absence ofmef2bindingtothent-rich element inthe muscle...

Received 6/3/96; revised 10/4/96; accepted 11/6/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was supported in part byTelethon-ltalia(Project 499; to S. F.),Consiglio Nazionale delle Aicerche Progetto Finalizzato. Ingegneria Ge-netica, Ministero dell’Universit#{225}e Ricerca ScientificaeTecnologica(40%),and Fondazione Cenci Bolognetti. A. K. was supported initially by a Well-come Travelling Fellowship and then by a European Community fellow-ship under the Human Capital and Mobility Program grant2 To whom requests for reprints should be addressed. Phone:(059) 428505; Fax: (059) 428524; E-mail: [email protected].

3 The abbreviations used are: d.p.c., days postcoitum; MHC, myosinheavy chain; MCK, muscle creatine kinase; HLH, helix-loop-helix; MEF2,myocyte enhancer factor 2; EMSA, electrophoretic mobility shift assay;AT-PCA, reverse transcription-PCA; nt, nucleotide(s); WA, 12-O-tetra-decanoylphorbol-i3-acetate; j3-gal, p-galactosidase; CAT, chloramphen-icol acetyltransferase.

Vol. 8, 23-34, January 1997 Cell Growth & Differentiation 23

Absence of MEF2 Binding to the NT-rich Element in theMuscle Creatine Kinase (MCK) Enhancer Correlateswith Lack of Early Expression of the MCK Genein Embryonic Mammalian Muscle1

Stefano Ferrari,2 Susanna Molinari,Roberta Melchionna, Maria Gabnella Cusella-De Angells, Renata Battini, Luciana Dc Angelis,Robert Kelly, and Giulio Cossu

Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica,Universit#{224}di Modena, Via G. Campi 287, 41 100 Modena, Italy [S. F.,S. M., A. B.]; Istituto di Istologia ed Embriologia Generale, Universit#{224}diAoma La Sapienza, 00161 Roma, Italy [A. M., M. G. C-D. A., L D. A.,G. C.]; and D#{233}partementdo Blologie Moleculaire, Institut Pasteur,75724 Paris Cedex 15, France [A. K.]

AbstractDuring skeletal muscle development, different types ofmuscle fibers are generated, which express differentcombinations of muscle-specific gene products. Forexample, the muscle creatine kinase gene (MCK) ishighly expressed in fetal but not embryonic myotubes.We performed transient transfections of CAT reporterconstructs, driven by the MCK promoter with variablelengths of 5’-flanking sequence, into primary culturesof embryonic and fetal muscle cells. Reporter activity

was observed in fetal but not embryonic muscle cells.We assayed the ability of nuclear extracts preparedfrom embryonic and fetal muscle and C2C12 myotubesto bind specific regulatory elements in the MCKenhancer. The profile of DNA/protein complexesresulting from electrophoretic mobility shift assays wasqualitatively the same with all extracts used when theoligonucleotide probes represented the MCK E-box,MHox site, CArG-box, and AP2 site. In contrast, nobinding activity to the MEF2 site was observed withembryonic nuclear extract. Interestingly, MEF2 mRNAsand proteins were detected in both fetal andembryonic muscle, with the exception of the MEF2DIbisoform, which is restricted to fetal muscle.Furthermore, we found that protein phosphataseinhibitors included in the preparation of embryonicnuclear extracts or added to the medium of transfected

embryonic myotubes can restore MEF2 DNA bindingactivity, as well as reporter activity driven by the MCKpromoter and partial transcriptional activation of theendogenous MCK gene. We propose thatphosphorylation of MEF2 regulates its activity andrepresents an important aspect of the mechanismcontrolling stage-specific transcription during skeletalmyogenesis.

Introduction

Skeletal muscle histogenesis is a very complex and asyn-chronous process that extends throughout pre- and postna-tal development. It involves different populations of myo-blasts and different intra- and extracellular signals to ensurethe formation of highly heterogeneous muscle fibers in thecorrect spatiotemporal context (i). The first multinucleatedmuscle fibers form, in the mouse, at around i 1 d.p.c.3 (2, 3)and are therefore defined as “primary” or “embryonic”; theyinitially express an embryonic muscle phenotype (4, 5). Dur-ing fetal development (from around i 5 d.p.c.), secondaryfibers appear, surrounding primary fibers (2, 3); they expressa “fetal” muscle phenotype (4, 5). With further development,both fibers begin to express a “neonatal” and then an “adult”muscle phenotype (6). For example, primary fibers expressboth slow and fast MHCs (7) but no muscle-specific isoformsof metabolic enzymes, such as MCK. Conversely, secondaryfibers initially express fast but not slow MHC and high levelsof MCK (8, 9). Interestingly, these phenotypic differences aremaintained in cultures of muscle cells derived from embry-onic and fetal myoblasts. Stage-specific expression of sev-eral muscle genes may depend on the presence of tissue-specific and/or ubiquitous transcription factors, on theirrelative concentrations, and on the mechanisms regulatingtheir activity.

The MCK gene represents a useful model for studyingdifferential gene expression during the transition from anembryonic to a fetal phenotype. The MCK gene is not tran-scribed at an appreciable rate in myotubes differentiated invitro from embryonic myoblasts, whereas it is highly ex-pressed in myotubes of fetal origin (9, i 0). Furthermore, theMCK gene has been studied extensively as a model forunderstanding the mechanisms that regulate skeletal muscle

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24 Lack of MEF2 Activity in Embryonic Muscle

gene transcription. It is well known that MCK gene transcrip-

tion depends, at least in cultured cells, on the presence of an

enhancer region, which lies between bp -1256 and -1050

relative to the transcription start site and contains two E-

boxes that are essential for enhancer activity in skeletal

muscle cells (i i-i 3). The E-box sequence is the target site ofthe HLH family of DNA binding proteins, which includes theskeletal muscle determination factors MyoD (14), myogenin

(i 5-i 7), myf-5 (i 8) and myf-6 (also called MRF-4 or herculin;Ref. i9).

