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Selective Repression of Myod Transcription by V-Myc Prevents

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Selective Repression of Myod Transcription by V-Myc Prevents Oncogene (2002) 21, 4838 – 4842 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc SHORT REPORTS Selective repression of myoD transcription by v-Myc prevents terminal differentiation of quail embryo myoblasts transformed by the MC29 strain of avian myelocytomatosis virus Severina A La Rocca1,3,5, Serena Vannucchi1,4,5, Monica Pompili1, Deborah F Pinney2, Charles P Emerson Jr2, Milena Grossi*,1 and Franco Tato` 1,6 1Istituto Pasteur-Fondazione Cenci-Bolognetti, Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Scienze Microbiologiche, Universita’ di Roma ‘La Sapienza’, 00185-Roma, Italy; 2Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104-6058, USA We have investigated the mechanism by which expression In vitro, after an initial proliferative phase, myogenic of the v-myc oncogene interferes with the competence of cells withdraw from the cell cycle, enter the post- primary quail myoblasts to undergo terminal differentia- mitotic stage and activate the transcription of a battery tion. Previous studies have established that quail of muscle-specific genes. Terminal differentiation of myoblasts transformed by myc oncogenes are severely skeletal myogenic cells is activated and maintained by impaired in the accumulation of mRNAs encoding the the concerted action of ubiquitous transcription myogenic transcription factors Myf-5, MyoD and factors, transcriptional coactivators (Puri and Sartor- Myogenin. However, the mechanism responsible for such elli, 2000) and muscle-specific transcription factors a repression remains largely unknown. Here we present belonging to two major groups: the Muscle Regulatory evidence that v-Myc selectively interferes with quail Factors (MRFs) of the MyoD family (Myf-5, MyoD, myoD expression at the transcriptional level. Cis- Myogenin and MRF-4) and members of the MEF2 regulatory elements involved in the auto-activation of family (Lassar et al, 1994; Li and Olson, 1992; qmyoD are specifically targeted in this unique example Molkentin and Olson, 1996). Thus, it is hardly of transrepression by v-Myc, without the apparent surprising that the interference exerted by many participation of Myc-specific E-boxes or InR sequences. oncogenes on the differentiation potential of myogenic Transiently expressed v-Myc efficiently interfered with cells in vitro is often associated with reduced expression MyoD-dependent transactivation of the qmyoD regula- and/or abnormal functioning of MRFs (Alema` and tory elements, while the myogenin promoter was Tato` , 1994; Maione and Amati, 1997). Independent unaffected. Finally, we show that forced expression of investigations on the effects of Myc proteins on MyoD in v-myc-transformed quail myoblasts restored myogenesis have led to different results that include: myogenin expression and promoted extensive terminal (i) complete inhibition of terminal differentiation in differentiation. These data suggest that transcriptional primary quail myogenic cells (Falcone et al, 1985; La repression of qmyoD is a major and rate-limiting step in Rocca et al, 1989, 1994); (ii) inhibition of fusion into the molecular pathway by which v-Myc severely inhibits myotubes, but not of biochemical differentiation and terminal differentiation in myogenic cells. cell cycle withdrawal, in the murine myogenic cell line Oncogene (2002) 21, 4838 – 4842. doi:10.1038/sj.onc. C2C12 (Crescenzi et al, 1994); (iii) severe inhibition of 1205586 MyoD- and/or Myogenin-initiated myogenic conver- sion of murine NIH3T3 cells (Miner and Wold, 1991). Keywords: Myc; qmyoD; myogenic differentiation; cell Indeed, Myc proteins can act as powerful modula- transformation tors of transcription through interaction with Max and/or other factors (Amati et al, 2001; Gartel et al, 2001; Grandori et al, 2000), and their action can be expected to vary, depending on the specific cellular context analysed. In this light, we decided to investigate further the mechanism by which v-Myc, *Correspondence: Milena Grossi, Dipartimento di Biologia Cellulare the product of the viral avian myc allele, severely e dello Sviluppo, Sezione di Sc. Microbiologiche, Via dei Sardi 70, perturbs the differentiation potential of primary quail 00185-Roma, Italy; E-mail: [email protected] Current addresses: 3Department of Immunology and Pathology, myoblasts, on the assumption that such a cellular Institute for Animal Health, Pirbright Surrey, GU24 ONF UK; model ought to be more likely to reflect muscle cell 4Istituto Superiore di Sanita` , Laboratory of Virology, 00161-Roma, physiological behavior, and hence, be informative on Italy the mechanism(s) of oncogenic processes in skeletal 5These authors