Vol. 11, No. 10

Myogenin Induces the Myocyte-Specific Enhancer Binding FactorMEF-2 Independently of Other Muscle-Specific Gene Products

PETER CSERJESI AND ERIC N. OLSON*Department ofBiochemistry and Molecular Biology, The University of Texas M. D. Anderson

Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030

Received 7 May 1991/Accepted 3 July 1991

The myocyte-specific enhancer-binding factor MEF-2 is a nuclear factor that interacts with a conservedelement in the muscle creatine kinase and myosin light-chain 1/3 enhancers (L. A. Gossett, D. J. Kelvin, E. A.Sternberg, and E. N. Olson, Mol. Cell. Biol. 9:5022-5033, 1989). We show in this study that MEF-2 isregulated by the myogenic regulatory factor myogenin and that mitogenic signals block this regulatoryinteraction. Induction of MEF-2 by myogenin occurs in transfected 1OT1/2 cells that have been converted tomyoblasts by myogenin, as well as in CV-1 kidney cells that do not activate the myogenic program in responseto myogenin. Through mutagenesis of the MEF-2 site, we further defined the binding site requirements forMEF-2 and identified potential MEF-2 sites within numerous muscle-specific regulatory regions. The MEF-2site was also found to bind a ubiquitous nuclear factor whose binding specificity was similar to but distinct fromthat of MEF-2. Our results reveal that MEF-2 is controlled, either directly or indirectly, by a myogenin-dependent regulatory pathway and suggest that growth factor signals suppress MEF-2 expression throughrepression of myogenin expression or activity. The ability of myogenin to induce MEF-2 activity in CV-1 cells,which do not activate downstream genes associated with terminal differentiation, also demonstrates thatmyogenin retains limited function within cell types that are nonpermissive for myogenesis and suggests thatMEF-2 is regulated independently of other muscle-specific genes.

Differentiation of skeletal myoblasts is accompanied bytranscriptional induction of a battery of unlinked muscle-specific genes. The control regions of several of these geneshave been characterized and shown to interact with complexsets of muscle-specific and ubiquitous nuclear factors thatcooperate to direct muscle-specific transcription (for a re-

view, see reference 50). The best-characterized muscle-specific transcription factors are the myogenic helix-loop-helix proteins of the MyoD family, which includes MyoD(19), myogenin (23, 68), Myf5 (4), and MRF4/herculin/Myf6(3, 43, 49). When introduced into a variety of nonmuscle celltypes, each of these factors has the ability to induce myo-genesis to different degrees (for reviews, see references 45and 58). Cells of mesodermal origin, and in particular 10T1/2fibroblasts, are especially permissive for myogenic conver-sion by the MyoD family, whereas cells derived from otherlineages show variable degrees of muscle-specific geneexpression in response to these factors (54, 66).

Activation of the myogenic program by members of theMyoD family is dependent on binding to a consensus se-quence CANNTG, known as an E box (6, 18). Numerousmuscle-specific genes contain E boxes within their controlregions and serve as direct targets for transcriptional activa-tion by the MyoD family (3, 6, 9, 12, 13, 24, 36, 40, 48, 51, 53,65, 67). Some muscle-specific genes, however, do not con-tain E boxes in their control regions, yet these genes can bespecifically expressed in muscle cells (1, 42, 64). Whetherthe latter types of genes are regulated indirectly by theMyoD family through induction of intermediate muscle-specific transcription factors remains to be determined.The myocyte-specific enhancer-binding factor MEF-2 is a

nuclear factor that interacts with the muscle-specific enhanc-ers of the muscle creatine kinase (MCK) and myosin light-

* Corresponding author.

chain 1/3 (MLC1/3) genes (25). These enhancers also containmultiple E boxes that are important for muscle-specifictranscription (6, 9, 10, 13, 30, 33, 36, 51, 57, 65, 67). Wepreviously identified MEF-2 as a nuclear factor that isundetectable in proliferating myoblasts and rapidly appearswhen myoblasts are induced to differentiate by withdrawalof serum (25). Results of in vitro DNA-binding assays havebeen confirmed by in vivo footprinting of the MCK en-hancer, which has shown the MEF-2 site to be occupied onlyin myotubes (44). Induction of MEF-2 activity can bedetected as early as 30 min after withdrawal of growthfactors from myoblasts and requires ongoing protein synthe-sis (25), suggesting that its appearance does not involvemodification of a preexisting protein. The kinetics for induc-tion of MEF-2 binding activity are similar to those forinduction of myogenin, which marks the activation of themuscle differentiation program (23). The factor MEF-2 bindsto an A+T-rich site that is highly conserved in the MCK andMLC1/3 enhancers. Although a single MEF-2 site does notappear to be sufficient to direct transcription when combinedwith a basal promoter, multimers of the MEF-2 site canactivate transcription from the MCK basal promoter indifferentiated C2 myotubes (25). Deletion of the MEF-2 sitefrom the MCK and MLC1/3 enhancers also impairs enhanceractivity, suggesting that transcriptional activation of thesegenes may involve cooperation between MEF-2 and mem-

bers of the MyoD family, which bind the enhancer cores (25,30, 67; see also reference 65).The nature of the factors that bind to the MEF-2 site has

been controversial. We have reported that MEF-2 is musclespecific and is the major factor that binds to this site (25).However, others have observed only ubiquitous bindingactivities with this site and have obtained no evidence forMEF-2 (10, 30, 31). To further define the regulation ofMEF-2 and clarify the relationship between the factors thatbind to the MEF-2 site, we have used nuclear extracts from

4854

MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 4854-48620270-7306/91/104854-09$02.00/0Copyright © 1991, American Society for Microbiology

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REGULATION OF MEF-2 4855

a variety of cell types to analyze binding activities for thissite. We show that ubiquitous and muscle-specific nuclearfactors bind to the MEF-2 site and that these factors showdistinct sequence specificities. Our results also demonstratethat MEF-2 can be regulated by myogenin through a mech-anism that requires withdrawal of mitogens. Activation ofMEF-2 expression by myogenin occurs in cells that can andcannot be converted to muscle, suggesting that MEF-2 isregulated independently from genes associated with terminaldifferentiation.

MATERIALS AND METHODS

Cell culture and stable transfections. All myogenic celllines were maintained in Dulbecco's modified Eagle's me-dium (DMEM) containing 20% fetal bovine serum (FBS) andwere induced to differentiate by transfer to differentiationmedium (DMEM containing 2% horse serum or 0.5% FBS),as indicated. Nonmyogenic cell lines were maintained inDMEM containing 10% FBS.