In the 3’ region, the MCK enhancer contains an NT-richsequence that has been shown to represent a high-affinitybinding site for MEF2 (20-24). The recent cloning of MEF2showed that it represents a family of four genes (Mef2A-D),which encode proteins that share homology within a MADS

(MCM1 , Agamous, Deficiens, Serum response factor) do-

main, which mediates DNA binding and dimerization (24-27).

Deletion or mutation of MEF2-binding sites in the regulatory

regions of several genes, such as, for example, those en-

coding MCK (i3, 20, 21), cardiac myosin light chain 2 (28),and the muscle-specific subunit of human phosphoglycerate

mutase (29), results in a dramatic reduction of transcription in

cultured muscle cells, indicating that the MEF2 sites arerequired for full transcriptional activity.

A second NT-rich element in the MCK enhancer, locatedimmediately 5’ of the left E-box, is required for full transcrip-

tional activity in skeletal and cardiac myocytes (i i , 1 3, 22,30). This element binds the homeodomain protein MHox and

Oct-i (3i , 32) and represents a weak but important site forbinding of MEF2, which was shown to be the functionally

relevant component (32). The MCK enhancer region also

contains a CArG-box, which is important for cardiac-specifictranscription (33), and an AP2 site, which appears to con-

tribute negatively to enhancer function (i3). Because the

regulatory elements of the MCK gene have been studied in

considerable detail, we decided to use such information to

explore the molecular basis for the differential transcription

of this gene during muscle development.

Here we report that MEF2 proteins, although present in the

nuclei of differentiated embryonic muscle cells, do not bind

their cognate site in the MCK enhancer and that MEF2 ele-ments cannot enhance transcription at the MCK promoter in

embryonic muscle cells. We also suggest that stage-depen-dent phosphorylation regulates MEF2 activity, thus providing

a mechanism by which skeletal muscle histogenesis is con-

trolled.

ResultsMCK Gene Expression in Embryonic and Fetal Myotubes.As previously shown by in situ hybridization experiments

(iO), MCK mRNA cannot be detected in embryonic muscle(Fig. i , Lane 1), whereas it represents an abundant transcript

in fetal muscle (Lane 2). This phenotype is retained in vitro

because, in keeping with recent observations (9), we ob-served that primary cultures of fetal (Lane 4), but not embry-

onic (Lane 3), differentiated muscle cells express MCKmRNA. It is well established that the transcriptional activity of

the MCK gene, in terminally differentiated myotubes, is

strongly dependent on the presence of an enhancer region

Fig. 1. Northern blot analysis of MCK mRNA in embryonic and fetalmuscle. A, i 0 �g oftotal ANA extracted from embryonic muscle (ME; Lane1), fetal muscle (MF; Lane 2), or in vitro differentiated muscle cells ofembryonic (MEM; Lane 3) and fetal (MFM; Lane 4) origin were analyzed forthe presence of MCK mANA. B, ethidium bromide staining of the 285rANA subunit present in the indicated ANAs.

located approximately 1 i 00 bp 5’ of the transcription startsite. To functionally identify the cis-acting elements that

might be involved in the fetal muscle-specific expression of

the MCK gene, primary cultures of embryonic and fetal myo-

blasts were transiently transfected with a series of CAT re-

porter constructs containing various subfragments of theDNA sequence flanking the 5-end of the MCK gene. Asexpected, fetal myoblasts transfected with the CAT plasmidcontaining the largest available 5’-flanking sequence (3300

bp) show, after withdrawal of growth factors, a progressive

increase of CAT activity as they progress to terminal differ-

entiation (Fig. 2A, filled bars). On the contrary, embryonic

myoblasts show no reporter gene activity detectable abovebackground (Fig. 2A, open bars), although they differentiate

at a comparable rate and, as a consequence, express a-gal

activity encoded by the IacZ gene present in the cotrans-

fected plasmid pi F-nlacZ-E. The MLC i F muscle-specificpromoter and the 3’ MLC1 F/3F enhancer present in thisplasmid direct �3-gal expression very early in muscle devel-

opment in vivo (34) and in myotubes of embryonic and fetal

origin in vitro (35). Serial deletions of the 5’-flanking se-

quence show, as expected, that in fetal myotubes, most ofthe transcriptional activity of the MCK promoter relies on the

enhancer region located between deletions - i 256 and

-776 (Fig. 2B, filled bars). Transfection experiments per-

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Fig. 2. Transfection of MCK/CAT constructs in primary cultures of em-bryonic and fetal myotubes. A, the reporter construct -3300MCKJCAT,which contains bp -3300 to +7 of the mouse MCK gene fused to CAT,was transfected into primary cultures of embryonic (LI) and fetal ($)myoblasts 1 day after plating. Sixteen h after transfection, medium con-taming i % FCS was added to the cultures, and cells were harvested at theindicated time points from growth factor withdrawal. The volume of celllysate used for CAT assays was corrected for transfection efficiency anddegree of myogenic differentiation by estimating the �3-gal activity con-ferred by the cotransfected plasmid pi F-nlacz-E. B, the deletion con-structs indicated were transfected into primary cultures of embryonic andfetal myoblasts and cells were harvested 72 h later. Normalization fortransfection efficiency and degree of myogenic differentiation was as in A.CAT activity of individual constructs is expressed relative to the activity of-2800MCK/CAT.

Cell Growth & Differentiation 25

24 46 72 96 hours

formed in parallel with embryonic myoblasts indicate that the

removal of sequences upstream of the enhancer region does

not cause any significant increase in the reporter activity (Fig.

2B, open bars), thus indicating that cis-acting elements with

a negative effect on transcription in embryonic muscle cells

are not likely to exist in the region between bp -3300 and

-1256.

EMSA Analysis of MEF2 Site Binding Proteins ShowsThat Distinct DNA/Protein Complexes Are Formed withNuclear Extracts from Embryonic or Fetal Muscle Cells.The MCK enhancer region consists of at least six elements

(CArG site, AP2 site, MHox site, left E-box, right E-box, and

MEF2 site), which have been shown to contribute differently

to full enhancer function and to bind specific protein factors.