contributed equally to this work 6Deceased on 7th July 2001 muscle tissues. Primary quail myoblasts in vitro have Received 22 January 2002; revised 4 April 2002; accepted 15 April been shown to express myoD, myf5 and myogenin in 2002 the proliferative phase before terminal differentiation; Transcriptional silencing of qmyoD by the v-myc oncogene SA La Rocca et al 4839 myogenin expression was further up-regulated upon terminal differentiation, whereas myf5 expression appeared to decline in coincidence with the progression of myotube maturation (Russo et al, 1997). In contrast, quail myoblasts infected with avian myelocytomatosis virus MC29, encoding a 110 kD Gag-v-Myc fusion protein, QM(myc), used in this study, failed to terminally differentiated when shifted to differentiation medium (DM), and displayed a strong reduction in the accumulation of myoD, myogenin and myf5 mRNA in proliferative condition, as compared to uninfected myoblasts (QMb) (data not shown; see Falcone et al, 1985; La Rocca et al, 1989, 1994). In order to elucidate the mechanism responsible for such a repression we analysed the functioning of qmyoD and myogenin control region in QM(myc), as compared to QMb. qmyoD expression is essentially dependent on a well defined, tissue-specific enhancer element, named R1, which is located 11.5 to 17.5 Kb upstream of the transcription start site. The R1 enhancer activity localizes to two adjoining fragments, P3 and P4, both containing myogenic E-boxes, putative MEF-2 sites and sequences identical to the corresponding human distal enhancer (Pinney et al, 1995). Transient transfec- tion assays were performed with CAT constructs driven by the following elements of the qmyoD control region: 72400 (promoter), R1 (entire enhancer) and its Figure 1 Transcriptional repression of myoD by v-Myc in quail myoblasts. (a) Schematic representation of the qmyoD R1 distal sub-elements P3, P4, sc1, sc3 and sc5 (Figure 1a), as enhancer. (b) Transient expression of CAT reporters driven by well as with a CAT construct controlled by the 5’ the qmyoD 72400 promoter region, the R1 control region and re- regulatory region 71565 of mouse myogenin, which lative sub-elements, and the myogenin regulatory region 71565, has a strong homology to the corresponding 5’ flanking analysed in QM(myc) versus QMb and (c) in QM(mycER) treated with b-estradiol (+) or left untreated (7). Quail embryo myo- region of avian myogenin (Malik et al, 1995). Figure 1b blasts (QMb) were prepared as previously described (Falcone et shows that in QM(myc), the expression level elicited by al., 1985). Quail cells were propagated in Growing Medium (D- the tissue specific enhancer R1 was significatively lower MEM containing 10% fetal calf serum (FCS), 10% tryptose (about eightfold) than in QMb. The analysis of R1 phosphate broth and 1% chicken serum) (GM). QM(myc) cells sub-elements P3 and P4 indicated that v-Myc could were obtained by high-multiplicity infection with the subgroup A pseudotype MC29(RAV1) and were propagated in GM on col- affect transcription from both these regions, particu- lagen-coated dishes. QM(mycER) were obtained by infection with larly from the P3 sub-element. Interestingly, the the mycER(RAV1) retroviral vector, generated by transfecting analysis of the P3 sub-fragments sc1, sc3 and sc5 chick embryo fibroblasts, producing the helper virus Rous asso- showed that the strongest inhibition (about 12-fold) in ciated virus 1 (RAV1), with the pmycER plasmid, derived by de- letion of the sea oncogene from pmycER-ts-sea vector (Pollerberg QM(myc) was associated with the construct controlled et al., 1995), and propagated in GM on collagen-coated dishes. by sc1 element, that is principally responsible for the Equimolar amounts of CAT constructs were cotransfected over- activity and muscle specificity of R1 enhancer in QMb night with the control plasmid RSVLacZ by the DNA-calcium phosphate precipitation method. Transfected cells were fed with (Pinney et al, 1995). Expression of the positive, but not 77 tissue-specific, sc5 control element was not appreciably GM, with the exception of QM(mycER)+, where 10 M b-estra- diol was also added, and harvested after 48 h. Expression levels of affected in QM(myc). In order to compare more CAT protein were determined in cell extracts using an enzymatic isogenic recipient cells, quail myoblasts transformed immuno-assay (Boeringher Mannheim). For all the experiments, by the conditional, chimaeric v-mycER oncogene RSVLacZ was also cotransfected with the plasmid RSVCAT in (QM(mycER)), where the hormone binding domain identical dishes, to normalize the results of independent experi- ments. Relative CAT amounts are presented as percentage of of the human estrogen receptor (ER) is tethered to v- the RSVCAT control construct expression and represent the aver- Myc
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