INTRODUCTION

An understanding of the molecular mechanisms that govern thedetermination and differentiation of skeletal muscle lineageshas progressed rapidly since the discovery of the

myoD familyof myogenic regulatory genes. These genes, myoD (Davis etal., 1987), myogenin (Edmondson and Olson, 1989; Wright etal., 1989), myf5 (Braun et al., 1989) and MRF4 (Rhodes andKonieczny, 1989; also known as myf6 [Braun et al., 1990] andherculin [Miner and Wold, 1990]), which are expressed exclu-sively in skeletal muscle, encode structurally related transcrip-tion factors of the basic helix-loop-helix (bHLH) class (Murreet al., 1989). These myogenic factors activate muscle-specificgene transcription in differentiating cells by binding to DNA

motifs known as E-boxes, sequences present in the regulatoryregions of most muscle-specific genes. Transfection assaysindicate that myoD family members also directly or indirectlypositively regulate each other’s expression (Thayer et al., 1989;Braun et al., 1989; Edmondson et al., 1991, 1992). These cross-and auto-regulatory functions may amplify expression of themyogenic factors and stabilize the muscle phenotype (Thayeret al., 1989). Importantly, these genes can induce myogenesisin a variety of non-muscle cell types when expressed from aconstitutive promoter (reviewed by Emerson, 1990; Olson,1990; Weintraub et al., 1991), a finding consistent with afunction in determination of the skeletal muscle lineage.

In vertebrates, skeletal muscle progenitor cells are derivedpredominantly from the somites, which are formed by seg-

637DevelopPrinted i

MyoD -lootranscr cle determ e ida tran embexpres

ancbeen l toupstre e. Wpresen enhTransg theenhanc genithis en ter skeleta ral indistin expdomain porthe ot thepromo nsebetwee ce distal r/hegous cle-expres thmyoD tionthe exception of E-boxes, the myoD enhancer has no

s ofula- not

stingent.tein

arerom

tioncer-

type. and thecoressedicates byther

elixfactors, transgenic mice

SUMM

Emb

oD

dist

David lexand C1Depa ive2Institu en3Institu t D

*Author al BioPhiladel†Presen Univ

p-helixlineageentifiedryonicer had 22 kbe nowancer.myoD

c mice,gene topatternressionted formyoD

rvationand itsterolo-

specificat the. With

apparent sequence similarity with regulatory regionother characterized muscle-specific structural or regtory genes. Mutation of these E-boxes, however, doesaffect the pattern of lacZ transgene expression, suggethat myoD activation in the embryo is E-box-independDNase I protection assays reveal multiple nuclear probinding sites in the core enhancer, although nonestrictly muscle-specific. Interestingly, extracts fmyoblasts and 10TG fibroblasts yield identical protecprofiles, indicating a similar complement of enhanbinding factors in muscle and this non-muscle cell However, a clear difference exists between myoblasts10TG cells (and other non-muscle cell types) inchromatin structure of the chromosomal myoDenhancer, suggesting that the myoD enhancer is repreby epigenetic mechanisms in 10TG cells. These data indthat myoD activation is regulated at multiple levelmechanisms that are distinct from those controlling ocharacterized muscle-specific genes.

Key words: myoD, transcription, myogenesis, basic helix-loop-h

gene is regulated by a highly conserved

ander Faerman3, Ayala King3, Moshe Shani3

rsity of Pennsylvania School of Medicine, Philadelphia, PA 19104, USAter, Philadelphia, PA 19111, USAagan 50250, Israel

logy, 219 Anatomy-Chemistry Building, University of Pennsylvania School of Medicine,

ersity of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

ment 121, 637-649 (1995)n Great Britain © The Company of Biologists Limited 1995

ryonic activation of the my

al control element

J. Goldhamer1,*, Brian P. Brunk2,†, Aharles P. Emerson, Jr2,†

rtment of Cell and Developmental Biology, Unte for Cancer Research, Fox Chase Cancer Cte of Animal Science, The Volcani Center, Be

for correspondence: Department of Cell and Developmentphia, PA 19104, USA. E mail: djg@pobox.upenn.edut address: Department of Cell and Developmental Biology,

belongs to a small family of basic helixiption factors implicated in skeletal musination and differentiation. Previously, w

scriptional enhancer that regulates the sion of the human myoD gene. This enhocalized to a 4 kb fragment located 18am of the myoD transcriptional start sitt a molecular characterization of this enic and transfection analyses localize er to a core sequence of 258 bp. In transhancer directs expression of a lacZ reporl muscle compartments in a spatiotempoguishable from the normal myoD, and distinct from expression patterns re

her myogenic factors. In contrast to ter, the myoD enhancer shows striking con humans and mice both in its sequenposition. Furthermore, a myoD enhancepromoter construct exhibits mus

sion in transgenic mice, demonstratingpromoter is dispensable for myoD activa

ARY

638

mentatiosequencetrunk andcells thamaturingDouarin,form facanterior s1991; Co

Gene-ration ofregulatormyoD anby both h1993). Inare devodesmin-pmice homno muscmyoD an(Braun ealthoughentiationnormal n1993; Nand myfmental plineagesmitmentnormal skeletal

Each tiotempoham, 1memberopmentain estab1993), awill requscriptionenhancepathwaytion in tanalyses gene is rfragmentmyoD trastudy, wregulatesThese dahighly cdistinct fulatory g

MATER

Unless otconductedwere purgradients.

D

n of the paraxial mesoderm in a rostral-to-caudal. The myotome gives rise to axial muscles, whereas limb muscles are derived from myogenic progenitor

t migrate away from the ventrolateral edge of the somite (Wachtler and Christ, 1992; Ordahl and Le 1992). Myogenic cells of the branchial arches, whichial, jaw and throat musculature, originate both fromomites and from cranial paraxial mesoderm (Noden,uly et al., 1992).

targeting experiments in mice showed that the elabo- the myogenic phenotype is dependent on myogenicy gene function. Mice homozygous null for bothd myf5 completely lack differentiated skeletal muscleistological and biochemical criteria (Rudnicki et al., these mice, muscle-forming regions of the embryoid of myoblasts, as evidenced by the absence ofositive cells (Rudnicki et al., 1993). Interestingly,

DNA constructs A 1.7 kb ApaI/PstI fragment derived from the 4 kb enhancer-con-taining fragment (fragment 3 in Goldhamer et al., 1992; see Fig. 2)was subcloned into pBluescript KS+ (KS+; Stratagene) by blunt-endligation into the unique EcoRV site of the multiple cloning site. Bi-directional nested deletions were created by exonuclease III digestionusing the Erase-a-base kit (USB), or by partial digestion with PvuIIat nucleotide 95, to create ∆15 (3′-5′). ∆14KS+ (3′-5′) contains 258base pairs (bp) of enhancer sequence, and is referred to as the coreenhancer throughout.

The reporter plasmid ptkCAT∆EH (derived from pBLCAT2[Luckow and Schutz, 1987] by deletion of the NdeI/HindII fragmentof pUC 18) was the parental plasmid used for transfection experi-ments (Goldhamer et al., 1992). A 2.7 kb genomic fragment contain-ing human myoD 5′ flanking sequences extending from an EcoRI siteat −2.5 kb to +198 relative to the TATA box (−37 relative to the trans-lational initiation codon) was generated from a sequencing deletionin KS+. −2.5CAT was produced by excising this promoter-contain-ing fragment from KS+ by digestion with SacI and KpnI, followed by

. J. Goldhamer and others

ozygous null for either myf5 or myoD alone exhibit

le defects at birth, indicating that the functions ofd myf5 in myogenesis are at least partially redundantt al., 1992; Rudnicki et al., 1992, 1993). In contrast, myogenin knockout mice show severe muscle differ- defects, myoblasts are present in approximatelyumbers and positions in these embryos (Hasty et al.,

abeshima et al., 1993). These data indicate that myoD5 serve upstream functions in the myogenic develop-rogram to determine, expand, or maintain myogenic

, whereas myogenin, although not required for com- of cells to the myogenic lineage, is required forbiochemical and morphological differentiation ofmuscle (reviewed by Weintraub, 1993).of the myogenic genes is expressed in a unique spa-ral pattern in developing skeletal muscle (Bucking-

992; Faerman and Shani, 1993), indicating thats of the myoD family are regulated by distinct devel-l signals. Given the formative role of myoD and myf5lishing the skeletal muscle lineage (Rudnicki et al., mechanistic understanding of lineage determinationire detailed information of how these genes are tran-ally controlled. Cis and trans analysis of the myoDr offers a powerful means to define upstream signalings and transcriptional events that govern myoD activa-

blunt end-ligation into the XbaI and BglII sites of ptkCAT∆EH. (XbaIand BglII digestion removes all thymidine kinase promotersequences.) myoD enhancer/promoter CAT constructs were generatedby digesting enhancer deletion constructs in KS+ with SalI and XbaIand cloning into unique SalI and XbaI sites of −2.5CAT. (The XbaIsite was derived from KS+ during the construction of −2.5CAT.)

