Proc. Natl. Acad. Sci. USAVol. 89, pp. 1291-1295, February 1992Biochemistry

Myb and Ets proteins cooperate in transcriptional activation of themim-1 promoter

(avian myeloblastosis virus/E26 vrus/trans-activation)

HENRYK DUDEK*, RAMANA V. TANTRAVAHI*, VEENA N. RAOt, E. SHYAM P. REDDYt, ANDE. PREMKUMAR REDDY*t*The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104; and lJefferson Cancer Institute, Life Sciences Building, ThomasJefferson University, 233 South 10th Street, Philadelphia, PA 19107

Communicated by Hilary Koprowski, November 13, 1991

ABSTRACT In the generation of the acutely transformingavian retrovirus E26, both myb and ets genes have beentransduced, leading to the production ofa Gag-Myb-Ets fusionprotein. This co-occurrence of v-myb and v-ets oncogenessuggests that the two might have a functional relationship. Tolook for such a relationship, we tested the transcriptionalactivation activity of Myb alone or with coexpressed Ets-1 orEts-2. Using the promoter of the v-Myb-inducible mim-1 geneas a target, we found that full-length c-Myb gene products werepoor activators of transcription, while an oncogenic (truncated)form of this protein was a strong trans-activator. However,coexpression of Ets-2 with full-length or truncated forms ofMyb greatly increased trans-activation. Coexpression of Ets-1,Fos, Jun, or Myc with Myb did not increase trans-activation ofthe mim-l promoter. The ability of Myb and Ets-2 to trans-activate was cooperative, since Ets-2 alone gave little or noactivation. Bacterially synthesized Ets-2 protein was found tobind specifically to the mim-1 promoter, suggesting that it maybe a target for both Myb and Ets proteins. Thus, Myb and Etsproteins can cooperate in transcriptional activation, and theirco-occurrence in the E26 virus may reflect a functional rela-tionship between these two oncoproteins. Truncated forms ofMyb may have a reduced need for cooperating factors such asEts-2, and this might constitute an important mechanismassociated with oncogenic activation.

The v-myb oncogene was first identified as the transformingcomponent of avian myeloblastosis virus (1, 2). Avian my-eloblastosis virus causes acute myeloblastic leukemia inchickens and transforms myelomonocytic cells in vitro (forreviews, see refs. 3 and 4). A second avian virus, termed E26,also was found to contain the myb oncogene but in associa-tion with a second transforming gene termed ets (5). E26 viruscauses predominantly erythroleukemia with a low level oc-currence of myeloblastic leukemia (6). c-myb, the normalcellular counterpart of v-myb, is expressed predominantly inhematopoietic cells (7, 8) and codes for at least two transla-tional products of 75 and 89 kDa (9, 10). A comparison of thesequence of the two v-myb genes with that of c-myb revealsthat the two viruses arose from the c-myb gene as a result ofextensive deletions at both the 5' and 3' ends of the codingregion (11). In addition to the two viral model systems, anumber ofmurine myeloid tumors have been characterized inwhich viral insertional mutagenesis has occurred at the c-myblocus, resulting in truncation of Myb protein (12, 13). Thus,a common route to myb activation appears to involve trun-cation of Myb protein.Myb proteins are localized in the nucleus (14) and bind

DNA with sequence specificity (15). Several studies have

now shown that Myb protein can function as an activator oftranscription (16-20). Most ofthese studies on transcriptionaltrans-activation by Myb have involved the use of syntheticpromoter/enhancer sequences, in which a basal promotersuch as the thymidine kinase (TK) gene promoter has beenlinked to tandem repeats of synthetic Myb-binding sites.While these studies have provided considerable useful infor-mation, they do not, however, represent a true in vivosituation, since no Myb-responsive genes have been foundthat contain such a tandem array of Myb-binding elements.Recently, Ness et al. (20) have identified a v-myb-inducedcellular gene termed mim-J, which contains Myb-bindingsites in its promoter and is transcriptionally trans-activatedby v-Myb protein. Identification of this naturally occurringMyb-responsive promoter/enhancer sequence provided uswith an opportunity to test the biochemical requirements fortranscriptional trans-activation by normal and activatedforms of the Myb protein.Our studies presented in this communication show that the

