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A cytosolic Ezh1 isoform modulates a PRC2–Ezh1 epigenetic adaptive response in postmitotic cells

Abstract

The evolution of chromatin-based epigenetic cell memory may be driven not only by the necessity for cells to stably maintain transcription programs, but also by the need to recognize signals and allow plastic responses to environmental stimuli. The mechanistic role of the epigenome in adult postmitotic tissues, however, remains largely unknown. In vertebrates, two variants of the Polycomb repressive complex (PRC2–Ezh2 and PRC2–Ezh1) control gene silencing via methylation of histone H3 on Lys27 (H3K27me). Here we describe a reversible mechanism that involves a novel isoform of Ezh1 (Ezh1β). Ezh1β lacks the catalytic SET domain and acts in the cytoplasm of skeletal muscle cells to control nuclear PRC2–Ezh1 activity in response to atrophic oxidative stress, by regulating Eed assembly with Suz12 and Ezh1α (the canonical isoform) at their target genes. We report a novel PRC2–Ezh1 function that utilizes Ezh1β as an adaptive stress sensor in the cytoplasm, thus allowing postmitotic cells to maintain tissue integrity in response to environmental changes.

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Figure 1: Ezh1 determines the increase in global H3K27me3 levels in disuse muscle atrophy.
Figure 2: The PRC2–Ezh1 complex is redistributed on muscle-specific targets.
Figure 3: PRC2–Ezh1 is required for the repression of atrophy-related genes after oxidative stress.
Figure 4: Ezh1 has a cytoplasmic protein isoform, Ezh1β, that interacts with Eed in the cytosolic compartment.
Figure 5: After oxidative stress, Ezh1β is proteolytically degraded while Eed relocalizes from the cytosol to the nucleus.
Figure 6: Ezh1β counterbalances PRC2–Ezh1's nuclear assembly and repressive function on target genes.

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Acknowledgements

We are grateful to C. Desplan, P. Sassone-Corsi, D. Gabellini, E. Battaglioli and S. Biffo for discussions and critical revision of the manuscript; G. Natoli (IFOM-IEO Campus, Milan, Italy) for sharing the Jmjd3 antibody; M. Mora (“Cells, tissues and DNA from patients with neuromuscular diseases” Telethon Biobank, Milan, Italy) for providing human primary myoblasts; R. Margueron (Institute Curie, Paris, France) for providing pCDNA-4TO-Ezh1α-HA plasmid; Sequentia Biotech SL, R. Bonnal and C. Cheroni for bioinformatical support; and M. Moro and M.C. Crosti for technical assistance with cell sorting. This work was supported by the EPIGEN Italian flagship program and King Abdullah University of Science and Technology (KAUST) (to V.O.).

Author information

Authors and Affiliations

Authors

Contributions

B.B. conceived the study, designed and performed experiments, analyzed the data and wrote the manuscript. F.M. designed and performed experiments, analyzed the data and wrote the manuscript. V.R. performed RNA-seq and ChIP-seq analyses. A.C. set up, performed and analyzed FRET and GST pulldown biochemical assays. F.D.V. performed and analyzed immunofluorescence assays and set up the in vivo model of atrophy. M.V.N. set up the protocol for obtaining histone extracts from muscle tissue. M.W. produced and shared the Ezh1α antibody (Margueron lab). A.Z. conceived and analyzed FRET and GST pulldown biochemical assays. C.L. conceived and analyzed immunofluorescence assays and carried out data analyses. M.P. conceived and analyzed RNA-seq and ChIP-seq analyses, and provided technical support for Ezh1β-C antibody design and the western blotting setup. V.O. conceived the study, designed experiments, analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Beatrice Bodega or Valerio Orlando.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Atrophic myotubes display a global increase in H3K27me3 histone marks.

