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Originally published In Press as doi:10.1074/mcp.M700070-MCP200 on July 20, 2007.
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Molecular & Cellular Proteomics 6:1917-1932, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry*,S

Débora Bonenfant{ddagger}, Harry Towbin, Michèle Coulot, Patrick Schindler, Dieter R. Mueller and Jan van Oostrum

From the Novartis Institutes for Biomedical Research, Lichtstrasse 35, CH-4056 Basel, Switzerland


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M. For all core histones, the modifications in the G1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G2/M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G2/M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.

The cell cycle constitutes a series of interrelated processes that includes accurate duplication of the vast amount of DNA in the chromosome and segregation of the copies into two genetically identical daughter cells. These events define the two major phases of the cell cycle: DNA replication, which occurs during synthesis (S) phase, and chromosome segregation followed by cell division, which take place during mitosis (M phase). The two gap phases, G1 and G2, before S and M phases, allow cells to grow. The fundamental unit of eukaryotic chromatin is the nucleosome core particle, which consists of DNA in close association with an octamer of the core histone proteins H2A, H2B, H3, and H4. The N-terminal tails of each of the four core histones are subject to several types of covalent modification, including acetylation and methylation of lysines, methylation of arginines, and phosphorylation of serines and threonines (1). The reversible histone modifications that affect the charge density of the flexible N-terminal and C-terminal domains or introduce bulky ubiquitin moieties have considerable potential to modulate histone/DNA interactions in chromatin. Histone modifications are also able to affect binding interactions with other proteins that may, in turn, alter the higher order structure of chromatin (2, 3).

Conceptually it may be useful to distinguish between modifications that occur locally at regions of chromosomes and at the entire set of chromosomes (4). Thus, acetylation and/or lysine as well as arginine methylation occurs locally on precisely defined sites such as a nucleosome in the vicinity of a transcription factor binding site. In fact, the activity of the transcription factor-recruited enzymes determines the modification patterns (5). Experimentally definition of modifications at single nucleosomes is so far only possible by chromosome immunoprecipitation. At a higher level of chromatin organization, modifications such as methylation of H3 at Lys9 are instrumental in silencing entire regions of chromosomes to form heterochromatin (6). Finally modifications that occur during mitosis affect a large proportion of nucleosomes on all chromosomes (7).

Phosphorylation of histone proteins is involved in the transition from interphase to mitotic chromatin. However, definitive roles of phosphorylation in this process have not yet been elucidated (8, 9). An increase in histone H3 Ser10 phosphorylation is a well known hallmark for mitosis and meiosis in various eukaryotic organisms (10). Recent reports have shown that H3 is also phosphorylated at Ser28, Thr11, Thr3, and H3.3 Ser31 during mitosis (11–14). For histones H4 and H2A, hyperphosphorylation was detected at their respective Ser1 residues during mitosis in worm, fly, and mammalian cells (15). For histone acetylation, one of the better understood histone modifications, it is now generally accepted that hyperacetylated histones are mostly associated with activated genomic regions, whereas deacetylation mainly results in repression and silencing (16, 17). In contrast, histone methylation appears to have multiple effects on chromatin function (18–20). Methylation of H3 on Lys9, for example, is largely associated with silencing and repression in many species (6, 21), and methylation of H3 Lys27 is suggested to be involved in transcriptional repression (22, 23). However, methylation of H3 Lys4, Lys36, and Lys79 were linked to actively transcribed genes (24–26). One challenge is to define the molecular events that link histone modifications with specific biological outcomes, such as transcriptional activation or repression.

Most of the studies describing histone modifications rely on the use of modification-specific antibodies to identify and monitor changes in modifications (e.g. Western blot, chromatin immunoprecipitation, and immunofluorescence). However, the use of these immunological reagents has limitations. First, it is not trivial to define the specificity of these antibodies (27, 28). Second, the influence of multiple modifications on a single histone is likely to impact the recognition by modification-specific antibodies. Finally except for a few antibodies, only one modification site is probed for at a time. Therefore, almost no information can be obtained in this way on the simultaneous presence of multiple modifications on one histone protein. Indeed it has been shown that some modifications have synergistic effects (29). In contrast, MS is a powerful technique that allows the identification of single post-translational modifications as well as multiple modifications on the same peptide (30–33). MS should hence be suitable for characterizing the modification patterns of histones that occur at a specific stage of the cell cycle. Combined with isotope labeling, mass spectrometry also allows quantitation of changes in modification occurring between different stages. In this study, we investigated post-translational modifications of the four human core histones of HeLa cells arrested in G1, S, and G2/M phases. In our strategy, histones were separated by HPLC and then digested with proteases (Glu-C, Arg-C, or trypsin) to produce fragments of suitable length for analysis by nano-ESI MS and MS/MS. For quantifying changes of modifications between S and G2/M phases we applied the technique of stable isotope labeling by amino acids in cell culture (SILAC)1 (34, 35).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Extraction of Histones—
HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% dialyzed FCS and 100 IU of penicillin/streptomycin to reach 40–60% confluence. For G1 phase synchronization, aphidicolin (Sigma-Aldrich) was added to the medium to a final concentration of 1 µg/ml and incubated for 24 h at 37 °C before harvesting the cells (36, 37). For S phase synchronization, cells were treated with aphidicolin as for G1 synchronization, and after 24 h incubation normal growth medium was added for an additional 6 h at 37 °C before harvesting the cells. For G2/M phase synchronization, nocodazole (Sigma-Aldrich) was added to the medium to a final concentration of 50 ng/ml and incubated for 24 h at 37 °C before harvesting the cells (38). HeLa cells were cultured in two 150-mm plates to reach a total of ~4 x 107 cells for each condition. To collect cells, the medium was removed from the plates, cells were washed twice with PBS, 10 ml of 10% trichloroacetic acid were added to the plate, and the cells were scratched off and centrifuged at 2000 x g for 8 min. For all three conditions, the cell pellet was suspended in the extraction buffer (80 mM NaCl, 20 mM EDTA, 1% Triton, 1 mM NaF, 10 mM ß-glycerophosphate, 1 mM sodium vanadate, 45 mM sodium butyrate, protease inhibitors) and centrifuged at 2000 x g for 8 min. For preparing histones (39), the pellets were resuspended in 900 µl of 0.2 M H2SO4 (2 h at 4 °C). After centrifugation at 16,100 x g, supernatants were precipitated by trichloroacetic acid (25% final concentration). The pellet was washed with 50 mM HCl in acetone, then with acetone, and subsequently dissolved in ß-mercaptoethanol (0.1%) in water. For each condition, ~50 µg of histone protein were obtained (Bio-Rad protein assay).

