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• W1965784018 abstract "Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1α, HP1β, and HP1γ play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1α and HP1β isoforms, but not HP1γ, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1α and HP1β, but not HP1γ, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner. Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1α, HP1β, and HP1γ play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1α and HP1β isoforms, but not HP1γ, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1α and HP1β, but not HP1γ, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner. Mammalian heterochromatin protein 1 (HP1) 5The abbreviations used are:HP1heterochromatin protein 1CSDchromoshadow domainH3K9histone H3 lysine 9HDAChistone deacetylaseMCKmuscle creatine kinaseHAhemagglutininGSTglutathione S-transferaselChIPchromatin immunoprecipitationIPimmunoprecipitation. isoforms are closely related non-histone proteins that are involved in transcriptional regulation and chromatin organization. In mammals, HP1 exists in three isoforms: α, β, and γ (Ref. 1Hediger F. Gasser S.M. Curr. Opin. Genet. Dev. 2006; 16: 143-150Crossref PubMed Scopus (126) Google Scholar and references therein). Each is composed of a conserved chromodomain that is important for heterochromatin binding and a chromoshadow domain (CSD), whose structure is similar to that of the chromodomain that is involved in dimerization and interaction with proteins containing the consensus sequence PXVXL (2Cavalli G. Paro R. Curr. Opin. Cell Biol. 1998; 10: 354-360Crossref PubMed Scopus (154) Google Scholar). The chromodomain and the CSD are separated by a less conserved region called the hinge region. HP1 isoforms exhibit different subnuclear localizations in interphasic nuclei: HP1α is mainly centromeric; HP1β is also centromeric but to a lesser extent; and HP1γ is located in both euchromatic and heterochromatic compartments (3Dialynas G.K. Terjung S. Brown J.P. Aucott R.L. Baron-Luhr B. Singh P.B. Georgatos S.D. J. Cell Sci. 2007; 120: 3415-3424Crossref PubMed Scopus (63) Google Scholar). HP1 proteins are known to bind methylated histone H3 lysine 9 (H3K9) and heterodimerize, contributing to the formation and maintenance of heterochromatic structures (4Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2177) Google Scholar, 5Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2184) Google Scholar). HP1 isoforms interact with a wide variety of proteins, mainly chromatin-associated proteins (Refs. 1Hediger F. Gasser S.M. Curr. Opin. Genet. Dev. 2006; 16: 143-150Crossref PubMed Scopus (126) Google Scholar and 6Lomberk G. Wallrath L. Urrutia R. Genome Biol. 2006; 7: 228-235Crossref PubMed Scopus (194) Google Scholar and references therein). heterochromatin protein 1 chromoshadow domain histone H3 lysine 9 histone deacetylase muscle creatine kinase hemagglutinin glutathione S-transferasel chromatin immunoprecipitation immunoprecipitation. HP1 proteins have been implicated in many differentiation pathways (7Panteleeva I. Boutillier S. See V. Spiller D.G. Rouaux C. Almouzni G. Bailly D. Maison C. Lai H.C. Loeffler J.P. Boutillier A.L. EMBO J. 2007; 26: 3616-3628Crossref PubMed Scopus (42) Google Scholar, 8Meshorer E. Yellajoshula D. George E. Scambler P.J. Brown D.T. Misteli T. Dev. Cell. 2006; 10: 105-116Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, 9Cammas F. Herzog M. Lerouge T. Chambon P. Losson R. Genes Dev. 2004; 18: 2147-2160Crossref PubMed Scopus (88) Google Scholar, 10Cammas F. Oulad-Abdelghani M. Vonesch J.L. Huss-Garcia Y. Chambon P. Losson R. J. Cell Sci. 2002; 115: 3439-3448Crossref PubMed Google Scholar), although their individual roles are not always well understood. During terminal differentiation of cells, chromatin undergoes dramatic morphological changes (reviewed in Refs. 11Francastel C. Schubeler D. Martin D.I. Groudine M. Nat. Rev. Mol. Cell