The lacZ vector pPD46.21 (kindly provided by Andrew Fire) wasused for transgene constructions. pPD46.21 is identical to pPD1.27(Fire et al., 1990) except that it lacks the sup-7 gene. It contains aninitiation codon and SV40 T antigen nuclear localization signal justupstream from the bacterial lacZ gene, and polyadenylation sequencesfrom the SV40 early region. Enhancer/myoD promoter lacZ constructswere prepared by liberating enhancer/promoter inserts from the CATvector by digestion with SalI and XhoI, and cloning into the SalI siteof pPD46.21. Orientation was determined by restriction digests.tklacZ was prepared by digesting ptkCAT∆EH with BamHI and BglIIand inserting the tk promoter fragment (from −105 to +51 of theHSVtk gene) into the BamHI site of pPD46.21. Orientation wasdetermined by sequencing. To produce 258tklacZ, the BamHI/BglIItk promoter fragment was cloned into the BamHI site of ∆14KS+.After orientation was determined by sequencing, the 258tk fusion wasexcised by digestion with HindIII and XbaI and cloned into the cor-responding sites of pPD46.21. The E-box mutant construct 258(E1-E3)/−2.5lacZ was cloned by excising the mutagenized coreenhancer (see below) from ∆14KS+ with SalI and XbaI, and cloningthe fragment into the SalI and SpeI (derived from the KS+ multiple

he embryo. Previously, we showed by transgenicthat the embryonic expression of the human myoDegulated by a distant enhancer localized to a 4 kb approximately 18 to 22 kb upstream from the start ofnscription (Goldhamer et al., 1992). In the presente define and characterize the distal enhancer that the embryonic activation of the human myoD gene.ta indicate that myoD activation is regulated by aonserved and complex regulatory system that isrom mechanisms that regulate the other myogenic reg-enes.

IALS AND METHODS

herwise noted, all molecular biological techniques were using standard methods (Sambrook et al., 1989). All DNAs

ified by double banding in cesium chloride equilibrium

cloning site during excision of the myoD promoter fragment) sites of −2.5lacZ.

Cloning of the mouse myoD enhancer Mouse sequences homologous to the human enhancer were detectedby Southern analysis of PstI-digested mouse genomic DNA. To createa size-limited library, the hybridizing band was excised from anagarose gel, ligated into PstI-digested KS+, and electroporated intoNM554 bacteria using the BioRad Gene Pulser. This library wasscreened by standard methods using a probe from the human coreenhancer to isolate the corresponding mouse sequences. Linkage tothe mouse myoD gene was verified by hybridization of enhancer-con-taining cosmid clones (cosmid library was kindly provided byYoshimichi Nakatsu) with a mouse myoD cDNA.

Sequence analysisThe sequence of the human and mouse myoD enhancer was deter-mined on both strands by dideoxy sequencing using the Sequenase kit(version 2.0; USB). The UWGCG sequencing package was used forsequence analysis, and transcription factor site searches utilized signalscan software (Prestridge, 1991). The nucleotide sequence of the

human GenBan

MutagE-box mmethodmerase,the temobtaineboxes CTNNTof bHLboth str

TransfCulturinfectionsviouslypendenIdentica(betweewhich ProteinBradforstandarwhich y

NucleaC2C12 were puand FCrespectirespecti(Gibco:10% (1Hyclon20% FBin RPMwith pewere feconfluenuclearto the mrinsing buffereNaCl, 2dithiothµg/ml lml tubeAll subpellets ml for ebuffer (0.5 mMwere cog for 5 mA contalysed wlysate w(60 mM0.2 mM2 mM Pwas centhe nucnuclear(0.42 M25% gl

639Regulation of the myoD gene in mouse embryos

and mouse myoD core enhancer has been submitted to thek database.

enesisutations were created by the PCR-based overlap extension

as previously described (Ho et al., 1989), using Vent poly- and ∆14KS+ as the template. By using mutant derivatives asplate for subsequent rounds of mutagenesis, a construct wasd in which all three conserved E-boxes were mutated. All E-were changed from the wild-type sequence CANNTG toA, which destroys the minimal sequence required for binding

H myogenic factors. Mutations were confirmed by sequencingands of the core enhancer in ∆14KS+.

ections and CAT assaysg of 23A2 myoblasts (Konieczny and Emerson, 1984), trans-, cell extract preparation and CAT assays were done as pre- described (Goldhamer et al., 1992). A minimum of three inde-t experiments were conducted for each DNA construct.l molar amounts (0.8 pmoles) of each plasmid were used

nuclear suspension was transferred to two microfuge tubes andincubated on ice for 30 minutes, gently inverting the tubes every 5minutes. Samples were microfuged at 3,000 revs/minute at 4°C for 5minutes, and the supernatants dialyzed (6,000 to 8,000 Mr cutoff) with1 liter of buffer D (60 mM KCl, 20 mM Hepes pH 7.9, 20% glycerol,0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF) with two changes. Afterdialysis, extracts were microfuged for 5 minutes at full speed at 4°C,and small aliquots were quick-frozen in liquid nitrogen. Protein con-centrations were determined by the modified Bradford assay (Bio-Rad) using bovine serum albumin as the standard.

DNase I protection assaysThe 258 bp core enhancer in ∆14 KS+ was 5′ end-labeled on theforward or reverse strand with [γ-32P]ATP (6,000 Ci/mmole; NEN)using unique SalI and XbaI restriction sites flanking the insert.Labeled fragments were purifed on non-denaturing 5% acrylamidegels, and after elution, were concentrated by ethanol precipitation. Foreach 50 µl reaction, 20 µg of nuclear protein and 5,000 to 10,000cts/minute (1.4 fmoles) of labeled fragment was incubated in bindingbuffer (12 mM Tris pH 8.0, 50 mM KCl, 1 mM DTT, 1 mM MgCl2,

n 2.2 µg and 6 µg, depending on the size of the plasmid),was adjusted to 25 µg with pUC 8 carrier plasmid DNA. concentrations in cell extracts were measured by the modifiedd assay (Bio-Rad) using bovine serum albumin as thed. 15 µg to 25 µg of protein was used in each CAT assay,ielded CAT activities within the linear range of the assay.

r extract preparationmyoblasts, 10TG fibroblasts and JEG-3 choriocarcinoma cellsrchased from the American Type Culture Collection. HMP8

1010 cells (kindly provided by Mark Lovell and Jerome Freed,vely) are primary human myoblasts and foreskin fibroblasts,vely. C2C12, 10TG, and JEG-3 cells were grown in DMEM with high glucose and sodium pyruvate) supplemented with0TG and JEG-3) or 15% (C2C12) fetal bovine serum (FBS;e). HMP8 cells were grown in Ham’s F-10 supplemented withS and 0.5% chick embryo extract. FC1010 cells were grown-I supplemented with 10% FBS. All media was supplementednicillin (100 units/ml) and streptomycin (100 µg/ml). Cellsd fresh medium every 3 days and harvested at about 50 to 80%nce. Typically, 20 to 40, 15 cm plates were used for each extract preparation. Nuclear extracts were prepared according

ethod of Zaret (personal communication) as follows. Aftercells twice with calcium- and magnesium-free phosphate-

d saline at room temperature, 5 ml of ice-cold PSDP (0.15 M0 mM sodium phosphate pH 7.4, 0.35 M sucrose, 0.5 mM

1 mM CaCl2, 5 mM NaCl, 100 µg/ml bovine serum albumin, 5%glycerol, 2% polyvinyl alcohol, and 0.5 µg poly(dI/dC)) on ice for 1hour. Reactions were equilibrated to room temperature, 50 µl ofbinding buffer was added and, 1 minute later, DNase I (Worthington)was added to a final concentration of 1 µg/ml (experimental lanes) or6 or 12 ng/ml (control lanes containing bovine serum albumin in placeof extract). After exactly 2 minutes, stop buffer (50 mM EDTA, 0.2%SDS, 100 µg/ml yeast tRNA) was added, proteinase K was added to75 µg/ml and tubes were incubated at 37°C for 30 minutes. Sampleswere extracted with an equal volume of phenol and the DNA was pre-cipitated with ethanol for 5 minutes at room temperature. After cen-trifugation, pellets were washed two times in 70% ethanol, dried in aSpeed-Vac, and resuspended in 8 µl of loading buffer (40%formamide, 1 mM EDTA). Samples were heated to 80°C and 3 µl ofeach sample was resolved on a 6% denaturing polyacrylamide gel.Dried gels were exposed to X-OMAT AR X-ray film (Kodak) withintensifier screens for 2 to 4 days. Positions of footprints were deter-mined by comparison with G+A sequencing ladders prepared bystandard Maxam and Gilbert chemical cleavages. At least two inde-pendently prepared extracts were tested for each cell type. DNase Iprotection assays were conducted a minimum of 3 times with eachextract.

