Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells
- Lovorka Stojic†1, 2,
- Zuzana Jasencakova†1, 3,
- Carolina Prezioso†1,
- Alexandra Stützer4,
- Beatrice Bodega1, 5,
- Diego Pasini3, 6,
- Rebecca Klingberg7,
- Chiara Mozzetta1, 8,
- Raphael Margueron9,
- Pier Lorenzo Puri1, 10,
- Dirk Schwarzer7,
- Kristian Helin3,
- Wolfgang Fischle4 and
- Valerio Orlando1Email author
© Stojic et al; licensee BioMed Central Ltd. 2011
Received: 30 June 2011
Accepted: 5 September 2011
Published: 5 September 2011
Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.
We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.
Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.
During development, differentiation programs require global rearrangements in repression and activation of lineage-specific genes. Chromatin-based epigenetic mechanisms ensure correct integration of developmental signals at gene regulatory regions, allowing the action of transcription factors and maintaining novel expression states in derived cell populations. Polycomb group (PcG) proteins are transcriptional repressors that remodel chromatin through epigenetic modifications that prevent changes in cell identity by maintaining transcription patterns, throughout development and in adulthood [1, 2]. They comprise two major multiprotein complexes, polycomb repressive complex (PRC)-1 and PRC-2. PRC1 is the larger-sized complex that contains several polypeptides whose functions include ubiquitination of histone H2A at lysine 119 (H2AK119), chromatin compaction and regulation of the basal transcription machinery . The core of the PRC2 complex is made up of three proteins, Suz12, Eed and Ezh2, the latter being the catalytic subunit that modifies histone H3 by trimethylation of lysine 27 (H3K27me3). Once H3K27me3 has been established, PRC2 is able to bind to this mark via the Eed subunit, which in turn activates the histone methyltransferase activity (HMT) of the complex [4, 5]. This process allows maintenance of the repressive mark and its transmission to daughter cells . Recently, it has been reported that in mammals HMTase Ezh2 can be replaced by another highly homologous polypeptide called Ezh1. However, whereas PRC2-Ezh2 catalyses H3K27me2/me3 and its knockdown affects global H3K27me2/me3 levels, PRC2-Ezh1 performs this function weakly [7, 8]. Although Ezh1 depletion does not impact global H3K27me2/me3 levels, the PRC2-Ezh1 complex robustly represses transcription from chromatinised templates and compact chromatin . Interestingly, while Ezh2 expression is closely associated with proliferation, Ezh1 is more abundant in non-proliferative adult organs, suggesting that these two PRC2 complexes may have different functions in dividing versus post-mitotic cells [9, 10]. Thus, replacement of the Ezh2 subunit with Ezh1 appears to be developmentally regulated. To date, however, the function of Ezh1 in differentiating cells remains elusive.
Vertebrate skeletal muscle formation constitutes an interesting model system to study the epigenetic signals and molecular mechanisms that govern cellular differentiation [11, 12]. Previous work revealed a crucial role of Ezh2 in skeletal muscle cell differentiation as its transcriptional and post-transcriptional downregulation is required to allow activation of muscle-specific genes [13, 14]. During myogenic differentiation, extracellular signals are transduced into the nucleus by mitogen-activated protein kinases (MAPKs), p38 or extracellular signal-regulated kinase (ERK) [15, 16]. The mitogen- and stress-activated protein kinases (Msk-1 and Msk-2), downstream targets of the p38 or ERK pathways , are responsible for the histone H3 phosphorylation at serine 28 (H3S28ph) and serine 10 (H3S10ph) [18, 19]. Recent data show that an H3K27/H3S28 methyl/phospho switch mechanism regulates gene activation via PRC2 chromatin displacement during neuronal differentiation, stress response and mitogenic signalling [20, 21]. If a similar mechanism is involved in muscle gene activation, allowing for PcG chromatin displacement, remains to be elucidated.
In the current work we report that two different PRC2 complexes contribute to skeletal muscle differentiation: PRC2-Ezh2, which is predominant in proliferating myoblasts, and PRC2-Ezh1, which contains Ezh1 but is devoid of Ezh2, and is specific for post-mitotic myotubes. Interestingly, these two PRC2 complexes are differentially associated with muscle regulatory regions. Indeed, while muscle creatine kinase (mCK) is a classic PRC2 target gene where its expression is associated with the displacement of PRC2-Ezh2 complex, myogenin (MyoG) shows a switch between PRC2-Ezh2/PRC2-Ezh1 complexes upon differentiation, suggesting a role of this dynamics in gene activation. In light of their different chromatin associations, we verified that a Msk1-dependent signalling that controls H3S28ph, is involved in the specific displacement of PRC2-Ezh2 from the MyoG and mCK regulatory regions, to result in muscle differentiation. This confirms the findings of previous reports that consider Ezh2 downregulation to be a necessary step in the myoblast-myotube transition [13, 14]. Surprisingly, we found that the PRC2-Ezh1 complex is insensitive to the H3S28ph activation mark. Indeed, this complex regulates the proper timing of MyoG transcriptional activation via recruitment of MyoD transcriptional factor in post-mitotic myotubes.
