Quantitative Proteomics Reveals Histone Modifications in Crosstalk with H3 Lysine 27 Methylation*

Methylation at histone H3 lysine 27 (H3K27me) is an evolutionarily conserved epigenetic mark associated with transcriptional repression and replication elongation. We have previously shown that in Tetrahymena thermophila, a unicellular eukaryote, the histone methyltransferases (HMTs) TXR1 and EZL2 are primarily responsible for H3K27 mono-methylation (H3K27me1) and di-/tri-methylation (H3K27me2/3), respectively. Using 15N metabolically labeled histones as the internal reference, we quantified global changes in histone post-translational modifications in ΔTXR1 and ΔEZL2 cells, to systematically identify potential crosstalk between H3K27 methylation and other PTMs across all four core histones as well as their variants. Most prominently, we observed hyper-acetylation of histones H2A, H2A.Z, and H4 in their N-terminal domains in response to decreased H3K27 methylation. We also provide additional evidence implicating hyper-acetylation in the DNA damage response pathway in replication-defective ΔTXR1 cells, in apparent contrast to the transcriptional role of hyper-acetylation in ΔEZL2 cells.

Methylation at histone H3 lysine 27 (H3K27me) is an evolutionarily conserved epigenetic mark associated with transcriptional repression and replication elongation. We have previously shown that in Tetrahymena thermophila, a unicellular eukaryote, the histone methyltransferases (HMTs) TXR1 and EZL2 are primarily responsible for H3K27 mono-methylation (H3K27me1) and di-/tri-methylation (H3K27me2/3), respectively. Using 15 N metabolically labeled histones as the internal reference, we quantified global changes in histone posttranslational modifications in ⌬TXR1 and ⌬EZL2 cells, to systematically identify potential crosstalk between H3K27 methylation and other PTMs across all four core histones as well as their variants. Most prominently, we observed hyper-acetylation of histones H2A, H2A.Z, and H4 in their N-terminal domains in response to decreased H3K27 methylation. We also provide additional evidence implicating hyper-acetylation in the DNA damage response pathway in replication-defective ⌬TXR1 cells, in apparent contrast to the transcriptional role of hyperacetylation in ⌬EZL2 cells. Molecular & Cellular Proteomics 13 In eukaryotic cells, nuclear DNA wraps around an octamer of two copies each of the four core histones H2A, H2B, H3, and H4, to form the nucleosome, the basic unit of chromatin. Many post-translational modifications (PTMs) 1 , including methylation, acetylation, phosphorylation, biotinylation, citrullination, ADP-ribosylation, and ubiquitylation, occur at numerous sites on histones (1). Dynamic changes in the chromatin structure and architecture, including the switch between condensed and decondensed states as well as interactions with a wide range of protein complexes, are modulated by these PTMs, deposited by histone modifying enzymes in a combinatorial pattern that is still being actively deciphered (2,3). Reflecting its crucial role in DNA-mediated transactions, the functionality of chromatin is relatively robust, tolerating substantial mutations in histone residues carrying PTMs as well as histone modifying enyzmes (4). The robustness is generally attributed to redundant or parallel roles played by many histone modifications, which often adapt compensatory changes to these perturbations. However, details of the crosstalk among histone PTMs and their physiological impacts are still incomplete (5).
Deposited by histone methyltransferases (HMTs) and removed by demethylases, histone lysine methylation (mono-, di-, and tri-methylated forms) is a dynamic epigenetic mark (6 -8). Boosted by the identification and characterization of the enzymatic machinery, histone lysine methylation has been the target of considerable research efforts, reflecting its key role in modulating histone functions. Unlike acetylation, methylation does not directly affect histone/DNA interactions by altering the net charge of histones, and its physiological impacts depend on the exact form and site of modification, as well as the effector proteins recognizing them (9).
