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The Myogenic Basic Helix-Loop-Helix Family of Transcription Factors Shows Similar Requirements for SWI/SNF Chromatin Remodeling Enzymes during Muscle Differentiation in Culture

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m205159200

ISSN

1083-351X

Autores

Kanaklata Roy, Ivana L. de la Serna, Anthony N. Imbalzano,

Tópico(s)

Cancer Mechanisms and Therapy

Resumo

The myogenic basic helix-loop-helix family of transcription factors, MyoD, Myf5, myogenin, and MRF4, can each activate the muscle differentiation program when ectopically expressed in non-muscle cells. SWI/SNF complexes are ATP-dependent chromatin remodeling enzymes. We demonstrated previously that SWI/SNF enzymes promote MyoD-mediated muscle differentiation. To ascertain the requirement for SWI/SNF enzymes in muscle differentiation mediated by different MyoD family members, we examined MyoD, Myf5, MRF4, and myogenin-mediated induction of muscle differentiation in cells expressing dominant negative versions of BRG1 or BRM-based SWI/SNF enzymes. We demonstrated that expression of dominant negative BRG1 or BRM inhibited the induction of muscle-specific gene expression by Myf5 and MRF4; however, myogenin failed to induce measurable quantities of muscle-specific mRNAs, even in cells not expressing dominant negative SWI/SNF. In contrast, all four myogenic regulators induced expression of the cell cycle regulators p21, Rb, and cyclin D3 and promoted cell cycle arrest independently of the SWI/SNF enzymes. We proposed that SWI/SNF enzymes are required for the induction of all muscle-specific gene expression by MyoD, Myf5, and MRF4, whereas induction of the cell cycle regulators, p21, Rb, and cyclin D3 occurred independently of SWI/SNF function. The myogenic basic helix-loop-helix family of transcription factors, MyoD, Myf5, myogenin, and MRF4, can each activate the muscle differentiation program when ectopically expressed in non-muscle cells. SWI/SNF complexes are ATP-dependent chromatin remodeling enzymes. We demonstrated previously that SWI/SNF enzymes promote MyoD-mediated muscle differentiation. To ascertain the requirement for SWI/SNF enzymes in muscle differentiation mediated by different MyoD family members, we examined MyoD, Myf5, MRF4, and myogenin-mediated induction of muscle differentiation in cells expressing dominant negative versions of BRG1 or BRM-based SWI/SNF enzymes. We demonstrated that expression of dominant negative BRG1 or BRM inhibited the induction of muscle-specific gene expression by Myf5 and MRF4; however, myogenin failed to induce measurable quantities of muscle-specific mRNAs, even in cells not expressing dominant negative SWI/SNF. In contrast, all four myogenic regulators induced expression of the cell cycle regulators p21, Rb, and cyclin D3 and promoted cell cycle arrest independently of the SWI/SNF enzymes. We proposed that SWI/SNF enzymes are required for the induction of all muscle-specific gene expression by MyoD, Myf5, and MRF4, whereas induction of the cell cycle regulators, p21, Rb, and cyclin D3 occurred independently of SWI/SNF function. myogenic regulatory factors retinoblastoma tumor suppressor protein reverse transcriptase myosin heavy chain Dulbecco's modified Eagle's medium fluorescence-activated cell sorter cyclin-dependent kinase thymidine kinase chloramphenicol acetyltransferase Mammalian SWI/SNF enzymes were first described as multiprotein complexes that could alter nucleosome structure in an ATP-dependent manner and facilitate the binding of transcriptional activators as well as the TATA-binding protein to nucleosomal DNA (1Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (656) Google Scholar, 2Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. 