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A phosphorylated subpopulation of the variant macroH2A1 is excluded from the inactive X and enriched during mitosis

Emily Bernstein*†, Tara L. Muratore-Schroeder‡, Robert L. Diaz*§, Jennifer C. Chow¶, Lakshmi N. Changolkarʈ, Jeffrey Shabanowitz‡, Edith Heard¶, John R. Pehrsonʈ, Donald F. Hunt‡**, and C. David Allis*††

*Laboratory of Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065; Departments of ‡Chemistry and **Pathology, University of Virginia, Charlottesville, VA 22901; ¶Curie Institute, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 218, 26 Rue d’Ulm, 75005 Paris, France; and ʈDepartment of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104

Contributed by C. David Allis, December 11, 2007 (sent for review November 26, 2007) Histone variants play an important role in numerous biological (5). Importantly, and unlike most other well studied variants, processes through changes in structure and stability mH2A is vertebrate-specific, consistent with the general view and possibly through mechanisms influenced by posttranslational that evolutionary expansion may correlate with increased needs modifications unique to a histone variant. The family of histone for functional specialization (6). Three isoforms exist in mam- H2A variants includes members such as H2A.Z, the DNA damage- mals: mH2A1.1, mH2A1.2, and mH2A2. The former two are associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body- alternatively spliced from the mH2A1 gene and differ only by deficient). Here, we have undertaken the challenge to decipher the one exon in the macro domain, whereas a second gene encodes posttranslational modification-mediated ‘‘histone code’’ of mH2A, mH2A2 (6, 7). All isoforms contain an N-terminal H2A-like a variant generally associated with certain forms of condensed region (65% homologous to H2A), a C-terminal macro domain, chromatin such as the inactive X chromosome in female mammals. and a short -rich hinge region that connects the two (5). By using female human cells as a source of mH2A, endogenous The functional differences between mH2A1.1 and 1.2 are un- mH2A was purified and analyzed by mass spectrometry. Although clear; however, only the macro domain of mH2A1.1 can bind mH2A is in low abundance compared with conventional , ADP-ribose (8). The in vivo consequences of this activity have we identified a site, S137ph, which resides within yet to be determined. the ‘‘hinge’’ region of mH2A. This lysine-rich hinge is an Ϸ30-aa mH2A has been shown to associate with different forms of stretch between the H2A and macro domains, proposed to bind condensed chromatin. In female mammals, for example, mH2A nucleic acids. A specific antibody to S137ph was raised; by using is preferentially concentrated on the inactive X chromosome this reagent, S137 phosphorylation was found to be present in (Xi), suggestive of a role in transcriptionally repressed chromatin both male and female cells and on both splice variants of the (9). During early mammalian development, one of two X

mH2A1 gene. Although mH2A is generally enriched on the inactive is silenced in females to achieve dosage compen- CELL BIOLOGY X chromosome in female cells, mH2AS137ph is excluded from this sation for X-linked gene products, and once inactivated, the heterochromatic structure. Thus, a phosphorylated subpopulation heterochromatic nature of the Xi is stably maintained (10). of mH2A appears to play a unique role in chromatin regulation Senescence-associated foci, specialized do- beyond X inactivation. We provide evidence that S137ph is en- mains of transcriptional repression, also contain mH2A (11). riched in mitosis, suggestive of a role in the regulation of mH2A Furthermore, mH2A has been associated with loci that are posttranslational modifications throughout the cell cycle. CpG-methylated, including imprinted loci and LTRs of Intra- cisternal A-particle retrotransposons (12, 13). Reconstituted macroH2A ͉ histone modifications ͉ phosphorylation mH2A-containing are resistant to ATP-dependent and factor binding in vitro istone variant proteins replace their conventional histone (14), and mH2A has recently been shown to repress RNA Hcounterparts within the chromatin template at specific polymerase II-driven transcription at the level of transcriptional genomic locations or during particular nuclear processes (1–4). initiation (15). When incorporated into chromatin, histone variants participate Histone tails and histone fold domains are rich in PTMs, in diverse nuclear functions, including centromeric regulation, including but not limited to phosphorylation, , ubiq- sensing DNA damage, and transcriptional activation and repres- uitylation, , and ADP-ribosylation. The ‘‘histone sion, and they potentially play a role in epigenetic inheritance of code’’ hypothesis has been proposed to help explain the intricate chromatin states (1–4). Histone variants also play an important patterns of PTMs and their biological outcomes (16–19). Phos- role in changing the structure and stability of the nucleosome (cis phorylation of proteins, in general, is thought to be important for mechanisms) and provide the cell with the potential to change its posttranslational modification (PTM) profile caused by amino acid sequence variations (4). In turn, differences in Author contributions: E.B., T.L.M.