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• W1995150531 abstract "Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity. Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity. In cardiac muscle differentiation, the fine regulation of temporal and spatial expression of heart-specific genes in response to developmental events may require precise interactions among various transactivators and repressors, giving rise to cardiomyocytes from primordial cardiomyoblasts. Hop (homeodomain only protein) is an example of fine regulation of cardiomyocyte-specific gene transcription (1Chen F. Kook H. Milewski R. Gitler A.D. Lu M.M. Li J. Nazarian R. Schnepp R. Jen K. Biben C. Runke G. Mackay J.P. Novotny J. Schwartz R.J. Harvey R.P. Mullins M.C. Epstein J.A. Cell. 2002; 110: 713-723Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 2Shin C.H. Liu Z.P. Passier R. Zhang C.L. Wang D.Z. Harris T.M. Yamagishi H. Richardson J.A. Childs G. Olson E.N. Cell. 2002; 110: 725-735Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Likewise, muscle-specific gene regulations are dependent on the precise orchestration between series of specific transcription factors and other ubiquitous factors. Hop encodes a 73-amino acid protein that includes a 60-amino acid motif homologous to the homeodomain of Hox transcription factors. Unlike other Hox homeodomains that regulate embryonic patterning, cell fate specification, and organ formation, however, Hop is unable to bind DNA. Nevertheless, Hop can function to modulate transcription by interfering serum-response factor (SRF) 4The abbreviations used are: SRF, serum-response factor; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; MHC, myosin heavy chain; VDCC, voltage-dependent calcium channel; MCK, muscle creatine kinase; PcG, polycomb group; E, embryonic day. 4The abbreviations used are: SRF, serum-response factor; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; MHC, myosin heavy chain; VDCC, voltage-dependent calcium channel; MCK, muscle creatine kinase; PcG, polycomb group; E, embryonic day.-dependent transcription of cardiac-specific genes. Thus, half of homozygous Hop mutant mouse embryos die of failure of cardiac muscle compaction (1Chen F. Kook H. Milewski R. Gitler A.D. Lu M.M. Li J. Nazarian R. Schnepp R. Jen K. Biben C. Runke G. Mackay J.P. Novotny J. Schwartz R.J. Harvey R.P. Mullins M.C. Epstein J.A. Cell. 2002; 110: 713-723Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), and the hearts of Hop mutant neonates elicited hypercellularity (2Shin C.H. Liu Z.P. Passier R. Zhang C.L. Wang D.Z. Harris T.M. Yamagishi H. Richardson J.A. Childs G. Olson E.N. Cell. 2002; 110: 725-735Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Enhancer of Polycomb1 (Epc1) is an unusual member of the polycomb group (PcG) gene family. Although homozygotic mutations of E(Pc) in Drosophila are lethal in the embryo, heterozygous mutations do not by themselves result in a zygotic homeotic phenotype. Rather, mutations in E(Pc) enhance the phenotypes in mutations of other PcG genes (3Hayes P.H. Sato T. Denell R.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 545-549Crossref PubMed Scopus (51) Google Scholar, 4Sato T. Denell R.E. Dev. Biol. 1985; 110: 53-64Crossref PubMed Scopus (24) Google Scholar). Although Epc1 itself does not have enzymatic activity, the complex, including Epc1, is reported to possess both activating and repressive activities as a transcription regulator. For example, human EPC1 was also shown to interact with the transcriptional repressor RET finger protein, RFP (similar to RING1) (5Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Likewise, transcriptional repressor complex E2F6 containing Epc1 has a function to regulate the cell cycle. As an effort to understand the role of Hop in muscle-specific gene regulation, we sought a Hop-binding candidate by the yeast two-hybrid screening technique and found that Epc1, a novel Hop-interacting partner, initiates skeletal muscle differentiation and that its interaction with Hop is required for the full activity. Animals and Plasmid Constructs—Pregnant CD1 mice were purchased from Daehan Biolink (Daejeon, Korea). Hop knock-out mice were kindly provided by Prof. Jonathan A. Epstein (University of Pennsylvania, Philadelphia). The experimental protocols were approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. pcDNA3.1-mouse Hop-myc was described previously (6Kook H. Lepore J.J. Gitler A.D. Lu M.M. Wing-Man Yung W. Mackay J. Zhou R. Ferrari V. Gruber P. Epstein J.A. J. Clin. Investig. 