The evolutionarily conserved H3K27 methylation has been traditionally associated with heterochromatin formation and transcriptional repression (10). Enhancer of zeste (E(z)), initially identified by its role in Polycomb repression, is the HMT required for H3K27 methylation in Drosophila (11)(12)(13). E(z) homologs are found in protozoa, metazoa, and plants, though they are conspicuously missing in fungi. All three forms of H3K27 methylation (H3K27me1/2/3) are catalyzed by E(z) homologs in metazoa (14). However, in protozoa and plants, a different family of HMTs are specific for H3K27me1, examplified by Arabidopsis ATXR5/ATXR6 (15). As a protozoan model organism, Tetrahymena thermophila features high levels of H3K27 methylation and several H3K27-specific HMTs, including EZL1, EZL2, EZL3 (all E(z) homologs), and TXR1 (ATXR5/ATXR6 homolog) (16 -19). In vegetatively growing cells, only TXR1 and EZL2 are significantly expressed. Our previous studies show that they jointly regulate H3K27 methylation at this physiological stage: TXR1 preferentially deposits H3K27me1 and affects replication elongation, whereas EZL2 is mainly responsible for H3K27me2/3 and transcription repression (18). Although H3K27 methylation is extensively studied, its relationship with other histone PTMs has not been systematically explored. In this study, we used MS-based quantitative proteomics methods to compare the global histone PTM levels in wild-type (WT), ⌬TXR1, and ⌬EZL2 cells. We identified potential crosstalk between H3K27 methylation and other histone PTMs, including prominent hyper-acetylation of histones H2A, H2A.Z, and H4 in their N-terminal tails. We also propose that two different pathways underlie the hyper-acetylation events observed in ⌬TXR1 and ⌬EZL2 cells. The results provide further support for the functional differentiation between TXR1-dependent H3K27me1 and EZL2-dependent H3K27me2/3.

EXPERIMENTAL PROCEDURES
Construction of HMT Knockout Strains-HMT mutants, ⌬TXR1 and ⌬EZL2, were generated from Tetrahymena wild-type CU428 cells as described previously (18). Briefly, genomic sequences flanking TXR1 or EZL2 were PCR amplified and fused with the neo4 cassette, which confers paromomycin resistance (20). Transformants were generated by standard biolistic bombardment and selected by paromomycin (21,22). After phenotypic assortment, complete replacement of endogenous copies in macronuclei was verified by quantitative PCR.
Cell Culture, Core Histone Preparation, and HPLC Purification-Media, cell culture conditions, and procedures used for nuclear preparation, acid extraction of histone, and histone purification were as described previously (17,18). The overall experimental design is illustrated in supplemental Fig. S1. Briefly, Tetrahymena wild-type, ⌬TXR1, and ⌬EZL2 cells were grown in 1ϫ SPP medium at 30°C with gentle shaking. Logarithmic-phase (2 ϫ 10 5 /ml) cells were collected for subsequent experiments. To prepare the [ 15 N] histones as internal standards, wild-type Tetrahymena cells were metabolically labeled by feeding on Escherichia coli BL21 cells grown in the [ 15 N] M9 minimal medium supplemented with 15 N-substituted Bioexpress (Cambridge Isotope Laboratories, Andover, MA). To isolate Tetrahymena macronuclei, 2 ϫ 10 8 cells were collected by centrifugation, resuspended in 200 ml of medium A (0.1 M sucrose, 2 mM MgCl 2 , 4% gum arabic, 10 mM Tris, 5 mM EDTA, 10 mM butyric acid, and 1 mM PMSF, pH 6.75), and disrupted by vigorous blending in the presence of 1-octanol (ϳ1 ml); macronuclei were subsequently separated by differential centrifugation. The purified macronuclei (1 ϫ 10 8 ) were extracted in 1 ml of sulfuric acid (0.4 N) overnight. The acid-extracted histone samples were precipitated by 20% trichloroacetic acid, washed with acetone, and dissolved in 500 l of water. To further purify individual histones, the histone samples were resolved on a C8 reversed-phase high performance liquid chromatography (HPLC) column. HPLC fractions containing individual histones were combined after evaluation by SDS-PAGE and quantified by the Bradford method.