-S., J.S., D.F.H., and C.D.A. designed research; E.B., T.L.M.-S., R.L.D., J.C.C., and L.N.C. performed research; E.H. and J.R.P. contributed new reagents/analytic PTM-based signatures of variants may lead to the differential tools; E.B., T.L.M.-S., J.C.C., L.N.C., J.S., E.H., J.R.P., and D.F.H. analyzed data; and E.B. and C.D.A. engagement of downstream binding effectors (trans mecha- wrote the paper. nisms), significantly affecting the biological readout of particular The authors declare no conflict of interest. genomic regions (4). †To whom correspondence may be addressed at the present address: Department of Histone variants have been identified primarily from the H2A Oncological Sciences, Mount Sinai School of Medicine, One Gustave L. Levy Place, New and H3 families, although H2B variants also exist in mammals (3, York, NY 10029. E-mail: [email protected]. 4). Variants often differ from conventional histones by subtle §Present address: Rosetta Inpharmatics, 401 Terry Avenue N., Seattle, WA 98109. amino acid changes. However, one histone variant in particular, ††To whom correspondence may be addressed. E-mail: [email protected]. macroH2A (hereafter mH2A), contains a large non-histone This article contains supporting information online at www.pnas.org/cgi/content/full/ domain (the macro domain) on its C terminus, resulting in a 0711632105/DC1. histone approximately three times the size of conventional H2A © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711632105 PNAS ͉ February 5, 2008 ͉ vol. 105 ͉ no. 5 ͉ 1533–1538 Downloaded by guest on September 25, 2021 regulatory control of signaling networks and docking sites for binding proteins (20, 21). Histone phosphorylation, in particular, has long been believed to play a direct role in mitotic chromatin compaction or chromosome condensation (e.g., H1 linker phos- phorylation; H3S10ph and H3S28ph), although causality relationships remain unclear (22–24). Connection of histone phosphorylation to other physiologically relevant processes that lie outside of mitosis, such as apoptosis (H2BS14), DNA damage and repair (␥H2A.X), and inducible gene activation (H3S10), are also well documented (25–27). Histone variants allow variation in the composition of indi- vidual nucleosomes and also allow the cell to expand its PTM profile (4). Although histone variants play significant roles in chromatin regulation, little is known about the PTMs that associate with them. To begin to decipher the PTM signature of the mH2A, a combination of purification, mass spectrometry (MS), and immunological approaches was taken. Here, we report the identification of two phosphorylation sites on endog- enously purified mH2A, both of which lie within the basic hinge region of mH2A: T128ph and S137ph, and we discuss the biological implications of this phosphorylation, in particular, S137ph. Results mH2A Is Phosphorylated in Its Basic Hinge Domain. To enrich for endogenous mH2A, we used HEK293 cells, a female cell line that contains multiple inactive X chromosomes as a result of aneuploidy. Because mH2A is enriched on the Xi, we rational- ized that this cell line would be a potentially enriched source of this minor variant. Isolation of histone variants from endoge- nous, nonoverexpressed sources can be challenging because of their low quantity in chromatin (Ͻ10% of total histone pool). To this end, mH2A was acid-extracted along with other core histones from HEK293 cells under asynchronous growth condi- tions. Total acid-extracted histones were fractionated by reverse- phase HPLC (Fig. 1a), and fractions containing mH2A were determined by using an antibody against mH2A1, which recog- Fig. 1. Purification of mH2A for PTM analysis. (a) Reverse-phase HPLC nizes both mH2A1.1 and mH2A1.2 (Fig. 1b). mH2A1 elutes late chromatogram of acid-extracted histones purified from HEK293 cells. Core in the HPLC chromatogram and is significantly less abundant conventional histone and mH2A peaks are labeled. Below is a blowup