HPLC Separation and Digestion of Histones—
The histone proteins were separated using reversed-phase HLPC (Agilent 1100 series, Agilent, Palo Alto, CA) (40). After elution from HPLC, 85% of histones were collected, and 15% were analyzed by mass spectrometry. Preparative HPLC fractions containing individual histones were pooled according to UV and MS chromatograms, dried (SpeedVac SC 110, Savant Instruments, Farmingdale, NY), and dissolved in 5–10 µl of 25 mM NH4HCO3 or 100 mM Tris-HCl, pH 8. The histones H2A and H2B were digested with endoproteinase Glu-C (sequence grade, Roche Diagnostics) in 25 mM NH4HCO3 at an enzyme ratio of 1:20 at 25 °C for 1–2 h. The histones H4 and H3 were digested either with endoproteinase Glu-C (sequence grade, Roche Diagnostics) in 25 mM NH4HCO3 at an enzyme ratio of 1:20 at 25 °C for 1–2 h or with endoproteinase Arg-C (sequence grade, Roche Diagnostics) at an enzyme ration of 1:100 at 37 °C for 1–2 h in 100 mM Tris-HCl, pH 8. The reaction was stopped by adding formic acid to a final concentration of 0.1%. For desalting, 5–10 µl of peptide mixtures were absorbed on a POROS R3 column, washed with 0.2% formic acid, and desorbed sequentially with 2 µl of 10, 20, 30, and 50% methanol in 0.2% formic acid. Peptides H3-(1–19), H3-(9–17), H3-(27–40), and H2B-(1–35) were eluted at 10% methanol. Peptides H2A-(1–41) (for the first and second HPLC peaks) and peptides H3-(73–83) were eluted at 50% methanol after 10, 20, and 30% step elutions. Peptides H4-(4–17) and H4-(20–35) were directly eluted at 50% methanol, and peptides H4-(1–24) and H4-(18–26) were directly eluted at 20% methanol.

SILAC Experiments—
HeLa cells were cultured in a custom-made medium (arginine-free Dulbecco's modified Eagle's medium, Invitrogen) supplemented with [12C6]arginine (Sigma-Aldrich) or [13C6]arginine (Cambridge Isotope Laboratories, Andover, MA) (34) with 10% FCS plus antibiotics. Arginine concentration was 0.120 mM. After 3 days of growth, cells were treated for S and G2/M phase synchronization. Histone purifications were performed as described previously (40). Two experiments were performed with cells containing [12C6]Arg synchronized at S phase and cells containing [13C6]Arg arrested at G2/M phase. Each [12C6]Arg- and [13C6]Arg-labeled sample was first analyzed individually by LC-MS for estimating the amount of protein. Then the samples were mixed in equal amounts, and the protein mixtures were fractionated and analyzed by LC-MS. After collecting histone fractions, the proteins were digested with endoproteinase Arg-C as described above.

To quantitate changes in post-translational modifications occurring in G2/M phase compared with S phase (set to 1), the ratio R* was calculated as follows. The intensities of the monoisotopic peaks of the labeled (G2/M) and the nonlabeled (S) peptides were considered for all calculations. First, the 13C atom enrichment ({lambda} = 0.99 for the arginine used) was taken into consideration, and the probability for six 13C atoms in [13C6]Arg ({lambda}6 = 0.996 = 0.94) and for twelve 13C atoms in peptides with two [13C6]Arg residues ({lambda}12 = 0.9912 = 0.88) was calculated. With these values, the summed isotopic cluster intensities for the heavy species could be derived directly from the monoisotopic peak containing six or 12 13C atoms (see supplemental data). Second, the fraction of incorporation ({Delta}) of [13C6]Arg into the proteins was calculated after independent peptide analysis of a [13C6]Arg-labeled sample. For the given experiment, the incorporation was 0.93. Third, the intensities of the monoisotopic peaks corresponding to [12C6]Arg- (a) and [13C6]Arg (b1 for one [13C6]Arg and c1 for two [13C6]Arg residues)-modified peptides were determined. From these data, the heavy to light ratios were calculated for one [13C6]Arg incorporated into peptides according to the equation R = 1/({lambda}6{Delta}(a/b1) + {Delta} – 1) and for two [13C6]Arg residues incorporated into peptides using R = 1/({lambda}12{Delta}2(a/c1) – (1 – {Delta})2) (see supplemental data). Finally a correction factor was applied to the heavy and light ratios to correct for deviations from the theoretical 1:1 mixing of labeled and nonlabeled samples. This correction factor was calculated using the intensities of the monoisotopic peaks corresponding to [12C6]Arg (x) and [13C6]Arg (y) peptides known to be devoid of modifications, Corr = 1/({lambda}6{Delta}(x/y + {Delta} – 1). The corrected ratios were given by R* = R/Corr.

Mass Spectrometry—
LC-MS of intact histones was conducted on an HPLC system (Agilent 1100 series) coupled to an LCQ electrospray ion trap mass spectrometer (LCQ, Thermo Finnigan, San Jose, CA). For optimal ESI conditions, spray voltage was set to 4000 V, capillary temperature was 200 °C, capillary voltage was set to 8 V, and the tube lens was set to 16 V. The instrument was scanning between 200 and 2000 Da. We estimated a mass accuracy of 1–2 Da for intact proteins. The molecular masses of the histones were determined after deconvolution of the multiply charged ion series (Bioworks software, Thermo Finnigan). Interpretation of MS spectra was done manually by comparing measured masses to calculated masses of histone sequences derived from Swiss-Prot.