DNase I hypersensitivity blotsAll cell types were from the American Type Culture Collection. TheNB41A3 neuroblastoma cell line was grown in F-10 medium supple-

reitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], 5eupeptin) was added per plate, and cells were scraped into 50s on ice. Cells were pelleted at 2,000 g for 5 minutes at 4°C.sequent procedures were conducted in a cold room on ice. Cellwere washed two times with PSDP and gently resuspended (2ach original 50 ml of cell suspension) in an ice-cold hypotonicbuffer A; 10 mM KCl, 10 mM Hepes pH 7.9, 1.5 mM MgCl2, DTT, 1 mM PMSF, and 5 µg/ml leupeptin). Cell suspensionsmbined, swelled on ice for 5 minutes, and centrifuged at 2,000

inutes at 4°C. Cell pellets were resuspended in 3 ml of bufferining 0.5% NP-40, incubated on ice for 5 minutes, and cellsith a dounce homogenizer (10-15 strokes, pestle A). The cellas transferred to a 15 ml disposable tube, 6 ml of buffer B KCl, 15 mM NaCl, 15 mM Tris-HCl pH 7.4, 0.2 mM EDTA, EGTA, 0.5 mM spermine, 0.15 mM spermidine, 1 mM DTT,MSF, and 5 µg/ml leupeptin) was added, and the cell lysatetrifuged at 1,600 g for 5 minutes at 4°C. After gently washing

lear pellet with 2 ml of buffer B and centrifuging as above, the pellet was gently resuspended in 2 ml of hypertonic buffer C NaCl, 20 mM Hepes pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA,ycerol, 1 mM DTT, 2 mM PMSF, 5 µg/ml leupeptin). The

mented with 15% horse serum and 2.5% FBS. All other cell typeswere grown in DMEM with 10% FBS. Cells were grown in 10 cmdishes and fed every 3 days to approximately 80% confluence. Cellswere harvested by scraping in calcium- and magnesium-freephosphate-buffered saline, permeabilized in NP-40, and treated witha range of DNase I concentrations (from 40-120 µg/ml) for 3 minuteson ice by the method of Rigaud et al. (1991). DNA was isolated bystandard methods. Restriction enzymes and probes used are shown inFig. 6. Hybridization and washing was done as described (Church andGilbert, 1984).

Whole-mount in situsWhole-mount in situs utilized a digoxigenin-labeled probe asdescribed (Conlon and Rossant, 1992). The probe was fromnucleotides 751 to 1785 of the mouse myoD cDNA (Sassoon et al.,1989; Faerman and Shani, 1993).

Transgenic micelacZ fusions were liberated from vector sequences and fragmentspurified as previously described (Goldhamer et al., 1992). Transgenicmice were produced by pronuclear injection of FVB/N 1-cell-stage

640

embryos genic lintransgeniFo mice, were anaby SouthDNA as sequence

RESUL

A distaembryoWe previn a 4 klocated 1binationexpressipartmenpattern genic mtemporacreated containi(containupstreamexpressithe endunpubliexpressi

In moall threecells are(Fig. 1Anately a10.5 d.anterior1A,C; sbud, howventral expressidorsal mcontrast these so1A,B,D)tosidaselation ofulations myotomsectionsmyoD mthat is dhighest and vent1E). Theregulatoof myoD

To furfragmenfection a

D

(Hogan et al., 1986; Shani, 1986). To produce stable trans-es, DNA-positive male mice, or male offspring of femalec mice were mated to FVB/N mice. For analysis of injected,at least four independently derived lacZ-positive embryos

lyzed for each construct. DNA-positive mice were detectedern blot analysis of tail (stable lines) or placental (Fo mice)described (Shani, 1986), using a probe specific to lacZ

s.

TS

l 258 bp core enhancer controls thenic activation of myoDiously identified a muscle-specific enhancer containedb fragment (fragment 3 in Goldhamer et al., 1992)8 to 22 kb upstream of the human myoD gene. In com-

with the myoD promoter, this enhancer drives

the myoD promoter in 23A2 myoblasts. The myoD promoteralone has a very low basal level of CAT reporter gene activity,only about 5-fold above a promoterless CAT construct.Fragment 3 typically increases this basal level of transcription10- to 20-fold in 23A2 myoblasts (Goldhamer et al., 1992). Aninternal 1.7 kb ApaI/PstI fragment was identified that yieldsapproximately 50% of the CAT activity of fragment 3 (Fig.2A). No other subfragment residing entirely outside of this 1.7kb fragment exhibits enhancer activity, although all of theactivity of fragment 3 could be reconstituted by the additiveactivities of two fragments that overlap the 1.7 kb fragment;the 2.1 kb KpnI fragment (see Fig. 2B), which includes the 5′half of the 1.7 kb fragment, exhibits approximately 70% of theactivity of fragment 3, and a 1.4 kb KpnI/EcoRI, whichincludes the 3′ half of the 1.7 kb fragment constitutes theremaining activity (data not shown). Importantly, the 1.7 kbfragment, and subfragments therein, yield the appropriate

. J. Goldhamer and others

pattern of muscle-specific expression in transgenic mice (see

on of a bacterial lacZ gene in all skeletal muscle com-ts of mouse embryos, conferring the endogenous myoDof expression in 11.5 days post-coitum (d.p.c.). trans-ice (Goldhamer et al., 1992). To analyze further thel and spatial pattern of activation of the transgene, westable transgenic lines that harbor the 4 kb enhancer-ng fragment juxtaposed to the myoD promotered in 2.5 kb of myoD 5′ flanking sequence) cloned of the lacZ gene. The spatiotemporal pattern of lacZ

on throughout embryogenesis coincides with that ofogenous mouse myoD gene (Fig. 1; Faerman et al.,shed data). Several interesting features of transgeneon patterns in somites are described below.st somites anterior to the level of the forelimb bud of independently derived transgenic lines, lacZ-positive scattered throughout the dorsal-ventral myotomal axis-C) and transgene expression appears to be coordi-

ctivated along the dorsal-ventral axis beginning at 10-p.c. (Fig. 1B). The most intense staining in these somites is in the dorsal region of the myotome (Fig.ee also Fig. 3A). In somites posterior to the forelimb

ever, lacZ-expressing cells initially accumulate in theportion of the myotome (Fig. 1B), followed byon in dorsal myotomal cells, including cells of the

below). Therefore, sequences outside of the 1.7 kb fragmentwere excluded from further analysis.

Transfection analysis of bi-directional nested deletions ofthe 1.7 kb fragment identified two non-contiguous DNAelements with modest enhancer activity (Fig. 2A). Enhancer 1,defined by the 3′ to 5′ deletion 14, was localized to a 258 bpsequence at the 5′ end of the 1.7 kb fragment (Fig. 2A, bottom).Further deletion to nucleotide +95 abolishes enhancer activity(Fig. 2A; deletion 15). Approximately 1 kb downstream, asecond enhancer was identified whose 5′ end falls between theendpoints of the 5′ to 3′ deletions, 7 and 8. The 3′ limit of thisenhancer lies between the endpoint of the 3′ to 5′ deletion 1,and the 3′ end of the 1.7 kb fragment. Precise definition of theenhancers’ boundaries is difficult because of their inherentlylow activity in transfection assays. No activity was detected insequences between enhancers 1 and 2 (data not shown). Aswith fragment 3 (Goldhamer et al., 1992), the 1.7 kb fragment,as well as subfragments containing enhancer 1 or 2, are activewhen transfected into both muscle and non-muscle cells inculture.

Deletion constructs containing either enhancer 1 or enhancer2 were tested transgenically for their ability to directexpression of a lacZ reporter gene in the typical myoDexpression pattern (Fig. 2B, 3). Transgenes driven by enhancer

edial lip of the dermamyotome (Fig. 1A,D). Into more rostral somites, the most intense staining inmites is in the ventral region of the myotome (Fig., which is likely due to a greater density of β-galac- (β-gal)-positive cells (Fig. 1D). Interestingly, a popu- myotomal cells between these ventral and dorsal pop-does not express the transgene (Fig. 1A,D) throughoutal stages, a result confirmed by analysis of serial. Whole-mount in situ analysis of endogenous mouseRNA yielded a similar pattern of myotomal expressionependent on axial position; message accumulation isdorsally in the myotome of the most anterior somitesrally in the myotome of more posterior somites (Fig.se data demonstrate the sufficiency of the identified

ry elements in recapitulating the normal spatial patternexpression in somites.

ther localize enhancer activity, overlapping restrictionts and nested deletions were tested in transient trans-ssays for their ability to enhance transcription from

2 and the myoD promoter are only ectopically expressed withno consistent pattern, which is typical of results observed withpromoter sequences alone (Goldhamer et al., 1992). Incontrast, both subfragments containing enhancer 1 (Fig. 2B)are expressed specifically in skeletal muscle in transgenicmice; all five Fo transgenic mice and a stable line containingthe minimal 258 bp enhancer 1 and the myoD promoter exhibita pattern of expression indistinguishable from the larger 1.7 kbfragment or the 4 kb fragment 3 (Fig. 3A). Because enhancer1 directs the myoD pattern of expression in transgenic mice,this 258 bp sequence will hereafter be referred to as the myoDcore enhancer.