of the than conventional histones (Fig. 1a). A slower-migrating, cross- mH2A-containing fractions. The y axis is UV214 absorbance, the x axis is minutes reacting band above the mH2A1 signal was repeatedly observed of collection, and the acetonitrile gradient is depicted across the top. (b) HPLC by immunoblotting (IB) and is consistent with the monoubiq- fractions were screened by using an mH2A antibody that recognizes mH2A1.1 uitylated form of mH2A given its size (an Ϸ8-kDa shift; Fig. 1b, and 1.2 (not mH2A2). Fractions at 83–86 min contained mH2A1 (see mH2A asterisk) (28, 29). Multiple HPLC runs were carried out, and peak in a, and compare peak size with core histones, e.g., H3 for relative abundance). Asterisk denotes a possible monoubiquitylated form of mH2A, peak fractions from all runs were pooled for MS; a small portion which elutes slightly earlier than nonubiquitylated mH2A. Size markers are of the total pool was examined by SDS/PAGE to determine the denoted on the left. (c) mH2A-containing fractions from multiple HPLC runs major constituents of this sample (Fig. 1c). MS analysis identified were pooled, and 1/50th was examined by SDS/PAGE (silver stain). The re- mH2A as the major component, and HSP60 as a likely contam- mainder was analyzed by both in-gel and in-solution MS methods for PTMs. inant (Fig. 1c). Both in-gel and in-solution enzymatic digestion mH2A was the major constituent of the pooled fractions (Hsp60 is a likely was performed for MS to identify PTMs of mH2A1 [see contaminant). Size markers are denoted on the left. supporting information (SI) Materials and Methods]. In the present work, particular attention was paid to the hinge region of mH2A1, a very basic stretch of amino acids that To investigate the biological implications of mH2AS137ph, a contains multiple potential phosphate acceptor residues (Fig. 2a, highly specific affinity-purified polyclonal antibody was gener- Upper). Because this region is lysine-rich, it is not amenable to ated. Peptide competitions demonstrated that a peptide phos- enzymatic digestion with trypsin and therefore was targeted with phorylated at S137 could compete antibody reactivity when endoproteinase GluC, generating a peptide of Ϸ40 aa (Fig. 2a, HEK293 histones were analyzed by IB, whereas the unmodified Upper and SI Fig. 5 a and b). Through our MS analysis, two peptide could not (Fig. 2b). Furthermore, peptide competitions phosphorylation sites were detected within the hinge of endog- with various phosphorylated peptides from other histones such enous mH2A1: T128ph and S137ph, the latter of which is a as H3S10ph, H3S28ph, and H3.3S31ph (SI Fig. 5c) and peptide previously unidentified modification of mH2A (for spectra, see dot blots and Luminex assays (data not shown) demonstrated SI Fig. 5 a and b). Intriguingly, a significant fraction (Ϸ5%) of specificity for mH2A phosphorylation. mH2A was phosphorylated at S137 in asynchronous cultures, a considerable amount given that there was no prior enrichment mH2AS137ph Is a Prevalent PTM. The hinge region of mH1A1.1 and for phosphopeptides. Of note, S137ph was detected on peptides 1.2 is identical, and we questioned whether both splice forms are that contained either KK or KKK C-terminal to S137, which are phosphorylated on S137. However, because our MS analyses forms of alternate splice selection (Fig. 2a, underlined) (6). The generated an identical peptide from mH2A1.1 and 1.2, we were significance of this splice selection is unknown. unable to confirm whether the phosphorylation is present on one

1534 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711632105 Bernstein et al. Downloaded by guest on September 25, 2021 to be phosphorylated at S137 may provide a unique role to this variant. We next used the S137ph antibody to screen cell lines of different origin for its presence, and all cells tested contain this PTM (Fig. 2d). Interestingly, differentiated mouse ES cells (and 3T3s) contain more mH2AS137ph than undifferentiated ES cells, suggesting a potential role for this modification in chro- matin changes throughout differentiation. S137ph is not exclu- sive to female cells because male ES cells also have significant amounts this modification (Fig. 2d, Right); it is also observed in male mouse liver (Fig. 2c). We note that although female liver has higher levels of S137ph than males, and the opposite trend occurs in ES cells, this difference may reflect variation in cell/tissue types. Taken together, these data suggested that the subpopulation of mH2A containing S137ph was not exclusive to the Xi, but rather that this modified form of mH2A has a distinct function within the chromatin template (see below).

