So~lu2tic Cell and Molecular Genetics. D)L 22, No. 5, 1990, pp. 349-361

Defective Myogenesis in NFB-s Mutant Associated with a Saturable Suppression of MYF5 Activity

Daniel K. Rohrer l,-~ and Helen M. Blau ~

t Del)a~fment oj'Molecular Pharmacology, Stat~fi)rd I/mvet:~'tty Medical Cenles; Sta~f(.,'d, Cal~/i)rma 94305-5332

Received 19 September 1996--Final 19 September t996

A b s t r a e t ~ M y o g e n i c cell lhTes have proved to be useful tools for #zvestigating the molecular mechanisms that control cellular d{ff'erentiation. NFB-s is a mutant myogenic cell lh, e which fitils to d(fferentiate in vitlv, and can repress d~ff'erentiation in normal myogenic cells when.fused to form hetetvkarvons, The NFB-s cell line was used here to study the molecular mechanisms underlying such myogenic repression. Using muscle-spec(fic reporter genes, we show that NFB-s cells fail to ac'ti~,'ate.fidly the muscle di[['erentiation program at a transcriptional level, although muscle-spec(fic transcription can be enhanced by regulators of" differentiation such as pertussis toxin. Paradoxi- cally, we find that the myogenic regulator m3!f5 is expressed at constitutively high levels in NFB-s cells, and retains DNA binding activi~..'. Expression plasmids encoding NFB-derived myf5 cDNA can rescue the myogenic phenol , pc in NFB-s cells, demonstrating that a threshold level q f positive regulators must be reached before the nw)genic program is acfivated. Thus, the dominant negative phenoO~pe does not appear to result from d~q'ective m).'[5, but is due to a dosage-dependent saturable mechanis'nz that inter, fetes with m~?f5 fi~nction. These studies demonstrate that the stoichiometric ratio q f positive and negative regulators is critical j b r determining the myogenic d~ff~erent.iation state.

I N T R O D U C T I O N

Several factors which control skeletd muscle cell differentiation have been described in recent years. Among these are the four members of the basic helix-loop-helix (bHLH) myoD family, including myoD, myogenin, myf5 and MRF-4 (1-4). bHLH factors activate muscle-specific genes through their interaction with myocyte enhancer factor-1 (MEF-1) sites, cis-e~.hancer elements found to be critical for expression of the vast majority of muscle- specific genes described to date. The myogenic bHLH proteins bind MEF- t sites as dimers, and

the preferred dimerization partners for muscle gene activation are El2 and E47 (5, 6), members of the E2A family (7). All muscle-specific bHLH members are capable of converting cultured fibroblasts such as C3H10TI/2 to a heritably myogenic phenotype when over- expressed (t-4), and together with factors such as MEF-2, MHox, and MCAT (8-121) are directly implicated in regulating the expression of virtually atl known muscle-specific genes.

Although expression of the myogenic bHLH proteins is generally correlated with progression to a differentiated phenotype, there are many examples of dimerization partners,

:Cucrcnt address: Deioarsmes~r of Pedialr~c~. Div~slo~l ~f Pediatr~c Cardiology. Sra~fi~rd U71iversizy, St~m/brd. Caiifi~rnla 94305

349

0740 7750/%/0900~0349513950/0 ~': 1':;96 Plenum Publishing Corporamm

350 Rohrer and Blau

growth factors, or culture conditions which block their action, preventing muscle celt differentiation. Such pathways of inhibition may be important for maintaining determined cells such as muscle satellite cells and prefusion myoblasts in an uncommitted state. For ex- ample, products of Id gene family members appear capable of antagonizing differentiation- specific transcription by heterodimerizing with bHLH factors (13-15): levels of Id are elevated under growth-promoting conditions, and their expression rapidly falls upon exposure to differentiation-promoting conditions (13, 14). Differentiation can also be blocked by specific growth factors such as FGF (16), which ultinmtely prevents bHLH proteins from associ- ating with their cognate MEF-I sites (117). Activated oncogenes and growth factors such as ras and TGF-t3 (18-21), or agents that directly or indirectly elevate intracellular cAMP also inhibit muscle cell differentiation, without affect- ing the ability of bHLH molecules to associate with MEF-1 sites (22, 23). These ,findings suggest that perturbations in one or several regulatory steps can influence the ultimate phenotype of a cell, and that "nodal points" of muscle cell differentiation exist (24-27), which are determined by the net balance, or stoichiom- etry of positive and negative regulators.

Study of the NFB cell type has revealed many important features of how the differenti- ated phenotype can be actively repressed and some of the requirements necessary to overcome that repression. Thus, this differentiation- defective mutant serves as a good model for elucidating the mechanislns by which cellular phenotypes are maintained and stabilized. NFB was shown previously to have acquired through mutagenesis a dominant negative phenotype: not only do NFB cells fail to undergo normal muscle cell differentiation, but they also inhibit muscle cell differentiation in trans when fused to normal myoblasts (28). This study extends those findings by demonstrating that the only myo- genic bHLH factor expressed in the subclone NFB-s is myf5, which is present at the protein

level and appears normal in its ability to bind DNA. The repression seen in NFB-s can be overcome by constitutive over-expression of NFB-s-derived myf5 cDNAs. Repression can also be partially relieved by activators of differentiation such as pertussis toxin. Thus, NFB-s is clearly a phenotype governed by the stoichiometric balance of positive and negative regulators. The observed repression of myogen- esis is mediated by a mechanism that impairs myf5 function, either directly or indirectly. Many experiments have focused primarily on the positive regulation of myogenesis by tran- scription factors. Here we explore some of the molecular determinants that repress the myo- genic phenotype, providing insight into the negative regulatory mechanisms that control differentiation and presumably play an impor- tant role in preventing its premature expression.

