Development 107, 945-956 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

945

Transactivation of the adenovirus Ella promoter in the absence of

adenovirus E1A protein is restricted to mouse oocytes and preimplantation

embryos

THOMAS P. DOOLEY1-*, MIRIAM MIRANDA2, NICHOLAS C. JONES1 and MELVIN L.

DePAMPHILIS2

'Gene Regulation Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England2Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110,USA

* Author for correspondence; current address: The Upjohn Company, Unit 7235-25-12, Kalamazoo, Michigan 49001, USA

Abstract

Undifferentiated mouse embryonal carcinoma (EC) cellsare capable of transactivating the adenovirus Ellapromoter in the absence of its normal transactivator,E1A protein, suggesting that EC cells contain an E1A-like activity. In an effort to identify where this activityappears during normal mouse development, mouseoocytes and preimplantation embryos were injected withplasmids containing the Ella promoter coupled to vari-ous reporter genes. These expression vectors were fullyactive in human 293 cells where E1A is present, but wereinactive in differentiated fibroblast cell lines unlesscotransfected with the El A gene. In mouse oocytes andpreimplantation embryos, Ella promoter activity in theabsence of adenovirus E1A protein was equivalent to orgreater than activity of the HSV thymidine kinasepromoter coupled to a strong enhancer. Coinjection ofthe E1A gene failed to stimulate Ella activity further,perhaps because c-myc protein, which has been reportedto transactivate this promoter, was already present athigh levels in mouse oocytes. Activation of the Ella

promoter in the absence of E1A was unique to mouseoocytes and preimplantation embryos because geneexpression from an Ella promoter introduced intotransgenic mice was observed only in the adult ovary,and particularly in the oocytes. In addition, post-implantation transgenic embryos failed to express theElA-activatable reporter gene, thereby indicating thatexpression from the Ella promoter is restricted to therelatively undifferentiated stages of oogenesis and pre-implantation development. These data suggest that cel-lular promoters of the class that can be transactivated byEl A may serve uniquely to initiate transcription of genesthat are needed for preimplantation development.

Key words: embryonal carcinoma cells (EC cells), El A,Ella, mouse oocytes and embryos, transcriptionalactivation, transgenic mice, microinjection, c-myc,adenovirus, chloramphenicol acetyltransferase,/3-galactosidase, luciferase.

Introduction

At present little is known about the transcriptionalregulatory mechanisms that govern the processes ofdifferentiation and early embryogenesis in mammals,although genetic and molecular approaches are begin-ning to reveal insights into some of these mechanisms incertain invertebrates, such as Drosophila and Caenor-habditis elegans. One approach to studying mammalianembryogenesis and differentiation involves the use ofembryonal carcinoma (EC) cell lines. An intriguingproperty of undifferentiated EC cells, such as F9 andPCC4, is that they are capable of activating transcrip-tion from the human adenovirus Ella promoter, in the

absence of its natural transcriptional activator, the El Agene product (Imperiale et al. 1984). The observationthat the Ella promoter is significantly more active inundifferentiated versus differentiated cells has resultedin the suggestion that certain EC cell lines express an'ElA-like' transcriptional regulatory property, which islost upon differentiation of EC cells (Imperiale et al.1984). Furthermore, EC cells fail to efficiently expressgenes linked to a number of viral enhancer elements,(derived from SV40, polyoma, MLV, etc.) which areknown to be repressed by E1A in differentiated celllines, consistent with the presence of an ElA-likeactivity (Schneider et al. 1987; Gorman et al. 1985;Sleigh and Lockett, 1985; Borrelli et al 1984; Jones,

946 T. P. Dooley and others

1986; Cremisi and Babinet, 1986; Barklis et al. 1986;Taketo and Tanaka, 1987). Whether the two ElA-likeactivities, transactivation and enhancer repression, areencoded for by the same endogenous gene productremains to be seen, for the identities of both cellularputative transcriptional regulators are as yet unknown.At present, no cellular gene has been reported thatencodes a cDNA homologous to El A. Therefore, theseputative cellular ElA-like activities are likely to becellular proteins whose properties are functionally anal-ogous to E1A, rather than a cellular homologue of thisviral oncogene. Furthermore, although the mechanismby which the Ella promoter is transactivated is not yetfully understood, it may represent a class of transcrip-tional promoters that is uniquely regulated duringdifferentiation.

