We have investigated the mechanism by which expression of the v-myc oncogene interferes with the competence of primary quail myoblasts to undergo terminal differentiation. Previous studies have established that quail myoblasts transformed by myc oncogenes are severely impaired in the accumulation of mRNAs encoding the myogenic transcription factors Myf-5, MyoD and Myogenin. However, the mechanism responsible for such a repression remains largely unknown. Here we present evidence that v-Myc selectively interferes with quail myoD expression at the transcriptional level. Cis-regulatory elements involved in the auto-activation of qmyoD are specifically targeted in this unique example of transrepression by v-Myc, without the apparent participation of Myc-specific E-boxes or InR sequences. Transiently expressed v-Myc efficiently interfered with MyoD-dependent transactivation of the qmyoD regulatory elements, while the myogenin promoter was unaffected. Finally, we show that forced expression of MyoD in v-myc-transformed quail myoblasts restored myogenin expression and promoted extensive terminal differentiation. These data suggest that transcriptional repression of qmyoD is a major and rate-limiting step in the molecular pathway by which v-Myc severely inhibits terminal differentiation in myogenic cells.
In vitro, after an initial proliferative phase, myogenic cells withdraw from the cell cycle, enter the post-mitotic stage and activate the transcription of a battery of muscle-specific genes. Terminal differentiation of skeletal myogenic cells is activated and maintained by the concerted action of ubiquitous transcription factors, transcriptional coactivators (Puri and Sartorelli, 2000) and muscle-specific transcription factors belonging to two major groups: the Muscle Regulatory Factors (MRFs) of the MyoD family (Myf-5, MyoD, Myogenin and MRF-4) and members of the MEF2 family (Lassar et al., 1994; Li and Olson, 1992; Molkentin and Olson, 1996). Thus, it is hardly surprising that the interference exerted by many oncogenes on the differentiation potential of myogenic cells in vitro is often associated with reduced expression and/or abnormal functioning of MRFs (Alemà and Tatò, 1994; Maione and Amati, 1997). Independent investigations on the effects of Myc proteins on myogenesis have led to different results that include: (i) complete inhibition of terminal differentiation in primary quail myogenic cells (Falcone et al., 1985; La Rocca et al., 1989, 1994); (ii) inhibition of fusion into myotubes, but not of biochemical differentiation and cell cycle withdrawal, in the murine myogenic cell line C2C12 (Crescenzi et al., 1994); (iii) severe inhibition of MyoD- and/or Myogenin-initiated myogenic conversion of murine NIH3T3 cells (Miner and Wold, 1991).
Indeed, Myc proteins can act as powerful modulators 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, the product of the viral avian myc allele, severely perturbs the differentiation potential of primary quail myoblasts, on the assumption that such a cellular model ought to be more likely to reflect muscle cell physiological behavior, and hence, be informative on the mechanism(s) of oncogenic processes in skeletal muscle tissues. Primary quail myoblasts in vitro have been shown to express myoD, myf5 and myogenin in the proliferative phase before terminal differentiation; 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 transfection assays were performed with CAT constructs driven by the following elements of the qmyoD control region: −2400 (promoter), R1 (entire enhancer) and its sub-elements P3, P4, sc1, sc3 and sc5 (Figure 1a), as well as with a CAT construct controlled by the 5′ regulatory region −1565 of mouse myogenin, which has a strong homology to the corresponding 5′ flanking region of avian myogenin (Malik et al., 1995). Figure 1b shows that in QM(myc), the expression level elicited by the tissue specific enhancer R1 was significatively lower (about eightfold) than in QMb. The analysis of R1 sub-elements P3 and P4 indicated that v-Myc could affect transcription from both these regions, particularly from the P3 sub-element. Interestingly, the analysis of the P3 sub-fragments sc1, sc3 and sc5 showed that the strongest inhibition (about 12-fold) in QM(myc) was associated with the construct controlled by sc1 element, that is principally responsible for the activity and muscle specificity of R1 enhancer in QMb (Pinney et al., 1995). Expression of the positive, but not tissue-specific, sc5 control element was not appreciably affected in QM(myc). In order to compare more isogenic recipient cells, quail myoblasts transformed by the conditional, chimaeric v-mycER oncogene (QM(mycER)), where the hormone binding domain of the human estrogen receptor (ER) is tethered to v-Myc and confers β-estradiol dependence to the transforming activity of v-MycER, were transiently transfected with selected representative constructs among which used for QM(myc). As shown in Figure 1c, β-estradiol-activated v-MycER inhibited the activity of the qmyoD R1 enhancer and the inhibition pattern was consistent with that observed in QM(myc). These data suggest that v-myc-induced transformation of quail myoblasts is associated with the repression of qmyoD transcription, through interference with the activity of the major tissue-specific control elements of the qmyoD R1 enhancer, and particularly with its sc1 sub-element. The low activity of the myogenin regulatory region (−1565 construct) in QM(myc) and QM(mycER)+β-estradiol indicates a transcriptional interference also with myogenin expression. We also investigated the kinetics of qmyoD mRNA downregulation upon v-MycER activation. Results from Northern blot analysis indicated that repression of qmyoD mRNA by v-Myc was evident within 2 h of β-estradiol treatment (data not shown), suggesting that interference with qmyoD expression is a very early event after v-MycER activation, rather than an epiphenomenon, secondarily occurring during the propagation of myc-transformed cells.
