Proc. Natl. Acad. Sci. USAVol. 87, pp. 5623-5627, August 1990Developmental Biology

MyoD binds cooperatively to two sites in a target enhancersequence: Occupancy of two sites is required for activation

(cooperativity/myogenesis/transcription)

HAROLD WEINTRAUB*, ROBERT DAVIS, DANIEL LOCKSHON, AND ANDREW LASSARFred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104

Contributed by Harold Weintraub, April 30, 1990

ABSTRACT MyoD is a master regulatory gene for myo-genesis. Its product, the MyoD protein, appears to act bybinding to muscle-specific enhancer sequences. We show thatMyoD binds cooperatively to two sites in the muscle-specificcreatine kinase enhancer; this is dramatically reflected indissociation-rate measurements. A deletion of the acidic Nterminus (residues 3-56) results in a protein that binds nor-mally to single sites but fails to bind cooperatively to twoadjacent sites, suggesting a role of the N terminus in cooper-ative interactions. In tranfection assays, a reporter geneflanked by a single MyoD binding site fails to be activated bycotransfected MyoD expression vectors. In contrast, a reporterwith two or more MyoD binding sites is activated by wild-typeMyoD but not by N-terminally deleted MyoD. A reporter genewith a single binding site, although not activated by MyoD, cannonetheless compete for expression with a reporter gene con-taining three sites. Thus, in vivo, a single site can bind MyoD,but occupancy of two or more sites is required for subsequenttranscriptional activation.

MyoD is a master regulatory gene for myogenesis. Expressionof MyoD from a viral long terminal repeat (LTR) activatesmany muscle-specific genes in a variety of differentiated celltypes (1) and the MyoD protein binds to many enhancers formuscle-specific structural genes (e.g., see ref. 2), either as ahomooligomer or as a heterooligomer with E2A proteins,ubiquitously expressed DNA-binding proteins (3-5).How MyoD expression is controlled during development is

not clear: MyoD transcription occurs only in presumptiveskeletal muscle (6-8); in frog embryos expression of MyoDoccurs rapidly after primary induction of mesoderm (8);MyoD activates its own transcription (9); somatic cell geneticexperiments (10) have shown that in nonmuscle cells atrans-acting factor from a specific chromosome negativelyregulates MyoD expression.There are also secondary controls on whether or not

expressed MyoD will activate downstream genes for myo-genesis. Thus, in many myoblast cell lines, MyoD RNA andprotein are present at the same levels when myoblasts areproliferating in high serum or when myoblasts begin toactivate the terminal myogenic program after removal ofserum (6). Similarly, in frog embryos, MyoD RNA appearswell before the appearance ofmuscle or muscle-specific actin(8). Possibly, the MyoD protein in myoblasts is in a form thatcan activate myoblast-specific genes (for example, the MyoDgene itself) but not myotube-specific genes (11).When proliferating myoblasts are induced to become mus-

cle by withdrawal of growth factors, individual cells seem tomake a decision to withdraw from the cell cycle and todifferentiate or to continue to grow and not to differentiate;that is, muscle-specific terminal differentiation genes are

usually activated as a program. The myogenic switch inducedby withdrawal of serum has analogies to the lysis/lysogenydecision in bacteriophage A (12) and the response of hunch-back to the bicoid gradient in Drosophila (13), both of whichare dependent on cooperative interactions between DNA-binding regulatory factors. Given this, the observation thatmyoblasts differentiate in an all-or-none way, and the factthat many MyoD-responsive enhancers contain severalMyoD binding sites, we decided to test whether MyoD canbind cooperatively to DNA. Here we describe the binding ofbacterially produced MyoD to the muscle-specific creatinekinase (MCK) enhancer (2, 14), which contains two MyoDbinding sites. We also show that activation of a chloram-phenicol acetyltransferase (CAT) reporter gene requires twoor more MyoD binding sites and that cooperative interactionsin vitro and synergistic activation of two MyoD binding sitesin vivo require the N-terminal 50 amino acids. This is anacidic region of MyoD distinct from the DNA-binding anddimerization domains-the basic (5) and helix-loop-helix(HLH; ref. 3) motifs (residues 100-162), respectively. Whileno function has previously been ascribed to the acidic Nterminus of MyoD, it is highly conserved among frogs, mice,and humans and therefore anticipated to be important.