Stable transfections were performed by using 10 ,ug ofEMSV-myogenin (23), which contains the mouse myogenincDNA linked to the Moloney sarcoma virus long terminalrepeat, and 200 ng of pSV2neo to confer neomycin resis-tance. After selection in the neomycin analog G418 for 14days, colonies were isolated and passaged into stable celllines. The CV8-1 cell line was derived by stable transfectionof CV-1 cells with EMSV-myogenin and was one of severalclones that expressed myogenin constitutively, but it failedto differentiate into muscle upon exposure to differentiationmedium. The 1OTFL2-3 cell line was derived by transfectionof 1OT1/2 cells with EMSV-myogenin and has been de-scribed previously (7-9). The C025 cell line was derived bystable transfection of C2 cells (69) with a steroid-inducibleN-ras allele (26).

Preparation of nuclear extracts and gel mobility shift assays.Nuclear extracts were prepared and gel mobility shift assayswere performed as described previously (25). Unless other-wise indicated, 10 ,ug of nuclear extract was used in each gelmobility shift assay. Oligomers corresponding to wild-typeor mutant MCK MEF-2 sites were synthesized by theMacromolecular Synthesis Facility at The University ofTexas M. D. Anderson Cancer Center and were end labeledwith 32P. For some experiments, an NcoI-BamHI fragmentencompassing the region between -1136 and -1048 of theMCK gene upstream region (57), which contains the MEF-2site, was used as a probe.

RESULTS

The MEF-2 site binds muscle-specific and ubiquitous nu-clear factors. To investigate the cell-type distribution ofbinding activities at the MEF-2 site, we tested nuclearextracts from a variety of nonmyogenic cell types for bindingto an oligomer corresponding to the MCK MEF-2 site in gelmobility shift assays. As reported previously (25), theMEF-2 complex, which migrates near the top of the gel, isgenerated only by myotube nuclear extracts (Fig. 1). Ex-tracts from myoblasts showed no evidence of MEF-2 butgave rise to two slightly faster migrating complexes, desig-nated complexes A and B, which were also detected inextracts from myotubes. Complexes A and B were alsodetected in different amounts in nuclear extracts from HeLa(human cervical carcinoma), cos (simian virus 40-trans-formed CV-1 cells), CV-1 (monkey kidney), HepG2 (humanhepatoma), and 1OT1/2 (mouse fibroblast) cells, whereas

E Ea) cs

0 0

MEF-2 ---MA _B _-o

NJcu '(.5Q) 0 > aI o01(.'

N

C

probe -_

1C T C T A A A A A T A A C C C T 10621077 G A G A T T T T T A T T G G GA -1062

FIG. 1. Muscle specificity of MEF-2. Nuclear extracts wereprepared from C2 myoblasts and myotubes and from HeLa, CV-1,cos, HepG2, and 1OT1/2 cells in differentiation medium. Gel mobil-ity shift assays were performed using 32P-labeled probes corre-sponding to the MCK MEF-2 site and 10 ,ug of nuclear extract fromeach cell type. Positions of the MEF-2-containing complex and theubiquitous complexes A and B are indicated. The sequence of theMEF-2 probe and the position of the MEF-2 site in the MCKupstream region are shown beneath the autoradiogram. SinceMEF-2 is induced during differentiation, its abundance relative tocomplexes A and B depends on the degree of differentiation. Thecultures used to prepare these myotube extracts were at an earlystage in the differentiation program. In fully differentiated myo-tubes, the level of MEF-2 can increase to a level at least 10-foldhigher than is shown here.

these extracts yielded no evidence of MEF-2 (Fig. 1). Wepreviously observed a rapidly migrating complex with myo-blast extracts that we attributed to the binding of a myoblast-specific factor, designated MBF-1, to the MEF-2 site (25).The relative intensity of the MBF-1 complex was less than1/10 that of the MEF-2 complex. For reasons that areunclear, we do not now observe the MBF-1 complex in allextracts from C2 myoblasts. Whether the variable presenceof this complex reflects subtle differences in the state ofgrowth or differentiation of cells, variations in preparation ofextracts, or both is presently unclear.

All of the complexes observed with the MEF-2 probe weresequence specific; the homologous probes, but not theheterologous probes, competed for their formation (seebelow). Each extract in Fig. 1 gave rise to a complex ofsimilar intensity with a probe containing a binding site for thewidely expressed transcription factor Oct-i (data notshown), indicating that equivalent amounts of nuclear ex-tract were present in each sample. These results confirm that

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4856 CSERJESI AND OLSON

A 1OTFL 10T B 1OTFL IOTC2 2-3 1 /2 C2 2-3 1/2 2

G DG DG D G DG DG D

MEF-2 _ -*

wild-type _--- mutant

probe probe

C _-C2G DDD G DG D

4- MEF-2

4- probe

FIG. 2. MEF-2 induction by myogenin in nonmyogenic cells. (A and B) Nuclear extracts were prepared from C2 cells, myogenin-transfected 1OT1/2 cells (1OTFL2-3), and parental 1OT1/2 cells in growth medium (G) and differentiation medium (D), as indicated. Gelmobility shift assays were performed using a 32P-labeled probe corresponding to the MCK MEF-2 site and 10 p.g of nuclear extract from eachcell type. In panel B, a mutant MEF-2 site, corresponding to mutant 6 in Fig. 4, was used to confirm the specificity of binding. (C) Nuclearextracts were prepared from C2 cells, CV-1 cells, and myogenin-transfected CV-1 cells (CV8-1) in growth medium (G) and differentiationmedium (D), as indicated. Gel mobility shift assays were performed with the MCK MEF-2 site as the probe as described above.

MEF-2 is muscle specific and suggest that the MEF-2 site isalso a site for binding of ubiquitous nuclear factors.

