MOLECULAR AND CELLULAR BIOLOGY, Sept. 1993, p. 5133-51400270-7306/93/095133-08$02.00/0Copyright C 1993, American Society for Microbiology

An E Box Mediates Activation and Repression of the

Vol. 13, No. 9

Acetylcholine Receptor b-Subunit Gene during MyogenesisALEXANDER M. SIMON AND STEVEN J. BURDEN*

Biology Department, 16-820, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 22 February 1993/Returned for modification 12 April 1993/Accepted 26 May 1993

The genes encoding the skeletal muscle acetylcholine receptor (AChR) are induced during muscledevelopment and are regulated subsequently by innervation. Because both the initiation and the subsequentregulation of AChR expression are controlled by transcriptional mechanisms, an understanding of the stepsthat regulate AChR expression following innervation is likely to require knowledge of the pathway thatactivates AChR genes during myogenesis. Thus, we sought to identify the cis-acting sequences that regulateexpression of the AChR B-subunit gene during muscle differentiation. We transfected muscle and nonmusclecell lines with gene fusions between 5'-flanking DNA from the AChR 8-subunit gene and the human growthhormone gene, and we show here that 148 bp of 5'-flanking DNA from the AChR 8-subunit gene contains tworegulatory elements that control muscle-specific gene expression. One element is an E box, which is importantboth for activation of the B-subunit gene in myotubes and for its repression in myoblasts and nonmuscle cells.Mutation of this E box, which prevents binding of MyoD-E2A and myogenin-E2A heterodimers, decreasesexpression in myotubes and increases expression in myoblasts and nonmuscle cells. An E-box binding activity,which does not contain MyoD, myogenin, or E2A proteins, is present in muscle and nonmuscle cells and maybe responsible for repressing the B-subunit gene in myoblasts and nonmuscle cells. An enhancer, which lacksE boxes, is also required for expression of the B-subunit gene but does not confer muscle-specific expression.

The skeletal muscle acetylcholine receptor (AChR) is amultisubunit, ligand-gated ion channel which is the neuro-transmitter receptor at all vertebrate neuromuscular junc-tions. The four different subunits (a, 1, -y, and b) are encodedby separate genes which are activated coordinately duringmuscle development (26). Transcription of these genes,unlike that of most other muscle genes, is regulated subse-quently by two innervation-dependent pathways. One path-way induces local transcription of AChR genes in myofibernuclei near the synaptic site (23, 39, 43), while the otherpathway inactivates AChR expression in nuclei throughoutthe myofiber (28, 43, 48). This pattern of AChR geneexpression is controlled by a signal in the synaptic basallamina that induces synapse-specific transcription (10, 20)and by signals associated with myofiber depolarization thatrepress AChR transcription throughout the myofiber (28, 43,48).

Little is known about the signalling pathways involved inneural regulation of AChR gene expression. The AChRsubunit genes are first expressed during myogenesis, whenmyoblasts withdraw from the cell cycle and fuse into multi-nucleated myofibers (11). Subsequently, these genes areregulated by innervation. Thus, an understanding of themechanisms that regulate AChR expression following inner-vation may require knowledge of the steps required toactivate AChR genes during myogenesis. In particular, cis-acting elements that are important for regulation duringmyogenesis may also be targets for regulation by innerva-tion. Therefore, we sought to identify the cis-acting se-quences that control AChR expression during myogenesisand to determine how these elements confer muscle-specificexpression.We analyzed the 5'-flanking DNA of the AChR b-subunit

gene to identify the cis-acting sequences that control muscle-

* Corresponding author.

specific expression. We show here that 148 bp of 5'-flankingDNA from the AChR b-subunit gene contains two regulatoryelements that control muscle-specific gene expression. Oneelement is an E box that is important both for activation ofthe 8-subunit gene in myotubes and for its repression inmyoblasts and nonmuscle cells. The other element is anenhancer that is required for expression of the b-subunitgene in muscle but does not confer muscle specificity, sinceit can activate a heterologous promoter in both muscle andnonmuscle cells.

MATERIALS AND METHODS

Plasmids. The 8(-840/+25)-CAT, b(-148/+25)-CAT,b(- 128/+25)-CAT, and b(- 107/+25)-CAT plasmids (3) con-tain 5'-flanking DNA from the AChR b-subunit gene fused tothe chloramphenicol acetyltransferase (CAT) gene. Pointmutations and internal deletions of the 8-subunit gene wereprepared by oligonucleotide-directed mutagenesis (47). AnEcoRI fragment containing b-subunit sequences extendingfrom nucleotides -840 to +25 and including a portion of theCAT gene was excised from the b(-840/+25)-CAT plasmidand ligated into the EcoRI site of M13mpl8. E boxes weremutated with oligonucleotides containing specific mis-matches or limited random mismatches, and deletion muta-tions were introduced with oligonucleotides which hybridizeto 15 bases on each side of the region targeted for deletion.The mutagenized EcoRI inserts were sequenced to verifythat the intended mutations were made and to determine thatadditional, unintended mutations had not occurred. Frag-ments containing the desired mutations were then religatedinto the parent vector.Mutated b-subunit sequences were transferred from the

CAT expression plasmid into a human growth hormone(hGH) expression vector (pOhGH) (41). A SacI-PstI frag-ment containing b-subunit sequences extending from -840to +25 and including polylinker sequences at its 3' end was

5133

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

5134 SIMON AND BURDEN

isolated from the CAT plasmid; protruding ends were re-moved, and the fragment was ligated into the HinclI site ofpOhGH. Plasmids containing 1,823 bp of 5'-flanking DNAfrom the b-subunit gene were constructed by ligating aHindIII-KpnI fragment from b(-1823/+25)-hGH (43) intoHindIII-ApnI-digested, mutated b(-840/+25)-hGH plas-mids.The b(-148/-53)-FBP-CAT and b(-53/-148)-FBP-CAT

plasmids were constructed by ligating a blunt-ended EcoRI-HaeIII fragment from 8(-148/+25)-CAT into a filled-in SalIsite of the c-fos basal promoter-(FBP)-CAT gene (6, 14). Theb(-148/-95)-FBP-CAT and 8(-95/-148)-FBP-CAT plas-mids were constructed by ligating oligodeoxynucleotidescontaining the appropriate b-subunit sequences and SalIends into the SalI site of FBP-CAT. The b(-94/-53)-FBP-CAT and b(-53/-94)-FBP-CAT plasmids were constructedby ligating a HaeIII-HphI fragment containing nucleotides-94 to -53 from the b-subunit gene into a filled-in Sall siteof FBP-CAT. Plasmid constructions were confirmed bydouble-strand-DNA sequencing. Plasmid DNA used intransfection experiments was purified with a Qiagen columnand ultracentrifuged through CsCl.