PROCEDURESBinding Assays. Protein-DNA complexes were separated

in 1.4% agarose minigels (SeaPlaque low-melting; FMC)containing TBE buffer (50mM Tris base/50mM boric acid/1mM EDTA). Electrophoresis was at 180 V for 30-60 min.

Preparation of MyoD-glutathione transferase fusion pro-teins was described previously (2). The "AN" protein is afusion protein missing residues 3-56 of wild-type MyoD (6).Protein, diluted from a 0.1-mg/ml stock solution, was addedlast to a 10-il binding reaction mixture containing 1 ng of32P-labeled DNA in 20mM Hepes, pH 7.6/50mM KCI/1 mMEDTA/3 mM MgCI2/1 mM dithiothreitol/8% (vol/vol) glyc-erol/0.5% (vol/vol) Nonidet P-40 with poly(dI-dC) at 4mg/ml. Methylation interference and CAT assays were doneas described (2, 5).DNA. The following synthetic double-stranded oligodeox-

yribonucleotides were used: R site, 5'-GATCCCCCCAAC-ACCTGCTGCCTGA-3'; L site, 5'-ATTAACCCAGACAT-GTGGCTGCCCC-3'; R+L site, 5'-AACCCAGACATGTG-GCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAG-3'.In addition, the above R+L sequence was synthesized with5 or 10 additional deoxycytidine residues in the oligo(dC)stretch to give the + 1/2 and + 1 derivatives, respectively, andwith 5 or 10 fewer deoxycytidines to give the -1/2 and -1derivatives, respectively. Labeling of these for use in bindingassays was done by using T4 kinase and [y-32P]ATP on one

Abbreviations: CAT, chloramphenicol acetyltransferase; HLH, he-lix-loop-helix; LTR, long terminal repeat; MCK, muscle-specificcreatine kinase; TK, thymidine kinase.*To whom reprint requests should be addressed.

5623

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

5624 Developmental Biology: Weintraub et al.

strand, followed by annealing with a 2-fold molar excess ofthe second strand.The restriction fragments used in the binding assays were

HindIII-BamHI fragments from plasmids 3300 CPK-CAT,110 CPK-CAT (R+L sites), AL-110 CPK-CAT (R site), andB3-110 CPK-CAT (L site) and 1100-base-pair (bp) EcoO109I-BamHI fragments ofAEA and pD12D, kindly provided by J.Buskin and S. Hauschka (14). The fragments were 32P-labeledusing T4 kinase after phosphatase treatment.

Expression Assays. The plasmid vector pt(18)TKCAT, de-rived from pTKCAT (a gift from G. Schutz, German CancerResearch Center, Heidelberg), was used as a cloning vehiclefor various oligonucleotides; these were cloned into thefilled-in Sal I site ofthe polylinker. The sequence between theflanking vector sequences was determined to be, readingdownstream toward the thymidine kinase (TK) promoter,AGCAGGTGTTGGGAG (one R site), GGCAGCAGGT-GTTGGGAGGCAGCAGGTGTTGGAG (two R sites),AGGCAGCAGGTGTTAGGCAGCAGGTGTTAGGCAG-CAGGTGTTAG (three R sites), or AGCAGGTGTTGG-GAGGCAGCAGGTGTTGGGAGGCAGCAGGTGTTGG-GAGGCAGCAGGTGT (four R sites). In addition the R+Lsite and the + Y2, + 1, - Y2, and -1 oligonucleotides describedabove were also cloned into the filled-in Sal I site of thevector in both orientations and the sequence was subse-quently determined.

Transfection assays were done as described (1).