mH2AS137ph Is Excluded from the Xi. To test the above hypothesis directly, we performed immunofluorescence (IF) analyses in HEK293 cells with antibodies against S137ph and mH2A (Fig. 3a). Although mH2A staining showed two or three intensely stained foci (Xis), S137ph staining did not produce similar results. We next generated a HEK293 cell line stably expressing mH2A1.2 fused to GFP, allowing direct visualization of Xis. Again, IF analysis, by using antibodies in direct comparison with the mH2A-GFP fusion, showed that, whereas mH2A was en- riched on the Xi, the phosphorylated form was not (Fig. 3b). This lack of enrichment is not caused by the inability of the GFP fusion protein to be phosphorylated on S137 (SI Fig. 5d). Although the localization of S137ph is not completely excluded from the Xi in all cases (Ϸ75% Xis show no enrichment, with 148 Xis counted), this modification is not enriched on the Xi as seen Fig. 2. mH2A1 is phosphorylated in the hinge region on S137. (a) MS analysis for general mH2A antibody (Fig. 3b). Furthermore, S137ph is identified two phosphorylation sites in the basic hinge region of mH2A1, T128 predominantly chromatin-associated as demonstrated by a chro-

and S137 (asterisks). Sequence shown represents the peptide generated by CELL BIOLOGY GluC endopeptidase digestion. Peptides with both forms of alternate splice matin fractionation assay and is released from chromatin by selection containing either KK or KKK were identified (underlined). The ARKS micrococcal nuclease digestion (Fig. 3c). motif conserved in H3 is shaded. ClustalW alignment of the phosphorylated To test the localization of S137ph mH2A during ES cell residues of mH2A 1.1, 1.2, and 2 is shown below. Note that phosphorylation differentiation, we used a female mouse ES cell line (LF2) that potential exists for all three isoforms in the case of T128 but only for mH2A1.1 inactivates one of its X chromosomes upon retinoic acid treat- and 1.2 at S137. (b) mH2AS137ph antibody is specific as demonstrated by ment (30). Because mH2A associates with the Xi late in the peptide competitions. HEK293 histones were used for peptide competitions differentiation process [Ϸday 5 or 6 in LF2 cells (31)], we with S137ph antibody. Membrane was cut into strips, probed with the anti- examined S137ph localization before (day 2) and after (day 9) bodies indicated, and reassembled; Prebl., prebleed serum; un, unmodified mH2A associates with the Xi (Fig. 3d). In both cases, peptide; and ph, phosphorylated peptide. mH2A IB was used as a control for size (last lane). Amido black staining of histones is used as a loading control. mH2AS137ph is absent from the Xi (marked by Xist RNA FISH; (c) Separation of mH2A isoforms demonstrates that both mH2A1 splice iso- Fig. 3d), indicating that a unique pool of phosphorylated mH2A forms, mH2A 1.1 and 1.2, are phosphorylated at S137 in adult female and male is excluded from this important epigenetically regulated hetero- mouse liver. (d)(Left) mH2AS137ph is found in all cell types examined: chromatic structure. Interestingly, S137ph stains interphase nu- HEK293, HeLa, LF2 ES cells, 3-day retinoic acid-differentiated LF2 cells, and clei in a highly punctate pattern, the significance of which is mouse 3T3 cells. (Right) Both male and female ES cells contain this PTM. unclear; however, we observed some overlap with regions of active transcription in LF2 cells (visualized by RNA polymerase II staining; SI Fig. 6). or both splice variants. Thus, we used SDS/PAGE to separate mH2A1 isoforms from adult mouse liver nuclei, a rich source of S137 Phosphorylation of mH2A Is Enriched During Mitosis. The mH2A. Interestingly, both mH2A1.1 and 1.2 are phosphorylated absence of S137ph on the Xi raises the possibility that phos- at S137 in male and female liver (Fig. 2c). Adult mouse liver is phorylation of mH2A in the hinge region might prevent its largely devoid of dividing cells, and the presence of S137ph in this association with the Xi or certain forms of heterochromatin. To tissue may represent a potential role of mH2A in senescence dissect the role of this PTM further, we examined three key because mH2A is also enriched in senescence-associated het- cellular mechanisms linked to phosphorylation of other histones: erochromatin foci (11). Of note, S137ph signal is absent from (i) DNA damage response, S139ph on H2A.X; (ii) apoptotic mH2A knockout liver in IB, again demonstrating specificity of chromatin, H2BS14ph; and (iii) chromatin condensation during this antibody (Fig. 2c). mitosis, S10ph and S28ph on H3 (22–26, 32). We examined the Although mH2A2 is not highly conserved with mH2A1 in the levels of S137ph after induction of apoptosis by etoposide hinge region, it is also lysine-rich. Sequence alignments of the treatment and DNA damage induced by ionizing irradiation, and three mammalian mH2A isoforms show that T128 is replaced by we observed no change in S137ph levels after either treatment a in mH2A2, a potentially phosphorylated residue (Fig. (SI Fig. 7 a and b). We did, however, observe a consistent change 2a, Lower). However, in the case of S137, mH2A2 contains a in the levels of S137ph during cell cycle progression. Although lysine residue (Fig. 2a, Lower). Thus, the ability of only mH2A1 asynchronous cell populations contain S137ph (Fig. 2d), the

