: 0-3 min, 0% solvent B; 3-85 min, 0-50% solvent B; 85-95 min, 50-100% solvent B; and 95-100 min, 100% solvent B. Solvent A contained 0.1% TFA in 98:2 water/ACN, and solvent B contained 0.1% TFA in 20:80 water/ACN. For MALDI MS/MS analysis, column effluent was directly mixed with MALDI matrix (3 mg/mL R-cyano-4hydroxycinnamic acid in 70% ACN/0.03% TFA) at a flow rate of 1.1 µL/min via a µ-Tee fitting. Fractions were automatically deposited every 20 s onto a MALDI target plate (Applied Biosystems, Framingham, MA) using a Probot micro fraction collector. In total, 192 spots were collected from each HPLC run. MALDI-TOF/TOF Mass Spectrometry. MALDI plates were analyzed on a 4700 Proteomics Analyzer MALDI TOF/TOF system (Applied Biosystems). The instrument was equipped with a Nd:YAG laser operating at 200 Hz. All mass spectra were recorded in positive reflector mode, and they were generated by accumulating data from 4000 laser shots (analysis of in-gel digests) or from 2000 laser shots (LC-MALDI experiments). The mass spectra were externally calibrated using peptide standards. First, MS spectra were recorded from each of the six calibration spots, and the default calibration parameters of the

research articles

instrument and the plate model for that plate were updated. Second, MS spectra were recorded for all 192 sample spots on that plate and then analyzed using software supplied with the instrument. Spectral peaks that met the threshold criteria and were not on the exclusion list were included in the acquisition list for the MS/MS spectra. The threshold criteria were set as follows: mass range, 700-4000 Da; minimum signal-to-noise (S/N), 60; peaks/spot, 10. Peptide CID was performed at collision energy of 1 kV and a collision gas pressure of approximately 2 × 10-7 Torr. During MS/MS data acquisition, a method with a stop condition was used. In this method, a minimum of 3000 shots (60 subspectra accumulated from 50 laser shots each) and a maximum of 5000 shots (100 subspectra) were allowed for each spectrum. The accumulation of additional laser shots was halted whenever at least 4 ions with a S/N of at least 50 were present in the accumulated MS/MS spectrum, in the region from m/z 200 to 90% of the precursor mass. Peptide and Protein Identification by Database Searching. For the LTQ-FTICR analysis, all MS/MS spectra files from each LC run were centroided and merged to a single file, which was searched using the MASCOT search engine version 2.1.042 (Matrix Science, London, U.K.). For MALDI-TOF/TOF experiments, the MS and MS/MS data were searched using Mascot version 2.1 (Matrix Science) as the search engine. All searches were performed against an A. thaliana database (30 690 sequences, released on September 9, 2006) consisting of respective data sets from The Arabidopsis Information Resource (TAIR).43 GPS (Global Proteome Server) Explorer Software (Applied Biosystems) was used for submitting data acquired with the MALDI-TOF/TOF mass spectrometer for database searching. The following search settings were used: maximum missed cleavages, 3; peptide tolerance, 60 ppm (in-gel digests) or 75 ppm (LC-MALDI) for MALDI-TOF/TOF and 3 ppm for LTQ-FTICR analyses; MS/MS tolerance, 0.2 Da for MALDI-TOF/ TOF and 0.5 Da for LTQ-FTICR analyses. Acetylation and mono- and dimethylation of lysine residues; mono-, di-, and trimethylation of the N-terminus of the protein; and ubiquitination of lysine residues were selected as variable modifications. Typically, acetylation of lysine and oxidation of methionine were selected as variable modifications for the analyses of in-gel-digested proteins. The 95 % significance thresholds correspond to Mascot scores of 26, 40, and 57 in database searches with LTQ-FT data, LC-MALDI-MSMS data, and MALDI-MS + MSMS data (in-gel digests), respectively. In addition, error tolerant searches were carried out for the major histone H2B variants HTB2, HTB9, and HTB11.

Results Isolation of Histones from Cultured Arabidopsis Cells. Histones were isolated from nuclei of cultured Arabidopsis cells by sulfuric acid extraction, and the different subclasses were separated by C4 reversed-phase HPLC (Figure 1A). Sample fractions were collected, and their purity was analyzed by SDSPAGE electrophoresis (Figure 1B). To identify the different histone types, bands were excised, in-gel-digested with trypsin, and analyzed by MALDI-TOF/TOF mass spectrometry. Figure 1A shows the HPLC chromatogram of histones extracted from cultured Arabidopsis cells. All four subclasses of Arabidopsis histones could be separated. H2B histones elute in three peaks, but in the third peak, H4 also begins to elute from the column. Another peak contains only histone H4. H2Avariants elute as two distinct peaks. H3 and H3.3-variants are Journal of Proteome Research • Vol. 6, No. 9, 2007 3657

research articles

Bergmu1 ller et al.

Figure 1. Purification and separation of histone classes by chromatography. (A) RP-HPLC chromatogram of histone extracts. The chromatogram shows the separation of histones extracted from cultured Arabidopsis cells. The 0.5 mL fractions were collected and used for further analysis. (B) SDS-PAGE analysis of the different histone fractions. One-tenth of the collected histone fractions was analyzed by gel electrophoresis to check for purity of the samples. The respective band were subjected to in-gel digestion with trypsin followed by mass spectrometry analysis.

observed much later in the chromatogram compared to the other histones. To examine the purity of the proteins, 1/10 of the collected fractions were analyzed on a 15% SDS-PAGE gel (Figure 1B). For the identification of the individual histones, the protein bands were excised from the gel and in-gel-digested with trypsin. The resulting peptides were analyzed by MALDITOF/TOF mass spectrometry and by combined database searches using peptide masses and fragment ion spectra. All four core histones (H2A, H2B, H3, and H4) were identified. 3658


This analysis shows that the respective histone classes are highly enriched-in the eluted fractions. Only the H4 fractions contain additional histone proteins, either H2B (fractions 35 and 36) or H2A (fractions 41-43). However, in fractions 42 and 43, H4 is still the most abundant protein. Histone H3 and the H3.3-variants elute together in fractions 77-82. Thus, the separation of the individual histone classes was very efficient, and the obtained profile was reproducible. H2B histones have high sequence similarity, and many tryptic peptides cannot be


research articles

Figure 2. Sequence alignment of H2B-variants shows high amino acid similarity. Amino acid sequences were aligned by CLUSTALW multiple sequence alignment. Differences mainly occur in the N-terminal region, while the C-terminus is highly conserved among the H2B-variants. The identified H2B-variants are highlighted in bold, and the observed unique peptides are underlined.