Cell culture and transfections. C2C12 cells (C2 cells) weregrown in Dulbecco's modified Eagle's medium supple-mented with 15% fetal calf serum and 50 ,ug of gentamicinper ml. NIH 3T3 cells were grown in Dulbecco's modifiedEagle's medium supplemented with 10% fetal calf serum and50 ,ug of penicillin and streptomycin per ml. To study geneexpression in myotubes, C2 cells were plated at -2.5 x 105per 60-mm-diameter dish. To study expression in myoblasts,C2 cells were plated at a lower density (-6.5 x 104 per60-mm-diameter dish) so that myoblast fusion would beminimized. 3T3 cells were plated at a density of -1.0 x 105per 60-mm-diameter dish. Cells were transfected by calciumphosphate precipitation (50): C2 cells were incubated withprecipitates overnight and glycerol shocked for 3 min,whereas 3T3 cells were incubated with precipitates for 6 hand glycerol shocked for 30 s. C2 myoblasts were eitherreturned to growth medium and assayed 36 to 48 h afterglycerol shock or maintained in growth medium until thecells became confluent, transferred to differentiation me-dium (Dulbecco's modified Eagle's medium supplementedwith 5% horse serum) to induce myotube formation, andassayed 4 to 5 days after glycerol shock. 3T3 cells wereassayed -48 h after glycerol shock. Duplicate dishes wereincluded in each experiment.To control for variability in transfection efficiency, cells

transfected with b-CAT or b-FBP-CAT plasmids werecotransfected with pXhGH5 DNA (41), which contains hGHunder the control of the metallothionein promoter, and CATactivity was normalized to the amount of secreted hGH (3,13). Similarly, cells transfected with b-hGH plasmids wetecotransfected with the pSV2CAT plasmid (16) and secretedhGH levels were normalized to the amount of CAT activity.To compare expression of the b-subunit gene in myotubesand myoblasts, we measured the level of CAT activity inmyotubes and myoblasts transfected with pSV2CAT or withb(-1823/+25)-CAT and normalized b-subunit expression topSV2 expression.

Mutations in E box 1 result in elevated expression ofb-CAT genes in myoblasts. Because CAT activity is rela-tively stable and CAT synthesized in myoblasts persists inthe cytoplasm of myotubes during muscle differentiation(42), we used b-hGH plasmids to determine the effect of theE-box 1 mutations on gene expression in myotubes. BecausehGH is secreted into the culture medium, which was

changed daily, hGH does not persist during muscle differen-tiation, and so the amount of secreted hGH more accuratelyreflects the rate of transcription.

Antibodies. Antisera to MyoD (46) and E2A proteins (30)have been described previously. Antibodies to E2A reactwith the carboxy-terminal 430 amino acids of E12 andcross-react with E47, E2-5(ITF-1), and E2-2(ITF-2) (29, 30).Hybridoma supernatant containing a monoclonal antibody tomyogenin (51) was concentrated 15-fold in an Amicon-100microconcentrator.

Gel shift experiments. 32P-labelled DNA probes were pu-rified on polyacrylamide gels. Nuclear extracts were pre-pared by the method of Dignam et al. (12) as modified byAbmayr and Workman (1). Nuclear extracts (3 to 7 ,g ofprotein) were preincubated in 20 ,ul of binding buffer [25 mMHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) (pH 7.5), 50 mM KCl, 12.5 ,uM ZnSO4, 5% glycerol,0.1% Nonidet P-40, 1 mg of bovine serum albumin per ml,0.5 mM dithiothreitol, 50 ,ug of poly(dI-dC) per ml] for 10min, incubated with labelled E-box probes (-30,000 cpm;0.1 to 0.5 ng) for 10 min at room temperature, and placed onice for 5 min, and the complexes were resolved by electro-phoresis (4 to 5 h at 200 V) at 4°C in a 5% polyacrylamide gel(0.5x Tris-borate-EDTA). Antibodies to basic helix-loop-helix (bHLH) proteins were used to identify the proteinspresent in protein-DNA complexes. Antibodies (1 to 2 RI ofserum or 0.5 ,ul of hybridoma supernatant that had beenconcentrated 15-fold) were incubated with nuclear extractsfor 10 min at room temperature before the addition oflabelled probe.Gel shift assays with the enhancer probe contained nu-

clear extracts (4 ,ug of protein) and labelled probe from -94to -53 (-3,000 cpm; 0.1 ng) in 20 ,ul of binding buffer [10mM Tris (pH 7.4), 40 mM KCl, 2 mM MgCl2, 1 mM EDTA,8% glycerol, 0.5 mM dithiothreitol, 50 ,g of poly(dI-dC) perml]. The reactions were allowed to proceed for 10 min atroom temperature, and complexes were resolved by electro-phoresis (2 h at 150 V) in a 5% polyacrylamide gel (0.5 xTris-borate-EDTA). The specificity of this binding reactionwas determined by addition of unlabelled competitor DNAto the reaction mixture. Gels were fixed, dried, and exposedto X-ray film.