Induction of MEF-2 by myogenin. We showed previouslythat MEF-2 activity was induced early in the muscle differ-entiation program with kinetics similar to those for theinduction of myogenin (25). Myogenin and MEF-2 areclearly distinct, however, as myogenin does not bind to theMEF-2 site of muscle-specific genes such as the MCK gene(9). Antibodies directed against myogenin also do not recog-nize the MEF-2 complex formed with the MCK enhancer(9). To further explore the regulation and muscle specificityofMEF-2 expression, we examined whether myogenin couldregulate MEF-2. We addressed this question by using astable line of 1OT1/2 cells, designated 1OTFL2-3, that con-stitutively expresses a transfected myogenin cDNA. Thelevel of expression of the myogenin protein in 1OTFL2-3cells is similar to that in C2 myotubes (7). As shown in Fig.2A, nuclear extracts from proliferating 1OTFL2-3 cells ingrowth medium showed no evidence of the MEF-2 complex.However, when the cells were transferred to differentiationmedium, MEF-2 was induced to high levels. Induction ofMEF-2 was dependent on myogenin and did not occur inparental 1OT1/2 cells under the same culture conditions. Anoligomer containing a point mutation within the MEF-2 sitecQnfirmed specificity of MEF-2 binding (Fig. 2B). We con-clude that myogenin can activate, either directly or indi-rectly, the expression of MEF-2 through a mechanism that isrepressed by mitogenic signals. In that myogenin also acti-*vates the expression of MyoD in 1OT1/2 cells (2, 8, 60), it ispossible that induction of MEF-2 by myogenin is indirectand is mediated by MyoD or other myogenic factors.We next examined whether myogenin could activate

MEF-2 expression in CV-1 cells, which have been shown tobe refractory to myogenic conversion by MyoD (66). Usinga clonal line of CV-1 cells, designated CV8-1, that stablyexpresses the myogenin cDNA, we detected little or no

MEF-2 activity when the cells were proliferating in growthmedium (Fig. 2C). However, the MEF-2 complex appearedfollowing transfer of CV8-1 cells to differentiation medium.There was no evidence of the MEF-2 complex in parentalCV-1 cells, even after prolonged exposure to differentiationmedium. As reported previously for MyoD (60, 66), myoge-nin failed to activate myogenesis in CV-1 cells. Northern(RNA) analysis showed constitutive myogenin expressionbut no detectable expression of MCK, MyoD, or troponin-T(cTnT) in the CV8-1 cell line (data not shown). The CV8-1cells also failed to stain for myosin heavy chain. Theseresults suggest that MEF-2 can be regulated by myogeninindependently from muscle-specific genes associated withterminal differentiation and that myogenin and MEF-2 arenot sufficient, by themselves, to activate the complete myo-genic program in all cell types.

Repression of MEF-2 by serum and ras. The observationthat MEF-2 could be induced in CV-1 cells by myogenin inthe absence of overt differentiation indicated that MEF-2could be regulated independently from other muscle genesand led us to examine MEF-2 expression under a variety ofconditions that have been shown to prevent induction ofmuscle-specific genes in myoblasts. Using the BC3H1 mus-cle cell line, which is defective for fusion and retainssensitivity to exogenous growth factors after initiation ofdifferentiation (8, 55, 59), we examined whether MEF-2induction was reversible. Figure 3A shows that MEF-2activity was induced following transfer of BC3H1 myoblastsfrom growth to differentiation medium. (The probe used inthese experiments was slightly larger than the oligomer usedpreviously and contains a binding site for an additionalubiquitous factor, designated C.) Conversely, stimulation ofdifferentiated BC3H1 myocytes with growth medium led tothe rapid disappearance of the MEF-2 complex. Myogenin isregulated in parallel with MEF-2 (22, 23, 25) under each ofthese conditions.

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REGULATION OF MEF-2 4857

probe(- 1 0 7 7 / - 1 0 6 2)

FIG. 3. Regulation of MEF-2 activity by serum and ras. (A)Nuclear extracts were prepared from BC3H1 cells in growth me-

dium (GM) and differentiation medium (DM). Following exposure todifferentiation medium for 3 days, one set of cultures was reexposedto growth medium for 16 h. The probe used in panel A correspondsto a fragment of the MCK enhancer (-1136/-1048 relative to thetranscription start site) and contains a binding site for an additionalfactor, designated C, which migrates slightly behind the free probe.The complex migrating between MEF-2 and C, which is present inall lanes, is nonspecific. (B) Nuclear extracts were prepared fromC025 cells in differentiation medium (DM) with and without 200 nMdexamethasone (DEX), which induces ras expression and blocksdifferentiation. The probe was an oligomer corresponding to theMCK MEF-2 site.

We also investigated whether MEF-2 was regulated byras, which has been shown to substitute for growth factorsand block induction of myogenin and other muscle-specificgenes (26, 37, 46, 56). Using the C025 cell line, we found thatthe MEF-2 complex was induced upon transfer to differen-tiation medium, whereas in the presence of dexamethasone,which induces N-ras expression, the MEF-2 complex failedto appear (Fig. 3B). Similarly, the C41 cell line, which was

derived from C2 cells by stable transfection with an acti-vated H-ras allele (46), withdrew from the cell cycle butfailed to differentiate after exposure to differentiation me-

dium and did not express MEF-2 (data not shown). Thus,MEF-2 was induced and repressed in parallel with myogeninin a variety of muscle cell types. The ability of ras tosuppress MEF-2 expression in quiescent myoblasts exposedto differentiation medium also shows that cessation of celldivision is not by itself sufficient to induce MEF-2 in musclecells.

Binding activities for the MEF-2 site can be distinguished bypoint mutations. To further investigate the relationship be-tween MEF-2 and the ubiquitous binding activities thatrecognize the same site, we tested a series of mutants of theMEF-2 site for binding to these factors. Each mutant was

tested in gel mobility shift assays with extracts from C2myotubes and CV8-1 cells in differentiation medium. Theseexperiments also allowed us to determine whether the MEF-2-like activity that was induced by myogenin in CV8-1 cells

was indeed equivalent to MEF-2 from differentiated musclecells. Using mutant MEF-2 sites as probes, we were able toidentify binding sites that could distinguish between MEF-2and the ubiquitous binding activities. For example, mutant 6bound the ubiquitous complex B strongly but did not bindMEF-2 or the ubiquitous complex A (Fig. 4A). This resultsuggests that complexes A and B result from the binding ofdifferent factors. Mutant 4 bound MEF-2 strongly but inter-acted weakly with the ubiquitous factors. The ubiquitousfactors and MEF-2 exhibited overlapping binding specifici-ties, as shown by the lack of complex formation with mutant2 or 5. Mutant 10 showed binding specificity similar to that ofthe wild-type MEF-2 site. Mutant 7, which corresponds toan A+T-rich element in the core of the MCK enhancer thatis essential for enhancer activity (15), failed to bind MEF-2or the ubiquitous factors but gave rise to several uniqueDNA-protein complexes. Mutant 9 also failed to bind MEF-2or complexes A and B but generated a site for a new factor.To allow direct comparison of factor binding to each mutantsite in Fig. 4A, we used equivalent amounts of nuclearextract in each assay. Shorter exposures were used toconfirm relative amounts of binding for assays that areoverexposed in the figure presented. Binding specificitieswere also confirmed by competition experiments using alabeled MEF-2 site as the probe and unlabeled mutantoligomers as competitors (data not shown). The MEF-2-likecomplex in CV8-1 cells showed the same sequence speci-ficity as MEF-2 from C2 myotubes. From these experiments,we conclude that MEF-2 and the ubiquitous factors that bindthe MEF-2 site have distinct but overlapping binding sitespecificities.The binding site experiments allowed us to expand the