a. Western blots detecting Ezh1, Ezh2, Myogenin, mCK, protein level during C2C12 differentiation (MB, myoblasts, MT1, myotubes day 1, MT2, myotubes day 2, MT4, myotubes day 4) in nuclear (Nuc) and cytosolic (Cyt) extracts. Lamin B and Gapdh were used as loading controls. (Independent experiments: n=2). b. Western blot detecting H3K27me3 levels in histone extracts during C2C12 differentiation (MB, myoblasts, MT2, myotubes day 2, MT4, myotubes day 4). H3 was used as the loading control. (Independent experiments: n=3). c. Left, expression levels of Atrogin1 gene in control (MT) and H2O2 (MT–H2O2) treated myotubes and Hsp70 gene in control (MT) and heat-shock-treated (MT-43°C) myotubes. Data were normalized on Gapdh expression. Mean and s.e.m. (independent experiments: n=7 for Atrogin1; n=4 for Hsp70). Two-tailed paired t-test: Atrogin1 p=0.0352. One-tailed paired t-test: Hsp70 p=0.046. Right, western blot detecting Myogenin and Myosin protein levels in control, H2O2 or 43°C heat shock treated myotubes. (Independent experiments: n=2). d. FACS profile in control and H2O2 treated myotubes; each panel represents percentage of apoptotic cells in G0. e. Western blots detecting H3K27me3 in control (MT) and H2O2 or heat shock (43°C) treatments quantified in Figure 1a. H3 was used as loading control. (Independent experiments: n=9 for control-H2O2; n=4 for control-43°C) f. Left, western blots detecting H3K27me3, H3K9me3, H3K27me1, H3K4me3 in histone extracts of control and H2O2 treated myotubes. Three biological replicates (I,II,III) were loaded on the same gel and signals were detected with Starion FLA 9000 through appropriate secondary antibody conjugated to fluorophores. H3 was used as loading control. Right, corresponding densitometric quantification of the blots. Mean and s.e.m. (independent experiments: n=3). Two-way ANOVA analysis of variance with Bonferroni post-tests correction, p<0.0001. F=5.619. g. Left, western blot detecting H3K27me3 levels in histone extracts of control (MT) and 43°C heat shock treated myotubes. Three biological replicates (I,II,III) were loaded on the same gel and signals detected with Starion FLA 9000 through appropriate secondary antibody conjugated to fluorophores. H3 was used as loading control. Right, corresponding densitometric quantification of the blots. Mean and s.e.m.(independent experiments: n=3). h. Left, expression levels of Atrogin1 gene in control and FeSO4 treated myotubes. Data were normalized on Gapdh expression. Mean and s.e.m. (independent experiments: n=4). Two-tailed paired t-test: Atrogin1 p=0.0007. Right, western blots detecting H3K27me3 in histone extracts of control (MT) and FeSO4 treated myotubes. H3 was used as loading control. (Independent experiments: n=2). i. Western blots detecting MYOGENIN and H3K27me3 levels in control and H2O2 treated human primary myotubes. LAMIN B and H3 were used as loading control. (Independent experiments: n=1). j. Western blots detecting H3K27me3 in control and H2O2 treated myotubes, depleted or not of Ezh1 quantified in Figure 1b. H3 was used as loading control. (Independent experiments: n=3). k. Western blots detecting Ezh1 and Ezh2 protein levels in control and H2O2 treated myotubes, depleted or not of Ezh1. α Tubulin was used as loading control. (Independent experiments: n=2).

Source data

Supplementary Figure 2 H3K27me3 levels increase in disuse muscle atrophy in vivo.