RESULTSCooperative Binding ofMyoD to theMCK Enhancer. MyoD

binds to two sites [the "right" (R) and the "left" (L) site] inthe muscle MCK enhancer (2, 14). Equilibrium titration withMyoD showed that the R site, the stronger site (2), became

ALR-

Wr

,x1: 0 1 2 3 4 5 1 2 3 4 5

L

MiltR

L-RR

WT

w

L gR -m eo

BOff-

Off-

cOf t _

!" 1kb

g_

FIG. 1. Cooperative binding ofMyoD to the two sites in the MCKenhancer. (A) Equilibrium binding. Increasing concentrations ofMyoD (100 Ag/ml; 0-5 pl added to a 10-pl reaction mixture) wereadded to either a wild-type (WT) or an R-site mutant (Mut-R)fragment from the MCK enhancer (1 ng per reaction mixture). After30 min, samples were electrophoresed. R, gel-shifted complex withpredominantly the R site filled; L+R, gel-shifted complex with the Land the R site filled; L, gel-shifted complex with the L site filled. (Band C) Dissociation of MyoD from the MCK enhancer. MyoD (5 Al)was allowed to equilibrate with either the wild-type (Lower) or R-sitemutant (Upper) fragment of the MCK enhancer as described above.In C, the same MCK sequence (R+L) was excised as a 1-kilobase(kb) fragment from the cloning vector. Competitor DNA (200 ng ofunlabeled R+L fragment) was added and at various times [0, 1, 2,4,8, 16, and 32 min (lanes left to right) in B; 0, 2, 4, 8, 16, and 32 minin C] samples were loaded directly onto a running agarose gel.

occupied first and then the L site filled (Fig. lA), as assayedpreviously by methylation protection and interference (2).There was no suggestion of cooperativity between sites sincethe L site filled, in this equilibrium assay, at the sameconcentrations of MyoD whether or not the R site wasmutated (Fig. 1A). In separate experiments (data not shown),the R site filled identically whether or not the L site wasmutated.Cooperative interactions were further studied by assaying

the dissociation rate ("off" rate) for MyoD binding. MyoDwas bound at relatively high concentration to either the entireMCK enhancer fragment (R+L) or a fragment containingonly the L site as a result of a mutation in the R site. After30 min, an excess of unlabeled enhancer fragment was addedand at successive time points, samples were loaded imme-diately onto agarose gels. The level of competitor was suchthat in equilibrium studies, it inhibited binding of labeledfragment by 20-fold. Increasing the level by a factor of 10 didnot alter the dissociation rate (see below), indicating that thecompetitor does not actively remove bound MyoD at theseconcentrations. Within a minute, MyoD dissociated from theL site ofa fragment where the R site was unoccupied becauseof mutation (Fig. 1B Upper). When the L site was mutated,dissociation occurred from the R site within 5 min (data notshown). In contrast, when both sites were simultaneouslyfilled in the wild-type fragment, molecules containing thedoubly occupied sites were stable for at least 30 min (Fig. 1BLower); within the same population, singly filled DNA mol-ecules (at the R site) dissociated within 10 min, although thisis likely to be an overestimate since dissociation ofthe doublyoccupied site probably involves a singly occupied interme-diate. For a 1-kb fragment containing two sites (Fig. 1C),there was also a much slower dissociation from doubly filledsites. Thus, filling both sites stabilizes the binding to the Lsite by a factor of 30 and the binding to the R site, by a factorof 3 or 4 as compared to the dissociation of MyoD bound tosimilar fragments containing either a mutated R site or amutated L site.

Since the dissociation-rate studies suggest a physical in-teraction between MyoD complexes bound at the R and Lsites, we conclude that MyoD binds cooperatively to the twosites in the MCK enhancer. To account for the fact thatcooperative binding of MyoD was not observed with equi-librium measurements (Fig. LA), we assume that MyoDbound at the R site inhibits the on rate for binding to the L siteand that the two effects-decrease in on rate and decrease inoff rate-essentially cancel each other. The molecular basisfor this presumed interference is not known and, as far as weare aware, such interference is unprecedented; however, itseems to be enhanced by MyoD mutants missing the MyoDN-terminal region (see Fig. 2 and text). Perhaps a singleMyoD complex bound at the R site can interact simultane-ously with the L site.The R and L sites of the MCK enhancer are normally

separated by a string of 13 deoxycytidine residues (14). Toinvestigate whether the spacing between the R and L siteswas critical to the cooperativity detected in the off-rate assay,fragments containing the R and L sites separated by an extra½2 turn or 1 turn of DNA were constructed with 5 or 10additional dC-dG base pairs; fragments deleted by Y/ or 1 turnwere also made. The off rate for all four constructs was notdramatically different, suggesting that cooperativity is notsensitive to helical pitch (data not shown; however, see Fig.2D below). Presumably, the interacting surfaces of MyoDcomplexes at the R site and MyoD complexes at the L site arerather flexible. Moreover, cooperativity is also apparentusing dimers of the R site, suggesting that neither thepoly(dC) stretch nor some unique property of the L site isrequired (data not shown).

