Dermo-1, a Multifunctional Basic Helix-Loop-Helix Protein, Represses MyoD Transactivation via the HLH Domain, MEF2 Interaction, and Chromatin Deacetylation-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文

DOI: 10.1074/jbc.m110228200

CAS-2 JCR-Q2 SCIE EI

Xue GongLi Li

Journal of Biological Chemistry

Apr 2002

73被引用

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摘要原文

Dermo-1 is a multifunctional basic helix-loop-helix (bHLH) transcription factor that has been shown to be a potent negative regulator for gene transcription and apoptosis. To understand the molecular mechanisms that mediate the function of Dermo-1, we generated a series of Dermo-1 mutants and used a MyoD-mediated transcriptional activation model to characterize the roles of its N-terminal, bHLH, and C-terminal structural domains in transcriptional repression. Both the C-terminal and HLH domains of Dermo-1 were essential for its repression of MyoD-mediated transactivation. Dermo-1 repressed, in a dose-dependent fashion, the transactivation activity of myocyte enhancer factor 2 (MEF2), a protein known to cooperate with MyoD in activating E-box-dependent gene expression. Both the N- and C-terminal domains of Dermo-1, but not the bHLH domain, were required for the inhibition of MEF2, suggesting that Dermo-1 inhibits both MyoD- and MEF2-dependent transactivation but through different mechanisms. Dermo-1 interacted directly with MEF2 and selectively repressed the MEF2 transactivation domain. An overall increase of histone acetylation induced by trichostatin A treatment reduced Dermo-1 transcriptional repression activity, suggesting that histone deacetylation is involved in Dermo-1-mediated transcriptional repression. Together, these results suggest that MEF2 is an important target in Dermo-1-mediated transcriptional repression and provide initial evidence of the involvement of histone acetylation in Dermo-1 transcriptional repression. Dermo-1 is a multifunctional basic helix-loop-helix (bHLH) transcription factor that has been shown to be a potent negative regulator for gene transcription and apoptosis. To understand the molecular mechanisms that mediate the function of Dermo-1, we generated a series of Dermo-1 mutants and used a MyoD-mediated transcriptional activation model to characterize the roles of its N-terminal, bHLH, and C-terminal structural domains in transcriptional repression. Both the C-terminal and HLH domains of Dermo-1 were essential for its repression of MyoD-mediated transactivation. Dermo-1 repressed, in a dose-dependent fashion, the transactivation activity of myocyte enhancer factor 2 (MEF2), a protein known to cooperate with MyoD in activating E-box-dependent gene expression. Both the N- and C-terminal domains of Dermo-1, but not the bHLH domain, were required for the inhibition of MEF2, suggesting that Dermo-1 inhibits both MyoD- and MEF2-dependent transactivation but through different mechanisms. Dermo-1 interacted directly with MEF2 and selectively repressed the MEF2 transactivation domain. An overall increase of histone acetylation induced by trichostatin A treatment reduced Dermo-1 transcriptional repression activity, suggesting that histone deacetylation is involved in Dermo-1-mediated transcriptional repression. Together, these results suggest that MEF2 is an important target in Dermo-1-mediated transcriptional repression and provide initial evidence of the involvement of histone acetylation in Dermo-1 transcriptional repression. Mouse Dermo-1 is a member of the basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLHbasic helix-loop-helixMEF2myocyte enhancer factor 2HDAChistone deacetylaseNLSnuclear localization signaltktyrosine kinaseTSAtrichostatin ADAB3,3′-diaminobenzidineMCKmuscle creatine kinase 1The abbreviations used are: bHLHbasic helix-loop-helixMEF2myocyte enhancer factor 2HDAChistone deacetylaseNLSnuclear localization signaltktyrosine kinaseTSAtrichostatin ADAB3,3′-diaminobenzidineMCKmuscle creatine kinase transcription factor family that was initially isolated using the yeast two-hybrid system with the bHLH domain of ubiquitously expressed E12 as bait (1.Li L. Cserjesi P. Olson E.N. Dev. Biol. 1995; 172: 280-292Crossref PubMed Scopus (214) Google Scholar). During embryogenesis, this gene is predominantly expressed in mesodermal- and ectodermal-derived tissues including somites, dermis, chondroblasts, limbs, teeth, and cranial structures and is believed to play important roles in the development and differentiation of these tissues and organs. hom*ologs of mouse Dermo-1 have been found in several other vertebrates such as humans, rats, and chicks with extensive sequence conservation during evolution (2.Lee M.S. Lowe G. Flanagan S. Kuchler K. Glackin C.A. Bone (NY). 