MATERIALS AND METHODS

Cell Lines and DNA Constructs. NFB cells were kindly provided by Dr. Zachary Hall, and have been described previously (28). A subclone of this original celt line, NFB-s, was generated in this laboratory and used for the studies outlined here. This subclone, unlike the original NFB, overexpresses myf5, but no longer expresses myogenin, and thus was selected for analysis of myf5-specific functions and mechanisms leading to its inhibition. C2C12 mouse myoblasts have been described previously (29). C3H10T1/2 mouse fibroblasts were generously provided by Dr. Andrew Lassar. Cells were grown at 37~ in humidified 10% CO2 incubators. Growth medium for NFB-s and 10TI/2 cells consisted of Dulbecco's modified Eagle's medium (DMEM) + 10% conditioned calf serum and penicillin/streptomy- cin. Growth medium l~r C2Cl2 cells consisted of DMEM + 20% fetal calf serum and penicillin/streptomycin. Differentiation media for all cells consisted of DMEM + 2% horse s e r u n l .

The myosin light chain 1/3 enhancer fragments were kindly provided by Dr. Nadia

Myogenic Repression in NFB Mutant 351

Rosenthal. These were cloned directly upstream of a minimal reporter gene construct consisting of a thymidine kinase promoter linked to firefly luciferase. CMV/3-galactosidase was a gift from Dr. Ed Mocarski.

The wild-type (WT) myf5 expression vector was created in this laboratory from a combination of human and mouse myf5 cDNAs. The cDNA encoded shares 100% identity with mouse myf5 sequence, and was created so that transfected and endogenous RNAs could be distinguished by RNase protection if needed. Mouse cDNA portions (cloned in this lab) corresponded to amino acids 1 through 81, and 167 through 255, while the central portion, amino acids 82 through 166 was encoded by the human cDNA (gift of Dr. Hans Arnold). This hybrid construct was created by insertion of the NcoI-SphI fragment of the human myf5 cDNA (3) in place of that in the mouse cDNA (based on sequence from Buonanno, et al. (30). This cDNA as well as the myf5 mutants which follow, were under the control of SV2 (SV40- derived) enhancer sequences as well as SV40 small intron and polyadenylation sequence.

NFB-s-derived myf5 cDNAs were ob- tained by PCR amplification using oligonucleo- tide primers located in the 5' untranslated and 3' untranslated regions respectively, based on mouse sequences (30): 5'UTR: 5'CGGAATTCT- GCTGAATCCAGGTATTC 3'. 3'UTR: 5'CGG- GAAITCTCATAAAGTGGCAGGAC3' . Itali- cized sequence is non-homologous, inserted for cloning puq~oses.

The various myf5 mutants were created as follows, all corresponding to published mouse myf5 sequence: WT = wild type myf5 cDNA; myfl31 = Ile insertion in helix 2 of HLH domain, amino acid 123; myfBam = Tyr-Arg-lle-Arg- Val insertion in helix 2 of HLH, amino acid 123; myfAC = truncation of C-terminal 107 amino acids; myfkN = truncation of N-terminal 47 amino acids; myfC 107 = C-terminal 107 mnino acids; mytN100 = N-terminal 99 amino acids.

Double stranded MEF-1 oligonucleotides used for EMSA were derived ti~om the human MLC 1/3 enhancer (31).

MEF-1 oligo: 5 'GATCAAGTAACAG- CAGGTGCAAAATAAAGT3' . Italicized por- tions indicate MEF-l binding E box. The corresponding opposite strand was synthesized such that a 5' overhanging G residue was present at both ends for Klenow fill-in with dCTP and/or 32PdCTR 32p labelled oligonucteo- tides were typically labelled to a specific activity of -- 108 cprrdug.

The 4R enhancer oligonucleotide was created by synthesizing the following oligo- nucleotide and its exact complement, based on sequences used by Weintraub, et al (32): 5 ' C G A T C A G C A G G T G T T G G G A G G C A G - CA G G TGTT GGG A GGC A G CA GG TGTT GGG AGGCAGCAGGTG~FGATC3 ' Following syn- thesis and annealing, the ds oligonucleotide was ligated, digested with Sau3A, then subcloned into the minimal TK-hiciferase construct.

Transjection and Staining of Cells to Assay Reporter Gene Activity. Supercoiled DNA for transfection was prepared by two successive CsC1 bandings. Cells were transfected by CaPO4 precipitation, and DNA precipitates were at- lowed to incubate on cells overnight. The following morning, cells were rinsed with PBS, then replaced with growth medium for 24 hours, followed by differentiation media for 48 hours. Pertussis toxin, when used, was added with differentiation media at a final concentration of 50 ng/ml (Sigma). To measure reporter gene expression, cell extracts were prepared by conventional means. Eqaal volumes of cell extract were used to measure [3-galactosidase activity by chemihiminescence. Cell extract volumes were then normalized to these values tbr determination of luciferase activity by standard methods.

Cells stained for immunofluorescent detec- tion of myosin heavy chain (MHC) and CAT protein were fixed in PBS + 1% formaldehyde at room temperature for 15 lninutes. The fixed cells were washed with PBS, followed by permeabilization with -20~ methanol for 5 minutes, Fixed and permeabilized cells were then incubated with anti-sarcomeric MHC monoclonal antibody 4A.1025 (developed in

352 Rohrer and Blau

our lab; (33)) and anti-CAT antibodies (Santa Cruz Biotechnologies), followed by washing and incubation with Texas Red conjugated anti-rabbit, and fluorescein isothyocyanate con- jugated anti-mouse secondalT antibodies. Immu- nofluorescence was visualized with a Zeiss Axiophot fluorescence microscope.