Biol. 2000; 1: 137-143Crossref PubMed Scopus (246) Google Scholar, 12Meshorer E. Misteli T. Nat. Rev. Mol. Cell Biol. 2006; 7: 540-546Crossref PubMed Scopus (554) Google Scholar, 13Guasconi V. Souidi M. Ait-Si-Ali S. Cancer Biol. Ther. 2005; 4: 134-138Crossref PubMed Scopus (14) Google Scholar), for example during muscle differentiation (14Moen Jr., P.T. Johnson C.V. Byron M. Shopland L.S. de la Serna I.L. Imbalzano A.N. Lawrence J.B. Mol. Biol. Cell. 2004; 15: 197-206Crossref PubMed Scopus (73) Google Scholar). These changes involve reorganization of constitutive heterochromatin (15Brero A. Easwaran H.P. Nowak D. Grunewald I. Cremer T. Leonhardt H. Cardoso M.C. J. Cell Biol. 2005; 169: 733-743Crossref PubMed Scopus (188) Google Scholar) and selective silencing and activation of specific groups of genes (Refs. 7Panteleeva I. Boutillier S. See V. Spiller D.G. Rouaux C. Almouzni G. Bailly D. Maison C. Lai H.C. Loeffler J.P. Boutillier A.L. EMBO J. 2007; 26: 3616-3628Crossref PubMed Scopus (42) Google Scholar, 16Ait-Si-Ali S. Guasconi V. Fritsch L. Yahi H. Sekhri R. Naguibneva I. Robin P. Cabon F. Polesskaya A. Harel-Bellan A. EMBO J. 2004; 23: 605-615Crossref PubMed Scopus (161) Google Scholar, and 17Narita M. Nunez S. Heard E. Narita M. Lin A.W. Hearn S.A. Spector D.L. Hannon G.J. Lowe S.W. Cell. 2003; 113: 703-716Abstract Full Text Full Text PDF PubMed Scopus (1713) Google Scholar; reviewed in Ref. 13Guasconi V. Souidi M. Ait-Si-Ali S. Cancer Biol. Ther. 2005; 4: 134-138Crossref PubMed Scopus (14) Google Scholar and references therein) and implicate both differential interactions of various proteins with chromatin and changes in the chromatin structure itself. Skeletal muscle terminal differentiation begins with an irreversible withdrawal from the cell cycle, followed by musclespecific marker expression with fusion of myoblasts into multinucleated myotubes (18Buckingham M. Biochem. Soc. Trans. 1996; 24: 506-509Crossref PubMed Scopus (37) Google Scholar). Cell cycle withdrawal corresponds to a definitive silencing of proliferation associated genes, such as E2F targets (Ref. 16Ait-Si-Ali S. Guasconi V. Fritsch L. Yahi H. Sekhri R. Naguibneva I. Robin P. Cabon F. Polesskaya A. Harel-Bellan A. EMBO J. 2004; 23: 605-615Crossref PubMed Scopus (161) Google Scholar; reviewed in Ref. 19Walsh K. Perlman H. Curr. Opin. Genet. Dev. 1997; 7: 597-602Crossref PubMed Scopus (265) Google Scholar and references therein). Terminal muscle differentiation is orchestrated by the myogenic basic helix loop helix transcription factors, such as MyoD, which is the master myogenic determination factor. In proliferating myoblasts, MyoD is expressed but is unable to activate its target genes even when it binds to their promoters (20Mal A. Harter M.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1735-1739Crossref PubMed Scopus (127) Google Scholar, 21Ohkawa Y. Marfella C.G. Imbalzano A.N. EMBO J. 2006; 25: 490-501Crossref PubMed Scopus (109) Google Scholar). Therefore, the requirement for MyoD to be continuously expressed in undifferentiated myoblasts is enigmatic. MyoD might have a repressive role at its target genes prior to initiating chromatin remodeling in differentiating cells (20Mal A. Harter M.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1735-1739Crossref PubMed Scopus (127) Google Scholar, 22Zhang C.L. McKinsey T.A. Olson E.N. Mol. Cell Biol. 2002; 22: 7302-7312Crossref PubMed Scopus (210) Google Scholar, 23de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar). In proliferating myoblasts, MyoD is associated with histone deacetylases (HDACs) and might actively suppress expression of its targets by inducing a locally repressive chromatin structure (20Mal A. Harter M.