The myoD core enhancer was tested in combination with theheterologous herpes simplex virus thymidine kinase (tk)promoter to assess the relative contributions of the enhancerand myoD promoter to the regulation of myoD expression. Asshown previously, the tk promoter alone exhibits only ectopicexpression due to integration site position effects (Allen et al.,1988; data not shown). In contrast, all seven DNA-positive

641Regulation of the myoD gene in mouse embryos

embryoin comspecific gradientbranchia3B). As thoracicdorsal howevecells scaall somi

Fig. 1. S11.5 d.p.of the malso seenThe nasaembryo farrows). myotomembryo fwith the cells venneural tuthoracic the myotpattern oventral relevel expbackgrou

in somites. (A-D) β-gal expression in stable F3/−2.5 lacZ transgenic embryos. (A) Whole-mountrn of β-gal expression. Transgene expression is predominantly in the dorsal region of the myotomend in the ventral region of myotomes (black arrows) posterior to the forelimb bud (fb). Staining is and in branchial arches (ba). Ectopic staining in the neural tube is likely due to position effects.s transgene expression in independent lines, for unknown reasons. (B) 10.5 d.p.c. whole-mountA. Transgene expression is most prominent in the ventral-most portion of thoracic somites (blackl-ventral axis of the most anterior somites. Weak staining in the dorsal-most region of the ectopic neural tube staining. (C) Transverse section anterior to the forelimb bud of an 11.5 d.p.c. Nuclear localized β-gal expression is observed throughout the dorsal-ventral axis of the myotomee arrow). asterisk, hypoglossal premuscle mass, corresponding to the more dorsal strip of stainedrman and Shani, 1993). Ectopic expression is restricted to dorsal root ganglia (drg) in this line. nt, to the forelimb bud from the same embryo shown in C. β-gal expression in the myotome of this

patial domains of myoD expression c. embryo showing the overall patteost anterior somites (white arrows) a in forelimb and hindlimb buds (hb)l epithelium (asterisk) usually showrom same transgenic line shown in Note staining along the entire dorsaes of thoracic somites is obscured byrom an independent transgenic line.most intense staining dorsally (whittral to anterior somites in A (see Faebe. (D) Transverse section posterior

s injected with a construct containing the core enhancerbination with the tk promoter show clear muscle-expression at 11.5 d.p.c., exhibiting a rostrocaudal

of expression in myotomes, as well as expression inl arches, limb buds, and other myogenic centers (Fig.with previous constructs, the most anterior somites and somites could be distinguished by their prominentand ventral staining, respectively. Interestingly,r, most of these transgenic mice exhibit lacZ-positivettered throughout the dorsal-ventral myotomal axis oftes, regardless of their axial position (Fig. 3B). This

indicates a contribution either of sequences in fragment 3outside of the core enhancer, or of myoD promoter sequences,in restricting the spatial expression of myoD within the somitemyotome.

The myoD core enhancer is highly conservedbetween humans and mice As a means of identifying critical regulatory motifs within theenhancer, we compared myoD enhancer sequences betweenhumans and mice, reasoning that important regulatorysequences would be most highly conserved. For this analysis,

somite is most prominent ventrally (black arrow). Note the population of β-gal negative cells (bracket) under the dorsal-most region ofome (white arrow). (E)Whole-mount in situ localization of endogenous mouse myoD mRNA in a 10.5 d.p.c. embryo. The spatialf myoD message accumulation closely matches the pattern of transgene expression. black arrows, localization of myoD mRNA in thegion of myotomes of thoracic somites. white arrows, myoD mRNA is most abundant in the dorsal region of anterior somites. Lowression in the forelimb bud and branchial arches is not apparent in this preparation. Staining of the otic vesicle (ov) is non-specificnd.

642

the mouplasmid RestricticontaininmyoD gethe humastart of tcore enidentity)enhancersimilaritalthoughkb ApaI(unpubli

The h

D

. J. Goldhamer and others

o

rh,

y

su

Fig. 2. (A) Localization of myoDenhancer activities by transienttransfection assays in 23A2myoblasts. The 1.7 kb ApaI/PstIfragment within fragment 3 (seeB) was the parental moleculeused to create nested deletions.All deletion constructs wereassayed for enhancer activity incombination with the myoDpromoter. Numbers are relative tothe CAT activity of the promoteralone, which was arbitrarily set toa value of 1. Values (± s.e.m.) arethe average of at least threeindependent experiments. Arrowsdenote the maximum limits of thetwo enhancers. (B) Summary oftransgenic data, localizing themuscle-specific regulatory regionto a 258 bp core enhancer. Allfragments shown were assayed incombination with the myoDpromoter. The approximate

se myoD enhancer was cloned from a size-selectedlibrary using the human core enhancer as a probe.n mapping and Southern blot analyses of enhancer-g cosmid clones demonstrated linkage with the mousene, and revealed that the mouse myoD enhancer, liken enhancer, is located approximately 20 kb 5′ of theanscription (data not shown). The human and mouseancer show extensive sequence similarity (89% particularly within the first 160 bp, in which the in the two species is 94% identical (Fig. 4). Sequence drops dramatically outside of the core enhancer,

small regions of homology exist throughout the 1.7/PstI fragment as well as 5′ of the core enhancerhed observations). man core enhancer sequence contains four E-boxes,

three of which (E-1, E-2 and E-3) are conserved in sequenceand position in mice (Fig. 4). Both central and flankingnucleotides, which strongly influence the affinity of bHLHprotein binding (Blackwell and Weintraub, 1990; Sun andBaltimore, 1991; Wright et al., 1991), are conserved betweenspecies in E-boxes 2 and 3. E-boxes 2 and 3 represent potentialhigh affinity binding sites for MyoD/E12-E47 and MyoDhomodimers, respectively (Blackwell and Weintraub, 1990;Sun and Baltimore, 1991). With the exception of E-boxes, themyoD core enhancer is distinguished by the lack of bindingsites for factors known to regulate the expression of othermuscle-specific genes (Fig. 4). Motifs that are absent includeMEF-2 (Gossett et al., 1989; Cserjesi and Olson, 1991), CArG(Minty and Kedes, 1986), MHOX (Cserjesi et al., 1992), andM-CAT (Mar and Ordahl, 1990) sites. The A-T rich sequences

positions of enhancer activitiesdefined by transient transfectionassays are shown (boxes).

from n4) resenucleotbindingCserjesdatabaswidelyproteinAP-1 ((Daileygene (F

MutatienhanTo testan enha1 throumice. Iwas chsequen(258(E1upstreaFo mou1 to 1.5nous m1993), vation of lacZmyogenAnalysapparenmutantthere aextent ois similto Figtransgetime coconstruthree csee Figlished core en

NuclesequeDNasesequenhumanC3H10cells umyoblaA repreHMP8 identichypersesensitivenhancwith DNA/pprotein

643Regulation of the myoD gene in mouse embryos

ucleotides 16 to 25, and from nucleotides 31 to 41 (Fig.mble consensus CArG and MEF-2 motifs, but differ atides required for serum response factor and MEF-2 (Treisman, 1986; Pollock and Treisman, 1990, 1991;i and Olson, 1991). A search of the transcription factore revealed consensus sequence binding sites for several

expressed transcription factors, including the Ets, PEA3 (Xin et al., 1992); nts 28-33, reverse strand),nts 143-149), NF-1 (nts 176 to 187), and H4TF-1 et al., 1988; nts 165-173), a regulator of the histone H4ig. 4).

on of the enhancer E-boxes does not affectcer activation in the embryo the functional significance of the conserved E-boxes,ncer construct in which all three conserved E-boxes (E-gh E-3; Fig. 4) were mutated, was tested in transgenicn this construct, the canonical E-box motif CANNTG

consensus binding sites for AP-1 (Lee et al., 1987) and H4TF-1 (Dailey et al., 1988), respectively, are produced with nuclearextracts from all cell types tested, including 23A2 aza-myoblasts, primary chicken liver cells and BNL liver cells(Fig. 5; data not shown). These sites represent the highestaffinity sites and/or are bound by the most abundant factorsbecause they typically exhibit complete protection, even undermore stringent conditions (10 µg protein, 4 µg poly(dI/dC);non-specific competitor; data not shown). Protection of E-boxes was not observed, however E-box binding is difficult todetect using standard DNase I protection assays (Buskin andHauschka, 1989).

Both primary human fibroblasts (FC1010) and JEG-3 cellextracts yield cell-specific qualitative and quantitative differ-ences in their footprinting profiles. Extracts from JEG-3 cells,a cell line in which the transfected enhancer is inactive(Goldhamer et al., 1992), does not show detectable DNase Iprotection over region 2, and only partial protection over

anged to CTNNTA, thereby destroying the minimalce required for bHLH factor binding. This construct-E3)/−2.5lacZ) contains the mutant core enhancer clonedm of the myoD promoter in the lacZ vector used above.se embryos were analyzed at 11.5 d.p.c., which is about days after the myotomal activation of both the endoge-yoD gene (Sassoon et al., 1989; Faerman and Shani,and myoD enhancer lacZ fusions. Defects in the acti-function of the enhancer would result in a delay or loss

expression, as observed with E-box mutations in thein promoter (Cheng et al., 1993; Yee and Rigby, 1993).

is of four lacZ-positive embryos, however, revealed not difference between the pattern of expression of the

construct and the wild-type constructs (Fig. 3C). Also,ppears to be no delay in activation, since the caudalf expression along the rostrocaudal expression gradient

ar to that of wild-type constructs at 11.5 d.p.c. (compares 1A, 3A). In addition, expression of the mutantne was detected in the hindlimb bud at 11.5 d.p.c., aincident with the initial detection of wild-type enhancercts. An enhancer construct entirely lacking E-boxes (the

onserved E-boxes and the fourth, non-conserved E-box;. 4), also is expressed normally at 11.5 d.p.c (unpub-observations). We conclude that E-boxes in the myoD

region 1. Note also the absence of a hypersensitive site betweenregions 1 and 2, and just 5′ of region 4 in JEG-3 cell extracts(Fig. 5, forward strand). Also JEG-3 extracts yield partial pro-tection of sequences between regions 2 and 3, and produced ahypersensitive site between regions 3 and 4 (Fig. 5, forwardstrand). Finally, protected region 3 extends slightly further 5′with JEG-3 extracts than with the other extracts. FC1010extracts exhibit partial or complete protection over all fiveregions, although DNase I hypersensitivity was reduced orabsent at most sites denoted in Figs 4 and 5. In addition, pro-tection over region 4 extends slightly further 3′ with FC1010extracts (Fig. 5), suggesting a qualitative difference inDNA/protein interactions over this site.