mim-J promoter is poorly trans-activated by full-lengthc-Myb gene products, while oncogenic (truncated) forms ofthis protein were strong trans-activators. This observationsuggested that optimal transcriptional trans-activation of anaturally occurring promoter such as mim-J might requireother factors in addition to Myb. The co-occurrence in E26of both v-myb and v-ets suggested the cellular homologues ofv-Ets as candidates for such cooperation with Myb. Wereport here that coexpression of Ets-2 with Myb significantlyincreased mim-J promoter activation. In addition, bacteriallyexpressed Ets-2 protein bound specifically to the mim-Jpromoter. Thus, Myb and Ets proteins can cooperate intranscriptional activation of mim-J.

MATERIALS AND METHODSPlasmids. myb vectors were generated by using pBC12/

CMV/IL-2 (21), which contained interleukin 2 sequencesunder the control of cytomegalovirus (CMV) early promoter.For the construction of Myb expression vectors, the inter-leukin 2 sequences were removed and the vector with CMVpromoter was used for cloning myb cDNAs. A mouse mybcDNA, encoding p75c-mYb, was subcloned to create pC75. Togenerate a myb cDNA containing exon 9A, a fragment of theABPL-2 myb cDNA (22) containing exon 9A was exchangedwith the corresponding fragment of c-myb cDNA lackingexon 9A. The resulting cDNA, encoding p89c-myb, was sub-cloned to create pC89. pCt encodes a C-terminal truncatedMyb protein (23). pCZ lacks myb coding sequences. Thesemyb expression vectors include a simian virus 40 origin ofreplication, and transfection into COS-1 cells, followed by invivo labeling and immunoprecipitation, confirmed expression

Abbreviations: TK, thymidine kinase; CMV, cytomegalovirus.tTo whom reprint requests should be addressed.

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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.

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of the expected proteins (H.D. and E.P.R., unpublisheddata). The mim-J promoter was isolated from chicken ge-nomic DNA by PCR and subcloned into pXP2 (24) to createpMIL. The promoter extends from the HindIII site at posi-tion -242 (20) to the transcription start site and was identicalto the previous isolate except at two locations: the G atposition -95 was deleted in our clone, and the sequence from-50 to -47 was 5'-GATC-3', rather than 5'-GTCC-3' (num-bering as in ref. 20). The reporter plasmid referred to here aspTA3 was kindly provided by Scott Ness and Thomas Graf(European Molecular Biology Laboratory, Heidelberg). Thisplasmid has three copies of the "A box" Myb binding sitefrom the mim-J promoter, cloned in tandem upstream of atruncated herpes simplex virus TK promoter, as described(20).For pSVfos, a rat c-fos cDNA (25), was subcloned into

pSV2neo (26) from which the neo gene had been removed.For pSVjun, a rat c-jun cDNA (27) was subcloned into pSG5(28). For pSVmyc, a human MYCcDNA from pCM8 (29) wassubcloned into pSG5. pSVets-1 was made by subcloning acDNA for human p54C'ts-1 (30) into the vector pSVTEXP(31). pSVets-2 was made by subcloning a human ETS2 cDNA(V.N.R. and E.S.P.R., unpublished data) into pSG5. ThepSVets-1 and pSVets-2 vectors have been shown to directhigh level Ets protein expression by transfection into COScells followed by in vivo labeling and immunoprecipitation(V.N.R. and E.S.P.R., unpublished data).To make the ets-2 bacterial expression vector, a human

ETS2 cDNA was amplified by PCR and subcloned intopDS56-6XHis (32). This construction removes the first sixamino acids ofEts-2, while adding six histidine residues, suchthat the N-terminal sequence is Met-rg-Gly-Ser-His-His-His-His-His-His-Gly-Ser-Lys-Asn-Met-Asp-Gln (non-Etsamino acids are underlined).