RESULTS

Positive and negative regulation at an E box. A 148-bpfragment of b-subunit 5'-flanking DNA is sufficient to confermuscle-specific expression in cell transfection experiments(3). This region contains a single binding site for bHLHproteins between nucleotides -24 and -15 (Fig. 1, El) (3).Because E boxes in some but not all muscle-specific genesare important for their activation in myotubes, we intro-duced point mutations into El of the b-subunit gene todetermine the role of this E box in b-subunit gene expres-sion.We transfected C2 myoblasts with b-hGH gene fusions,

induced myotube differentiation, and measured the amountof hGH secreted into the medium. Mutation of El reduceshGH expression in myotubes (3- to 25-fold), suggesting thatthis site is important for activation of the b-subunit geneduring myogenesis (Fig. 2). Two additional E boxes, E2(nucleotides -270 to -261) and E3 (nucleotides -368 to-359), are present in the b-subunit 5'-flanking DNA (Fig. 1);however, mutations of E2 and E3, individually or together,have little or no effect on expression (Fig. 3). These resultssuggest that the position and/or sequence context of El

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

E-BOX-MEDIATED REPRESSION 5135

region sufficient formuscle-specific expression

-350 -300 -250 -200 -150 -100 -50 -25

TACAGGTGGAE3

/\GACAGGTGTA

E2

IL[I mm IIIAP-2 SpiCBF40

AGCACCTGTCEl

FIG. 1. The 5'-flanking DNA of the AChR b-subunit gene con-tains an E box near the transcription start site and an enhancer thatlacks E boxes. Three binding sites for bHLH proteins, El (nucle-otides -24 to -15), E2 (-270 to -261), and E3 (-368 to -359), arepresent in the 5'-flanking DNA of the AChR b-subunit gene. Thecore sequences of these E boxes (underlined) match the consensusbinding site for myogenic bHLH-E2A complexes (8). E2 and E3 arein the orientation opposite that of El. An additional E box (notshown) is centered at nucleotide -275, but this site does not fit theconsensus sequence and is not detected in binding site selectionassays (8, 45, 51). The positions of an initiator element (I), apotential TATA element (T), and potential binding sites for AP-2,CBF40, and Spl are also indicated. The arrow indicates the direc-tion of transcription from the initiation site.

ADelta -18231+25a-a-aa.a. a; .s....s.. [ hGH

E3 E2 El

Delta -18231+25;;.m.*.asSsssla hGH

mutation 1

Delta -18231+25177-77'77 .: sl t.::::'>*.;II* :sB ; hGH

mutation 2

Delta - 18231+251B:: ES."." hGH

mutation 3

Defta - 18231+25-::s:SsB.s::: e E:B1 I ;-hGH

mutatfon 4

Defta - 18231+25hGH

mutation 5

El MYOTUBESEQUENCE EXPRESSION

GCACCTGTC 100

GAACCTIrC 28 ± 9 (5)

GCACGGTAA 11 ±3 (6)

G.QACCTGTC 4.2 ±0.9 (3)

GCAACTGTC 36±17 (3)

GCACCAGTC 4.5 ±1.8 (3)

distinguishes it from E2 and E3 as a site for activation inmyotubes.

Surprisingly, mutation of El elevates gene expression inmyoblasts (5- to 14-fold) and fibroblasts (3- to 7-fold),indicating that El is important for repressing gene expres-sion in these cell types (Fig. 2). Because several mutationshave the same effect, it is unlikely that a site for geneactivation in myoblasts and fibroblasts was created inadvert-ently. Mutations of E2 and E3 have little or no effect onexpression in myoblasts (Fig. 3). These results suggest thatthe position and/or sequence context of El also distinguishesit from E2 and E3 as a site for repression. These results showthat El has a dual role, since it is a site for activation inmyotubes and a site for repression in nonmuscle cells. El istherefore a critical element that controls muscle-specificexpression of the b-subunit gene in muscle cells grown in cellculture.

Nuclear extracts from myotubes and myoblasts containmultiple E-box binding activities. Our transfection experi-ments show that El is a site for activation in myotubes. Toidentify El binding activities in myotubes, we performed gelshift experiments with myotube nuclear extracts and an Elprobe. Myotube nuclear extracts contain three El bindingactivities (Fig. 4 and 5). The binding of these activities isspecific, since point mutations in El eliminate or greatlyreduce binding (Fig. 4).

Candidates for the activators that bind to El includeheterodimeric complexes of myogenic and E2A bHLH pro-teins. These complexes have been shown to activate expres-sion of muscle-specific genes by binding to E boxes presentin their cis-acting regulatory regions (49). Antibodies toMyoD disrupt the formation of complex 1, and a monoclonalantibody to myogenin supershifts complex 2 (Fig. 5). Bothcomplex 1 and complex 2 are disrupted by antibodies to E2Aproteins (Fig. 5). These results indicate that complex 1contains MyoD-E2A heterodimers and that complex 2 con-tains myogenin-E2A heterodimers. Since MyoD-E2A andmyogenin-E2A heterodimers can activate the expression ofother genes by binding to E boxes, one or both of theseheterodimeric complexes are likely to bind El in vivo andactivate 8-subunit expression in myotubes. A third activity,which is also present in myoblasts and nonmuscle cells (seebelow), forms a complex with El (Fig. 5, complex 3) that is

B EXPRESSIONEl SITE MYOBLASTS

wild-type 1.0

mutation 1 14±4 (4)

-mutation 2 8.0 ±1.9 (4)

mutation 3 4.5 ±1.0 (3)

mutation 4 10 ±1 (3)

FIBROBLASTS

1.0

6.7±2.2 (2)

2.5 ± 0.2 (2)

2.8 ± 0.3 (2)

3.4 ±0.3 (2)

mutation 5 7.1 ± 0.4 (3) | 2.5 ±0.01 (2)

FIG. 2. E box 1 regulates expression in myotubes, myoblasts,and fibroblasts. (A) C2 cells were cotransfected with pSV2CAT andb-hGH gene fusions that contain either the wild-type (open box) ora mutated (solid box) El site. Expression (hGH-CAT activity) inmyotubes transfected with b(-1823/+25)-hGH was assigned a valueof 100, and all other values are expressed relative to this. For eachEl sequence, the altered nucleotides are underlined. (B) C2 myo-blasts and 3T3 fibroblasts were cotransfected with pXhGH5 andb(-840/+25)-hGH gene fusions that contain either the wild-type ora mutated El site. Expression (CAT-hGH activity) from the wild-type b-hGH plasmid was assigned a value of 1.0, and all other valuesare expressed relative to this. The means ± the standard errors ofthe mean are given, and the number of repetitions for each experi-ment is shown in parentheses. The level of CAT expression is-20-fold greater in myotubes than in myoblasts transfected withb(-1823/+25)-CAT (see Materials and Methods).

not affected by any of the antibodies, indicating that thisactivity does not contain MyoD, myogenin, or E2A proteins.Our experiments suggest that heterodimeric complexes of

myogenic and E2A bHLH proteins activate expression inmyotubes by binding to El. The identity of a factor thatcould repress expression in myoblasts and fibroblasts is lessclear. E-box binding activities, which have not been wellcharacterized, have been detected in myoblasts and nonmus-cle cells (9, 19, 25, 31). To identify potential repressoractivities, we performed gel shift experiments with myoblastnuclear extracts and the El probe. Myoblast extracts con-tain two activities that comigrate with myotube complexes 1

VOL. 13, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

5136 SIMON AND BURDEN

A

Delta - 18231+25

E3 E2 El

Delta - 18231+25

mutation 6

Delta -18231+25

mutation 7

Delta - 18231+25.. uon.......8

mutation 8

E3 E2 MYOTUBESEQUENCE SEQUENCE EXPRESSION

IhGH ACAGGTGT ACAGGTGG

IhGH ACIGGTGT ACAGGTGG

IhGH ACAGGTGT ACIGGTGG

IhGH ACIGGTGT ACIGGTGG

100

El ~ .O~~.-~probe: .