Proc. NatL Acad Sci. USA 87 (1990)

Proc. NatL Acad. Sci. USA 87 (1990) 5625

The N-terminal 50 Residues of MyoD Are Required forCooperativity. Previous work showed that bacterial MyoDdeleted of either the basic region (residues 102-121) or helix2 of the HLH region (residues 143-162) failed to bind DNA(2, 5). Mutants with deletions of the acidic N terminus(residues 3-56; AN-MyoD) or the C terminus (residues167-318; AC-MyoD) bind DNA and also activate myogenesis(2, 5). In a more thorough investigation ofthese mutants, bothAN-MyoD and AC-MyoD bound the isolated R and L sitesequivalently for both equilibrium (Fig. 2A and B) and off-rateassays (data not shown), and the AC-MyoD mutant (whichmigrates much faster as a complex due to the smaller size ofAC-MyoD) bound to the R+L fragment like wild-type MyoD(Fig. 2C). In contrast, at these concentrations AN-MyoD(Fig. 2C) filled the L site only marginally in the R+L fragment.Attempts to overcome the failure ofAN-MyoD to fill the L siteby changing the helical relationship between the R and L sitesby 5-bp or 10-bp insertions and deletions were not successful(Fig. 2D).

Cooperative binding of MyoD requiring the N terminus isalso suggested from methylation interference results (Fig. 3).When MyoD binds to the individual R and L sites (onseparate DNA fragments), specific methylation interferenceand protection patterns were seen (2). Similarly, methylationprotection was observed over both the R and the L site indoubly occupied fragments containing both sites (2). Inmethylation interference experiments using fragments thatcontained both sites, the typical R-site interference patternwas seen for singly or doubly occupied species with wild-typeMyoD, AN-MyoD, and AC-MyoD (Fig. 3, vertical bar R).Surprisingly, for doubly occupied fragments the typical L-site interference pattern was not seen forMyoD or AC-MyoD(arrows) even though both the R and L sites were occupiedas determined by mobility shift, methylation protection, andgenetic deletion analysis (2). In contrast, with doubly occu-pied fragments, using concentrations of AN-MyoD higherthan those shown in Fig. 2 (but equivalent to those used forwild-type MyoD and AC-MyoD in these experiments), inter-ference was seen over both the R site and (weakly, butreproducibly) the L site (Fig. 3, arrowheads). We interpretthese data to mean that doubly occupied fragments withMyoD or AC-MyoD can occupy methylated L sites becauseof cooperative interactions with MyoD at occupied R sites;hence, no interference over the L site is seen. With AN-MyoD, cooperative interactions are weaker. However, whendouble occupancy is forced with high levels of AN-MyoD,more of the binding energy depends upon proper contacts ofAN-MyoD with specific functional groups in the L site;hence, methylation interference is observed when these

groups are modified. Le Bowitz et al. (15) have made similarobservations for cooperative binding of the Oct-2 protein toadjacent octamer (strong) and heptamer (weak) sites, wheremethylation interference is more dramatic at the heptamersite when the octamer site is deleted.The bacterial MyoD we used for these studies was a fusion

protein with glutathione transferase (2, 5); however, coop-erative binding ofMyoD was probably not a consequence ofthe glutathione transferase moiety since AN-MyoD, also afusion protein, failed to show cooperative binding and inrecent experiments, a non-fusion MyoD protein made inbacteria also showed cooperative binding that depended onthe N terminus.MyoD Activation ofMCK in Vivo Requires Multiple Binding

