Interplay between proliferation and differentiation within the myogenic lineage

DEVELOPMENTAL

BIOLOGY

154,261-272

(19%)

Interplay between Proliferation and Differentiation within the Myogenic Lineage ERICN.OLSON Department of Biochemistry

and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030 Accepted August 7, 1992

In muscle cells, as in a variety of cell types, proliferation and differentiation are mutually exclusive events controlled by a balance of opposing cellular signals. Members of the MyoD family of muscle-specific helix-loop-helix proteins which, in collaboration with ubiquitous factors, activate muscle differentiation and inhibit cell proliferation function at the nexus of the cellular circuits that control proliferation and differentiation of muscle cells. The activities of these myogenic regulators are negatively regulated by peptide growth factors and activated oncogenes whose products transmit growth signals from the membrane to the nucleus. Recent studies have revealed multiple mechanisms through which intracellular growth factor signals may interfere with the functions of the myogenic regulators. When expressed at high levels, members of the MyoD family can override mitogenic signals and can cause growth arrest independent of their effects on differentiation. The ability of these myogenic regulators to inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that they interact with conserved components of the cellular machinery involved in cell cycle progression and that similar types of regulatory factors participate in differentiation and cell cycle control in diverse cell types. 0 1992 Academic Press, Inc. INTRODUCTION

Cell proliferation and differentiation are mutually exclusive events in a variety of cell types. However, despite years of intense investigation, the molecular basis for this antagonism remains largely unknown. Skeletal muscle provides an auspicious system for approaching this problem because differentiation of skeletal myoblasts is accompanied by transcriptional activation of a battery of muscle-specific genes whose expression is inhibited by defined peptide growth factors or activated oncogenes that transmit growth signals from the cell membrane to the nucleus. The recent identification of the MyoD family of muscle-specific transcription factors, which can activate the muscle differentiation program and inhibit cell proliferation, has also provided insight into the mechanisms for cross-talk between the regulatory pathways that control myoblast proliferation and differentiation (reviewed in Olson, 1990; Emerson, 1990; Weintraub et aL, 1991). In muscle cells, as in other cell types, the decision to divide or differentiate is determined by a balance of opposing cellular signals. This review focuses on the mechanisms through which growth factor signals regulate the muscle differentiation program and considers the ways in which the cellular circuits that control myoblast proliferation and differentiation may interact. PEPTIDE GROWTH FACTORS MYOBLAST DIFFERENTIATION

are committed to a myogenic fate but do not express the phenotypic markers of muscle until they receive the appropriate environmental cues. The exact signals that induce myogenesis in vivo are unknown. However, when skeletal myoblasts are placed in tissue culture, their differentiation is tightly controlled through a repressiontype mechanism by serum and exogenous peptide growth factors that prevent entry into the differentiation pathway until their concentration is reduced below a critical threshold (Fig. 1). Activation of the muscle differentiation program in response to growth factor withdrawal is accompanied by fusion to form multinucleate myotubes, irreversible commitment of muscle cells to the postmitotic state, and transcriptional activation of muscle-specific genes (reviewed in Florini et al, 1991; Olson, 1992). Two of the most potent inhibitors of myoblast differentiation in vitro are fibroblast growth factor (FGF)l and transforming growth factor type-/3 (TGF-P) (Clegg et aL, 1987; Olson et a&, 1986). Whether these growth factors regulate the timing of myogenesis in vivo remains to be determined, but both FGF and TGF-P are expressed at appropriate times and places during embryogenesis to play a role in the regulation of myogenesis. Repression of muscle gene expression by FGF and TGF-P requires their continuous presence, suggesting

INHIBIT ’ Abbreviations used: TGF-P, transforming fibroblast growth factor; HLH, helix-loop-helix; loop-helix; IGF, insulin-like growth factor.

During vertebrate embryogenesis, myogenic progenitor cells arise within the somites. These progenitor cells 261

00121606/92 Copyright All rights

growth factor+; bHLH, basic

FGF, helix-

$5.00

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

262

DEVELOPMENTAL BIOLOGY Myogenic Progenitor Cells

Determinated Myoblasts

Determination

VOLUME X4,1992

F

Peptide Growth Factors I

Differentiation

Differentiated Myotubes

b

FIG. 1. Schematic representation of the steps involved in determination and differentiation of the myogenic lineage. Mesodermal progenitor cells become determined to form myoblasts, which are restricted to a myogenic fate. Myoblasts continue to divide but do not express phenotypic markers of differentiated muscle until they are placed in an environment lacking peptide growth factors. Upon exposure to medium lacking growth factors, myoblasts exit the cell cycle, fuse to form multinucleate myotubes, and activate transcription of a battery of muscle-specific genes.

that occupancy of their corresponding cell surface receptors elicits a short-lived intracellular signal that interferes with the mechanism for muscle-specific gene activation. The requirement for ongoing protein synthesis, for growth factor-dependent repression of differentiation is consistent with the notion that growth factorinducible early gene products may be essential mediators of the inhibitory effects of growth factors on muscle transcription (Spizz et ak, 1986). Indeed, several protooncogenes that are rapidly induced by growth factors have been shown to repress muscle-specific gene activation (see below). Following fusion, muscle cell nuclei lose the ability to reinitiate DNA synthesis, and muscle-specific genes become refractory to repression by exogenous growth factors (Nadal-Ginard, 1978). The molecular mechanism responsible for the loss in sensitivity to growth factors is unknown, but it appears to be coupled to fusion because fusion-defective muscle cell lines retain the ability to downregulate muscle-specific genes and reenter the cell cycle in response to mitogenic stimulation (Nguyen et aZ., 1983; Spizz et ah, 1986). A likely explanation for the loss in growth factor responsivness in terminally differentiated myocytes is the disappearance of one or more components of the signal transduction pathways that link the cell membrane with the nucleus. Indeed, several cell surface growth factor receptors are downregulated following myoblast fusion, although the delayed kinetics for their disappearance suggests that additional events are involved in irreversible commitment to the postmitotic state (Olwin and Hauschka, 1988; Hu and Olson, 1990). Evidence against an alteration in the basic cellular machinery required for DNA synthesis as a cause for the lack of growth factor responsiveness is provided by the observation that forced expression of SV40 large T antigen in terminally differentiated myotubes is sufficient to induce DNA synthesis (Endo and Nadal-Ginard, 1989; Iujvidin et al, 1990). An alternate explanation for the loss in cell growth potential is that

one or more tumor suppressor genes, whose products inhibit cell growth, is activated or expressed following myoblast fusion. The retinoblastoma gene product (Rb), for example, is upregulated during myogenesis (Coppolla et aZ., 1990) and acts as an inhibitor of cell growth (Weinberg, 1990). The fact that SV40 large T binds to Rb and inactivates its growth-inhibitory activity is therefore probably not circumstantial. GROWTH FACTOR-MEDIATED REPRESSION OF MYOGENESIS OCCURS IN THE G, PHASE OF THE CELL CYCLE AND IS INDEPENDENT OF CELL PROLIFERATION

The muscle differentiation program is intimately coupled to the cell cycle, such that muscle-specific transcription is initiated only when myoblasts are growth arrested in the G,/G, phase. Arrest of myoblasts at other points in the cell cycle does not result in musclespecific gene activation, suggesting that the myogenic regulatory program is dependent on gene products that are only expressed or functional in Go/G, or that inhibitory factors expressed at other phases of the cell cycle are incompatible with the events required for activation of the myogenic program. Even within Go/G,, there appears to be a specific compartment that is essential for activation of the myogenic program. FGF and TGF-P inhibit muscle-specific gene expression in Go/G, without stimulating cell proliferation, indicating that growth factor-mediated repression of the myogenic program is not a secondary consequence of cell proliferation (Olson et ah, 1986; Spizz et ab, 1986). Inhibition of differentiation of the fusion-defective muscle cell line BC3Hl by FGF has been shown to be associated with movement of cells to a position 4 hr closer to the G/Sphase boundary than the position of differentiated myocytes in G,/G, (Lathrop et al., 1985). Considering that the immediate early gene products c-Myc, C-FOS, and c-Jun are induced by serum and FGF during this period and can inhibit myogenesis when expressed au-