A

104 ±5 (3)

3 -- -

72 ±7 (3)

B94 ±9 (3)

1 _ 1O

3 -_- .--

MYOBLASTE2/E3 SITES EXPRESSION

wild-type 1.00

mutation 6 0.74 ±0.05 (3)

mutation 7 0.97 ±0.06 (3)

mutation 8 1.37 ±0.19 (3)

FIG. 3. E box 2 and E box 3 do not regulate b-subunit expres-sion. (A) C2 cells were transfected with 8-hGH gene fusions thatcontain wild-type (open box) or mutated (solid box) E boxes, andexpression in myotubes was determined as described for Fig. 2.Nucleotides which differ from the wild-type sequence are under-lined. (B) C2 cells were transfected with 8-hGH gene fusions thatcontain wild-type or mutated E boxes, and expression in myoblastswas determined as described for Fig. 2. The means ± the standarderrors of the mean are given, and the number of repetitions of eachexperiment is shown in parentheses.

and 3 (Fig. 4). The binding of these activities is specific,since point mutations in El eliminate or reduce bindingseverely (Fig. 4). Complex 3, but not complex 1, is alsodetected in nuclear extracts from 3T3 and HeLa cells (42).Complex 1, as expected given our results with myotubeextracts, reacts with antibodies to MyoD and E2A proteins(Fig. 5), indicating that this complex contains MyoD-E2Aheterodimers. Complex 3 is not affected by any of theantibodies tested, suggesting that this complex does notcontain MyoD, myogenin, or E2A proteins (Fig. 5).The protein(s) contained in complex 3 is a good candidate

for an activity that could repress b-subunit expression inmyoblasts and fibroblasts, since mutations in El prevent theformation of complex 3 and result in elevated expression inmyoblasts and fibroblasts (Fig. 2 and 4). Although MyoD-E2A heterodimers, which we detect in myoblast nuclearextracts, could conceivably function as a repressor in myo-blasts, MyoD is not present in 3T3 cells, yet repression ofthe b-subunit gene in 3T3 cells is mediated by El. Thus, wefavor the idea that the protein(s) in complex 3 repressesb-subunit expression in myoblasts and 3T3 cells. Althoughcomplex 3 could contain a bHLH protein, it does not reactwith antibodies to E2A proteins; thus, the protein(s) incomplex 3 must be distinct from E12, E47, E2-5(ITF-1) andE2-2(ITF-2). Alternatively, complex 3 may contain a DNA-binding protein that is distinct from bHLH proteins.

FIG. 4. Myotubes and myoblasts contain multiple E-box 1 bind-ing activities. Gel shift analysis was performed with nuclear extractsprepared from C2 myotubes (A) and C2 myoblasts (B). Each probeextends from nucleotides -25 to -14 of the b-subunit gene andcontains either the wild-type or a mutated El site (Fig. 2). Threecomplexes (arrows) were detected with myotube nuclear extracts,and two complexes (arrows) that comigrate with myotube com-plexes 1 and 3 were detected with myoblast nuclear extracts. Thepresence of an additional, weaker complex, which migrates more

rapidly than complex 3, was variable. No binding with E-boxmutations 1, 2, 3, and 5 was observed; mutation 4, however,retained -10% of complexes 2 and 3.

Nucleotides -148 to -53 are necessary for expression inmyotubes and contain an enhancer that is active in muscle andnonmuscle cells. Deletion analysis shows that El is not theonly element in the 8-subunit promoter that is critical forexpression in myotubes. Important sequences are presentthroughout the region extending from nucleotides -148through -53: deletion of nucleotides -148 to -108 reducesexpression -50-fold, deletion of nucleotides -95 to -68reduces myotube expression -100-fold, and deletion ofnucleotides -67 to -53 reduces myotube expression -600-fold (Fig. 6). We determined whether this region had en-hancer activity by testing whether the sequence from -148to -53 could stimulate expression of the FBP. In eitherorientation, the sequence from -148 to -53 enhances ex-pression (-25-fold) of the FBP in myotubes (Fig. 7). Thesequence from -148 to -53, however, is not a muscle-specific enhancer, since it also enhances expression of theFBP (5- to 15-fold) in myoblasts and fibroblasts (Fig. 7).Thus, this enhancer is not sufficient to confer muscle-specificexpression on a heterologous promoter. Reduced enhanceractivity is present between nucleotides -94 and -53,whereas no enhancer activity is present between nucleotides-148 and -95 (Fig. 7).To determine whether myotubes, myoblasts, or fibroblasts

contain activities that bind to the enhancer, we performedgel shift experiments with nuclear extracts from these cells.Each cell type contains an activity with identical electro-phoretic mobility that binds to nucleotides -94 to -53 (Fig.8). This DNA-protein complex results from sequence-spe-cific interactions, since binding is inhibited by the unlabelled

B

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

E-BOX-MEDIATED REPRESSION 5137

-150 -100 -75 -50,,Delta -8401+25

Delta -1481+25 ..........