2003; 112: 863-871Crossref PubMed Scopus (267) Google Scholar). pGBKT7-human HOP and pcDNA3.1-human HOP-V5/His were prepared by subcloning after PCR amplification of coding region of Hop from pcDNA3.1-HOP-myc or from human adult heart cDNA library (BD Biosciences). All constructs were confirmed by sequencing. pCMV-myc-mouse Epc1 was kindly provided by Dr. Kristian Helin (Biotech Research and Innovation Centre, Copenhagen, Denmark). Structures of human EPC1 and its truncated mutants were described previously (5Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Four Epc1 truncated mutants of the EPcA domain-containing region (EPC A, amino acids 2-285), EPcB domain-containing region (EPC B, amino acids 280-496), EPcC domain- and glutamine-rich-containing region (EPC CQ, amino acids 493-620), or CQ and carboxyl-terminal region (EPC CQCT, amino acids 493-836) are shown in Fig. 2A. For myogenin minimal promoter (7Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (347) Google Scholar), -185 to +45 bp from the transcription start sequence was amplified from mouse genomic DNA and subcloned into pGL3 basic vector (Promega, Madison, WI). For antisense strategy, complementary sequences of the 1-516-nucleotide region of rat Epc1 was amplified and subcloned into pcDNA6/myc-HisA (Invitrogen) containing the blasticidine resistance gene. Yeast Two-hybrid Screening for Binding Candidates—All assays were carried out according to the protocols of MATCHMAKER GAL4 two-hybrid system 3 (BD Biosciences). pGBKT7-human HOP plasmid was co-transfected with human adult heart cDNA library into the AH109 strain of Saccharomyces cerevisiae. Approximately 1250 independent clones were found to grow on a minimal medium lacking leucine, tryptophan, and histidine with 2.5 mm 3-amino-1,2,4-triazole. To confirm the positive reactions, a yeast one-to-one hybrid was performed in the AH109 yeast strains to detect the initiation of the lacZ reporter gene transcription qualitatively. Colony-lift filter assay was used to check the activity of β-galactosidase. Plasmid DNA from positive yeast clones were further characterized by sequencing and analyzed for gene homology by the BLAST data base. Epc1 domain mapping study was done by yeast one-to-one match experiments. The pGBKT7-human HOP plasmid was co-transfected with pACT2-EPC A, pACT2-EPC B, pACT2-EPC CQ, or pACT2-EPC CQCT into the AH109 strain of S. cerevisiae, and the histidine “jump-start” method was utilized. Antibodies, Cell Cultures, and Transfection Study—Epc1 antibody was generated from Peptron (Daejeon, South Korea), with the epitope region of human Epc1 195-209 amino acids. The antibodies able to recognize myogenin (Sc-576, Santa Cruz Biotechnology), FLAG (M2, Sigma), V5 (Invitrogen) embryonic myosin heavy chain (MHCemb, MF20, Developmental Studies Hybridoma Bank), and desmin (Chemicon, Temecula, CA) were utilized. H9c2, HeLa, COS7, and 293T cells were obtained from Seoul Korean Cell Line Bank (Seoul, Korea) and maintained with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Differentiation of H9c2 cells was induced by depriving the serum with 1% FBS as described previously (8L'Ecuyer T. Horenstein M.S. Thomas R. Heide R.V. Mol. Genet. Metab. 