263

REVIEWS

tonomously, it is tempting to speculate that their induction plays a role in repression (see below). HIERARCHICAL MUSCLE-SPECIFIC

REGULATION TRANSCRIPTION

OF

How can a single growth factor coordinately repress an array of genetically unlinked muscle-specific genes, many of which do not share common &s-acting regulatory sequences? The most likely explanation is that inhibitory growth factor signals are targeted at a single regulator that controls, directly or indirectly, all downstream events within a regulatory cascade. Indeed, members of the MyoD family of myogenic regulators function within the regulatory pathway leading to myogenesis and appear to serve as targets for negative regulation of myogenesis by growth factor signals. The MyoD family includes MyoD (Davis et ah, 198’7), myogenin (Wright et ah, 1989; Edmondson and Olson, 1989), myf5 (Braun et ab, 1989a), and MRFUherculin/ myf6 (Rhodes and Konieczny, 1989; Miner and Wold, 1990; Braun et al. 1990), each of which can activate myogenesis when expressed in a variety of nonmuscle cell types. Members of the MyoD family are characterized by a 70-amino-acid segment of homology that encompasses a basic region and adjacent helix-loop-helix (HLH) motif. Similar basic-HLH (bHLH) motifs have recently been discovered to be present in a number of proteins that participate in cell fate specification, in several basic transcription factors, and in the myc family of oncogenes (Murre et aZ. 1989a). Mutagenesis studies have shown that the HLH motif mediates oligomerization, which brings together the basic regions of HLH proteins to form a joint DNA-binding domain (Voronova and Baltimore, 1990; Davis et ah, 1990). The target sequence for DNA binding of bHLH proteins is CANNTG, known as an E-box. Individual bHLH proteins show distinct binding site preferences that depend on the variable nucleotides within and surrounding the invariant nucleotides of the consensus sequence (Blackwell and Weintraub, 1990). Oligomerization through the HLH motif allows for combinatorial interactions among heterologous HLH proteins and dramatically expands the regulatory potential of this family of transcriptional regulators. There is, however, selectivity among different bHLH proteins with respect to oligomerization partners. Myogenic HLH proteins, for example, can homodimerize, but they preferentially form heterodimers with certain widely expressed HLH proteins, such as the E2A gene products El2 and E47 and the related HEB gene product (Murre et ah, 198913; Brennan and Olson, 1990; Chakraborty et aZ., 1991; Hu et aZ., 1992). E-boxes are found within numerous muscle-specific promoters and enhancers where they serve as direct targets for transcriptional activation by myogenic HLH

proteins. However, several genes that are activated in response to the MyoD family do not contain E-boxes within their control regions, suggesting that they are regulated through indirect mechanisms. In this regard, the myocyte-specific enhancer factor-2 (MEFB) is a muscle-specific nuclear factor that binds an A+T-rich motif found within many muscle-specific promoters and enhancers and can activate muscle-specific transcription in the absence of an E-box (Gossett et al, 1989). MEF2 is induced by myogenin and MyoD, suggesting that it lies “downstream” of myogenic HLH proteins in a dependent regulatory pathway (Cserjesi and Olson, 1991; Lassar et ah, 1991). Activation of muscle transcription by myogenic HLH proteins or MEF2 also requires collaboration with other cellular transcription factors that are more widely expressed (Gossett et ak, 1989; Sartorelli et al., 1990; Lin et aZ., 1991; Cserjesi et al, 1992). The requirement of these accessory factors for activation of muscle transcription provides an additional point for potential regulation of the myogenic program. In addition to activating the expression of genes associated with terminal differentiation, members of the MyoD family positively autoregulate their own expression and cross-activate expression of one another (Thayer et ab, 1989; Braun et al, 1989b). It has been postulated that these autoregulatory interactions amplify the expression of these factors above a threshold necessary to activate the muscle differentiation program and to stabilize the myogenic phenotype. A hypothetical view of the position of the MyoD family within a myogenic regulatory gene pathway is shown in Fig. 2. According to this model, one or more lineagedetermining genes may be activated in mesodermal progenitor cells, causing them to become committed to a myogenic fate. The myd locus, which can activate expression of myogenin and MyoD in lOT1/2 cells, would be a candidate for such a lineage determination gene (Pinney et al., 1988). However, until myd is characterized at the molecular level, its exact mechanism of action will remain conjectural. As a result of myogenic lineage commitment, members of the MyoD family are expressed and the muscle differentiation program acquires the potential to be activated when myoblasts become deprived of growth factors. The final decision of a myoblast to divide or differentiate appears to be determined by a balance between growth and differentiation signals. Overexpression of factors that activate or inhibit either regulatory pathway can tip the balance in one direction or the other. PROTO-ONCOGENES CIRCUIT

FOR

IN THE REGULATORY MYOGENESIS

There are multiple points within the myogenic regulatory gene pathway in which growth factor signals im-

264

DEVELOPMENTAL BIOLOGY Lineage Determination Genes?

Determination

Growth Factor and Oncogenic Signals

Differentiation Intermediate

E-boxdependent Muscle Genes FIG. 2. Schematic

representation in the myogenic

Myogenic

E-box-independent Muscle Genes of a hypothetical lineage. One or

hierarchy

of

more lineage-determining genes may be activated in mesodermal progenitor cells, which leads to formation of myoblasts. Members of the MyoD family are expressed in myoblasts and are able to positively autoregulate their own expression. Growth factor and oncogenic signals prevent activation of genes associated with terminal differentiation by members of the MyoD family and can, under certain situations, inhibit the autoregulatory interactions among these genes, which leads to a loss in their expression. Myoblasts that no longer express members of the MyoD family retain their position in the myogenic lineage, possibly through the action of lineage-determining genes. Upon release from growth factor repression, members of the MyoD family activate expression of intermediate myogenic regulators such as MEF-2 and genes associated with terminal differentiation. MEF-2 may collaborate with myogenic HLH proteins to activate muscle-specific genes or may regulate some genes independently. regulatory

genes

pose negative control over muscle-specific genes. Growth factors can inhibit expression of myogenic HLH proteins in determined myoblasts (Vaidya et al., 1989; Wright et al., 1989; Edmondson and Olson, 1989; Braun et ab, 1989b) and can also silence the activity of myogenic HLH proteins, such that they are unable to activate downstream muscle-specific genes (Davis et aZ., 1987; Tapscott et ab, 1988; Brennan et ab, 1991b; Edmondson et aZ., 1991; Martin et ah, 1992). Since members of the MyoD family activate and maintain their own expression, transcriptional repression of these genes by growth factors is likely to be a manifestation of the inhibitory effects of growth signals on the activities of these proteins. It is well established that the products of proto-oncogenes constitute a regulatory circuit for transduction of growth factor signals from the cell membrane to the nucleus. Transformation of myoblasts with oncogenes