Thus, our study reveals a novel important layer of PcG-mediated epigenetic regulation of skeletal muscle cell differentiation, in which the different dynamics and chromatin regulated switch between PRC2-Ezh2 and PRC2-Ezh1 complexes are coordinated to induce the transition from myoblast to myotube transcriptional programs.
Two PRC2 complexes, PRC2-Ezh2 and PRC2-Ezh1, are present during myogenic differentiation
PRC2-Ezh2 and PRC2-Ezh1 complexes are differentially associated with muscle gene regulatory regions
Taken together, these results suggest that the binding of the PRC2-Ezh1 complex at the MyoG promoter in differentiating cells could play a role in the regulation of the proper transcriptional profile of this gene.
A H3K27/H3S28 methyl/phospho switch regulates muscle gene activation via PRC2-Ezh2 chromatin displacement
In light of the known role that Msk1 plays in the phosphorylation of H3S10 , we asked whether H3S10ph was also involved in muscle gene activation. However, because we did not observe any increase of this modification at the MyoG and mCK regulatory regions during muscle differentiation, we ruled out the possibility that H3S10ph functions in muscle gene activation (Additional file 2E). Furthermore, we examined whether Msk1 can phosphorylate H3S28 in an environment including pre-existing H3K27me3. Recombinant Msk1 kinase was incubated with a histone H3 (residues 21-33) peptide, which was either unmodified or modified with K27me3 or S28ph. Although the H3K27me3 substrate was phosphorylated under similar kinetic conditions as the unmodified peptide, no phosphorylation of the H3S28ph substrate was observed (Figure 3D), indicating that the serine 28 is the only residue phosphorylated by Msk1. Taken together, these data suggest that displacement of the PRC2-Ezh2 complex from MyoG and mCK promoters is regulated by a H3K27me3/H3S28ph switch via Msk1 recruitment onto chromatin.
PRC2-Ezh2 and PRC2-Ezh1 chromatin dynamics are differentially regulated by a H3K27/H3S28 methyl/phospho switch
Correct timing of myogenin transcriptional activation requires the PRC2-Ezh1 complex
PRC2-Ezh1 is required for the recruitment of MyoD on myogenin promoter
Different dynamics of PRC2-Ezh2 and PRC2-Ezh1 complexes allow the correct timing of skeletal muscle gene transcriptional activation
PcG proteins contribute to differentiation through their ability to repress transcription of developmental regulators in committed cells, including skeletal muscle cell lines. Previous analysis of Ezh2 dynamics during myogenic differentiation has lead to a two-step activation model defining PcG-dependent muscle gene expression and cell differentiation . However, a broad analysis of other PRC2 core components (Suz12 and Eed), including Ezh1, has not yet been attempted. Our data show that Ezh1 is the only PRC2 component that is maintained at constant levels during myogenic differentiation, while levels of Ezh2, Suz12 and Eed, to different extent, decrease from undifferentiated to differentiated states (Figure 1B, C and Additional file 1). We propose that skeletal muscle differentiation could be regulated by two distinct PRC2 complexes, PRC2-Ezh2 in myoblasts and PRC2-Ezh1 in myotubes. Existence of two partially redundant PRC2 complexes has been previously reported [7–10]. However, our data suggest that Ezh1 is more than just a substitute for Ezh2. Indeed, observations regarding the chromatin dynamics of the PRC2-Ezh1 complex on the MyoG promoter raise questions as to its functionality during skeletal muscle cell differentiation. Insight regarding the function of Ezh1 in skeletal muscle differentiation can be derived from the evidence that, unlike Ezh2 , Ezh1 is required for myogenic differentiation (Figure 5). In regard to this, we detected Ezh1 on the MyoG promoter when the gene is activated and RNA Pol II is recruited (Figure 2A). Indeed, Ezh1 depletion led to a delay of MyoG transcriptional activation due to the impairment of MyoD recruitment on the MyoG promoter (Figure 8). However, at the later stages of differentiation, the binding of Ezh1 and Suz12 (Figure 2A) could indicate that this complex has a role in the subsequent resilencing of MyoG in terminally differentiated myotubes. In agreement with this hypothesis, a recent report showed that MyoG upregulation during the initial stages of skeletal muscle differentiation is followed by subsequent repression . Notably, MyoG is activated in the early stages of neurogenic muscle atrophy and failure in later downregulation is causally correlated with disease progression .
Surprisingly, our data showed that a PcG protein, such as Ezh1, is recruited on muscle specific gene when it is activated. Indeed, previous reports provided evidences that other PcG proteins bind actively transcribed genes [36, 37]. The coexistence of active (AcH3, H3S28ph and H3K4me3) and repressive marks (H3K27me3) at the MyoG promoter could be similar to the bivalent domains of embryonic stem (ES) cells, as it has been shown that these domains are not limited to these cells . Indeed, 10% to 20% of reported PcG target genes in ES cells are transcriptionally active [31, 39]. The presence of PcG on active genes may be comparable to the presence of trithorax (trxG) proteins on repressed genes as this dual configuration of PcG and trxG proteins on active and repressed regions may provide a given gene with the flexibility to rapidly change its expression profile upon developmental or environmental stimuli.