The endogenous myoD enhancer exhibits muscle-specific DNase I hypersensitivityThe identical DNase I protection profiles produced usingnuclear extracts from non-myogenic 10TG cells (in which theendogenous gene is inactive), C2C12 myoblasts and HMP8myoblasts suggest that the constellation of enhancer bindingproteins are highly similar in these cell types. In addition, themyoD enhancer is active in 10TG cells when introduced bytransfection (Goldhamer et al., 1992). These data raise the pos-sibility that repression of the endogenous myoD gene in 10TG

hancer are not required for myoD gene activation.

ar trans factors interact with multiplence elements in the core enhancer I protection assays were used to identify enhancerces that interact in vitro with nuclear factors from and mouse cells. Mouse cell types analyzed wereTG (10TG) fibroblasts and C2C12 myoblasts. Humansed were primary fibroblasts (FC1010), primarysts (HMP8) and the choriocarcinoma cell line, JEG-3.sentative experiment is shown in Fig. 5. Extracts frommyoblasts, C2C12 myoblasts and 10TG fibroblasts showal DNase I footprinting profiles (protected regions andnsitive sites). Five protected regions and many hyper-e sites were detected, nearly spanning the coreer (Figs 4, 5). The hypersensitive sites not associatedprotected regions likely reflect lower affinityrotein interactions, or binding of lower abundances. Protected regions 4 and 5, which encompass

cells is mediated by epigenetic mechanisms, such as packaginginto inactive chromatin, that could restrict accessibility ofcritical cis-acting enhancer sequences to positive trans factors.We used DNase I hypersensitivity as an indicator of chromatinstructure to assess whether chromatin structural differencesexist between myogenic and non-myogenic cells. For thisanalysis, the endogenous mouse myoD enhancer was assayedin C2C12 and 23A2 myogenic cells in addition to several non-myogenic cell lines, while the endogenous human myoDenhancer was assayed in primary human fibroblasts (FC1010)and myoblasts (HMP8). Permeabilized cells were treated withincreasing concentrations of DNase I, and the presence andposition of hypersensitive sites determined by Southernblotting using indirect end-labeled probes. Three distincthypersensitive sites are present in chromatin derived fromC2C12 and 23A2 myoblasts, two of which map to the coreenhancer, with the third mapping just 5′ of the enhancer (Fig.6). In contrast, no hypersensitive sites were detected inchromatin from non-myogenic mouse cells, including 10TG

644

cells, BNL liver cells, or NB41A3 neuroblastoma cells (Fig.6). Similarly, chromatin from primary human myoblastsexhibits DNase I hypersensitivity within the core enhancer,whereas chromatin from primary human fibroblasts is 5- to 10-fold more resistant to DNase I digestion, although a very weaksignal was detected (data not shown). These data provideevidence that the chromatin structure of the core enhancer isaltered in myogenic cells, perhaps reflecting greater accessi-bility to trans factors.

DISCUSSION

A distal core enhancer governs myoD geneactivation in the embryoWe have identified and characterized regulatory sequencesresponsible for myoD expression to begin to define upstreamsignaling pathways and transcriptional events that governmyoD activation in the embryo. Cis-acting sequences thatregulate the embryonic expression of the human myoD genewere localized to a 258 bp core enhancer, which is locatedapproximately 20 kb upstream from the start of myoD tran-scription (Goldhamer et al., 1992). A lacZ reporter gene, underthe control of the myoD enhancer and promoter, is expressedin a muscle-specific and spatiotemporal pattern that coincideswith the endogenous mouse myoD expression domain, asrevealed by in situ hybridization (present study; Buckingham,1992; Faerman and Shani, 1993; Faerman et al., in prepara-tioapptribsecprospe25emmy

protheshoof alsthascrmytiobechettio

D. J. Goldhamer and others

Figpatexpression of 11.5 d.p.c. whole-mount mouse embryo derived from astable line harboring the construct, 258/−2.5lacZ. β-gal expression isdetected in all myogenic compartments, including somites, limbbuds, and branchial arches. The spatial pattern of lacZ-expressingcells is similar to that of larger enhancer constructs (see Fig. 1). (B) β-gal expression of 11.5 d.p.c. Fo whole-mount mouse embryoinjected with the construct, 258/tklacZ. Expression with thisheterologous promoter construct is observed in all myogeniccompartments. Expression is most prominent in the dorsal portion ofthe most anterior somites and in the ventral portion of thoracicsomites, similar to constructs with the myoD promoter. Somitesposterior to the forelimb bud, however, show continuous lacZexpression along the dorsal-ventral myotomal axis, normallyobserved only in the most anterior somites. Ectopic, position effectstaining in the brain and dorsal root ganglia is observed in thisembryo. (C) β-gal expression of 11.5 d.p.c. Fo whole-mount mouseembryo injected with the E-box mutant construct, 258(E1-E3)/−2.5lacZ. The pattern of β-gal expression is the same as that of wild-type constructs. Ectopic staining in the neural tube is observed in thisembryo. (A-C) Arrows; β-gal expression in branchial arches. hb;hindlimb bud.

n). Transfection assays revealed a second, weak enhancerroximately 1 kb 3′ of the core enhancer, which may con-ute to quantitative levels of myoD expression in vivo. Thisond region, however, in combination with the myoDmoter, was neither necessary nor sufficient for muscle-cific expression of the lacZ transgene. We conclude that the

8 bp core enhancer mediates myoD activation in allbryonic skeletal muscle compartments, including somiticotomes, limb buds and branchial arches.

The relative contributions of the myoD core enhancer andmoter in directing myoD expression was tested by replacing myoD promoter with the heterologous tk promoter. Wewed that the myoD promoter is dispensable for activation

the myoD gene in the embryo, as this heterologous constructo yields muscle-specific expression of a lacZ reporter genet closely resembles the endogenous pattern of myoD tran-ipt accumulation. This experiment also establishes thatoD mRNA accumulation is dictated primarily by transcrip-nal control mechanisms rather than by mRNA turnover,ause no transcribed human sequences were present in theerologous promoter construct. Consistent with these func-nal data, the core enhancer is highly conserved in sequence

. 3. The 258 bp core enhancer directs the appropriate, myoDtern of expression in transgenic mouse embryos. (A) β-gal

45Regulation of the myoD gene in mou

(89% overall; 94% in the first 160 nucleotidebetween humans and mice, whereas 5′ flankingonly 66% similar within 250 bp of the start owith no extended regions of sequence simupstream of the TATA box (unpublished obser

eight regions with one or more hypersensSeveral hypersensitive sites are not assocprotection, probably reflecting interactiolower abundance or affinity. Only protecencompass consensus binding sites for k

Fig. 4. Sequence alignmencore enhancer and the corr ofthe mouse. Sequence similand 94% in the first 160 bp Iprotection assays (Fig. 5) iThe positions of DNase I p wsequence) and hypersensitabove sequence; the JEG-3hypersensitive site is denoshown. Not all of the hype nare produced with FC1010extracts (see Fig. 5). Proteproduced with JEG-3 nuclConsensus sequence bindi rknown transcription factorregions of protection, or th orpostulated to function in m n.

Fig. 5. DNase I protection assay of the humanusing nuclear extracts from myogenic (C2C12myogenic (10TG, FC1010, JEG-3) cell types. F(boxes) and multiple hypersensitive sites (astespecific hypersensitive site is denoted by a ‘+’extract does not protect region 2 and yields paregion 1 (most apparent on forward strand). FCextracts exhibit substantially different patternshypersensitive sites compared to the other cellextract yields the same footprinting profile as Cmyoblast nuclear extracts. The forward strandsequence shown in Fig. 4. Protected region 1 onot very apparent due to band compression neBSA, control lanes in which bovine serum albextract. G+A, purine-specific sequence ladder.

6se embryos

t of the human myoDesponding sequencearity is 89% overall,. Data from DNase s also summarized.rotection (bars belo

ive sites (asterisks-specific

ted by a ‘+’) arersensitive sites show and JEG-3 nuclearcted region 2 is notear extracts.ng sites are shown fos that reside withinat have been shownuscle gene regulatio

ofasallheofisDDl.,re

er-l.,he is.

ndofDn.nd

s) and position sequences aref transcription,ilarity presentvations). When

the tk promoter was used, however, the spatial patterning transgene expression was partially disrupted; the transgene wexpressed along the entire dorsal-ventral myotomal axis in somites, regardless of axial position (see below). Thus, tmyoD promoter may function to refine spatial domains myoD expression within the somite myotome. Also, it possible that the promoter regulates aspects of postnatal myoexpression; a mouse genomic clone consisting of the myopromoter and a regulatory element at −5 kb (Tapscott et a1992) that shares no obvious sequence similarity with the coenhancer, appropriately confers transgene expression prefentially in fast glycolytic fibers of adult muscle (Hughes et a1993). Our results, however, clearly establish that neither tpromoter nor the more proximal control element at −5 kbrequired for muscle-specific activation of myoD in embryos

myoD is subject to complex transcriptionalregulationThe extensive sequence similarity between the human amouse myoD enhancer and the complex patterns DNA/protein interactions observed in vitro indicate that myoexpression in the embryo is subject to complex regulatioDNase I protection assays identified five protected regions a

itive sites (Figs 4, 5).iated with detectablens with proteins ofted regions 4 and 5

nown factors; region

myoD core enhancer, HMP8) and non-ive protected regionsrisks; the JEG-3-) were detected. JEG-3rtial protection of1010 and JEG-3

of DNase I types. 10TG nuclear

2C12 and HMP8 corresponds to then the reverse strand is

ar the top of the gel.umin replaced nuclear

646

4 includetarget fowhereas rhistone Hnot knowsubstitutioaffect enhGoldham

With thnon-myogdifferent observed choriocarmouse spprimary similar toqualitativeexist withresults. Inyield a uencompas5). Thus, to FC1010shown to ‘ubiquitou(Andrewsmyogenicthe lack ofover regioJEG-3 celin JEG-3 cwith tissuenhancer adermal ce

vious transfection datachanisms repress myoDfootprinting gels revealsypersensitivity profilesC12, HMP8 and 23A2wn). Assuming that lessere not missed in this

plement of regulatoryer are similar, if not cells. In addition, thecells (Goldhamer et al., endogenous myoD genef these trans factors byeterodimerization withver, that the chromatinhe myoD core enhanceryogenic cells, but not in

D.