2000; 27: 591-602Crossref PubMed Scopus (63) Google Scholar, 3.Tamura M. Noda M. J. Cell. Biochem. 1999; 72: 167-176Crossref PubMed Scopus (31) Google Scholar, 4.Scaal M. Fuchtbauer E.M. Brand-Saberi B. Anat. Embryol. 2001; 203: 1-7Crossref PubMed Scopus (40) Google Scholar). Interestingly, Dermo-1 hom*ologs are also expressed in a subset of mesodermal- and ectodermal-derived tissues such as subectodermal mesenchyme, osteoblasts, and limb buds (2.Lee M.S. Lowe G. Flanagan S. Kuchler K. Glackin C.A. Bone (NY). 2000; 27: 591-602Crossref PubMed Scopus (63) Google Scholar, 3.Tamura M. Noda M. J. Cell. Biochem. 1999; 72: 167-176Crossref PubMed Scopus (31) Google Scholar, 4.Scaal M. Fuchtbauer E.M. Brand-Saberi B. Anat. Embryol. 2001; 203: 1-7Crossref PubMed Scopus (40) Google Scholar), which potentially act as negative regulators of differentiation. It has been shown that Dermo-1 functions as a potent transcriptional repressor for MyoD and as an anti-apoptotic agent for Myc- and p53-dependent cell death (1.Li L. Cserjesi P. Olson E.N. Dev. Biol. 1995; 172: 280-292Crossref PubMed Scopus (214) Google Scholar, 5.Maestro R. Dei Tos A.P. Hamamori Y. Krasnokutsky S. Sartorelli V. Kedes L. Doglioni C. Beach D.H. Hannon G.J. Genes Dev. 1999; 13: 2207-2217Crossref PubMed Scopus (453) Google Scholar). However, the precise biological roles of Dermo-1 during embryogenesis and its molecular mechanisms of action remain largely unknown. basic helix-loop-helix myocyte enhancer factor 2 histone deacetylase nuclear localization signal tyrosine kinase trichostatin A 3,3′-diaminobenzidine muscle creatine kinase basic helix-loop-helix myocyte enhancer factor 2 histone deacetylase nuclear localization signal tyrosine kinase trichostatin A 3,3′-diaminobenzidine muscle creatine kinase In general, members of the bHLH transcription factor family are critical regulators of important biological processes such as cell lineage determination, proliferation, and differentiation (6.Littlewood, T., and Evan, G. I. (1998) Oxford University Press, London, UKGoogle Scholar). There is ample evidence of interplay between different members of bHLH proteins in the regulation of myogenesis, cardiogenesis, neurogenesis, and hematopoiesis (7.Jan Y.N. Jan L.Y. Cell. 1993; 75: 827-830Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 8.Kadesch T. Cell Growth & Differ. 1993; 4: 49-55PubMed Google Scholar, 9.Olson E.N. Klein W.H. Genes Dev. 1994; 8: 1-8Crossref PubMed Scopus (606) Google Scholar, 10.Srivastava D. Annu. Rev. Physiol. 2001; 63: 451-469Crossref PubMed Scopus (84) Google Scholar). It is well documented that bHLH proteins regulate the differentiation of many other cell types during development either as transcriptional activators or repressors of gene expression (6.Littlewood, T., and Evan, G. I. (1998) Oxford University Press, London, UKGoogle Scholar). The bHLH proteins are characterized by two distinct motifs: (i) the basic region that mediates specific binding to the E-box consensus sequence (CANNTG) and (ii) the HLH domain that mediates heterodimerization with E proteins such as E12 (11.Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1846) Google Scholar, 12.Lassar A.B. Skapek S.X. Novitch B. Curr. Opin. Cell Biol. 1994; 6: 788-794Crossref PubMed Scopus (310) Google Scholar). In addition, the bHLH proteins have also been shown to form complexes with non-bHLH proteins such as the myocyte enhancer factor 2 (MEF2), SRF, and p300 (13.Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 14.Biesiada E. Hamamori Y. Kedes L. Sartorelli V. Mol. Cell. Biol. 1999; 19: 2577-2584Crossref PubMed Scopus (73) Google Scholar, 15.Groisman R. Masutani H. Leibovitch M.P. Robin P. Soudant I. Trouche D. Harel-Bellan A. J. Biol. Chem. 1996; 271: 5258-5264Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16.Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (321) Google Scholar). It has been established that MEF2, a MADS-domain protein, interacts directly with bHLH proteins to regulate gene transcription (13.Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Abstract Full Text PDF PubMed Scopus (700) Google Scholar). Recently, genetic studies have shown that members of the MEF2 family, as transcription factors and coactivators for a battery of gene regulation, are critical in controlling the development of multiple tissues including heart, vasculature, neural tubes, and skeletal muscle (17.Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (843) Google Scholar, 18.Lin Q. Schwarz J. Bucana C. Olson E.N. Science. 1997; 276: 1404-1407Crossref PubMed Scopus (774) Google Scholar, 19.Bi W. Drake C.J. Schwarz J.J. Dev. Biol. 1999; 211: 255-267Crossref PubMed Scopus (158) Google Scholar). In tissue culture, MEF2 can also induce gene expression in response to a calcium calmodulin-dependent protein kinase signal via the dissociation from HDAC factors (20.Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (414) Google Scholar). The function of MEF2 has been shown to be modulated via chromatin remodeling by recruiting HDAC 4/HDAC5/HDAC7 and their related protein MITR (20.Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (414) Google Scholar, 21.Zhang C.L. McKinsey T.A. Lu J.R. Olson E.N. J. Biol. Chem. 2001; 276: 35-39Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 22.Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Many important attributes of the bHLH factors have been initially defined in the past using muscle differentiation as a model and members of myogenic bHLH proteins as regulators (6.Littlewood, T., and Evan, G. I. (1998) Oxford University Press, London, UKGoogle Scholar). MyoD is the first and most well characterized bHLH protein that behaves as a transcriptional activator; it provides a paradigm for defining the function of other bHLH proteins in cell determination and differentiation during development (23.Olson E.N. Genes Dev. 1990; 4: 1454-1461Crossref PubMed Scopus (553) Google Scholar). The molecular mechanisms that mediate the transcriptional activation of MyoD during myogenesis require both basic and HLH domains (24.Davis R.L. Cheng P.F. Lassar A.B. Weintraub H. Cell. 1990; 60: 733-746Abstract Full Text PDF PubMed Scopus (526) Google Scholar). Furthermore, the transcriptional activation activity of MyoD can be synergistically stimulated by members of the MEF2 family (13.Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Abstract Full Text PDF PubMed Scopus (700) Google Scholar). Such cooperativity requires direct interactions between MyoD and MEF2, but only one of them is needed to bind to DNA (25.Molkentin J.D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9366-9373Crossref PubMed Scopus (373) Google Scholar). The transcriptional activity of MyoD during myogenesis is also regulated by Id and Twist, members of the HLH superfamily (6.Littlewood, T., and Evan, G. I. (1998) Oxford University Press, London, UKGoogle Scholar). Both Id and Twist are transcriptional repressors for MyoD. However, the molecular mechanisms mediating their action are distinct. The Id proteins belong to a class of HLH proteins that lack a basic region and have a greater affinity for E proteins (26.Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1797) Google Scholar). Therefore, Id functions as a dominant negative mutant to inhibit the function of tissue-restricted bHLH proteins such as MyoD. Twist, sharing significant hom*ology with Dermo-1, contains both basic and HLH domains and has been shown to be a potent transcriptional repressor for MyoD (1.Li L. Cserjesi P. Olson E.N. Dev. Biol. 1995; 172: 280-292Crossref PubMed Scopus (214) Google Scholar, 27.Hebrok M. Fuchtbauer A. Fuchtbauer E.M. Exp. Cell Res. 1997; 232: 295-303Crossref PubMed Scopus (57) Google Scholar). The repression mechanisms of Twist on MyoD transactivation have been demonstrated to be mediated by direct interaction with the basic domain of MyoD, sequestering E proteins, inhibiting MEF2 activation, and inhibiting the histone acetylase activity of MyoD coactivators such as pCAF and CBP (28.Spicer D.B. Rhee J. Cheung W.L. Lassar A.B. Science. 