An important feature of MRFs is their ability to transcriptionally activate their own and each other's expression. To assess whether the v-Myc oncoprotein could interfere with qmyoD autoactivation we analysed the activity of R1 enhancer control elements, regulated by the exogenous qmyoD, in uninfected or v-myc transformed quail fibroblasts, QEF and QEF(myc) respectively. Cells were transiently cotransfected with a qmyoD expression vector and various CAT reporters driven by the following elements: R1, P3, sc1, sc3, sc5, and the myogenin regulatory regions, −1565 and −84. Expression from R1 and its muscle specific subfragments P3, sc1 and sc3, in QEF, strictly required the presence of exogenous qMyoD (Figure 2a), confirming the role of the sc1 and sc3 elements in qmyoD autoactivation (Pinney et al., 1995). Interestingly, qMyoD was severely impaired in activating expression from all R1-tissue-specific elements in QEF(myc), in particular, from the sc3 sub-fragment. Unexpectedly, in the same conditions, the myogenin regulatory region resulted equally activated by exogenous qMyoD either in QEF or in QEF(myc), thus indicating that transformation by v-myc does not affect MyoD transactivating function per se; rather, v-Myc interferes specifically with the proper functioning of MyoD on qmyoD 5′ regulatory sequences.
To demonstrate that the effect of v-Myc on the qmyoD autoactivation circuit was specific and direct, CAT constructs containing the qmyoD sc1 and sc3 elements or the two myogenin control region −1565 and −84 were cotransfected in QEF along with the qmyoD expression vector and with myc expression vectors, encoding either wt-Myc or the Δ48-Myc mutant, which lacks the entire leucine zipper domain. Such a mutant is unable to heterodimerize with Max (Crouch et al., 1993), and can neither cause cell transformation nor inhibit myogenic differentiation (La Rocca et al., 1994). The results in Figure 2b show that sc1 and sc3 activation by qMyoD was strongly inhibited by transient co-expression of Myc, whereas myogenin regulatory regions were activated by qMyoD independently from functional Myc expression. Interestingly, the Δ48-Myc mutant could not repress transactivation by MyoD on either sc1 or sc3, suggesting that Max binding was required, and that the aminoterminal transmodulatory domain was not sufficient to interfere with qmyoD autoregulatory circuit. These results strongly indicate that suppression of endogenous myoD transcription in quail myoblasts is a direct and specific effect of v-Myc that might represent a major, rate-limiting step in Myc-induced block of terminal differentiation in QM(myc). On this basis, it would be predicted that forced expression of MyoD in QM(myc) should override the v-Myc-mediated qmyoD repression, and thus activate endogenous myogenin expression; this, in turn, might allow the execution of the terminal differentiation program.
Accordingly QM(myc) were super-infected with an empty adenoviral vector (Ad-null) or with an adenoviral vector encoding murine MyoD (Ad-MyoD) and, after cultivation in DM, super-infected cultures were examined for the expression of Myogenin, skeletal Myosin Heavy Chain (MHC) and fusion into multinucleated myotubes. Figure 3 shows that Ad-MyoD superinfection triggered quantitative expression of endogenous Myogenin as well as MHC in QM(myc), as compared to Ad-null super-infected cells (Figure 3a), and that differentiated cells could fuse into multinucleated myotubes (Figure 3b).