consensus for MEF-2 binding (Fig. 4B). MEF-2 appears tohave the potential to recognize 9-bp elements with A's or T'sat most positions, although there is at least one T that cannotbe replaced with an A (mutant 8). The 16-nucleotide wild-type sequence shown in Fig. 4B is sufficient for MEF-2binding; however, we are not yet certain of the boundaries ofthe MEF-2 site or the exact role of the nucleotides flankingthe A+T-rich region. These flanking nucleotides are lesswell conserved among different potential MEF-2 sites (seebelow) and did not appear to interact with MEF-2, assayedby diethylpyrocarbonate interference of MEF-2 binding (25).Nevertheless, the flanking nucleotides appear to influencebinding to the A+T-rich core, as demonstrated by mutants 1and 11.

Using the preliminary consensus, we were able to identifyseveral potential MEF-2 sites in other muscle-specific con-trol regions (Fig. 5). Most of the sites shown in Fig. 5 arelocated within regions that have been shown to be function-ally important for transcription of the associated genes, andin cases where these genes have been sequenced frommultiple species, there is strong conservation of these se-quences and divergence of surrounding nucleotides. We didnot identify potential MEF-2 sites outside of the regulatoryregions of these genes, supporting the notion that this sitemay be associated with elements that mediate muscle-specific transcription. Interestingly, several of these genesare expressed in cardiac as well as skeletal muscle. We didnot compare the MEF-2 site with sequences associated withnon-muscle-specific genes, but this site has been shown to beimportant for expression of at least one nonmuscle gene, thebrain creatine kinase gene (31).

A BC3H1

a)_-CD

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B C025xw

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MEF-2

hto

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probe -_(- 1 136/1048)

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4858 CSERJESI AND OLSON

A C2 myotubes CV8-1 (DM)

a) °aC- N cD SV 4C CD P- OD a)- CL N Cf "gt U') CO r- 0 a) 1

ccc c c c: c cr c c ccca c C C r-CCo C CC CZ CO C Ca Ca C C V C ig ig2 C C C5 C,_ Z3 3 :3 : :3 :3 :3 = D m m nn._ =:: n m D

3 E E E E E E E E E E E ¢ E E E E E E E E E E E

II

t.S

I_IihIwiMEF-2 Binding Site Mutations

_ [- - -C C C T

RelativeBinding

++

+

--A -

_-++

FIG. 4. Gel mobility shifts using wild-type and mutant MEF-2 sites. (A) Gel mobility shift assays. Nuclear extracts were prepared fromC2 myotubes or CV8-1 cells in differentiation medium (DM), and gel mobility shift assays were performed using end-labeled oligomerscorresponding to mutants of the MEF-2 site shown in panel B. Ten micrograms of nuclear extract was used in each assay. (B) Summary ofbinding data obtained with mutant MEF-2 sites. A dash within the sequence indicates the wild-type nucleotide at that position. Binding isindicated as + + (wild-type binding), + (detectable but weak binding), or - (no detectable binding). A preliminary consensus for the core ofthe MEF-2 site, which is consistent with the binding data, is shown. The region with the most profound effect on MEF-2 binding is shaded.Flanking nucleotides may affect binding to this central region, but they appear to be less important. The "t" indicates that this nucleotide ispermissive but leads to diminished binding relative to the wild-type sequence.

DISCUSSIONOur results show that the MEF-2 site in the MCK en-

hancer interacts with muscle-specific and ubiquitous nuclearfactors that are functionally distinct. In agreement with our

previous studies (25), we found that MEF-2 represents a

muscle-specific DNA-binding activity that is induced duringthe myoblast-to-myotube transition. Less abundant ubiqui-tous factors that interact with the same site show subtly

MEF-2 -A -*

B -_

probeb_

4- MEF-2

4- A4- B

*- probe

B

c T Cwild-type

mutant 1

mutant 2

mutant 3

G G G

mutant 4

mutant 5

mutant 6

G G Tj

T A A A A A T A A

G-

- T-

- - T- G

- - - T -____

- - - - c- - - -

- -T- -T - -

T A A T AA A A A

mutant 7

mutant 8

mutant 9

mutant 10

mutant 1 1

consensus

G-

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REGULATION OF MEF-2 4859

mouse MCK

human MCK

rat MCK

rabbit MCK

mouse MCK (intron)

rat MLC1/3

human MLC1/3

chicken MLC2A

rat MLC2A

chicken MLC3f

mouse MLC3f

rat MLC3f

human MLC3f

mouse sTnc

mouse GLUT4

human desmin

hamster desmin

mouse desmin

chicken cardiac Tn

human PGAM-M

rat muscle AMPD

human muscle ANPD

c

c

c

c

A

T

C

G

G

C

T

T

A

A

A

C

C

T

T

T

T

T

T

G

G

T

C

C

T

A

A

T

T

A

mouse cardiac MBC

conserved

nucleot ides

A AA0-Ai.A IATAA

AT A....i

T' :A ':AA::'A ::A-::

A~:^:.A :::A A

:::: :~:-̀ :: :: AAE

:TA:A: A- AEA X-^:-T.::: A.A.TT.::'::AA-::::::::::::::::

:::::::::-'---: :: :::::::: ::::

_:"::.. .:::.....: ......:::::::

A TC ~:'T A''L...'' A: -0A-: A0:::T .'-Ad.:.-G C----- --

.-.-.-.-.. ....a".-`X'

:aTw:aG:sATAG::-^-"vA TA::G::::-:-:-:A TAG:::E-::::::

-E - _ 7T. ......... - --.-- -- . , . - .. - E -. . _:- r . :: - . . .. . _- E - 7 E - : E : - - 7 : _c T

CT.