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The Myf5 cDNA was excised by digestion with BamHI and EcoRI and cloned intoBamHI/EcoRI-digested pBABE to create pBABE-mouse Myf5. Cell lines that inducibly express the Tet-VP16 regulator (Tet-VP16), dominant negative BRM (H17), or dominant negative BRG1 (B22) were maintained and induced by removal of tetracycline (Sigma) as described previously (31de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (280) Google Scholar, 35de la Serna I.L. Carlson K.A. Hill D.A. Guidi C.J. Stephenson R.O. Sif S. Kingston R.E. Imbalzano A.N. Mol. Cell. Biol. 2000; 20: 2839-2851Crossref PubMed Scopus (141) Google Scholar). To generate retrovirus, BOSC23 cells were cultured in 100-mm dishes and transfected at 80% confluence with FuGENE (Roche Molecular Biochemicals) with 10 μg of pBABE-MyoD, pBABE-mouse Myf5, pBABE-MRF4, pBABE-Myogenin, or the empty vector (pBABE). Viral supernatants were harvested 48 h after transfection. Dishes (60 mm) of B22, H17, and Tet-VP16 cells grown in the presence of tetracycline at 50% confluence were infected individually with equal volumes of each different virus in DMEM containing 10% calf serum, 4 μg/ml of Polybrene, and 2 μg/ml tetracycline in a final volume of 5 ml. Each dish was split 1:3 48 h after infection, and the media were replaced with DMEM containing 10% calf serum, 2 μg/ml puromycin, and 2 μg/ml tetracycline. Following drug selection, each infected cell line was split 1:4 into DMEM plus 10% calf serum containing or lacking tetracycline and differentiated as described (31de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (280) Google Scholar). Isolation of proteins and Western blotting were as described (35de la Serna I.L. Carlson K.A. Hill D.A. Guidi C.J. Stephenson R.O. Sif S. Kingston R.E. Imbalzano A.N. Mol. Cell. Biol. 2000; 20: 2839-2851Crossref PubMed Scopus (141) Google Scholar). Antibodies against the FLAG epitope (M2) and Rel B were from Sigma and Santa Cruz Biotechnology, respectively. The anti-cyclin D3 antibody was obtained from Signal Transduction Laboratories. Total cellular RNA was isolated by Trizol (Invitrogen) as described by the manufacturer. Northern analysis was performed as described (32de la Serna I.L. Roy K. Carlson K.A. Imbalzano A.N. J. Biol. Chem. 2001; 276: 41486-41491Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Probes for myogenin, myosin heavy chain, p21, and troponin T were described (32de la Serna I.L. Roy K. Carlson K.A. Imbalzano A.N. J. Biol. Chem. 2001; 276: 41486-41491Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The entire cDNAs encoding MyoD, MRF4, and Myf5 were excised from pEMSV-MyoD, pEMSV-MRF4, and pIC 19H and used as probes. RT-PCR procedures and Rb and hypoxanthine-guanine phosphoribosyltransferase primers were described previously (32de la Serna I.L. Roy K. Carlson K.A. Imbalzano A.N. J. Biol. Chem. 2001; 276: 41486-41491Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). For the transfection experiments, the 4RtkCAT reporter gene plasmid and a control CMV-βgal plasmid were transfected using Superfect (Qiagen) 24 h prior to the onset of differentiation. CAT and β-galactosidase assays were performed as described (31de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (280) Google Scholar, 64Javed A. Gutierrez S. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Mol. Cell. Biol. 1999; 19: 7491-7500Crossref PubMed Scopus (135) Google Scholar, 65Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 9. John Wiley & Sons, Inc., New York1996: 7.14-7.20Google Scholar). CAT signal was normalized to β-galactosidase signal. Cells were grown and differentiated as described above and then fixed as described (66Kowalik T.F. DeGregori J. Schwarz J.K. Nevins J.R. J. Virol. 1995; 69: 2491-2500Crossref PubMed Google Scholar). Incorporation of propidium iodide was determined by flow cytometry. We previously created NIH 3T3-based cell lines that stably incorporate inducible, ATPase-deficient alleles of either the BRG1 or BRM catalytic subunit of mammalian SWI/SNF chromatin remodeling enzymes. In the absence of tetracycline, these cells express the mutant ATPases, which associate with other SWI/SNF subunits and generate non-functional SWI/SNF complexes that act as dominant negatives (35de la Serna I.L. Carlson K.A. Hill D.A. Guidi C.J. Stephenson R.O. Sif S. Kingston R.E. Imbalzano A.N. Mol. Cell. Biol. 2000; 20: 2839-2851Crossref PubMed Scopus (141) Google Scholar). Previous studies utilizing these cell lines revealed a requirement for SWI/SNF complexes for full induction of the