VOLUME 154,1992

whose products act at different steps in growth factor signal transduction pathways has therefore shed light on the mechanisms involved in repression of the myogenie program by growth factors (reviewed in Olson, 1992). Some oncogene products substitute for peptide growth factors and generate intracellular signals that indirectly repress the expression or activity of the myogenie regulators. Members of the Ras family of oncogene products, for example, which bind GTP and couple specific growth factor receptors with intracellular effectors, inhibit myoblast differentiation when activated by missense mutations that diminish their endogenous GTPase activity (Olson et ab, 1987; Payne et al, 1987). Transformation of myoblasts by activated Ras oncogenes extinguishes MyoD and myogenin expression (Lassar et al., 1989; Konieczny et ab, 1989). However, overexpression of MyoD can overcome the block imposed by Ras, suggesting a stoichiometric mechanism for repression that is dependent on the relative levels of MyoD and Ras. The membrane-associated protein kinases, v-&c, v-Fps, and v-ErbB can also mimic the inhibitory actions of peptide growth factors on myogenesis, presumably by activating protein kinase cascades that culminate in the nucleus (Falcone et al, 1985; Holtzer et ah, 1975; Fiszman and Fuchs, 1975; West and Boettinger, 1983). In contrast to Ras, v&c does not seem to interfere with expression of MyoD, but does suppress MyoD’s ability to activate terminal differentiation (Falcone et al., 1991). Other proto-oncogene products, such as c-Myc, c-Fos, and c-Jun, which act in the nucleus, may interfere more directly with members of the MyoD family (see below). It is likely that many of the above oncogene products that inhibit myogenesis act in series, with those in the nucleus mediating the effects of those in the membrane or cytoplasm. In some cases, the repression imposed by oncogenes is a secondary consequence of deregulated growth control (LaRocca et ah, 1989), while in others, repression occurs in G,/G, and is independent of cell proliferation (Olson et al, 1987; Gossett et ab, 1988). Alterations in growth factor-signaling pathways may also contribute to the variations among different cell types in responsiveness to the actions of myogenic HLH proteins. Whereas some cell lines, such as lOT1/2, activate virtually the entire muscle differentiation program in response to the myogenic regulators, others, such as HeLa and HepG2 cells, show little or no expression of either endogenous or exogenous muscle-specific genes (Weintraub et aZ., 1989; Schafer et aZ., 1990). These differences in myogenic potential may reflect variations in the expression of positive- and negative-acting cellular factors that modulate the activities of the myogenic regulators. Activated oncogenes or the loss of tumor suppressor genes in many immortalized cell lines may also account for the failure of myogenic HLH proteins to

265

REVIEWS

1989). The molecular basis for the differing responsiveness of MyoD to growth signals in different cell types is unclear. It is interesting to note that myoblasts in which MyoD expression has been extinguished by growth factors or activated Ras retain their position in the myogenie lineage and rapidly reactivate the expression of these myogenic regulators upon termination of the growth signal (Vaidya et ah, 1989; Gossett et ah, 1988). This suggests that regulatory factors in addition to those of the MyoD family may be responsible for maintaining myoblasts in the lineage. The identities of “upstream” regulators of the MyoD family that could fulfill this role are unknown aside from myd (Pinney et al, 1988), which remains uncharacterized. Repression of myogenin transcription by growth factor signals could, in principle, be mediated by a negative regulatory element in the myogenin promoter, or it MYOGENIC HLH PROTEINS INHIBIT CELL PROLIFERATION could reflect the lack of expression or activity of posiThe antagonism between growth and differentiation tive-acting factors essential for activation of myogenin in the myogenic lineage is illustrated by the ability of transcription. Analysis of the cis- and trans-regulatory myogenic HLH proteins to inhibit cell proliferation system required for myogenin expression has revealed when expressed at high levels (Davis et ak, 1987). that the myogenin promoter contains a MEF-2 site and Growth inhibition has been examined most extensively a high-affinity E-box that collaborate to impart musclewith MyoD and has been shown to occur in the G, phase specificity, positive autoregulation, and growth factor of the cell cycle (Crescenzi et aZ., 1990; Sorrentino et aZ., responsiveness to the myogenin gene (Edmondson et al, 1990). Inhibition of cell growth by MyoD is not a second1992). The finding that myogenin transcription is reguary consequence of differentiation because basic domain lated by MEF-2 and myogenic HLH proteins, which are mutants of MyoD that cannot activate myogenesis re- activated only after withdrawal of growth factors, sugtain the ability to inhibit proliferation. Moreover, sev- gests that repression of myogenin transcription by eral cell types that are refractory to myogenic convergrowth factor signals is due to the absence of activators sion can be induced to exit the cycle by MyoD. MyoD-inof myogenin transcription in proliferating myoblasts, duced growth arrest requires relatively high levels of with no need to invoke a mechanism for active represMyoD, suggesting that MyoD may compete for a limitsion of the locus. These results also suggest that represing cellular factor required for cell proliferation. Given sion of myogenin transcription by oncogenic transcripthat the HLH motif is essential for growth inhibition by tion factors such as c-Fos and c-Jun (see below) does not MyoD, it is tempting to speculate that the target for this involve direct binding of these factors to the promoter activity is another HLH protein or a protein that an- but rather reflects an indirect effect of these factors on other HLH protein utilizes to activate cell proliferation. the activity of other myogenic HLH proteins. activate muscle transcription in some cell types. Indeed, expression of a c-Fos antisense expression vector in HeLa cells, which do not respond to MyoD, can partially restore the ability of MyoD to activate an exogenous muscle-specific target gene in this cell type (Weintraub et al., 1989). HeLa cells, as well as several other cell lines of human origin, are also known to express the human papilloma virus E7 protein, which exhibits properties similar to the adenovirus ElA protein (Chellappan et ah, 1992), and may account for their failure to activate muscle-specific genes in response to the myogenic regulators (Webster et al, 1988; Enkemann et ah, 1990). ElA has been shown to interfere with the functions of myogenie HLH proteins through multiple mechanisms (Braun et al., 1992).

GROWTH

FACTOR OF

SIGNALS MYOGENIC

CAN EXTINGUISH HLH PROTEINS

EXPRESSION

Among the myogenic regulatory genes, myogenin is the most sensitive to repression by growth factor signals. In contrast to MyoD and myf5, which are often expressed in proliferating myoblasts prior to differentiation (Davis et al, 1987; Braun et ah, 1989a), myogenin is not upregulated until cell proliferation has ceased in response to growth factor withdrawal or contact inhibition of cell growth (Wright et al., 1989; Edmondson and Olson, 1989; Edmondson et ah, 1991; Salminen et al., 1991). MyoD expression can also be repressed by high serum levels, FGF, and TGF-0 in some muscle cell lines, whereas in others it appears to be nonresponsive to growth factor signals (Lassar et al,, 1989; Vaidya et al,

GROWTH FACTOR SIGNALS ACTIVITY OF MYOGENIC

CAN EXTINGUISH HLH PROTEINS

THE

In addition to inhibiting the expression of myogenic HLH proteins, growth factor signals can silence the transcriptional activities of these factors. This type of inhibition is apparent during myogenesis, when MyoD and myf5 proteins are present in myoblast nuclei but are unable to activate muscle-specific genes, as long as cells are exposed to peptide growth factors (Davis et aZ., 1987; Braun et ah, 1989a). The delayed activation of muscle differentiation genes relative to expression of the myogenic regulators during embryogenesis may also reflect post-translational repression of the activity of these factors by exogenous growth factors (Hopwood et

266

DEVELOPMENTAL

BIOLOGY

VOLUME

154,1992

Actlvatlon

E-box

FIG. 3. Potential points for negative control of myogenic HLH proteins by growth factor signals. Myogenic HLH proteins (myo) dimerize with El2 or other ubiquitously expressed HLH proteins. Id, which is induced by serum, also dimerizes with El2 and, to a lesser degree, with MyoD, resulting in inhibition of muscle gene expression. Serum may also inhibit DNA binding of myo:ElZ heterodimers, possibly through induced phosphorylation. Activation of muscle-specific transcription by myogenic HLH proteins requires collaboration with other transcription factors that bind surrounding sites in muscle-specific enhancers and promoters. These factors could potentially be repressed by growth factor signals, resulting in repression of the activity of myogenic HLH proteins independent of DNA binding. Putative coregulators that cooperate with myogenic HLH proteins to induce muscle transcription may also be targets for repression by growth factor signals. TGF-P blocks muscle-specific gene activation by myogenic HLH proteins but does not affect their DNA-binding activity, suggesting that its effects could be targeted at a putative coregulator required for activation of muscle transcription.