For peptide mass fingerprints, ESI mass spectra were recorded on a QStar Pulsar hybrid quadrupole time-of-flight mass spectrometer equipped with a nanospray ion source (Applied Biosystems, Foster City, CA). The needles (Proxeon, Odense, Denmark) containing the peptide mixtures were adjusted in front of the orifice, and the spray voltage was set between 900 and 1300 V. MS spectra were acquired by scanning over m/z range 200–2000. Mass accuracy of at least 0.02 Da was achieved with external calibration. Parent ions were selected for CID with a mass window of ±0.5 Da. Interpretation of MS and MS/MS spectra was done manually by comparing the measured peptide and fragment masses with those calculated according to histone sequences (Supplemental Table I).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
General Strategy of MS Analysis of Histones from Cell Cycle-arrested Cells—
For obtaining cell populations arrested at defined points in the cell cycle, HeLa cells, which are widely used for such studies, were exposed to aphidicolin, an inhibitor of DNA polymerase, to enrich for cells at the G1/S boundary (36, 37). Release from the aphidicolin block for 7 h yielded a population rich in S phase cells. Nocodazole, a microtubule depolymerizing agent, results in cells accumulating at the G2/M boundary (38). Histones from these three cell preparations were resolved by reversed-phase chromatography into fractions that contained histones H2A, H2B, H3, and H4 (Supplemental Fig. 1) (40). 15% of the histones eluting from HPLC were analyzed directly by MS, and 85% were digested with specific proteases. The peptide mixtures were subsequently analyzed by MS and MS/MS. This strategy allowed us to characterize the post-translational modifications present on all histone variants. Analysis by LC-MS of intact histones isolated from the different phases gave us indications of changes in post-translational modifications (Supplemental Figs. 2–5). However, analysis of peptides generated after histone digestion was necessary to pinpoint changes at individual amino acid positions. To achieve full sequence coverage for all histones, different proteases were used for the digestions. Endoproteinase Glu-C and Arg-C were used for H2A, Glu-C and trypsin were used for H2B (40), and Arg-C and Glu-C were used for H3 and H4 (Supplemental Table I). After digestion, the peptide mixtures were analyzed by nano-ESI MS. The nanospray MS allowed us to optimize the quality of MS/MS spectra in particular for multiply modified large peptides. This optimization would have been difficult to achieve with true HPLC separation coupled on line to the mass spectrometer where typically acquisition of MS/MS spectra is performed in seconds rather than minutes for manual nanospray MS. Full sequence coverage was achieved for H2A, H2B, and H4, and almost 99% sequence coverage was reached for H3 and its variants. The post-translational modifications of histones H2A, H2B, H3, and H4 and their variants from HeLa cells were identified by MS and MS/MS (Table I). For histones H4 and H3, results are summarized in Tables II, III, and IV.


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  		TABLE I List of H2A and H2B peptides

 

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  		TABLE II List of modifications identified on H4 peptides

 

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  		TABLE III List of modifications identified on H3 peptides

 

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  		TABLE IV List of modifications identified on H3 peptides

 
To confirm the modification changes during the cell cycle, a quantitative mass spectrometric analysis using a SILAC strategy was used (35). The labeled amino acids, in this case arginine, were incorporated into all proteins as they were synthesized (34). Cells grew normally in the presence of labeled medium, and incorporation was nearly complete after three days (93% of incorporation). The changes in post-translational modifications occurring in G2/M phase compared with S phase were calculated as ratios of the intensities of the monoisotopic peaks of the labeled (G2/M) versus nonlabeled (S) peptides. These ratios were calculated taking into account the incorporation of isotopic labeled arginine into proteins (here 93%) and the incorporation of 13C into arginine (here 99%). Furthermore these ratios were then corrected for variation from the theoretical 1:1 mixing of labeled and nonlabeled samples by using the monoisotopic peaks of the labeled (G2/M) versus nonlabeled (S) non-modified peptides. The SILAC experiments were done twice, and each biological experiment was analyzed by MS several times. MS quantitation was only performed for histones H3 and H4. For histones H2A and H2B, MS quantitation was difficult to perform due to the low ion intensities of modified peptides, the overlapping of the non-labeled and isotopically labeled peptide ions, and the presence of numerous variants.

We focused our MS and MS/MS analysis on the histone peptides in which we detected modifications by MS; as expected the majority of them were located on the N-terminal tails. In the following, we give details on our findings for each of the core histones.

Acetylation on Histones H2A and H2B Was Reduced in G2/M Phase—
In HeLa cells, the same variants of histones H2A and H2B were found as in Jurkat cells (Table I and Ref. 40). Also the types of modifications in HeLa cells were identical to those of Jurkat cells although the abundance of the modifications were changing during the cell cycle. The molecular masses of histones H2A (first and second HPLC peaks, see Supplemental Fig. 1) and H2B were determined after deconvolution of multiply charged ion series (Supplemental Figs. 2–4). Inspection of the mass spectra of intact H2A and H2B histones for G1, S, and G2/M phases indicated a decrease in acetylation for H2BQ/Q5QNW6 and H2AO between S and G2/M phases. However, for the major H2AC and H2BA histone variants, it was difficult to observe changes in acetylation during the cell cycle due to the presence of various variants with close molecular masses. Therefore, peptides generated after histone digestion were analyzed.

Thus, the major H2A variants H2AO (first HPLC peak) and H2AC (second HPLC peak) were acetylated on Lys5 and phosphorylated on Ser1 (Fig. 1, A and B, and Table I). In addition, acetylation on Lys5 was confirmed for both H2A variants by MS/MS analysis of Arg-C peptides 1–11 (Supplemental Figs. 14 and 20). The ion intensities corresponding to modified peptides 1–41 were low compared with the non-modified peptides. Still comparison of the ion intensities of these peptides for G1, S, and G2/M phases indicated absence of acetylation on Lys5 in the G2/M phase, whereas phosphorylation on Ser1 was apparently not changed. This observation was confirmed for histone H2A (second HPLC peak) by analyzing ions corresponding to peptide 1–41 with a different charge state (Supplemental Fig. 6). For histone H2B, most of the variants in the G1 experiment were found to be monoacetylated and diacetylated for the most intense variant, H2BA (Fig. 1C and Table I). In all three analyses, the ion intensities of acetylated peptides were decreased in S phase, and most of these ions were absent in G2/M phase. For the monoacetylated H2BJ/N and H2BA variants, MS/MS analysis of the corresponding peptides localized acetylation on Lys12, Lys15, and Lys20 (Supplemental Figs. 16 and 18 and Ref. 40). In conclusion, these results for histone H2A and H2B variants indicated a relative decrease of acetylation in the mitotic phase, whereas phosphorylation on H2A remained unchanged. All MS/MS spectra of peptides listed in Table I are shown in the supplemental data.