assigned to a single variant. The Top Down approach has been applied to perform gene-specific identification of human histone H2B-variants.44 Here, we identified unique peptides for four different H2B-variants from the SDS gels that correspond to the proteins annotated as At3g45980 (HTB9, 16 305 kDa), At5g22880 (HTB2; 15 601 kDa), At3g46030 (HTB11; 15 780 kDa), and At5g59910 (HTB4; 16 318 kDa). While the first three H2Bvariants eluted in the first and second peak, the latter one was mainly observed in the third peak of the H2B elution profile. Furthermore, we identified a fifth H2B-variant (HTB1, At1g07790) from the tryptic in-solution digest of the collected HPLCfraction. Figure 2 shows the sequence alignment of all 11 H2B proteins. The detected H2B variants and peptides unique to these variants are indicated. Isolation of Histones from Euchromatin. To analyze Arabidopsis histone modifications on transcriptionally active chromatin, nuclear fractions enriched in euchromatin were isolated after mild MNase treatment of freshly prepared nuclei.38 This

procedure yielded a soluble S1 fraction that was enriched in euchromatin, a heterochromatin-containing S2 fraction, and a pellet (P) with the matrix-containing chromatin.38 DNA was isolated from all three fractions and analyzed on a 1% agarose gel. The gel image in Figure 3A clearly shows the mononucleosomes of the euchromatin-enriched fractions, the nucleosome ladder of heterochromatin, and the non-digested, matrixcontaining chromatin. To further confirm the enrichment of euchromatin in the S1 fraction, proteins extracted from all three fractions were immunoblotted with anti-H3K4diMe, which is a marker for transcriptionally active chromatin, and with antiH3K9diMe, which is a marker for transcriptionally silent chromatin.45 Figure 3B shows that all fractions contained the H3K4diMe modification, but the signal was most abundant in the S1 fraction. Furthermore, anti-H3K9diMe was associated only with the S2 fraction but not with the S1 fraction, suggesting that the S1 fraction was highly enriched in transcriptionally active chromatin. Histones were subsequently extracted from Journal of Proteome Research • Vol. 6, No. 9, 2007 3659

research articles

Bergmu1 ller et al.

MS using an LTQ-FT mass spectrometer to identify peptides containing post-translational modifications. Modifications were identified by the observation of mass increments of tryptic peptides in MS spectra, and the sites of modification were determined based on mass changes of individual amino acids in MS/MS spectra, which result in specific shifts of b- and y-ions. Mono- and dimethylation of peptides involve characteristic mass gains of 14 and 28 Da, respectively. Acetylation and trimethylation, however, both increase the nominal mass by 42 Da. The exact mass difference between these modifications is only 0.03639 Da with acetylation resulting in a mass shift of 42.01056 Da and trimethylation in an increase of 42.04695 Da. The distinction between these two modifications requires either high mass accuracy measurements or detection of modification-specific marker ions or neutral losses. Collisioninduced dissociation (CID) of peptides containing acetylated lysine residues results in a modification-specific ion of m/z 126,24,48 while a neutral loss of trimethylamine (-59 Da) is characteristic for trimethylated lysine.49

Figure 3. Isolation of bulk euchromatin from cultured Arabidopsis cells. (A) 1% agarose gel of different chromatin fractions after MNase digestion. DNA from the obtained chromatin fractions of the MNase digest was isolated and loaded on a 1% agarose gel and stained with ethidium bromide. (B) Western blot analysis of proteins from various chromatin fractions. A total of 20 µg of total protein of each fraction was loaded on the gel. Anti-H3MeK4 was used as a marker for euchromatin and anti-H3MeK9 for heterochromatin. S1, soluble fraction 1 from MNase digest, corresponding to euchromatin enriched fraction; S2, soluble fraction from MNase digest, corresponding to heterochromatin enriched fraction; P, pellet fraction from MNase digest, corresponding to matrix containing chromatin; Nc, total nuclei extracts, M, 1 kb DNA ladder.

the S1 euchromatin-enriched fraction and separated, and the post-translational modifications of histone H2B were analyzed. Expression of Arabidopsis H2B-Variants. Arabidopsis encodes 11 genes for different H2B histones that show an amino acid sequence similarity between 44% and 97%, whereas 10 of the proteins have a similarity of more than 80%.46 The differences in their amino acid sequence are mostly confined to the N-terminal regions of the proteins, while their C-terminal regions are highly conserved. Compared to H2B histones from animals, plant H2B histones have an N-terminal extension, and the first amino acid after the initiator-methionine is alanine instead of proline. This organization of H2B proteins is conserved among different plant organisms. Gene expression data from Genevestigator revealed that most of the H2B genes are highly transcribed in the shoot apex and in pollen, while certain H2B-variants are only expressed in floral tissue or seeds (Supplementary Figure 1 in Supporting Information). The five most abundant H2B-variants in cell suspension culture where HTB9, HTB2, HTB11, HTB4, and HTB1.47 These are also the variants we detected by mass spectrometry. Post-Translational Modifications Identified in TrypticDigested H2B-Variants. To characterize post-translational modifications of H2B-variants, H2B proteins were extracted from total chromatin, separated by reversed-phased HPLC, and analyzed by MALDI-TOF/TOF and LTQ-FTICR mass spectrometry. Peptides resulting from the partial trypsin digestion were separated offline by nanoLC and spotted onto a MALDI plate for MALDI-TOF/TOF analysis or analyzed by on-line LC-ESI3660