As Ezh1 methyltransferase activity on histones is found to be modest , it will be interesting to investigate whether this PcG protein has targets in addition to histone H3, such as RNA Pol II enzyme. Indeed, a very recent report reveals that the C-terminal domain (CTD) of RNA Pol II is methylated by the coactivator-associated arginine methyltransferase 1 (CARM1) .
Future genome-wide analysis coupled to loss-of-function experiments will be required to address EZH1 function in myofibres.
H3K27/H3S28 methyl/phospho switch mechanism is the basis of PRC2-Ezh2 target gene activation during myogenic differentiation
If PRC2-Ezh1 is required for the correct timing of MyoG transcriptional activation, removal of PRC2-Ezh2 from this gene would be necessary to guarantee its activation. One way of doing this would be to reduce intracellular PcG levels. In regard to this, Juan et al.  provided evidence that miR-214 regulates Ezh2 protein levels in skeletal muscle and ES cells. Recent studies raise interesting questions concerning the assumption that PcG derepression must be accompanied by the loss of the H3K27me3 repressive mark. Seenundun and coworkers  showed that the histone demethylase UTX is targeted to muscle-specific genes by the transcriptional activator Six4 to mediate removal of the repressive H3K27me3 mark during myogenesis. Recent reports suggest that demethylation of H3K27 may not be the only mechanism for derepression of PcG target genes [20, 21]. A novel mechanism regulating PcG displacement from chromatin has been identified, in which phosphorylation of H3S28, via mitogen and stress-activated kinases Msk1 and 2, is able to neutralise the H3K27me3 repressive mark to result in PRC2 removal and gene activation [20, 21]. Our data show that a similar mechanism appears to operate in differentiating myoblasts, in which Msk1 regulates a H3K27/H3S28 methyl/phospho switch to allow removal of the PRC2-Ezh2 complex and muscle gene activation (Figure 3). Notably, our in vitro experiments indicate that the Msk1-methyl/phospho switch pathway is specific to the PRC2-Ezh2 complex, while it appears that PRC2-Ezh1 is not regulated by this mechanism (Figure 4). Our ChIP analysis shows that the H3K27me3 mark is not alternative to H3S28ph and we can detect them independently. The in vivo presence of a phospho group at H3S28 may interfere with epitope recognition of H3K27me3 antibodies, raising potential concerns about the interpretation of the existing H3K27me3 ChIP genome-wide database . In our ChIP experiments we did not encounter this problem as H3K27me3 was efficiently detected, even in the presence of adjacent H3S28ph mark. Previous studies suggest that PRC2 function is required during S-phase to guarantee maintenance of silenced state . A recent genome-wide analysis of histone modifications performed in C2C12 myotubes revealed that the H3K27me3 mark on repressed non-muscle genes is not associated with PRC2, but with PRC1 complexes . Thus, the function of the PRC2 complex in post-mitotic myotubes may not be linked to the maintenance of the H3K27me3 mark. Indeed, our data suggest that the PRC2-Ezh1 complex, and in particular the Ezh1 subunit, is required for proper MyoG activation when H3K27me3 mark is not removed, suggesting that Ezh1 function is linked to promoter setting of terminally differentiating cells. Future experiments will be required to test the hypothesis that while some genes are permanently inactive and do not require PRC2-Ezh2 activity once cells have stopped proliferating, other genes remain active and maintain their competence to resilence by using chromatin bound PRC2-Ezh1, as a security measure.
Our work addresses the role of PRC2 complexes during skeletal muscle cell differentiation.
We report that two different PRC2 complexes, PRC2-Ezh2 and PRC2-Ezh1, are differentially associated with muscle gene regulatory regions and play distinct roles in the terminal differentiation process. We show that as Ezh2 is removed from MyoG and mCK, high levels of Ezh1 persist in differentiating muscle cells and PRC2-Ezh1 is recruited at MyoG, a step that is essential for activation of the early myogenic program. These events are required for regulation of the correct timing of MyoG transcriptional activation, and loss of Ezh1 affects recruitment of the MyoD transcription factor on its promoter in post-mitotic myotubes. Further, we report that Msk1-signalling controls H3S28ph and is involved in the specific displacement of PRC2-Ezh2 from muscle regulatory regions, triggering muscle gene activation and thereby muscle cell terminal differentiation. Consistent with its role involving MyoG transcriptional activation, we show that the PRC2-Ezh1 complex is insensitive to the H3S28ph activation mark. Thus, our study reveals a novel important layer of PcG-mediated epigenetic regulation of skeletal muscle cell differentiation, in which the coordinated different dynamics and chromatin-regulated switch between PRC2-Ezh2 and PRC2-Ezh1 complexes are required to initiate the transition from myoblast to myotube transcriptional programs. Notably, our data suggest a novel and unexpected role for PRC2-Ezh1 in promoter setting. Further, based on published data concerning MyoG regulation in muscle fibres, we speculate that PRC2-Ezh1 may be required for subsequent developmentally regulated resilencing of MyoG and perhaps other skeletal muscle genes. Our study provides new epigenetic insights into the process of terminal differentiation, in which the regulated and coordinated chromatin dynamics of two PRC2 complexes is required for the correct timing of muscle gene activation and thereby muscle differentiation.