2001; 74: 370-379Crossref PubMed Scopus (24) Google Scholar). To establish antisense Epc1 cell lines, pcDNA6/myc-HisA-antisense Epc1 and vector was transfected to H9c2 cells. The cells were treated with 5 μg/ml blasticidine (Invitrogen), and positive colonies were selected 2 weeks later. Epc1-overexpressing cell lines were generated by co-transfecting pCMV-myc-Epc1 and pSV2-hygro (Invitrogen). Positive cells were selected by adding 50 μg/ml hygromycin (Invitrogen) for 2 weeks. For transient transfection of Hop and Epc1, pcDNA3.1-HOP-V5/His and/or pCMV-myc-Epc1 was introduced to H9c2 cells by Lipofectamine Plus reagent (Invitrogen). The differentiation rates were determined morphologically by analyzing multinucleated myotube formation and nuclei clustering using phalloidin staining. The nuclear fusion indices were measured as described (9Kashour T. Burton T. Dibrov A. Amara F. J. Mol. Cell. Cardiol. 2003; 35: 937-951Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The rate of myoblast fusion was expressed as the percentage of nuclei in fused cells of the total number of nuclei in randomly chosen fields as viewed under a microscope. Cells containing more than three nuclei were regarded as fused cells. The effect of Hop and Epc1 on cell proliferation/survival was measured by direct cell counting. Transfection of pcDNA3.1-HOP-V5/His and/or pCMV-myc-Epc1 to H9c2 cells was repeated every other day; the cells were lifted at the 5th days, and the numbers were counted as described previously (10Koh J.T. Kook H. Kee H.J. Seo Y.W. Jeong B.C. Lee J.H. Kim M.Y. Yoon K.C. Jung S. Kim K.K. Exp. Cell Res. 2004; 294: 172-184Crossref PubMed Scopus (66) Google Scholar). Primary myoblasts from either wild type or Hop knock-out mice were prepared as described (11Rawls A. Morris J.H. Rudnicki M. Braun T. Arnold H.H. Klein W.H. Olson E.N. Dev. Biol. 1995; 172: 37-50Crossref PubMed Scopus (111) Google Scholar) with a slight modification. Muscle tissues from hindlimbs were dissociated by mincing with microdissecting scissors followed by treatment with collagenase (2 mg/ml; Worthington) for 60 min and trypsin/EDTA (0.25% Invitrogen) for 20 min. Then the enzymatic digestion was stopped by the addition of DMEM containing 15% FBS. The cell suspension was filtered through a 100-μm and then a 70-μm sieve (Cell Strainer Nylon, Falcon) and collected. The cells were resuspended, and the contaminating fibroblasts were removed by preplating the cell suspension for 1 h at 37°C, 5% CO2. The myoblasts were plated on culture dishes coated with 0.2% collagen and grown in 15% FBS in DMEM supplemented with 5 nm basic fibroblast growth factor (BIOSOURCE). Forty eight hours later, the cells were preplated once more to remove the extra fibroblast. To induce differentiation, the growth medium was changed to 2% horse serum in DMEM. Promoter analysis was described previously (1Chen F. Kook H. Milewski R. Gitler A.D. Lu M.M. Li J. Nazarian R. Schnepp R. Jen K. Biben C. Runke G. Mackay J.P. Novotny J. Schwartz R.J. Harvey R.P. Mullins M.C. Epstein J.A. Cell. 2002; 110: 713-723Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). H9c2 cells were plated in 24-well plates, and transfections were carried out. Total DNA used in each transfection was adjusted by adding the pcDNA3.1 vector. Cells were harvested 48 h after transfection, and luciferase activity was measured (Promega). Transfection rate was normalized by β-galactosidase activity. Immunoprecipitation and Western Blot—Protocols for immunoprecipitation and Western blot were described previously (6Kook H. Lepore J.J. Gitler A.D. Lu M.M. Wing-Man Yung W. Mackay J. Zhou R. Ferrari V. Gruber P. Epstein J.A. J. Clin. Investig. 2003; 112: 863-871Crossref PubMed Scopus (267) Google Scholar). For immunoprecipitation, 293T cells were transfected with pCMV-FLAG-Epc1 full or truncated mutants together with pcDNA3.1-HOP-V5/His by FuGENE 6 (Roche Diagnostics). The cells were harvested with lysis buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mm EDTA, with protease inhibitor mixture (Roche Diagnostics), and 0.5% v/v Igepal CA-630. Whole cell lysates were incubated with specific antibody or normal mouse IgG. Immunocomplex was pulled down by incubating the samples with protein A/G-agarose (Santa Cruz Biotechnology). After washing the agarose beads three times, the immunoprecipitates were separated by