al., 1989; Sassoon et al., 1989; Scales et ak, 1991; Venuti et a& 1991; Rupp and Weintraub, 1991). Several potential mechanisms through which growth factor signals might negatively regulate the activity of myogenic HLH proteins can be envisioned, including (1) induction of inhibitory HLH proteins, (2) induction of immediate early gene products that negatively regulate muscle-specific genes, and (3) modification of myogenic HLH proteins through changes in phosphorylation. These different types of growth factor-dependent signals could then interfere with the activities of myogenic HLH proteins by impairing dimerization or DNA binding or they could inhibit transcriptional activation independent of DNA binding (Fig. 3). Evidence for the potential involvement of these mechanisms in growth factor-dependent repression of myogenic HLH proteins is summarized below. THE

INHIBITORY HLH PROTEIN REGULATES THE MyoD

Id NEGATIVELY FAMILY

The discovery of the HLH protein Id, which lacks a basic region and inhibits the activity of other HLH proteins by forming biologically inactive hetero-oligomers, provided an attractive model to explain the inhibitory effect of serum on myogenesis (Benezra et al., 1990). Id is expressed at high levels in proliferating cells and is downregulated upon withdrawal of serum. Id oligomerizes preferentially with E2A products and, to a lesser extent, with myogenic HLH proteins. It has been proposed that the decline in Id levels during myogenesis releases E2A proteins to oligomerize with MyoD, which then leads to activation of the myogenic program. Indeed, the DNA-binding activity of myogenic HLH proteins is attenuated in serum-stimulated myoblasts (Buskin and Hauschka, 1989; Brennan et al., 1991b), and the high-affinity E-box in the MCK enhancer is unoccupied under these conditions, despite high levels of MyoD

(Mueller and Wold, 1989). It has not yet been shown, however, whether Id is indeed expressed at a high enough level in myoblasts to consume all available dimerization partners for MyoD and whether Id alone is sufficient to mediate repression by serum. In contrast to serum, which induces expression of Id and inhibits the DNA binding activity of myogenic HLH proteins, TGF-/3 inhibits the activity of myogenin and MyoD without affecting their ability to bind DNA (Brennan et al., 1991b). Id is also downregulated in myoblasts that are arrested in the differentiation pathway by TGF-P. The mechanism through which TGF-/3 represses the transcriptional activity of myogenic HLH proteins is unclear but could conceivably involve repression of the expression or activity of cellular factors required by the myogenic regulators to activate musclespecific transcription. Using a series of myogenin deletion mutants, Martin et al. (1992) showed that repression by TGF-P was targeted, directly or indirectly, at the bHLH region and did not require either the N- or C-terminal transcriptional activation domains. Repression by TGF-/3 is specific for myogenic bHLH proteins and is not observed with the E2A product E47. TGF-P-mediated repression of muscle transcription also maps to the E-box motif and is independent of other DNA sequence elements. In this regard, it has been suggested that the basic regions of myogenic HLH proteins may interact with a coregulator that contributes to muscle-specific gene activation but is not required for DNA binding (Davis et al., 1990; Brennan et ah, 1991a; Weintraub et ab, 1991). The possibility that such a coregulator might be a target for repression by TGF-P is presently under investigation. INHIBITION

OF MYOGENESIS INDUCIBLE EARLY

BY GENE

GROWTH PRODUCTS

FACTOR-

Several immediate early genes that are induced when quiescent cells are stimulated with growth factors have

267

REVIEWS

been shown to repress muscle-specific gene expression. For example, c-Fos and c-Jun are rapidly induced by serum, FGF, and TGF-~3 and individually can inhibit myogenesis. Both Fos and Jun dimerize through their leucine zippers to form the AP-1 complex, which confers growth factor responsiveness to numerous genes (for review, see Karin, 1990). Whereas Jun can bind the AP-1 DNA consensus sequence as a homodimer, Fos fails to bind DNA specifically on its own. Forced expression of the cellular or viral forms of Fos and Jun leads to inhibition of myogenesis (Ball et al., 1988; Lassar et ah, 1989; Rahm et al., 1989; Bengal et ah, 1992; Li et al, 1992). Inhibition of myogenesis by v-Fos is associated with repression of MyoD expression (Lassar et al., 1989). The block to myogenesis imposed by Fos can be overcome by overexpression of MyoD, suggesting that the decision to differentiate is determined by the balance between these antagonistic factors (Lassar et ah, 1989). tramdominant negative mutants of Fos that fail to bind DNA lead to accelerated differentiation upon withdrawal of serum from myoblasts (Bengal et al., 1992), suggesting that Fos is in the pathway for repression of myogenesis by serum. Because Fos and Jun are thought to function downstream of Ras (Imler et ah, 1988), they may therefore also mediate the inhibitory effects of Ras on the myogenic program. Recent experiments indicate that members of the Jun family may act through multiple mechanisms to inhibit myogenesis. The basic region and leucine zipper of c-Jun have been reported to interact directly with the bHLH region of MyoD and inhibit its activity (Bengal et al., 1992). This interaction also inhibits trans-activation of AP-l-containing target genes by c-Jun. The most likely mechanism for this repression would seem to be through mutual inhibition of DNA binding, in a manner analogous to the interactions between c-Jun and the glucocorticoid receptor (Yang-Yen et al., 1990; Schiile et ah, 1990; Jonat et aZ., 1990; Diamond et al., 1990), but this remains to be demonstrated. In addition, the N-terminal activation domain of c-Jun has been shown to act as a potent inhibitor of muscle transcription by MyoD and myogenin (Li et ah, 1992). Repression by the N-terminus of c-Jun is targeted at the bHLH motif of the myogenic regulators. This form of repression appears to involve a competition-type mechanism in which c-Jun competes with the myogenic bHLH for interaction with a third factor required for activation of muscle-specific transcription. This type of “squelching” is specific because cJun does not inhibit general transcription nor does it block trans-activation by the widely expressed bHLH protein E47, which recognizes the same DNA sequence as members of the MyoD family. The latter observation suggests that c-Jun can discriminate between different HLH proteins. Myogenic HLH proteins also appear to

have the ability to discriminate between different members of the Jun family because c-Jun and JunB, which are induced by growth factors, are potent repressors of muscle transcription, whereas JunD, which is expressed ubiquitously (Li et ah, 1990) has no effect on the activity of these proteins. The potential role of c-Myc as a negative regulator of myogenesis has also been intensely investigated. Expression of c-Myc rapidly declines when myoblasts are induced to differentiate, whereas differentiation-defective muscle cells fail to downregulate c-Myc (Sejersen et ah, 1985; Endo and Nadal-Ginard, 1986; Spizz et al, 1987). c-Myc levels also remain elevated following transfer of Ras-transformed myoblasts to serum-deficient medium (Olson et aZ., 1987). The decline in c-Myc appears to be coupled to activation of the differentiation program because deregulated expression of c- or v-Myc interferes with myoblast differentiation (Falcone et ah, 1985; Schneider et ah, 1987; Denis et ah, 1987). Coexpression of c-Myc with myogenin and MyoD indicates that Myc can block the myogenic activity of these proteins, with the extent of inhibition being dependent on the ratio of Myc to the myogenic regulator (Miner and Wold, 1991). These results support the notion that Myc and the myogenic regulators play opposing roles in the control of proliferation and differentiation in the myogenic lineage and that the ultimate fate of a cell faced with these two classes of regulatory proteins is dictated by their relative levels. Because these proteins belong to the bHLH family, it suggests that they exert their effects through a common mechanism mediated by binding to the E-box consensus sequence. However, these two classes of bHLH proteins recognize E-boxes with different nucleotides in the central and flanking variable regions, and they have distinct oligomerization preferences (Blackwood and Eisenman, 1991; Prendergast et al, 1991), making it unlikely that competition for DNA binding or for a common HLH-containing partner provides the basis for their antagonistic activities. It is possible that Myc could induce the expression of a cellular factor that inhibits the activity of myogenic HLH proteins. In this regard, myoblasts that are arrested in the differentiation pathway by Myc have been shown to downregulate Id levels normally, indicating that Id does not act as a mediator of Myc and that Myc and Id represent distinct pathways for negative regulation of myogenesis (Miner and Wold, 1991). PHOSPHORYLATION THE ACTIVITY