Table 1 shows a list of acetylated and methylated peptides from H2B subtypes isolated from total chromatin and analyzed by LC-MALDI-TOF/TOF mass spectrometry. Peptides at m/z 2107.17, 2149.25, and 2191.28, which showed similar fragmentation patterns in the MS/MS spectra, were observed with a mass increment of 42 and 84 Da, respectively, and assigned to peptide 4AEKKPAEKKPAAEKPVEEK22 of histone HTB9. While peptide 2107.17 was unmodified, peptide 2149.25 was found to contain one and peptide 2191.28 two acetyl groups (Figure 4). The first acetylation site of the peptide could be clearly assigned to Lys-6, and the second one was identified at Lys11. In both spectra (m/z 2149 and 2191), a peak at m/z 126 was detected, indicating acetylation and not trimethylation of the peptides. Further proof that these peptides contain acetylated and not trimethylated lysine residues was obtained from ESI-FTICR experiments. The acetylated peptides were detected in H2B isolated from total chromatin as well as in fractions isolated from euchromatin. Analogous peptides for HTB11 were also observed. Further acetylation sites established for HTB9 and HTB11 were Lys-27 and Lys-32 or Lys-22 and Lys-27, depending on the H2B-variant. Both acetylations were found to be simultaneously present on peptide 18SKAEKAPAEK27. Although still very similar, the N-terminus of HTB2 could be more easily distinguished from the other H2B-variants. Four HTB2 acetylation sites were assigned to Lys-5, Lys-10, Lys-15, and Lys-27 (Table 1). Furthermore, a doubly acetylated peptide AGK(Ac)K(Ac)LPK was observed. This sequence is found in five of the Arabidopsis H2B proteins. The peptide was also observed with a single acetyl group at the first lysine residue only. All MS/MS spectra of the acetylated peptides showed the diagnostic ion signal at m/z 126. A lysine residue at position 3 of HTB11 and HTB4 was found to be partially monomethylated (Table 2). Monomethylated lysine forms a characteristic immonium ion at m/z 98.49 A dimethylated lysine residue was discovered in peptide 6KPAEKKPAEK15 from HTB2 at position 11, adjacent to an acetylated lysine at position 10 (Supplementary Data in Supporting Information). An ubiquitination site was assigned to Lys-145 of peptide which is the C-terminal tryptic peptide of most H2B-variants of Arabidopsis (Figure 5). The modified peptide was detected by searching for a mass tag of 114 Da corresponding to two glycine residues attached to the -amino group

142AVTKFTSS,149

research articles


Table 1. List of Acetylated and Methylated Peptides Identified from Total Chromatin by LC-MALDI-TOF/TOFa ion detected

MH+calc

ppm

1723.0436 1920.1248 2149.2510 2191.2832 195.6801 1694.9628 1324.7744 1821.0612 1721.9513 2117.2190 1100.5905 1495.8837 1521.8640 910.5438 783.5078 825.5193

1722.9747 1920.0800 2149.1863 2191.1968 1195.7050 1694.9323 1324.7471 1821.0116 1721.9067 2117.1599 1100.5946 1495.8478 1521.8270 910.5356 783.5086 825.5192

40 23 30 39 -21 18 21 27 26 28 -4 24 24 9 -0.1 0

sequence 1AKADKKPAEKKPAEK15 2PKAEKKPAEKKPVEEK17 4AEKKPAEKKPAAEKPVEEK22 4AEKKPAEKKPAAEKPVEEK22 6KPAEKKPAEK15 4AEKKPAEKKPVEEK17 7KPAEKKPVEEK17 7KPAEKKPAAEKPVEEK22 11KPAEKTPAAEPAAAAEK27 11KPAEKTPAAEPAAAAEKKPK30 18SKAEKAPAEK27 18SKAEKAPAEKKPK30 16TPAAEPAAAAEKKPK30 23APAEKKPK30 31AGKKLPK37 31AGKKLPK37

Mascot score

modification

Ac (K5), Ac (K10) Ac (K6), Ac (K11) Ac (K6) Ac (K6), Ac (K11) Ac (K10), di-Me (K11) Ac (K6), Ac (K11) Ac (K11) Ac (K11) Ac (K15) Ac (K15), Ac (K27) Ac (K22) Ac (K22), Ac (K27) Ac (K27) Ac (K27) Ac (K33) Ac (K33), Ac (K34)

HTB2 HTB11 HTB9 HTB9 HTB2 HTB11 HTB11 HTB9 HTB2 HTB2 HTB9, HTB11 HTB9, HTB11 HTB2 HTB9, HTB11 all all

82 118 80 114 36 80 79 139 105 63 59 n. a. 89 41 n. a. 26

a Ions were detected as singly charged peptides [M + H]+. The spectra were analyzed by Mascot database searches and manual interpretation. If more than one histone variant is mentioned, the peptide could not be assigned to one specific H2B histone. In this case, the sequence numbering refers to the first H2B variant mentioned in the last column of the table. n.a., the Mascot score of these peptides is not applicable. The spectra were interpreted manually (see supplementary data in Supporting Information).

of lysine. The two glycine residues represent the C-terminal fragment of ubiquitin resulting from digestion with trypsin. We did not detect mono- or dimethylated arginine residues in any of the H2B-variants. The absence of these modifications was supported by the absence of neutral losses and marker ions usually observed in MALDI-TOF/TOF spectra of peptides containing methylated arginine.50,51 We also analyzed H2B for phosphorylation of serine and threonine residues, but we were unable to detect any phosphorylation sites. This might have been due to the low abundance of phosphopeptides, but also to the histone extraction method, which did not include phosphatase inhibitors. Our results did also not reveal lysine methylation or acetylation sites in the C-terminal region of H2B. H2B Modifications in Transcriptionally Active Chromatin Include Unusual Mono, Di-, and Trimethylation of N-terminal Alanine. The H2B-variants HTB9/HTB1 and HTB11/HTB4 differ only slightly in their N-terminal peptides (amino acid sequence 1-11, Figure 2). They show a single amino acid substitution at position 3 (Lys f Arg). Since the mass difference between dimethyllysine and arginine is only 0.0112 Da, it is relatively difficult to distinguish the corresponding peptides from each other. Accurate mass measurements by FTICR-MS and the detection of diagnostic ions in MALDI-MS/MS data enabled this differentiation. Fragmentation of peptides containing arginine residues results in fragment ions at m/z 87 and 112, which can be detected by MALDI-TOF/TOF mass spectrometry. Table 2 summarizes all modified N-terminal peptides of HTB9/HTB1 and HTB11/HTB4 isolated from euchromatin-enriched fractions that were identified by MALDITOF/TOF and LTQ-FTICR mass spectrometry measurements. The detection of longer unique N-terminal peptides allowed the direct assignment of some post-translational modifications to HTB9 and HTB11. The peptide m/z 626.87272+ corresponds to the sequence 1A(diMe)PRAEKKPAEK11 of HTB9/HTB1 (Figure 6A). To confirm that this peptide is dimethylated at Ala-1 and not at Arg-3 or at one of the Lys residues of 1APRAEKKPAEK11, the CID spectra of the MALDI-TOF/TOF and the LTQ-FTICR were manually interpreted (Figure 6A). In the acquired CID spectra, a signal was detected at m/z 1153.74, which corresponded to the y10ion without any modification. This suggests that the mass gain of + 28 Da results from dimethylation of the N-terminal Ala.