Cell Culture and Transient Gene Expression. QT6 cells (33)were grown in Dulbecco's modified Eagle's medium with10% fetal bovine serum and 10 mM Hepes (pH 7.4) andtransfected by calcium phosphate precipitation (34). Cells (2x 105) were seeded into 60-mm plates 1-2 days beforetransfection. Typically, 4 jug of each expression vector and0.5 ug of reporter plasmid were used. In addition, 0.2 ;kg ofreference plasmid, containing the lacZ gene driven by theRous sarcoma virus long terminal repeat, was included fornormalization. The total amount ofDNA in each transfectionwas kept constant by the addition of pBluescript (Strata-gene), and 1-2 days after the precipitate was removed, cellswere harvested, lysed by resuspension in lysis buffer (100mM potassium phosphate, pH 7.8/1 mM dithiothreitol/1%Triton X-100), and assayed for luciferase activity (35). f3-Ga-lactosidase activity was assayed by using chlorophenol redfB-D-galactopyranoside as the substrate, following the man-ufacturer's instructions (Boehringer Mannheim).

Bacterial Protein Expression. MC15 cells producing Ets-2were lysed in 50 mM sodium phosphate/6 M guanidinehydrochloride, pH 8.0, and Ets-2 protein was purified bynickel-chelate affinity chromatography (36). Protein concen-tration was determined by the Bradford procedure (37), usingBio-Rad dye and bovine serum albumin as a standard.SDS/PAGE was as described (38). The production of bac-terial t-Myb protein has been described (39).DNA Binding Assays. Mobility-shift assays were done

essentially as described (40). Each binding reaction mixturecontained 1 ,ug of poly(dI-dC)poly(dI-dC) and 3 ,ug of bovineserum albumin.

RESULTSTrans-Activation of the mim-l Promoter by Truncated but

Not Full-Length c-Myb Protein. Fig. 1 shows plasmid con-structs used to measure trans-activation in transient cotrans-

MYB EXPRESSION PLASMIDS

TA NR;!l_pC75

PC89 ZLI ,li~Ni! i"-JTA E9A NRMin I_

CMiV DNrAB TA

pCt ___....

REPORTER PLASMIDS

mpLrn- IRA

pMIL F FV i - LU1C.iERSR Ei--_

pTA3 __ 1lF'-l LUCIFERASE.

FIG. 1. Plasmid constructs used for trans-activation assays. Thinlines, plasmid backbone; boxes, promoters or coding regions.Hatched boxes, promoters (CMV immediate early for expressionplasmids; mim-1 or herpes simplex virus TK for reporters). DNAB,DNA-binding domain; TA, trans-activation domain; NR, negativeregulatory domain; E9A, exon 9A-derived sequences. Solid verticalbars in the promoters of pMIL and pTA3 indicate the Myb bindingsites.

fection assays. The expression vectors pC75 and pC89 en-code, respectively, p75c-mYb and p89c mYb, two normal mouseMyb proteins that differ in the presence of sequences en-coded by alternatively spliced exon 9A (9, 10). pCt encodesa C-terminal truncated c-Myb protein (t-Myb) analogous tothat expressed in the NFS-60 cell line (13, 41), in whichretroviral insertional mutagenesis has occurred at the myblocus. This protein lacks the negative regulatory domain (17)while retaining the DNA binding and trans-activation do-mains of Myb (16, 17). A control expression vector, pCZ,lacks myb sequences. The Myb-inducible promoter of themim-l gene (20) was cloned into a luciferase vector to yieldthe reporter plasmid pMIL. The mim-l promoter in pMILextends from -242 to the transcription start site, and includesthree separated Myb binding sites (20). The reporter plasmidpTA3 contains three copies of one of the strong Myb bindingsites from the mim-1 promoter, cloned in tandem upstream ofa truncated TK promoter (20).

Fig. 2A illustrates the levels of trans-activation seen withfull-length c-Myb (p75) and with t-Myb when used with themim-l promoter reporter. In these experiments, little trans-activation ofthe mim-l promoter was observed for full-lengthc-Myb, while t-Myb trans-activated expression of the lu-ciferase gene by 9-fold. To test whether higher trans-activation could be obtained with a synthetic target promoter,we tested the ability of c-Myb and t-Myb to trans-activatetranscription from reporter plasmid pTA3, containing threetandem Myb-binding sites upstream ofthe TK promoter. Theresults of this experiment are shown in Fig. 2B. In theseexperiments, we find that both c-Myb and t-Myb trans-activate this synthetic target promoter more strongly than theintact mim-l promoter. These results suggest that for anatural promoter such as mim-J, c-Myb alone is insufficientto achieve detectable levels of trans-activation.