NanoLC-MS Analysis of Core Histone PTMs-Chemical derivatization, trypsinization, and nanoLC-MS/MS analysis of histone samples (in biological triplicates) were performed as previously described (17,23). Briefly, 3 g of each core histone from WT, ⌬TXR1, or ⌬EZL2 cells, were equally mixed with the internal standards, the [ 15 N] core histones prepared from WT cells as described above. Before mass spectrometry analysis, samples were chemically derivatized with propionic anhydride before and after trypsin digestion (23). All histones were in-solution digested with sequencing-grade trypsin and the fully propionylated peptide mixtures were vacuum-dried and dissolved in 0.1% formic acid before the LC-MS analysis. Acid extracted bulk histones (for H2A.Y identification) and histone H2A.Z (for N-terminal acetylation quantification) collected from HPLC were directly submitted to trypsin digestion without propionylation. The peptide mixtures were separated with a C18 analytical column (3 m, 300Å, 150 mm x 100 m; CVC Technologies, Fontana, CA), eluted, and introduced into a Thermo Fisher Orbitrap XL mass spectrometer with a 15 m PicoTip emitter (New Objective, Woburn, MA) at 200 nl/min flow rate. Mass spectrometer was connected with a manufacturer's nano-ESI source operated in both positive ion mode and data dependent mode at a resolution of 30,000 on MS1. The 8 strongest peaks were collision induced dissociation (CID) fragmented with 30% normalized collision energy. To achieve the best LC-MS/MS performance, a 2-min dynamic exclusion and internal mass lock were also executed.
Raw data processing, database searching, and peptide quantification were all done in Mascot Distiller (Matrix Sciences, Version 2.4 for Distiller and Version 2.2.07 for search engine). The NCBI Tetrahymena database (created from NCBI on 5/25/2010 with a total of 51,502 sequence entries) was used in performing the database searching. Precursor ion tolerance was 10 ppm and 0.8 Da for the fragment ions. N-terminal propionylation was set as fixed modification and the following PTMs were considered as variable modifications: Acetylation (Protein N terminus, K), Methylation (Protein N terminus, KR), Propionylation (K, unmodified or monomethylated), Phosphorylation (STY), Formylation (K), Citrullination (R), and Ubiquitination (K). Up to five missed cleavages were allowed for trypsin digestion as the propionylation and PTMs on lysyl residues blocks trypsin digestion. Several peptides from each of the core histones were selected for normalization by their average values: GKTASSKQVSR, GQASQDL, FLKHGR, AGLQFPVGR, HLLLAIR, ATIAGGGVIPHIHK from H2A and its variants; LLLPGELAR, IALESSKLVR, EVQTAVKLLLPGELAR from H2B; FRPGTVALR, VTIMTKDMQLAR, YQKSTDLLIR from H3; and SFLENVVR, QVLKSFLENVVR, TLYGFGG from H4. We assumed that the levels of those peptides were unchanged across different epigenetic features as their ratios (L/H) were close to 1 and quite consistent (coefficient of variation, CV Ͻ15%) in all experiments. Student t test was done in Microsoft Excel and heatmaps were generated from R software. All peptides with scores above mascot identity or homology threshold were considered as positive hits when a confidence threshold is set at p ϭ 0.05. Spectra assigned with PTMs were manually validated based on the same criteria we published previously (17). Our unambiguous assignment of PTM sites in the H2B N-terminal tail was facilitated by a previous MS study of Tetrahymena H2B (24).
Analysis of Acetylation States in Histone H2A and H4 by Acetic Acid-Urea Polyacrylamide Gel Electrophoresis-To analyze the acetylated isoforms of H2A and H4, HPLC-purified intact proteins were separated by continuous acetic acid-urea (AU) gel system as described (25). H2A and H4 samples prepared from WT and mutants were treated with protein phosphatase (New England Biolabs, Ipswich, MA), re-pelleted, and dissolved in 10 l of loading buffer containing 2 M urea, 2% ␤-mercaptoethanol, and 5% acetic acid, before they were loaded onto the gel. The gel was stained using the blue silver staining method for 30 min and visualized by destaining in deionized water for several times (26).
Chromatin Immunoprecipitation (ChIP)-Cells were fixed with 0.5% paraformaldehyde (PFA) and treated with micrococcal nuclease (MNase). Mono-nucleosomes were purified by sucrose gradient as described (27). ChIP was performed as described (16), using purified mono-nucleosome as input. The primary antibody (Penta, used at 1:500 dilution) has been validated to recognize acetylated-lysine in H2A.Z and H4 N-terminal tails (28). Salmon sperm DNA covered protein A beads (Millipore) were used to recover the immuno-complex.