All together, the results here presented are consistent with the following molecular pathway to explain how transcription of myogenin is severely reduced and competence for terminal differentiation is lost in QM(myc): v-Myc-induced silencing of qmyoD transcription would lead to inactivation of myogenin transcription; in the absence of functional Myogenin, neither muscle-specific gene expression nor permanent withdrawal from cell cycle can be activated in QM(myc). This model is supported by the observation that forced expression of an exogenous MyoD in QM(myc) could rescue endogenous myogenin expression and the execution of the myogenic differentiation program. Although at the moment we cannot exclude the possibility that other defects in the myogenic regulatory circuit may contribute to v-myc-induced interference with differentiation of quail myoblasts, the present results strongly indicate that transcriptional repression of qmyoD is a critical, rate-limiting step that can largely account for the inhibition of myogenic differentiation by v-Myc, and led to the identification of qmyoD distal enhancer components (sc1, sc3), previously shown to be involved in qmyoD autoactivation (Pinney et al., 1995), as the regions sensitive to v-Myc induced transcriptional down-regulation.
Myc proteins are known to affect transcription by dimerization with Max and binding to DNA via the specific E-box ‘CACGTG’; in addition, Myc can also interfere with gene transcription via the InR elements and by interacting with several transcription factors (Amati et al., 2001; Gartel et al., 2001; Grandori et al., 2000). Surprisingly, the sc1/sc3 enhancer elements of qmyoD regulatory region do not ostensibly contain myc-specific E boxes or InR elements, although the presence of an intact leucine zipper was required for v-Myc inhibitory activity. Here we have presented evidence that MyoD can transactivate the minimal myogenin promoter, which contains a single myogenic E-box, even in the presence of v-Myc. This finding strongly suggests that myogenic E-boxes, present also in the sc1/sc3 elements, are not involved in the transcriptional silencing by v-Myc, and confirms the contention that v-Myc is not simply interfering with MyoD transactivating function per se, but does so in the context of quail myoD autoregulatory circuit.
Although Myc and MyoD are unable to heterodimerize with each other, we cannot exclude the possibility that Myc and MyoD share a transcriptional or co-transcriptional partner(s), specifically required in the MyoD autoregulatory circuit. Alternatively, an unknown v-Myc target gene might be activated that interferes with MyoD transactivating function on the sc1/sc3 elements.
The implication of the myoD autoregulatory circuit as a specific Myc target might reflect the existence of a similar, reversible mechanism during muscle formation in the embryo. Proliferation and migration of committed muscle precursor cells from the somites to distal sites is required during embryogenesis, and these processes are under the control of several genes including HGF/SF and c-met (Birchmayer and Gherardi, 1998). In migrating precursor cells, presumably derived from somitic cells expressing Myf-5 and/or MyoD, myogenic bHLH factor expression is repressed during the migratory phase and yet the myogenic identity is retained (Tajbakhsh and Buckingham, 1994). We have shown that QM(myc) can be induced to terminally differentiate when co-cultured with various normal cell types, thus implying that v-Myc-induced transformation does not erase the myogenic identity (La Rocca et al., 1989). A c-Myc-dependent, transcriptional repression circuit, akin to the one here illustrated for v-Myc, might be responsible for the reversible suppression of MyoD family member expression in migrating myogenic cells. Consistent with this view, expression of c-myc is known to be elevated in embryonic and proliferating cells (Zimmerman et al., 1986), and the consequences of high c-Myc levels on quail embryo myoblasts are fully comparable to those of v-Myc (La Rocca et al., 1994). Accordingly, QM(myc) represent an attractive model system to identify MyoD-specific target genes in myogenic cells as well as factors, acting upstream of the MyoD family, involved in the establishment and maintenance of the myogenic identity.
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We thank S Alemà (CNR, Roma, Italy) for the adenoviral vectors, D Fischman (Cornell University, New York, USA) for the MF20 antibody, D Gillespie (CRC Beatson Laboratories, Glasgow, UK) for the 237 antibody, E Olson (UT Southwestern Medical Center, Dallas, USA) for the myogenin constructs, B Paterson (NIH, Bethesda, USA) for the anti-Myogenin antibody, G Piaggio (Istituto Regina Elena, Roma, Italy) for the M4 construct and M Zenke (MDC, Berlin, Germany) for the pmycER-ts-sea vector. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), MURST (Cofin 1999) and CNR/MURST (Legge 95/95).
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v-Src inhibits myogenic differentiation by interfering with the regulatory network of muscle-specific transcriptional activators at multiple levels
Journal of Biological Chemistry (2003)