a. Left, Histone methyltransferase activity on H3K27me3 peptide associated to immunoprecipitated Ezh1 and Ezh2 proteins from control (MT) and H2O2 treated myotubes (MT-H2O2) nuclear extracts. Mean and s.e.m. (Independent experiments: n=3 for Ezh1, n=4 for Ezh2). One-tailed paired t-test: Ezh1 MT versus MT–H2O2, p=0.048. Right, corresponding immunoprecipitation and western blot analyses for Ezh1 and Ezh2 in nuclear extracts from myotubes and myotubes treated with H2O2. Isotype-matched IgG is the control antibody used for IPs. The assay was optimized testing different concentrations of Ezh1 antibody for coIP (Ezh1i and Ezh1ii) and using Suz12 IP as control. Please refer to ‘Online Methods’. b. Representative immuno-staining of tibialis anterior muscle transverse sections of control, disuse atrophy and recovery mice. Nuclei are counterstained in DAPI (blue) and stained for Laminin (red). Original magnification 20X. The scale bar is indicated. Right, anti-Laminin-stained sections were used to measure cross-sectional areas of the fibers (CSA); the graph represents the distribution of fibers (% of Fibers) with respect to their area (μm2); 400-600 fibers for each replicate were evaluated. Mean ± s.e.m. (mice: n=4 for control, n=2 for Disuse and Recovery, plotted with single values). c. Expression levels of Atrogin1, Murf1, Myh3 genes in extensor digitorum longus muscles of control, disuse atrophy and recovery mice. Data were normalized on Gapdh expression. Mean and s.e.m. (mice: n=3). One-tailed paired t-test: Atrogin1 control versus Disuse p=0.0068, control versus Recovery p=0.0274; Murf1 control versus Recovery p=0.0017. d. Western blots detecting H3K27me3 histone mark in histone extracts of tibialis anterior muscles of control (nc), disuse atrophy (DA) and recovery mice (R). Three mice (I, II, III) are represented. H3 was used as the loading control e. Schematic representation of recovery experiment in C2C12 myotubes: after H2O2 treatment, the medium was replaced with fresh medium; cells were collected 24 h after the treatment (Atrophy, H2O2) and 48 h after medium replacement (Recovery). f. Western blots detecting H3K27me3 level in histone extracts of control (MT), H2O2-treated cells (H2O2) and recovered cells (R); H3 was used as loading control. (Independent experiments: n=2). g. Integrated fluorescence intensity expressed in H3K27me3 fluorescence units (FU) in function of nuclei area, referred to Fig. 1d. Relative regression curves are represented.

Source data

Supplementary Figure 3 Additional information for ChIP-seq and RNA-seq data sets.

a. Schematic representation of the strategy adopted to analyze ChIP seq and RNA seq datasets; processing, analysis and integration steps are listed. b. Venn diagrams representing target gene integration between Ezh1, Eed and Suz12 gene targets identified by ChIP-seq in control (MT) and H2O2-teated myotubes (MT-H2O2). c. Global chromosomal distribution of Ezh1, Eed, and Suz12 ChIP peaks in myotubes (MT) and oxidative stress treated Myotubes (MT-H2O2). d. Targets from “Organismal injury and abnormalities” and “Cellular growth and proliferation” networks reported in Figure 2b are bound by PRC2-Ezh1 complex (Ezh1, Suz12 and Eed) in MT but not in MT-H2O2 (Cd244 and Sfi1). e. PRC2-Ezh1 target genes which are bound by PRC2-Ezh1 complex in MT and MT-H2O2 (belonging to the 206 common genes indicated in the Venn diagram in Fig. 2a) (Krt78 and Mroh2a).

Supplementary Figure 4 PRC2–Ezh1 complex is redistributed on muscle-specific targets that become repressed; validations and controls.