Off-site Hyper-methylation Potentially Compensates H3K27 Methylation Deficiency in ⌬TXR1 Cells-H3K27 methylation was significantly affected in ⌬TXR1 and ⌬EZL2 cells, as previously reported (supplemental Table S2-H3) (17,18). In ⌬TXR1 cells, the mono-methylation of H3K27 and H3K36 were reduced at least 80%, whereas the unmethylated form of the peptide increased significantly. The dimethylated form was also significantly reduced, whereas the trimethylated form was only slightly affected. In ⌬EZL2 cells, H3K27me2/3 levels declined by at least 80%, whereas H3K27me1 levels increased, consistent with H3K27me1 being a feedstock for the HMT activity of EZL2. However, H3K27 methylation was not completely eliminated even in the ⌬TXR1/⌬EZL2 double knockout cells (supplemental Table S3-EZL2/TXR1), suggesting the presence of other HMTs with similar specificity but reduced efficiency. In contrast to the decreased levels of H3K27me1, increased off-site mono-methylation was observed for H3 at K4, K23, and particularly K56 in ⌬TXR1 (Table  S2-H3). Changes in lysine mono-methylation on histones other than H3 were less significant (p Ն 0.05, supplemental Table S2, H2A, H2B, H4). Also observed was increased arginine mono-methylation at H3R83 and possibly at H3R128 (p Ն 0.05). It is possible that hyper-methylation at these sites at least partially compensates for H3K27me1 deficiency in ⌬TXR1 cells.
Hyper-acetylation in Histone H2A, H2A.Z, and H4 in Response to H3K27 Methylation Deficiency-Controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs), histone acetylation contributes to a more open chromatin structure and recruits effector proteins leading to downstream events (29). Acetylation neutralizes the positively charged lysyl residues on histones and attenuates electrostatic interactions with the negatively charged DNA, facilitating chromatin decondensation. Acetyl-lysine is also recognized by bromodomains, which are ubiquitous in eukaryotes and widely distributed in pathways involving DNA transactions (30). Hyper-acetylation is traditionally linked to transcription activation (31,32), and more recently implicated in DNA repair (33).
Elevated acetylation levels were observed on many sites in histones from ⌬TXR1 and ⌬EZL2 cells (supplemental Table  S2). Hyper-acetylation of H2A (including the canonical H2A and the variant H2A.X, sharing the same N-terminal sequence), H2A.Z, and H4 in their N-terminal tails is the most prominent feature in the H3K27 methylation-deficient mutants (Figs. 1, 2, and 3). There are four acetylatable lysine residues in the N-terminal domains of H2A and H2A.X, which share the same amino acid sequence and are not distinguishable by MS. The presence of mono-, di-, tri-, and tetra-acetylated forms, with their various permutations, generated complicated tryptic peptides, which were resolved by MS (Fig. 1A,  1B). As determined from the quantitative proteomics results, the mutant/WT ratios of the acetylated forms were gradually increased from low to high degree of acetylation (Fig. 1A). The fully tetra-acetylated peptide showed a more than threefold increase in H2A from ⌬TXR1 and ⌬EZL2 cells (Fig. 1A). This increase in hyper-acetylated H2A was also clearly visualized by the acetic acid-urea gel electrophoresis (Fig. 4), which separates different protein isoforms by their net charges, directly reflecting acetylation levels in the absence of phosphorylation (on phosphatase treatment). Future analysis of separated H2A and H2A.X will address whether they have different patterns of N-terminal acetylation.
There are six acetylatable lysine residues in the N-terminal tail of H2A.Z. We observed a similar trend in the gradual buildup of the hyper-acetylated forms, culminating in dramatic accumulations of penta-and hexa-acetylated peptides in ⌬TXR1 and ⌬EZL2 cells ( Fig. 2A, 2B). There are four acetylatable lysine residues in the conserved N-terminal tail of H4 (K4, K7, K11, and K15, corresponding to K5, K8, K12, and K16 in higher eukaryotes). Accumulations of hyper-acetylated forms in the two mutants were observed, coupled to corresponding diminishment of unacetylated forms (Fig. 3A, 3B). In addition to hyper-acetylation of H2A, H2A.Z, and H4 in their N-terminal domains, K9ac, K14ac, K18ac, K23ac, K27ac, and K56ac levels were also moderately elevated in H3 (Tab. S2-H3). It is worth noting that ⌬TXR1 cells appeared to exhibit increased levels of hyper-acetylation over ⌬EZL2 cells for H4, and to a lesser degree, H2A.Z, whereas the two mutants were virtually indistinguishable in H2A hyper-acetylation levels.