Electrol)horetic Mobility Sh!fl Assays and Antibody Supershifts. Nuclear extracts were prepared as described by Dinghaln, and modi- :fled by Olson (34). Briefly, cells were scraped off of tissue culture dishes in ice-cold PBS, followed by resuspension in low salt buffer (10 mM HEPES, 1.5 mM MgCI> 10 mM KCI, 0.5 mM DTT, pH 7.9) and dounced with a loose fitting pestle. Low speed centfifugation was performed to recover the nuclear pellet, which was resuspended in low salt buffer, dounced, and re-centrifuged. The nuclem: pellet was then resuspended in glycerol-containing high salt buffer with protease inhibitors (20 mM HEPES, 1.5 mM MgCI2, 550 mM NaC1, 0.2 mM EDTA, 25% glycerol, 0.5 mm DTT. 0,5 mM phm~y- methyl sulfonyl flouride (PMSF), 2 uM leupep- tin, 20 in TIU aprotinin/ml, pH 7.9), and rocked gently at 4~ for 30 minutes. Following microcentrifngation for 10 minutes at 4~ the supernatant was desalted into binding buffer (15 mM HEPES, 40 mM KCI, 1 mM EDTA, 0.5 mM PMSF, 0.5 mM DTTo 20% glycerol, pH 7.9) by column chromatography over P6-DG (Bio- Rad), and frozen in aliquots at -80~ Protein concentration was determined by the method of Bradtbrd (gio-Rad). Binding reactions were carried out for 30 minutes at room temperature, in a mixture containing 4 big of nuclear extract, 2 ~ag poly dl:dC, 15,000 cpm 32p labelled MEF-t oligonucleotide, 0.05% NP-40, in 1• binding buffer, plus or minus I big of the IgG fraction of pre-immune serum or myf5 antiserum. EMSAs were performed as follows: 4% acrylamide gels containing 1 • TBE buffer (1 X TBE = 0.089 M Tris-borate, 2 mM EDTA) were pre-run at room temperature for 1 hr at 120V, followed by loading of the binding reactions. Samples were electrophoresed 2 hours at 120V, followed by moun!i~lg and drying of the gel on Whatman

3MM chromatography paper. Autoradiographs were produced by exposing these gels to Kodak XAR-5 film in cassettes with Lightning Plus enhancer screens.

Production q# re)f5 Antiserum. A fusion protein was created between maltose binding protein (MBR New England Biolabs) mad the C-terminal 107 anaino acids of mouse myf5. This region of mytLS has very poor homology to the other myogenic bHLH proteins, and was selected to avoid any potential cross-reactivity. An in-frame fusion was ascertained by sequenc- ing and increased molecular weight (compared to wild type MBP) of the hybrid on SDS- polyacryamide gels. Several milligrams of batch-electrophoresed MBP-(myf5) fusion pro- tein were electroeluted and used as immunogen in two New Zealand White rabbits, using RIBI adjuvant. Animals were immunized with 250 tag of fusion protein, followed by 250 ~g boosters 4 weeks later. Booster immunizations were re- peated every 4 weeks. One rabbit produced antisermn which specifically recognized myf5 in the EMSA, and was used for further study and boosting, The IgG fraction of pre-immmae and immune serum was isolated by protein A affinity chromatography.

R E S U L T S

mRNA Profile of NFB-s Cells. Northern blot analysis was performed on total RNA samples from boda C2C12 cells and NFB-s cells. Figure 1 shows that transcripts encoding myoD, myogenin, and lnyf5 are all highly expressed in C2C12 cells, whereas the NFB-s cells appear to express only myf5 mRNA at a significant level. Indeed, NFB-s cells overexpress myf5 relative to the parental C2C12 cells. This blot was also probed with cDNA for the HLH inhibitor of differentiation Id-l, which is expressed in C2C12 cells in growth media, but not differentia- tion media (Fig. 1, lower panel). Comparable mnounts of NFB-s RNA show Tittle if any expression of ld-1 mRNA in either culture condition. This result would argue against overexpression of Id-1 as being causal in

Myogenic Repression in NFB Mutant 353

the NFB-s phenotype. The observed overexpres- sion of myf5 led to the hypothesis that the myf5 in NFB-s cells was defective mid acted in a dominant negative manner to suppress myogenesis.

t~'f5 Protein is Expressed in NFB-s Cells and Binds the MEF-] Site. Polyclonal antibod- ies were raised against the C-terminus of mouse myfS, which is distinct from other bHLH C-termini, in order to assess patterns of myf5 expression in NFB-s cells. The C-terminal 107 anmlo acids of mouse tnyf5 were fused in-frame to the 37 kDa maltose binding protein (New England Biolabs), and the resultmat fusion protein was used to immunize rabbits, Antisera were then characterized for their ability to supershift mouse myf5 bound to MEF-1 ele- ments, using the human myosin light chain I/3 enhancer (3 l) in electrophoretic mobility shift assays (EMSAs). As can be seen in Fig. 2, the IgG fi-action of rabbit antiserum against mouse myf5 fails to specifically retard DNA-protein complexes ibrmed between C3H 10T I/2 ( 10T 1/2) fibroblast cell nuclear extracts and .~2p labelled MEF-1 oligonuc[eotide. Thus the 10T1/2 cell which does not express any muscle-specific proteins serves as a negative control. However, a speci tic DNA-protein complex t}om NFB-s cell nuclear extracts is supershifted by anti-myf5 IgG. As expected, nuctear extracts from C2C12 cells also exhibit a MEF-1 binding activity which can be supershifted with the myf5 antiserum. Neifller of these cell types contains nuclear proteins which are supershifted by pre-immune serum. The profile of supershifted nuclear proteins from C2Ct2 and NFB-s cells closely parallels the profile of bHLH mRNA

Fig. L mRNA expression Wofiles m C2C12 a~d NFB-s cells. Top panel: lt) pg of total RNA extracted from C2C12(C) and NFB-s cells (N) grown under differentiation promoting condiUons (DMEM + 2% horse serum) was electropboresed and blotted t o Nylon membranes, then we/bed with cDNAs coding for myoD, myogenin, and myl3. Equal RNA lnading was verified by a photograph of the ethidium bronude stained gel before mmsfer 128S). Bottom panel: Expression of Id-I mRNA under growth promtmng (G: DMEM + 20% fetal calf serum) or differentmtion promoting (F: DMEM + 2% horse serum) condiuons was assessed in C2C12 and NFB-s cells. Equal RNA loading was v eri tied by a photograph of the ethidi u m bromide s tained gel before transfer (28S),

expression patterns. C2C12 cells express myf5, myoD, and myogenin at high levels, and this is reflected in the ability of myf5 antisera to supershifi a subset of the MEF-I complex, whereas in NFB-s cells, myf5 is the only bHLH appreciably expressed, resulting in a complete supershifl of MEF-1 activity. These results demonstrate the specificity of the myf5 antise-