No y-type ion at m/z 1181.70 was detected, which would have been expected if Arg-3 was dimethylated. Furthermore, no diagnostic fragment ions indicative of dimethylated arginine at m/z 46, 71, or 88 were detected.50,51 The N-terminal peptide 1APKAEKKPAEK11 of HTB11/HTB4 showed different methylation states of Ala-1. The precursor ion at m/z 612.86972+ had a mass increment of +28 Da as compared to the mass of the unmodified peptide, indicating the presence of two methyl groups. The CID spectra obtained from MALDI-TOF/TOF and LTQ-FT analyses showed a b3-ion at m/z 325.22, which suggested that the two methyl groups were located at the N-terminus or at Lys-3 (Figure 6B). The fact that an ion at m/z 1125.66 corresponding to an unmodified y10-ion was observed, unambiguously indicated that the Nterminal Ala of HTB11/HTB4 was dimethylated. We identified a peptide, showing that HTB9/HTB1 can be trimethylated at the N-terminal alanine too. The MALDI-MS/ MS spectrum of the peptide at m/z 1266.756, corresponds to 1 A(triMe)PRAEKKPAEK11 of HTB9 (Figure 7). We observed a neutral loss of 59 Da from the precursor ion and from the ions b5, b7, and b10, which was consistent with a trimethylation of the peptide (Figure 8). Since a mass increase of 42 Da was already observed for the ions b2, b3, b4, and b5, although at very low intensity, the N-terminal Ala-1 was identified as the trimethylated residue. The LTQ-FT and MALDI MS/MS spectra recorded from a peptide of 1237 Da could be assigned either to 1A(diMe)PK(Me)AEKKPAEK11 or to 1A(triMe)PKAEKKPAEK11 of HTB11/ HTB4 (Figure 8). Since all observed N-terminal sequence ions from b3 to b10 were shifted by 42 Da, the modification must be located at the first three amino acids. The data resulting from the LTQ-FTICR and the MALDI-TOF/TOF measurements showed an ion at m/z 1139, suggesting that there was an additional methyl group at Lys-3 and not a trimethylated N-terminal amino group. Furthermore, we detected a low mass ion at m/z 98, which is a diagnostic ion of methyl-lysine. However, a weak signal at m/z 1179.97 was present in the MALDI-TOF/TOF spectrum that we interpreted as a neutral loss of trimethylamine (-59 Da) from the precursor ion (Figure 8B). A neutral loss of trimethylamine from trimethylated lysine was recently observed.49 A similar release of trimethylamine from a trimethylated N-terminal amino group seems to occur Journal of Proteome Research • Vol. 6, No. 9, 2007 3661

research articles

Bergmu1 ller et al.

Figure 4. MS/MS spectra, obtained by LC-MALDI-TOF/TOF experiments, of peptide 4-22 of HTB9 show different acetylation levels. The CID spectrum recorded from the ion at m/z 2149.221 showed an acetylation site at Lys-6 (A), and the spectrum recorded from the ion at m/z 2191.205 was assigned to the same peptide containing two acetylation sites at Lys-6 and Lys-11 (B). The marker ion signals at m/z 126 indicate the presence of acetyl-lysine in these peptides.

as well. Together, these results suggest that a small fraction of N-terminal Ala residues of HTB11/HTB4 were also trimethylated in euchromatin. In addition, an error tolerant search of HTB11, isolated from total chromatin, identified the N-terminal peptide 2PKAAEK(Ac)KPAEK(Ac)KPVEEK17 from which the Nterminal alanine residue has been cleaved off (Table 1). Longer N-terminal peptides of HTB9 and HTB11 containing up to four missed tryptic cleavage sites were also identified. 3662


The N-terminal peptide 1APKAEKKPAEKKPVEEK18 of HTB11 was observed in various different forms with different combinations of modifications. Table 2 summarizes the type and position of the modifications of this peptide. On the basis of the sequence-specific fragment ions, it was possible to assign two acetylation sites, Lys-6 and Lys-11, together with three methyl groups, on one peptide. Similar results were obtained for HTB9 (Table 2).

research articles


Table 2. N-terminal Modifications of H2B-Variants from Euchromatin-Enriched Fractions Identified by (A) LC-MALDI-TOF/TOFa and (B) LTQ-FTICRb ion detected

MH+calc

ppm

1238.7518 1252.7377 1266.7556 1280.7485

1238.7472 1252.7386 1266.7534 1280.7578

3.8 0.72 1.74 -7.3

1294.7625 1921.1599 1935.1715 1949.1863 1991.1603

1294.7483 1921.1122 1935.1279 1949.1435 1991.1541

11 24 22 21 3.5

2033.1686

2033.1646

1.97

2459.4404 2473.4612 2501.4092 2543.4519

2459.3986 2473.4142 2501.4084 2543.4197

17 19 0.32 12.7

1223.7238 1237.7394 1265.7456 1279.7462 1251.7299

0.90 1.2 1.66 2.19 0.72

612.86972+ 619.87622+ 633.87552+ 640.88092+ 626.87272+

sequence

Mascot score

modification

(A) LC-MALDI-TOF/TOF diMe (N-term), Me (K3)/triMe (N-term) 1APRAEKKPAEK11 diMe (N-term) 1APRAEKKPAEK11 triMe (N-term) 1APKAEKKPAEK11 diMe (N-term), Me (K3), Ac (K6)/triMe (N-term), Ac (K6) 1APRAEKKPAEK11 diMe (N-term), Ac (K6) 1APKAEKKPAEKKPVEEK17 Me (N-term) 1APKAEKKPAEKKPVEEK17 diMe (N-term) 1APKAEKKPAEKKPVEEK17 diMe (N-term), Me (K3)/triMe (N-term) 1APKAEKKPAEKKPVEEK17 diMe (N-term), Me (K3), Ac (K6)/triMe (N-term), Ac (K6) 1APKAEKKPAEKKPVEEK17 diMe (N-term), Me (K3), Ac (K6), Ac (K11)/ triMe (N-term), Ac (K6), Ac (K11) 1APRAEKKPAEKKPAAEKPVEEK22 diMe (N-term) 1APRAEKKPAEKKPAAEKPVEEK22 triMe (N-term) 1APRAEKKPAEKKPAAEKPVEEK22 diMe (N-term), Ac (K6), 1APRAEKKPAEKKPAAEKPVEEK22 diMe (N-term), Ac (K6), Ac (K11) (B) LTQ-FTICR 1APKAEKKPAEK11 diMe (N-term) 1APKAEKKPAEK11 diMe (N-term), Me (K3) 1APKAEKKPAEK11 diMe (N-term), Ac (K6) 1APKAEKKPAEK11 diMe (N-term), Me (K3), Ac (K6) 1APRAEKKPAEK11 diMe (N-term) 1APKAEKKPAEK11