Figure 1
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  		FIG. 1. Analysis of peptides 1–41 of first and second H2A HPLC fraction and analysis of H2B peptides 1–35 during cell cycle. A, nano-ESI MS spectra of endoproteinase Glu-C peptide 1–41 of the first H2A HPLC peak purified from HeLa cells arrested in G1, S, or G2/M phases. The monoisotopic ions at m/z 560.59, 565.83, and 570.58 were identified as [M + 8H]8+ of unmodified, acetylated, and phosphorylated peptides 1–41 from histone H2AO, respectively. MS/MS analysis identified Ser1 and Lys5 as phosphorylated and acetylated sites, respectively. B, nano-ESI MS spectra of endoproteinase Glu-C peptide 1–41 of the second H2A HPLC peak purified from HeLa cells arrested in G1, S, or G2/M phases. The monoisotopic ions at m/z 562.34, 567.57, and 572.29 were identified as [M + 8H]8+ of unmodified, acetylated, and phosphorylated peptides 1–41 from histone H2AC; the monoisotopic ion at m/z 560.58 was identified as [M + 8H]8+ of peptide 1–41 from histone H2AL; and the monoisotopic ion at m/z 564.33 was identified as [M + 8H]8+ of peptide 1–41 from histone H2AA/G. MS/MS analysis identified Ser1 and Lys5 as phosphorylated and acetylated sites, respectively. The asterisks represent peptides with a different charge state unrelated to peptide 1–41 of histone H2A. The amino acids X represent residues that vary in the H2A sequence. C, nano-ESI MS spectra of endoproteinase Glu-C peptide 1–35 of H2B purified from HeLa cells arrested in G1, S, or G2/M phases. The monoisotopic ions at m/z 550.05, 556.05, and 562.05 were identified as [M + 7H]7+ of the unmodified, mono-, and diacetylated peptides 1–35 from histone H2BA/K/Q/R; the monoisotopic ions at m/z 548.06 and 554.05 were identified as [M + 7H]7+ of unmodified and monoacetylated peptides 1–35 from histone H2BJ/N; the monoisotopic ions at m/z 552.05 and 558.05 were identified as [M + 7H]7+ of unmodified and monoacetylated peptides 1–35 from histone Q52LW6; the monoisotopic ions at m/z 552.33 and 558.34 were identified as [M + 7H]7+ of unmodified and monoacetylated peptides 1–35 from histone H2BC; the monoisotopic ions at m/z 554.33 and 560.33 were identified as [M + 7H]7+ of unmodified and monoacetylated peptides 1–35 from histone H2BB; and the ion at m/z 561.92 was identified as [M + 7H]7+ of peptide 1–35 from histone H2BE. MS/MS analysis identified Lys12, Lys15, and Lys20 as acetylated sites. The asterisks represent peptides with a different charge state unrelated to peptide 1–35 of histone H2B. The amino acids X represent residues that vary in the H2B sequence. ac, acetyl group; ph, phosphoryl group.

 
Complex Pattern of Modification Changes on Histone H4 during the Cell Cycle—
The molecular masses of histone H4 were determined after deconvolution of multiply charged ion series. Inspection of H4 mass spectra indicated changes in methylation and acetylation between G1, S, and G2/M phases (Supplemental Fig. 5). Analysis of peptides generated after histone digestion was then performed to confirm these changes.

Indeed the ion intensities corresponding to mono- and diacetylated peptides 4–17 were intense compared with the non-acetylated peptide (Fig. 2A). Comparison of the ion intensities of these peptides derived from G1-, S-, and G2/M-arrested cells showed a relative decrease in acetylation in G2/M phase, such as for histones H2A and H2B. In addition, MS quantitation by SILAC confirmed this decrease in acetylation in G2/M phase for mono- and diacetylated peptides (10 and 50% reduction, respectively; see Fig. 2B). The non-modified and triacetylated peptides 4–17 were difficult to quantitate due to their low intensities. Analysis by MS/MS of the mono-, di-, and triacetylated peptides identified isomeric peptides with acetyl residues distributed over Lys5, Lys8, Lys12, and Lys16 in the three phases (Table II). The comparison of the fragment ion intensities in the MS/MS spectra of mono- and diacetylated peptides 4–17 for the different cell cycle phases did not indicate which lysine residue (Lys5, Lys8, Lys12, or Lys16) was preferentially affected (Supplemental Figs. 36 and 38). Histone H4 was also methylated on Lys20, and the ion intensities corresponding to the mono- and dimethylated peptides 20–35 were changing during the cell cycle (Supplemental Fig. 7). In contrast to histone H4 acetylation, MS quantitation revealed an increase of methylated peptides 20–35 in G2/M phase compared with S phase (150% monomethylation and 40% dimethylation; see Fig. 2C). Phosphorylation on Ser1 was investigated by MS analysis of Glu-C peptide 1–24 (Fig. 3). The ions at m/z [525.91]5+ and m/z [534.31]5+ were prominent in G2/M phase and barely detected in G1 and S. These ions were identified as peptides containing phosphorylation on Ser1 in addition to the methylation and acetylation already described for peptides 4–17 and 20–35 (Fig. 3 and Table II). All MS/MS spectra of peptides listed in Table II are shown in the supplemental data.


Figure 2
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  		FIG. 2. Analysis of H4 peptides during cell cycle. A, nano-ESI MS spectra of endoproteinase Arg-C peptide 4–17 of H4 purified from HeLa cells arrested in G1, S, or G2/M phases. The ions at m/z 424.27, 438.27, 452.26, and 466.27 were identified as [M + 3H]3+ of unmodified, mono-, di-, and triacetylated peptides. MS/MS analysis identified Lys5, Lys8, Lys12, and Lys16 as acetylated sites. B, MS quantitation of unmodified, mono-, and diacetylated peptides 4–17 in G2/M compared with S phase. C, MS quantitation of unmodified, mono-, and dimethylated peptides 20–35 in G2/M compared with S phase. ac, acetyl group; me, methyl group.

 

Figure 3
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  		FIG. 3. Analysis of H4 peptides 1–24 during cell cycle. Nano-ESI MS spectra of endoproteinase Glu-C peptide 1–24 H4 purified from HeLa cells arrested in G1, S, or G2/M phases are shown. The ions at m/z 504.30, 512.70, 521.11, and 529.51 were identified as [M + 5H]5+ of unmodified, mono-, di-, and triacetylated peptides. The ions at m/z 507.11 and 515.51 were identified as [M + 5H]5+ of monomethylated peptides with zero or one acetyl group, respectively. The ions at m/z 509.91, 518.31, 526.71, and 535.11 were identified as [M + 5H]5+ of dimethylated peptide with zero, one, two, or three acetyl groups, respectively. In G2/M phase, the ions at m/z 525.91 and 534.31 were identified as [M + 5H]5+ of dimethylated and monophosphorylated peptides with zero or one acetyl group. MS/MS analysis identified Lys5, Lys8, Lys12, and Lys16 as acetylated sites, Lys20 as mono- and dimethylated sites, and Ser1 as the phosphorylated site. ac, acetyl group; ph, phosphoryl group; me, methyl group.