Sites. Reporter constructs with one or two R sites linked 30bp upstream of a herpes virus TK promoter-CAT reportergene (1R vs. 2R) were cotransfected into C3H/1OTY2 mousefibroblasts with LTR expression vectors encoding wild-typeMyoD, AN-MyoD, or AC-MyoD (Table 1). A significantsignal was observed only with the reporter construct con-taining two R sites. Constructs with three or fourR sites wereslightly more active (2-fold) than those with 2 R sites (data notshown); orientation of the R sites (i.e., two or three R sitesfacing toward or away from the promoter) seemed to havelittle effect on activity or lack of activity (data not shown).MyoD and AC-MyoD gave comparable signals; significantly,the AN-MyoD construct showed low activity, even on re-porter constructs with four sites. As with the DNA-bindingstudies, we also inserted and deleted S or 10 bp between theR and L sites in these CAT reporters and found very littleeffect on MyoD-dependent expression (data not shown). Asa control, the AN-MyoD construct can activate a morecomplicated reporter where the entire 3.3-kb MCK upstreamregion and promoter drove the CAT gene (Table 1). AN-MyoD can convert C3H/1OTY2 cells to muscle and it forms atight heterooligomer with E12 that binds specifically to theMCK enhancer (5). Thus, it seems that in the minimalconstruct with two (or even three or four) R sites driving theCAT gene, AN-MyoD cannot activate in vivo; however, inthe more complex case, with the 3.3-kb MCK upstreamregion, presumably additional MyoD binding sites or, morelikely, additional factors [e.g., MEF-2, another muscle-specific DNA-binding activity with sites adjacent to theMCKenhancer (16)] stabilize AN-MyoD binding. Interactions withthese components would presumably depend upon regions ofMyoD other than the N terminus.To determine whether MyoD could actually bind in vivo to

a single site, we tested the ability ofsuch a plasmid containinga single R site to compete for CAT expression from a vector

A

0 0 :

RC

&N-mm f AC_-

C

R-L

B.. :

II-L

AIN*N_ acC--

D

R-L_

R.L FIG. 2. The N terminus ofR MyoD is needed for joint occu-

pancy of the R and L sites. (A andB) AN-MyoD or AC-MyoD (100p g/ml; 0, 1, 2, 3, or 4 ,ul) wasadded to 1 ng of oligonucleotide(25 bp) containing either the R orthe L site. (C) AN-MyoD or AC-MyoD (0, 1, 2, 3, 4, orS LI) was

iA~j &1tC' ifi iadded to 5 ng of fragment (48 bp)AL'" j containingboth Rand L sites. (D)

R and L sites separated by inser-tions or deletions of 1/2 or 1 turn insuccessive groups of three laneswere mixed with 3 ,ul of buffer,AN-MyoD, or AC-MyoD. In allcases in D, AN-MyoD gave a sin-gle-band shift whereas AC-MyoD

+1/2 0 -1/2 -gave a double shift.

Developmental Biology: Weintraub et al.

OIL

5626 Developmental Biology: Weintraub et al.

A C MyoD

R R

L R F R L R F_ _

- t -9 *am T -' a. w

amamat

RI

dwm ,41 *So *-

_am gmM-._y __X_ _

_ am t _ _ -_ _ _

4

Table 1. Two R sites, but not one, can lead toMyoD-dependent activation

Reporter Relative CAT activitytemplate EMSV MyoD AN-MyoD AC-MyoD

1R-CAT 3 6 0 42R-CAT 1 54 3 45MCK-CAT 0 100 89 95

Expression vectors containing one or two R sites linked topTK-CAT (1R-CAT and 2R-CAT) or containing 3.3 kb of MCKpromoter and upstream region linked to CAT (MCK-CAT) werecotransfected into C3H/1OTY2 cells with Moloney murine sarcomavirus LTR enhancer-promoter vectors driving expression of wild-type MyoD, AN-MyoD, or AC-MyoD, or as a control, with theparental expression vector (EMSV). CAT activities have been nor-malized to those obtained using MyoD to activate MCK-CAT andrepresent averages from three separate experiments.

containing three binding sites. Fig. 4 shows that the plasmidcontaining a single R site driving TK-CAT, which itself willnot express CAT above background (Table 1), can, never-theless, inhibit expression from a vector containing threesites; the identical control vector without a MyoD bindingsite (TK-CAT) does not inhibit.We conclude that in the minimal situation with R sites

driving TK-CAT, two or more R sites are needed for MyoDactivation and that the N terminus ofMyoD is required for thetranscription machinery to recognize these multiple occupiedsites.