HTB11/HTB4 HTB9/HTB1 HTB9/HTB1 HTB11/HTB4

n. a. 35 16 n. a.

HTB9/HTB1 HTB11 HTB11 HTB11 HTB11

41 82 n. a. n. a. n. a.

HTB11

n. a.

HTB9 HTB9 HTB9 HTB9

102 n. a. n. a. n. a.

HTB11/HTB4 HTB11/HTB4 HTB11/HTB4 HTB11/HTB4 HTB9/HTB1

28 35 35 35 36

a The observed singly charged peptides are represented as [M + H]+. The spectra were analyzed by Mascot database searches and manual interpretation. n.a., the Mascot score of these peptides is not applicable. The spectra were interpreted manually (see supplementary data in Supporting Information). b MS/ MS spectra obtained from the LTQ were analyzed by Mascot database searches and manual interpretation.

Figure 5. An ubiquitination site of H2B-variants has been detected, by the identification of a GlyGly diepeptide, a characteristic footprint of ubiquitinated proteins digested with trypsin. The CID spectrum from the FTICR experiment identified a GlyGly residue at Lys-145 in all H2B-variants.

In H2B samples isolated from total chromatin extracts, we detected three N-terminal peptides, which are modified at the N-terminus. Peptides m/z 1280.7589, 1991.2195, and 2033.2346 can be di- or trimethylated at Ala-1. All three peptides were assigned to HTB11.

Discussion Over the past several years, many studies examining histones and their role in gene expression have been reported, but our Journal of Proteome Research • Vol. 6, No. 9, 2007 3663

research articles

Bergmu1 ller et al.

Figure 6. MS/MS spectra of peptides 1-11 of histones HTB9/HTB1 and HTB11/HTB4 recorded by LTQ-FTICR show dimethylation of the N-terminal alanine. (A) The CID spectrum of m/z 626.87682+ corresponds to peptide A(diMe)PRAEKKPAEK of HTB9/HTB1. The y10-ion detected at m/z 1153 confirms dimethylation of Ala-1, since it does not have a mass increment of 28 Da. (B) The fragmentation spectrum of ion m/z 612.86972+ corresponds to peptide A(diMe)PKAEKKPAEK of HTB11/HTB4.

understanding of the status and function of their modifications is still incomplete. Since post-translational modifications appear to affect the functional properties of histones, a comprehensive investigation of the modifications on all H2B variants isolated from cultured Arabidopsis cells is required. Mass spectrometry analysis has become an important approach to analyze posttranslational modifications of histones.23,33,34,52 Utilizing this technique, we identified acetylated, mono- and dimethylated, 3664


and ubiquitinated lysine residues, as well as mono-, di-, and trimethylated N-terminal alanine residues in H2B-variants (Table 3). Most of these modification sites are only partially occupied. Our results demonstrate that Lys 6, 11, 27, 32, 38, and 39 represent acetylation sites and Lys-145 an ubiquitination site in the HTB9-variant. HTB11 contains analogous modifications, and an additional lysine at position 3 was found to be partially monomethylated. Furthermore, a dimethylated lysine was detected at position 11 of histone HTB2. Interestingly, the


research articles

Figure 7. MS/MS spectrum of peptide m/z 1266.756 of HTB9/HTB1. Fragmentation spectrum of a precursor ion at m/z 1266.756, which was assigned to peptide A(triMe)PRAEKKPAEK of HTB9/HTB1. The signal 59 Da below the parent ion mass indicates loss of trimethylamine.

N-terminal alanine residues of HTB9/HTB1 and HTB11/HTB4 occur free or mono-, di-, or trimethylated, a modification that has not been previously described for histone proteins of Arabidopsis. For HTB11, we also detected a peptide where Ala-1 has been removed. Because Arabidopsis H2B histones share significant sequence identity, it was not always possible to assign lysine acetylation sites to a specific H2B-variant. Acetylation is the best understood post-translational modification of histones, and it is now generally accepted that hyperacetylation is associated with transcriptionally active chromatin while hypo-acetylation results in a silent state.1 Histone acetylation neutralizes the positive charge of Lys, which alters the charge state of the N-terminal histone amino acid sequences. This charge alteration is typically correlated with a more open chromatin conformation that is required for the activation of gene expression.53 However, the model that post-translational modifications of histones change DNA accessibility in chromatin simply by changing the histone charge states has been advanced by the histone code theory.11,12,54 In most cases, we observed acetylation of lysines in an AEKmotif and, in one case, in the sequence ADK, whereas aspartate and glutamate are both acidic amino acids. Only the additional AEK-motif of the five amino acid extension of HTB9 was not observed in an acetylated state. It might be possible, that this duplicated section is only weakly acetylated. Two adjacent acetylation sites, identified in peptide AGKKLPK (Table 1) do not comply with this sequence motif. This AEK-motif seems to be plant- and H2B histone-specific. Methylation of lysine residues can exist as mono-, di-, or trimethylation.55 Not only the sites of these modifications, but also the number of methyl groups added at a particular site are important for regulating chromatin structure and function. We identified one mono- and one dimethylation site on H2Bvariants close to the N-terminus of the proteins. A dimethylated lysine was identified in the sequence motif 6KPAEKKPAEK15 with the latter lysine (K11) being modified. A study of post-