1996; 272: 1476-1480Crossref PubMed Scopus (275) Google Scholar, 29.Hamamori Y. Wu H.Y. Sartorelli V. Kedes L. Mol. Cell. Biol. 1997; 17: 6563-6573Crossref PubMed Scopus (136) Google Scholar, 30.Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). In the present study, we investigated the molecular mechanisms by which Dermo-1 represses transcription. Since our first report on the cloning of Dermo-1 in a mouse and its function as a transcriptional repressor for MyoD (1.Li L. Cserjesi P. Olson E.N. Dev. Biol. 1995; 172: 280-292Crossref PubMed Scopus (214) Google Scholar), no further studies have been published on the characterization of the molecular mechanisms of Dermo-1 in transcriptional repression. Here we have focused on the roles of each domain of Dermo-1 in transcriptional repression using MyoD transactivation as a model. Our results demonstrate that the HLH and C-terminal domains and not the N-terminal and the basic regions of Dermo-1 are essential for its transcriptional repression activity. Further, Dermo-1 directly associates with MEF2 to repress its transactivation domain, and histone deacetylation is involved in Dermo-1-mediated gene repression. Dermo-1 cDNA was inserted into pcDNA3.1 vectors (Invitrogen) containing a FLAG epitope at the C terminus (pcDNA3.1FLAG, constructed by M. Yang in the laboratory of L. Li). Point mutants were generated using primers encoding several different mutations by a QuikChangeTM site-directed mutagenesis kit (Stratagene). The resulting point mutants (DermoHLH−, Dermob−, DermoNls1−, DermoNls2−, and DermoNls1&2−) were mutated at F86P, R74A/E75A/R76A, R31A/K32A/R33A/R34A, K52A/K53A, and R31A/K32A/R33A/R34A/K52A/K53A, respectively. To minimize the introduction of errors by PCR, the sequences of these point mutants were confirmed and then inserted into wild type pcDNA3.1FLAG vectors for additional subcloning. The deletion mutants were generated by PCR using the following 5′ and 3′ primers (the 5′ primers all contained an EcoRI or HindIII site; the 3′ primers all contained an XhoI site). DermoΔN-(1–28): 5′ primer, 5′-GGAATTCATGGGCCGGAAGCGGCGCTACAG-3′ and 3′ primer, 5′-CGGCTCGAGGTGGGAGGCGGACATGGAC-3′; DermoΔN-(1–65): 5′ primer, 5′CCCAAGCTTAGCCAGCGCATCCTGGCCAAC-3′ and 3′ primer, 5′-CGGCTCGAGGTGGGAGGCGGACATGGAC-3′; DermoΔC-(121–160): 5′ primer, 5′-GGAATTCGGGCGCCATGGAGGAGG-3′ and 3′ primer, 5′-CGGCTCGAGCTGGTAGAGGAAGTCTATGTACCTG-3′; DermoΔC-(134–160): 5′ primer, 5′-GGAATTCGGGCGCCATGGAGGAGG-3′ and 3′ primer, 5′-CGGCTCGAGGCAGCTGGTCATCTTATTGTC-3′; DermoΔC-(145–160): 5′ primer, 5′-GGAATTCGGGCGCCATGGAGGAGG-3′ and 3′ primer, 5′-CGGCTCGAGCCACACGGAGAAGGCGTAG-3′; DermoHLHC: 5′ primer: 5′-CCCAAGCTTACCCAGTCGCTCAACGAGG-3′ and 3′ primer, 5′-CGGCTCGAGGTGGGAGGCGGACATGGAC-3′. DermoΔN-(1–65) and DermoHLHC mutants were fused in-frame with the SV40 nuclear localization signal (NLS) at the N terminus of Dermo-1 in the pcDNA3.1FLAG vector. To ensure the accuracy of mutagenesis, all resulting mutants were confirmed by sequencing. Transient transfection assays were performed using LipofectAMINE PlusTM (Life Science) according to the manufacturer's instructions. The reporter gene MCK-luc contains two E-boxes (MyoD binding sites) and two MEF2 binding sites in the promoter region normally inactivated in 10T1/2 cells. 4R-tk-luc is a simplified MyoD-dependent reporter containing four E-boxes upstream of the minimal thymidine kinase promoter. Briefly, 10T1/2 cells were grown in 6-well plates in Dulbecco's modified Eagle's medium containing 10% fetal calf serum until they reached 80% confluence. Then, 10T1/2 cells were cotransfected with 0.5 μg of reporter gene (either MCK-luc or 4R-tk-luc or MEF2x3-luc or pGln5-luc (Refs. 31.Sternberg E.A. Spi G. Perry W.M. Vizard D. Weil T. Olson E.N. Mol. Cell. Biol. 1988; 8: 2896-2909Crossref PubMed Scopus (175) Google Scholar, 32.Lassar A.B. Davis R.L. Wright W.E. Kadesch T. Murre C. Voronova A. Baltimore D. Weintraub H. Cell. 1991; 66: 305-315Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 33.Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Mol. Cell. Biol. 1996; 16: 2627-2636Crossref PubMed Scopus (165) Google Scholar)) and 0.5 μg of activator (either EMSV-MyoD or pcDNA-MEF2C or pGal4-MEF2C or pMyoD-VP16 or pMEF2C-VP16 (Refs. 32.Lassar A.B. Davis R.L. Wright W.E. Kadesch T. Murre C. Voronova A. Baltimore D. Weintraub H. Cell. 1991; 66: 305-315Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 34.Martin J.F. Schwarz J.J. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5282-5286Crossref PubMed Scopus (218) Google Scholar)) in the presence of the regulator (Dermo-1 or a Dermo-1 mutant in pCDNA3.1FLAG vector or pcDNA-M-twist (Ref. 29.Hamamori Y. Wu H.Y. Sartorelli V. Kedes L. Mol. Cell. Biol. 1997; 17: 6563-6573Crossref PubMed Scopus (136) Google Scholar) or EMSV-E12 (Ref. 32.Lassar A.B. Davis R.L. Wright W.E. Kadesch T. Murre C. Voronova A. Baltimore D. Weintraub H. Cell. 1991; 66: 305-315Abstract Full Text PDF PubMed Scopus (686) Google Scholar) or EMSV-Id (Ref.26.Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1797) Google Scholar)). Empty vectors (EMSV or pCDNA3.1) were included to verify that equal amounts of DNA were available for each transfection. 3–4 h after transfection, cells were placed in a mixture of 10% fetal calf serum in Dulbecco's modified Eagle's medium and incubated overnight. Then the medium was changed to 0.5% fetal calf serum for 2 days to induce differentiation. Next, the transfected cells were harvested and assayed for luciferase activity using a commercial luciferase assay kit (Promega). Luciferase activities were normalized to the protein content in each sample. To test whether Dermo-1-mediated transcriptional repression requires deacetylase activity, 10T1/2 cells were treated overnight with 330 nm deacetylase inhibitor trichostatin A (TSA), beginning 24 h after transfection. For all transfection experiments, the luciferase activities were the average of the results of three independent duplicate experiments. To determine the ability of Dermo-1 and its mutants to inhibit MyoD transactivation, 10T1/2 cells were plated onto 6-well plates and transiently transfected with 1 μg of MyoD as described above in the absence or presence of 1 μg of Dermo-1 or each mutant. Empty vectors (pcDNA3.1) were used to normalize the amounts of DNA transfected in each well. After 5 days in differentiation medium, the expression of skeletal myosin was detected by incubating cells first with an anti-myosin antibody (Sigma) for 1 h at room temperature, then with biotinylated anti-mouse secondary antibody for 30 min, and finally with a horseradish peroxidase-strepavidin conjugate (ABC system; Vector Laboratories) for 30 min. Positive cells were visualized by DAB staining for 30 min at room temperature (Roche Molecular Biochemicals). The expression level of MyoD was assayed by standard Western blot analysis. 10T1/2 cells plated on 6-well plates were transiently transfected as described above but with 2 μg of pcDNA3.1FLAG-Dermo-1 or its mutants. Forty-eight hours after transfection, cells were rinsed two times in phosphate-buffered saline buffer and lysed in sample buffer (15 mm Tris HCl, 2% SDS, 4% glycerol, 1% 2-mercaptoethanol). Cell extracts were prepared by boiling for 5 min and brief centrifugation. Equal amounts of each sample were then subjected to 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). FLAG antibody was used to detect the expression of each protein, which was visualized using a commercial chemiluminescence Western blotting kit (Roche Molecular Biochemicals). For immunoprecipitation assays, Dermo-1 and MEF2C were cotransfected into COS cells and, 48 h later, harvested in lysis buffer containing 20 mmTris-Cl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 10% glycerol, and proteinase inhibitor (Roche Molecular Biochemicals). Harvested cells were then transferred to a 1.5-ml Eppendorf tube using a 21-gauge needle and centrifuged to remove debris. FLAG-tagged Dermo-1 proteins were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma) at 4 °C for 3–4 h and then washed five times in 0.1% Nonidet P-40 lysis buffer with gentle agitation at 4 °C. Immunoprecipitated proteins were then separated out by SDS-PAGE, transferred to a