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Fig. 4. mH2AS137ph is enriched during mitosis. (a) Asynchronous HeLa cells contain mH2AS137ph, and its levels increase after nocodazole treatment (mitotic cells). Comparably lower levels are seen after hydroxyurea (HU) treatment (S phase). H3S10ph is used as a mitotic marker. Amido black staining of histones is used as a loading control. (b) A double-thymidine block and subsequent release of synchronized HeLa cells throughout the cell cycle Fig. 3. mH2AS137ph is absent from the inactive X chromosome. (a) HEK293 demonstrate an increase in S137ph levels during mitosis (see mitotic marker cells were stained with antibodies to S137ph and mH2A. Arrows depict Xi H3S10ph). Amido black staining of histones is used as a loading control. (c)IF chromosomes. DAPI stains are shown to the left of each staining. Note that of U2OS cells shows staining of S137ph throughout interphase and different HEK293 cells have multiple Xis. (b) S137ph is not enriched on the Xi in HEK293 stages of mitosis. (Top) DAPI stains. (Middle) S137ph. (Bottom) Merges. (d) In cells. HEK293 cell lines containing stably expressed mH2A1.2-EGFP to mark the vitro kinase assays demonstrate Cdk2/cyclin E and Cdk1/cyclin B kinase activity Xi (green) were stained with S137ph and mH2A antibodies (red). DAPI and toward an unmodified mH2A1 peptide containing S137. Peptides with the merged images are shown; arrows depict Xis. (c) Chromatin fractionation and same backbone phosphorylated at S139 prevent S137 phosphorylation. An micrococcal nuclease (Mnase) digestion of chromatin in HEK293 cells. Anti- S137ph peptide was used as a positive control for S137ph antibody signal. bodies to Mek2 (microtubule-associated kinase) and H3 are used as cytoplas- Amido black staining of peptides is used as a loading control. mic and nuclear fractionation controls, respectively. (d) Female mouse ES cells display exclusion of S137ph from the Xi. ES cells were stained on day 2 and day 9 of retinoic acid-induced differentiation (before and after mH2A association during interphase, we suggest that the role of this mark is not with the Xi, respectively) for S137ph (red) and Xist RNA (by RNA FISH, green). limited to mitosis. In keeping, other well characterized histone Arrows depict Xis; DAPI (blue) merged images are shown. phosphorylation events exhibit complex relationships during the cell cycle. For example, H3S10 and S28 are phosphorylated during periods of immediate–early gene induction or mitosis (23, levels increase significantly upon nocodazole treatment, whereas 27). The timing of mH2AS137 phosphorylation resembles that of an S phase block with hydroxyurea results in very low levels linker histone H1, which is present during interphase and (Fig. 4a). Analyses of synchronized HeLa cells induced by a double thymidine block procedure confirmed that the levels of becomes hyperphosphorylated during mitosis (22). Because mH2AS137ph increase during mitosis (Fig. 4b). These IB results S137ph is next to a proline residue (Fig. 2a) and appears similar were confirmed by IF studies, which showed strong staining of in timing to cyclin-dependent kinase (Cdk)-mediated H1 phos- mitotic chromosomes in U2OS cells compared with interphase phorylation, we suspected that S137 phosphorylation is also nuclei (Fig. 4c); similar results were seen with HeLa cells (SI catalyzed by Cdks. In vitro kinase assays demonstrate that Fig. 7c). recombinant Cdk complexes, including Cdk2/cyclin E and Cdk1/ Phosphorylation and dephosphorylation of many substrates cyclin B (S phase and mitotic regulators, respectively) can are well documented to mediate large-scale changes in cellular phosphorylate an mH2A peptide containing an unmodified S137 organization during the cell cycle. Because mH2AS137ph is residue (Fig. 4d). Interestingly, phosphorylation of nearby S139 observed by IB throughout the cell cycle and in IF staining (a currently unidentified mH2A PTM) prevents S137 phosphor-