Delta -1071+25

Delta A(-95/-81)

Delta A(-95/-68)

MYOTUBEEXPRESSION

ICAT 100

85 ±1 (2)

1.7 ±0.2 (2)

51 ±5 (3)

0.82 ±0.39 (3)

N . _

B

FIG. 5. Binding activities 1 and 2 contain heterodimers of myo-genic bHLH proteins and E2A proteins. Gel shift analysis was

performed with C2 myotube (A) and C2 myoblast (B) nuclearextracts and an El probe. Binding reactions were performed in theabsence of antibodies (No ab) or after preincubation of nuclearextracts with antibodies to MyoD, myogenin, or E2A proteins. Amonoclonal antibody to vinculin was used to control for the speci-ficity of the monoclonal antibody to myogenin, and preimmune sera(PreImm) were used to control for the specificity of the MyoD andE2A antisera. (A) Complex 1 is disrupted by antibodies to MyoDand E2A, and complex 2 is supershifted by antibodies to myogeninand E2A. Complex 3 is not affected by antibodies to MyoD,myogenin, or E2A (the decreased amount of complex 3 caused byantibodies to myogenin and E2A was not observed in other exper-iments). Arrowheads indicate supershifted complexes resulting fromspecific interactions between antibodies and complexes 1 and 2. (B)Complex 1 is disrupted by antibodies to MyoD and E2A, whereascomplex 3 is not affected by antibodies to MyoD, myogenin, orE2A. The arrowhead indicates the supershifted complex resultingfrom specific interaction between antibodies and complex 1. TheMyoD-E2A complex that we observe in myoblast nuclear extractsindicates incomplete titration by Id in our nuclear extracts.

DNA sequence from -94 to -53, but not by the sequencefrom -148 to -95 (Fig. 8). Although binding is observedwith a nucleotide -148 to -95 probe, this binding wasconsidered nonspecific, since it is not inhibited by theunlabelled DNA sequence from -148 to -95 (42). Thepresence of an apparently identical enhancer-binding activ-ity that is similarly abundant in myotubes, myoblasts, andfibroblasts is consistent with our expression data whichshow that the enhancer is active in each cell type when fusedto a heterologous promoter.

DISCUSSION

Our experiments show that muscle-specific expression ofthe b-subunit gene is controlled by two elements that arelocated within 148 bp immediately upstream of the transcrip-tion start site. One element is an E box that is located nearthe transcription initiation site, and the other element is anenhancer located between nucleotides -148 and -53. The Ebox has a dual role: it is important for activation of expres-

Delta A(-95/-53) 1 :..:.....:.I...1 ....

Delta A(-67/-53) 2-E.

0.28 ±0.08 (3)

0.17 ±0.08 (4)

FIG. 6. The nucleotide sequence from -148 to -53 is importantfor myotube expression. C2 cells were cotransfected with b-CATand pXhGH5 plasmids, and CAT and hGH activities were measuredfollowing myotube differentiation. Expression (CAT-hGH activity)from C2 cells transfected with b(-840/+25)-CAT was assigned a

value of 100, and all other values are expressed relative to this.Constructs containing internal deletions have nucleotide -840 astheir 5' end. The position of El is indicated by an open box. Themeans + the standard errors of the means are given, and the numberof repetitions of each experiment is shown in parentheses.

sion in myotubes and for repression in myoblasts andnonmuscle cells. Thus, this site acts as a critical cell speci-ficity element that restricts b-subunit gene expression tomyotubes. The enhancer is required for expression in myo-tubes, but it does not confer muscle specificity, since it canactivate expression of a heterologous promoter in myoblastsand fibroblasts as well as in myotubes.The experiments described here indicate that positive and

negative regulations of the b-subunit gene act through thesame site (Fig. 9). The mechanisms for activation andrepression are not clear, but distinct activators and repres-sors may share a common DNA recognition site, as has beenobserved for homeodomain proteins (18, 32) and steroidhormone receptors (2, 33). This model predicts that therelative concentrations of activator and repressor woulddetermine the transcriptional state of the gene.An E box (,uE5 site) in the immunoglobulin heavy-chain

enhancer, like El in the AChR 8-subunit, is important forboth activation and repression: the ,uE5 site is involved inrepression in non-B cells and in activation in B cells (38).Thus, an E box can be a target for both positive and negativeregulators in cell types other than skeletal muscle cells. Theidentity of the factors that mediate negative regulation,however, remains unclear.

In myoblasts, repression through El may be required tosupplement negative regulation mediated by Id. AlthoughMyoD and E2A proteins are expressed in myoblasts (46), Id,an HLH protein that lacks a basic region, can preventfunctional interaction of MyoD and E2A in myoblasts bybinding to E2A proteins (5, 19). The efficiency of Id-medi-ated repression in vivo, however, is not known. Thus, arepressor that binds El in myoblasts may attenuate the effectof low levels of MyoD-E2A heterodimers.

Mutations of E2 and E3 have little or no effect onb-subunit expression in myotubes or myoblasts, indicatingthat these sites are not required for either activation or

repression. Furthermore, these E boxes do not substitute forEl, since the effect of mutations in El is evident even whenE2 and E3 are present. One explanation for these results isthat the context of these E boxes may be important. Al-

Antibody:

A >

1 -

2-3-

VOL. 13, 1993

.4

lp R -_le,.p e~. . .. - JIM r-o-, \-.. ',%w PM4

90-

-0-

-0-

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

5138 SIMON AND BURDEN

EXPRESSION

l- l CATFBP

Delta -1481-53 \ \

Delta -531-148 v//Vm 3

Delta -941-53 _''

Delta -531-94 U/ |

Delta -1481-95 I

Delta -951-148

MT

1.0

26±0.2 (2)

23 ±2 (3)

7.7 +2.0 (2)

5.6 ±1.6 (2)

0.80 ±0.19 (3)

1.2 ±.06 (2)

MB

1.0

15±3 (3)

11 ±4 (3)

4.0 ±1.3 (3)

5.5 ±0.8 (3)

0.59 ±0.10 (3)

1.1 ±0.04 (2)

FB

1.0

12±2 (7)

5.1 ±1.2 (3)

3.0±0.4 (3)

3.2 ±0.3 (2)

0.65 ±0.13 (2)

0.89 (1)