C:::::.T

IL-A- -A" A I

;At00i-0A .:AA

AAAA.... ..

c c c

C T C

C T C

C T C

C C A

C T T

C T T

C C C

C C C

C A G

C C C

C C C

C T C

C C T

C C A

C C G

A C G

G

c

c

;A G

:A A

'C C

;T C

;T C

:T G

FIG. 5. Potential MEF-2 sites in muscle-specific regulatory re-

gions. On the basis of the binding data in Fig. 4, several potentialMEF-2 sites were identified in the control regions of muscle-specificgenes. All of these sites are contained within regions that have beenshown to be important for expression of the associated genes inskeletal or cardiac muscle or both. References for sequences are as

follows: mouse MCK 5' enhancer (33, 57), human MCK 5' enhancer(61), rat MCK 5' enhancer (30), rabbit MCK 5' enhancer (63), mouseMCK first-intron enhancer (57), rat MLC1/3 3' enhancer (20),human MLC1/3 3' enhancer (51), chicken MLC2A promoter (5, 52),rat MLC-2A promoter (70), chicken MLC3f promoter (17), mouse,rat (16), and human MLC3f promoters (54a), mouse fast skeletalmuscle troponin C (sTnc) first-intron enhancer (47), mouse GLUT4upstream region (34), human and hamster desmin 5' enhancers (38),mouse desmin 5' enhancer (11), chicken cardiac TnT 5' enhancer(32), rat and human muscle-specific AMP deaminase (AMPD) pro-moters (29), human muscle-specific phosphoglycerate mutase(PGAM-M) upstream region (62), and cardiac myosin heavy chain(MHC) upstream region (27). Conserved nucleotides are shaded.The consensus determined in Fig. 4 is contained within the shadedregion.

different sequence specificities from MEF-2. These factorsalso appear to lack the ability to activate transcription froma multimerized MEF-2 site placed at a distance from theMCK basal promoter because, as we showed previously,such a reporter plasmid is active in differentiated C2 myo-

tubes but not in 1OT1/2 or 3T3 fibroblasts (25). Whether theseubiquitous factors can negatively regulate the MEF-2 siteremains to be determined.Forced expression of myogenin in nonmuscle cell types

can lead to the appearance of MEF-2, indicating that MEF-2activity can be induced by a myogenin-dependent regulatorypathway. Mitogenic signals elicited by high serum interferewith this regulatory interaction and prevent induction ofMEF-2 activity by myogenin, even when myogenin is ex-pressed at high levels from an exogenous expression vector,as in the 10TFL2-3 cell line. Until MEF-2 is cloned, it willnot be possible to distinguish whether mitogenic signals aretargeted at the myogenin protein and prevent it from induc-ing MEF-2 or whether they prevent detection of MEF-2 byinhibiting its DNA-binding activity. In this regard, serum canrepress myogenin's ability to activate genes associated withterminal differentiation, indicating that at least some of itsactivities are negatively regulated by serum (7, 8, 23). Inagreement with the apparent ability of myogenin to regulateMEF-2, we found that myogenin and MEF-2 show similarpatterns of expression in C2 and BC3H1 cells; both areundetectable in proliferating myoblasts and are rapidly in-duced following initiation of differentiation in response toserum withdrawal. Myogenin and MEF-2 are also reversiblyregulated by serum in BC3H1 cells and are subject tonegative control by ras.

It is intriguing that MEF-2 can be induced in cells that can(lOT1/2) and cannot (CV-1) be converted to muscle bymyogenin. The ability of myogenin to induce MEF-2 in CV-1cells suggests that myogenin retains some degree of functionin cells that are refractory to myogenic conversion and thatcells such as CV-1 are blocked from differentiating at a pointdownstream of the mechanisms that control MEF-2 induc-tion. Forced expression of myogenin and other members ofthe MyoD family in CV-1 cells does not lead to autoactiva-tion of the endogenous myogenic regulatory factor genes (2,60, 66). Thus, the ability of myogenin to induce MEF-2activity in CV-1 cells also indicates that myogenin canregulate MEF-2 in the absence of other members of theMyoD family.We do not yet know whether induction of MEF-2 by

myogenin is direct, or whether one or more intermediatesteps intervene. It is also unclear whether myogenin nor-mally regulates MEF-2 expression in myoblasts. The factthat the kinetics of induction of myogenin and MEF-2 duringmyogenesis are virtually superimposable (23, 25) suggests tous that MEF-2 may normally be regulated by another mem-ber of the MyoD family that is expressed in myoblasts beforemyogenin and MEF-2. In this regard, MyoD also can induceMEF-2 in transfected 1OT1/2 cells after withdrawal of serum(35). Paradoxically, MEF-2 sites are present in the promot-ers of the mouse myogenin and MRF4 (21) and XenopusMyoD (39) genes and are important for muscle-specifictranscription of these genes. Thus, the precise hierarchicalrelationship between MEF-2 and the MyoD family is likelyto be complex.There has been controversy regarding the factors that bind

the MEF-2 site. Horlick et al. (30, 31), using the MEF-2 sitefrom the rat MCK enhancer as a probe in gel mobility shiftassays, have identified a heterogeneous set of ubiquitouscomplexes. The factor responsible for formation of the majorubiquitous complex has been referred to as TARP, forT+A-rich binding protein. Our demonstration of ubiquitousbinding factors that recognize the MEF-2 site is consistentwith their studies. However, these ubiquitous binding spe-cies are considerably less abundant than MEF-2. Although

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we cannot absolutely rule out the possibility that MEF-2represents a modified form of the ubiquitous factors thatbind this site, we believe it is unlikely for several reasons: (i)the binding site specificity of MEF-2 is clearly distinct fromthat of the ubiquitous factors; (ii) the apparent abundance ofMEF-2 is at least an order of magnitude greater in differen-tiated muscle cells than the ubiquitous factors in myoblastsand nonmuscle cells; and (iii) induction of MEF-2 appears torequire new protein synthesis (25), making it seem less likelythat it arises through modification of a preexisting protein.The ability of myogenin to induce MEF-2 activity in trans-fected 1OT1/2 and CV-1 cells, which would otherwise neverexpress this activity, also argues that MEF-2 is a bindingactivity unique to cells that have entered a myogenic path-way. The basis for the apparent failure to detect MEF-2 inother studies is currently unclear but could be explained byproteolysis in nuclear extracts, differences in assay condi-tions, or both.