 
Histone H3 Variants—
Mammalian cells contain three different histone H3 protein variants (H3.1, H3.2, and H3.3) that are almost identical in their amino acid sequence. Although the H3.1 and H3.2 variants differ by only one amino acid at position 96, H3.1 and H3.3 variants differ at positions 31, 87, 89, 90, and 96. Deconvoluted mass spectra of the three last peaks of the histone HPLC chromatogram were attributed to full intact H3 protein modified with several post-translational modifications (Supplemental Fig. 1) (40). However, the change in post-translational modifications during the cell cycle was difficult to observe from mass spectra of intact histone H3 and its variants due to the complexity of the spectra. Also assignment of the variants was only possible by peptide mapping of the three HPLC peaks. Analysis by MS/MS identified the specific Arg-C peptides 84–116 from H3.1, H3.2, and H3.3 and the specific peptides 27–40 for H3.1 or H3.2 and H3.3 (Supplemental Table I). This enabled attribution of the histone H3.2, H3.3, and H3.1 variants to the peaks 1, 2, and 3 of the HPLC chromatogram. The H3 modified peptides were analyzed by MS and MS/MS for the three variants at G1, S, and G2/M phases. For the SILAC H3 experiments, quantitation was performed on a mixture of the three variants.

Histone H3 Acetylation on Lys18 and Lys23 Was Reduced in G2/M Phase—
The peptides 18–26 were found to be mono- and diacetylated on Lys18 and/or Lys23 on all three variants. Ions corresponding to mono- and diacetylated peptides were intense in the spectrum, whereas ions corresponding to non-modified peptides were detected by MS/MS but difficult to discriminate from contaminating ions in MS experiments (Table III and Supplemental Fig. 8). Based on the MS analysis, slight intensity changes for the ions corresponding to the diacetylated peptides were observed between the three phases for the three variants. SILAC experiments confirmed a decrease in Lys18 and Lys23 acetylation for mono- and diacetylated peptides of 30 and 55%, respectively (Fig. 4, A and B) in G2/M phase. All MS/MS spectra of peptides 18–26 listed in Table III are shown in the supplemental data.


Figure 4
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  		FIG. 4. Analysis of H3 peptides 18–26 during cell cycle. A, MS quantitation of mono- and diacetylated peptides 18–26 in G2/M compared with S phase. The quantitation values represent the average of the three H3 variants. MS/MS analysis identified Lys18 and Lys23 as acetylated sites. B, SILAC MS spectrum of peptide 18–26. For the S phase experiment, the ions at m/z 514.82 and 585.82 were identified as [M + 2H]2+ of mono- and diacetylated [12C6]Arg-labeled peptides. For G2/M experiment, the ions at m/z 517.83 and 538.83 were identified as [M + 2H]2+ of mono- and diacetylated [13C6]Arg-labeled peptides. ac, acetyl group.

 
Histone H3: Ser10 Was Phosphorylated in G2/M Phase for the Three H3 Variants—
MS analysis of peptide 9–17 revealed six peptide ions with mass shifts corresponding to methylations (14 Da) or acetylation (42 Da) (Fig. 5, Table III, and Supplemental Fig. 9, A and B). Unexpectedly the endoproteinase Arg-C cleaved after Lys36 to generate peptide 27–36. MS/MS analysis of ions corresponding to modified peptides 9–17 identified the presence of contaminating ions corresponding to modified peptides 27–36. The modified peptides 9–17 and 27–36 had identical masses for the H3.1 and H3.2 variants, whereas for the H3.3 variant, mass shifts of 2 Da were observed for peptides 27–36 due to variation on H3.3 at residue 31 (Supplemental Fig. 9B). For peptide 27–36, mono-, di-, and trimethylations were observed on Lys27 and Lys36 for the three cell cycle phases. These modifications were further described in the analysis of peptide 27–40. For H3 variants in the three cell cycle phases, mono- and dimethylations were identified on Lys9 and Lys14 (Table III). However, trimethylation and acetylation were preferentially detected on Lys9 and Lys14, respectively (Table III). MS/MS analysis with high mass accuracy allowed us to differentiate between acetyllysine and trimethyllysine. In this study, we used an electrospray ionization tandem MS instrument (hybrid quadrupole time-of-flight mass spectrometer) with a mass resolution around 10,000 (full width at half-maximum) and a mass accuracy of at least 0.02 Da with external calibration. To differentiate between acetylation (mass increment, 42.0106 Da) and trimethylation (mass increment, 42.0470 Da) of a modified lysine, we compared measured and calculated mass differences (in ppm) between two specific fragment ions from MS/MS spectra of the modified peptides 9–17 (Supplemental Tables II, III, and IV).


Figure 5
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  		FIG. 5. Analysis of H3.1 peptides 9–17 during cell cycle. Nano-ESI MS spectra of endoproteinase Arg-C H3.1 peptide 9–17 purified from HeLa cells arrested in G1, S, or G2/M phases are shown. The ions at m/z 458.28, 465.27, 472.29, 479.29, and 486.29 were identified as [M + 2H]2+ of mono-, di-, tri-, tetra-, and pentamethylated peptides. Also the ions at m/z 472.29, 479.29, 486.29, and 493.29 were identified as monoacetylated peptide with zero, one, two, or three methyl groups, respectively. Exclusively in G2/M phase, the ions at m/z 498.28, 505.28, 512.28, 519.28, and 526.29 were identified as [M + 2H]2+ of monophosphorylated peptide with one, two, three, four, or five methyl groups, respectively. Also the ions at m/z 512.28, 519.28, and 526.29 were identified as [M + 2H]2+ of monoacetylated/trimethylation and monophosphorylated peptide with in addition zero, one, or two methyl groups, respectively. MS/MS analysis identified Lys9 as mono-, di-, and trimethylated sites; Lys14 as mono-, di-, and trimethylated/acetylated sites; and Ser10 as the phosphorylated site. The asterisks indicate peptides with a different charge state unrelated to peptide 9–17 of histone H3. ac, acetyl group; ph, phosphoryl group; me, methyl group.

 
Specifically for the G2/M phase, five additional peptide ions were detected and identified as phosphorylated peptides 9–17 for each of the H3 variants (Fig. 5 and Supplemental Fig. 9, A and B). Also the presence of contaminating ions corresponding to phosphorylated modified peptides 27–36 was detected. For H3.1 and H3.2, MS/MS analysis of peptides 9–17 identified phosphorylation on Ser10 in addition to methylation of Lys9 and methylation or acetylation of Lys14 (Table III). Also due to the low amount of H3.3 variant protein, MS/MS analysis of the corresponding 9–17 phosphopeptides in G2/M phase was difficult to perform. SILAC quantitations were not performed for the ions corresponding to modified peptides 9–17 due to contamination by peptides 27–36. All MS/MS spectra of peptides 9–17 listed in Table III are shown in the supplemental data.