translational modifications on the Arabidopsis histone H3 revealed several major mono- and dimethylation sites (Lys-4, Lys-9, Lys-27, and Lys-36).35 The different numbers of methyl groups can have functional significance, as shown for H3K27. While mono- and dimethylated H3K27 are mainly associated with heterochromatin, trimethylated H3K27 is more evenly distributed.56 Also the position of the methyl groups has an effect on the chromatin state. For example, methylation of H3K9 and H3K27 is associated with gene repression, but methylated H3K4 was found to enhance gene expression.57 It would be interesting to determine the biological function of methylated lysine residues in H2B histones of Arabidopsis. Methylation of the N-terminal alanine has been observed in histone proteins of Tetrahymena and recently also of Trypanosoma brucei.28,58,59 In Tetrahymena, there is evidence that the modification state of the N-terminus and a specific lysine residue are mutually exclusive.28 The physiological function of the methylation of the N-terminal alanine is still a matter of speculation. It might be involved in membrane insertion, regulation of stability and solubility, and protection of the exposed protein ends against aminopeptidases. We actually identified peptide 2-17 of histone variant HTB11, from which the N-terminal alanine residue had been removed probably by an aminopeptidase (Table 1). Determining whether this N-terminal modification plays a role as an epigenetic mark will provide new insights into this class of histones. In the previously mentioned comprehensive analysis of H3 protein modifications,35 no phosphorylation at serine residues and no arginine methylation were observed, which is in agreement with our own analysis of Arabidopsis H2B histones. Most Arabidopsis H2B-variants contain only a single potential phosphorylation site (Ser-23, Thr-16, and Ser-18) in the Nterminal tail domain, which carries most other identified posttranslational modifications. We were further able to identify a ubiquitination site on the lysine residue of peptide AVTKFTSS, which represents the Journal of Proteome Research • Vol. 6, No. 9, 2007 3665

research articles

Bergmu1 ller et al.

Figure 8. The CID spectrum of peptide 1-11 from HTB11/HTB4 identifies three methyl groups. (A) The CID spectrum of ion at 619.87622+ indicates dimethylation of the N-terminus and methylation of Lys-3. The high mass accuracy of the parent mass excludes acetylation of the peptide. The y10-ion at m/z 1139.96 shows a mass increase of +28 Da, while y8 does not. (B) Fragmentation of ion at 1238.747, obtained by MALDI-TOF/TOF measurements, seems to be a mixture of two differentially modified peptides. The ions at m/z 1139.69 (y10) and 339.22 (b3) suggest dimethylation of Ala-1 and methylation of Lys-3. The loss of -59 Da from the precursor ion indicates trimethylated Ala-1.

C-terminus of most H2B-variants. This ubiquitination site, which was established on mammalian histones H2B already in 1987,7 seems to be highly conserved among organisms. Biochemical studies suggest that in yeast, monoubiquitination of Lys-123 of H2B, which corresponds to Arabidopsis Lys-145, is an important prerequisite for global methylation of Lys-4 of H3 in a so-called “trans-tail” manner.60 Shahbazian and colleagues61 showed that Lys-123 ubiquitination is dispensable for H3 Lys-4 monomethylation but essential for the transfer of additional methyl groups on Lys-123. Recent results revealed 3666


that ubiquitination of H2B Lys-123 regulates trimethylation of H3 Lys-4 but not dimethylation.62 Compared to H3 and H4, H2B histones are less conserved among different organisms. It is not clear whether these minor sequence differences have any effect on chromatin function, as it is the case for H3.3-variants. Incorporation of different H2B isoforms could provide specific modification sites and thus selective binding sites for transcription factors and regulatory proteins. The major differences in amino acid sequence, in

research articles

Arabodopsis Histone H2B-Variants Post-Translational Modifications Table 3. Summary of Post-Translational Modifications of H2B-Variants HTB9, HTB2, and HTB11 histone

HTB9

HTB2

HTB11

position

modification

A1 K6 K11 K27 K32 K38 K39 K145 K5 K10 K11 K15 K27 K33 K34 K140 A1 K3 K6 K11 K22 K27 K33 K34 K140

Me; Me2; Me3 Ac Ac Ac Ac Ac Ac Ubiquitin Ac Ac Me2 Ac Ac Ac Ac Ubiquitin Me; Me2, Me3 Me Ac Ac Ac Ac Ac Ac Ubiquitin

particular sequence extensions/reductions and variations of single amino acids, occur in the first 50 amino acids.

Conclusion Post-translational modifications of Arabidopsis H2B histones were characterized using a strategy based on the separation of intact histone proteins by chromatography, partial trypsin digestion of a mixture of H2B variants, and LC-MS analyses of the resulting peptides. The LC-separated peptides were analyzed by MALDI-TOF/TOF and by high accuracy LTQ-FT mass spectrometry. The high accuracy FTMS measurements allowed the distinction of different types of modifications displaying the same nominal masses, and the MALDI-TOF/TOF spectra often showed marker ions or neutral losses characteristic of certain modifications. In addition, the combination of data from both instruments increased protein sequence coverage. These mass spectrometric techniques allowed the identification of various post-translational modifications on five expressed Arabidopsis histones H2B. In this investigation, we identified several sites of lysine acetylation, lysine mono- and dimethylation, and lysine ubiquitination. The N-terminal alanine occurs in four different forms: mono-, di-, or trimethylated or unmodified. The identification and mapping of covalent histone modifications of histone H2B proteins provides the basis for the understanding of how histones H2B are involved in triggering chromatin structure and gene regulation. In the future, interest will increasingly be directed to quantitative changes of histone modifications. For example, it will be interesting to compare the distribution of histone modifications and histone variants in heterochromatin and euchromatin to investigate their physiological function. Further investigations may also focus on the identification and characterization of the corresponding modifying enzymes and on proteins that bind to histone modifications. We expect that unraveling the functions of these modifications will improve the understanding of the epigenetic code and increase our knowledge about the regulation of gene expression.

Acknowledgment. We would like to thank Dr. Bernd Roschitzki and Dr. Bertran Gerrits (Functional Genomics Center

Zurich) for help with the LTQ-FTICR analysis. This work was supported by a fellowship of the Roche Research Foundation and by Marie-Curie Intra-European Fellowship, FP6 to E.B.