a. Ezh1, Eed and Suz12 ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT) and H2O2-treated myotubes (MT-H2O2). Results are represented as fold enrichment over the mock. Mean and s.e.m. (Independent experiments: n=4, except for Eed, n=3), One-tailed paired t-test: for Ezh1 ChIP: Myogenin MT versus MT-H2O2 p=0.014, Myh3 MT versus MT-H2O2 p=0.021; for Eed ChIP: Myh3 MT versus MT-H2O2 p=0.003; for Suz12 ChIP: Myog MT versus MT-H2O2 p=0.006. b. Ezh2 ChIP-qPCR analysis on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT) and H2O2-teated myotubes (MT-H2O2). Results are represented as fold enrichment over the mock. Mean and s.e.m. (Independent experiments: n=6). c. Left, western blots detecting HA-Ezh1 and Myogenin protein levels in nuclear (Nuc) and cytosolic (Cyt) extracts of C2C12 stable line overexpressing HA tagged-Ezh1. (Independent experiments: n=1). Right, HA ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control and H2O2-treated myotubes overexpressing HA tagged Ezh1. Results are represented as fold enrichment over the mock. Mean is indicated (Independent experiments: n=2, plotted with single values). d. H3K27me3 ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT), H2O2-treated (MT-H2O2) and Ezh1 depleted myotubes treated with H2O2 (MT-H2O2 Ezh1 kd). ChIP results are represented as percentage of immunoprecipitated H3. Mean and s.e.m. (Independent experiments: n=4 in MT and MT-H2O2, n=2 in MT-H2O2 Ezh1 kd, plotted with single values). One-tailed paired t-test: Myh3, MT versus MT-H2O2 p=0.041. e. Expression levels of Myogenin, Myh3, Desmin and NeuroG1 genes in control (MT) and FeSO4 treated (MT-FeSO4) myotubes. Data were normalized on Gapdh expression. Mean and s.e.m. (Independent experiments: n=3). Two-tailed paired t-test: Myogenin p=0.018. f. Expression levels of Myogenin, Myh3, Desmin, and NeuroG1 genes in control (MT) and 43°C heat-shock-treated (MT-43°C) myotubes. Data were normalized on Gapdh expression. Mean and s.e.m. (Independent experiments: n=4 for Myog, n=5 for Myh3 and Des, n=2 for NeuroG1, plotted with single values). Two-tailed paired t-test: Myh3, p=0.022. g. Expression levels of Myogenin and Myh3 genes, in control (MT), H2O2-treated (MT-H2O2) and recovered myotubes (MT Recovery). Data were normalized on Gapdh expression. Mean is indicated (Independent experiments: n=2, plotted with single values). h. Jmjd3 ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT) and H2O2 treated myotubes (MT-H2O2). ChIP results are represented as fold enrichment over the mock. Mean and s.e.m. (Independent experiments: n=3).

Source data

Supplementary Figure 5 Ezh1α and Ezh1β isoform expression levels in adult tissues, and specific antibody characterization.

a. PCR performed on C2C12 myotubes cDNA with primers specific for Ezh1β coding sequence. The amplicon is detected at the expected molecular weight (1740 bp); M: 1kb ladder, 1: Ezh1β amplicon, 2: no template. (Independent experiments: n=1). b. Expression levels of genes encoding Ezh1α, Ezh1β and Ezh2 in different mouse tissues (St: stomach, Lu: lung, B: brain, Li: liver, Spl: spleen and Sk.M: skeletal muscle). Data were normalized on Gapdh expression. Mean is indicated (mice: n=2, plotted with single values). c. Western blots detecting EGFP or Ezh1β protein in total extract of NIH3T3 transfected or not transfected with Ezh1α-YFP or Ezh1β-YFP. Ezh1β C-term and EGFP antibodies were used for detection. Actin was used as the loading control. (Independent experiments: n=2). d. Immunoprecipitation with Ezh1 β C-term antibody and western blot in NIH3T3 total extract transfected with Ezh1α-YFP or Ezh1β-YFP. Ezh1β C-term and EGFP antibodies were used for detection. (Independent experiments: n=2). e. Western blots detecting HA or Ezh1αin NIH3T3 total extract transfected or not transfected with Ezh1β-EGFP or Ezh1α-HA. Ezh1 antibody from Raphael Margueron’s lab (Ezh1α RM) and EGFP antibodies were used for detection. Actin was used as loading control. (Independent experiments: n=1). f Co-immunoprecipitation (co-IP) and western blot analyses for Ezh1β and Eed in myotube nuclear extracts. Isotype-matched IgG represent a control antibody used for IPs. Eed isoform is specified. (Independent experiments: n=2). g. Co-immunoprecipitation (co-IP) and western blot analyses for Ezh1β, Ezh1α and Suz12 in cytosolic and nuclear extracts from myotubes. Isotype-matched IgG is the control antibody used for IPs. (Independent experiments: n=2).