1536 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711632105 Bernstein et al. Downloaded by guest on September 25, 2021 ylation (Fig. 4d) and may represent a switching mechanism for the Xi, and ectopic expression of Xist on autosomes results in the potential binding effectors. This effect is not caused by epitope recruitment of mH2A to these ectopic sites (39–41). occlusion by S139ph because S137 phosphorylation is also Intriguingly, we also recognized a motif within the hinge of reduced on this peptide in radioactive kinase assays (data not mH2A1 that is similar to the H3 tail, a conserved ‘‘ARKS’’ cassette shown). Other Cdk/cyclin combinations can also phosphorylate in which H3K9/S10 and H3K27/S28 residues reside (Fig. 2a, the unmodified peptide, whereas S/T kinases such as casein shaded). If modified by methylation (K156) and/or phosphorylation kinase II and tyrosine kinases such as Wee1 are incapable of (S157), respectively, the mH2A ARKS motif might be analogous to phosphorylating the same peptide in parallel assays (data not the well studied modifications on that mediate HP1 shown). Because Cdks can compensate for the loss of other Cdks (K9me3) and Polycomb (K27me3) binding, with phosphorylation during the cell cycle, it has been difficult to assess which Cdk is at adjacent (S10 and S28) playing a role in mitosis (23, 24, responsible for S137 phosphorylation in vivo, and it remains an 42). To date, we have not detected these modifications by MS important challenge for the future. analysis, but we cannot exclude the possibility that these PTMs exist in low abundance (Ͻ0.1% of total mH2A) or take place only during Discussion certain cell cycle phases. Interestingly, a ‘‘methyl/phos histone Histone variants have great potential to alter the nucleosome in mimic’’ corresponding to the above motif has recently been iden- what has recently been described as a potential ‘‘nucleosome tified in the histone methyltransferase G9a, suggesting a broader code’’ (4). Such changes can be structural and/or charge-related role for this motif (43). in nature and derived from unique sequence elements in the Our principal interest in mH2A stems from the fact that in variants themselves and/or effects of their PTMs. PTMs of mammals, it is preferentially concentrated on the Xi in females, certain histone variants have been well documented, such as suggestive of a role in transcriptionally repressed chromatin (9). phosphorylation of H2A.X in response to DNA damage (26). However, the studies reported here point to a potential role of Nevertheless, PTMs of other H2A variants, such as mH2A, are mH2A and its hinge-directed phosphorylation in a role(s) out- just beginning to be uncovered. For example, MS approaches side Xi biology. Based on the emerging literature, we favor the have recently uncovered monoubiquitylation of K115 on over- view that mH2AS137ph and potentially other hinge-based phos- expressed mH2A1.2, a site analogous to K119ub of phorylation sites regulate critical interactions with effector (28, 29). Methylation of K17, K122, and K238 has been reported, molecules that, in turn, govern the association of mH2A with key and so has phosphorylation of T128, a site verified by us in this genetic elements like the Xi. A stated prediction of the histone work (29). mH2A is also ADP-ribosylated, as demonstrated by code hypothesis is that effector-binding proteins engage the immunological approaches (33). However, it now remains a appropriately modified histone tails (18). The incorporation or challenge to understand the biological roles of these mH2A removal of histone variants, each potentially decorated with its PTMs and those of other histones, variant or nonvariant. own PTM signature, might greatly increase the regulatory Although a role for mH2A in transcriptional repression and X options of the epigenome, and we look forward to experimental inactivation has been well documented, recent studies suggest tests of this hypothesis in the future. that mH2A has roles beyond these processes. Although mH2A