H3K27 methylation, especially the di-and tri-forms, is generally associated with heterochromatin and hypo-acetylation. It is therefore not surprising to see increased acetylation levels in the H3K27me-deficient mutants, especially ⌬EZL2 cells. Nonetheless, we were intrigued by the specific hyper-acetylation of H2A, H2A.Z, and H4. The patterns of acetylation were very similar: an inverted gradient of accumulation tilting toward the highly acetylated forms rather than the less modified forms. This implies potential synergistic effects of lysine acetylation and a positive feedback mechanism. The readerwriter coupling, represented by the wide-spread association of bromodomains with HATs, may be a key contributing factor. In addition, genetic studies in yeast have demonstrated the hierarchical order of the lysine acetylation events on the conserved H4 N-terminal tail: relative to K5ac, K8ac, and K12ac, H4K16ac is especially important and has major effects on chromatin structure (34,35). Generally, our observations provide a more detailed picture of the combinatorial response. Importantly, we only detected H4K15ac (equivalent to yeast H4K16ac) in association with other acetylation events, further supporting that it plays a rate-limiting and possibly founding role in establishing hyper-acetylation. This hierarchical buildup of H4 hyper-acetylation may be physiologically significant, as the tetra-acetylated form of H4 is highly correlated with transcription and the most highly acetylated forms have the highest affinity for transcription factors (31,36).
It is intriguing that we only observed hyper-acetylation on H2A, H2A.Z, and H4, but not on H2B and H3. This substrate specificity suggests the involvement of NuA4-family HATs. Initially characterized in yeast, the NuA4 complex is evolutionarily conserved (37) and NuA4-like HAT activities have been reported in Tetrahymena (38). Furthermore, the Tetrahymena genome encodes several homologs of the MYST/MOZ family HAT, as well as homologs to key structural components of NuA4 complexes (data not shown). NuA4 has been implicated in transcription activation and DNA repair (37, 39 -41). The former is consistent with de-repression associated with inactivation of the Polycomb pathway, as in ⌬EZL2 cells, whereas the latter is consistent with the observed chromatin decon-densation associated with the DNA damage response to replication stress in ⌬TXR1 cells (18).
Other Histone Modifications Potentially in Crosstalk with H3K27 Methylation-All core histones and the linker histone H1 have been found to be phosphorylated across species and implicated in gene activation, chromosome condensation, DNA repair, apoptosis, among other cellular functions (42)(43)(44)(45)(46). Phosphorylation of H1, H2A, and H2A.Z from Tetrahymena macronuclei has been previously reported (47)(48)(49). In this study, we identified phosphorylation in H2A and H2B from Tetrahymena macronuclei without prior enrichment of phospho-proteins/peptides. H2A and H2A.X are the most highly phosphorylated core histones in Tetrahymena macronuclei (47). Mutagenesis studies have pinpointed phosphorylation events at the C-terminal tail of H2A.X, including constitutive phosphorylation (at S122, S124, T127, and S129 of H2A.X; the equivalent modifications in H2A are not studied) as well as DNA damage signaling (at S134 of H2A.X; a conserved phosphorylation mark generally referred to as ␥H2A.X) (49). However, we were unable to recover the corresponding phosphopeptides in this study, possibly because of their poor retention on RP-HPLC, very low levels not easily observed in unenriched samples, or ion suppression effects. Intriguingly, we found that the N-terminal domain of H2A was also phosphorylated, although we could not identify the specific site because of incomplete ion fragmentation and we could not quantify effectively because of sample-to-sample variation. Our finding is consistent with the identification of H2A serine 1 phosphorylation in mammalian cells, with implications in transcription, mitosis and S phase-associated events (50,51). We also identified H2B phosphorylation in the C-terminal tail, FIG. 4. Validation of histone hyper-acetylation by acid-urea gel electrophoresis. Increased levels of acetylation from low-degree to high-degree in H2A and H4 N-terminal domains were also demonstrated by acid-urea gel electrophoresis in the HMT mutants. Histone samples were treated with protein phosphatase to remove phosphorylations before loading onto the gel. Five micrograms of total protein (either H4 or H2A) were loaded onto a 15% acid urea gel and run at 100V for 30 min followed by 400V for 10 h. Note: ⌬E2ϩT1 is the double knockout strain of EZL2 and TXR1.