Cell lines and reagents
C2C12 mouse myoblasts cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS) (Euroclone, Devon, UK). Differentiation was induced when cells reached approximately 80% confluency using DMEM containing ITS media supplement (Sigma, St Louis, MO, USA) or 2% horse serum (HS) (Euroclone). Human primary myoblasts from healthy donors were obtained from the Telethon BioBank (Neuromuscular Diseases and Neuroimmunology Unit, Muscle Cell Biology Laboratory, C Besta Neurological Institute). The cell lines were cultured in DMEM supplemented with 20% FBS (Lonza, Basel, Switzerland), insulin 10 mg/ml, human fibroblast growth factor (hFGF) 25 ng/ml, human epidermal growth factor (hEGF) 10 ng/ml (proliferating medium), and then induced to differentiate by means of DMEM supplemented with 2% HS (differentiating medium). H89 (Alexis Corporation, Farmingdale, NY, USA) was replaced every 24 h.
Satellite cell isolation and culture
Single muscle fibres were isolated by standard procedures. In brief, the hind limb muscles were digested with collagenase and single myofibres were cultured in GM1 (DMEM supplemented with 10% HS (GIBCO, Invitrogen, Carlsbad, CA, USA), 0.5% chick embryo extract (MP Biomedicals, Illkirch, France), and penicillin-streptomycin (GIBCO)) at 37°C in suspension for 72 h, and then plated on matrigel (Sigma, 1 mg/ml ECM gel)-coated dishes for satellite cell culture. Then, 3 days later, the fibres were removed and the medium replaced with proliferation medium (GM2: 20% FBS, 10% horse serum, 1% chick embryo extract in DMEM). After 4-5 days, the medium was replaced with differentiation medium (DM: 2% HS and 0.5% chick embryo extract in DMEM).
RNA isolation and quantitative real-time PCR
RNA was extracted from cells using TriReagent (Sigma) according to the manufacturer's instructions. cDNA synthesis was performed using the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany). Quantitative real-time PCR reactions were performed in triplicate using QuantiTect SYBR Green master mix (Qiagen) on a DNA Engine Opticon 2 machine (MJ Research) controlled by Opticon Monitor 2 software. C(T) values were calculated by Opticon Monitor 2 software. Gapdh, MHCIIB and mCK primers have been previously described . The remaining primer sequences are available upon request.
C2C12 cell line and satellite cells: siRNA EZH1 no. 1 (SI00997766), siRNA EZH1 no. 2 (SI00997773), siRNA SUZ12 no. 1 (SI01438416), siRNA SUZ12 no. 2 (SI01438402), as well as negative control siRNA (scrambled sequence not targeting mouse genome, 1027313) were purchased from Qiagen. The remaining siRNA sequences are as follows: siRNA Ezh2 no. 1: AAGGAAAGAACTGAAACTTA; siRNA Ezh2 no. 2: AAGCTGAAGCCTCCATGTTTA.
Cells were transfected with HiPerfect (Qiagen) following the manufacturer's instructions. At 48 h after transfection the cells were induced to differentiate and collected at the indicated timepoints. All siRNAs were used at a final concentration of 20 nM.
Human myoblasts: cells were transfected with DharmaFECT (Thermo-Scientific, Waltham, Massachusetts, USA) following the manufacturer's instructions. At 48 h after transfection the cells were induced to differentiate and collected at the indicated timepoints. All siRNAs were used at a final concentration of 6 nM (Ambion/Applied Biosystems, USA). The siRNA sequences are available upon request.
Cell lysis and immunoblot
Cells were harvested and washed twice with PBS. Cell lysis of total cell extracts was performed on ice in 50 mM Tris-HCl pH 8, 125 mM NaCl, 1% NP-40, 2 mM ethylenediaminetetra-acetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitory cocktail (Roche, Madison, WI, USA)) for 25 min. Insoluble material was pelleted by centrifugation at 16000 g for 3 min at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). The proteins were denatured, reduced, separated by SDS-PAGE and transferred to nitrocellulose transfer membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween (Sigma) (TBST) and incubated with primary antibodies overnight at 4°C. Following three washes with TBST, membranes were incubated with the peroxidase-conjugated secondary antibody, in TBST with 2.5% non-fat dry milk, and immunoreactive proteins were detected using Supersignal West Dura HRP Detection Kit (Thermo-Scientific). For cytoplasmic and nuclear extracts preparation the cells were resuspended first in buffer A (10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.9, 10 mM KCl, 0.1 mM EDTA and 0.1 mM ethylene glycol tetra-acetic acid (EGTA)) supplemented with protease inhibitory cocktail (Roche), 1 mM dithiothreitol (DTT) and 1 mM PMSF. After incubation on ice for 10 min, NP-40 was added to a final concentration of 0.5% and the samples were vortexed for 5 s. Nuclei were pelleted at 13,200 rpm for 10 s and the cytoplasmic proteins were collected. The pellet was then washed five times with buffer A and resuspended in buffer C (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitory cocktail (Roche) and 1 mM PMSF). After 10 min on ice, the samples were sonicated and centrifuged at 13,200 rpm for 10 min and nuclear proteins were collected.