Fig. 6. Soumuscle-spemyoD coreand 23A2)the core en(arrowheadneuroblasthypersensicomparablstaining ofthe restrict

Footprinting experiments and presuggest that cis-acting epigenetic meexpression in 10TG cells. Inspection of no differences in protection or hproduced from 10TG extracts and C2myoblast extracts (Fig. 5; data not shostable muscle-specific interactions wassay, these data suggest that the comfactors that bind the myoD enhancidentical, in 10TG cells and myogenictransfected enhancer is active in 10TG

1992), indicating that repression of theis not a consequence of inactivation opost-translational modification or hnegative regulators. We found, howewithin and immediately surrounding texhibits DNase I hypersensitivity in m

J. Goldhamer and others

thern blot of DNase I treated chromatin, revealingcific DNase I hypersensitive sites in the endogenous enhancer. Chromatin from mouse myogenic cells (C2C12

10TG or other non-myogenic cells. We propose, therefore, that

s a consensus AP-1 site, representing a potentialr fos/jun complexes (Curran and Franza, 1988),egion 5 contains a consensus sequence important in4 gene expression (Dailey et al., 1988). While we do the molecular species bound to these sites, linker-n mutants encompassing these regions adverselyancer activity in transfection assays (Lukitsch and

er, unpublished observations). e exception of 10TG cells (see below), extracts fromenic cells yield footprinting profiles substantiallyfrom those of myogenic cells. These differenceswith human primary fibroblasts (FC1010) and humancinoma cells (JEG-3) do not simply reflect human andecies differences because extracts from human

myoblasts (HMP8) exhibit a footprinting profile

the myoD core enhancer is packaged in inactive chromatin in10TG cells, rendering it inaccessible to positive trans factorsrequired for gene activation. In theory, regulated, muscle-specific changes in accessibility of trans factors to myoDcontrol elements, mediated by chromatin structural changes orother epigenetic modifications such as DNA methylation,could be a critical prerequisite for myoD activation, with suchchanges being stably passed on to progeny cells (see Groudineand Weintraub, 1982; Razin and Cedar, 1991). In this regard,demethylation of specific CpGs in the mouse myoD enhancercorrelates with myoD expression both in cell culture and inmouse embryos (Brunk et al., unpublished data). Analysis ofthe temporal relationship between DNA demethylation,changes in chromatin structure, and the expression of myoDwill address whether these epigenetic modifications are a causeor an effect of myoD gene activation.

E-boxes in the core enhancer are not required formyoD activationThe presence of E-boxes in the myoD core enhancer raised thepossibility that myoD activation is regulated by direct transac-tivation by other bHLH myogenic factors. Auto- and cross-reg-ulatory interactions between the myogenic factors have beenwell-documented in cell culture systems (reviewed by

exhibits three DNase I hypersensitive sites; two map tohancer (arrows) and one maps just 5′ of the core enhancer). Non-myogenic mouse 10TG cells, NB41A3

oma cells, and BNL liver cells do not exhibit DNase Itivity. The degree of DNAse I digestion of total DNA ise in all cell types, as determined by ethidium bromide agarose gels. The relative position of the enhancer, andion enzymes and probe used are shown diagrammatically.

mouse C2C12 (and 23A2) myoblasts. Numerous and quantitative differences in hypersensitive sites FC1010 and JEG-3 extracts, as detailed in the addition, extracts from primary human fibroblastsnique pattern of protection and hypersensitivitysing and adjacent to the consensus AP-1 site (Figs 4,the protein species bound to this site may be unique cells. Recently, a hematopoietic-specific factor wasregulate globin gene expression by binding to as’ AP-1 site within the β-globin locus control region et al., 1993). The most dramatic difference between and non-myogenic binding activities, however, was protection over region 2 and only partial protectionn 1 with nuclear extracts from ectodermally derivedls (Figs 4, 5). As the transfected enhancer is inactiveells (Goldhamer et al., 1992), region 2 may interacte-restricted positive trans factors required forctivity; whether the protein(s) is restricted to meso-

lls will require further analysis.

Emerson, 1990; Olson, 1990). In addition, myogenin promoterfunction in embryos is E-box dependent, suggesting thatmyogenic factors regulate myogenin expression in vivo (Chenget al., 1993; Yee and Rigby, 1993). We found, however, thatenhancer constructs lacking the three conserved E-boxes(present study) or lacking all four E-boxes (see Fig. 4; unpub-lished observations), exhibit the wild-type pattern ofexpression at 11.5 d.p.c. In addition, both the wild-type andmutant myoD enhancers are activated in somites to the samecaudal extent, indicating no delay in transgene activation.Although, we cannot formally rule out the possibility that E-boxes in promoter sequences functionally substitute for theenhancer E-boxes in this construct, this is unlikely because themyoD promoter shows no muscle specificity in transgenic micewhen assayed alone (Goldhamer et al., 1992) or in combina-tion with enhancer 2 (Fig. 2B), and is dispensable for muscle-specific transgene expression. Thus, unlike many muscle-specific genes (see below), activation of myoD is not likely tobe mediated by E-boxes. Importantly, the present experiments

do not anance orelativemutant developmyoD.

myoDmechamusclemyoDexpressShani, 1anisms anteriorfactors d.p.c. tomyoD (Buckinand Rigactivatiexpressthe foreexpressration).somitesof exprtransgethe myotransgepositionfor endare fouof the mthe forpositiveof myomyf5 reentire d(ChengBuckinthese u

Clearregulatimyogenexpresskb upsthumansproximaet al., 1start of Yee andleast in genes cthe expr(Patapoexhibitsmyogenpromoteexpressdefined)

647Regulation of the myoD gene in mouse embryos

ddress the possible function of E-boxes in the mainte-f myoD expression, once activated; because β-gal is aly stable protein (see Paterson et al., 1991), E-boxconstructs will need to be investigated at later stages ofment further removed from the initial activation of

expression is controlled by regulatorynisms distinct from other characterized-specific genes

exhibits a distinct temporal and spatial pattern ofion (reviewed by Buckingham, 1992; Faerman and993; present results), indicating that regulatory mech-controlling myoD expression are unique. In the most somites of the mouse, for example, the myogenicare activated sequentially over a 2.5 day period from 8 10.5 d.p.c. in the order; myf5, myogenin, MRF4 and

Buckingham, 1992). Also, while myf5 (Tajbakhsh and

to regulate other muscle genes, including E-boxes, MEF-2sites, CArG boxes, and MCAT sites, only E-boxes are presentin the core enhancer. The E-boxes, however, are not requiredfor myoD activation. In striking contrast, the myogeninpromoter contains an E-box and a MEF-2 site, both of whichare critical for normal myogenin expression in embryos (Chenget al., 1993; Yee and Rigby, 1993). Together with possible epi-genetic mechanisms regulating the myoD enhancer by controlof chromatin structure and methylation status noted above,these data indicate that myoD is regulated by a control systemdistinct from the other characterized muscle-specific genes.

Analysis of the regulation and expression of the myogenicfactors has revealed heterogeneity among myotomal cell pop-ulations. Mutational studies of myogenin promoter functionrevealed MEF-2-dependent and -independent populations ofcells (Cheng et al., 1993; Yee and Rigby, 1993). In the presentstudy, we document the existence of spatially restricted popu-lations of myoD-positive and myoD-negative myotomal cells.

gham, 1994) and myogenin (Cheng et al., 1992; Yeeby, 1993) follow a strict rostral to caudal sequence of

on in somites, myoD transcripts and myoD transgeneion are first detected in thoracic somites at the level oflimb bud, followed approximately a half day later byion in more anterior somites (Faerman et al., in prepa- In addition, spatial domains of myoD expression within exhibit a distinctive axial position-dependent patternession. In somites anterior to the forelimb bud, myoDne expression is most prominent in the dorsal region oftome, whereas in somites posterior to the forelimb bud,

ne expression is most prominent ventrally. This axial-dependent patterning of expression is also observed

ogenous myoD transcripts. Finally, lacZ-positive cellsnd scattered throughout the dorsoventral myotomal axis

ost anterior somites, whereas in somites posterior toelimb bud, dorsal and ventral populations of lacZ- cells are separated by a spatially restricted population

D-negative myotomal cells. In contrast, myogenin andgulatory elements drive expression of lacZ along theorsal-ventral myotomal axis regardless of axial position et al., 1992; Yee and Rigby, 1993; Tajbakhsh andgham, 1994). The embryological signals that dictatenique expression patterns remain to be defined.