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1735-1739Crossref PubMed Scopus (127) Google Scholar, 24Fulco M. Schiltz R.L. Iezzi S. King M.T. Zhao P. Kashiwaya Y. Hoffman E. Veech R.L. Sartorelli V. Mol. Cell. 2003; 12: 51-62Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 25Mal A. Sturniolo M. Schiltz R.L. Ghosh M.K. Harter M.L. EMBO J. 2001; 20: 1739-1753Crossref PubMed Scopus (208) Google Scholar). It is known that histone deacetylation contributes to H3K9 methylation on these same promoters (22Zhang C.L. McKinsey T.A. Olson E.N. Mol. Cell Biol. 2002; 22: 7302-7312Crossref PubMed Scopus (210) Google Scholar). It has been shown that some MyoD target promoters, including p21 and myogenin, are methylated at H3K9 specifically in proliferating myoblasts (20Mal A. Harter M.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1735-1739Crossref PubMed Scopus (127) Google Scholar, 22Zhang C.L. McKinsey T.A. Olson E.N. Mol. Cell Biol. 2002; 22: 7302-7312Crossref PubMed Scopus (210) Google Scholar), a modification known to be bound by HP1 proteins. Here we show that HP1 proteins associate directly with MyoD in an isoform-dependent manner and inhibit its transcriptional activity. Indeed, MyoD interacts preferentially with the isoforms HP1α and HP1β, but not HP1γ. In addition, HP1 proteins are recruited to MyoD target promoters preferentially in proliferating myoblasts. Finally, overexpression of HP1 isoforms interferes with muscle terminal differentiation in an isoform-dependent manner. Taken together, these data strongly suggest that, in addition to its role as an activator of differentiation-specific genes, MyoD can also act as a transcriptional repressor in proliferating myoblasts in cooperation with specific isoforms of HP1 protein. Cell Culture and Transfection—C2C12, HEK 293, and HeLa cells were maintained using standard conditions. C2C12 cells were differentiated as described in Ref. 16Ait-Si-Ali S. Guasconi V. Fritsch L. Yahi H. Sekhri R. Naguibneva I. Robin P. Cabon F. Polesskaya A. Harel-Bellan A. EMBO J. 2004; 23: 605-615Crossref PubMed Scopus (161) Google Scholar. The biotin-streptavidin interaction studies were performed as described in Ref. 26Viens A. Mechold U. Lehrmann H. Harel-Bellan A. Ogryzko V. Anal. Biochem. 2004; 325: 68-76Crossref PubMed Scopus (48) Google Scholar. Stable Cell Lines Establishment—A HeLa cell line stably expressing MyoD was established with a transgene encoding for full-length MyoD, and C2C12 cell lines expressing HP1 isoforms were established with transgenes encoding for full-length HP1α, HP1β, and HP1γ as described in Ref. 27Robin P. Fritsch L. Philipot O. Svinarchuk F. Ait-Si-Ali S. Genome Biol. 2007; 8: R270Crossref PubMed Scopus (32) Google Scholar. The transgenes were tagged with double hemagglutinin (HA) and double FLAG epitopes at the N terminus as described in Ref. 27Robin P. Fritsch L. Philipot O. Svinarchuk F. Ait-Si-Ali S. Genome Biol. 2007; 8: R270Crossref PubMed Scopus (32) Google Scholar. HeLa and C2C12 control cell lines transduced with the empty vector were also established. Protein Complex Purification—MyoD complex characterization was performed as described in Ref. 27Robin P. Fritsch L. Philipot O. Svinarchuk F. Ait-Si-Ali S. Genome Biol. 2007; 8: R270Crossref PubMed Scopus (32) Google Scholar. Protein Extraction, Coimmunoprecipitations, and Western Blotting—The biotin-streptavidin interaction studies were performed as described in Ref. 26Viens A. Mechold U. Lehrmann H. Harel-Bellan A. Ogryzko V. Anal. Biochem. 2004; 325: 68-76Crossref PubMed Scopus (48) Google Scholar. Expression vectors for biotinylatable proteins are kind gifts from Dr. V. Ogryzko (26Viens A. Mechold U. Lehrmann H. Harel-Bellan A. Ogryzko V. Anal. Biochem. 