Histone H3: Ser10 and Thr3 Phosphorylation Was Only Detected in G2/M—
To cover the first eight amino acids of H3, digestion with endoproteinase Glu-C was necessary. Unexpectedly this protease cleaved histone H3 after Gln residues and generated peptides 1–19. Analysis of peptides 1–19 could only be reported for the H3.2 variant (Fig. 6A and Table III). The modification patterns of peptide 1–19 were very similar for G1 and S phases, consisting of four peptide ions with mass increments corresponding to methylation (14 Da) and/or acetylation (42 Da). In G2/M, we observed peptide ions with mass increments corresponding to one or two phosphorylations in addition to methylation and/or acetylation. The MS/MS analyses of these modified peptides were very complicated due to the presence of several modifications on peptide 1–19 (Lys9, Ser10, Lys14, and Lys18). We concentrated our analysis on the first residues of peptide 1–19 because sequence 9–19 had been covered already by the analysis of peptides 9–17 and 18–26. In the MS/MS spectrum, the predominant triply charged y14 ions and singly charged b2 ions indicated the presence of modifications on the N-terminal part of peptide 1–19. In highly methylated peptides (m/z [431.66]5+ in G1 and S phases) and phosphopeptides (m/z [447.66]5+ in G2/M phase), multiple methylations were detected between amino acids 1 and 5 indicating methylation of Arg2 and Lys4. In G2/M, monophosphorylated peptides were identified with phosphates on either Thr3 or Ser10 in addition to methylation or acetylation of Arg2, Lys4, Lys9, Lys14, and Lys18. Also doubly phosphorylated peptide ions were detected at m/z [452.45]5+ and m/z [455.25]5+ with phosphorylated Ser10 and Thr3 (Fig. 6B). SILAC quantitation was difficult to perform due to the low intensity of the peptide ions in S and G2/M phases. However, in addition to MS analysis, MS/MS analysis of the selection ion with the hypothetical parent masses could clearly confirm the absence of phosphorylated peptides in G1 and S phases, indicating the exclusive presence of phosphopeptides in the mitotic phase. MS/MS spectra of peptides 1–19 at m/z [431.67]5+, [447.66]5+, and [455.25]5+ are shown in the supplemental data.


Figure 6
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  		FIG. 6. Analysis of H3.2 peptides 1–19 during cell cycle and localization of Thr3 and Ser10 phosphorylation sites. A, nano-ESI MS spectra of endoproteinase Glu-C H3.2 peptide 1–19 purified from HeLa cells arrested in G1, S, or G2/M phases. In G1 and S phases, the ions at m/z 423.27, 426.08, 428.88, and 431.68 were assigned to modified peptides with three, four, five, or six methyl groups, respectively. Also these ions were assigned to modified peptides with one acetyl group and zero, one, two, or three methyl groups, respectively; and the ion at 431.68 was assigned as diacetylated peptide. Exclusively in G2/M phase, the ions at m/z 433.65, 436.46, 439.25, 442.02, 444.86, and 447.66 were assigned to monophosphorylated peptides with one, two, three, four, five, or six methyl groups, respectively. Also the ions at m/z 439.25, 442.02, 444.86, and 447.66 were assigned to monoacetylated and monophosphorylated peptide with zero, one, two, or three methyl groups, respectively. The ion at m/z 447.66 was assigned to diacetylated and monophosphorylated peptide. The ions at 452.45 and 455.25 were assigned to diphosphorylated and di- or trimethylated peptides, respectively; and the ion at 455.25 was assigned to diphosphorylated and monoacetylated peptide. MS/MS analysis identified methylation on Arg2 and Lys4 and phosphorylation on Ser10 and Thr3. The asterisks indicate peptides with a different charge state unrelated to peptide 1–19 of histone H3. B, the ion at m/z 452.44 corresponding to [M + 5H]5+ of diphosphorylated and dimethylated peptide 1–19 (molecular mass, 2257.21 Da) of histone H3.2 was subjected to fragmentation. The y14*3+ and {Delta}y14*3+ ions at m/z 531.96 and 499.30 indicated the presence of phosphorylation and dimethylation in positions 6–19 and therefore the presence of one phosphorylation on Thr3. The b3*1+, {Delta}b3*1+, b5*1+, {Delta}b5*1+, and {Delta}b5*2+ ions at m/z 409.23, 311.18, 437.27, 439.80, and 284.18 confirmed the presence of phosphorylation on Thr3. The y13*3+ ions at m/z 498.27 excluded the presence of phosphorylation on Thr6. The {Delta}y10*2+ ions at 506.30 indicated the presence of phosphorylation in positions 10–19. The {Delta}{Delta}b10**3+ ions at m/z 374.23 confirmed the identification of Ser10 phosphorylation and Lys9 dimethylation. The {Delta}b18**4+ and {Delta}{Delta}b18**4+ ions at m/z 504.29 and 479.80 and the b14**3+, {Delta}b14**3+, and {Delta}{Delta}b14**3+ ions at m/z 553.93, 521.29, and 488.62 confirmed the presence of two phosphorylations and dimethylation in positions 1–14. The y4 ions at m/z 528.34 confirmed that amino acids between positions 16 and 19 are not modified. Phosphorylated fragment ions are indicated by asterisks, and ions that underwent neutral loss of phosphoric acid are labeled by {Delta}. ac, acetyl group; ph, phosphoryl group; me, methyl group.

 
Histone H3: at G2/M Lys27 and Lys36 Methylation Were Decreased whereas Ser28 as Well as H3.3 Ser31 Were Phosphorylated—
For the three variants, MS analysis of peptide 27–40 revealed six peptide ions with mass shifts corresponding to methylations (Fig. 7A and Supplemental Fig. 10, A and B). Mono-, di-, and trimethylation of Lys27 and/or Lys36 was identified in all three phases (Table IV). In the MS analysis of the three variants, the ion intensities of methylated peptides in G1 and S phases were similar, whereas those of mono-, di-, and trimethylated peptides increased and those of tetra- and pentamethylated peptides decreased in the G2/M phase. To confirm this change in methylation, quantitative MS analysis was performed using the SILAC approach. Increased ion intensities for di- and trimethylated peptides 27–40 were observed (35 and 17%, respectively), whereas ion intensities of tetra- and pentamethylated peptides were decreased in G2/M phase (50 and 35%, respectively; see Fig. 7, B and C). These results indicated a decrease of Lys27 and Lys36 methylation in G2/M phase. The comparison of the fragment ion intensities in the MS/MS spectra of di-, tri-, tetra-, and pentamethylated peptides 27–40 for the different cell cycle phases did not indicate which lysine residue (Lys27 or/and Lys36) was preferentially affected (Supplemental Figs. 75, 77, 79, and 81).