ChIP was performed as previously described (Breiling A and Orlando V, doi:10.1101/pdb.prot4560, with adaptations) using a crosslinking time of 10 min. Antibodies were coupled to Dynal magnetic beads (Invitrogen) by overnight incubation at 4°C. The following day, chromatin was added to antibody-bead complexes and incubated overnight at 4°C. The bound complexes were washed twice in Low Salt Solution, twice in High Salt Solution, once in LiCl and once in Tris/EDTA (TE) buffer. DNA was extracted from beads by standard phenol/chloroform extraction, precipitated and resuspended in 30 μl TE. To quantify the results, quantitative (q)PCR reactions were performed in triplicate (precipitated DNA samples as well as serially diluted input DNA) using QuantiTect SYBR Green master mix (Qiagen) on a DNA Engine Opticon 2 machine (MJ Research) controlled by Opticon Monitor 2 software. C(T) values were calculated by Opticon Monitor 2 software. To calculate relative enrichment the signal from the control immunoprecipitation experiment (Mock) was subtracted from that observed with the antibody of interest. Myoblasts values (GM) were set as 1 and values from differentiated cells in DM with or without inhibitor display relative enrichment or reduction to those observed in GM. ChIP primers are available upon request.
For immunoblot: EZH2 (3147) was from Cell Signaling (Danvers, MA, USA). SUZ12 (46264), MyoG (12732), and MHCIIB (2064) were from Santa Cruz (Santa Cruz, CA, USA). β-Tubulin (T0198) was from Sigma. mCK antibody was kindly provided by Hidenori Ito (Aichi Human Service Center, Kasugai, Aichi, Japan). Ezh1 and EED antibodies were previously characterised (, ). For ChIP: H3K4me3 (8580), RNA polymerase II (5408) and SUZ12 (12073) were from Abcam (Cambridge, UK) while Ezh2 (07-400), H3K27me3 (07-449), H3S28ph (07-145), H3S10ph (05-817) and Acetyl H3 (06-599) were purchased from Millipore (Billerica, Massachusetts, USA). MSK1 (9392, 25417) and MyoD (760) were from Santa Cruz.
Size exclusion chromatography
Size exclusion chromatography was performed using C2C12 cell nuclear extracts on a Superose 6 PC 3.2/30 gel filtration column (GE Healthcare, Little Chalfont, UK) using an AEKTA purifier system (GE Healthcare) in IP (300) buffer (50 mM Tris-HCl at pH 7.5, 300 mM NaCl, 5% glycerol, 0.2% Igepal (Sigma), Aprotinin, Leupeptin, 100 mM PMSF, 1 mM DTT). Immunodepletion was performed as described . Briefly, protein extracts were subjected to five serial depletions within 24 h at 4°C using the AC22 EZH2 monoclonal antibody  precoupled to Protein-A beads.
Histone tail peptides
Histone H3 peptides were synthesised in unmodified and modified form using Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid-phase synthesis. Peptides used for kinase assays corresponded to amino acids 21-33 of H3 containing an artificial Y at the C-terminus: H3 unmodified, ATKAARKSAPATGY; H3K27me3, ATKAARK(me3)SAPATGY; H3S28ph, ATKAARKS(ph)APATGY. Peptides used for precipitation experiments corresponded to amino acids 1-40 of H3 and contained a C-terminal non-native YCK sequence with the lysine biotinylated at the e-amino group: H3 unmodified, ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHR-YCK (biotin); H3K27me3, ARTKQTARKSTGGKAPRKQLATKAARK(me3)SAPATGGVKKPHR-YCK (biotin); H3K27me3S28ph, ARTKQTARKSTGGKAPRKQLATKAARK(me3)S(ph)APATGGVKKPHR-YCK (biotin).
In vitro peptide kinase assay
Recombinant MSK1 (Millipore) was used to phosphorylate H3 histone tail peptides (21-33). Kinase assays were performed according to the manufacturer's protocol by incubating 15 ng of MSK1 with 1 μg of peptide for 30 min at 30°C. The reaction was stopped by adding 0.5% phosphoric acid, spotted on P81 paper and washed three times with 0.5% phosphoric acid and once with acetone. Filter circles were air dried and counted in a scintillation counter.