DISCUSSIONOur results show that, as measured by dissociation rate,MyoD binds cooperatively to the two binding sites in theMCK enhancer; that cooperativity requires (either directly orindirectly) the acidic N-terminal 50 amino acid residues ofMyoD; and that, in vivo, even though a single site can beoccupied by MyoD, two or more binding sites, as well as theN terminus ofMyoD, are required for activation ofa minimalmuscle-specific reporter gene containing multimerized Rsites.Myogenesis requires activation of a large battery of struc-

tural genes. How a MyoD-activated switch coordinatelyturns on all of these genes is unclear. One possibility is thatonce effective MyoD levels reach a critical threshold (i.e., theswitch is activated), MyoD first activates the myogenic genessuch as MyoD itself (9), myogenin (17), Myf-S (18), andherculin (19)/Myf-6 (18)/Mrf4 (20), so that very high, effec-tive levels of these myogenic activators are rapidly achieved(i.e., the switch is stabilized). This would initiate, and committhe cell to, a programmed expression of downstream genes.In this model, the role of multiple myogenic activators is topromote high-level expression once the switch is activated,

w.___wW

B i* _ __~~~~~~Opo-4400.Nw

myoD - ttk-cat1R-cat3R-cat

FIG. 3. Cooperativity revealed by methylation interference. AN-MyoD, AC-MyoD, or wild-type MyoD (10 Al) was mixed withmethylated fragments (1 ng) containing both the R and the L site.Vertical bars indicate the interference patterns over the R site andover the L site. Shown are the fragments associated with the free(lanes F), singly occupied (lanes R), or doubly occupied (lanes R+L)complexes. Arrowheads indicate methylated guanosine residues thatinterfere with AN-MyoD binding to L site; methylation ofthese sameresidues does not interfere with binding of AC-MyoD or wild-typeMyoD (see arrows).

FIG. 4. A single R site can compete in vivo with a vectorcontaining three R sites. The MyoD expression vector (5 ,ug) wascotransfected in duplicate into C3H/10T½/2 cells together with a

control TK-CAT vector (25 psg); with a competing vector containinga single R site (lR-CAT, 20.ug) and a reporter containing three R sites(3R-CAT, 5 ,ug); or with a control competing TK-CAT vector (20 ,g)together with a reporter containing three R sites (5 1%g). Cell lysateswere incubated with radioactive chloramphenicol and CAT activitywas detected by TLC separation of the labeled substrate and itsacetylated products.

A N

R

IF R L R F R

L

Proc. NatL Acad. Sci. USA 87 (1990)

Proc. Natl. Acad. Sci. USA 87 (1990) 5627

which might occur at any of the myogenic determining genesdepending on the particular developmental situation.A major distinction (e.g., see ref. 21) is whether the

requirement for multiple MyoD binding sites for in vivoexpression reflects the need for the activation machinery (oran adaptor) to "touch" more than one MyoD-containingcomplex simultaneously, or whether "touching" a singlecomplex will do, but extra sites are needed to assure that atleast one site remains filled. The two alternatives above mightbe resolved by overexpressing MyoD and asking whether areporter with a single R site will respond. Thus far, this typeof experiment has shown no activation. Since a plasmid witha single MyoD binding site can specifically compete intransfection assays with the CAT vector containing three Rsites, it is likely that MyoD can bind a single site in vivo.Consequently, we favor the notion that activation requiressimultaneous recognition of at least two occupied MyoDbinding sites. This would be compatible with cooperativebinding of MyoD (see also refs. 22-24); otherwise, a singlebound MyoD site might activate transcription and the poten-tial regulatory advantages provided by cooperativity wouldbe lost. This is a particularly acute problem with MyoD sinceits recognition sequence, CANNTG, is expected to be pres-ent once every 250 bp.Given that MyoD binds cooperatively to multiple sites, it