2004; 325: 68-76Crossref PubMed Scopus (48) Google Scholar). For the study of the endogenous protein interactions, nuclear extracts were used for immunoprecipitation overnight, after which the immunoprecipitates were incubated with Ultralink immobilized protein A/G (Pierce) for 2 h at room temperature and washed with the dilution buffer 4–10 times. Plasmids, GST Fusions, and GST Pulldown—Expression vectors derived from pGEX for GST fusions of wild type HP1α, β, γ, and HP1α deletion mutants GST-HP1α(1–119), GST-HP1α(1–66), and GST-HP1α(67–119) were described in Ref. 28Nielsen A.L. Sanchez C. Ichinose H. Cervino M. Lerouge T. Chambon P. Losson R. EMBO J. 2002; 21: 5797-5806Crossref PubMed Scopus (73) Google Scholar. All of the plasmid constructs were expressed in Escherichia coli strain BL21 and purified using glutathione-Sepharose beads according to the manufacturer (Sigma). Purified proteins were quantified by Coomassie staining after SDS-PAGE separation and by the Bradford protein assay. BL21 cells were also used for bacterial expression of untagged MyoD as described in Ref. 29Polesskaya A. Duquet A. Naguibneva I. Weise C. Vervisch A. Bengal E. Hucho F. Robin P. Harel-Bellan A. J. Biol. Chem. 2000; 275: 34359-34364Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar. Beads coated with equal amounts of GST fusion proteins (1 μg) were incubated with 100 ng of recombinant MyoD in NTEN buffer (100 mm NaCl, 20 Mm Tris-HCl, pH 8, 0.5 mm EDTA, 0.1% Nonidet P-40) completed with protease inhibitors (Roche Applied Science) for 4 h at 30 °C. The beads were then washed four times with washing buffer (150 mm NaCl, 10 mm Tris-HCl, pH 7.5, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride) and resuspended, and the proteins were resolved by SDS-PAGE gel for Western blot analysis. For this GST pulldown assays using MyoD deletion mutants, HEK 293 cells were transfected with 10 μg of expression plasmids of tagged MyoD (wild type MyoD) or its deletion mutants (Cter, Nter, ΔCter, and ΔNter), using Lipofectamine (Qiagen). 48 h post-transfection, the cells were lysed in lysis buffer (300 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0,4% Nonidet P-40, 10 mm MgCl2) to extract MyoD or its deletion mutants. GST pulldown assays were then performed as described above. Gene Reporter Assays—HEK 293 cells at 60% confluence were cotransfected by calcium phosphate coprecipitation. 24 h post-transfection, the cells were lysed in a reporter lysis buffer (Promega, Charbonnières, France). Luciferase activity (Promega luciferase assay system) was determined and normalized to the level of β-galactosidase (Promega β-galactosidase enzyme assay system) and to the total protein amount. The plasmids used in the assay were pEMSV-MyoD, pEMSV, pcDNA3-HP1α, pcDNA3-HP1β, pcDNA3-HP1γ, pcDNA3, pMCK-Luciferase reporter gene, and pCMV-β-galactosidase. Antibodies—The C-20 anti-MyoD, M-225 anti-myogenin, normal rabbit IgG, and normal mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HP1 antibodies (2HP2G9, 1MOD1A9AS, and 2MOD1GC) were obtained from Euromedex (Souffelweyersheim, France). Rabbit polyclonal anti-Suv39h1 (suppressor of variegation 39h1) antibody (catalog number 07-550) was obtained from Upstate Biotechnology, Inc.. Rabbit polyclonal anti-MCK antibody was developed by Dr. H. Ito (30Ito H. Kamei K. Iwamoto I. Inaguma Y. Kato K. Exp. Cell Res. 2001; 266: 213-221Crossref PubMed Scopus (59) Google Scholar). The horseradish peroxidase-streptavidin conjugate (Sigma; catalog number S 2438), anti-FLAG and anti-α-tubulin antibodies were purchased from Sigma. Rat anti-HA antibody was purchased from Roche Applied Science. Chromatin Immunoprecipitation (ChIP)—ChIP protocol and primers have been described in Ref. 26Viens A. Mechold U. Lehrmann H. Harel-Bellan A. Ogryzko V. Anal. Biochem. 2004; 325: 68-76Crossref PubMed Scopus (48) Google Scholar. HP1 Proteins Interact with MyoD—To exhaustively characterize MyoD protein partners, we first purified MyoD complex from a HeLa cell line ectopically expressing a double tagged form of MyoD. To this end, we performed double affinity purification of the HA-FLAG-MyoD complex (Fig. 1A) from chromatin enriched in mononucleosomes (as described in Ref. 27Robin P. Fritsch L. Philipot O. Svinarchuk F. Ait-Si-Ali S. Genome Biol. 2007; 8: R270Crossref PubMed Scopus (32) Google Scholar). Mass spectrometry analysis of MyoD complex confirmed some “historical” partners of MyoD such as Id, E12/E47, and MEIS1, and other partners that have never been described to interact with MyoD, among them HP1 proteins, as confirmed by Western blotting (Fig. 1B). Thus, MyoD can coexist with HP1 proteins in the same complex, at least in an artificial cell system. To further characterize these interactions, we used HEK 293 human cells to express biotinylatable forms of either MyoD or HP1 proteins α, β, or γ (as described in Ref. 26Viens A. Mechold U. Lehrmann H. Harel-Bellan A. Ogryzko V. Anal. Biochem. 