To search for differences between embryonic and fetal dif-

ferentiated muscle cells in the binding activity of specific

trans-acting factors, we analyzed nuclear extracts for the

ability to bind oligonucleotides representing individual ele-

ments of the MCK enhancer. EMSA analysis of E-box, MHox

site, CArG-box, and AP2 site binding proteins did not revealany significant difference among nuclear extracts of embry-

onic muscle, fetal muscle, and differentiated C2C12 cells(data not shown). In contrast, a different profile of DNN

protein complexes was observed with the MEF2 site probe.Fig. 3A shows that a retarded complex (complex b) is ob-

served in EMSAs in which nuclear extracts from fetal muscle

(MF, Lane 6) and C2Ci 2 myotubes (C2C12, Lane 10) were

incubated with the MEF2 site oligonucleotide probe. Corn-

plex b contains a member (or members) of the MEF2 family

of transcription factors because, as Fig. 3B shows, a poly-

clonal antibody directed to human MEF2A supershifts the

complex (MEF2 Ab, Lanes 8 and 1 1). No effect on complex

b was observed when an antibody to MHox (MHox Ab, Lanes

9 and 12) or preimmune serum were used (ol, Lanes 10 and

13). Complex b is drastically reduced by the addition of a

molar excess of unlabeled MEF2 site probe (A, MEF2, Lanes

8 and 12) and partially reduced by the addition of the MHox

oligonucleotide (MHox, Lanes 7 and 1 1); no effect was

caused by a molar excess of the E-box oligonucleotide

(MCKE, Lanes 9 and 13). This finding suggests that the MEF2

site binding activity is capable of binding structurally related

A/l-rich sequences, although with different affinity, which is

in keeping with recently published observations (32). Strik-

ingly, complex b is absent in EMSAs in which the nuclear

extracts prepared from embryonic muscle were used (A, ME,

Lane 2) and is replaced by a faster mobility complex, mdi-

cated by complex a in Fig. 3A. Complex a is not competed

by a molar excess of added oligonucleotides representing

MHox and E-box (Lanes 3 and 5, respectively), and is spe-

cific, because it is reduced by the unlabeled MEF2 site probe

added as competitor (Lane 4). The embryo-specific complex

a is unaffected by antibodies to either MEF2 or MHox (Fig.

3B, Lanes 5 and 6). Consistent with the results obtained withthe antiserum specifically directed to MEF2A, antibodies

raised against the RSRF (Related to Serum Response Fac-

tor) family of transcription factors (which includes MEF2)

cause a supershift of complex b (Fig. 3B, RSRFAb, Lanes

19 and 20); no effect, however, was observed on complex

a (Lane 18). In addition to complexes a and b, at least two

other intense complexes with fast electrophoretic mobility

can be observed in all lanes in Fig. 3. Although they appear

to be affected by the MEF2 oligonucleotide added in molar

excess (Fig. 3A, Lanes 4, 8, and 12), they are not recog-

nized by antibodies directed to MEF2, they are present

with roughly equal intensities in nuclear extracts prepared

from embryonic and fetal muscle as well as from C2Ci2

myotubes, and finally, they form with the same efficiency

when the oligonucleotide probe contains a mutated MEF2

site, which does not bind MEF2 (data not shown).

To determine the affinity of the MEF2 binding activities,

nuclear extracts from C2Ci 2 myotubes and embryonic mus-

cle were incubated with increasing amounts of MEF2 site

probe. Bands corresponding to complexes a and b were

excised and counted. To obtain Kd values, the data were

plotted by the Scatchard method. As Fig. 4A shows, the

MEF2 site binding activity contained in C2Ci2 myotube nu-

clear extract (complex b, immunologically related to MEF2A)

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is represented by a single, high-affinity component with a Kdof 5.68 :t 0.23 nM. The MEF2 site binding activity observed

in embryonic muscle nuclear extract (complex a; Fig. 4B)

appears to be represented by two components of low and

very low binding affinity (Kdl = i8.65 ± 5 nM and Kd2 =

62.39 � 20 nM, respectively).

We further investigated whether the lack of MEF2 binding

Fig. 3. EMSA analysis of pro-teins binding to the MEF2 site. A,a 32P-labeled oligonucleotiderepresenting the MEF2 site lo-cated in the MCK enhancer wasused as a probe in EMSA mixescontaining nuclear extracts pre-pared from embryonic muscle(ME; Lane 2), fetal muscle (MF;Lane 6) and C2Ci2 myotubes(C2C12; Lane 10). Competing ol-igonucleotides, added in 100-fold molar excess to the extractsas indicated, were as follows:MHox site (MHox; Lanes 3, 7, and1 1), MEF2 site (MEF2; Lanes 4, 8,and 12), and E-box (MCKE;Lanes 5, 9, and 13). B, antisera toMEF2 (MEF2 Ab; Lanes 5, 8, and1 1), MHox (MHox Ab; Lanes 6, 9,and 12), RSAF (RSRFAb; Lanes18-20) or preimmune serum (ol;

Lanes 7, 10, and 13) were addedto the nuclear extracts indicated.In both panels, the probe with noadded extract is indicated as C(A, Lane 1; B, Lanes 1 and 14). aand b, specific protein/DNAcomplexes.

presence of an inhibitor. To this end, in vitro translated hu-

man MEF2A was assayed for ability to bind the MCK MEF2

site in the presence of increasing amounts of embryonic

muscle nuclear extract. As Fig. 5 shows, even in the pres-

ence of 1 5 jLg of embryonic muscle (Lane 5) or STO fibroblast

(Lane 8) nuclear proteins, no detectable decrease of MEF2

binding activity was observed. In keeping with this observa-

tion, we found that embryonic muscle nuclear proteins do

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-� Fig. 5. EMSA analysis of in vitro translated human MEF2A in the pres-ence of embryonic muscle and STO uibroblast nuclear extracts. In vitrotranslated hMEF2A was incubated with 32P-labeled MEF2 site probe,either alone (Lane 2) or in the presence of increasing amounts of embry-onic muscle (ME; Lanes 3-5) or STO fibroblast (STO; Lanes 6-8) nuclear

- � proteins. Lane 1, probe added with unprogrammed lysate (U.L.).