AS A MECHANISM OF MYOGENIC HLH

FOR SILENCING PROTEINS

Several serine-, threonine-protein kinases have also been implicated in the signal transduction pathways through which growth factors inhibit myogenesis. Among these is protein kinase C (PKC), which is acti-

268

DEVELOPMENTAL

BIOLOGY

vated by mitogens that induce phosphatidylinositol hydrolysis (for review see Bell, 1986). Treatment of avian myoblasts with tumor-promoting phorbol ester (TPA), which activates PKC, inhibits muscle-specific gene expression (Cohen et ab, 197’7; Cossu et ah, 1983). PKC mutants that are constitutively activated also can substitute for growth factors and silence the activity of myogenie HLH proteins (L. Li and E. Olson, unpublished data). PKC has been implicated as a downstream target of Ras (Imler et ab, 1988), raising the possibility that it may mediate several of the effects of Ras on myogenesis (Vaidya et al, 1991). The ability of PKC to induce Fos, Jun, and Myc also provides a mechanism through which a PKC-dependent signal could indirectly repress the myogenic program. The observation that activated PKC-a can translocate to the nucleus strengthens the possibility that direct phosphorylation of myogenic HLH proteins by PKC may play a role in growth factormediated repression of myogenesis (James and Olson, 1992). CAMP-dependent signal-transducing pathways have also been shown to lead to repression of muscle-specific gene expression (Hu and Olson, 1990). Signal transduction via the CAMP pathway is mediated by the catalytic (C) subunit of CAMP-dependent protein kinase (PKA). In the absence of CAMP, the C-subunit associates with a regulatory subunit as a tetramer RR:CC in an inactive form. Upon elevation of intracellular CAMP, CAMP binds to the R-subunit and causes its dissociation from the C-subunit, which then becomes activated and phosphorylates numerous cellular substrates (reviewed in Edelman et al, 1987). Activated PKA inhibits the ability of myogenic HLH proteins to activate endogenous and exogenous muscle-specific genes (Li et aL, 1992). Repression of the transcription-activating functions of these proteins by PKA is determined by the ratio of R- to C-subunit, such that an excess of R-subunit can attenuate repression imposed by PKA. The mechanism through which PKA inhibits the activities of the myogenie regulators is not yet clear but could involve direct phosphorylation of these proteins or their partners. Alternatively, or in addition, repression could be mediated indirectly by induction of c-Fos or JunB, both of which are induced by the CAMP signal transduction pathway (Muller et aZ., 1989; Chiu et ab, 1989). The transcriptional activator CREB is also induced by CAMP (Yamamoto et CLL,1988) and could conceivably participate in repression through direct interaction with the myogenic regulators or by inducing the expression of one or more negative regulators of the myogenic program. INSULIN-LIKE

GROWTH REGULATORS

OF

FACTORS AS MYOGENESIS

POSITIVE

Whereas peptide growth factors are generally considered to inhibit myogenesis, there is also evidence that

VOLUME

154,199Z

insulin-like growth factors (IGFs) -1 and -11 can stimulate differentiation (Florini and Ewton, 1990; Florini et al., 1991). Intriguingly, myoblast differentiation is accompanied by induction of IGFs -1 and -11 as well as their cell surface receptors (Brunetti et al., 1989; Brunetti and Goldfine, 1990; Tollefsen et aZ., 1989a,b; Szebenyi and Rotwein, 1991), supporting the notion that an autocrine loop, mediated by IGFs amplifies myoblast differentiation, Stimulation of myogenic differentiation by IGFs is preceded by induction of myogenin transcription, which is likley to mediate the effects of IGFs on muscle gene expression (Florini et aZ., 1991). While the myogenin gene is unlikely to be a direct target for the IGF signal transduction pathway, defining the cis-acting elements and trans-acting factors that confer IGF responsiveness to the myogenin locus should shed light on mechanisms for IGF-dependent intracellular signaling. DISRUPTION

OF THE MYOGENIC REGULATORY IN RHABDOMYOSARCOMA

PATHWAY

The skeletal muscle malignancy rhabdomyosarcoma also provides an example of the antagonism between signals for growth and differentiation within the myogenie lineage. Rhabdomyosarcoma cells express MyoD and myogenin to varying degrees, but they show only limited expression of genes associated with terminal differentiation (Hiti et al., 1989; Dias et al., 1991; Tonin et ak, 1991). The phenotype of these cells suggests that they are derived from myogenic progenitors committed to a myogenic fate but are arrested at an early step in the differentiation pathway due to altered growth control. The etiology of rhabdomyosarcoma is heterogeneous and complex. Activated Ras has been implicated in some types of these tumors (Hall et al., 1983). There is also evidence for the loss of a tumor suppressor gene that maps to chromosome 11 in some rhabdomyosarcomas (Scrable et al., 1989). Although MyoD also maps to human chromosome 11 (Tapscott et al, 1988), its expression in rhadomyosarcomas rules out the possibility that its deletion is responsible for the aberrant growth of these cells. Whether the loss of a putative tumor suppressor results in persistent activation of inhibitory growth-signaling pathways or perhaps plays a more direct role in muscle-specific gene activation in this tumor remains to be determined. The cause for tumorigenicity in rhabdomyosarcoma is more complex than a simple decrease in MyoD activity because overexpression of MyoD in rhabdomyosarcoma cells can increase the extent of differentiation but does not seem to diminish tumorigenicity in viva (Hiti et aZ., 1989). PERSPECTIVES

The decision of a myoblast to differentiate is intimately linked to the cell cycle and is influenced by a

REVIEWS

balance between antagonistic signals for growth and differentiation. It is apparent that a wide range of growth factor and oncogenic signals can negatively regulate the muscle differentiation program and that multiple mechanisms are involved in this repression. This redundancy may ensure that the differentiation program is not activated prematurely and thereby allows the population of myogenic progenitor cells to be amplified during embryogenesis. Members of the MyoD family appear to function at the nexus of the regulatory circuits that control myoblast proliferation and differentiation and thereby control the decision of a myoblast to divide or differentiate. While much has been learned about the mechanisms through which the MyoD family members activate muscle-specific transcription, the mechanisms through which these proteins inhibit proliferation remain largely unknown. The observation that MyoD can inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that the myogenic regulators interact with the basic cellular machinery involved in cell cycle progression. Thus, it is probable that insights into the molecular basis for antagonism between proliferation and differentiation within the myogenic lineage will shed light on the interrelationship between these events in a variety of cell types. Work in the author’s laboratory is supported by the National Institutes of Health, The Muscular Dystrophy Association, The Council for Tobacco Research, and the Robert A. Welch Foundation.

