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. 2012 Oct 15;11(20):3828-36.
doi: 10.4161/cc.22025. Epub 2012 Sep 14.

The ablation of EZH2 uncovers its crucial role in rhabdomyosarcoma formation

Irene Marchesi  1 , Francesco Paolo Fiorentino, Flavio Rizzolio, Antonio Giordano, Luigi Bagella
Affiliations

The ablation of EZH2 uncovers its crucial role in rhabdomyosarcoma formation

Irene Marchesi et al. Cell Cycle. .

Abstract

Rhabdomyosarcoma (RMS) is a pediatric tumor that arises from muscle precursor cells. RMS cells express several markers of early myogenic differentiation, but they fail to complete both differentiation program and cell cycle arrest, resulting in uncontrolled proliferation and incomplete myogenesis. Previous studies showed that EZH2, which is involved in both differentiation and cancer progression, is overexpressed in RMS, but a functional binding between its expression and its functional role in tumor formation or progression has not yet been demonstrated. We hypothesized that EZH2 is a key regulator of muscular differentiation program in RMS cells. In this study, we demonstrated that EZH2 directly binds muscle specific genes in RD cells. Silencing of EZH2 promotes the recruitment of a multiprotein complex at muscle-specific promoters, their transcriptional activation and protein expression. Moreover, we demonstrated that EZH2 is directly involved in transcriptional repression of MyoD, the main factor promoting myogenesis. EZH2 ablation induces MyoD activation the recovery of its binding on muscle-specific genes.

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Figures

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Figure 1. RD cells fail to complete the differentiation program and show high levels of EZH2. Myoblasts (C2C12) and RMS cells (RD) were grown to 90% and induced to differentiate by serum withdrawal and addition of 2% horse serum (DM) for 96 h. Pellets were collected each 24 h. (A) Immunoblot analysis reveals the expression of EZH2 in concurrence with decreased levels of MyoD, Myogenin and Myh expression. HSP70 was used as loading control. (B) C2C12 and RD phenotype after 96 h of differentiation induction. C2C12 cells form myotubes, whereas no phenotypic changes can be seen in RD. (C) EZH2 transcription at various times after differentiation induction. Although in differentiation conditions, RD cells maintain EZH2 expression.
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Figure 2. EZH2 binds muscle-specific gene promoters in RD cells. Chromatin immunoprecipitation revealed that EZH2 binds muscle-specific gene promoters in RD cells. The chromatin-immunoprecipitated DNA was amplified by real-time PCR using specific primers for myogenin (A), Myh (B) and MyoD (C) promoter regions. Input DNA was used as loading reference and normal IgG control as calibrator.
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Figure 3. EZH2-knockdown RD cells show partial reversion of tumor phenotype. Real-time PCR (A) and immunoblotting (B) show a decrease of EZH2 levels of 80% in EZH2 knockdown RD (kd) compared with RD wild type (wt) and scramble (scr). (C) RD wild type, RD scramble and RD EZH2-knockdown morphology in GM and after 96 h in DM. RD EZH2 knockdown show the formation of several myotube-like cells. Black arrows indicate myotube-like morphology.
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Figure 4. EZH2 inhibits the expression of skeletal muscle-specific genes. Differentiation induction in absence of EZH2 results in a significant increase of muscle-specific genes. (A) Real-time qPCR was performed using cDNA from RD wild type and EZH2 knockdown at several stage of differentiation (24h-96 h). Reported data were normalized to GAPDH levels. EZH2-kd RD cells showed increase of muscle specific gene transcription following differentiation induction. (B) Immunoblot analysis reveals that protein levels of muscle specific genes increase in EZH2-knockdown RD cells following differentiation induction. (C) Luciferase assay shows that EZH2-knockdown cells have a greater MyoD promoter activation following differentiation promotion. The assay was conducted on RD wild type and RD EZH2-knockdown cells transfected with MyoD-luc reporter and RL-TK plasmid as calibrator. Transfected cells were cultured in DM for 72 h. Luciferase activity was normalized to TK-directed Renilla expression. The results are expressed in arbitrary units relative to the activity of the basic luciferase vector control. Student’s t-test was used to evaluate statistical significance: * p < 0.05, ** p < 0.01. Values were obtained by the average of at least three independent experiments.
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Figure 5. EZH2 ablation restores MyoD binding on muscle-specific genes. ChIP assays, performed in RD wild type and EZH2 knockdown 72 h following differentiation induction, show (A) the recovery of MyoD binding at myogenin and Myh regulatory regions in absence of EZH2, (B) a strong decrease of H3K27me3 enrichment at myogenin and Myh promoters. Student’s t-test was used to evaluate statistical significance: * p < 0.05, ** p < 0.01. Values were obtained by the average of at least three independent experiments.
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Figure 6. EZH2 ablation restores the phosphorylation of RNA polymerase II on muscle-specific genes promoting transcriptional activation. Silencing of EZH2 promotes the recruitment of transcriptional activation complex at MyoD-dependent genes promoting phosphorylations of RNA PolII CTD. Chromatin immunoprecipitation assay was performed 72 h after induction of myogenesis. Samples were analyzed with real-time PCR using specific primers for the myogenin and Myh promoter regions (A and B, respectively). Input DNA was used as reference and normal IgG immunoprecipitated DNA as calibrator. The data were compared with RD wild type precipitated DNA. Student’s t-test was used to evaluate statistical significance. * p < 0.05, ** p < 0.01. Values were obtained by the average of at least three experiments.
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References

    1. Merlino G, Helman LJ. Rhabdomyosarcoma--working out the pathways. Oncogene. 1999;18:5340–8. doi: 10.1038/sj.onc.1203038. - DOI - PubMed
    1. Otten AD, Firpo EJ, Gerber AN, Brody LL, Roberts JM, Tapscott SJ. Inactivation of MyoD-mediated expression of p21 in tumor cell lines. Cell Growth Differ. 1997;8:1151–60. - PubMed
    1. Sebire NJ, Malone M. Myogenin and MyoD1 expression in paediatric rhabdomyosarcomas. J Clin Pathol. 2003;56:412–6. doi: 10.1136/jcp.56.6.412. - DOI - PMC - PubMed
    1. Saab R, Spunt SL, Skapek SX. Chapter 7 - Myogenesis and Rhabdomyosarcoma: The Jekyll and Hyde of Skeletal Muscle. 1sted. Elsevier Inc; 2011. - PubMed
    1. Puri PL, Sartorelli V. Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and post-transcriptional modifications. J Cell Physiol. 2000;185:155–73. doi: 10.1002/1097-4652(200011)185:2<155::AID-JCP1>3.0.CO;2-Z. - DOI - PubMed

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