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Fig. 4. Determination of an equilibrium dissociation constant for MEF2binding activities present in differentiated C2C12 cells and embryonicmuscle. Nuclear extracts prepared from C2C12 myotubes (top panel) andfrom embryonic muscle (bottom panel) were incubated with increasingamounts of 32P-labeled MEF2 site oligonucleotide, and the DNAlproteincomplexes were resolved from free DNA by gel electrophoresis. Afterautoradiography of the dried gel, the bands were excised and counted ina liquid scintillation counter. Control mixtures not containing nuclear ex-tract were incubated with each probe concentration, and the region of thegel corresponding to the shifted complex was used to subtract back-ground. The data obtained from saturation curves were plotted by theScatchard method to obtain Kd values.

not inhibit the binding of fetal muscle nuclear extract to the

MEF2 site (not shown).Detection of MEF2 mRNA and Protein in Embryonic

and Fetal Muscle Cells. The data obtained from EMSA

experiments collectively indicate that the embryo-specific

lack of MEF2 site binding activity is the only significant dif-

ference between embryonic and fetal muscle nuclear ex-

tracts in their ability to bind to defined cis-acting elements


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present in the MCK enhancer. To further explore this finding,

RT-PCR experiments were performed to assess whether

significant changes in the many splicing MEF2 mRNA iso-

forms exist in embryonic versus fetal muscle. Fig. 6A shows

that the MEF2A mRNA sequence from nt 424 to nt 841 (24)

is present in both embryonic (Lane 1) and fetal (Lane 2)muscle, as is the splice variant referred to as aMEF2 (Lanes

3 and 4; Ref. 24); in addition, isoforms either containing ornot containing the SEEEELEL mini-exon are present at both

developmental stages (Lanes 5 and 6). Fig. 68 shows that the

MEF2C mRNA sequence from nt 342 to nt 730 (26) is present

in the RNA of both embryonic (Lane 1) and fetal (Lane 2)origin. In addition, direct PCR sequencing of the 388-bp

fragments obtained from both amplification reactions sug-

gests that no alternate exon exists in the analyzed fragment

(not shown). The two MEF2C isoforms generated by the

presence of alternate exon B (36) are equally represented in

embryonic (Lane 3) and fetal (Lane 4) muscle, whereas alter-

nate exon A appears to be missing in both (Lanes 5 and 6).

A prominent difference was observed in the choice of alter-

nate exons in MEF2D (Fig. 6C). In fact, oligonucleotides DP2

A

B

C

CP3+

CP4

304-210-

184-

34

MEF2A

MEF2C

MEF2D

D ProbeA ProbeB

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Fig. 6. Analysis of MEF2 mRNA isoforms and proteins. A, RNA extractedfrom 11-d.p.c. (Lanes 1, 3, and 5) and 17-d.p.c. (Lanes 2, 4, and 6) limbswas subjected to RT-PCR analysis for providing evidence of MEF2A-specific sequences (primers APi and AP2), the alternative exon present inaMEF2 (APi +AP3), and the alternative exon containing the SEEEELELmotif (primers AP4+AP5). B, as in A, but oligonucleotide primers werechosen to provide evidence of MEF2C-specific sequences (CP1 + CP2)and alternate exons B (CP3+CP4) and A (CP5+CP6). C, RNA from11-d.p.c. (Lanes 1 and 4) and 17-d.p.c. (Lanes 3 and 6) limbs was sub-jected to RT-PCR analysis to show MEF2D-specific sequences (primersDP2+DP3) and alternate exon 2 (primers DP4+DP5); control reactionswith no added reverse transcriptase were run in Lanes 2 and 5. D,amplified DNA from RT-PCRs obtained with primers DP2 + DP3 was run intriplicate and hybridized to 32P-labeled oligonucleotide probes specific toMEF2D exon 1 a (probe A), exon 1 b (probe B), or a region common to bothamplified DNA fragments. Lanes 1, 4, and 7, DNA from RT-PCRs of11-d.p.c. limb RNA; Lanes 3, 6, and 9, DNA from RT-PCRs of 17-d.p.c.limb RNA; Lanes 2, 5, and 8, control RT-PCRs with no added reversetranscriptase. E, Western blot analysis of MEF2 proteins present in 1 1 -

d.p.c. limbs (Lanes 1 and 4), 17-d.p.c. limbs (Lanes 2 and 5), and C2C12myotubes (Lanes 3 and 6); antibodies specific to MEF2A (MEF2A Ab) andMEF2D (MEF2D Ab) were used.


and DP3 allowed the amplification of a DNA fragment of

identical size in both embryonic (Lane 1) and fetal (Lane 3)

muscle; however, hybridization of probes specific to isoform

i a (Fig. 6D, probe A) or i b (orobe B) showed that the muscle-

specific isoform 1 b (27) is missing in embryonic muscle (Lane

4). Western blot experiments (Fig. 6E) indicated that MEF2A

and MEF2D proteins are present and have the same appar-

ent molecular weight in nuclear extracts of embryonic (Lanes

1 and 4) and fetal (Lanes 2 and 5) origin, as well as in

differentiated C2C12 nuclear extract (Lanes 3 and 6). RT-

PCR on MEF2B mRNA or Western blots on the correspond-

ing protein were negative in both embryonic and fetal muscle

(not shown). Finally, the polyclonal antibody to hMEF2A was

used in immunological experiments aimed at evaluating

whether proteins of the MEF2 family were localized in the

nuclei of embryonic and fetal muscle cells. Differentiated

myotubes derived in culture from embryonic and fetal myo-

blasts were double-stained by indirect immunofluorescence

with antibodies to sarcomeric MHC and MEF2A. Fig. 7

shows that all myosin-positive (i.e. , differentiated) muscle

cells from both embryonic (Fig. 7C) and fetal (Fig. 7D) cul-

tures contain nuclei that are stained by the anti-MEF2A an-

tibody (Fig. 7, A and B, respectively).