FIG. 7. The nucleotide sequence from -148 to -53 is an enhancer that is active in myotubes, myoblasts, and fibroblasts. C2 or 3T3 cellswere cotransfected with pXhGH5 and a CAT expression vector containing B-subunit 5'-flanking DNA fused to the FBP-CAT plasmid. hGHand CAT activities were measured for C2 myoblasts (MB), C2 myotubes (MT), and 3T3 fibroblasts (FB). FBP-CAT contains nucleotides -56to + 109 from the c-fos gene, which includes a TATA box and a transcription start site. Expression (CAT-hGH activity) from cells transfectedwith FBP-CAT was assigned a value of 1.0, and all other values are expressed relative to this. In either orientation, the nucleotide sequencefrom -148 to -53 enhances expression of the FBP in myotubes, myoblasts, and fibroblasts. The sequence from -94 to -53 has enhanceractivity that is weaker than that of -148 to -53. The sequence from -148 to -95 has no enhancer activity in any cell type. The means ±

the standard errors of the mean are given, and the number of repetitions of each experiment is shown in parentheses.

though El, E2, and E3 contain the consensus binding site formyogenic bHLH-E2A heterodimers (8), E2 and E3 are in theorientation opposite that of El, and the 2-bp sequences thatflank these three E boxes are not identical (Fig. 1). Flankingnucleotides are important in determining the DNA bindingsite preferences of bHLH proteins (8, 45, 51), and in somecases, bHLH proteins are capable of distinguishing betweenE-box sequences that differ by only a single base pair in theirflanking sequences (17). Further experiments will be re-

Nuclear extract: MT MB FBol 7 / ~ >t11 -

Competitor: c:P D? § W- > If D;9)>I

_-W w- ~ W) WX w

* * %O % Wm

FIG. 8. Myotubes, myoblasts, and fibroblasts contain an activitythat binds to the enhancer. Nuclear extracts from C2 myotubes(MT), C2 myoblasts (MB), or 3T3 fibroblasts (FB) were incubatedwith a probe extending from nucleotides -94 to -53 of the B-subunitgene. An activity binds to the sequence from -94 to -53 (arrow),and the binding is inhibited efficiently by a 100-fold molar excess ofthe unlabelled DNA sequence from -94 to -53 but not by thesequence from -148 to -95. The first lane contains probe (asterisk)that was incubated without nuclear extract.

quired to determine whether sequences that flank El have arole in determining its properties.

Alternatively, the position of El relative to the enhanceror the transcription initiation site may functionally distin-guish El from E2 and E3. The sequence surrounding thetranscription initiation site fits the consensus for the pyrim-idine-rich initiator element (44) in seven of eight positions.This raises the possibility that factors bound at El mightinteract with activities bound at the initiator element. In this

A MYOBLAST

-200 .150 ( 7)

Enhancer El

B MYOTUBE

orS-2W10 .0 _

Enhancer El

FIG. 9. A model for muscle-specific expression of the AChRB-subunit gene. (A) Expression of the AChR B-subunit gene is low inmyoblasts and nonmuscle cells. A repressor protein (R) binds to Eland represses transcription. Id binds to E2A proteins and minimizesformation of heterodimers with MyoD. (B) Expression of the AChRB-subunit gene is high in myotubes. Id levels are low, allowingformation of MyoD-E2A and myogenin-E2A heterodimers, whichdisplace the repressor and activate transcription. The enhancer,which is necessary for myotube expression, binds proteins (X) thatare present in myoblasts and myotubes.

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

E-BOX-MEDIATED REPRESSION 5139

regard, it is noteworthy that an initiator element-bindingtranscription initiation factor, TFII-I, interacts coopera-tively with an E-box binding activator, USF, a member ofthe bHLH-Zip family (37). TFII-I is related to USF byimmunological criteria and contains repeated domains withsequences that are homologous to HLH domains (36), sug-gesting that TFII-I may be capable of interacting with otherproteins that have HLH domains. Thus, positive or negativeregulation through El may result from interactions betweenbHLH proteins bound at El and general initiation factors,such as TFII-I.Myotube and myoblast nuclear extracts contain multiple

El binding activities. We show that one binding activity,present in myotubes and myoblasts, contains MyoD-E2Aand that another activity, present only in myotubes, containsmyogenin-E2A. MyoD-E2A and myogenin-E2A are likely tobe required for activation of the b-subunit in myotubes. Athird binding activity is present in myotubes, myoblasts, 3T3cells, and HeLa cells and does not contain MyoD, myoge-nin, or E2A proteins. It is possible that this third activitycontains a bHLH protein that does not react with antibodiesto MyoD, myogenin, or E2A. HeLa E-box binding factor(HEB) is a recently identified bHLH protein related to E2Aand E2-2(ITF-2) (17). We do not know whether E2A anti-serum cross-reacts with HEB, but the bHLH domains inthese proteins are 80 to 90% identical (17). HEB can formhomodimers and heterodimers with myogenin, E12, or E2-2(ITF-2) in vitro (17). If HEB is present in complex 3, itsdimerization partner must be distinct from E12, E47, E2-5(ITF-1), and E2-2(ITF-2), because complex 3 is not affectedby the E2A antiserum. Although it is possible that complex3 contains HEB homodimers, Hu et al. (17) report that HEBhomodimers are not detectable in HeLa nuclear extracts bygel shift analysis. Complex 3 is unlikely to contain a memberof the bHLH-Zip family of transcription factors (c-Myc,Max, USF, TIFE-3, TFEB, and AP-4), which contain abHLH domain adjacent to a leucine zipper domain (24),since these proteins bind poorly or not at all to E boxes withthe core sequence CACCTG (7, 22, 35).Although many muscle-specific genes require more than

one E box for maximal expression (49), several genes (27,40), like the b-subunit gene, require only a single E box. Inthese genes, expression also depends on the presence ofother activator sites. Expression of the human cardiac actingene, for example, requires a CArG box and a Spl site inaddition to an E box (40). Similarly, the b-subunit El site isnecessary for maximal expression in myotubes but is notsufficient to activate expression in the absence of enhancersequences.The b-subunit enhancer is required for expression in

myotubes but is not muscle specific, since it can also activateexpression of a heterologous promoter in myoblasts andfibroblasts. Deletion analysis indicates that the enhancerspans a relatively large region (nucleotides -148 to -53),suggesting that the enhancer is composed of multiple ele-ments, including a potential binding site for AP-2 and apotential binding site for Spl. Overlapping the potentialAP-2 site is a sequence, CCCACCCCC, which is essentialfor muscle-specific expression of the human myoglobinpromoter and which binds a 40-kDa protein (CBF40) that ispresent in myotubes, myoblasts, and fibroblasts (4). Multiplecopies of this sequence activate expression of a heterologouspromoter in both muscle and nonmuscle cells (4). Thus, theb-subunit enhancer contains several potential binding sitesfor widely expressed factors which may regulate the activityof this enhancer.