Mutations in the MEF-2 site enabled us to further definethe binding consensus and to identify several muscle-specificgenes that contain potential MEF-2 sites in their controlregions. Since the MEF-2 site appears to be degenerate, andwe have not yet fully defined the range of sequences that arepermissive for MEF-2 binding, it is likely that additionalMEF-2 sites exist in other muscle-specific control regions. Itis intriguing that several genes that contain MEF-2 sites intheir control regions are expressed in cardiac as well asskeletal muscle (e.g., MCK and cTnT). Both MCK and cTnTrequire the MEF-2 site for cardiac muscle expression (28,41), suggesting that MEF-2 may play a role in regulation ofcardiac as well as skeletal muscle-specific transcription.Indeed, a MEF-2-like binding activity that recognizes theMEF-2 site in the MLC2A promoter has been detected innuclear extracts from cardiac myocytes (14). Since membersof the MyoD family have not been found in the heart, it willbe interesting to determine how MEF-2 becomes induced incardiac cells.Members of the MyoD family have been shown to activate

transcription of numerous muscle-specific genes throughbinding to the E-box consensus sequence CANNTG (3, 6, 9,12, 13, 24, 36, 40, 48, 51, 53, 65, 67). The observation thatmyogenin can induce MEF-2 suggests that members of theMyoD family also might indirectly activate transcription ofgenes that contain MEF-2 sites but lack E boxes within theircontrol regions. Indeed, recent results confirm that myoge-nin can transactivate a reporter gene linked to a basalpromoter and five copies of the wild-type MEF-2 site, butnot a multimerized mutant site corresponding to mutant 6 inFig. 4 (15). The ability of MEF-2 to direct muscle-specifictranscription would be consistent with previous resultswhich showed that a multimerized MEF-2 site could directhigh levels of expression in C2 myotubes when combinedwith the MCK basal promoter, which is developmentallyneutral. We suggest therefore that myogenin (and othermembers of the MyoD family) may regulate some muscle-specific genes indirectly, as a consequence of induction ofMEF-2 and perhaps other intermediate muscle-specific fac-tors. Such a hypothetical regulatory pathway is shown inFig. 6. According to this idea, MEF-2 would be induced inresponse to myogenin or other myogenic helix-loop-helixproteins after myoblasts have been induced to differentiateby withdrawal of growth factors. Myogenin and MEF-2could then regulate specific target genes independently,while other muscle-specific genes such as those encodingMCK and MLC1/3 would be regulated through cooperationbetween myogenin and MEF-2. Cloning of MEF-2, which is

(other

Growth factorsignals

Intermediate Myogenic Factors(MEF2)

AChR- a MCK, MLC-1/3

E-BOX DEPENDENT GENES E-BOX INDEPENDENTGENES

FIG. 6. Hypothetical model for regulation of MEF-2 expressionand its role in muscle-specific transcription. Myogenin activatesexpression of MEF-2, and possibly other intermediate muscle-specific factors, upon withdrawal of growth factors. Myogenin andother members of the MyoD family also activate their own expres-sion (2, 60), making it equally likely that other members of theMyoD family also activate MEF-2 expression. In at least some celltypes, these autoregulatory interactions are suppressed by growthfactor signals. Myogenin and MEF-2 may regulate some muscle-specific genes independently and may cooperate to activate othergenes such as those encoding MCK and MLC1/3. Several muscle-specific genes contain multiple E boxes that bind members of theMyoD family (designated M). Ubiquitous transcription factors thatbind other sites within muscle-specific control regions and areimportant for muscle-specific transcription are not shown. AChR,acetylcholine receptor (48).

currently under way, will allow these predictions to be testeddirectly.

ACKNOWLEDGMENTS

This work was supported by grants to E.N.O. from the NationalInstitutes of Health and the American Cancer Society. E.N.O. is anEstablished Investigator of the American Heart Association. Oligo-nucleotides were synthesized by the Macromolecular SynthesisFacility at the University of Texas M. D. Anderson Cancer Centerthrough the support of NIH grant CA16672.We are grateful to S. Jasser and J. Toma for assistance with tissue

culture, L. Gossett for performing initial gel mobility shifts, and E.Madson for secretarial assistance. We also thank the followingindividuals for communicating sequences and results prior to publi-cation: Y. Capetenaki, K. Chien, M. Crow, S. Hauschka, E.Holmes, A. Lassar and H. Weintraub, J. Mar and C. Ordahl, M.Perry, and K. Walsh.

REFERENCES1. Bouvagnet, P. F., E. E. Strehier, G. E. White, M.-A. Strehler-

Page, B. Nadal-Ginard, and V. Mahdavi. 1987. Multiple positiveand negative 5' regulatory elements control the cell-type-spe-cific expression of the embryonic skeletal myosin heavy-chaingene. Mol. Cell. Biol. 7:4377-4389.

2. Braun, T., E. Bober, G. Buschhausen-Denker, S. Kotz, K.Grzeschik, and H. H. Arnold. 1989. Differential expression ofmyogenic determination genes in muscle cells: possible autoac-tivation by the Myfgene products. EMBO J. 8:3617-3625.

3. Braun, T., E. Bober, B. Winter, N. Rosenthal, and H. H. Arnold.1990. Myf-6, a new member of the human gene family ofmyogenic determination factors: evidence for a gene cluster on

MOL. CELL. BIOL.

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Nov

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247.

REGULATION OF MEF-2 4861

chromosome 12. EMBO J. 9:821-831.4. Braun, T., G. Buschhausen-Denker, E. Bober, E. Tannich, and

H. H. Arnold. 1989. A novel human muscle factor related to butdistinct from MyoDl induces myogenic conversion in 1OT1/2fibroblasts. EMBO J. 8:701-709.

5. Braun, T., E. Tannich, G. Buschhausen-Denker, and H. H.Arnold. 1989. Promoter upstream elements of the chickencardiac myosin light-chain 2-A gene interact with regulatoryfactors for muscle-specific transcription. Mol. Cell. Biol.9:2513-2525.

6. Brennan, T. J., T. Chakraborty, and E. N. Olson. 1991. Muta-genesis of the myogenin basic region identifies an ancientprotein motif critical for activation of myogenesis. Proc. Natl.Acad. Sci. USA 88:5675-5679.

7. Brennan, T., D. G. Edmondson, L. Li, and E. N. Olson. 1991.TGF-4 represses the actions of myogenin through a mechanismindependent of DNA binding. Proc. Natl. Acad. Sci. USA88:3822-3826.

8. Brennan, T. J., D. G. Edmondson, and E. N. Olson. 1990.Aberrant regulation of MyoDl contributes to the partiallydefective myogenic phenotype of BC3H1 cells. J. Cell Biol.110:929-937.

9. Brennan, T. J., and E. N. Olson. 1990. Myogenin resides in thenucleus and acquires high affinity for a conserved enhancerelement on heterodimerization. Genes Dev. 4:582-595.

10. Buskin, J. N., and S. D. Hauschka. 1989. Identification of amyocyte-specific nuclear factor which binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.Mol. Cell. Biol. 9:2627-2640.