2004; 325: 68-76Crossref PubMed Scopus (48) Google Scholar). The system is based on the coexpression of the target protein fused to a short biotin acceptor domain together with the biotinylating enzyme BirA from E. coli. The strength of the biotin-streptavidin interaction allows a robust characterization of protein-protein interactions, even in stringent conditions. Using biotinylated HP1 proteins, detected with streptavidin-horseradish peroxidase (Fig. 1C, bottom panel), we could precipitate MyoD with HP1α and HP1β but not with HP1γ (Fig. 1C, lanes 1–3). The signal is specific for HP1α and β; we did not detect MyoD in the HP1γ precipitate, nor in the control cells, which do not express any biotinylatable protein (Fig. 1C, lanes 1 and 4, respectively), nor in the cells that express biotinylated HP1 proteins but not MyoD (Fig. 1C, lanes 5–7). The level of ectopic MyoD was normalized by Western blotting, and that of biotinylated HP1 proteins was normalized by streptavidin-horseradish peroxidase (Fig. 1C, bottom panel). The reverse experiment using biotinylated MyoD and FLAG-tagged HP1 proteins (Fig. 1D, top panel) led to the same conclusion: a preferential interaction of MyoD with HP1α and HP1β, but not with HP1γ. The precipitation of biotinylated MyoD by streptavidin beads coprecipitated HP1α and HP1β but never HP1γ (Fig. 1D, lanes 4–6). The coprecipitation of HP1α and HP1β with biotinylated MyoD was specific, because there was no signal in cells not transfected with the biotinylated MyoD expressing vector (Fig. 1D, lanes 1–3). The absence of an interaction between MyoD and HP1γ is not due to a low expression of this isoform, because this is shown in the bottom panel of Fig. 1D (normalization of the inputs). Taken together, these results confirm, in cells, the specific interaction of MyoD with HP1α and HP1β, but not HP1γ. MyoD Interacts with HP1α and HP1β, but Not with HP1γ, in Proliferating Myoblasts—To confirm the interaction between MyoD and HP1 proteins in a more physiological context, we used C2C12 myoblasts. In this system, MyoD is expressed at a low level in proliferating myoblasts, and its expression increases upon initiation of terminal differentiation. Immunoprecipitation (IP) experiments were performed using isoform-specific anti-HP1 or anti-MyoD antibodies or an anti-Suv39h1 antibody as a positive control, because this H3K9 methylase is known to interact with HP1. Indeed, Suv39h1 coprecipitated the three isoforms of HP1 as expected (Fig. 2A). Endogenous MyoD specifically coprecipitated endogenous HP1α and HP1β, but not HP1γ (Fig. 2A), and the IgG control did not show any HP1 signal (Fig. 2A). The results of the reciprocal IP confirmed these conclusions: IP of HP1α or HP1β, but not HP1γ, coprecipitated MyoD (Fig. 2B), and the control IP did not give any detectable signal (Fig. 2B). The absence of the interaction between MyoD and HP1γ is not due to a low expression of the latter, because this isoform is highly detected by Western blotting. Taken together, these results show that MyoD interacts preferentially with the α and β isoforms of HP1, but not with the γ isoform, in myoblasts. MyoD Interacts Directly with HP1α and HP1β in Vitro—We studied the possible direct interaction between MyoD and HP1 proteins. GST-HP1 fusion proteins produced in bacteria were immobilized on agarose-glutathione beads, and an untagged recombinant form of MyoD (purification protocol described in Ref. 29Polesskaya