electrophoresis on 10% SDS-PAGE and subjected to immunoblot. The antibodies for Western blot analysis were anti-myogenin (1:500), Epc1 (1:200), actin (1:2000), and tubulin (1:1000). Immunohistochemistry and Fluorescent Immunocytochemistry—To examine the tissue distribution of Epc1 in the mouse embryo, immunohistochemistry was performed. Mouse embryos were obtained from pregnant mice at 13.5, 15.5, and 17.5 days post-coitum. Sagittal sections were obtained and utilized for immunohistochemistry as described (12Kee H.J. Sohn I.S. Nam K.I. Park J.E. Qian Y.R. Yin Z. Ahn Y. Jeong M.H. Bang Y.J. Kim N. Kim J.K. Kim K.K. Epstein J.A. Kook H. Circulation. 2006; 113: 51-59Crossref PubMed Scopus (271) Google Scholar). To test the specificity of the α-Epc1 antibody, an excess amount of blocking peptide that had been used to raise the antibody was simultaneously incubated with α-Epc1 antibody. Protocol for fluorescent immunocytochemistry was described previously (6Kook H. Lepore J.J. Gitler A.D. Lu M.M. Wing-Man Yung W. Mackay J. Zhou R. Ferrari V. Gruber P. Epstein J.A. J. Clin. Investig. 2003; 112: 863-871Crossref PubMed Scopus (267) Google Scholar). Northern Blot Analysis and RT-PCR—Northern blot analysis was performed using a specific probe spanning 432 bp in EPC A region (13Kee H.J. Ahn K.Y. Choi K.C. Won Song J. Heo T. Jung S. Kim J.K. Bae C.S. Kim K.K. FEBS Lett. 2004; 569: 307-316Crossref PubMed Scopus (49) Google Scholar). Total RNA from embryonic mouse heart and differentiated H9c2 cells was extracted using TRIzol reagent (Invitrogen) and subjected to reverse transcription reaction followed by semi-quantitative PCR amplification. The amplified DNA fragment was confirmed by sequencing after cloning into pCR2.1 TOPO TA vector (Invitrogen). The primer sequences for RT-PCRs can be provided upon request. In Vivo Wound Healing Assay—Seven- or 8-week-old wild type or Hop knock-out mice were utilized for in vivo wound healing assay as described previously (14Takahashi T. Ishida K. Itoh K. Konishi Y. Yagyu K.-I. Tominaga A. Miyazaki J.-I. Yamamoto H. Gene Ther. 2003; 10: 612-620Crossref PubMed Scopus (36) Google Scholar). Under anesthesia, right hamstring muscles were cut transversely using a 15 scalpel and then sutured with a modified Kessler stitch using nylon 7-0 wires. After 1 or 3 weeks, the mice were sacrificed, and the muscles were isolated. The transverse sections at suture sites were obtained and utilized for hematoxylin and eosin staining or desmin immunohistochemistry. Identification of Hop-interacting Partners—We postulated that Hop might be involved in muscle differentiation by interacting with other transcription regulators. By utilizing a yeast two-hybrid technique, we screened adult human heart cDNA library with a bait of full-length human HOP. From this screen we identified 300 clones that were His- and β-galactosidase-positive. Among them ∼100 clones were sequenced and blasted to the public data base. Six positive clones were proven to encode sequences of the amino-terminal part of Epc1. Enhancer of polycomb is highly conserved and is known to have several subtypes by gene duplication or alternative splice in mice as follows: Epc1L, Epc1S, Epc2L, and Epc2S (5Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 15Stankunas K. Berger J. Ruse C. Sinclair D.A.R. Randazzo F. Brock H.W. Development (Camb.). 1998; 125: 4055-4066Crossref PubMed Google Scholar). In our yeast two-hybrid screening experiments, Epc1L was detected. To confirm the interaction further, in vivo interaction assay in yeast was performed with pGBKT7-HOP and pACT2-Epc1. HOP did show a strong association with Epc1, although it had no apparent interaction with an empty pACT2 vector (Fig. 1A). To test if the interaction takes place in mammalian cells, we performed immunoprecipitation and found that Hop successfully recruited Epc1 (Fig. 1B). We next tried to see the subcellular localization of Epc1 in relation to that of Hop by immunofluorescent staining. After transient transfection of both pCMV-myc-Epc1 and pcDNA3.1-HOP-V5/His, COS7 