polyvinylidene difluoride membrane immunoblotted with MEF2 polyclonal antibody (no. sc-313, Santa Cruz Biotechnology), and finally visualized using the chemiluminescence Western blotting kit (Roche Molecular Biochemicals). The MEF2 antibody is made against the carboxyl terminus of MEF2A and recognizes MEF2A, MEF2C, and MEF2D. Previously, we reported that Dermo-1 was able to inhibit the transactivation activities of myogenic bHLH factors (1.Li L. Cserjesi P. Olson E.N. Dev. Biol. 1995; 172: 280-292Crossref PubMed Scopus (214) Google Scholar). To determine whether Dermo-1 could repress the role of MyoD in initiating the myogenic program, we transiently transfected 10T1/2 cells with MyoD alone or MyoD plus Dermo-1 expression plasmids. As expected, MyoD converted 10T1/2 fibroblast cells into differentiated myogenic cells, as indicated by the expression of skeletal muscle myosin protein detected using an anti-myosin antibody (Fig. 1A). However, when Dermo-1 was cotransfected with MyoD, the number of skeletal muscle myosin-positive cells decreased about 80% (Fig. 1A). As confirmation that the reduction of myogenic cells was not due to a Dermo-1-mediated down-regulation of MyoD expression, the expression level of MyoD was examined in 10T1/2 cells transfected with MyoD alone versusMyoD plus Dermo-1. The expression level of MyoD was comparable in 10T1/2 cells transfected with or without Dermo-1 (Fig. 1B), suggesting that Dermo-1 does not affect the expression of MyoD in pEMSV vector but represses the ability of MyoD to initiate the myogenic program. To investigate whether Dermo-1 inhibits myogenic differentiation through a mechanism similar to another myogenic inhibitor, Id, we examined the inhibitory activities of Dermo-1 and Id in a model of MyoD-mediated gene activation using the E-box-dependent reporters (MCK-luc and 4R-tk-luc). Both Dermo-1 and Id repressed MyoD-dependent transactivation, but Dermo-1 was a more potent inhibitor than Id (Fig. 2, A and B). However, Dermo-1 and Id responded differently when E12 was cotransfected into 10T1/2 cells. The Id-dependent repression of MyoD was almost completely relieved by exogenous E12. In contrast, excess E12 only rescued 5–10% of Dermo-1-mediated repression of MyoD transactivation on 4R-tk-luc and MCK-luc reporters (Fig. 2, A and B). These findings suggested that sequestration of E12 protein is not the major mechanism for Dermo-1-mediated repression. To dissect the functional domains in Dermo-1 required for transcriptional repression, we generated a series of Dermo-1 mutants with mutations in its N-terminal, bHLH, or C-terminal regions (Fig. 3). The inhibitory ability of each mutant was determined using the same model system described above. Wild type Dermo-1 almost completely abolished MyoD transactivating activity, whereas deletion mutant DermoΔC-(121–160) (created by completely deleting the C-terminal region) repressed 45% of the MyoD activity (Fig. 4A). This suggested that the C-terminal region is essential for Dermo-1-mediated gene repression. However, mutants generated by limited deletions within the C-terminal region (i.e. DermoΔC-(134–160) and DermoΔC-(149–160)) did not reduce its ability as a transcriptional repressor (Fig. 4A), suggesting that the critical amino acids mediating Dermo-1 repression lie in the sequence immediately C-terminal to the HLH domain.Figure 4Both C-terminal and helix 1 domains are required for Dermo-1-mediated transcriptional repression of E-box-dependent reporters. 10T1/2 cells were transfected with 0.5 μg of expression vectors encoding MyoD, Dermo-1, or the indicated mutants in the presence of 0.5 μg of reporter MCK-luc (A) or 4R-tk-luc (B). Cells were harvested 48 h after transfection. The transactivation activities of MyoD were assigned a value of 100%. The luciferase activities were the average of the results of three independent duplicate experiments. C, Western blot analysis using the anti-FLAG antibody was done to examine the protein level of Dermo-1 and its mutants. 