A. Duquet A. Naguibneva I. Weise C. Vervisch A. Bengal E. Hucho F. Robin P. Harel-Bellan A. J. Biol. Chem. 2000; 275: 34359-34364Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) was detected by Western blotting using an anti-MyoD antibody. GST pulldown experiments showed that MyoD interacts with HP1α and HP1β but not with HP1γ (Fig. 3A, lanes 1–3). The interaction of MyoD with HP1α and HP1β was specific; we did not detect any MyoD signal in the presence of GST protein alone (Fig. 3A, lane 4). The quantity of each GST-HP1 protein was checked by Western blotting using an anti-GST antibody (Fig. 3A, lower panel). The interaction of HP1 proteins with MyoD was specific and was not seen with myogenin, another myogenic basic helix loop helix factor (Fig. 3B). These results show that, very interestingly, MyoD, but not myogenin, interacts specifically and directly with the isoforms α and β of HP1. The Chromoshadow Domain of HP1α and the C-terminal Domain of MyoD Are Required for Their Interaction—In an attempt to delimit the domain of HP1 responsible for interaction with MyoD, we used deletion mutants of HP1α fused to GST. A GST pulldown experiment was performed using bacterially produced MyoD as described above. GST pulldown revealed an interaction of MyoD with the wild type HP1α as expected (Fig. 3C, lane 1) and with a HP1α 119–189 mutant, which retains the CSD (Fig. 3C, lane 2). MyoD failed to interact with truncated HP1α versions lacking the CSD, i.e. mutants 1–119, 67–119, and 1–67 (Fig. 3C, lanes 3–5). The amounts of the different GST-HP1α mutants were checked by Western blotting (Fig. 3C, bottom panel). These experiments clearly show that the CSD of HP1α is required for interaction with MyoD. The same experiments were performed using tagged deletion mutants of MyoD ectopically expressed in HEK 293 cells. The results show that the C-terminal domain of MyoD is required for the interaction with HP1 (Fig. 3, D and E). HP1 Represses MyoD Transcriptional Activity—To investigate the effects of HP1 proteins on MyoD transcriptional activity, we used a Luciferase reporter gene under the control of the muscle creatine kinase (MCK) promoter, which is a direct target promoter of MyoD. Cotransfection experiments were performed in different nonmuscle cells lines that do not express MyoD endogenously. We observed that the cotransfection of either HP1α- or HP1β-expressing plasmid together with MyoD expression vector resulted in the inhibition of MCK promoter activity in a dose-dependent manner (Fig. 4, A and B, black bars). Interestingly, cotransfection of a HP1γ expression vector had no effect on the activity of MyoD (Fig. 4C, black bars). The expression of transfected MyoD was not influenced by cotransfection with HP1 expression vectors as determined by Western blotting (data not shown). The inhibitory effect of HP1α and HP1β is specific and is MyoD-dependent. Indeed, it was not seen with β-galactosidase expression under a cytomegalovirus promoter (used as a normalization control for transfection efficiency) nor with a pMCK-luciferase vector in the absence of MyoD (Fig. 4, gray bars). Thus, HP1α and HP1β, but not HP1γ, directly inhibit MyoD activity. HP1 Protein Isoforms Are Preferentially Recruited to MyoD Target Genes in Proliferating Myoblasts—To test the recruitment of HP1 isoforms into MyoD target promoters, we performed ChIP experiments using specific antibodies to each HP1 isoform. Our results show a preferential enrichment in all the three HP1 isoforms on both p21 and MCK promoters, which are MyoD targets activated early and late in differentiating cells, respectively, in proliferating myoblasts compared with differentiating myotubes (Fig. 5). Thus, even HP1γ, which does not interact directly with MyoD, is recruited on MyoD target promoters, suggesting that HP1γ may regulate myogenesis independently of any interaction with MyoD (see below). These results suggest that all the three HP1 isoforms regulate MyoD