Supporting Information Available: A PDF file containing all fragmentation spectra of peptides containing modifications (Supplementary mass spectra). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Grunstein, M. Histone acetylation in chromatin structure and transcription. Nature 1997, 389 (6649), 349-52. (2) Zhang, Y.; Reinberg, D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 2001, 15 (18), 2343-60. (3) Kouzarides, T. Histone methylation in transcriptional control. Curr. Opin. Genet. Dev. 2002, 12 (2), 198-209. (4) Gary, J. D.; Clarke, S. RNA and protein interactions modulated by protein arginine methylation. Prog. Nucleic Acid Res. Mol. Biol. 1998, 61, 65-131. (5) Nathan, D.; Sterner, D. E.; Berger, S. L., Histone modifications: Now summoning sumoylation. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (23), 13118-20. (6) Davie, J. R.; Murphy, L. C. Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription. Biochemistry 1990, 29 (20), 4752-7. (7) Thorne, A. W.; Sautiere, P.; Briand, G.; Crane-Robinson, C. The structure of ubiquitinated histone H2B. EMBO J. 1987, 6 (4), 1005-10. (8) Kadam, S.; Emerson, B. M. Mechanisms of chromatin assembly and transcription. Curr. Opin. Cell. Biol. 2002, 14 (3), 262-8. (9) Fischle, W.; Wang, Y.; Allis, C. D. Histone and chromatin crosstalk. Curr. Opin. Cell. Biol. 2003, 15 (2), 172-83. (10) Brownell, J. E.; Zhou, J.; Ranalli, T.; Kobayashi, R.; Edmondson, D. G.; Roth, S. Y.; Allis, C. D. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 1996, 84 (6), 843-51. (11) Strahl, B. D.; Allis, C. D. The language of covalent histone modifications. Nature 2000, 403 (6765), 41-5. (12) Turner, B. M. Histone acetylation and an epigenetic code. BioEssays 2000, 22 (9), 836-45. (13) Jenuwein, T.; Allis, C. D. Translating the histone code. Science 2001, 293 (5532), 1074-80. (14) Martienssen, R. A.; Colot, V. DNA methylation and epigenetic inheritance in plants and filamentous fungi. Science 2001, 293 (5532), 1070-4. (15) Goodrich, J.; Tweedie, S. Remembrance of things past: chromatin remodeling in plant development. Annu. Rev. Cell. Dev. Biol. 2002, 18, 707-46. (16) Fischle, W.; Tseng, B. S.; Dormann, H. L.; Ueberheide, B. M.; Garcia, B. A.; Shabanowitz, J.; Hunt, D. F.; Funabiki, H.; Allis, C. D. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 2005, 438 (7071), 1116-22. (17) Eissenberg, J. C.; Elgin, S. C. Molecular biology: antagonizing the neighbours. Nature 2005, 438 (7071), 1090-1. (18) Kamakaka, R. T.; Biggins, S., Histone variants: deviants? Genes Dev. 2005, 19 (3), 295-310. (19) Henikoff, S.; Ahmad, K. Assembly of variant histones into chromatin. Annu. Rev. Cell. Dev. Biol. 2005, 21. 133-53. (20) Henikoff, S.; Ahmad, K.; Platero, J. S.; van Steensel, B. Heterochromatic deposition of centromeric histone H3-like proteins. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (2), 716-21. (21) Ahmad, K.; Henikoff, S. The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 2002, 9 (6), 1191-200. (22) McKittrick, E.; Gafken, P. R.; Ahmad, K.; Henikoff, S., Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (6), 1525-30. (23) Burlingame, A. L.; Zhang, X.; Chalkley, R. J. Mass spectrometric analysis of histone posttranslational modifications. Methods 2005, 36 (4), 383-94. (24) Zhang, K.; Tang, H.; Huang, L.; Blankenship, J. W.; Jones, P. R.; Xiang, F.; Yau, P. M.; Burlingame, A. L. Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionizationpostsource decay, and nanoelectrospray ionization tandem mass spectrometry. Anal. Biochem. 2002, 306 (2), 259-69.

Journal of Proteome Research • Vol. 6, No. 9, 2007 3667

research articles (25) Zhang, K.; Williams, K. E.; Huang, L.; Yau, P.; Siino, J. S.; Bradbury, E. M.; Jones, P. R.; Minch, M. J.; Burlingame, A. L. Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry. Mol. Cell. Proteomics 2002, 1 (7), 500-8. (26) Cocklin, R. R.; Wang, M. Identification of methylation and acetylation sites on mouse histone H3 using matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization tandem mass spectrometry. J. Protein Chem. 2003, 22 (4), 327-34. (27) Zhang, K.; Tang, H. Analysis of core histones by liquid chromatography-mass spectrometry and peptide mapping. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2003, 783 (1), 1739. (28) Medzihradszky, K. F.; Zhang, X.; Chalkley, R. J.; Guan, S.; McFarland, M. A.; Chalmers, M. J.; Marshall, A. G.; Diaz, R. L.; Allis, C. D.; Burlingame, A. L. Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS). Mol. Cell. Proteomics 2004, 3 (9), 872-86. (29) Bonaldi, T.; Imhof, A.; Regula, J. T. A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications. Proteomics 2004, 4 (5), 138296. (30) Ren, C.; Zhang, L.; Freitas, M. A.; Ghoshal, K.; Parthun, M. R.; Jacob, S. T. Peptide mass mapping of acetylated isoforms of histone H4 from mouse lymphosarcoma cells treated with histone deacetylase (HDACs) inhibitors. J. Am. Soc. Mass Spectrom. 2005, 16 (10), 1641-53. (31) Zhang, L.; Eugeni, E. E.; Parthun, M. R.; Freitas, M. A. Identification of novel histone post-translational modifications by peptide mass fingerprinting. Chromosoma 2003, 112 (2), 77-86. (32) Chu, F.; Nusinow, D. A.; Chalkley, R. J.; Plath, K.; Panning, B.; Burlingame, A. L. Mapping post-translational modifications of the histone variant macroH2A1 using tandem mass spectrometry. Mol. Cell. Proteomics 2006, 5 (1), 194-203. (33) Bonenfant, D.; Coulot, M.; Towbin, H.; Schindler, P.; van Oostrum, J., Characterization of histones H2A and H2B variants and their post-translational modifications by mass spectrometry. Mol. Cell. Proteomics 2006, 5, 541-52. (34) Beck, H. C.; Nielsen, E. C.; Matthiesen, R.; Jensen, L. H.; Sehested, M.; Finn, P.; Grauslund, M.; Hansen, A. M.; Jensen, O. N. Quantitative proteomic analysis of post-translational modifications of human histones. Mol. Cell. Proteomics 2006, 5 (7), 131425. (35) Johnson, L.; Mollah, S.; Garcia, B. A.; Muratore, T. L.; Shabanowitz, J.; Hunt, D. F.; Jacobsen, S. E. Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of posttranslational modifications. Nucleic. Acids Res. 2004, 32 (22), 6511-8. (36) Loidl, P. A plant dialect of the histone language. Trends. Plant. Sci. 2004, 9 (2), 84-90. (37) Tariq, M.; Paszkowski, J. DNA and histone methylation in plants. Trends. Genet. 2004, 20 (6), 244-51. (38) Fass, E.; Shahar, S.; Zhao, J.; Zemach, A.; Avivi, Y.; Grafi, G. Phosphorylation of histone h3 at serine 10 cannot account directly for the detachment of human heterochromatin protein 1gamma from mitotic chromosomes in plant cells. J. Biol. Chem. 2002, 277 (34), 30921-7. (39) van Blokland, R.; ten Lohuis, M.; Meyer, P. Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing. Mol. Gen. Genet. 1997, 257 (1), 1-13. (40) Remboutsika, E.; Lutz, Y.; Gansmuller, A.; Vonesch, J. L.; Losson, R.; Chambon, P. The putative nuclear receptor mediator TIF1alpha is tightly associated with euchromatin. J. Cell. Sci. 1999, 112 ( Pt 11), 1671-83. (41) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850-8. (42) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 3551-67. (43) Rhee, S. Y.; Beavis, W.; Berardini, T. Z.; Chen, G.; Dixon, D.; Doyle, A.; Garcia-Hernandez, M.; Huala, E.; Lander, G.; Montoya, M.; Miller, N.; Mueller, L. A.; Mundodi, S.; Reiser, L.; Tacklind, J.; Weems, D. C.; Wu, Y.; Xu, I.; Yoo, D.; Yoon, J.; Zhang, P. The