C N

myoD

G

: i : i " # ..... myogenin

myf5

28S

C N F G F

Id-1

28S

354 Rohrer and Blau

Ext rac t 10T 10T NFB NFB C2 C2 NFB C2

Antibody PI Myf PI Myf PI Myf - o

C o m p e t i t o r . . . . . . + +

MEF-1 Supershift

MEF-1

Free Probe

Fig. 2. Myf-5 expression and DNA binding capaciw as demonstrated by e/ectrophoreUc mobility shift assay (EMSA) on C3HI 0T1/2, NFB-s, and C2C12 nuclear extaacts. Nuclear extracts prepared from C3H I 0TI/2 (10), NFB-s (NFB), or C2CI 2 (C2) cells were incubated with 32p labeled MEF-1 oligonuclcotide 13 lmer derived from the human myosin light chain 1/3 enhancer) in tbe presence of either pre-immune (PI) or immune sera to myf5(Myf), or an excess of unlabeled MEF-1 oligonucleotide. The presence of supershifted protein-DNA complexes in the NFB-s and C2C12 cell extracts only in the presence of immune sera to myf5 demonstrates the specificity of the antisera, as C3H10TI/2 cells do not express any myogemc bHLH genes. The overexpressmn of myf5 mRNA m NFB-s cells is also reflected in the greater intensity of supe~hifted DNA~protein complex. The specificity of complex formation was tested by incubating reactioT~s with a 50-fold excess of tmlabeled MEF-I oligonncleolide. MEF-1 complexcs are efficleJltty competed for by the witd type MEF-1 oligonucleottde. By the criteria of DNA binding and competitmn by specific oligonucleoudes, NFB-s derived myf5 behaves ,denucally to C2Ct2 derived myf5.

rum. They also show that the dominan t negat ive

phenotype which character izes N F B - s cells does

not disrupt expression o f the myt25 protein or its

abil i ty to bind DNA. The profi le seen here is

identical whether NFB-s is g rown under growth

promot ing or differentiat ion promot ing condi-

tions, demonst ra t ing that myf5 D N A binding

act ivi ty is not regulated in NFB-s cel ls (data not

shown).

The D N A binding specificity o f NFB-s -

der ived M E F - I complexes was also tested by

adding in excess unlabel led M E F - 1 o l igonucleo-

tides. As can be seen in the last two lanes o f Fig.

2, a 50- tb ld excess of unlabel led MEF-1

o l igonucleot ide reduces M E F - I b inding activity

to near ly undetectable levels in both C2C12 and

NFB-s cells. The behavior o f NFB-s -de r ived

MEF- I binding activity is qual i ta t ively sinfilar

Myogenic Repression in NFB Mutant 355

to that seen for C2C 12 cells, arguing against the possibility that a novel MEF-1 binding activity is associated with NFB-s cells. By contrast with the slower migrating MEF-I complexes, the fastest migrating complex is likely to represent a non-specific interaction. This is supported by the observation that this complex is also observed in incubations of 32p MEF-1 with 10TI/2 nuclear extracts. Thus, we conclude that the myf5 mRNA present in NFB-s cells is translated and is capable of binding its DNA consensus site.

Expression of MLC1/3 Reporter Genes in NFB-s Cells. To test whether the myf5 protein present in NFB-s cells was functional, its potential to induce expression from a myosin light chain 1/3 muscle-specific reporter gene construct was tested by transfection. The MLC1/3 enhancer was placed upstream of a basal TK promoter controlling luciferase cDNA, and was transiently co-transfected together with a constitutively active reporter construct (CMV [3-galactosidase) to correct t!or transfection efficiency. Various fragments of the MLCI/3 enhancer region have been used extensively as muscle-specific reporters, and the tissue re-

Fig. 3. Extent of myogenic determinatmn and muscle speci tic signal transducfion m NFB-s, 10TI/2, C2C 12 cells. NFB-s cells were tested for thmr level of myogenic determinauon by transfecfing in muscle-specific reporter genes, Various muscle specific enhancer fragments derived ti'om the human myosin light chain 1/3 gene (gill, N, Rosenthal) or a synthetic oligomer containing four MEF-I sites in tmxtem were tizsed t~ TK-luciferase, These constructs werc transfected ~dong with CMV-I3gal, a constitutively active reporter gene used to standardize transfection effic,ency. Cell extracts were prepared m~d assayed from such transfectants wh.lch had been m low serum (differentmtiou) medium ('~r 48 hours following trmasfecdon, The upper panel is ~ schematic of the muscle-specific enhancer fragments linked to the TK- luciferase cassette. The middle pm~el demonstTates that MLCI/3 enhancer fragments are expressed (relative to TK-lucilerase) significantly Ingher il~ NFB-s ceils than the uncomuutted IOTI/2 cell, illustrating some level of myogemc determinatton (Gt7, n := 5; .4/.5. n = 2" mean • S.E.). The boltom panel demonstrates that pertus- sis mxm (50 ng/ml, 48 hrs), has a significant effect m tim NFB-s celt, upregulatmg several muscle specific constructs. The uncommitted, non-myogemc 10TI/2 celt displays no such muscle specific signaling activity (GI7, n = 4; all others, n = 2: mean -+ S.E.),

stricted expression of these elenaents has been well characterized (35-37). This enhancer is composed of several well characterized muscle- specific elements (see Fig. 3, top): The 550 bp .4/.5 fragment contains three MEF-I sites that bind the myoD family of proteins, a MEF-2 binding site, CArG box, and an MHox site.