FIG . 5. Preferential H2A.Z and H4 hyper-acetylation of rDNA in ⌬TXR1 cells. Hyper-acetylation of H2A.Z and H4 was associated with the rDNA in ⌬TXR1 cells, demonstrated by cross-linking chromatin immuno-precipitation (X-ChIP) using antisera recognizing acetylated H4 and H2A.Z. ChIP samples were quantified by Q-PCR and normalized with input. The numbers on the x-axis denote the median positions of the PCR products. rDNA mini-chromosome sequence (corresponding to half of the palindrome, numbered from the center to one end) was retrieved from GenBank (accession No. X54512).
although again, we could not precisely localize the site (supplemental Fig. S4 and Table S2-H2B). This modification was significantly down-regulated (by as much as 80%) in the mutants in variant H2B.1. Mammalian H2B is phosphorylated at three sites (S113, T116, T120) near the C-terminal tail, but their biological functions remain unclear (52).
We also identified several formylation sites in H2A and H3, two of which (H2A K39, and H3 K23) were slightly elevated in the mutants (supplemental Table S2). N-formylation of lysine is a common but under-studied PTM in histones, high mobility group proteins, and other nonhistone proteins (53,54). Similar to acetylation in structure and chemical properties, formylation can potentially share some common functions with acety-lation and histone formylation has been implicated in the DNA damage response (53).
DNA Damage Response to Replication Stress May Reshape the Epigenetic Landscape in ⌬TXR1 Cells-In metazoa, homologs of Drosophila E(z) control all three forms of H3K27 methylation, contributing to the Polycomb repression pathway essential for Hox gene silencing and X-inactivation (55). In plants, H3K27me1 is largely delegated to homologs of Arabidopsis ATXR5 and ATXR6, which constitute another pathway involved in heterochromatin formation and DNA replication (15,56). The homologous HMTs and corresponding pathways responsible for H3K27 methylation have also been identified in the protozoan Tetrahymena (16 -19). As de- scribed in our previous publications (17,18), there is clear division of labor between TXR1-dependent H3K27me1 and EZL2-dependent H3K27me2/H3K27me3: the former is required for efficient replication elongation, whereas the latter is likely involved in transcriptional gene silencing. Here we focus on ⌬TXR1 cells, and explore how the epigenetic landscape is reshaped by replication stress and DNA damage response.
We have previously reported that ␥H2A.X (H2A.X S134 phosphorylation) is highly induced in ⌬TXR1 cells (18), as a sensitive indicator of replication stress (57). ␥H2A.X is also known as an upstream signal for the DNA damage response (DDR) (58). Recent studies in yeast and mammalian systems have demonstrated that the NuA4 HAT complex can be recruited to sites of DNA damage by ␥H2A.X (37, 39 -41). Further, NuA4 targets nucleosomal H2A, H2A.Z, and H4, with further enhanced HAT activities for the latter two, and modulates local chromatin structure and accessibility to the DNA repair machinery (37). Indeed, we observed a small but consistent increase in hyper-acetylation levels in H2A.Z and H4, but not H2A, in ⌬TXR1 over ⌬EZL2 cells. We also analyzed the genomic distribution of H2A.Z and H4 hyper-acetylation by ChIP, using a well-established antibody specific for acetylated lysyl residues in the N-terminal domains of H2A.Z and H4 (28). We focused on the acetylation levels in the rDNA mini-chromosome. In Tetrahymena, the genomic region containing the 35S pre-rRNA (transcribed by RNA polymerase I) is rearranged to form a palindromic dimer and the ϳ20 kb mini-chromosome is replicated from origins near the center (59,60). rDNA is under severe replication stress in ⌬TXR1 cells (18), but is not tran-scribed by the NuA4-coupled RNA polymerase II, allowing us to differentiate the DNA repair functionality of NuA4 from its transcriptional counterpart. Here we observed a significant enrichment of the acetylation signals in ⌬TXR1 cells, but not in WT and ⌬EZL2 cells (Fig. 5). This result is consistent with the general macronuclear chromatin decondensation and the specific nucleolar abnormality observed in ⌬TXR1 cells (18). In conclusion, we posit that the particular hyper-acetylation pattern in ⌬TXR1 cells can be attributed to ␥H2A.X-mediated recruitment of NuA4 to regions under replication stress.