REFERENCES Ball, A. R., Jr., Bos, T. J., Loliger, C., Nagata, L. P., Nishimura, T., Su, H., Tsuchie, H., and Vogt, P. K. (1988). Jun: Oncogene and transcriptional regulator. Cold Spring Harbor Symp. @ant. Biol. 53,687693. Bell, R. M. (1986). Protein kinase C activation by diacylglycerol second messengers. Cell 45,631-632. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990). The protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell 61,49-59. Bengal, E., Ransone, L., Scharfmann, R., Dwarki, V. J., Tapscott, S. J., Weintraub, H., and Verma, I. M. (1992). Functional antagonism between cJun and MyoD proteins: A direct physical association. Cell 68,507519. Blackwell, K. T., and Weintraub, H. (1990). Differences and similarities in DNA-binding preferences of MyoD and E2A protein eomplexes revealed by binding site selection. Science 250,1104-1110. Blackwood, E. M., and Eisenman, R.N. (1991). Max: A helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251,1211-1217. Braun, T., Bober, E., and Arnold, H. H. (1992). Inhibition of muscle differentiation by the adenovirus Ela protein: Repression of the transcriptional activating function of the HLH protein Myf-5. Genes Dev. 6,888-902. Braun, T., Bober, E., Winter, B., Rosenthal, N., and Arnold. H. H. (1990). Myf-6, a new member of the human gene family of myogenic determination factors: Evidence for a gene cluster on chromosome 12. EMBO J. 9,821-831. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich E., and Ar-

269

nold, H. H. (1989a). A novel human muscle factor related to but distinct from MyoDl induces myogenic conversion in lOT1/2 fibroblasts. EMBO J. 8,701-709. Braun, T., Bober, E., Buschhausen-Denker, G., Kotz, S., Grzeschik, K., and Arnold, H. H. (1989b). Differential expression of myogenic determination genes in muscle cells: Possible autoactivation by the Mdgene products. EMBO J. 8,3617-3625. Brennan, T. J., Chakraborty, T., and Olson, E. N. (1991a). Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis. Proc. NatL Acad. Sci. USA 88, 5675-5679. Brennan, T. J., Edmondson, D. G., Li, L., and Olson, E. N. (1991b). TGF-P represses the actions of myogenin through a mechanism independent of DNA binding. Proc. Natl. Acad. Sci. USA 88,3822-3826. Brennan, T. J., and Olson, E. N. (1990). Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimerization. Genes Dev. 4,582-595. Brunetti, A., and Goldfine, I. D. (1990). Differential effects of fibroblast growth factor on insulin receptor and muscle specific protein gene expression in BCBH-1 myocytes. Mol. Endocrinol. 4,880-885. Brunetti, A., Maddux, B. A., Wong, K. Y., and Goldfine, I. D. (1989). Muscle cell differentiation is associated with increased insulin receptor biosynthesis and messenger RNA levels. J. Clin. Invest. 83, 192-198. Buskin, J. N., and Hauschka, S. D. (1989). Identification of a myocytespecific nuclear factor which binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene. Mol. Cell. Biol. 9, 26272640. Chakraborty, T., Brennan, T. J., Li, L., Edmondson, D., and Olson, E. N. (1991). Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding. Mel Cell. Biol. 11, 3633-3641. Chellappan, S., Kraus, V. B., Kroger, B., Munger, K., Howley, P. M., Phelps, W. C., and Nevins, J. R. (1992). Adenovirus ElA, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. Proc. Natl. Acad. 5%. USA 89,4549-4553. Chiu, R., Angel, P., and Karin, M. (1989). Jun-B differs in its biological properties from, and is a negative regulator of, C-Jun. Cell 59,979986. Clegg, C. H., Linkhart, T. A., Olwin, B. B., and Hauschka, S. D. (1987). Growth factor control of skeletal muscle differentiation occurs in G,-phase and is repressed by fibroblast growth factor. J. Cell. Biol. 105,949-956. Cohen, R., Pacifici, M., Rubinstein, N., Biehl, J., and Holtzer, H. (1977). Effect of a tumor promoter on myogenesis. Nature (London) 226, 538-539. Coppolla, J. A., Lewis, B. A., and Cole, M. D. (1990). Increased retinoblastoma gene expression is associated with late stages of differentiation in many different cell types. Oncogene 5,1731-1733. Cossu, G., Molinaro, M., and Pacifici, M. (1983). Differential response of satellite cells and embryonic myoblasts to a tumor promoter. Dev. Biol. 98, 520-524. Crescenzi, M., Fleming, T. P., Lassar, A. B., Weintraub, H., and Aaronson, S. A. (1990). MyoD induces growth arrest independent of differentiation in normal and transformed cells. Proc. Natl. Acad Sci. USA 87,8442-8446. Cserjesi, P., and Olson, E. N. (1991) Myogenin induces muscle-specific enhancer binding factor MEF-2 independently of other muscle-specific gene products. Mol. Cell. BioL 11, 4854-4862. Cserjesi, P., Lilly, B., Bryson, L., Wang, Y., Sassoon, D. A., and Olson, E. N. (1992). MHox: A mesodermally-restricted homeodomain pro-

270

DEVELOPMENTALBIOLOGY

tein that hinds an essential site in the muscle creatine kinase enhancer. Development 115,1087-1101. Davis, R. L., Weintraub, H., and Lassar, A. B. (1987). Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987-1000. Davis, R. L., Cheng, P.-F, Lassar, A. B., and Weintraub, H. (1990). The MyoD DNA binding domain contains a recognition code for musclespecific gene activation. Cell 60.733-746. Denis, N., Blanc, S., Leibovitch, M. P., Nicolaiew, N., Dautry, F., Raymondjean, M., Kruh, J., and Kitzis, A. (1987). c-myc oncogene expression inhibits the initiation of myogenic differentiation. Ezp. Cell Res. 172,212-217. Diamond, M. I., Miner, J. M., Hoshinaga, S. K., and Yamamoto, K. R. (1990). Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 249,12661272. Dias, P., Parham, D. M., Shapiro, D. N., Weber, B. L., and Houghton, P. J. (1991). Myogenic regulatory protein (MyoDl) expression in childhood solid tumors: Diagnostic utility in rhabdomyosarcoma. Am. J. PathoL 137,1283-1291. Edelman, A. M., Blumenthal, D. K., and Krebs, E. G. (1987). Protein serinejthreonine kinases. Annu. Rev. Biochem. 56,567-613. Edmondson, D. G., and Olson, E. N. (1989). A gene with homology to the mvc similarity region of MyoDl is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev.

3,628-640.

Edmondson, D. G., Brennan, T. J., and Olson, E. N. (1991). Mitogenic repression of myogenin autoregulation. J. Biol. Chem 266, 2134321346. Edmondson, D. G., Cheng, T.-C., Cserjesi, P., Chakraborty, T., and Olson, E. N. (1992). Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2. Mel Cell. BioL 12,3665-3677. Emerson, C. P. (1990). Myogenesis and developmental control genes. Cum-. Opin. CellBioL 2,1065-1075. Enkemann, S. A., Konieczny, S. F., and Taparowsky, E. J. (1990). Adenovirus 5 ElA represses muscle-specific enhancers and inhibits expression of the myogenic regulatory factor genes, MyoD and myogenin. Cell Growth mer. 1,375-382. Endo, T., and Nadal-Ginard, B. (1986). Transcriptional and posttranscriptional control of c-myc during myogenesis: Its mRNA remains inducible in differentiated cells and does not suppress the differentiated phenotype. Mel CeU. BioC 6,1412-1421. Endo, T., and Nadal-Ginard, B. (1989). SV40 large T-antigen induces re-entry of terminally differentiated myotubes into the cell cycle. In “Cell and Molecular Biology of Muscle Development” (L. H. Kedes and F. E. Stockdale, Eds.), pp. 95-104. A. R. Liss, New York. Falcone, G., Tato, F., and Alema, S. (1985). Distinctive effects of the viral oncogenes myc, erb, fps, and src on the differentiation program of quail myogenic cells. Proc. N&l. Acad Sci. USA 82,426-430. Faleone, G., Alema, S., and Tato, F. (1991). Transcription of musclespecific genes is repressed by reactivation of p~60”-~ in postmitotic quail myotubes. Mol. Cell. Biol. 11,3331-3338. Fiszman, M. Y., and Fuchs, P. (1975). Temperature-sensitive expression of differentiation in transformed myoblasts. Nature (London) 254,429-431. Florini, J. R., and Ewton, D. Z. (1990). Highly specific inhibition of IGF-I-stimulated differentiation by an antisense oligodeoxyribonucleotide to myogenin mRNA. No effects on other action of IGF-I. J. Biol.