In addition, previous immunocytochemical studies identifiedmyosin-positive cells that express either MyoD or Myogenin,neither protein, or both proteins (Cusella-De Angelis et al.,1992). Finally, Myf5 protein appears to be expressed by morecells than MyoD in cultures derived from somites (Smith et al.,1993), although individual cells have not been assayed for bothmarkers. Whether these differential patterns of myogenic geneexpression reflect different fates or developmental potentialsamong these cell populations is presently unclear. Given theformative but functionally redundant roles of myf5 and myoDin skeletal muscle formation, it will be important to determinethe degree of concordance of myoD and myf5 expression inindividual myogenic cells. This will help distinguish whetherfunctional redundancy arises because myf5 and myoD havesimilar biochemical properties within the same cell or whetherdistinct myf5-positive and myoD-positive myogenic popula-tions can regulate their size and compensate for the loss of theother (see Emerson, 1993; Weintraub, 1993). Using myoDenhancer-lacZ transgenes to track myoD expression domainsin myf5 knockout mice will also address cellular compensatorymechanisms.

Activation of myogenic gene expression is initiated veryearly in presumptive myotomal cells (Ott et al., 1991; Pownalland Emerson, 1992), whereas presumably committed

differences also are emerging in the organization andon of cis sequences that control expression of theic genes, consistent with their distinct patterns ofion. While myoD is controlled by a distal enhancer 20ream of the gene, which is highly conserved between and mice, myogenin is regulated by highly conservedl promoter elements (compare sequences in Salminen

991 and Edmondson et al., 1992) within 200 bp of thetranscription (Salminen et al., 1991; Cheng et al., 1992; Rigby, 1993). myf5 and myf6 are also regulated, atpart, by 5′ flanking sequences (within 6 kb) as trans-

ontaining these sequences recapitulate some aspects ofession pattern of the corresponding endogenous genesutian et al., 1993). In addition, the myoD core enhancer no extended regions of sequence identity with thein promoter or other muscle-specific enhancers orrs (specific sequences controlling the embryonic

ion of myf5 and MRF4 genes have not yet been. Furthermore, among the specific DNA motifs known

myogenic cells migrate from the lateral somite (Ordahl and LeDouarin, 1992) into the developing limb before expression ofthe myogenic genes is detected (Sassoon et al., 1989; Chenget al., 1992; Yee and Rigby, 1993; Tajbakhsh and Bucking-ham, 1994). This raises the questions of whether these distinctskeletal muscle lineages are established and maintained bysimilar mechanisms, and whether myogenic cell fate in pre-sumptive limb muscle is governed by additional unknownfactors. As the myoD core enhancer functions as a moleculartarget to integrate ‘upstream’ embryonic signals and activatemyoD in all skeletal muscle lineages, functional dissection ofthe myoD enhancer will serve as a directed molecular approachto investigate upstream regulatory processes, including themechanisms of skeletal muscle lineage determination in theembryo.

We thank Yoshimichi Nakatsu for providing the mouse cosmidlibrary, and Marisa Bartolomei and Kristen Lukitsch for criticalcomments on the manuscript. This work was supported by grants from

648

the Amerof Healthfrom the sylvania,tion and United Sment Fun

REFER

Allen, N. DTransgedevelop

Andrews,Orkin, haemato

BlackwellDNA-bibinding

Braun, TArnoldgenes EMBO

Braun, TMyf-6, factors

Braun, TH. (198induce

Braun, TTargeteabnorm

Buckingh148.

Buskin, Jfactor creatin

Cheng, TMappinlinked

Cheng, TSeparaembryo

Church, Acad. S

Conlon, Ranterio116, 357

Couly, GdevelopDevelop

Cserjesi, P(1992). an essen1087-11

Cserjesi, Penhanceproduct

Curran, TCell 55,

Cusella-Ddifferen1243-12

Dailey, L.histone Dev. 2,

Davis, R. transfec

Edmondsrepressi

D

ican Cancer Society (IRG-135N) and the National Institutes (NIH; AR-42644) to D. J. G., by a CORE grant (CA-06927)NIH and an appropriation from the Commonwealth of Penn- by research grants from the Muscular Dystrophy Associa-the NIH (HD-07796) to C. P. E., and by a grant from thetates-Israel Binational Agricultural Research and Develop-d (BARD) to C. P. E. and M. S.

ENCES

., Cran, D. G., Reik, S. C. B. S. H. R. and Surani, M. A. H. (1988).nes as probes for active chromosomal domains in mousement. Nature 333, 852-855. N. C., Erdjument-Bromage, H., Davidson, M. B., Tempst, P. and

S. H. (1993). Erythroid transcription factor NF-E2 is apoietic-specific basic-leucine zipper protein. Nature 362, 722-728., T. K. and Weintraub, H. (1990). Differences and similarities in

Edmondson, D. G., Cheng, T. C., Cserjesi, P., Chakraborty, T. and Olson,E. N. (1992). Analysis of the myogenin promoter reveals an indirect pathwayfor positive autoregulation mediated by the muscle-specific enhancer factorMEF-2. Mol. Cell. Biol. 12, 3665-3677.

Edmondson, D. G. and Olson, E. N. (1989). A gene with homology to the mycsimilarity region of MyoD1 is expressed during myogenesis and is sufficientto activate the muscle differentiation program. Genes Dev. 3, 628-640.

Emerson Jr., C. P. (1990). Myogenesis and developmental control genes.Curr. Op. Cell Biol. 2, 1065-1075.

Emerson Jr., C. P. (1993). Skeletal myogenesis: genetics and embryology tothe fore. Curr. Opin. Genet. Dev. 3, 265-274.

Faerman, A. and Shani, M. (1993). The expression of the regulatory myosinlight chain 2 gene during mouse embryogenesis. Development 118, 919-929.

Fire, A., White-Harrison, S. and Dixon, D. (1990). A modular set of lacZfusion vectors for studying gene expression in Caenorhabditis elegans. Gene93, 189-198.

Goldhamer, D. J., Faerman, A., Shani, M. and Emerson Jr., C. P. (1992).Regulatory elements that control the lineage-specific expression of myoD.Science 256, 538-542.

Gossett, L. A., Kelvin, D. J., Sternberg, E. A. and Olson, E. N. (1989). A newmyocyte-specific enhancer-binding factor that recognizes a conserved

. J. Goldhamer and others

nding preferences of MyoD and E2A protein complexes revealed by element associated with multiple muscle-specific genes. Mol. Cell. Biol. 9,

site selection. Science 250, 1104-1110.., Bober, E., Buschhausen-Denker, G., Kotz, S., Grzeschik, K. and, H.-H. (1989). Differential expression of myogenic determination

in muscle cells: possible autoactivation by the Myf gene products. J. 18, 3617-3625.., Bober, E., Winter, B., Rosenthal, N. and Arnold, H.-H. (1990).a new member of the human gene family of myogenic determination: evidence for a gene cluster on chromosome 12. EMBO J. 9, 821-831.., Buschhausen-Denker, G., Bober, E., Tannich, E. and Arnold, H.9). A novel human muscle factor related to but distinct from MyoD1

s myogenic conversion in 10TG fibroblasts. EMBO J. 8, 701-709.., Rudnicki, M. A., Arnold, H.-H. and Jaenisch, R. (1992).d inactivation of the muscle regulatory gene Myf-5 results inal rib development and perinatal death. Cell 71, 369-382.am, M. (1992). Making muscle in mammals. Trends Genet. 8, 144-

. N. and Hauschka, S. D. (1989). Identification of a myocyte nuclearthat binds to the muscle-specific enhancer of the mouse musclee kinase gene. Mol. Cell. Biol. 9, 2627-2640..-C., Hanley, T. A., Mudd, J., Merlie, J. P. and Olson, E. N. (1992).g of myogenin transcription during embryogenesis using transgenes

to the myogenin control region. J. Cell Biol. 119, 1649-1656..-C., Wallace, M. C., Merlie, J. P. and Olson, E. N. (1993).

ble regulatory elements governing myogenin transcription in mousegenesis. Science 261, 215-218.G. M. and Gilbert, W. (1984). Genomic sequencing. Proc. Natl.ci. USA 81, 1991-1995.. A. and Rossant, J. (1992). Exogenous retinoic acid rapidly induces

r ectopic expression of murine Hox-2 genes in vivo. Development

5022-5033.Groudine, M. and Weintraub, H. (1982). Propagation of globin DNAase I-

hypersensitive sites in absence of factors required for induction: a possiblemechanism for determination. Cell 30, 131-139.

Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M.,Olson, E. N. and Klein, W. H. (1993). Muscle deficiency and neonatal deathin mice with a targeted mutation in the myogenin gene. Nature 364, 501-506.

Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989).Site-directed mutagenesis by overlap extension using the polymerase chainreaction. Gene 77, 51-59.

Hogan, B., Costantini, F. and Lacy, E. (1986). Manipulating the MouseEmbryo: A Laboratory Manual. Cold Spring Harbor Press, Cold SpringHarbor, NY.