target genes in proliferating myoblasts using different mechanisms. Note that HP1 proteins are not found on coding regions of MyoD target genes (data not shown). HP1 Levels Are Crucial for Muscle Terminal Differentiation—To test more generally the role of HP1 proteins in muscle terminal differentiation, we employed gain-of-function strategy. To ectopically express HP1 protein isoforms, we used a retrovirus expressing HA and FLAG-tagged isoforms of HP1 or an empty vector as a negative control to transduce C2C12 cells (see “Experimental Procedures”). Immunofluorescence studies using anti-HA antibody showed that the exogenous HP1 isoforms had normal subnuclear localization (Fig. 6A). Overexpression of any of the HP1 isoforms, α, β, or γ, resulted generally in the inhibition of differentiation compared with the control cell line. HP1-overexpressing cells did not express the differentiation markers myogenin and MCK (Fig. 6B) and did not fuse into myotubes (data not shown). The faint expression of MCK and myogenin in HP1α-expressing cells cultured in differentiation medium was due to the fact that this cell line was not pure for the expression of tagged HP1α, ∼90%; during culture, some cells lost tagged HP1α expression. We presume that these cells differentiated normally and expressed myogenin and MCK. Overexpression of HP1 proteins could have broad effects (31Sharma G.G. Hwang K.K. Pandita R.K. Gupta A. Dhar S. Parenteau J. Agarwal M. Worman H.J. Wellinger R.J. Pandita T.K. Mol. Cell Biol. 2003; 23: 8363-8376Crossref PubMed Scopus (83) Google Scholar). Thus, differentiation inhibition we have seen in cells overexpressing HP1 proteins could be a result of these broad effects. To check the specificity of the effects seen when we overexpress HP1 isoforms (Fig. 6, A and B), we performed ChIP experiments using anti-FLAG resin to test whether the tagged HP1 isoforms are recruited to MyoD target genes. Our results show that the exogenous HP1 isoforms are indeed physically recruited into MyoD target genes (Fig. 6C), but unlike the endogenous HP1 (Fig. 5), these exogenous isoforms remain on MyoD target promoters even in myoblasts cultured in differentiation conditions (Fig. 6C). This result suggests that the differentiation defect seen in HP1-overexpressing myoblasts could be due, at least in part, to a lack of de-repression of MyoD target genes. Taken together, these results suggest that overexpression of HP1 proteins impairs MyoD target genes expression and thus muscle terminal differentiation, regardless of the HP1 isoform. Repression of MyoD target genes in proliferating myoblasts involves H3K9 methylation (20Mal A. Harter M.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1735-1739Crossref PubMed Scopus (127) Google Scholar, 22Zhang C.L. McKinsey T.A. Olson E.N. Mol. Cell Biol. 2002; 22: 7302-7312Crossref PubMed Scopus (210) Google Scholar), which is normally recognized by HP1 proteins (4Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2177) Google Scholar, 5Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2184) Google Scholar). This suggests the formation of local facultative heterochromatin on MyoD target promoters, insuring their stable repression until the cells undergo terminal differentiation. We tested the hypothesis that HP1 proteins are involved in the repression of MyoD target genes and explored their role in muscle terminal differentiation in general. Physical and Functional MyoD/HP1 Interaction—Immunoprecipitation assays showed that MyoD int" @default.
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• W1965784018 date "2008-08-01" @default.
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• W1965784018 title "Differential Cooperation between Heterochromatin Protein HP1 Isoforms and MyoD in Myoblasts" @default.
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• W1965784018 doi "https://doi.org/10.1074/jbc.m802647200" @default.
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