is concentrated on the Xi, it is also widely distributed throughout Materials and Methods CELL BIOLOGY chromatin and localizes to other nuclear domains, exists in Histone Isolation and Purification. Histone isolation was performed essentially vertebrate species that do not undergo X inactivation, and is as described in ref. 44 and SI Materials and Methods. Histones were purified expressed to similar degrees in males and females (9, 33, 34). by RP-HPLC and screened for mH2A by IB, and positive fractions were pooled Moreover, whereas mH2A1 knockout mice are viable, fertile, for MS analysis (see SI Materials and Methods). and do not display X inactivation defects (mH2A2 is still present), a set of genes does exhibit increased expression levels IB and IF. For antibodies used in this work, see SI Materials and Methods. during the transition from newborn to young-adult animals (35). Peptide competitions for IB were performed by incubating S137ph antibody This finding suggests that mH2A1 is not restricted to large-scale with each peptide at 2 ␮g/ml for2hatroom temperature before overnight gene silencing and chromosome inactivation, but it also fine incubation with the membrane. IF analysis on HEK293 cells was carried out by tunes expression levels of specific genes through development. using poly-L-lysine (Sigma)-covered slips, fixed in 2% formaldehyde, perme- One way in which these diverse roles of mH2A might be abilized, and stained. Cells were analyzed on Axioskop 2 plus and Axioplan 2 orchestrated is through its PTMs. microscopes (Zeiss). IF and RNA FISH on female ES cells (LF2) were performed as described in ref. 31, and cells were analyzed on a Deltavision microscopy Here, we report the identification of mH2A phosphorylation system (Applied Precision). sites. S137ph appears to contribute to Xi-independent chromatin changes, such as chromatin condensation during mitosis. An- Chromatin Fractionation and Separation of mH2A Isoforms. Chromatin frac- other intriguing possibility is that phosphorylation of mH2A tionation was performed as described in ref. 45. Mouse liver mH2A isoforms regulates RNA binding. S137ph is found in the hinge region of were separated as described in ref. 35. mH2A, which has been suggested to bind nucleic acids (6). Interestingly, heterochromatin protein 1 (HP1) also has a lysine- Cell Culture and Cell Cycle Arrest. Cell lines were grown under standard rich hinge domain (between its chromo- and chromoshadow conditions (SI Materials and Methods). Rat mH2A1.2 (95% similar to human; domains) that is phosphorylated in vivo (36). The HP1 hinge GenBank accession no. U79139) was cloned into EGFP-N1 (Clontech), gener- domain has been shown to bind RNA in vitro, and RNase ating a C-terminal EGFP tag. Stable HEK293 cell lines were created by neomy- treatment of mammalian cells releases HP1 from chromatin (37, cin selection. Nocodazole-arrested HeLa cells were treated with 200 ng/ml 38). In keeping, we propose that mH2A may bind RNA through inocodazol (Sigma) for 16 h and harvested by mitotic shake off. HeLa cells were its hinge domain, and the resulting negative charge caused by arrested in G1/S by double-thymidine block with 2 mM thymidine for 16 h, released for 9 h, and blocked for 16 h. Hydroxyurea block was used to phosphorylation (T128ph, S137ph, and/or yet unidentified sites) synchronize cells in S phase by treating for 20 h with 2 mM hydroxyurea. might abrogate its ability to bind RNA, consistent with its exclusion from the Xi. Although the ability of mH2A (and its Kinase Assays. Unmodified and phosphorylated mH2A peptides (amino acid hinge domain) to bind RNA is unclear, several studies have 132–142) were incubated with 10–40 ng of recombinant Cdk/cyclin complexes shown that its localization to the Xi depends on the Xist RNA: (Millipore) and ATP (500 ␮M)for1hat30°C. Recombinant H1 was used as a conditional deletion of Xist during X inactivation or abolishing positive control for Cdk reactions (data not shown). Detailed reaction condi- the ability of Xist to bind the Xi prevent mH2A association with tions can be found at the distributor’s website.

Bernstein et al. PNAS ͉ February 5, 2008 ͉ vol. 105 ͉ no. 5 ͉ 1537 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank David Shechter, Andrew Xiao, Sandra Hake, 09-018). This work was funded by High-Throughput Epigenetic Regulatory Elizabeth Duncan, Ping Chi, and Monika Kauer (all presently or previously at Organization in Chromatin (HEROIC), an integrated project funded by the The Rockefeller University) for advice and reagents; Holger Dormann and European Union under Framework Program LSHG-CT-2005-018883 (J.C.C. and Sandra Hake for critical reading of the manuscript; The Rockefeller University E.H.), by a Fondation pour la Recherche Me´dicale fellowship (to J.C.C.) and Bio-Imaging Facility for microscopy assistance and Proteomics Resource Center Equipe Fondation pour la Recherche program (E.H.), and by National Insitu- for peptide synthesis; Weronika Prusisz for technical assistance; and Millipore tutes of Health Grants GM49351 (to J.R.P), GM37537 (to D.F.H), and GM40922 antibody development scientists for generation of ␣mH2AS137ph (catalog no. (to C.D.A).

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