Cooperative Trans-Activation of the mim-1 Promoter byMyb and Ets. The low transcriptional trans-activation of themim-I promoter by full-length c-Myb suggested the possibil-ity that efficient transcription of this promoter requires otherfactors in addition to c-Myb. The co-occurrence ofv-myb andv-ets in E26 suggested that the c-Ets proteins might interactfunctionally with Myb. Ets proteins have been shown to be

1292 Biochemistry: Dudek et al.

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A

pC75pCtpCZ

BpC75pCtpCZ

Activation, -fold

5 10 15

1.5

19.2J 0.7

=1.4

FIG. 2. Transcriptional trans-activation by full-length or trun-cated Myb proteins. Trans-activation assays were performed asdescribed in the text. Data are presented as -fold activation, definedas luciferase activity in the presence of the indicated expressionplasmids divided by luciferase activity for reporter alone. Luciferasevalues were normalized for PS-galactosidase activity before calcula-tion of -fold activation. Data are means of at least three separateexperiments. (A) Trans-activation of the reporter plasmid pMILcontaining the mim-1 promoter. (B) Trans-activation of reporterplasmid pTA3 containing three tandem Myb binding sites upstreamof the TK promoter.

nuclear proteins that bind DNA with sequence specificity andfunction in transcriptional activation (42-46). In addition,myb and ets are both expressed in hematopoietic tissues (7,8, 47, 48). To test whether coexpression ofEts proteins wouldaffect trans-activation, we cotransfected expression vectorsfor c-ets-J or c-ets-2 along with the different myb expressionvectors. Expression of ets genes was directed by the simianvirus 40 early promoter to avoid promoter competition ef-fects. As shown in Fig. 3A, cotransfection of an ets-) vector(pSVetsl) along with pC75 (expressing p75c-myb) caused noincrease in trans-activation above that obtained for Mybalone. However, cotransfection of an ets-2 vector (pSVets2)along with pC75 increased trans-activation by 9.2-fold. Sim-ilar results were obtained for p89CmYb. Coexpression of Ets-1with p89 did not increase trans-activation, while coexpres-sion of Ets-2 with p89 yielded 6-fold trans-activation. t-Myb,expressed from pCt, gave 9.2-fold trans-activation by itself,and this was not increased by Ets-1 coexpression. Coexpres-sion of Ets-2 with t-Myb, however, dramatically increased

trans-activation by 56-fold. Expression of Ets-1 or Ets-2individually yielded little or no trans-activation, indicatingthat the interaction between Ets-2 and Myb is cooperative.The control expression vector pCZ had no effect on mim-Ipromoter activity, either alone or with coexpressed Ets-1 orEts-2.To further determine whether the cooperation in mim-)

promoter activation was specific to Ets-2, we tested the effectofcotransfecting other protooncogenes with myb. In additionto ets-) and ets-2, we used vectors expressing fos, jun, ormyc. Fig. 3B shows the -fold activation oftranscription, whenexpression vectors were transfected either individually (Up-per) or along with pCt (expressing t-Myb) (Lower). Wheneach vector was transfected individually, only pCt yieldedmim-J promoter activation. When the other expression vec-tors were cotransfected with pCt, only cotransfection of theEts-2 vector (pSVets2) increased trans-activation over thatobtained for pCt alone. Cotranszfection offos, jun, myc, orets-1 vectors with pCt did not increase the level of trans-activation. In addition, the empty expression vector pSG5,which was used to make the ets-2 expression vector, had noeffect either individually or with pCt. Thus, Myb and Ets-2cooperate specifically in transcriptional trans-activation ofthe mim-J promoter.

Binding of Ets-2 to the mim-1 Promoter. To address themechanism of cooperation between Ets-2 and Myb, weexpressed the Ets-2 protein in bacteria and purified theprotein by nickel chelate chromatography (36). To determinewhether Ets-2 protein binds to the mim-J promoter, a 260-base-pair fragment (corresponding to the promoter se-quences present in pMIL) was labeled and used in mobility-shift assays with recombinant Ets-2 protein. As shown in Fig.4 (lane 2), Ets-2 protein bound strongly. This binding wassequence specific, since it could be efficiently blocked bycompetition with unlabeled mim-J promoter fragment (lanes3-5), while a heterologous fragment showed little or nocompetition (lanes 6-8).