11. Capetanaki, Y. (Baylor College of Medicine). Personal commu-nication.

12. Chakraborty, T., T. J. Brennan, L. Li, D. G. Edmondson, andE. N. Olson. 1991. Inefficient homooligomerization cont-ributesto the dependence of myogenin on E2A products for efficientDNA binding. Mol. Cell. Biol. 11:3633-3641.

13. Chakraborty, T., T. J. Brennan, and E. N. Olson. 1991. Differ-ential trans-activation of a muscle-specific enhancer by myo-genic helix-loop-helix proteins is separable from DNA binding.J. Biol. Chem. 266:2878-2882.

14. Chien, K. (University of California, San Diego). Personal com-munication.

15. Cserjesi, P., and E. N. Olson. Unpublished data.16. Crow, M. (National Institute of Aging). Personal communica-

tion.17. Daubas, P., B. Robert, I. Garner, and M. Buckingham. 1985. A

comparison between mammalian and avian fast skeletal musclealkali myosin light chain genes: regulatory implications. NucleicAcids Res. 13:4623-4643.

18. Davis, R. L., P.-F. Cheng, A. B. Lassar, and H. Weintraub.1990. The MyoD DNA binding domain contains a recognitioncode for muscle-specific gene activation. Cell 60:733-746.

19. Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Expressionof a single transfected cDNA converts fibroblasts to myoblasts.Cell 51:987-1000.

20. Donoghue, M., H. Ernst, B. Wentworth, B. Nadal-Ginard, andN. Rosenthal. 1988. A muscle-specific enhancer is located at the3' end of the myosin light chain 1/3 gene locus. Genes Dev.2:1779-1790.

21. Edmondson, D. G., T.-C. Cheng, and E. N. Olson. Unpublisheddata.

22. Edmondson, D. G., and E. N. Olson. Unpublished data.23. Edmondson, D. G., and E. N. Olson. 1989. A gene with homol-

ogy to the myc similarity region of MyoDl is expressed duringmyogenesis and is sufficient to activate the muscle differentia-tion program. Genes Dev. 3:628-640.

24. French, B. A., K.-L. Chow, E. N. Olson, and R. J. Schwartz.1991. Heterodimers of myogenic helix-loop-helix regulatoryfactors and E12 bind a complex element governing myogenicinduction of the avian cardiac a-actin promoter. Mol. Cell. Biol.11:2439-2450.

25. Gossett, L. A., D. J. Kelvin, E. A. Sternberg, and E. N. Olson.1989. A new myocyte-specific enhancer-binding factor thatrecognizes a conserved element associated with multiple mus-

cle-specific genes. Mol. Cell. Biol. 9:5022-5033.26. Gossett, L. A., W. Zhang, and E. N. Olson. 1988. Dexametha-

sone-dependent inhibition of differentiation of C2 myoblastsbearing steroid-inducible N-ras oncogenes. J. Cell Biol. 106:2127-2137.

27. Gulick, J., A. Subramaniam, J. Neumann, and J. Robbins. 1991.Isolation and characterization of the mouse cardiac myosinheavy chain genes. J. Biol. Chem. 266:9180-9185.

28. Hauschka, S. (University of Washington, Seattle). Personal com-munication.

29. Holmes, E. (Duke University). Personal communication.30. Horlick, A., and P. A. Benfield. 1989. The upstream muscle-

specific enhancer of the rat muscle creatine kinase gene iscomposed of multiple elements. Mol. Cell. Biol. 9:2396-2413.

31. Horlick, R. A., G. M. Hobson, J.-H. Patterson, M. T. Mitchell,and P. A. Benfield. 1990. Brain and muscle creatine kinase genescontain common TA-rich recognition protein-binding regulatoryelements. Mol. Cell. Biol. 10:4826-4836.

32. lanello, R. C., J. H. Mar, and C. P. Ordahl. 1991. Characteri-zation of a promoter element required for transcription inmyocardial cells. J. Biol. Chem. 266:3309-3316.

33. Jaynes, J. B., J. B. Johnson, J. M. Buskin, C. L. Gartside, andS. D. Hauschka. 1988. The muscle creatine kinase gene isregulated by multiple elements, including a muscle-specificenhancer. Mol. Cell. Biol. 6:2855-2864.

34. Kaestner, K. H., R. J. Christy, and M. D. Lane. 1990. Mouseinsulin-responsive glucose transporter gene: characterization ofthe gene and trans-activation by the CCAAT/enhancer bindingprotein. Proc. Natl. Acad. Sci. USA 87:251-255.

35. Lassar, A. B. (Fred Hutchinson Cancer Research Center, Seat-tle). Personal communication.

36. Lassar, A. B., J. N. Buskin, D. Lockshon, R. L. Davis, S. Apone,S. D. Hauschka, and H. Weintraub. 1989. MyoD is a sequence-specific DNA binding protein requiring a region of myc homol-ogy to bind to the muscle creatine kinase enhancer. Cell58:823-831.

37. Lassar, A. B., M. J. Thayer, R. W. Overell, and H. Weintraub.1989. Transformation by activated RAS or FOS prevents myo-genesis by inhibiting expression of MyoDl. Cell-5:659-667.

38. Li, Z., and D. Paulin. 1991. High level desmin expressiondepends on a muscle-specific enhancer. J. Biol. Chem. 266:6562-6570.

39. Liebham, D., and W. M. Perry (The University of Texas M. D.Anderson Cancer Center). Personal communication.

40. Lin, H., K. Yutzey, and S. F. Konieczny. 1991. Muscle-specificexpression of the troponin I gene requires interactions betweenhelix-loop-helix muscle regulatory factors and ubiquitous tran-scription factors. Mol. Cell. Biol. 11:267-280.

41. Mar, J. H., and C. P. Ordahl (University of California, SanFrancisco). Personal communication.

42. Mar, J. H., and C. P. Ordahl. 1990. M-CAT binding factor, anovel trans-acting factor governing muscle-specific transcrip-tion. Mol. Cell. Biol. 10:4271-4283.

43. Miner, J. H., and B. Wold. 1990. Herculin, a fourth member ofthe MyoD family of myogenic regulatory genes. Proc. Natl.Acad. Sci. USA 87:1089-1093.

44. Mueler, P. R., and B. Wold. 1989. In vivo footprinting of amuscle-specific enhancer by ligation mediated PCR. Science246:780-786.