was surprising that a single site was able to compete soeffectively in the transfection experiments (Fig. 4) at such amoderate excess; however, the in vitro equilibrium bindingresults (Fig. 1) demonstrating marginal effects of cooperativ-ity at equilibrium are consistent with this result. Both oftheseresults might suggest that cooperativity (as measured by thedissociation rate) is not important in vivo. Rather, we proposethat expression from a MyoD-driven reporter requires twoevents in series. The first is equilibrium binding to multiplesites. In this step, vectors with a single site can compete forMyoD binding. The second event is recognition of multipleMyoD complexes by the transcription machinery as dis-cussed above. This seems to require simultaneous occupancyof two or more sites. The demonstration in vitro that thedissociation rate decreases by a factor of 30 when two sitesare simultaneously occupied with MyoD complexes suggeststhe hypothesis that when functional MyoD reaches highenough levels to fill multiple sites, cooperativity keeps thesesites jointly occupied for a long enough period of time so theycan be jointly recognized by the transcription machinery.

We thank Hazel Sive for suggestions and comments. This work

was supported by the National Institutes of Health. A.L. wassupported by a grant from the Lucille Markey Foundation.

1. Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J.,Adam, M. K., Lassar, A. B. & Miller, A. D. (1989) Proc. Natl.Acad. Sci. USA 86, 5434-5438.

2. Lassar, A. B., Buskin, J. N., Lockshon, D., Davis, R. L.,Apone, S., Hauschka, S. D. & Weintraub, H. (1989) Cell 58,823-831.

3. Murre, C., McCaw, P. S & Baltimore, D. (1989a) Cell 56,777-783.

4. Murre, C., McCaw, P. S., Vassin, H., Caudy, M., Jan L. Y.,Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D.,Lassar, A. B., Weintraub, H. & Baltimore, D. (1989b) Cell 58,537-544.

5. Davis, R. L., Cheng, P.-F., Lassar, A. B. & Weintraub, H.(1990) Cell 60, 733-746.

6. Davis, R. L., Weintraub, H. & Lassar, A. B. (1987) Cell 51,987-1000.

7. Sasoon, D., Wright, W., Lin, U., Lassar, A., Weintraub, H. &Buckingham, M. (1989) Nature (London) 341, 303-307.

8. Hopwood, N. D., Pluck, A. & Gurdon, J. B. (1989) EMBO J.8, 3409-3417.

9. Thayer, M. J., Tapscott, S. J., Davis, R. L., Wright, W. E.,Lassar, A. B. & Weintraub, H. (1989) Cell 58, 241-248.

10. Thayer, M. J. & Weintraub, H. (1990) Cell, in press.11. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. &

Weintraub, H. (1990) Cell 61, 49-59.12. Ptashne, M. (1987) A Genetic Switch (Blackwell, Palo Alto,

CA).13. Driever, W. & Nusslein-Volhard, C. (1989) Nature (London)

337, 138-143.14. Buskin, J. N. & Hauschka, S. D. (1989) Mol. Cell. Biol. 9,

2627-2640.15. Le Bowitz, J. H., Clarc, R. G., Brenowitz, M. & Sharp, P.

(1989) Genes Dev. 3, 1625-1638.16. Gossett, L., Kelvin, D. J., Sterberg, E. A. & Olson, E. N.

(1989) Mol. Cell. Biol. 9, 5022-5033.17. Wright, W. E., Sasoon, D. A. & Lin, V. K. (1989) Cell 56,

607-617.18. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E. &

Arnold, H. H. (1989) EMBO J. 8, 701-709.19. Miner, J. H. & Wold, B. (1990) Proc. Natl. Acad. Sci. USA 87,

1089-1093.20. Rhodes, S. J. & Konieczny, S. F. (1989) Genes Dev. 3, 2050-

2061.21. Giniger, E. & Ptashne, M. (1988) Proc. Natl. Acad. Sci. USA

85, 382-386.22. Jansen-Durr, P., Boeuf, H. & Kedinger, C. (1989) EMBO J. 8,

3365-3370.23. Tsai, S. Y., Tsai, M. & O'Malley, B. (1989) Cell 57, 443-448.24. Schmid, W., Strahle, U., Schfitz, G., Schmitt, J. & Stunnen-

berg, H. (1989) EMBO J. 8, 2257-2263.

Developmental Biology: Weintraub et al.