Since the Myb protein is known to bind to the mim-Jpromoter (20), we tested whether Myb and Ets-2 proteinswould cooperate in binding. In this experiment, labeledmim-J promoter fragment was incubated with recombinantMyb or Ets-2 proteins either individually or in combinationwith each other. Fig. 5 shows the band shifts obtained forEts-2 alone (lane 2), t-Myb alone (lane 3), and Ets-2 andt-Myb together (lane 4). As expected, t-Myb yielded two

A

pC75pC75 + pSVetslpC75 + pSVets2

pC89pC89 + pSVetslpC89 + pSVets2

pCtpCt + p$VetslpCt + pSVets2

pCZpCZ + pSVetslpCZ + pSVets2

Activation, -fold10 20 30 40 50

]1.5]1.7

9.2

] 1.5] 1.7

16.1

19.211.8

56.0

10.70.4J0.9

pSVetsl ~0.6

pSVets2 ] 1.5

B

pCtpSVetslpSVets2pSVfospSVjunpSVmyc

pSG5

pCt + pSVetslpCt + pSVets2pCt + pSVfospCt + pSVjun

pCt + pSVmycpCt + pSG5

Activation, -fold10 15

14.1] 0.4

= 0.9=o0.8=1.0] 0.4

J0.6

13.3

2.3

13.512.8

13.3

FIG. 3. Synergistic trans-activation by Myb and Ets proteins. Trans-activation assays were performed as described in the text and in thelegend to Fig. 2, with pMIL used as reporter plasmid. (A) Myb and Ets expression vectors were transfected either individually or in combination.Ets-2 coexpression increased the trans-activation obtained for all three forms of Myb. Data are means of five separate experiments, except forpCZ transfections, which are from four experiments. (B) Specificity ofcooperation between Ets-2 and Myb. Expression vectors were transfectedeither individually (Upper) or with pCt (expressing t-Myb) (Lower). Individually, only t-Myb trans-activated the mim-) promoter. Only Ets-2coexpression increased t-Myb trans-activation. Data are means of four independent experiments.

Biochemistry: Dudek et al.

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0 0

0)c'J 0 O O

~00 ~-Q0

cN rid ~UJ -

< >x+ X X o X X)L)O 0 L O 0CMJ LO CM LO r-

U.

LiA ,l li ,ii i I

1 2 3 4 56 7 8

FIG. 4. Sequence-specific binding of Ets-2 to the mim-1 pro-moter. Lanes: 1, free probe; 2-8, binding obtained with 800 ng ofbacterial Ets-2 protein; 3-5, competition with unlabeled probe, in25x, 50x, and 100x molar excess, respectively; 6-8, competitionwith control fragment, in 25x, 50x, and 1OOX molar excess, respec-tively. Probe was the mim- promoter from pMIL, as a 262-base-pairBamHI/Sal I fragment. The control fragment was a 98-base-pair PstI fragment from the human ETS2 cDNA. Lane 2 shows strongbinding by Ets-2 protein, which was blocked by competition with themimn- fragment (lanes 3-5).

slow-migration complexes, since the mim- promoter has twohigh-affinity Myb binding sites (20). When Ets-2 and t-Mybwere present together, the band shift obtained was primarilythe sum of the band shifts obtained for Ets-2 alone and t-Mybalone. A single shifted band observed only when Ets-2 andt-Myb were both present is indicated by the arrow andprobably represents simultaneous binding by both Ets-2 andMyb to the mim-J promoter fragment. The low abundance ofthis species suggests that under these conditions recombinantEts-2 and t-Myb proteins do not heterodimerize or directlycooperate in binding. In addition, we have been unable todetect any physical association between Myb and Ets-2 inexperiments in which the two proteins were in vitro trans-lated, mixed, and tested for coimmunoprecipitation, despiteusing a variety of anti-Myb antibodies and immunoprecipi-tation buffer conditions (H.D. and E.P.R., unpublished data).These results suggest that the Myb-Ets-2 cooperativity intranscriptional activation may not be due to direct bindingbetween Myb and Ets-2 proteins.