Chem.

265,13435-13437.

Florini, J. R., Ewton, D. Z., and Magri, K. A. (1991). Hormones, growth factors and myogenic differentiation. Annu. Rev. Physiol. 53, ZOl216. Florini, J. R., Ewton, D. Z., and Roof, S. L. (1991). Insulin-like growth

V0~~~~154,1992 factor-l stimulates terminal myogenic differentiation hy induction of myogenin gene expression. Mol Endocrinol. 5, '718-724. Florini, J. R., Magri, K. A., Ewton, D. Z., James, P. L., Grindstaff, K., and Rotwein, P. S. (1991). “Spontaneous” differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II. J. Biol. Chem. 266,15917-15923. Gossett, L. A., Zhang, W., and Olson, E. N. (1988). Dexamethasone-dependent inhibition of differentiation of C2 myoblasts bearing steroid-inducible N-ras oncogenes. J. Cell Biol. 106,2127-2137. Gossett, L. A., Kelvin, D. J., Sternberg, E. A., and Olson, E. N. (1989). A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol.

Cell. Biol.

9,5022-5033.

Hall, A., Marshall, C. J., Spurr, N. K., and Weiss, R. A. (1983). Identification of transforming gene in two human sarcoma cell lines as a new member of the ras gene family located on chromosome 1. Nuture(London)303,396-400. Hiti, A., Bogenmann, L. E., Gonzales, F., and Jones, P. A. (1989). Expression of the MyoDl muscle determination gene defines differentiation capability but not tumorigenicity of human rhabdomyosarcomas. Mol. CelL Biol 9,4722-4730. Holtzer, H., Biehl, J., Yeoh, G., Meganathan, R., and Kaji, A. (1975). Effect of oncogenic virus on muscle differentiation. Proc Natl. Acad.

Sci. USA 72,4051-4055.

Hopwood, N. D., Pluck, A., and Gurdon, J. (1989). MyoD expression in the forming somites is an early response to mesoderm induction in Xenows embryos. EMBO J 8,3409-3417. Hu, J.-S., and Olson, E. N. (1988). Inhibition of differentiation of the BC3H 1 muscle cell line through CAMP-dependent and -independent pathways. J. Biol. Chem. 263,19670-19677. Hu, J.-S., and Olson, E. N. (1990). Functional receptors for transforming growth factor-p are retained biochemically differentiated C2 myocytes in growth factor-deficient medium containing EGTA but down-regulated during terminal differentiation. J. Biol. Chem 265, 7914-7919.

Imler, J., Schatz, C., Wasylyk, C., Chatton, B., and Wasylyk, B. (1988). A Harvey-ras responsiveness transcription element is also responsive to a tumour-promoter and to serum. Nature (Lonclon) 332,275278. Iujvidin, S., Fuchs, O., Nudel, U., and Yaffe, D. (1990). SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiation myotubes. Differentiation 43,192~203. James, G., and Olson, E. N. (1992). Deletion of the regulatory domain of protein kinase C (Yexposes regions in the hinge and catalytic domains that mediate nuclear targeting. J. Cell Biol. 116,863-874. Jonat, C., Rahmsdorf, H. J., Park, K.-K., Cato, A. C. B., Gebel, S., Ponta, H., and Herrlich, P. (1990). Antitumor promotion and antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62,1189-1204. Karin, M. (1990). The AP-1 complex and its role in transcriptional control by protein kinase C. In “The Hormonal Control Regulation of Gene Transcription” (P. Cohen and J. G. Foulkes, Eds.), pp. 235253. Elsevier New York. Konieczny, S. F., Drobes, B. L., Menke, S. L., and Taparowsky, E. J. (1989). Inhibition of myogenic differentiation by the H-ras oncogene is associated with the down regulation of the MyoDl gene. Oncogene4,473-481. LaRocca, S. A., Grossi, M., Falcone, G., Alema, S., and Tato, F. (1989). Interaction with normal cells suppresses the transformed phenotype of v-myc-transformed quail muscle cells. Cell 58,123-131. Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, C., Voronova, A., Baltimore, D., and Weintraub, H. (1991). Functional activity of myogenic HLH proteins requires heterooligomerization with E12/E47-like proteins in viva. Cell 66,305-315.

REVIEWS Lassar, A. B., Thayer, M. J., Overell, R. W., and Weintraub, H. (1989). Transformation by activated RAS or FOS prevents myogenesis by inhibiting expression of MyoDl. Cell 58,659-667. Lathrop, B. K., Thomas, K., and Glaser, L. (1985). Control of myogenic differentiation by fibroblast growth factor is mediated by position of the G, phase of the cell cycle. J. Cell Biol. 103,1’799-1805. Li, L., Chambard, J-C., Karin, M., and Olson, E. N. (1992). Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression. Genes Dev. 6, 676689.

Li, L., Heller-Harrison, R., Czech, M., and Olson, E. N. (1992). CAMPdependent protein kinase inhibits the activity of myogenic helixloop-helix proteins. Mol Cell. Biol, in press. Li, L., Hu, J.-S., and Olson, E. N. (1990). Different members of the jun proto-oncogene family exhibit distinct patterns of expression in response to type fl transforming growth factor. J. Biol. Chem 265, 1556-1562. Lin, H., Yutzey, K. E., and Konieczny, S. F. (1991). Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors. Mol. Cell. Biol. 11,267-280. Martin, J. F., Li, L., and Olson, E. N. (1992). Repression of myogenin function by TGF-61 is targeted at the basic helix-loop-helix motif and is independent of E2A products. J. Biol Chem 267,10956-10960. Miner, J. H., and Wold, B. J. (1990). Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc. Natl. Acad Sci. USA 87,1089-1093.

Miner, J. H., and Wold, B. J. (1991). c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation. Mol. Cell. Biol. 11, 2842-2851. Mueller, P. R., and Wold, B. J. (1989). In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246,780-786. Muller, U., Roberts, M. P., Engel, D. A., Doerfler, W., and Shenk, T. (1989). Induction of transcription factor AP-1 by adenovirus ElA and CAMP Genes Dev. 3,1991-2002. Murre, C., McCaw, P. S., and Baltimore, D. (1989a). A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56, ‘777-783. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, L. Y., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., Lassar, A. B., Weintraub, H., and Baltimore, D. (1989b). Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58,537-544. Nadal-Ginard, B. (1978). Commitment, fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis. Cell 15,855-864. Nguyen, H. T., Medford, R. M., andNadal-Ginard, B. (1983). Reversibility of muscle differentiation in the absence of commitment: Analysis of a myogenic cell line temperature-sensitive for commitment. Cell 34,281-293. Olson, E. N. (1990). MyoD family: A paradigm for development? Genes Dev. 4,1454-1461. Olson, E. N. (1992). Proto-oncogenes in the regulatory circuit for myogenesis. Sem Cell Biol. 3,127-136. Olson, E. N., Spizz, G., and Tainsky, M. (1987). The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation. Mol. Cell Biol.

7,2104-2111.

Olson, E. N., Sternberg, W., Hu, J. S., Spizz, G., and Wilcox, C. (1986). Regulation of myogenic differentiation by type fl transforming growth factor. J. Cell Biol. 103,1799-1805. Olwin, B. B., and Hauschka, S. D. (1988). Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle termination differentiation in culture. J. Cell Biol. 107,761-769.