cells were stained with anti-Epc1 polyclonal and anti-V5 monoclonal antibodies. As shown in Fig. 1C, the majority of Epc1 (Fig. 1C, panel a) and Hop (Fig. 1C, panel b) was co-localized in the nucleus of COS7 cells, as demarcated by 4,6-diamidino-2-phenylindole nucleus staining (Fig. 1C, panel c). The merged image is shown in Fig. 1C, panel d. Identification of Hop-interacting Domain of Epc1 by Deletion Mapping—Epc1 has four distinct structures named EPcA, EPcB, EpcC, and a Qx domain; all of them are conserved in many species, including Drosophila, Caenorhabditis elegans, yeast, mouse, and human (15Stankunas K. Berger J. Ruse C. Sinclair D.A.R. Randazzo F. Brock H.W. Development (Camb.). 1998; 125: 4055-4066Crossref PubMed Google Scholar). To identify the interacting domains, pACT2 vector versions of the truncated human Epc1 (Fig. 2A) were used for yeast one-to-one match experiments. Strong interaction was detected in the EPC A region. In contrast, the EPC B, EPC CQ, and EPC CQCT regions showed no activity or very weak interacting activity (Fig. 2B). Each experiment was done by using four independent colonies, and the results are summarized in Fig. 2C. Hop-interacting domain of Epc1 was further confirmed by immunoprecipitation in mammalian cells. FLAG-tagged full or truncated human EPC1 plasmids were co-transfected with pcDNA3.1-human HOP-V5/His to 293T cells. The upper panel of Fig. 2D shows the expression of each truncated mutant of Epc1 with diverse molecular weights, and the middle panel shows the constant expression of V5-tagged HOP in each experimental condition. After immunoprecipitation with anti-FLAG antibody, HOP was detected with anti-V5 antibody. HOP was pulled down by EPC full or EPC A, confirming that EPC A is responsible for the interaction with HOP (Fig. 2D). We next investigated whether the lack of interactions of truncated mutants was caused by loss of nuclear localization. Epc1 fragments as well as full clone were transfected into HeLa cells and stained with α-FLAG antibody (Fig. 2E). EPC A (Fig. 2E, panel b) and EPCB(panel c) were still localized in the nucleus as was EPC full (panel a), but EPCs, which have carboxylterminal regions only, were seen in the cytoplasm (panels d and e), suggesting that alteration in the structure and thereby failure of nuclear localization might be one of the causes of losing the interaction with Hop in the mammalian cells. Expression Patterns of Epc1—The tissue distribution of Epc1 was examined. Northern blot analysis for Epc1 showed expression of the 4-kb transcript. Although it was expressed ubiquitously, Epc1 was abundant in heart and skeletal muscles (data not shown). In Western analysis with mouse adult tissue blot, 90-kDa bands were detected (Fig. 3A, upper panel), which was confirmed by the presence of the same size band in pCMV-myc-Epc1-transfected 293T cells (rightmost lane). Those bands completely disappeared when the antibody was premixed with blocking peptide at excess amounts. Muscle tissues such as skeletal or ventricular muscles were strongly positive for Epc1 expression. We further investigated the time course of Epc1 expression at the ages ranging from embryonic day 11.5 to postnatal week 8. Northern blot analysis showed that Epc1 expression in the heart is gradually decreased with aging (Fig. 3B). The changes in the Epc1 expression as well as cardiac specific genes were further examined by semi-quantitative RT-PCR, and the typical gel pictures are shown in Fig. 3C. As expected, α-myosin heavy chain (α-MHC), one of the cytoskeletal proteins in the differentiated cardiomyocytes, was turned on in late embryonic stage and after birth, whereas β-MHC, a fetal cardiac protein, was expressed only in the embryonic period and in postnatal day 1, as suggested in previous reports (16Davis F.J. Pillai J.B. Gupta M. Gupta M.P. Am. J. Physiol. 