10T1/2 cells were transfected with equal amounts of FLAG-tagged expression plasmids encoding wild type Dermo-1, DermoΔN, DermoHLH−, Dermob−, and vector pcDNA3.1. For DermoΔ-(C121–160), 1.25 μg of plasmid DNA expressed similar amount of protein compared with 0.5 μg of wild type plasmid DNA. Please note that this amount of DermoΔC-(121–160) protein could repress 45% of the MyoD activity in Fig. 4A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the HLH domain in Dermo-1 was essential for its repression ability, we introduced a proline into the helix 1 region to disrupt the helix structure. The resulting mutant (DermoHLH−) completely lost its ability to repress transcription (Fig. 4A). This suggested that the HLH domain was required for the inhibitory function of Dermo-1. Consistent with this notion, a fusion protein of HLH and C-terminal domains at the C-terminal of the SV40 nuclear localization signal (i.e.DermoHLHC) is sufficient to repress 80% of the MyoD transactivation activities (Fig. 4A). However, the basic region mutant (i.e. Dermob−) and the N-terminal deletion mutant (i.e. DermoΔN-(1–65)) retained most of the repressor function (Fig. 4A). This result suggested that neither the DNA binding region nor the N terminus is essential for Dermo-1-mediated transcriptional repression. Data obtained using MCK-luc as a reporter were similar to results obtained using 4R-tk-luc as a reporter (Fig. 4B). However, Dermo-1 was a less potent repressor of 4R-tk-luc than of MCK-luc. Because MCK-luc contains the two MEF2 sites in this promoter, the results suggest the potential importance of the MEF2 sites in mediating Dermo-1 transcriptional repression. To ensure that loss of the inhibitory function by the mutants was not due to the absence of protein expression, the protein expression levels of all of the mutants described above were examined by Western blot analysis. Because all mutants were cloned in expression vectors containing the FLAG epitope tag, FLAG antibody immunostaining was used to visualize mutant proteins. Similar levels of protein expression were detected for all mutants except DermoΔC-(121–160) (Fig. 4C). Equal amounts of DermoΔC-(121–160) and wild type Dermo-1 proteins were observed when DermoΔC-(121–160) plasmid DNA was transfected 2.5-fold more than wild type plasmid DNA (Fig. 4C). This amount of DermoΔC-(121–160) protein could repress 45% of the MyoD activity (Fig. 4A). To determine whether a putative NLS in the N terminus of Dermo-1 contributes to cellular localization of Dermo-1 protein, three NLS mutants (DermoNls1−, DermoNls2−, and DermoNls1&2−) were generated and transfected into 10T1/2 cells (Fig. 5A). Immunostaining for the FLAG antibody demonstrated that the NLS mutations did not significantly block the translocation of Dermo-1 protein from the cytoplasm into the nucleus (Fig. 5B). Whereas wild type Dermo-1 protein expression was exclusively nuclear, DermoNls1&2− protein expression was mostly but not completely nuclear (Fig. 5B), suggesting that amino acids other than the putative NLS sequences in the N terminus were also providing the nuclear localization signal. Mutation at both NLS sequences did not affect the expression and stability of these mutants compared with that of the wild type Dermo-1, as assessed by Western blot analysis (Fig. 5C). Consistent with the observation that all the NLS mutants remain cytoplasmic, DermoNls1−, DermoNls2−, and DermoNls1&2− mutants all retained the ability to inhibit MyoD-activated reporter genes in transient transfection assays (Fig. 5D). Because MEF2C cooperates with MyoD to transactivate MCK and 4R-tk promoters, the effect of Dermo-1 on the transcriptional activity of MEF2C was examined. MEF2C significantly transactivated the MEF reporter (3xMEF-luc), which contains three MEF2 binding sites upstream of the basal tk promoter. However, such a transactivation was repressed by Dermo-1 in a dose-dependent manner (Fig. 6A). Dermo-1-mediated inhibition was also observed when MEF2C was replaced with MEF2A, another member of the MEF2 family (data not shown). For confirmation that Dermo-1 did not down-regulate the expression of MEF2C, the protein

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Dermo-1, a Multifunctional Basic Helix-Loop-Helix Protein, Represses MyoD Transactivation via the HLH Domain, MEF2 Interaction, and Chromatin Deacetylation-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文 | 图表同屏 (2024)