The G17 construct is expressed in NFB-s cells several fold higher than the enhancerless TK luciferase construct. Moreover, these rela- tive expression levels are significantly elevated by comparison to those seen in non-myogenic 10T1/2 cells (Fig. 3B). The level of expression observed with the G17 construct is on par with

Reporter Gene Control Regions

. 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4, .5 C,~rG M~ox

I I l~176 Legend

G l r CArG ~r - MEF-1 Site J

Dra ~) - MEF-2 Site I1 - MLC 1/3 Enhancer

4 R M E F ~ - Synthet c 4X MEF-1

2oo T o c2cz2 :~ ~ / B NFB ,'o ~ 1 5 0 I I I , ' I T I / ' ~ -6 ,~ 100

~ 50

"~ --I 6

E ~- 4 me

2 ,,o o

GI7 .4/.5 Construct

c~ 3 C 0 OF-

O , - Z~ v) 2

L~

o

13C2C12 I~NFB

GI7 Dra 4R Construct

356 Rohrer and Blau

the larger and more complex .4/.5 enhancer construct, and together these demonstrate a consistent but small level of muscle determina- tion in NFB-s cells relative to 10T1/2 fibro- blasts. Nonetheless, the expression levels of these constructs in NFB-s cells is ~5-10% of the levels in the pm'ental C2Ct2 cells. It is likely that the overexpressed myf5 is giving rise to the muscle-specific expression seen here. However, it is impossible to segregate the contributions of individual bHLH factors from other muscle specific bHLH factors in the committed C2C12 cells, and there is no way to make a direct comparison between the activities of NFB-s- derived myf5 and C2C 12-derived myf5.

Pertussis toxin treatment of myogenic cells is known to enhance diflerentiation potential mad expression of muscle specific genes (38). As a test of whether NFB-s cells would respond to such a stinmlus, we treated MLC1/3 transfec- rants with pertussis toxin at 50 ng/ml. Figure 3C demonstrates that two different MLC1/3 con- structs as well as the 4R construct m'e all positively regulated by pertussis toxin treat- ment. The 4R construct represents an artificial enhancer containing an array of four MEF-I sites in tandem (4R-Luc, adapted from Wein- traub, et al., (32)). These tandem MEF-1 sites are known to interact specifically and exclu- sively with myogenic bHLH proteins, and are thus thought to reflect the activity of myogenic bHLH proteins in a given cell.

NFB-s cells upregulate expression from all three enhancers in the presence of pertussis toxin, and these results are informative in several respects. First, a signal transduction pathway known to operate in muscle cells to control nmscle gene expression is intact in NFB-s cells, presumably through its efl:ects on G-protein coupling or protein phosphorylation. Also. since all constructs including the 4R are upregulated in the presence of pertussis toxin, the activity of myf5 itself is likely to be increased in NFB-s cells. The fold increase in expression induced by pertussis toxin is similar when comparing C2C12 cells and NFB-s cells, suggesting that the G-protein pathway stimu-

lated by pertussis toxin in both cell types is intact. As pertussis toxin is known to ADP- ribosylate inhibitory G-proteins, and G-proteins are known mediators of protein phosphoryla- tion, it is possible that the phosphorylation state of myf5 in NFB-s cells is altered in some way by the action o f pertussis toxin. Note that control 10TI/2 fibroblast cells do not increase expres- sion of the reporter genes under the influence of pertussis toxin, providing further evidence that pertussis toxin modulates muscle-specific gene expression, but does not promiscuously upregu- late these reporter genes in all cell types.

The presence of pertussis toxin does not lead to phenotypic rescue of NFB-s cells, however. The absolute level of reporter gene expression, even following pertussis toxin treat- ment, never approaches the values seen for C2C 12 cells. As a further measure of differentia- tion, NFB-s cells were scored tbr fusion microscopically, and for endogenous myosin heavy chain expression by Western blot and immunofluorescence. Neither of these pm-am- eters were significantly stimulated in pertussis toxin treated NFB-s cells (data not shown). These results suggest that although stimulated by pertussis toxin, myf5 is only partially active in NFB-s cells, possibly due to constitutive phosphorylation. Coupled with the lack of myoD, myogenin, or MRF4 expression in NFB-s, these two conditions may suffice to prevent terminal differentiation.

Rescue of Myogenic PheltoO,pe in NFB-s Cells by no!/5 0verexpression. One hypothesis to explain the inability of N FB-s ceils to differentiate in spite of high myf5 expression is the possibility that myf5 itself is mutated. If this were the case. irrespective of its exwession levels, the mutant form would lack the necessaw regulmory function required to establish a differentiated phenotype. Several myf5 cDNA ch:mes derived fi'om NFB-s mRNA were iso- lated by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequenced. Howevel; there were no consistent mutations found in the bHLH regions of these cDNAs following sequencing. As a further test of the above

Myogenic Repression in NFB Mutant 357

hypothesis, several RT-PCR derived myf5 cDNAs were cloned into expression vectors and transfected into both 10TI/2 and NFB-s cells. The assay for myogenic conversion was to co-transfect a myf5 cDNA along with a reporter gene which expressed chloramphenicol acetyl transferase (CAT) at a 5: l ratio (myf5:CAT), and score for expression of CAT and myosin heavy chain (MHC) by innnunofluorescence. The CAT reporter was used as an indicator of those cells which had been successfully transfected, and the ratio of myf5:CAT was biased to ensure that cells taking up CAT DNA would be likely to take up myf5 DNA. Myogenic conversion was scored as the percentage of CAT-positive cells which were also myosin heavy chain positive. Fig. 4 demonstrates that NFB-s-derived myf5 cDNAs effectively convert NFB-s cells as well as naive 10TI/2 cells to a myogenic phenotype (see myf PCR). Furthermore, these NFB-s derived cDNAs convert 10TI/2 cells at a frequency on par with that seen with a wild type mouse myf5 cDNA (myl:-5 WT). The data from PCR-derived cDNAs are the pooled results of four independent RT-PCR clones, arguing against the possibility that NFB-s cells may have one mutant and one normal myf5 allele. These data, coupled with the earlier EMSA studies on myf5 expression and activity suggest that NFB-s- derived myf5 mRNA and protein are nolanal.