Protein Phosphatase Inhibitors Restore the Ability ofMEF2 Proteins to Bind Their Cognate Site and EnhanceMEF2-dependent Transcription in Embryonic Myotubes.Because MEF2 proteins appear to be present and correctly

localized in the nuclei of embryonic muscle cells, we ex-

plored the possibility that posttranslational modifications

might be responsible for the lack of binding to the MEF2 site.

As Fig. 8A shows, the presence of phosphatase inhibitors inthe buffers used for the preparation of embryonic muscle cell

nuclei leads to a nuclear extract capable of producing a

shifted complex related to MEF2 (Lane 7). Both sodium or-

thovanadate at high concentration (i 0 mM) and okadaic acid

(1 j.tg/ml, not shown) caused the same effect. In addition,

alkaline phosphatase treatment of embryonic muscle nuclear

extracts prepared in the presence of sodium orthovanadate

(Fig. 8B, Lane 4) or of fetal muscle nuclear extracts (Fig. 8C,

Lane 4) led to a loss of MEF2 binding activity. Interestingly,

the same MEF2 proteins that can be detected in the shifted

complex produced by C2Ci 2 myotube nuclear extract are

present in the corresponding complex produced by the em-

bryonic muscle nuclear extract. Specifically, as is demon-

strated by specific supershifting antibodies, MEF2A appears

to be the major species (Fig. BA, Lanes 3 and 8), MEF2D is

present in lower proportion (Lanes 5 and 10), whereas

MEF2B is not detected (Lanes 4 and 9). These results sug-gest that MEF2 proteins bind DNA preferentially as phos-

phoproteins and that phosphorylation might represent a

mechanism by which the activity of MEF2 is regulated.

Therefore, we were prompted to investigate MEF2 protein

phosphorylation in muscle cells. As Fig. 9 shows, treatment

of nuclear extracts with alkaline phosphatase reduces the

heterogeneity of polypeptides recognized by MEF2A-spe-

cific antibody in immunoblotting experiments (see Lanes 1, 3,

5, and 7 versus Lanes 2, 4, 6, and 8). Also, MEF2D appears

to be phosphorylated in vivo, although to a lesser extent

(data not shown). However, no obvious differences can be

Fig. 7. Double immunofluorescence of a 4-day-old culture from embryonic and fetal limbs stained with MF2O (C and D, respectively) and embryonic andfetal limbs stained with anti-MEF2A (A and B, respectively). Arrows indicate that the same embryonic or fetal myotube that shows positive staining with MF2Ois also positive with anti-MEF2A. Bar, 10 pm.


observed in the electrophoretic profile of phosphorylatedMEF2A in embryonic muscle nuclear extracts prepared in the

absence (Fig. 9, Lane 1) or in the presence (Fig. 9, Lanes 3

and 6) of protein phosphatase inhibitors. Finally, protein

phosphatase inhibitors and protein kinase activators were

assayed for the ability of activating MEF2-dependent tran-

scription and possibly the expression of the endogenous

MCK gene in embryonic myotubes. For this purpose, the

reporter plasmid pMCKCAT/MEF2x2 was transfected in pri-

mary cultures of embryonic myoblasts. After differentiation,

cell cultures were incubated with sodium orthovanadate at

low concentration, okadaic acid, TPA, or dibutyryl cyclic

AMP for 6 h prior to harvesting. As Fig. iOA shows, okadaic

acid treatment causes a small but significant (3-fold) increase

in reporter activity as compared to untreated myotubes. So-

dium orthovanadate at low concentration (inhibitory for tyro-

sine but not for serine/threonine phosphatases) is not effec-

tive. We could not incubate cells with sodium orthovanadate

at the high concentration required for restoring MEF-2 bind-

ing activity in embryonic nuclear extracts because it pro-

duces a clearly toxic effect even over a 6-h period. TPA is

equally ineffective, whereas dibutyryl cyclic AMP reduces

basal reporter activity. Okadaic acid and not TPA treatment

appears to activate the entire transcriptional program leading

to MCK expression, because RT-PCR analysis provides ev-

idence of MCK mRNA-specific sequences in differentiated

embryonic myotubes, treated for 6 h with the phosphatase

inhibitor prior to harvesting (Fig. i OB, Lane 1).

Discussion

This study investigated the basis for the stage-specific ex-

pression of the MCK gene during the development of mam-

malian skeletal muscle.

Transcription of the MCK Gene Occurs at a Very LowRate in Muscle Cells of Embryonic Origin. We have

shown that transcription of a reporter gene, under the control

of the MCK promoter and variable extensions of the 5’ flank-

ing sequence, is barely above background level in trans-

fected myotubes of embryonic origin. In contrast, differenti-

ated muscle cells offetal origin both contain MCK mRNA and

actively transcribe a reporter gene from the MCK promoter.

Because it is well established that transcriptional activity of

the MCK gene depends on the presence of a composite

enhancer located between bp -1256 and bp -1050, we

have investigated whether any difference can be revealed in

embryonic versus fetal myotubes, with respect to the trans-

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#{149}#{149} . . . . C


Fig. 8. EMSA analysis of MEF2 site binding proteinsprepared in the presence of phosphatase inhibitors. A,embryonic muscle nuclear extracts prepared either in theabsence (ME; Lane 12) or in the presence (ME+ Van;Lane 7) of 1 0 mM sodium orthovanadate were assayedfor ability to bind a 32P-labeled oligonucleotide repre-senting the MEF2 site located in the MCK enhancer.Nuclear extracts from C2C1 2 myotubes were assayed inparallel (C2; Lane 2). The identities of MEF2 proteinsparticipating in the shifted complex indicated by the ar-row were determined by using antibodies specificallydirected to MEF2A (MEF2A Ab; Lanes 3, 8, and 13),MEF2B (MEF2B Ab; Lanes 4, 9, and 14), and MEF2D(MEF2D Ab; Lanes 5, 10, and 15). In Lanes 6 and 1 1, allthree antibodies were added to the extracts; C, oligonu-cleotide probe with no added extract. B, embryonic mus-cle nuclear extracts prepared in the presence of sodiumorthovanadate were directly assayed in a gel-shift exper-iment (Lane 2) or after incubation for 90 mm at 37CC withno added alkaline phosphatase (A.P. ; Lane 3) or in thepresence of 40 units alkaline phosphatase (Lane 4). Lane1 , the internally labeled 32P-oligonucleotide representingthe MEF2 site located in the MCK enhancer with noadded extract. C, as in B, except that nuclear extractsprepared from fetal muscle were used.