2005; 288: H1477-H1490Crossref PubMed Scopus (42) Google Scholar). As shown in Northern blot, Epc1 transcript amount was abundant in early embryonic heart and gradually decreased after birth. The expression patterns of Epc1 in embryonic tissue were further investigated by immunohistochemistry with E13.5 to E17.5 embryo sections. Strong positive immunoreactivity was detected in E13.5 heart (Fig. 3D, panel a), which disappeared completely by premixing the excess blocking peptides (Fig. 3D, panel b). However, Epc1 expression in the heart (arrow in Fig. 3D, panel c, at E13.5) was gradually decreased by aging (Fig. 3D, panel d, E15.5, and Fig. 3D, panel e, E17.5 day). Interestingly, skeletal muscles such as tongue were gradually increased by aging (arrow in Fig. 3D, panel e), suggesting the differences in the expression patterns in both types of sarcomeric muscles. Epc1 was also well expressed in the endothelium, spinal ganglia, and cartilage. Although choroidal plexus and meninges were positive, brain parenchymal structures did not show any significant immunoreactivities. Changes of Epc1 Expression in Differentiating H9c2, a Myoblast Cell Line—Changes in expression patterns of Epc1 in skeletal and cardiac muscle led us to speculate that Epc1 is involved in the process of muscle differentiation. H9c2, a cardiomyoblast cell line originated from rats, is widely employed for the in vitro model of muscle differentiation (17Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 367-381Crossref PubMed Scopus (453) Google Scholar). Differentiating H9c2 cells could have either cardiac or skeletal phenotypes, depending on the presence of retinoic acid in differentiation media (18Menard C. Pupier S. Mornet D. Kitzmann M. Nargeot J. Lory P. J. Biol. Chem. 1999; 274: 29063-29070Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In our experimental model, serum starvation with 1% FBS induced elongation of H9c2 cells and multinucleation. Typical serial changes in morphology are shown in Fig. 4A, which is accompanied by induction of myogenin (Fig. 4C). In our model, H9c2 seemed to undergo phenotype switch from cardiac to skeletal muscles during differentiation; the expression of cardiac type of voltage-dependent calcium channel (VDCC)α1 was decreased by differentiation, whereas those of muscle creatine kinase (MCK) and skeletal type of VDCCα1 were gradually up-regulated by differentiation (Fig. 5B). Interestingly, Epc1 was transiently up-regulated in the early phase of differentiation, whereas Hop was not changed (Fig. 4, B and C), suggesting that Epc1 plays an important role in the initiation of H9c2 differentiation. To delineate the role of Epc1 in the differentiation, we generated the Epc1-knockdown cell lines with antisense strategy as well as the stable cell lines overexpressing Epc1 (Fig. 5A) and induced the differentiation by serum starvation. As evidenced in Fig. 4A in which H9c2 differentiation is markedly impaired in knockdown cell lines, the increases in differentiation markers such as MCK and skeletal VDCCα1 were significantly reduced (Fig. 5C). In Epc1-overexpressing cell lines, however, induction of those markers was greatly enhanced (Fig. 5D). Skeletal muscle fate determination/differentiation processes are governed by several distinct transcription factors such as MyoD, Myf5, myogenin, and Myf6/Mrf4 (19Buckingham M. Bajard L. Chang T. Daubas P. Hadchouel J. Meilhac S. Montarras D. Rocancourt D. Relaix F. J. Anat. 2003; 202: 59-68Crossref PubMed Scopus (620) Google Scholar). Therefore, to investigate whether H9c2 cells elicit the skeletal muscle phenotype during its differentiation process, we tried to examine the expression of those four myogenic transcription factors. Myf5 and Myf6/Mrf4 were increased in the late phase of H9c2 differentiation, whereas MyoD was transiently up-regulated in the early phase of differentiation (Fig. 5B). Myogenin, a key step regulator of differentiation of skeletal muscle (20Biederer C.H. Ries S.J. Moser M. Florio M. Israel M. McCormick F. Buettner R. J. Biol. Chem. 2000; 275: 26245-26251Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), was abruptly increased in the beginning of the serum starvation. Strikingly, all of those changes in