Figure 7
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  		FIG. 7. Analysis of H3.1 peptides 27–40 during cell cycle. A, nano-ESI MS spectra of endoproteinase Arg-C H3.1 peptide 27–40 purified from HeLa cells arrested in G1, S, or G2/M phases. The ions at m/z 359.25, 362.75, 366.25, 369.75, 373.25, and 376.75 were identified as [M + 4H]4+ of non-modified, mono-, di-, tri-, tetra-, and pentamethylated peptides. Exclusively in G2/M phase, the ions at m/z 386.25 and 389.75 were identified as [M + 4H]4+ of monophosphorylated peptide with two or three methyl groups, respectively. MS/MS analysis identified Lys27 and Lys36 as mono-, di-, and trimethylated sites and Ser28 as the phosphorylated site. The asterisks indicate peptides with a different charge state unrelated to peptide 27–40 of histone H3.1. B, MS quantitation of unmodified, mono-, di-, tri-, tetra-, and pentamethylated peptides 27–40 in G2/M compared with S phase. The quantitation values represent the average of H3 variants. C, SILAC MS spectrum of peptide 27–40. For the S phase experiment, the ions at m/z 379.22, 362.73, 366.23, 369.73, 373.23, and 376.74 were identified as [M + 4H]4+ of mono-, di-, tri-, tetra-, and pentamethylated [12C6]Arg-labeled 27–40 peptides. For the G2/M experiment, the ions at m/z 360.73, 364.23, 367.73, 371.24, 374.74, and 378.28 were identified as [M + 4H]4+ of mono-, di-, tri-, tetra-, and pentamethylated [13C6]Arg-labeled 27–40 peptides. ph, phosphoryl group; me, methyl group.

 
For H3.1 and H3.2 variants, two ions at m/z [386.25]4+ and m/z [389.75]4+ were exclusively detected in G2/M phase (Fig. 7A and Supplemental Fig. 10A), and Ser28 was found to be phosphorylated in addition to mono-, di-, and trimethylation on Lys27 and/or Lys36 (Table IV). Also for histone H3.3 containing a Ser in position 31, phosphopeptides were detected exclusively in G2/M phase (Supplemental Fig. 10B). Phosphorylation was identified, in addition to methylation on Lys27 and/or Lys36, either at Ser28 as previously found in H3.1 and H3.2 variants or at Ser31, the specific amino acid found only on the H3.3 sequence (Table IV and Supplemental Fig. 10C). All MS/MS spectra of peptides 27–40 listed in Table IV are shown in the supplemental data.

Histone H3: Methylation of Lys79 Does Not Change during the Cell Cycle—
Finally analyses of peptides 73–83 for all three histone H3 variants were performed. Mono- and dimethylation were identified on Lys79 (Table IV) and quantified by SILAC. No change in methylation was observed (Supplemental Fig. 11). All MS/MS spectra of peptides 73–83 listed in Table IV are shown in the supplemental data.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Elucidation of the modification patterns of histones that occur at a specific stage of the cell cycle has been a field of research for many years, and advances in analytical techniques have brought stepwise progress in these efforts. Here we present results of our approach, which introduces the combination of SILAC/mass spectrometry to the histone field. As an alternative method to quantify changes in histone modifications, LC-MS/MS has been suggested (31). Recently Beck et al. (41) studied relative levels of acetylation and methylation under the influence of a histone deacetylase inhibitor (PXD101) by such an approach. This method based on non-isotope labeling quantitation had to address the issues of uneven ionization efficiencies, differences in sample loading, and discrepancies in chromatographic pattern by using internal standard peptides across LC-MS/MS experiments for data normalization. Although this method then becomes applicable to complex samples like tissues or blood, the SILAC approach has in many studies shown its accuracy, reliability, and ease of use in a cell-based system (34, 35). Because our aim was solely the analysis of dynamic changes of histone modifications in HeLa cells, a combination of SILAC and nanospray MS analysis was the most direct and reliable strategy.

We aimed at providing an extensive analysis of the modifications that occur on the four core histones (H2A, H2B, H3, and H4) during the cell cycle. Our MS experiments allowed recognition of modifications (acetylation, methylation, or phosphorylation) that coexist on the same histone. For example, on H4, all combinations of acetylation, methylation, and phosphorylation were found. Peak intensities of MS spectra gave information about the extent of modification. Based on MS experiments with non-labeled cells, the modifications in the G1 and S phases were largely identical for all core histones but underwent drastic changes during mitosis. This allowed us to limit the SILAC experiments to a comparison between S and G2/M phases. The SILAC data yielded quantitative information about relative changes of particular modified peptides (but not about the extent of modification). Cell cycle-dependent H2A and H2B modifications, which have not been investigated previously, showed a decrease in acetylation of H2A Lys5 and H2B Lys12, Lys15, and Lys20 in G2/M. Acetylation of the H3 peptide containing Lys18 and Lys23 was also decreased in mitotic cells, and acetylation was similarly decreased on H4 peptide carrying Lys5, Lys8, Lys12, and Lys16. The presence of phosphorylation on H4 Ser1, H3 Thr3, H3 Ser10 and Ser28, and H3.3 Ser31 in G2/M phase was also observed. In contrast to H3 and H4, phosphorylation on H2A Ser1 remained unchanged during cell cycle. H4 Lys20 methylation was increased in the G2/M phase by a factor of 2.5 for monomethylated H4 Lys20. However, the complex methylation of Lys27 and Lys36 on non-phosphorylated H3 was decreased and most evident by a 50% reduction of the tetramethylated and a 35% reduction of the pentamethylated peptide in the mitotic phase. Finally methylation on Lys79 did not change during cell cycle. The individual modifications and cycle-dependent alterations are in general agreement with the literature. Other reported types of modification such as ubiquitination or sumoylation were not found in this study most likely because of their low abundance.