Peptide affinity purification
For preparation of nuclear extracts, cells were lysed in buffer A (10 mM Hepes-KOH pH 7.8, 60 mM KCl, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail (Roche), 0.075% NP-40). After incubation on ice for 15 min, nuclei were pelleted and washed once with buffer A without NP-40. The nuclear pellet was suspended in buffer B (20 mM Tris pH 8.0, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, protease inhibitor cocktail, PhosSTOP (Roche)) and sonicated on ice in a Branson Sonifier (duty cycle 20%, output 7.5). Extract was left on ice for 30 min before centrifugation for 15 min at 16,000 g. The supernatant was supplemented with 0.1% Triton X-100 and used for precipitation experiments.
For H3 peptide precipitation experiments, 10 μg of biotinylated histone peptides (1-40) were coupled to 50 μl streptavidin-coated paramagnetic beads in PBS/bovine serum albumin (BSA) (1 mg/ml) for 4 h at 4°C. Beads were washed three times with PD150 (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 20% glycerol, 1 mM DTT, protease inhibitor cocktail, PhosSTOP) to remove unbound peptides. Peptide-bound beads were incubated with nuclear extract for 2 h and washed four times with PD300. Bound proteins were eluted with SDS sample buffer, separated by SDS-PAGE and analysed by immunoblotting.
Cells were grown on coverslips, washed in PBS, fixed in 3.7% formaldehyde/PBS (15 min, 4°C) and permeabilised in 0.2% Triton X-100/PBS (5 min, 4°C). The coverslips were then washed in PBS, and blocked with 3% low-fat milk/PBS for 1 h at room temperature. Following overnight incubation with primary antibodies at 4°C, the coverslips were washed and incubated with secondary antibodies (Molecular Probes, Eugene, OR, USA) for 60 min at 37°C, and then washed again and counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1 μg/μl, Vectashield, Vector Laboratories Inc., Burlingame, CA, USA)). Pictures were captured using epifluorescence microscopy (Leica DM6000B) using Leica Application Suite software.
Fluorescence-activated cell sorting (FACS) analysis
C2C12 myoblasts were cultured in growing conditions and collected at 24 h, 48 h, 72 h and 96 h after plating. Cells were divided in aliquots of 1.2 × 106 cells per tube, washed with cold PBS 1 ×, fixed by 70% cold ethanol and incubated for 30 min on ice. After incubation, cells were washed with PBS 1 ×, resuspended in 0.5 ml of PBS 1 ×/RNase A (100 μg/ml) and incubated at 37°C for 30 min. Finally, propidium iodide (20 μg/ml) was added and the cells were incubated in the dark for 30 min at 4°C. The samples were then analyzed for the cell cycle profile and the cell death profile using a Becton Dickinson Instrument.
We thank all the members of the Orlando laboratory for their constructive comments and suggestions. We are also grateful to Massimiliano di Pietro for his careful reading of the manuscript and to Holly Bream for manuscript editing. The work was supported by Deutsche Forschungsgemeineschft (DFG) within the Emmy-Noether program (SCHW 1163/3-1) to DS; the Danish National Research Foundation to KH; the Max Planck Society to WF; Telethon (S00094), AIRC (Associazione Italiana Ricerca sul Cancro), The Epigenome NoE FP6 to VO; LS was supported by an EMBO long-term fellowship; ZJ was supported by an EMBO long-term and a Human Frontier Science Program (HFSP) fellowship; DP was a recipient of a postdoctoral fellowship from the Danish Medical Research Council.
- Schuettengruber B, Cavalli G: Recruitment of polycomb group complexes and their role in the dynamic regulation of cell fate choice. Development. 2009, 136: 3531-3542. 10.1242/dev.033902.View ArticlePubMedGoogle Scholar
- Simon JA, Kingston RE: Mechanisms of polycomb gene silencing: knowns and unknowns. Nat Rev Mol Cell Biol. 2009, 10: 697-708.View ArticlePubMedGoogle Scholar
- Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS, Zhang Y: Role of histone H2A ubiquitination in polycomb silencing. Nature. 2004, 431: 873-878. 10.1038/nature02985.View ArticlePubMedGoogle Scholar
- Margueron R, Justin N, Ohno K, Sharpe ML, Son J, Drury WJ, Voigt P, Martin SR, Taylor WR, De Marco V, Pirrotta V, Reinberg D, Gamblin SJ: Role of the polycomb protein EED in the propagation of repressive histone marks. Nature. 2009, 461: 762-767. 10.1038/nature08398.PubMed CentralView ArticlePubMedGoogle Scholar
- Montgomery ND, Yee D, Chen A, Kalantry S, Chamberlain SJ, Otte AP, Magnuson T: The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr Biol. 2005, 15: 942-947. 10.1016/j.cub.2005.04.051.View ArticlePubMedGoogle Scholar