45. Olson, E. N. 1990. MyoD family: a paradigm for development?Genes Dev. 4:1454-1461.

46. Olson, E. N., G. Spizz, and M. Tainsky. 1987. The oncogenicforms of N-ras or H-ras prevent skeletal myoblast differentia-tion. Mol. Cell. Biol. 7:2104-2111.

47. Parmacek, M. S., A. R. Bengur, A. J. Vora, and J. M. Leiden.1990. The structure and regulation of expression of the murinefast skeletal troponin C gene. J. Biol. Chem. 265:15970-15976.

48. Piette, J., J.-L. Bessereau, M. Huchet, and J.-P. Changeux. 1990.Two adjacent MyoDl-binding sites regulate expression of theacetyl choline receptor a-subunit. Nature (London) 345:353-355.

49. Rhodes, S. J., and S. F. Konieczny. 1989. Identification ofMRF4: a new member of the muscle regulatory factor gene

VOL . 1l, 1991

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from

http

s://j

ourn

als.

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.org

/jour

nal/m

cb o

n 26

Nov

embe

r 20

21 b

y 42

.116

.94.

247.

4862 CSERJESI AND OLSON

family. Genes Dev. 3:2050-2061.50. Rosenthal, N. 1989. Muscle cell differentiation, review. Curr.

Opin. Cell Biol. 1:1094-1101.51. Rosenthal, N., E. N. Berglund, B. M. Wentworth, M. Donoghue,

B. Winter, E. Bober, T. Braun, and H. H. Arnold. 1990. A highlyconserved enhancer downstream of the human MLC1/3 locus isa target for multiple myogenic determination factors. NucleicAcids Res. 18:6239-6246.

52. Ruoquian-Shen, S., K. Goswami, E. Mascareno, A. Kumar, andM. A. Q. Siddiqui. 1991. Tissue-specific transcription of thecardiac myosin light-chain 2 gene is regulated by an upstreamrepressor element. Mol. Cell. Biol. 11:1676-1685.

53. Sartorelli, V., K. A. Webster, and L. Kedes. 1990. Muscle-specific expression of the cardiac a-actin gene requires MyoDl,CArG-box binding factor, and Spl. Genes Dev. 4:1811-1822.

54. Schafer, B. W., B. T. Blakely, G. J. Darlington, and H. M. Blau.1990. Effect of cell history on response to helix-loop-helixfamily of myogenic regulators. Nature (London) 344:454-458.

54a.Seidel, U., and H. Arnold. 1989. Identification of the functionalpromoter regions in the human gene encoding the myosin alkalilight chains MLC1 and MLC3 of fast skeletal muscle. J. Biol.Chem. 264:16109-16117.

55. Spizz, G., D. Roman, R. Strauss, and E. N. Olson. 1986. Serumand fibroblast growth factor inhibit myogenic differentiationthrough a mechanism dependent on protein synthesis and inde-pendent of cell proliferation. J. Biol. Chem. 261:9483-9488.

56. Sternberg, E., G. Spizz, M. E. Perry, and E. N. Olson. 1989. Aras-dependent pathway abolishes activity of a muscle-specificenhancer upstream from the muscle creatine kinase gene. Mol.Cell. Biol. 9:594-601.

57. Sternberg, E. A., G. Spizz, W. M. Perry, D. Vizard, T. Weil, andE. N. Olson. 1988. Identification of upstream and intragenicregulatory elements that confer cell-type-restricted and differ-entiation-specific expression on the muscle creatine kinasegene. Mol. Cell. Biol. 8:2896-2909.

58. Tapscott, S. J., and H. Weintraub. 1991. MyoD and the regula-tion of myogenesis by helix-loop-helix proteins. J. Clin. Invest.87:1133-1138.

59. Taubman, M. B., C. W. J. Smith, S. Izumo, J. W. Grant, T.Endo, A. Andreadis, and B. Nadal-Ginard. 1989. The expressionof sarcomeric muscle-specific contractile protein genes inBC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are

defective for commitment to terminal differentiation. J. CellBiol. 108:1799-1806.

60. Thayer, M. J., S. J. Tapscott, R. L. Davis, W. E. Wright, A. B.Lassar, and H. Weintraub. 1989. Positive autoregulation of themyogenic determination gene MyoDl. Cell 58:241-248.

61. Trask, R. V., A. W. Strauss, and J. J. Billadello. 1988. Devel-opmental regulation and tissue-specific expression of the humanmuscle creatine kinase gene. J. Biol. Chem. 263:17142-17149.

62. Tsujino, S., S. Sakoda, R. Mizuno, T. Kobayashi, T. Suzuki, S.Kishimoto, S. Shanske, S. DiMauro, and E. A. Schon. 1989.Structure of the gene encoding the muscle-specific subunit ofhuman phosphoglycerate mutase. J. Biol. Chem. 264:15334-15337.

63. Walsh, K. (Case Western Reserve Medical School, Cleveland).Personal communication.

64. Walsh, K., and P. Schimmel. 1987. Two nuclear factors competefor the skeletal muscle actin promoter. J. Biol. Chem. 262:9429-9432.

65. Weintraub, H., R. Davis, D. Lockshon, and A. Lassar. 1990.MyoD binds cooperatively to two sites in a target enhancersequence: occupancy of two sites is required for activation.Proc. Natl. Acad. Sci. USA 87:5623-5627.

66. Weintraub, H., S. J. Tapscott, R. L. Davis, M. J. Thayer, M. A.Adam, A. B. Lassar, and A. D. Miller. 1989. Activation ofmuscle specific genes in pigment, nerve, fat, liver and fibroblastcell lines by forced expression of MyoD. Proc. Natl. Acad. Sci.USA 86:5434-5438.

67. Wentworth, B. M., M. Donoghue, J. C. Engert, E. B. Berglund,and N. Rosenthal. 1991. Paired MyoD binding sites regulatemyosin light chain gene expression. Proc. Natl. Acad. Sci. USA88:1242-1246.

68. Wright, W. E., D. A. Sassoon, and V. K. Lin. 1989. Myogenin,a factor regulating myogenesis, has a domain homologous toMyoDl. Cell 56:607-617.

69. Yaffe, D., and 0. Saxel. 1977. Serial passaging and differentia-tion of myogenic cells isolated from dystrophic mouse muscle.Nature (London) 270:725-727.

70. Zhu, H., A. V. Garcia, R. S. Ross, S. M. Evans, and K. R. Chien.1991. A conserved 28-base-pair element (HF-1) in the ratcardiac myosin light-chain-2 gene confers cardiac-specific anda-adrenergic-inducible expression in cultured neonatal rat myo.cardial cells. Mol. Cell. Biol. 11:2273-2281.

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