3668


Bergmu1 ller et al.

(44)

(45)

(46)

(47)

(48)

(49)

(50)

(51)

(52)

(53) (54)

(55) (56)

(57)

(58)

(59)

(60)

(61)

(62)

Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 2003, 31 (1), 224-8. Siuti, N.; Roth, M. J.; Mizzen, C. A.; Kelleher, N. L.; Pesavento, J. J., Gene-specific characterization of human histone H2B by electron capture dissociation. J. Proteome. Res. 2006, 5 (2), 2339. Jasencakova, Z.; Soppe, W. J.; Meister, A.; Gernand, D.; Turner, B. M.; Schubert, I. Histone modifications in Arabidopsis- high methylation of H3 lysine 9 is dispensable for constitutive heterochromatin. Plant J. 2003, 33 (3), 471-80. Chenna, R.; Sugawara, H.; Koike, T.; Lopez, R.; Gibson, T. J.; Higgins, D. G.; Thompson, J. D. Multiple sequence alignment with the Clustal series of programs. Nucleic. Acids Res. 2003, 31 (13), 3497-500. Zimmermann, P.; Hennig, L.; Gruissem, W. Gene-expression analysis and network discovery using Genevestigator. Trends Plant Sci. 2005, 10 (9), 407-9. Kim, J. Y.; Kim, K. W.; Kwon, H. J.; Lee, D. W.; Yoo, J. S. Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation. Anal. Chem. 2002, 74 (21), 5443-9. Zhang, K.; Yau, P. M.; Chandrasekhar, B.; New, R.; Kondrat, R.; Imai, B. S.; Bradbury, M. E. Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3. Proteomics 2004, 4 (1), 1-10. Rappsilber, J.; Friesen, W. J.; Paushkin, S.; Dreyfuss, G.; Mann, M. Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode. Anal. Chem. 2003, 75 (13), 3107-14. Gehrig, P. M.; Hunziker, P. E.; Zahariev, S.; Pongor, S. Fragmentation pathways of N(G)-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS. J. Am. Soc. Mass Spectrom. 2004, 15 (2), 142-9. Freitas, M. A.; Sklenar, A. R.; Parthun, M. R. Application of mass spectrometry to the identification and quantification of histone post-translational modifications. J. Cell. Biochem. 2004, 92 (4), 691-700. Wade, P. A.; Pruss, D.; Wolffe, A. P. Histone acetylation: chromatin in action. Trends. Biochem. Sci. 1997, 22 (4), 128-32. Margueron, R.; Trojer, P.; Reinberg, D. The key to development: interpreting the histone code? Curr. Opin. Genet. Dev. 2005, 15 (2), 163-76. Dutnall, R. N. Cracking the histone code: one, two, three methyls, you’re out! Mol. Cell 2003, 12 (1), 3-4. Lindroth, A. M.; Shultis, D.; Jasencakova, Z.; Fuchs, J.; Johnson, L.; Schubert, D.; Patnaik, D.; Pradhan, S.; Goodrich, J.; Schubert, I.; Jenuwein, T.; Khorasanizadeh, S.; Jacobsen, S. E. Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3. EMBO J. 2004, 23 (21), 4286-96. Lachner, M.; O’Sullivan, R. J.; Jenuwein, T. An epigenetic road map for histone lysine methylation. J. Cell. Sci. 2003, 116 (Pt 11), 2117-24. Janzen, C. J.; Fernandez, J. P.; Deng, H.; Diaz, R.; Hake, S. B.; Cross, G. A. Unusual histone modifications in Trypanosoma brucei. FEBS Lett. 2006, 580 (9), 2306-10. Nomoto, M.; Kyogoku, Y.; Iwai, K., N-Trimethylalanine, a novel blocked N-terminal residue of Tetrahymena histone H2B. J. Biochem. (Tokyo) 1982, 92 (5), 1675-8. Sun, Z. W.; Allis, C. D. Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 2002, 418 (6893), 104-8. Shahbazian, M. D.; Zhang, K.; Grunstein, M. Histone H2B ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1. Mol. Cell 2005, 19 (2), 271-7. Shukla, A.; Stanojevic, N.; Duan, Z.; Shaale, T.; Bhaumik, S. R. Functional analysis of H2B-Lys-123 ubiquitination in regulation of H3-Lys-4 methylation and recruitment of RNA polymerase II at the coding sequences of several active genes in vivo. J. Biol. Chem. 2006, 281 (28), 19045-54.

PR0702159

Characterization of Post-Translational Modifications of Histone H2B

Recommend Documents