- Hansen KH, Bracken AP, Pasini D, Dietrich N, Gehani SS, Monrad A, Rappsilber J, Lerdrup M, Helin K: A model for transmission of the H3K27me3 epigenetic mark. Nat Cell Biol. 2008, 10: 1291-1300. 10.1038/ncb1787.View ArticlePubMedGoogle Scholar
- Margueron R, Li G, Sarma K, Blais A, Zavadil J, Woodcock CL, Dynlacht BD, Reinberg D: Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol Cell. 2008, 32: 503-518. 10.1016/j.molcel.2008.11.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, Yuan GC, Orkin SH: EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell. 2008, 32: 491-502. 10.1016/j.molcel.2008.10.016.PubMed CentralView ArticlePubMedGoogle Scholar
- Ezhkova E, Pasolli HA, Parker JS, Stokes N, Su IH, Hannon G, Tarakhovsky A, Fuchs E: Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. 2009, 136: 1122-1135. 10.1016/j.cell.2008.12.043.PubMed CentralView ArticlePubMedGoogle Scholar
- Ezhkova E, Lien WH, Stokes N, Pasolli HA, Silva JM, Fuchs E: EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 2011, 25: 485-498. 10.1101/gad.2019811.PubMed CentralView ArticlePubMedGoogle Scholar
- Sawarkar R, Paro R: Interpretation of developmental signaling at chromatin: the polycomb perspective. Dev Cell. 2010, 19: 651-661. 10.1016/j.devcel.2010.10.012.View ArticlePubMedGoogle Scholar
- Palacios D, Puri PL: The epigenetic network regulating muscle development and regeneration. J Cell Physiol. 2006, 207: 1-11. 10.1002/jcp.20489.View ArticlePubMedGoogle Scholar
- Caretti G, Di Padova M, Micales B, Lyons GE, Sartorelli V: The polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation. Genes Dev. 2004, 18: 2627-2638. 10.1101/gad.1241904.PubMed CentralView ArticlePubMedGoogle Scholar
- Juan AH, Kumar RM, Marx JG, Young RA, Sartorelli V: Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. Mol Cell. 2009, 36: 61-74. 10.1016/j.molcel.2009.08.008.PubMed CentralView ArticlePubMedGoogle Scholar
- Simone C, Forcales SV, Hill DA, Imbalzano AN, Latella L, Puri PL: p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci. Nat Genet. 2004, 36: 738-743. 10.1038/ng1378.View ArticlePubMedGoogle Scholar
- Wu Z, Woodring PJ, Bhakta KS, Tamura K, Wen F, Feramisco JR, Karin M, Wang JY, Puri PL: p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps. Mol Cell Biol. 2000, 20: 3951-3964. 10.1128/MCB.20.11.3951-3964.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Deak M, Clifton AD, Lucocq LM, Alessi DR: Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 1998, 17: 4426-4441. 10.1093/emboj/17.15.4426.PubMed CentralView ArticlePubMedGoogle Scholar
- Thomson S, Clayton AL, Hazzalin CA, Rose S, Barratt MJ, Mahadevan LC: The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J. 1999, 18: 4779-4793. 10.1093/emboj/18.17.4779.PubMed CentralView ArticlePubMedGoogle Scholar
- Soloaga A, Thomson S, Wiggin GR, Rampersaud N, Dyson MH, Hazzalin CA, Mahadevan LC, Arthur JS: MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14. EMBO J. 2003, 22: 2788-2797. 10.1093/emboj/cdg273.PubMed CentralView ArticlePubMedGoogle Scholar
- Gehani SS, Agrawal-Singh S, Dietrich N, Christophersen NS, Helin K, Hansen K: Polycomb group protein displacement and gene activation through MSK-dependent H3K27me3S28 phosphorylation. Mol Cell. 2010, 39: 886-900. 10.1016/j.molcel.2010.08.020.View ArticlePubMedGoogle Scholar
- Lau PN, Cheung P: Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing. Proc Natl Acad Sci USA. 2011, 108: 2801-2806. 10.1073/pnas.1012798108.PubMed CentralView ArticlePubMedGoogle Scholar
- Buckingham M: Skeletal muscle development and the role of the myogenic regulatory factors. Biochem Soc Trans. 1996, 24: 506-509.View ArticlePubMedGoogle Scholar
- Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K: Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. EMBO J. 2004, 23: 4061-4071. 10.1038/sj.emboj.7600402.PubMed CentralView ArticlePubMedGoogle Scholar
- Li D, Niu Z, Yu W, Qian Y, Wang Q, Li Q, Yi Z, Luo J, Wu X, Wang Y, Schwartz RJ, Liu M: SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin. Nucleic Acids Res. 2009, 37: 7059-7071. 10.1093/nar/gkp773.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferri P, Barbieri E, Burattini S, Guescini M, D'Emilio A, Biagiotti L, Del Grande P, De Luca A, Stocchi V, Falcieri E: Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem. 2009, 108: 1302-1317. 10.1002/jcb.22360.View ArticlePubMedGoogle Scholar
- Davies SP, Reddy H, Caivano M, Cohen P: Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000, 351: 95-105. 10.1042/0264-6021:3510095.PubMed CentralView ArticlePubMedGoogle Scholar