-�.

1234

-�-

1234

acting factors capable of binding to defined elements of the

MCK enhancer.

MEF2 Binding Activity to the 3’ NT-rich Element of theMCK Enhancer Is Lacking in Nuclear Extracts of Embry-onic Myotubes, despite the Presence of MEF2 mRNAs

and Proteins. Members of the MEF2 family have been

shown to participate in a complex regulatory circuit involving

positive feedback loops that amplify the expression of HLH

myogenic determinants and stabilize the myogenic program

(23, 37-39). It is known, for instance, that the expression of

ME ME ME C2C12+ +

____ Van. Okad. ____A.P_+_#{247} + - +

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: -30

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Fig. 9. Phosphorylation of MEF2A. Fifty �g of nuclear proteins preparedfrom embryonic muscle as previously indicated (ME; Lanes 1 and 2) or inthe presence of sodium orthovanadate (ME+ Van; Lanes 3 and 4) orokadaic acid (ME+Okad; Lanes 5 and 6) and from C2C12 myotubes(C2C12; Lanes 7 and 8) were subjected to Western blot analysis withantibody specifically directed to MEF2A. Prior to loading, samples wereincubated at 30CC for 90 mm either in the absence (-) or in the presence(+) of 40 units of alkaline phosphatase (A.P.).

A 3.5#{149}

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.� 2.5

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B MEM

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Vanadate Okadaic acid IPA CAMP Control

MEM+

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Fig. 10. Effect of protein phosphatase inhibitors and and protein kinaseactivators on MEF2-dependent transcription. A, the reporter constructpMCKCAT/MEF2x2 was transfected in primary cultures of embryonicmyoblasts as described previously. Six h prior to harvesting, cultures wereincubated with 0.1 mM sodium orthovanadate, 1 �g/ml okadaic acid, 0.1�M WA, or 1 mM dibutiryl cAMP. CAT activities, normalized as previouslyindicated, are expressed relative to the activity of unincubated cell culture.B, RT-PCA analysis of MCK mRNA sequences present in total ANAextracted from primary cultures of embryonic myotubes treated withokadaic acid (Lanes 1 and 2), TPA (Lanes 3 and 4), or untreated (Lanes 5and 6), and from primary cultures of fetal myotubes (Lanes 7 and 8). Whereindicated (-), reverse transcriptase (R.T.) was omitted from reaction mixesto exclude amplification due to contaminating DNA. The position of mo-lecular weight markers is shown at right (expressed in number of basepairs).


myogenin in the correct spatiotemporal context requires

MEF2 and a basic HLH protein (most probably Myf-5, theonly myogenic basic HLH factor present in the embryo when

myogenin is activated; Ref. 40). Furthermore, transcription of

the minimal Xenopus MyoDa promoter in nonmuscle cells is

activated by expression of X. MEF2 and requires binding of

both MEF2 and TFIID to the TATA element (41). Finally,

inactivation of the MEF2 site, in genes that are expressed in

terminally differentiated muscle cells, such as MCK (13, 20-

22), cardiac myosin light chain 2 (28), and phosphoglyceratemutase (29), causes a dramatic reduction of transcription.

These data indicate that MEF2 is a major determinant inmuscle-specific transcription and suggest that modulation of

MEF2 activity might represent an efficient mechanism for

controlling the expression of muscle-specific genes. Indeed,

the potential for an exquisitely tuned regulation of MEF2

activity is provided by several observations: (a) multiple Mef2

genes and isoforms exist (24-27, 36); (b) the expression of

MEF2 mRNAs does not parallel the expression of MEF2proteins, suggesting that posttranscriptional mechanisms

regulate MEF2 accumulation (21 , 24); (c) MEF2 proteins bindas dimers (36), and MEF2D can form heterodimers with both

MEF2A and MEF2C (27); and (d) a common property of

MADS proteins is their ability to cooperate with other tran-scriptional regulators to control gene expression (42-45).

Our EMSA analysis has shown that the only relevant differ-

ence between embryonic and fetal muscle nuclear extracts

consists in the absence of MEF2 binding to its cognate site

in embryonic extracts. A detailed RT-PCR analysis of alter-

native exon sequences present in MEF2A, MEF2C and

MEF2D mRNAs has revealed that no major differences existbetween embryonic and fetal MEF2 species with the excep-

tion of the muscle-specific MEF2D isoform, which is missing

1�L J�

‘�i-3OO�-

-100

in the embryonic muscle. The physiological relevance of this

variation is difficult to assess, particularly in light of the

observation that all four MEF2D isoforms bind to the MCK

MEF2 site with similar affinity (27). Western blot and immu-nofluorescence experiments indicate that MEF2 proteins are

present, in comparable amounts, in both embryonic and fetal

muscle and that they are localized in the nuclei of myosin

positive cells. These findings indicate that a selective insta-

bility of MEF2 proteins is not likely to be the basis for the lack

of MEF2 binding activity in embryonic muscle.

Inhibition of Protein Phosphatases Correlates withTranscriptional Activity at the MCK Promoter. Most in-terestingly, we found that MEF2 binding activity can be de-

tected in embryonic muscle nuclear extracts when protein

phosphatase inhibitors, such as okadaic acid and vanadate,

were added to the tissue prior to purification of the nuclei.