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Supplementary Figure 6 In atrophic myotubes, Eed redistributes in the nucleus, and Ezh1β is proteolytically degraded.

a. Western blots detecting Eed protein levels in nuclear and cytosolic extracts of C2C12 muscle cells depleted or not of Eed. H3 and α Tubulin were used as the loading controls. (Independent experiments: n=1). Eed isoforms are specified. b. Left, representative immuno-staining for Suz12 (green) and Eed (red) in control (MT) and H2O2-treated myotubes (MT-H2O2) (nuclei are counterstained in DAPI, blue). Original magnification 63X. The scale bar is indicated. Right, correlation between Suz12 nuclear mean intensity (FU) (Y axis) and Eed nuclear mean intensity (FU) (X axis) in control (MT) and H2O2-treated myotubes (MT-H2O2). Regression curves are represented (nuclei: n=458 in MT, n=360 in MT-H2O2). Fisher’s Z test: p=0.0262. c. Mean intensity of nuclear and cytosolic Eed and Suz12 in control (MT) and H2O2-treated myotubes (MT-H2O2). The fluorescence signal for each nucleus was calculated; to derive cytosolic signal, we subtracted the sum of the mean nuclear fluorescence values, from the mean fluorescence of the field. Mean and s.e.m. (for Nuc, nuclei: n=458 in MT, n=360 in MT-H2O2; for Cyt, fields: n=8 in MT, n=9 in MT-H2O2). Two-tailed unpaired t-test: Nuclear Eed MT versus MT-H2O2 p=0.0045; Cytosolic Eed MT versus MT-H2O2 p=0.0466. d. Scatter plot representing Pearson correlation values of Suz12-Eed nuclear co-staining in control (MT) and H2O2-treated myotubes (MT-H2O2). Mean ± s.e.m. (nuclei: n=458 in MT, n=360 in MT-H2O2). Two-tailed unpaired t-test: p=0.0279. e. Left, representative image of PLA detecting Eed-Suz12 interaction in control and H2O2-treated myotubes (See Methods). The scale bar is indicated. Right, scatter plot representing the corresponding PLA nuclei intensity. Mean ± s.e.m. (nuclei: n=153). Two-tailed unpaired t-test: p < 0.0001. f. Right, luminescence measurement of Ezh1β-Nanoluc or Eed-Nanoluc in control and H2O2 treated NIH3T3. Mean and s.e.m. (independent experiments: n=8 for Ezh1β, n=6 for Eed). One-tailed paired t-test: p=0.0279. g. Left, western blot detecting Eed, Suz12, Ezh1α and Ubiquitin in total extracts of control, H2O2, MG132 and MG132-H2O2-treated myotubes. β Tubulin is used as the loading control. Right, corresponding Eed, Suz12 and Ezh1α densitometric quantification. Mean and s.e.m. (independent experiments: n=3). Eed isoform is specified. h. Western blots detecting Ezh1β and Ubiquitin in total extract of control, H2O2, MG132 and MG132-H2O2-treated NIH3T3 cells. Actin is used as the loading control. (Independent experiments: n=2, represented on the same blot). i. Co-IP and western blot analyses for Ezh1β and Ubiquitin (Ubq) in cytosolic extracts of MG132 and MG132-H2O2-treated myotubes. Isotype-matched IgG is the control antibody used for IPs. (Independent experiments: n=2). j. Expression levels of Ezh1β or Eed in YFP (Mock), Eed-CFP or Ezh1β-YFP transfected myotubes treated or not with H2O2. Data were normalized on Gapdh expression. Mean and s.e.m. (Independent experiments: n=3).

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Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 1664 kb)

Supplementary Table 1

ChIP-seq and RNA-seq results ChIP-seq peaks, IPA networks, differentially expressed genes and Gene Ontology enrichment analysis terms are reported in different sheets of the table. (XLSX 121 kb)

Supplementary Table 2

List of oligonucleotides and antibodies Different sheets of the table show a list of oligonucleotide sequences for RT-qPCR, ChIP and siRNA, and a list of antibodies used for western blotting, immunoprecipitation and immunofluorescence. (XLSX 43 kb)

Supplementary Data Set 1

Uncropped western blots. Uncropped images of the immunoblots displayed in the main figures. The figure number and panel to refer back to are indicated, and molecular weights are specified. Dashed boxes indicate the areas reported in the main figures. (PDF 15694 kb)

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Bodega, B., Marasca, F., Ranzani, V. et al. A cytosolic Ezh1 isoform modulates a PRC2–Ezh1 epigenetic adaptive response in postmitotic cells. Nat Struct Mol Biol 24, 444–452 (2017). https://doi.org/10.1038/nsmb.3392

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