- Vicent GP, Ballare C, Nacht AS, Clausell J, Subtil-Rodriguez A, Quiles I, Jordan A, Beato M: Induction of progesterone target genes requires activation of Erk and Msk kinases and phosphorylation of histone H3. Mol Cell. 2006, 24: 367-381. 10.1016/j.molcel.2006.10.011.View ArticlePubMedGoogle Scholar
- Brami-Cherrier K, Lavaur J, Pages C, Arthur JS, Caboche J: Glutamate induces histone H3 phosphorylation but not acetylation in striatal neurons: role of mitogen- and stress-activated kinase-1. J Neurochem. 2007, 101: 697-708.View ArticlePubMedGoogle Scholar
- Aoto T, Saitoh N, Sakamoto Y, Watanabe S, Nakao M: Polycomb group protein-associated chromatin is reproduced in post-mitotic G1 phase and is required for S phase progression. J Biol Chem. 2008, 283: 18905-18915. 10.1074/jbc.M709322200.View ArticlePubMedGoogle Scholar
- Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K: Genome-wide mapping of polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 2006, 20: 1123-1136. 10.1101/gad.381706.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K, Koseki H, Fuchikami T, Abe K, Murray HL, Zucker JP, Yuan B, Bell GW, Herbolsheimer E, Hannett NM, Sun K, Odom DT, Otte AP, Volkert TL, Bartel DP, Melton DA, Gifford DK, Jaenisch R, Young RA: Control of developmental regulators by polycomb in human embryonic stem cells. Cell. 2006, 125: 301-313. 10.1016/j.cell.2006.02.043.PubMed CentralView ArticlePubMedGoogle Scholar
- Weintraub H, Tapscott SJ, Davis RL, Thayer MJ, Adam MA, Lassar AB, Miller AD: Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci USA. 1989, 86: 5434-5438. 10.1073/pnas.86.14.5434.PubMed CentralView ArticlePubMedGoogle Scholar
- Tapscott SJ: The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development. 2005, 132: 2685-2695. 10.1242/dev.01874.View ArticlePubMedGoogle Scholar
- Berghella L, De Angelis L, De Buysscher T, Mortazavi A, Biressi S, Forcales SV, Sirabella D, Cossu G, Wold BJ: A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle. Genes Dev. 2008, 22: 2125-2138. 10.1101/gad.468508.PubMed CentralView ArticlePubMedGoogle Scholar
- Moresi V, Williams AH, Meadows E, Flynn JM, Potthoff MJ, McAnally J, Shelton JM, Backs J, Klein WH, Richardson JA, Bassel-Duby R, Olson EN: Myogenin and class II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases. Cell. 2010, 143: 35-45. 10.1016/j.cell.2010.09.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Papp B, Muller J: Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 2006, 20: 2041-2054. 10.1101/gad.388706.PubMed CentralView ArticlePubMedGoogle Scholar
- Ringrose L, Ehret H, Paro R: Distinct contributions of histone H3 lysine 9 and 27 methylation to locus-specific stability of polycomb complexes. Mol Cell. 2004, 16: 641-653. 10.1016/j.molcel.2004.10.015.View ArticlePubMedGoogle Scholar
- Mohn F, Weber M, Rebhan M, Roloff TC, Richter J, Stadler MB, Bibel M, Schubeler D: Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol Cell. 2008, 30: 755-766. 10.1016/j.molcel.2008.05.007.View ArticlePubMedGoogle Scholar
- Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK, Bell GW, Otte AP, Vidal M, Gifford DK, Young RA, Jaenisch R: Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature. 2006, 441: 349-353. 10.1038/nature04733.View ArticlePubMedGoogle Scholar
- Sims RJ, Rojas LA, Beck D, Bonasio R, Schuller R, Drury WJ, Eick D, Reinberg D: The C-terminal domain of RNA polymerase II is modified by site-specific methylation. Science. 2011, 332: 99-103. 10.1126/science.1202663.PubMed CentralView ArticlePubMedGoogle Scholar
- Seenundun S, Rampalli S, Liu QC, Aziz A, Palii C, Hong S, Blais A, Brand M, Ge K, Dilworth FJ: UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis. EMBO J. 2010, 29: 1401-1411. 10.1038/emboj.2010.37.PubMed CentralView ArticlePubMedGoogle Scholar
- Asp P, Blum R, Vethantham V, Parisi F, Micsinai M, Cheng J, Bowman C, Kluger Y, Dynlacht BD: PNAS Plus: genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci USA. 2011Google Scholar
- Villa R, Pasini D, Gutierrez A, Morey L, Occhionorelli M, Viré E, Nomdedeu JF, Jenuwein T, Pelicci PG, Minucci S, Fuks F, Helin K, Di Croce L: Role of the polycomb repressive complex 2 in acute promyelocytic leukemia. Cancer Cell. 2007, 11: 513-525. 10.1016/j.ccr.2007.04.009.View ArticlePubMedGoogle Scholar
- Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K: EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 2003, 22: 5323-5335. 10.1093/emboj/cdg542.PubMed CentralView ArticlePubMedGoogle Scholar
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