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E-Box- and MEF-2-Independent Muscle-Specific Expression

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E-Box- and MEF-2-Independent Muscle-Specific Expression MOLECULAR AND CELLULAR BIOLOGY, Aug. 1994, p. 5474-5486 Vol. 14, No. 8 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology E-Box- and MEF-2-Independent Muscle-Specific Expression, Positive Autoregulation, and Cross-Activation of the Chicken MyoD (CMD1) Promoter Reveal an Indirect Regulatory Pathway CLAUDE A. DECHESNE,t QIN WEI, JUANITA ELDRIDGE, LEILA GANNOUN-ZAKI,t PHILIPPE MILLASSEAU,t LYDIE BOUGUELERET,§ DOMINIQUE CATERINA,§ AND BRUCE M. PATERSON* Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received 15 February 1994/Returned for modification 17 March 1994/Accepted 23 May 1994 Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMDI) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken MyfS and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the oth r myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, &utoregulation, or cross activation depends upon the presence of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors. The transcriptional cascade that establishes the muscle cell It has also been assumed that these regulatory interactions lineage in the somites of the newly forming vertebrate embryo involve the direct binding of the myogenic factor proteins to and ultimately leads to terminal myogenesis and the formation the promoters of the responding genes; however, little is of muscle has been only partially defined by the isolation and known about the promoter regulatory regions that control the characterization of the MyoD family of muscle-specific gene- transcription of these myogenic regulatory proteins. This regulatory proteins: MyoD, myogenin, Myf5, and MRF4 (8, 16, leaves open the formal possibility that these interactions are 51, 74). Members of this family of gene regulators have the indirect and may involve the induction or action of additional unusual property that when any one factor is introduced into a regulatory factors. The differences in the in vivo and in vitro variety of nonmuscle cells of different germ layers or tissue expression patterns for the myogenic factors during myogen- origins, myogenesis is activated (7, 13, 16, 71). Closely related esis and in different muscle cell lines suggest that additional proteins have also been isolated from Drosophila melanogaster cellular factors are involved in the myogenic regulatory path- (35, 44), Caenorhabditis elegans (25), and sea urchins (65), and way (10, 23, 56, 58). all have the ability to convert 1OT1/2 mouse fibroblasts into To begin to understand the regulatory circuitry involved in muscle. This conversion involves not only the activation of the the regulation of these myogenic factors, we have analyzed the downstream muscle-specific genes but also the positive activa- tissue-specific expression, autoregulation and cross activation tion of one or more of the endogenous MyoD-related genes, of the chicken MyoD (CMDJ) promoter in various cell back- through poorly understood mechanisms. It is thought that this grounds. Here we demonstrate that the muscle specificity, regulatory circuitry between the myogenic genes plays an positive autoregulation, and cross activation of CMD1 tran- important role in the activation and maintenance of the muscle scription do not involve either E-box or MEF2 sites in the cell phenotype (for reviews, see references 10, 40, 56, and 67). CMD1 promoter and, therefore, that the putative autoregula- tory/cross-activating circuits utilize either additional regulatory regions in the gene and/or ancillary cellular factors yet to be * Corresponding author. Phone: (301) 496-1746. Fax: (301) 402- identified. 3095. t Present address: INSERM U 300, Faculte de Pharmacie, 34090 MATERIALS AND METHODS Montpellier Cedex 01, France. t Present address: Genethon, 91002 Evry Cedex, France. Cell cultures. All of the mammalian cell lines and the § Present address: Centre d'Ptude du Polymorphisme Humain, primary chick fibroblasts (SL-29) were from the American 75010 Paris, France. Type Culture Collection (Rockville, Md.) and were grown and 5474 VOL. 14, 1994 AUTOREGULATION OF THE MyoD (CMDJ) PROMOTER 5475 maintained as previously described (30, 43). The chick fibro- RNA was prepared from embryonic chicken muscle and liver blast cell strain SL-29 was developed by standard trypsinization as described previously (17). tRNA or embryonic chicken liver of a decapitated 11-day SPAFAS Leghorn embryo. The fibro- RNA was used as a negative control. Primer extension was blast-like population can be propagated through a total of carried out under previously described conditions (42), with an about 35 doublings, according to the American Type Culture end-labeled double-stranded 89-bp CMD1 cDNA restriction Collection. Primary cultures of chicken embryonic breast mus- fragment (ApaI-NcoI) labeled at the NcoI site. A total of cle were also prepared and maintained by previously described 25,000 cpm of 32P-labeled probe was hybridized with 20 ,ug of methods (43). Under standard conditions, 50 to 70% of the total RNA at 50°C and was extended with avian myeloblastosis cells in the muscle cultures would fuse. When required, virus reverse transcriptase. For Si nuclease protection studies chicken myoblast cultures were treated with 5-bromo-2'-de- (32), 20 ,ug of total RNA was hybridized at 60°C with 20,000 oxyuridine (BrdU) at a concentration of 5 ,ug/ml for 3 to 5 days cpm of 32P-labeled probe, a 1-kb XhoI-NcoI genomic fragment (3, 41). labeled at the NcoI site. For RNase protection, the 1.2-kb NcoI Cell transfections, CAT, and histochemical I0-galactosidase fragment (+ 174 to -955) was subcloned into pKS+II (Strat- assays. Transient transfections of mammalian and chick cells agene), and the antisense probe was transcribed from the T7 were carried out by the calcium phosphate precipitation pro- promoter. The hybridization and digestion were carried out cedure as described before (30, 48). Forty-eight hours after according to the protocol in the RPAII RNase protection kit transfection, the mammalian cultures were switched from 10% (Ambion). Digestion, extension, and protection products were fetal calf serum to either 2 or 10% horse serum, to trigger analyzed on a 6% sequencing gel along with the G reaction for differentiation. After a further 48 to 72 h, cell extracts were the Si probe (32). prepared from 60- or 100-mm-diameter plates for the analysis Gel mobility shift assays. All of the chicken myogenic of chloramphenicol acetyltransferase (CAT) activity, either on factors for MyoD (CMD1), myogenin, Myf5, MRF4, and E12 thin-layer plates (30, 48) or by using the nonchromatographic were prepared in Escherichia coli, purified by histidine tag procedure (Promega). Assays were normalized to equivalent affinity chromatography (Qiagen), and used in band shift amounts of protein. Transfection efficiencies were determined assays as previously described (57). MEF-2 protein was pre- by cotransfection with either a ,B-galactosidase or a luciferase pared by in vitro transcription-translation of the MEF-2 cDNA expression plasmid (Promega). Conversion values represent clone, obtained from Eric Olson (34). Nuclear extracts were four to seven independent assays. The 1.2-kb NcoI fragment prepared from embryonic chicken muscle or cultured cells by (+174 to -955) and the 322-bp PstI-SmaI fragment (-327 to previously well-established methods (21, 33). Five to ten -5) were subcloned into the 3-galactosidase expression vector micrograms of nuclear extract protein was used in each band pNASS-,B (Clontech) for transfection studies, and the histo- shift reaction. Oligonucleotide probes were synthesized on a chemical ,-galactosidase staining of the cultures was carried Milligen 8750 by Juanita Eldridge in our department and were out as described previously (4). end labeled by fill-in reaction with 32P[dCTP] and Klenow Isolation and mapping of genomic clones. A chicken polymerase (32). The complementary oligonucleotide strand is genomic library in lambda EMBL3 (Clontech) was screened offset enough to allow the fill-in of 1 to 3 added G nucleotides. with the full-length CMD1 cDNA
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