The retarded complex generated by embryonic nuclear ex-

tracts obtained in the presence of phosphatase inhibitors

contains the same MEF2 proteins found in fetal and C2C12


nuclear extracts. Consistent with this finding is the observa-

tion that the addition of okadaic acid to embryonic myotubestransfected with pMCKCAT/MEF2x2 leads to a 3-fold in-crease of reporter activity. Along the same lines, and perhapsmore significantly, okadaic acid treatment appears to be

effective in activating the genetic program that leads to theappearance of MCK mRNA sequences even in embryonicmyotubes. It should be noted that although okadaic acid hasbeen reported to inhibit myogenesis and suppress the ex-pression of muscle-specific genes (46), embryonic cells weretreated with this molecule only after differentiation and for ashort period, only sufficient to alter protein phosphorylation.Collectively, these data suggest that phosphorylation mightrepresent a major control mechanism for MEF2 activity. In-deed, phosphatase treatment of muscle nuclear extractsprovided direct evidence for MEF2 phosphorylation, inagreement with recently published observations (47). How-ever, the demonstration that changes in phosphorylation are

responsible for the lack of MEF2 binding activity in embry-onic muscle will require a detailed mapping of phosphoryl-ated residues, particularly in light of the fact that no grossdifferences in the phosphorylation profile of MEF2 proteinswere observed in embryonic extracts treated with phospha-tase inhibitors or in nuclear extracts from C2C1 2 myotubes.At present, we can only speculate that phosphorylation ofspecific residues is functionally relevant, as has been re-

ported, for example, in the case of SRF (serum responsefactor), a protein that is structurally related to MEF2 (48, 49).

The actual control of the transition from an embryonic to afetal phenotype is likely to be very complex and possiblydiversified for each gene differentially expressed in embry-

onic and fetal muscle. In fact, although many different genesare expressed in fetal but not in embryonic muscle, eachshows some distinct features in the control of this differentialexpression. For example, the �-enoIase gene is already ex-pressed, albeit at low levels, in embryonic muscle, and itsexpression increases in the fetus (50, 51). In contrast, MCKexpression cannot be detected in the embryo. These differ-ences suggest that each gene has a distinct regulation oftissue-specific, as well as stage-specific, transcription, al-though it is possible that a basic common mechanism exists.

In conclusion, this report provides the first indication thatthe developmentally regulated phosphorylation of MEF2might be responsible for differential gene expression in em-

bryonic versus fetal muscle, although additional work will berequired to fully elucidate the molecular basis of this phe-nomenon.

Materials and MethodsCell Culture and Transfections. Primary cultures of embryonic and fetalmyoblasts were prepared from the limbs of 11-d.p.c. and 16-d.p.c. mouseembryos, respectively, as previously described (9). Twenty-four h afterplating, cells were transfected with plasmid DNAs by the calcium phos-phate coprecipitation method. Plasmids -2800pMCKCAT, -1256pMCK-

CAT, -776pMCKCAT, and -8OpMCKCAT (1 1) were a kind gift of S.Hauschka. pMCKCAT/MEF2x2 was obtained by inserting a double-stranded oligonucleotide representing two copies of the MEF2 site 10-cated in the mouse MCK enhancer into the Hindlll/SaIl sites of -8OpM-CKCAT. p1 F-nlacZ-E contains the IacZ gene with a nuclear locationsignal, driven by the mouse MLC1 F muscle-specific promoter (1 .2 kb) and

the mouse 3’ MLC1 F/3F enhancer sequence (34). Ten �ig of CAT reporter

plasmid DNA and 5 �g of p1 F-nlacZ-E DNA were cotransfected per

65-mm-diameter culture dish. Approximately 16 h after transfection, dif-

ferentiation was initiated by reducing the serum concentration to 1 % FCS.

Cells were harvested 64 h after transfection (or at the indicated timepoints). Where indicated, 0.1 m� sodium orthovanadate, 1 j�g/ml okadaic

acid, iO� M WA, or 1 mM dibutyryl cyclic AMP were added to transfectedmyotubes 6 h prior to harvesting. After resuspension in 0.1 ml of 0.25 M

Tris-HCI (pH 7.8), whole cell extracts were obtained by ultrasonic treat-ment followed by centrifugation. The amount of (3-gal activity in eachlysate was determined colorimetrically and used to normalize the amountof extract used for CAT assays.

RNA Analysis. Total ANA was extracted from cultured cells or fromthe limbs of 11- and 17-d.p.c. mouse embryos by the guanidinium/cesiumchloride method (52). Ten �g of total ANA were fractionated by electro-phoresis on denaturing agarose gels, transferred to nylon membranes,and hybridized as described previously (53). The 32P-labeled cDNA insertof plasmid pMCK-m3’-5P65 (54) was used as probe for MCK mANA.

The presence of alternative exons in MEF2 mANAs was investigated byAT-PCR. Three jzg of total ANA extracted as above were used in each

AT-PCA assay. The foflowing primers were used for amplification: APi,5’-MGAAAATACA&ATCACACGC-3’ (nt 10-30 in MEF2A�; AP2, 5’-ACAT-TGAGMGUCTGAGGTG-3’ (nt 421-401. in MEF2A); AP3, 5’-MTCAC-TATCUCAmAGTr-3’ (nt 370-350 in MEF2A); AP4, 5’-AGGCTCTGM-CMGM-3’ (nt 671-686 in MEFZA4; AP5, 5’- mATGATrCCGCATC-3’ (nt804-789 in MEF2A); CP1, 5’-AGTACACCGAGTACMCGAGC-3’ (nt 342-362 in MEF2C); CP2, 5’-GCCTGTGTTACCTGCACTTGG-3’ (nt 730-710 inMEF2C); CR3, 5’-CATGCCGCCATCTGCCCTCAG-3’ (nt 1186-1206 inMEF2C); CP4, 5’-CCC1TFCGTCCGGCGMGGTC-3’ (nt 1490-1470 inMEF2C); CP5, 5’-GCG1TCUATCCCACCTGGC-3’ (nt 906-925 in MEF2C);