With respect to acetylation, it is generally accepted that changes in histone acetylation levels are associated with changes in chromatin structure (16, 17). Thus, deacetylation leads to repression of transcription and is likely required for correct packaging of nucleosomes into metaphase chromosomes. In vitro, multiple acetylations on histone tails were shown to destabilize the chromatin fiber and to decondense chromatin fibers (42). This phenomenon has recently been traced down to be due to the acetylation of histone H4 Lys16 (43). Also several studies with acetylation-specific antibodies have shown deacetylation of histones H3 and H4 in the mitotic phase (29, 44, 45). In our study, we individually identified the G2/M phase decrease in acetylation of all acetylated sites for individual core histones (Fig. 8). Our findings corroborate the findings made earlier exclusively by antibodies. In addition, we also quantified acetylated peptide ratios for H3 and H4.


Figure 8
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  		FIG. 8. Summary of results found by SILAC and MS. Methyl, phosphoryl, and acetyl groups are represented by me, ph, and ac, respectively. Trapezoid-shaped boxes indicate differences between S and G2/M phase. The boxes widening toward the bottom represent a decrease of the modification during G2/M and vice versa. The triangles indicate the exclusive presence of phosphorylations in G2/M phase. Modifications without boxes indicate the lack of cell cycle-dependent change and questionable cases; see "Discussion" for details. X represents residues that vary in the H2A and H2B sequences.

 
Phosphorylation of histone proteins is known to be important for proper segregation and condensation of chromosomes during mitosis; mechanistic roles of phosphorylation in this process are beginning to evolve (8, 9, 46). In our study, phosphorylation of histone H4 and multiple phosphorylation sites on the H3 variants were exclusively detected in G2/M phase. By contrast, phosphorylation of histone H2A did not change between G1, S, and G2/M phases. Largely in agreement with our results, Garcia et al. (47) have recently identified H3 phosphorylation sites by MS on mitotically arrested HeLa cells (Ser10, Ser28, and Thr3 for the H3.1, H3.2, and H3.3 variants and H3.3 Ser31). In their study, all N termini of the peptides were acylated by propionylation and all acidic residues were converted to methyl esters, and the samples were enriched for phosphopeptides using IMAC prior to tandem mass spectrometry. Neither derivatization nor enrichment steps were needed to identify phosphopeptides in our experiments. Other previous studies using phosphospecific antibodies have also found the phosphorylation sites on the H3 histones described above in mitotic phase (10, 11, 13, 14). Phosphorylation on Thr11 is an additional modification occurring at mitosis that has been found by immunoblotting and confocal immunofluorescence analyses (12). Thr11 phosphorylation was not detected with either our MS method or with that of Garcia et al. (47). Because this modification is limited to centromeres it is likely that the amounts of Thr11 phosphorylation are insufficient for detection by MS. Concerning histones H4 and H2A, a high level of phosphorylation on both Ser1 residues could be observed by phosphospecific antibodies during mitosis, whereas a low level of phosphorylation was detected in asynchronized HeLa cells (15). The results obtained for Ser1 H4 are in accordance with our H4 MS study. Also MS showed that phosphorylation occurred in all combinations of methylation (Lys20) and acetylation (Lys5, Lys8, Lys12, and Lys16). Phosphorylation on Ser1 of H2A, which contains a sequence with high similarity to that of H4, was not changing during the cell cycle. In addition to this result, we have identified the H2A Ser1 phosphorylation on asynchronized HeLa and Jurkat cells (data not shown and Ref. 40). However, H2A Ser1 phosphorylation was not detected using specific antibody on asynchronized HeLa cells compared with mitotic arrested cells (15).

Methylation has been observed so far on six lysine residues of H3 and H4 histones. Recently methylation of H4 Lys20 was described to increase specifically during mitosis, consistent with an increase in histone H4 Lys20 methyltransferase (PR-set7) activity during G2/M phase (48, 49). Our findings corroborate the findings made earlier. In addition, we also quantified the increase of Lys20 methylation to 2.5-fold for monomethylation and 1.5-fold for dimethylation. Here the high methylation on Lys20 was found to be inversely correlated with deacetylation on H4 Lys5, Lys8, Lys12, and Lys16 in mitotic phase indicating that these modifications have antagonizing functions. We can postulate that Lys20 methylation serves as a recognition motif for the binding of chromatin-associated proteins that are involved in higher order chromatin structure during mitosis.

To date, there has been no study examining the cell cycle regulation of histone H3 Lys27 and Lys36 methylation levels. Here independently of phosphorylation, methylation of H3 Lys27 and H3 Lys36 were decreasing in the mitotic phase compared with S phase. The methylation of H3 on Lys36 is known to be associated with transcriptional activity. Indeed the enzyme responsible for its modification has been shown to physically associate with RNA polymerase II during elongation (25). Recently JHDM1 protein was identified to specifically demethylate histone H3 at Lys36 in HeLa cells (50). Thus the JHDM1 protein is probably activated in G2/M phase, therefore playing a specific role during mitosis. On the other hand, methylation of H3 Lys27 was thought to be a marker for repressive chromatin (22, 23). In our study, methylation on Lys27 was unexpectedly decreasing in mitosis. It has been recently shown that the location of the Ser10 residue in close proximity to Lys9 enables the possibility that phosphorylation on Ser10 influences Lys9 methylation in G2/M phase (46, 51). Indeed phosphorylation on Ser28 can also affect methylation on Lys27 in G2/M phase.

For the first time, methylation on H3 Arg2 was identified by mass spectrometry in G1, S, and G2/M phases of HeLa cells (19). This site is known to be modified by the protein-arginine methyltransferase CARM1, which has been linked to active transcription (52). Also methylation on H3 Lys4, known to be associated with gene activity (24), was identified in the three phases. However, our method failed to indicate any change in methylation on Arg2 and Lys4 during the cell cycle. As previously discussed, Arg2 and Lys4 methylation may also be affected during mitosis when Thr3 is phosphorylated.

Recently it has been found that phosphorylation of H3 Ser10 by aurora B during mitosis required the previous deacetylation of H3 histones by histone deacetylase 3 (53, 54). As for acetylation, changes in methylation occurring on residues nearby phosphorylation sites may also be required before kinase phosphorylation. Indeed methyltransferases or demethylases involved in regulation of methylation on H3 and H4 should also play important mechanistic roles in mitosis (55).


  FOOTNOTES
 
Received, February 15, 2007, and in revised form, June 22, 2007.

Published, MCP Papers in Press, July 20, 2007, DOI 10.1074/mcp.M700070-MCP200

1 The abbreviation used is: SILAC, stable isotope labeling by amino acids in cell culture. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} To whom correspondence should be addressed: Novartis Insts. for Biomedical Research, WSJ-88.706, Lichtstrasse 35, CH-4056 Basel, Switzerland. Tel.: 41-61-32-44-175; Fax: 41-61-32-44-331; E-mail: debora.bonenfant{at}novartis.com


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