Hughes, S. M., Taylor, J. M., Tapscott, S. J., Gurley, C. M., Carter, W. J.and Peterson, C. A. (1993). Selective accumulation of MyoD and MyogeninmRNAs in fast and slow adult skeletal muscle is controlled by innervationand hormones. Development 118, 1137-1147.

Konieczny, S. F. and Emerson Jr., C. P. (1984). 5-azacytidine induction ofstable mesodermal stem cell lineages from 10TG cells: evidence forregulatory genes controlling determination. Cell 38, 791-800.

Lee, W., Mitchell, P. and Tjian, R. (1987). Purified trancription factor AP-1interacts with TPA-inducible enhancer elements. Cell 49, 741-752.

Luckow, B. and Schutz, G. (1987). CAT constructions with multiple uniquerestriction sites for the functional analysis of eukaryotic promoters andregulatory elements. Nucleic Acids Res. 15, 5490.

Mar, J. H. and Ordahl, C. P. (1990). M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol. Cell. Biol. 10,4271-4283.

Miner, J. H. and Wold, B. (1990). Herculin, a fourth member of the MyoD

-368.. F., Coltey, P. M. and Le Douarin, N. M. (1992). Themental fate of cephalic mesoderm in quail-chick chimeras.ment 114, 1-15.., Lilly, B., Bryson, L., Wang, Y., Sassoon, D. A. and Olson, E. N.

MHox: a mesodermally restricted homeodomain protein that bindstial site in the muscle creatine kinase enhancer. Development 115,01.. and Olson, E. N. (1991). Myogenin induces the myocyte-specific

r binding factor MEF-2 independently of other muscle-specific genes. Mol. Cell. Biol. 11, 4854-4862.. and Franza Jr., B. R. (1988). Fos and jun: The AP-1 connection.

395-397.e Angelis, M., et al. (1992). MyoD, myogenin independenttiation of primordial myoblasts in mouse somites. J. Cell Biol. 116,55., Roberts, S. B. and Heintz, N. (1988). Purification of the humanH4 gene-specific transcription factors H4TF-1 and H4TF-2. Genes1700-1712.L., Weintraub, H. and Lassar, A. B. (1987). Expression of a singleted cDNA converts fibroblasts to myoblasts. Cell 51, 987-1000.on, D. G., Brennan, T. J. and Olson, E. N. (1991). Mitogenicon of myogenin autoregulation. J. Biol. Chem. 266, 21343-21346.

family of myogenic regulatory genes. Proc. Natl. Acad. Sci. USA 87, 1089-1093.

Minty, A. and Kedes, L. (1986). Upstream regions of the human cardiac actingene that modulate its transcription in muscle cells: presence of anevolutionarily conserved repeated motif. Mol. Cell. Biol. 6, 2125-2136.

Murre, C., McCaw, P. and Baltimore, D. (1989). A new DNA binding anddimerisation motif in immunoglobulin enhancer binding, daughterless,MyoD, and myc proteins. Cell 56, 777-783.

Nabeshima, Y., Hanaoka, K., Hayasaka, M., Esumi, E., Li, S., Nonaka, I.and Nabeshima, Y. (1993). Myogenin gene disruption results in perinatallethality because of severe muscle defect. Nature 364, 532-535.

Noden, L. (1991). Vertebrate cranial facial development. Brain Behav. Evol.38, 190-210.

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

Ordahl, C. P. and Le Douarin, N. M. (1992). Two myogenic lineages withinthe developing somite. Development 114, 339-353.

Ott, M.-O., Bober, E., Lyons, G., Arnold, H. and Buckingham, M. (1991).Early expression of the myogenic regulatory gene, myf-5, in precursor cellsof skeletal muscle in the mouse embryo. Development 111, 1097-1107.

Patapoutian, A., Miner, J. H., Lyons, G. E. and Wold, B. (1993). Isolatedsequences from the linked Myf-5 and MRF4 genes drive distinct patterns ofmuscle-specific expression in transgenic mice. Development 118, 61-69.

Regulation

Paterson, B. M., Walldorf, U., Eldridge, J., Dubendorfer, A., Frasch, M.and Gehring, W. J. (1991). The Drosophila homologue of vertebratemyogenic-determination genes encodes a transiently expressed nuclearprotein marking primary myogenic cells. Proc. Natl. Acad. Sci. USA 88,3782-3786.

Pollock, R. and Treisman, R. (1990). A sensitive method for thedetermination of protein-DNA binding specificities. Nucleic Acids Res. 18,6197-6204.

Pollock, R. and Treisman, R. (1991). Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 5, 2327-2341.

Pownall, M. E. and Emerson Jr., C. P. (1992). Sequential activation of threemyogenic regulatory genes during somite morphogenesis in quail embryos.Devel. Biol. 151, 67-79.

Prestridge, D. S. (1991). SIGNAL SCAN: A computer program that scansDNA sequences for eukaryotic transcriptional elements. CABIOS 7, 203-206.

Razin, A. and Cedar, H. (1991). DNA methylation and gene expression.Microbiol. Rev. 55, 451-458.

Rhodes, S. J. and Konieczny, S. F. (1989). Identification of MRF4: a newmember of the muscle regulatory factor gene family. Genes Dev. 3, 2050-2061.

Rigaud, G., Roux, J., Pictet, R. and Grange, T. (1991). In vivo footprinting ofrat TAT gene: dynamic interplay between the glucocorticoid receptor andliver-specific factor. Cell 67, 977-986.

Rudnicki, M. A., Braun, T., Hinuma, S. and Jaenisch, R. (1992).Inactivation of MyoD in mice leads to up-regulation of the myogenic HLHgene Myf-5 and results in apparently normal muscle development. Cell 71,383-390.

Rudnicki, M. A., Schnegelsberg, P. N. J., Stead, R. H., Braun, T., Arnold,H.-H. and Jaenisch, R. (1993). MyoD or Myf-5 is required for the formationof skeletal muscle. Cell 75, 1351-1359.

Salminen, A., Braun, T., Buchberger, A., Jurs, S., Winter, B. and Arnold,H.-H. (1991). Transcription of the muscle regulatory gene MYF4 is regulatedby serum components, peptide growth factors and signaling pathwaysinvolving G proteins. J. Cell Biol. 115, 905-917.

Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY.

Sassoon, D., Lyons, G., Wright, W. E., Lin, V., Lassar, A., Weintraub, H.and Buckingham, M. (1989). Expression of two myogenic regulatoryfactors myogenin and MyoD1 during mouse embryogenesis. Nature 341,303-307.

Shani, M. (1986). Tissue-specific and developmentally regulated expression of

a chimeric ac2631.

Smith, T. H., B(1993). A unproteins distimyogenic cel

Sun, X.-H. antranscription heterodimers

Tajbakhsh, S.determined inAcad. Sci. US

Tapscott, S. J.,enhancer elem5003.

Thayer, M. J., TWeintraub, determination

Treisman, R. (transcriptiona

Wachtler, F. anformation in v

Weintraub, Hnetworks, an

Weintraub, Hspecification

Wright, W. Eselection ofMol. Cell. B

Wright, W. Eregulating m617.

Xin, J. H., Cocloning andfamily that i6, 481-496.

Yee, S.-P. anexpression d1277-1289.

649 of the myoD gene in mouse embryos

tin-globin gene in transgenic mice. Mol. Cell. Biol. 6, 2624-

lock, N. E., Rhodes, S. J., Konieczny, S. F. and Miller, J. B.ique pattern of expression of the four muscle regulatory factornguishes somitic from embryonic, fetal and newborn mousels. Development 117, 1125-1133.d Baltimore, D. (1991). An inhibitory domain of E12

factor prevents DNA binding in E12 homodimers but not in E12. Cell 64, 459-470. and Buckingham, M. E. (1994). Mouse limb muscle is the absence of the earliest myogenic factor myf-5. Proc. Natl.A 91, 747-751. Lassar, A. B. and Weintraub, H. (1992). A novel myoblast

ent mediates MyoD transcription. Mol. Cell. Biol. 12, 4994-

apscott, S. J., Davis, R. L., Wright, W. E., Lassar, A. B. andH. (1989). Positive autoregulation of the myogenic

gene MyoD1. Cell 58, 241-248.1986). Identification of a protein-binding site that mediatesl response of the c-fos gene to serum factors. Cell 46, 567-574.d Christ, B. (1992). The basic embryology of skeletal muscleerterbrates: the avian model. Semin. Dev. Biol. 3, 217-227.

. (1993). The MyoD family and myogenesis: redundancy,d thresholds. Cell 75, 1241-1244.., et al. (1991). The myoD gene family: nodal point during of the muscle cell lineage. Science 251, 761-766.., Binder, M. and Funk, W. (1991). Cyclic amplification and targets (CASTing) for the myogenin consensus binding site.iol. 11, 4104-4110.., Sassoon, D. A. and Lin, V. K. (1989). Myogenin, a factoryogenesis, has a domain homologous to MyoD. Cell 56, 607-

wie, A., Lachance, P. and Hassell, J. A. (1992). Molecular characterization of PEA3, a new member of the Ets oncogenes differentially expressed in mouse embryonic cells. Genes Dev.

d Rigby, P. W. J. (1993). The regulation of myogenin geneuring the embryonic development of the mouse. Genes Dev. 7,

(Accepted 30 November 1994)