271

Payne, P. A., Olson, E. N., Hsiau, P., Roberts, R., Perryman, M. B., and Schneider, M. D. (1987). A mutationally activated c-Ha-ras allele blocks the induction of muscle-specific genes in BC3Hl cells following mitogen withdrawal. Proc Natl. Ad 5%. USA 84,8956-8960. Pinney, D. F., Pearson-White, S. H., Konieczny, S. F., Latham, K. E., and Emerson, C. P., Jr. (1988). Myogenic lineage determination and differentiation: Evidence for a regulatory gene pathway. Cell 53, 781-793. Prendergast, G. C., Lawe, D., and Ziff, E. B. (1991). Association of Myn, the murine homolog of Max with c-myc stimulates methylationsensitive DNA binding and ras cotransformation. Cell 65,395-407. Rahm, M., Jin, P., Sumegi, J., and Sejersen, T. (1989). Elevated c-fos expression inhibits differentiation of L6 rat myoblasts. J. Cell. Physiol 139,237-244. Rhodes, S. J., and Konieczny, S. F. (1989). Identification of MRF4: A new member of the muscle regulatory factor gene family. Genes Deu. 3,2050-2061.

Rupp, R. A. W., and Weintraub, H. (1991). Ubiquitous MyoD transcription at the mid-bastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X luevis. Cell 65, 927-937.

Salminen, A., Braun, T., Buchberger, A., Jurs, S., Winter, B., and Arnold, H. H. (1991). Transcription of the muscle regulatory gene MYF4 is regulated by serum components, peptide growth factors and signaling pathways involving G proteins. J. Cell Biol. 15, 905917. Sartorelli, V., Webster, K. A., and Kedes, L. (1990). Muscle-specific expression of the cardiac alpha-a&in gene requires MyoDl, CArGbox binding factor, and Spl. Genes Dev. 4,1811-1822. Sassoon, D., Lyons, G., Wright, W. E., Lin, V., Lassar, A., Weintraub, H., and Buckingham, M. (1989). Expression of two myogenic regulatory genes with homology to MyoDl are expressed before somite formation in early embryogenesis. Nature (London) 341,303-307. Scales, J. B., Olson, E. N., and Perry, M. (1991). Differential expression of two distinct MyoD genes in Xenopus. Cell Grmth D$2,619-629. Schafer, B. W., Blakely, B. T., Darlington, G. J., and Blau, H. M. (1990). Effect of cell history on response to helix-loop-helix family of myogenie regulators. Nature @m&m) 344,454-458. Schneider, M. D., Perryman, M. B., Payne, P. A., Spizz, G., Roberts, R., and Olson, E. N. (1987). Autonomous expression of c-myc in BC3Hl cells partially inhibits but does not prevent myogenic differentiation. MoL Cell. Biol. 7,1973-1977. Schtile, R., Rangarajan, P., Kliewer, S., Ransone, L. J., Bolado, J., Yang, N., Verma, I. M., and Evans, R. M. (1990). Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62,1217-1226.

Scrable, H., Witte, D., Shimada, H., Seemayer, T., Wang-Wuu, S., Soukup, S., Koufos, A., Houghton, P., Lampkin, B., and Cavenee, W. (1989). Molecular differential pathology of rhabdomyosarcoma. Genes Chrom. Cancer 1,23-35. Sejersen, T., Sumegi, J., and Ringertz, N. R. (1985). Density-dependent arrest of DNA replication is accompanied by decreased levels of c-myc mRNA in myogenic but not in differentiation-defective myoblasts. J. Cell Phgsiol. 125,465-470. Sorrentino, V., Pepperkok, R., Davis, R. L., Ansorge, W., and Philipson, L. (1990). Cell proliferation inhibited by MyoDl independently of myogenic differentiation. Nature (London) 345,813-815. Spizz, G., Roman, D., Strauss, A., and Olson, E. N. (1986). Serum and fibroblast growth factor inhibit myogenic differentiation through a mechanism dependent on protein synthesis and independent of cell proliferation. J. BioL Chem. 261, 9483-9488. Spizz, G., Hu, J.-S., and Olson, E. N. (1987). Inhibition of myogenic differentiation by fibroblast growth factor or type P-transforming

272

DEVELOPMENTAL BIOLOGY

growth factor does not require persistent c-myc expression. Dev. B&L 123.500-50’7. Szebenyi, G., and Rotwein, P. (1991). Differential regulation of mannose 6-phosphate receptors and their ligands during the myogenic development of C2 cells. J. BioL Chem. 266,5534-553$X Tapscott, S. J., Davis, R. L., Thayer, M. J., Cheng, P.-F., Weintraub, H., and Lassar, A. B. (1988). MyoDl: A nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts. Science 242,405-411. Thayer, M. J., Tapscott, S. J., Davis, R. L., Wright, W. E., Lassar, A. B., and Weintraub, H. (1989). Positive autoregulation of the myogenic determination gene MyoDl. Cell 58,241-248. Tollefsen, S. E., Lajara, R., McCusker, R. H., Clemmons, D. R., and Rotwein, P. (1989a). Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation. J. BioL Chem 264, 13801-13817. Tollefsen, S. E., Sadow, J. L., and Rotwein, P. (1989b). Coordinate expression of insulin-like growth factor II and its receptor during muscle differentiation. Proc. NatL Acnd. Sci. USA 86,1543-1547. Tonin, P. N., Scrable, H., Shimada, H., and Cavenee, W. K. (1991). Muscle-specific gene expression in rhabdomyosarcoma and stages of human fetal skeletal muscle development. Cancer Res. 51,51005106. Vaidya, T. B., Rhodes, S. J., Taparowsky, E. J., and Konieczny, S. R. (1989). Fibroblast growth factor and transforming growth factor fi repress transcription of the myogenie regulatory gene MyoDl. Mol. Cell. BioL 9, 3576-3579.

Vaidya, T. B., Weyman, C. M., Teegarden, D., Ashendel, C. L., and Taparowsky, E. J. (1991). Inhibition of myogenesis by the H-ras oncogene: Implication of a role for protein kinase C. J. Cell BioL 114, 809-820. Venuti, M. J., Goldberg, L., Chakraborty, T., Olson, E. N., and Klein,

VOLUME 154,199z

W. H. (1991). A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells. Proc NatL Acad. Sci. USA 88,6219-6223. Voronova, A., and Baltimore, D. (1990). Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains. Proc. NatL Acad. Sci USA 87,4722-4726. Webster, K. A., Muscat, G. E. O., and Kedes, L. (1988). Adenovirus ElA products suppress myogenic differentiation and inhibit transcription from muscle-specific promoters. Nature &ndon) 332,553-557. Weinberg, R. A. (1990). The retinoblastoma gene and cell growth control. Trends B&hem. Sci. 15,199-202. Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adam, M. A., Lassar, A. B., and Miller, A. D. (1989). Activation of muscle specific genes in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD. Proe. NatL Acad Sci. USA 86,5434-5438. Weintraub, H., Davis, R., Tapscott, 8, Thayer, M., Krause, M., Benezra, R., Blackwell, T., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., and Lassar, A. B. (1991). The myoD gene family: Nodal point during specification of the muscle cell lineage. Science 251,761-766. West, C. M., and Boettiger, D. (1983). Selective effect of Rous sarcoma virus src gene expression on contractile protein synthesis in chick embryo myotubes. Cancer Res. 43,2042-2048. Wright, W. E., Sassoon, D. A., and Lin, V. K. (1989). Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56, 607-617. Yamamoto, K. K., Gonzalez, G. A., Biggs, W. H., III, and Montminy, M. R. (1988). Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature &m&m) 334,494-498. Yang-Yen, H.-F., Chambard, J.-C., Sun, Y.-L, Smeal, T., Schmidt, T. J., Drouin, J., and Karin, M. (1990). Transcriptional interference between c-Jun and the glucocorticoid receptor: Mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62, 1205-1215.