Several myf5 mutants were constructed to test the hypothesis that rescue of the mutant phenotype was occurring by a titrating mecha- nism, whereby the rescue of NFB-s cells by exogenous myf5 occurred because excess myf5 protein saturated the activity of a negative regulator such as a kinase. A schematic of these mutants is shown in Fig. 4, As can be seen, many different myf5 mutants were tested, and none of them possessed rescue activity significantly different from an expression vector lacking any myf5 cDNA (Fig. 4, SV2neo). A basal level of MHC positivity is seen for NFB-s cells in this assay, due to low level, constitutive reversion (see SV2neo, Fig. 4). This background reversion is at a much lower level than what can be achieved by transfection of myf5 or myoD, and

has been noted previously (28). The mutations tested ranged fl'om gross deletions of either N-terminal or C-terminal information (myfAN, myfAC), to subtle mutations of one or a few amino acids (myfgl, myfBam). Based on prior studies assessing mutations tolerated in myo- genic bHLH factors, none of these mutants would be expected to possess conversion activity on naive cell types such as 10T1/2 (39), and this is confirmed in Fig. 4. It is clear, however, that multiple myf5 mutants which as a group present all possible myf5 epitopes do not rescue the phenotype, suggesting that rescue is not occurring by titrating a negative regulator of myf5 activity.

D I S C U S S I O N

In this report we have explored the molecular mechanisms that serve to repress myogenesis in NFB-s cells. This differentiation defective muscle cell line expresses myf5, but lacks expression of the other three myogenic bHLH factors. Despite the high level of myf5 mRNA expression and avid DNA binding exhibited by myf5 protein, the level of muscle determination and reporter gene expression is remarkably low in NFB-s. Yet, the repressed phenotype in NFB-s can be overcome via the myogenic bHLH signalling patbway, since myf5 overexpression rescues the phenotype, resulting in muscle differentiation. Indeed, the overex- pressed myf5 can be derived from NFB-s mRNA, indicating that there are no apparent defects in NFB-s myf5 mRNA or protein. These findings suggest that the NFB-s phenotype results from a specific inhibition of the transcrip- tional activity of myf5 protein.

To characterize the mechanistic basis tbr the repression of myf5 activity in NFB-s, several types of experiments were carried out. Pertussis toxin was capable of upregulating both complex muscle-specific reporter genes derived from the MLCI/3 gene, as well as defined E-box containing reporter genes, suggesting that pertus- sis toxin's known myogenic activity (38) may be due to direct effects on the proteins involved in

358 Rohrer and Blau

b H L H

~-/.~/..../...-/.., ~'/h....-.-.'....h-.-_~ myf-5 WT

l v myf BI I f / . f f / . , ~ , f . # " . f . f . f ~ ' l r i / J ' ~ . , f , f . f . . f i . , / ' .S ' ~ / " ~ i

TRIRV

~/,,,-/.~ myf 6107

myf NIO0

"~ 3 0 ~" P'/I �9 10T~/,_

,0 lil

O

oJ '-?, ~ , E E - -

Construct

Fig. 4. Exogenously expressed mys cDNAs can rescue the repressed phenotype. NFB-s or 10T1/2 cells were transfccted with expression constructs harboring either wild type mouse myf5, specific mulants ot mouse myf5, or myf5 cDNAs derived from R%PCR of NFB-s mRNA. The upper panel shows a schematic of the myl:5 cDNA and specific mutants used. myf-5 WT = wikt type myf5 eDNA" myfBl = lie mseruon in helix 2 of HLH domain: mytBam = Tyr-Arg-Ile-Arg-Val inse,mon m helix 2 of HLH; myfAC = truncation of C-termm,d 107 amino acids: myfAN = mmcafion o:[: N-terminal 65 amino acids: myfC]07 = C-terminal 107 amino acids: mytNt00 = N-terminal 100 amino acids. The lower panel shows the results of several experiments m which lhese constructs were transfected into 10TI/2 and NFB-s cells along with a marker for trmls[ected cells, SV2-CAT. Following transfeclton, cells were switched to dilTerent~atmn medim]] for 48 hours. SV-2 CAT posture cells, scored by redirect immtmofluorescence, were secondarily scored for sarcomenc myosin heavy chain (MHC) expression by means of :redirect nnmunofluorescence as well. Myogemc conversion ~as scored as the ~A of CXl:posltive cells which were also MHC positive. The only constructs with slgnilicant conversion pntenual are the myf-5 WT and

) myfl CR, which represenls the pooled scol ings of foctr independent RT-PCR clones from NFB-s mRNA.

ti le b H L H s i g n a l l i n g c a s c a d e . T h e f a c t t ha t

N F B - s ce l l s fai l to d i f f e r e n t i a t e f u l l y f o l l o w i n g

p e r t u s s i s t o x i n t r e a t m e n t i n d i c a t e s t ha t t he

G - p r o t e i n t a r g e t m e d i a t i n g the e f f e c t s o f p e r t u s -

s i s t o x i n is n o t t h e p r i m a r y s o u r c e o f m y o g e n i c

r e p r e s s i o n in N-FB-s . To a n a l y z e t h e m e c h a n i s m

o f r e p r e s s i o n fu r the r , v a r i o u s m y f 5 m u t a n t s

w e r e c r e a t e d as p o t e n t i a l d e c o y s in a n a t t e m p t to

Myogenic Repression in NFB Mutant 359

saturate the activity of modifiers of myf5. From previous studies, we anticipated that these mutants would not be capable of converting 10T1/2 cells to muscle (39). However, these mutants also failed to titrate negative regulators of myf5 activity and rescue the myogenic phenotype in NFB-s cells, suggesting that post-translational modifications directed at myf5 such as phosphorylation are probably not the sole source of repression. A caveat to this interpretation is that the relative translational efficiency or stability of the mutant myf5 molecules remains unknown, The results of Sarbassov, et al. (40), show that treatment of NFB cells with IGF-1 both enhances myogenic differentiation and specifically alters the phos- photyrosine labelling pattern towards one more closely resembling the C2Cl 2 parental cell line. Together, these results suggest that protein phosphorytation patterns in NFB cells are likely to play an important role in the repressed phenotype, although our experiments using myf5 mutants argues against a direct modifica- tion of myf5.