endogenous Hsp70gene in response to some but not all environmental stresses and demonstrated that the expression of several constitutively expressed genes were unaffected by the dominant negative complexes (35de la Serna I.L. Carlson K.A. Hill D.A. Guidi C.J. Stephenson R.O. Sif S. Kingston R.E. Imbalzano A.N. Mol. Cell. Biol. 2000; 20: 2839-2851Crossref PubMed Scopus (141) Google Scholar). Subsequent studies demonstrated that SWI/SNF enzymes were absolutely required for induction of the myogenic differentiation pathway mediated by introduction of the MyoD regulator (31de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (280) Google Scholar, 32de la Serna I.L. Roy K. Carlson K.A. Imbalzano A.N. J. Biol. Chem. 2001; 276: 41486-41491Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To further understand the initiation of myogenesis and the myogenic regulatory proteins that mediate this process in this in vitro differentiation model, we sought to compare the requirement for SWI/SNF enzymes when differentiation was induced by each of the four myogenic regulators MyoD, Myf5, MRF4, and myogenin. Our previous studies examining MyoD-induced differentiation utilized a retroviral vector (pBABE (67Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1921) Google Scholar)) to introduce MyoD. For these comparative studies, we cloned the Myf5, MRF4, and myogenin cDNAs into this vector and used the derived retroviruses to initiate the myogenic program in cell lines expressing or not expressing dominant negative BRG1 (B22 cells) or dominant negative hBRM (H17 cells). The parental cell line tet-VP16, which encodes the tetracycline-regulated transactivator but no target gene under tetracycline operator control, was used as a control. Cells infected with the empty pBABE vector served as an additional control. Fig.1 demonstrates that FLAG-tagged mutant BRG1 was inducibly expressed in the differentiated B22 cells, and FLAG-tagged mutant hBRM was inducibly expressed in the differentiated H17 cells. Expression levels of the mutant ATPase alleles were similar for each cell line regardless of the retroviral vector utilized. As expected, no FLAG immunoreactivity was observed in the tet-VP16 cells. Because these cell lines are derived from NIH 3T3 cells, they do not fuse upon differentiation to form myotubes as do many other fibroblasts, thus our analysis involved molecular detection of myogenic markers. Among the four myogenic regulators, myogenin can be induced by the other three factors; however, MyoD, Myf5, and MRF4 typically do not induce the expression of each other in cell culture models of muscle differentiation. Initial experiments therefore analyzed levels of MyoD, Myf5, and MRF4 in tet-VP16 control and B22 and H17 dominant negative cell lines that were infected with retroviruses encoding each of the four MRFs. Tet-VP16 cells infected with the Myf5, MyoD, or MRF4 retrovirus produced only mRNA for the gene encoded by the retrovirus (Fig. 2, A andB). Identical results were obtained upon infection of the dominant negative cell lines (Fig. 2, A and B). The presence or absence of tetracycline did not affect the level of MRF mRNA, indicating that the dominant negative SWI/SNF enzymes did not affect retroviral integration or expression from the viral promoter (Fig. 2, A and B; see also Ref. 31de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (280) Google Scholar). As expected, infection with the empty pBABE vector did not result in expression of any of these MRFs. In each of these blots we observed only one band for each MRF mRNA, and each was slightly larger than the size expected for mRNA transcribed from the endogenous gene (data not shown). Because the retrovirus generates a transcriptional fusion between retroviral gag gene and the MRF cDNA, this indicates that the ectopic expression of Myf5, MyoD, or MRF4 did not activate transcription from the respective endogenous loci. Previous work (31de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (280) Google Scholar,68Thayer M.J. Tapscott S.J. Davis R.L. Wright W.E. Lassar A.B. Weintraub H. Cell. 1989; 58: 241-248Abstrac

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