expression levels were significantly blunted or deranged in Epc1-knockdown cells (Fig. 5C), although they were enhanced in Epc1-overexpressing cells (Fig. 5D). Forced Expression of Hop and Epc1 Induces H9c2 Differentiation—The roles of Epc1 and Hop in the differentiation of H9c2 cells were further investigated by transient transfection, followed by the evaluation of cell morphology (Fig. 6A) or myogenin expression (Fig. 6B). Hop itself did not induce significant changes in the cell morphology (Fig. 6A, panel b), compared with mock-transfected H9c2 cells (Fig. 6A, panel a). Epc1 transfection, however, caused the cells to be thinned or elongated and often fused to form multinucleated syncytia appearing like myotubes (Fig. 6A, panel c). The changes of the cell shapes became more prominent by co-transfection of Epc1 and Hop (Fig. 6A, panel d). The number of myogenin-positive nuclei as well as the expression level were increased in Epc1-transfected H9c2 cells (Fig. 6B, panel g) compared with mock- (Fig. 6B, panel e) or Hop-transfected cells (Fig. 6B, panel f). The myogenin staining was much stronger in Hop/Epc1-co-transfected H9c2 cells (Fig. 6, panel h). The expression of myogenin was quantified by Western blot analysis after transfection of Epc1 or Hop. Myogenin expression was increased when Epc1 or Epc1 + Hop was transfected to H9c2 cells (Fig. 6C). The transcript level of myogenin was also increased by Epc1 or Hop + Epc1 transfection (Fig. 6D). Interestingly, skeletal VDCCα1 was significantly increased by the co-transfection of Hop and Epc1 (Fig. 6D). On the contrary, the absence of Epc1 reduced myogenin and skeletal VDCCα1, and Hop did not affect the expression (Fig. 6, C and D). We next investigated if Epc1 can activate myogenin promoter. Because the myogenin promoter, which spans from -185 to +48 bp of its transcription initiation site, has been reported to have essential regulatory functions of the gene (7Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (347) Google Scholar), we subcloned the minimal promoter region to pGL3 basic vector to drive luciferase activity. By testing the basal promoter activity, 20 ng of myogenin minimal promoter (-185 to +45)-luciferase construct was selected for optimal reporter constructs (data not shown). Epc1 dramatically activated the myogenin minimal promoter activity in a dose-dependent fashion (Fig. 6E), although Hop did not increase the activity in the range fro" @default.
• W1995150531 created "2016-06-24" @default.
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• W1995150531 date "2007-03-01" @default.
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• W1995150531 title "Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation" @default.
• W1995150531 cites W1848888865 @default.
• W1995150531 cites W1990786422 @default.
• W1995150531 cites W2003128644 @default.
• W1995150531 cites W2008352344 @default.
• W1995150531 cites W2009262960 @default.
• W1995150531 cites W2026325417 @default.
• W1995150531 cites W2035779163 @default.
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• W1995150531 cites W2054895660 @default.
• W1995150531 cites W2054944874 @default.
• W1995150531 cites W2056897025 @default.
• W1995150531 cites W2058225109 @default.
• W1995150531 cites W2059088759 @default.
• W1995150531 cites W2059978662 @default.
• W1995150531 cites W2068482643 @default.
• W1995150531 cites W2078831708 @default.
• W1995150531 cites W2079315297 @default.
• W1995150531 cites W2081480500 @default.
• W1995150531 cites W2085927442 @default.
• W1995150531 cites W2098412336 @default.
• W1995150531 cites W2108136318 @default.
• W1995150531 cites W2108374314 @default.
• W1995150531 cites W2114318954 @default.
• W1995150531 cites W2114815592 @default.
• W1995150531 cites W2121628947 @default.
• W1995150531 cites W2128782542 @default.
• W1995150531 cites W2130562212 @default.
• W1995150531 cites W2137499286 @default.
• W1995150531 cites W2142491779 @default.
• W1995150531 cites W2155908077 @default.
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