One hypothesis is that in order to rescue NFB-s, a critical E box target must be accessed. Under normal conditions, occupation of such a critical E box by a repressorqike molecule would prevent myf5 from acting as a transcrip- tional activator. Under conditions of myf5 excess, however, the stoichiometric balance shifts to favor myf5 occupation and muscle activation. Although our data from EMSAs argue against the presence of such a repressor in NFB-s cells, it is possible that the MEF-1 oligonucleotide used here from the human MLC1/3 enhancer may not effectively mimic such a critical E-box target, as muscle-specific E-boxes display some sequence heterogeneity. Given the fact that MEF-t complexes exhibit some flexibility in their affinity for binding site sequences and that tissue-specific bHLH pro- teins from other tissues bind related yet distinct E box-containing elements, it is quite possible that such a repressor would not be detected by our methods. Such inequality between E-boxes has been noted by others (41). Experiments which have shown cooperativity between myo-

genic bHLH proteins and MEF2 also raise the possibility that the critical target(s) involved in NFB conversion may be MEF2 sites, or a combination of MEF-I and MEF2 sites. MEF2 cDNA constructs lacking transactivation do- mains are capable of mediating muscle-specific transcription through the MEF2 site when myoD and El2 are present (42), suggesting that the two classes of proteins functionally interact, and raising the possibility that en- trainment of multiple myogenic pathways must occur before commitment to the muscle lineage.

There are several examples of growth factors, oncogenes, transcription factors, and signalling pathways which inhibit myogenesis, and many of the resulting phenotypes are remarkably similar to that observed in NFB-s cells. Agents that elevate cAMP or activate cyclic AMP dependent protein kinase (PKA) are known inhibitors of myogenesis (22, 23, 38, 43). It is of interest that similar to our findings, these agents do not interfere with the ability of bHLH proteins to bind DNA (23), and there is even evidence which suggests that PKA-mediated phosphorylation of bHLH proteins is indepen- dent of the observed myogenic inhibition (22). TGF-[3 can also inhibit myogenesis without affecting the DNA binding capacity of myogenic regulators (44).

A striking parallel between the phenotype seen in NFB-s cells is also seen in E1-A transforlned L6 myoblasts, where myf5 expres- sion is normal or elevated, while myogenin expression is extinguished (45). Furthermore, the myf5 in these transformed cells can still bind DNA, yet transactivation of muscle-specific reporter genes is greatly reduced. More recent studies have suggested that the EI-A 12S protein may dimerize with the bHLH domain of myogenin, leading to repression (46). What remains unclear in all of these cases is why myf5 is persistently expressed and how its activity is repressed.

A well known modulator of cell type specification, notch, has also been implicated mechanistically in an NFB-s-like repressed phenotype (47). Activation of this putative

360 Rohrer and Blau

receptor protein in Drosophila appears to inhibit neural fate in cells which otherwise would become neural cells due to their expression of achae te - scu te . The mouse homologs of notch

and myf5 can he co-localized in presomitic mesoderm, at a t ime preceding overt myogen- esis and expression of the other myogenic bHLH regulators. Furthermore, ectopic expres- sion in cultured cells or embryos of a constitu- tively active notch protein results in myogenic repression (47), Again, although repression of differentiation is observed, myogenic bHLH protein binding to DNA consensus sites is apparently unaffected.

Addit ional insight into the mechanisms of myogenic repression comes from a vm'iety of mutagenized muscle cell lines der ived in our laboratory: virtually "all mutagenized C2C12 derivatives maintain expression of at least one myogenic bHLH, and in each mutant the bHLH proteins expressed retain the abili ty to bind DNA. For instance, NMU-2 cells are muta- genized C2C12 derivatives that express myoD 148), whereas NMU-7 and NQO-1 cells express myf5, In each case, the cells are differentiation defective, yet the expressed bHLH protein forms MEF-1 complexes with the MLCI /3 enhancer oligonucleotide (data not shown). Possibly, like the NFB-s mutant cell type, the repression of myogenesis in these mutants reflects a critical target or set of targets that must be saturated, or smTnounted, before the cascade of myogenesis can ensue, The many examples of inhibitors and unique cell lines which share phenotypic trNts with NFB-s speak to a preserved mechanism or set of mechanisms involved in cell specification which are essential in preventing cells from promiscuous determina- tion or differentiation. Muscle satellite cells are but one example of determined, uncommitted cells in vivo which must be prevented from premature differentiation, so that they are available to proliferate in response to muscle dmnage. Taken together, these findings under- score the importance of exploring the mecha- nisms that underlie rewessed differentiation, like those characteristic of NFB-s, in order to

better understand how multiple signalling cas- cades converge to ultimately determine cell fate.

A C K N O W L E D G M E N T S

We are grateful to Dr, Nadia Rosenthal for the gift of myosin light chain 1/3 enhancer fragments, and Dr. Charlotte Peterson for helpful comments and discussion of this manu- script. D.R, was supported by a U.S.RH.S. postdoctoral fellowship (ARO 8196), and HMB received grants from NIH (AG 09521 and HD 18179) and from the Muscular Dystrophy Associat ion of America.

L I T E R A T U R E C I T E D

1. Davis, R.L., Wetntraub, H,, and Lassar, A.B. 1987. Cell 51:987-t000.

2. Wright, W.E, Sassoon= D.A., and Lin, V.K. 1989. Cell 56:607-6 t 7.

3. Braun, T., Buschhausen, D.G.. Bober, E., Tmmtch, E., and Arnold, H.H, 1989. Embo. ,1. 8:701-709.

4, Rhodes. S.J. and Konieczny, S.E 1989. Genes Dev, 3:2050-2(}61,

5. Mun'e. C., McCaw, PS., Vaessm, It,, Caudy, M., Jan, I.Yi, Jan. Y.N.. Cabrera, C.V., Buskin, J.N., Hauschka, S,D,, Lassar, A.B., and et al, 1989. (Tell 58:537-544.

6. Lassar, A.B., Dax, Ls, R.L., Wright, W.E., Kadesch, T., Murre, C., Voronova, A., Batmnore, D., and Wem- traub. H, 1991. Cell 66:305-3 [5.

7. Mellemm, J.D.. Mum'e, C., Donlon. T.A., McCaw. RS.. Smith. S.D., Carroll, A.J., McDonald, M.E., Baltt- more, D.. and Cleary, M.L. 1989. Sctence 246:379- 382,

8, Gossett, L.A,, Kelvin, D.J., Sternberg, E.A., and Olson, E.N. 1989. Mol. C)ll Biol. 9:5022-5033.