We also quantified acid-extracted nuclear proteins (mostly histones) by MS. Intriguingly, we observed that H2A.Y, a recently identified H2A variant interacting with protein phosphatase 1 (PP1) (61), was highly induced only in ⌬TXR1 cells (Fig. 6A). The MS result is consistent with our microarray data, which showed a ninefold increase of H2A.Y mRNA levels in ⌬TXR1 cells (relative to WT or ⌬EZL2 cells) (Fig. 6B). Indeed, H2A.Y was present at very low levels in WT cells and could not be detected in our MS analysis, despite repeated trials. It is important to note that levels of all other histones, including H2A.Z, were not significantly changed in the mutants (data not shown), consistent with our microarray results (Fig. 6B). H2A.Y has been reported to regulate H3 S10 phosphorylation in mitotic micronuclei (a mark completely missing in the amitotic macronuclei in this study) (61). However, the macronuclear target(s) of H2A.Y, which most likely accounts for its essential function (61), remains elusive. Based on our results, we propose that H2A.Y plays a critical role in regulating levels of ␥H2A.X. It may also play a role in other phosphorylation FIG. 7. Epigenetic changes in ⌬TXR1 cells: response to replication stress. Multiple histone PTMs in crosstalk with H3K27 methylation, including hyper-acetylation (H2A, H2A.Z, and H4), hyper-methylation (particularly at H3K56), and ␥H2A.X (H2A.X S134 phosphorylation). They are potentially underlain by DNA damage response to replication stress in ⌬TXR1 cells, modulated by ATR, NuA4, and H2A.Y. ac: acetylation; me1: mono-methylation; ph: phosphorylation. See text for details. events associated with the DNA damage response (C.Z., P.C.A., and Y.L., unpublished results).
Conclusions and Implications-The highly conserved H3K27 methylation is intensively studied because of its longestablished biological significance in heterochromatin formation (55), with implications in Polycomb repression (55), long noncoding RNA (lncRNA) biogenesis (62,63), and DNA replication in heterochromatic regions (56). Its misregulation results in abnormal development and tumorigenesis (64,65). This work represents a systematic investigation of the global level crosstalk between H3K27 methylation and other histone PTMs. Despite the dramatic deficiency in H3K27 methylation in the two HMT deletion Tetrahymena mutants, viability is not affected, implying the existence of effective compensatory mechanisms. In the context of ⌬TXR1 cells, which are deficient in H3K27me1 and also defective in replication elongation, we want to re-emphasize several adaptations at the chromatin level (summarized in Fig. 7). First, there is increasing off-site lysine mono-methylation. Particularly interesting is H3K56me1, which has recently been shown to interact with PCNA and play a role in DNA replication (66). However, H3K56me1 in mammalian cells is reportedly catalyzed by G9a, which has no homolog in Tetrahymena. This may be another case for conserved histone PTMs with alternative modifying enzymes, to be sorted out in future studies. Second, the epigenetic landscape is reshaped by the DNA damage response. There is dramatic induction of ␥H2A.X (H2A.X S134 phosphorylation), which has been well-documented in our previous study (18), but was not detected by this MS study because of technical limitations. We also demonstrated H2A, H2A.Z, and H4 hyper-acetylation, which we infer is elicited by ␥H2A.X and mediated by NuA4-like HAT activities in Tetrahymena (38). In addition, H2A.Y is highly induced, potentially recruiting more protein phosphatase 1 (PP1) to chromatin (61). This may represent an effort to modulate levels of ␥H2A.X, as well as other phosphorylation events in the DNA damage response. In summary, our study has revealed possible mechanisms and potential pathways involved in crosstalk with H3K27 methylation, and raised more questions about the network relationships of histone PTMs. □ S This article contains supplemental Figs. S1 to S4 and Tables S1 to S3.
Data availability: The MS raw data can be downloaded from Pep-tideAtlas (http://www.peptideatlas.org/) with data set Identifier: PASS00209&Password: RH4425to, The microarray data can be downloaded from the National Center for Biotechnology Information (NCBI) GEO database with the accession number GSE44990.