DISCUSSIONUsing the promoter of the cellular mim-1 gene as a target forMyb trans-activation, we show here that coexpression ofEts-2 with Myb yields significantly greater activation thanthat obtained by Myb expression alone. This was observedwith two normal isoforms of c-Myb and with a truncated,activated form ofMyb (t-Myb). The level ofmim-1 activationby Ets-2 plus t-Myb was similar to that obtained previouslyfor the E26 Myb-Ets fusion protein (20). The interactionbetween Myb and Ets-2 is cooperative, since Ets-2 expres-sion alone yields little or no mim-1 activation. In addition, the

FIG. 5. Independent binding by Myb and Ets-2. Probe is the sameas in Fig. 4, but it contains additional flanking polylinker sequences.Lanes: 1, free probe; 2-4, binding obtained with, respectively, 370ng of Ets-2 (lane 2), 100 ng of t-Myb (lane 3), and 370 ng of Ets-2 plus100 ng of t-Myb (lane 4). Arrow indicates a complex observed onlywhen both Ets-2 and t-Myb were present.

effect is specific to Ets-2, since coexpression of Ets-1, Fos,Jun, or Myc proteins with Myb yielded approximately thesame mim-) promoter activity as that obtained with Mybalone. The ability of Ets-2, but not Ets-1, to cooperate withMyb is surprising, since v-ets in E26 is derived from c-ets-1,and not c-ets-2. It is possible that Ets-1 can interact with Mybwhen covalently linked to Myb, as in E26. Indeed, a recentstudy by Metz and Graf (49) shows that covalent linking ofMyb and Ets sequences is essential for the transformingactivity of E26 in vivo. It is also possible that intact Ets-1might interact with Myb under conditions different from ours,or with a target promoter other than mim-). The specificitywe have observed for Ets-2 is interesting, however, in viewof previous reports that ets-2 expression is more closelycorrelated with cell proliferation than is ets-) expression (48).

Since Ets proteins are known to be DNA-binding proteinsthat function in transcription activation (42-46), Ets-2 couldcooperate with Myb by binding to the mim-1 promoter. Ourresults show that recombinant Ets-2 protein does bind spe-cifically to the mim-1 promoter. However, we observed noheterodimerization or cooperativity in binding between Myband Ets-2, suggesting that the transcriptional cooperationdoes not involve direct Myb-Ets-2 heterodimerization. Onepossibility is that Myb and Ets-2 participate in a promotercomplex that includes additional bridging factors. This wouldbe consistent with models in which promoters are regulatedby multiple, synergizing inputs. While many eukaryotic tran-scription factors show modest binding specificity or affinityindividually, high overall binding specificity and affinity maybe provided by cooperative interactions between the multiplecomponents of a complex (for reviews, see refs. 50 and 51).Optimal activity by a transcriptional activator would there-fore require the presence of other components. In thiscontext, it is interesting that oncogenic versions ofMyb, suchas t-Myb, activate the mim-J promoter without exogenousEts-2 and may therefore be somewhat relieved of a normalregulatory requirement for other factors. This could consti-

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1294 Biochemistry: Dudek et al.

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Proc. Natl. Acad. Sci. USA 89 (1992) 1295

tute an important mechanism associated with the oncogenicactivation of this protooncogene.

Further support for a Myb-Ets functional relationshipcomes from recent studies by Metz and Graf (49, 52), whohave reported that the v-myb and v-ets components of E26contribute cooperatively to cell transformation. Our resultssuggest that the molecular basis for this may be cooperationin transcriptional activation. The presence of both v-myb andv-ets in E26 may not be coincidental; rather, this co-occurrence may represent the selected juxtaposition of twooncogenes whose products can cooperate in function.

We thank Dr. Bryan Cullen for the plasmid pBC12/CMV/IL-2;Dr. Grace Ju for pCM8; Dr. Tom Curran for c-fos and c-jun cDNAsand for pDS56-6XHis; and Drs. Scott Ness and Thomas Graf for theA box Myb reporter plasmid. We also thank Dr. Marcel Baluda forthe QT6 cell line and Dr. Frank Rauscher for many helpful discus-sions. This work was supported by National Institutes of HealthGrants CA52009 and CA21124 to E.P.R.; H.D. and R.V.T. weresupported under National Institutes of Health Training GrantCA09171.

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