9. Merrill LIE, Miano, J.M., Huslad, C.M., Copeland, N,G., Jenkins, N.A., and Ol,~on, E.N. 1994. tVIol, Celt Biol. 14:1647-t656.

t1). Cserjesl, R, Lilly, B., Bryson, L., Wang, Y., Sassoon, D.A., and Olson, E.N. 1992, Devetopmem 115:1087- 1 t0l.

II. Mar, J.H., and Ordahl, C.R 1990. Mol. Cell Biol, 10:4271--4283.

12. Farrance, I.K,, Mar, J.H, and Ordahl, C.P. 1992. d. Biol, Chem. 267:17234-17240.

13. Benezra, R., Davis, R.L., Lesser, A., Tapscott, S., Thayer, M., Lockshon, D., and Weintmub, H. 1990. Ann. N.Y Acad. Sc~. 599:1-1 I~

t4. Benezra, R., Davis, RL., Lockshon, D., ~lhrner, D.L., and Weintranb, H. 1990. Celt 61:49-59.

15. Jen, Y., Weintranb, H., and Benezra, R. 1992. Genes. Dev 6:1466-1479.

16. Spizz, G., Roman, D., Strauss, A., and Olson, E.N. 1986. J. Biol. Chem. 261:9483-9488.

Myogenic Repression in NFB Mutant 361

17. Li, L., Zhou, J., James, G., Heller, H.R., Czech, M.E, and Olson, E.N. 1992. Cell 71: I 181-1194.

I8. Otson, E.N., Steinberg, E., Hu, J.S., Spizz, G., and Wilcox, C. 1986. J. Cell Biol. 103:1799-1805.

19. Olson, E.N., Splzz, G., and Tainsky, M.A, 1987. Mol. Cell Biol. 7:2104-2111.

20. Lassar, A.B., Thayel; M.J., Overell, R.W., and Weintraub, H. 1989. Cell 58:659-667.

21. Martin, J.E, L~, L., and Olson, E.N. 1992. J. Biol. Chem. 267: t 0956-10960.

22. Li, L., Heller. H.R., Czech, M., and Olson, E.N. 1992. Mol. Cell Biol. 12:4478-4485.

23. Winter, B., Braun, T., and Arnold, H.H. 1993. J. Bicd. Chem, 268:9869-9878.

24. Miller, S.C., Pavlath, G.K., Blakely, B.T., and Blau, H.M. 1988. Genes Dev. 2:330-340.

25, Schaf?r, B.W., Blakely, B.T., Dartington, G.J., mad Blau, H.M. 1990. Nature 344:454-458.

26. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T,K., Turner, D., Rupp, R., Hollenberg, S., and et al. 1991. Science 251:761-766.

27. Blau, H.M. 1992. Anm~. Rm: Biochem. 61:12t3-1230. 28, Peterson, C.A., Gordon, H.. Hall, Z.W., Pmerson,

B.M., and Blau, H,M. 1990. Cell 62:493-502. 29 Yaffe, D., and Saxel, O. 1977. Nature 270:725-727. 30_ Buonanno, A., Apone, L., Morasso, M.I., Beers, R.,

Brenner, H.R., and EftlmJe, R. 1992. NuclezcAeids Re,~ 20:539-544.

31. Rosenthal, N., Bergtund, E.B., Wentworth, B.M., Donoghue, M., Winter, B.. Bober, E., Braun, %, and Arnold, H.H. 1990. Nuclen:Acids Res. 18:6239-6246.

32. Weinlraub, H., Daws, R,. Lockshon, D., ~md Lassar, A. 1990. Proc. Natl. Acad.. Set. U.S.A. 87:5623-5627.

33. Silberstein, L., Webster, S.G., Travls, M, and Blau. H.M. 1986. Ce1146:1075-t081.

34. Gossett, L.A., Kelvin, D.J., Sterngerg, E.A., and Olson, E.N. 1989. Mol. (Jell Biol. 9:5022-5033.

35. Donoghue, M.J., Merlie, J.R, Rosenthal, N,, and Sanes, J.R. 1991. Proc. Natl. Acad. Sci. U,S.A. 88:5847-5851,

36. Ernst, H, Watsh, K., Han-ison, C.A., and Rosenthal, N. 199 t. Mol. Cell Biol. 11:3735-3744.

37. Wentworth, B.M,, Donoghue, M., Engert, J.C., Ber- glund, E.B., and Rosenthal, N. t 991. Proc. Notl. Aead. Scz. U.S.A. 88:t242-1246.

38. Amo[d, H.H,, and Salmlnen, A. 1993. Cell Mot. Biol. Res'. 39:195-208.

39. Winter, B.. Braun, T., and Arnold, H.H. 1992. Embo. J. 11:1843-1855.

40. Sarbassov, D.D., Stefanova, R., Gfigoriev, V.G., and Peterson, C.A. 1995. Ptvc. Natl. Acad. Sci. U.S,A. 92:10874-10878.

41. Yutzey, K.E., and Konieczny, S.F. 1992. NucleieAc~ds Res. 20:5105-5113.

42. Molkentin, LD,, Black, B.L., Martin, J.F., ~md Olson, E.N. 1995. Celt 83:1 [25-1136.

43. Hu. J.S., and Olson. E.N. 1988. dl Biol. Chem. 263:19670- 19677.

44. Brennan, TJ,, Eatmondson, I).G., Li, L., and Olson, E.N. 1991. Proe. Natl. Acad. Scl. U.S.A. 88:3822-3826.

45. Braun. T_ Bober, E., and Arnold, H.H. 1992. Genes Dev. 6:888-902.

46. Taylor~ D.A., Kraus, V.B., Schwarz, J.J., Olson, E.N., and Kraus, W.E. 1993. Mol. Cell. Biol. 13:4714-4727.

47. Kopan, R., Nye, J.S,, and Wemtraub, H. 1994. Development 12t1:2385-2396.

48. Rastinejad, F., and Blau, H.M. 1993. Cell 72:9(13-917.