The AChR a-, -y-, and b-subunit genes contain E boxes intheir 5'-flanking regions that are important for expression inmyotubes (15, 31, 34). The e gene, however, contains an Ebox that is not necessary for expression in myotubes but isnecessary for repression in nonmuscle cells: mutation of thisE box increases expression in HeLa cells and lOTl/2 cellsbut does not alter expression in myotubes (31). The reportergene product used in these experiments (CAT), however, isrelatively stable, and it is possible that CAT synthesized inmyoblasts persists in the cytoplasm of myotubes duringmuscle differentiation and obscures a decrease in £-geneexpression in myotubes.The AChR b-subunit gene is expressed initially during

myogenesis and is regulated subsequently by innervation.Signals associated with the synaptic basal lamina activateb-subunit transcription in nuclei near the synaptic site,whereas propagated electrical activity represses 8-subunitexpression in nuclei throughout the myofiber (20, 43). Thecis-acting sequences for both aspects of innervation-depen-dent expression are located in the same 181 bp of 8-subunit5'-flanking DNA that confers muscle-specific expression (13,21, 43). Thus, it is possible that El and the enhancer aretargets not only for regulating expression of the b-subunitgene during muscle differentiation but also for controllingexpression of the b-subunit expression following innerva-tion.

ACKNOWLEDGMENTS

We thank A. Lassar for providing us with antiserum to MyoD (160to 307). We thank C. Murre for providing us with antiserum to E2Aproteins, and W. Wright for providing us with the monoclonalantibody to myogenin (F5D). We thank Stephen Dyer for excellenttechnical assistance and C. Jennings for his comments on themanuscript.

This work was supported by research grants from the NationalInstitutes of Health (NS27963) and the Muscular Dystrophy Asso-ciation.

REFERENCES1. Abmayr, S. M., and J. L. Workman. 1990. Preparation of

nuclear and cytoplasmic extracts from mammalian cells. InF. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G.Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols inmolecular biology. Wiley Interscience, New York.

2. Akerblom, I. E., E. P. Slater, M. Beato, J. D. Baxter, and P. L.Mellon. 1988. Negative regulation by glucocorticoids throughinterference with a cAMP responsive enhancer. Science 241:350-353.

3. Baldwin, T. J., and S. J. Burden. 1988. Isolation and character-ization of the mouse acetylcholine receptor delta subunit gene:identification of a 148-bp cis-acting region that confers myotube-specific expression. J. Cell Biol. 107:2271-2279.

4. Bassel-Duby, R., M. D. Hernandez, M. A. Gonzalez, J. K.Krueger, and R. S. Williams. 1992. A 40-kilodalton protein bindsspecifically to an upstream sequence element essential formuscle-specific transcription of the human myoglobin promoter.Mol. Cell. Biol. 12:5024-5032.

5. Benezra, R., R. L. Davis, D. Lockshorn, D. L. Turner, and H.Weintraub. 1990. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49-59.

6. Berkowitz, L. A., K. T. Riabowol, and M. Z. Gilman. 1989.Multiple sequence elements of a single functional class arerequired for cyclic AMP responsiveness of the mouse c-fospromoter. Mol. Cell. Biol. 9:4272-4281.

7. Blackwell, T. K., L. Kretzner, E. M. Blackwood, R. N. Eisen-man, and H. Weintraub. 1990. Sequence-specific DNA bindingby the c-Myc protein. Science 250:1149-1151.

8. Blackwell, T. K., and H. Weintraub. 1990. Differences andsimilarities in DNA-binding preferences of myoD and E2A

VOL. 13, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.

5140 SIMON AND BURDEN

protein complexes revealed by binding site selection. Science250:1104-1110.

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. Brenner, H. J. R., A. Herezeg, and C. R. Slater. 1992. Synapse-specific expression of acetylcholine receptor genes and theirproducts at original synaptic sites in rat soleus muscle fibres re-generating in the absence of innervation. Development 116:41-53.

11. Buonanno, A., and J. P. Merlie. 1986. Transcriptional regulationof nicotinic acetylcholine receptor genes during muscle devel-opment. J. Biol. Chem. 261:11452-11455.

12. Dignam, J. D., R. D. Liebovitz, and R. H. Roeder. 1983.Accurate transcription initiation by RNA polymerase II in asoluble extract from isolated mammalian nuclei. Nucleic AcidsRes. 11:1475-1489.

13. Dutton, E. K., A. M. Simon, and S. J. Burden. 1993. Electricalactivity-dependent regulation of the acetylcholine receptorb-subunit gene, MyoD, and myogenin in primary myotubes.Proc. Natl. Acad. Sci. USA 90:2040-2044.

14. Gilman, M. Z., R. N. Wilson, and R. A. Weinberg. 1986.Multiple protein-binding sites in the 5'-flanking region regulatec-fos expression. Mol. Cell. Biol. 6:4305-4316.

15. Gilmour, B., G. R. Fanger, C. Newton, S. M. Evans, and P. D.Gardner. 1991. Multiple binding sites for myogenic regulatoryfactors are required for expression of the acetylcholine receptory-subunit gene. J. Biol. Chem. 266:19871-19874.

16. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982.Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051.

17. Hu, J.-S., E. N. Olson, and R. E. Kingston. 1992. HEB: ahelix-loop-helix protein related to E2A and ITF2 that canmodulate the DNA-binding ability of myogenic regulatory fac-tors. Mol. Cell. Biol. 12:1031-1042.

18. Jaynes, J. B., and P. H. O'Farrell. 1988. Activation and repres-sion of transcription by homeodomain-containing proteins thatbind a common site. Nature (London) 336:744-749.

19. Jen, Y., H. Weintraub, and R. Benezra. 1992. Overexpression ofId protein inhibits the muscle differentiation program: in vivoassociation of Id with E2A proteins. Genes Dev. 6:1466-1479.

20. Jo, S. A., and S. J. Burden. 1992. Synaptic basal lamina containsa signal for synapse-specific transcription. Development 115:673-680.

21. Jo, S. A., and S. J. Burden. Unpublished data.22. Kato, G. J., W. M. F. Lee, L. Chen, and C. V. Dang. 1992. Max:

functional domains and interaction with c-Myc. Genes Dev.6:81-92.