CP6, 5’-CAGAGAGTACTCAGTACCATAT-3’ (nt 1090-1069 in MEF2C); DP2,5’-A�GGGATGATGTCACCAGGGA-3’ (nt 752-732 in MEF2D); DP3, 5-AT-GGGGAGGAAAAAGA1TCAG-3’ (nt 234-254 in MEF2D); DP4, 5’-GCMGC-CTGATCTGCGGGTCA-3’ (nt 1030-1961 in MEF2D); and DP5, 5’-CTrGGT-

G1TGCCACGG.4AACC-3’ (nt 1i86-1 166).The following oligonucleotides were used as probes for discriminating

muscle-specific exons in MEF2D: probe A, 5’-AAGTACCGGCGGGC-CAGTGAGGA-3’ (nt 579-601 ; detects non-muscle-specific isoform);probe B, 5’-CCCCGAGGTGGACGAGGCGmGC-3’ (nt 524-547; de-

tects the muscle-specific isoform); and probe C, 5’-lTCAGCAATCCAAG-TAGCTCT-3’ (nt 699-719; detects both isoforms).

The following primers were used for amplification of MCK mANA-specific sequences (from nt 1 179 to nt 1393; Aef. 55): MCKP1 , 5’-GGC-CAGTCCATCGACGACATGATC-3’; and MCKP2, 5’-TAAGGCAGGGCAT-

GGAGAAGCAGC-3’.

EMSAS. Nuclear extracts were prepared from cultured cells and mus-cle tissue as described previously (56). Where indicated, 10 m� sodiumorthovanadate or 1 �g/ml okadaic acid were included in the buffer used

for the preparation of nuclei. Dephosphorylated nuclear extracts wereprepared by incubating 100 �g of extract proteln with 40 units of calfintestine alkaline phosphatase (Promega) for 90 mm at 37#{176}Cin 30 pi of 50mM Tris-HCI, pH 8.5, 10 mM MgCI2, 0.1 mp,i ZnCl2. The sequences of thesense strands of the oligonucleotides used as probes and competitorswere as follows: MCK E-box, 5’-mAACCCAGACATGTGGCTGCCCC-3’; MCK MEF2 site, 5’-AAGCTCGCTCTAAAAATAACCCTGTCCCTGGT-

3’; MCK MEF2 mut6, 5’-AAGCTCGCTCTAMCATAACCCTGTC-CCTGGT-3’; MCK MHox site, 5’-GGTrATM1TMCCCAG-3’; MCKCArG-box, 5’-GGCTGCCCATGTAAGGAGGCA-3’; and MCK AP2 site,5’-GGC�GGCCTGGGGACACCCGAGATGCC-3’. Double-stranded oh-gonucleotides were purified and 32P-labeled by T4 polynucleotide kinase(specific radioactivity, 1-3 x 108 cpm/mg) as described (56). For bandshift experiments in which alkaline phosphatase-treated nuclear extracts

were used, oligonucleotide probes with 5’ overhangs were labeled by endfilling with Klenow enzyme. Nuclear extracts (10 �g protein) were incu-bated with 5 �z9 poly(deoxyinosinic-deoxycytidylic acid) (Pharmacia) for20 mm at room temperature in 10 m� Tris-HCI (pH 7.5), 50 m�.i KCI, 5 m�i

MgCI2, 1 mM Dli, 1 mM EDTA, and 15% glycerol. 32P-labeled oligonu-cleotide was then added (0.5.-i ng) and incubation was prolonged for 30mm (final volume, 20 p1). In vitro translated MEF2A was obtained using theTnT rabbit reticulocyte lysate system (Promega) with the MEF2A cDNA

plasmid construct pGMEF2-X/R (24). Two �l of lysate were used in theEMSA assay, either alone or in combination with the indicated amount ofnuclear extracts. Where indicated, specific antibodies or competing oh-


gonucleotides (1 00-fold molar excess) were added to the incubationmixture 1 0 mm before the addition of the probe. Polyclonal antibodies toMyoD (57), myogenin (58), MEF2 (24, 59), MHox (60), and ASAF (61) wereused at a final dilution of 1 :20. Electrophoresis of DNA/protein complexesand autoradiography were as described previously (56).

hmmunocytochemhstry and Westrn BlOb. The following antibodieswere used: (a) MF2O, a monoclonal antibody that recognizes all sarcom-eric myosins (62); (b) a rabbit polyclonal antibody to MEF2 (24); and (c)rabbit polyclonal antibodies specific to MEF2A (partially cross-reactive toMEF2C), MEF2B, and MEF2D (59). Cells were fixed and processed forimmunocytochemistry as described previously (58). The same nuclear

extracts used in EMSA were subjected to Western blot analysis (50 �gprotein/sample; Aef. 63) with anti-MEF2 antisera (final dilution, 1:1000).Secondary antibody conjugated to horseradish peroxidase (Pierce) was

added (final dilution, 1:3000), and the Immunoperoxidase reaction wasvisualized by the chemiluminescence method (Boehringer Mannheim).

AcknowledgmentsWe thank S. Hauschka and V. Mahdavi for providing the MCK/CAT plas-mid constructs and the MEF2 cDNA clone pGMEF2-XIR, respectively. Weare indebted to the following scientistsfor making avallablethe antibodiesto the various DNA-binding proteins examined: S. Ahem#{224}(MyoD), V.Mahdavi (MEF2), E. Olson (MHox), W. Wright (myogenmn), T. Mohun(RSAF), and A. Prywes (MEF2A, B, and D). We are also indebted to D.Fishman, who provided the antibody to sarcometic myosin. We thank E.Olson for providing unpublished mouse MEF2A cDNA sequence and M.Buckingham and M.S. Moruzzi for critically reading the manuscript andproviding helpful suggestions.

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absence ofmef2bindingtothent-rich element inthe muscle...

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