23. Klarsfeld, A., J.-L. Bessereau, A.-M. Salmon, A. Triller, C.Babinet, and J.-P. Changeux. 1991. An acetylcholine receptora-subunit promoter conferring preferential synaptic expressionin muscle of transgenic mice. EMBO J. 10:625-632.

24. Lamb, P., and S. L. McKnight. 1991. Diversity and specificity intranscriptional regulation: the benefits of heterotypic dimeriza-tion. Trends Biochem. Sci. 16:417-422.

25. Lassar, A. B., R. L. Davis, W. E. Wright, T. Kadesch, C. Murre,A. Voronova, D. Baltimore, and H. Weintraub. 1991. Functionalactivity of myogenic HLH proteins requires hetero-oligomeriza-tion with E12/E47-like proteins in vivo. Cell 66:305-315.

26. Laufer, R., and J.-P. Changeux. 1989. Activity-dependent reg-ulation of gene expression in muscle and neuronal cells. Mol.Neurobiol. 3:1-53.

27. Lin, H., K. E. Yutzey, and S. F. Konieczny. 1991. Muscle-specific expression of the troponin I gene requires interactionsbetween helix-loop-helix muscle regulatory factors and ubiqui-tous transcription factors. Mol. Cell. Biol. 11:267-280.

28. Merlie, J. P., and J. M. Kornhauser. 1989. Neural regulation ofgene expression by an acetylcholine receptor promoter in mus-cle of transgenic mice. Neuron 2:1295-1300.

29. Murre, C. (University of California, San Diego). 1992. Personalcommunication.

30. Murre, C., P. S. McMaw, H. Vaessin, M. Caudy, L. Y. Jan,Y. N. Jan, C. V. Cabrera, J. N. Buskin, S. D. Hauschka, A. B.Lassar, H. Weintraub, and D. Baltimore. 1989. Interactions

between heterologous helix-loop-helix proteins generate com-plexes that bind specifically to a common DNA sequence. Cell58:537-544.

31. Numberger, M., L. Duirr, W. Kues, M. Koenen, and V. Witzemann.1991. Different mechanisms regulate muscle-specific AChR -y- ande-subunit gene expression. EMBO J. 10:2957-2964.

32. Ohkuma, Y., M. Horikoshi, R. G. Roeder, and C. Desplan. 1990.Binding site-dependent direct activation and repression of invitro transcription by Drosophila homeodomain proteins. Cell61:475-484.

33. Oro, A. E., S. M. Hollenberg, and R. M. Evans. 1988. Transcrip-tional inhibition by a glucocorticoid receptor-,-galactosidasefusion protein. Cell 55:1109-1114.

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

35. Prendergast, G. C., and E. B. Ziff. 1991. Methylation-sensitivesequence-specific DNA binding by the c-Myc basic region.Science 251:186-189.

36. Roeder, R. G., A. Roy, M. Meisterernst, P. Pognonec, Y. Luo,and H. Fuji. 1992. Structure and functional interactions ofgeneral initiation factors, regulatory factors and cofactors.FASEB J. 6:A274.

37. Roy, A. L., M. Meisterernst, P. Pognonec, and R. G. Roeder.1991. Cooperative interaction of an initiator-binding transcrip-tion initiation factor and the helix-loop-helix activator USE.Nature (London) 354:245-248.

38. Ruezinsky, D., H. Beckmann, and T. Kadesch. 1991. Modulationof the IgH enhancer's cell type specificity through a geneticswitch. Genes Dev. 5:29-37.

39. Sanes, J. R., Y. R. Johnson, P. T. Kotzbauei, J. Mudd, T.Hanley, J.-C. Martinou, and J. P. Merlie. 1991. Selectiveexpression of an acetylcholine receptor-lacZ transgene in syn-aptic nuclei of adult muscle fibers. Development 113:1181-1191.

40. 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.

41. Selden, R. F., K. B. Howie, M. E. Rowe, H. M. Goodman, andD. D. Moore. 1986. Human growth hormone as a reporter genein regulation studies employing transient gene expression. Mol.Cell. Biol. 6:3173-3179.

42. Simon, A. M., and S. J. Burden. Unpublished data.43. Simon, A. M., P. Hoppe, and S. J. Burden. 1992. Spatial

restriction of AChR gene expression to subsynaptic nuclei.Development 114:545-553.

44. Smale, S. T., and D. Baltimore. 1989. The "Initiator" as atranscription control element. Cell 57:103-113.

45. Sun, X.-H., and D. Baltimore. 1991. An inhibitory domain ofE12 transcription factor prevents DNA binding in E12 ho-modimers but not in E12 heterodimers. Cell 64:459-470.

46. Tapscott, S. J., R. L. Davis, M. J. Thayer, P.-F. Cheng, H.Weintraub, and A. B. Lassar. 1988. MyoDl: a nuclear phospho-protein requiring a myc homology region to convert fibroblaststo myoblasts. Science 242:405-411.

47. Taylor, J. W., J. Ott, and F. Eckstein. 1985. The rapid genera-tion of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA. Nucleic Acids Res.13:8765-8785.

48. Tsay, H.-J., and J. Schmidt. 1989. Skeletal muscle denervationactivates receptor genes. J. Cell Biol. 108:1523-1526.

49. Weintraub, H., R. Davis, S. Tapscott, M. Thayer, M. Krause, R.Benezra, T. K. Blackwell, D. Turner, R. Rupp, S. Hollenberg, Y.Zhuang, and A. Lassar. 1991. The myoD gene family: nodalpoint during specification of the muscle cell lineage. Science251:761-766.

50. Wigler, M. A., A. Pellicer, S. Silverstein, R. Axel, G. Urlaub, andL. Chasin. 1979. DNA mediated transfer of the adenine phos-phoribosyltransferase locus into mammalian cells. Proc. Natl.Acad. Sci. USA 76:1373-1376.

51. Wright, W. E., M. Binder, and W. FunL 1991. Cyclic amplifi-cation and selection of targets (CASTing) for the myogeninconsensus binding site. Mol. Cell. Biol. 11:4104-4110.

MOL. CELL. BIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/m

cb o

n 21

Dec

embe

r 20

21 b

y 18

6.56

.34.

70.