The purpose of the experiments described in thispaper was to determine whether or not the ElA-likeenvironment exhibited by undifferentiated EC cells isrepresentative of normal mouse embryonic cells. If so,is the activity restricted to one or more specific stages inmouse development? We have addressed these ques-tions by introducing various Ella-reporter gene con-structions into cultured cell lines, mouse oocytes andpreimplantation embryos, and transgenic mice to moni-tor expression from the Ella promoter. Our resultsdemonstrate that an ElA-activatible promoter is ex-pressed, in the absence of E1A, specifically during earlyembryonic development and in the adult mouse ovary.

Materials and methods

Construction of Ella-reporter genespEUa-CAT and pEII-65/75 Is-CAT (this linker-scanningmutant promoter is referred to as pEU65-CAT) were pro-vided by B. Thimmapaya (Murthy et al. 1985). These con-structs contain the Ad5 Ella early promoter fused to thebacterial chloramphenicol acetyltransferase gene (CAT). Thewild-type Ella promoter was fused to the Escherichia coli/3-galactosidase lacZ gene to form pEW-lacZ. This wasaccomplished by insertion of a 3 kb Bg/II-linked lacZ frag-ment from pZip ras /Sgal (Thompson et al. 1989) into Bg/II-cutpEII-G47\ pEIl-luciferase (pEII-/wc) was constructed byinserting the 350 bp Ella promoter {Xho-BglU. fragment frompEU-CAT) into a pML-1 vector containing the firefly lucifer-ase gene encoded by a 2572 bp HindlH-BamHI fragmentfrom pSV232AL-AD5 along with SV40 splicing and polyaden-ylation signals (de Wet et al. 1987). The Ad5 ElA-expressionplasmid, pCE, has been previously reported (Haley et al.1984).

Microinjection of DNA into mouse oocytes andembryosIsolation, culture and microinjection of mouse oocytes andembryos were carried out as previously described (DePam-philis et al. 1988; Martinez-Salas et al. 1989). Oocytes wereobtained from 16 to 18 day old CD1 mice and cultured inminimal essential medium in the presence of 100 jig ml"1

dbcAMP so that about 85 % retained their germinal vesicle. 1-cell embryos were isolated 17 to 18 h after hCG was injectedinto CD1 or B6SJL females 7 to 8 weeks old. Embryos werecultured in Whitten and Bigger's medium supplemented with40fiM-EDTA and 2^gml of aphidicolin. 2-cell embryos

were isolated 40 to 42 h post-hCG and cultured in Whitten andBigger's medium plus EDTA.

Groups of 100 to 150 embryos at a time were injected with2 pi of DNA sample into one of their nuclei using the sameneedle and an automated injection system (Eppendorfmicroinjector 5242). The amount of DNA injected wasdetermined as previously described (Martinez-Salas et al.1988). Surviving oocytes were cultured for 24 h, and surviving1-cell and 2-cell embryos for 42 h before measuring luciferaseor /3-galactosidase activity in individual oocytes and embryos.

Transfection of promoter - luciferase constructs intocultured cell linesMouse NIH/3T3 and human 293 cells grown in Dulbecco'smodified Eagle's Medium (DMEM) and 10 % fetal calf serumon 60 mm dishes to 40-60 % confluency were transfected withplasmid DNA using the calcium phosphate technique (Wigleret al. 1978). At 6 to 18 h after addition of DNA, the cells wereincubated in 20% glycerol in DMEM for 2min at 37°C. Thissolution was removed and fresh medium was added. At 48hafter addition of DNA, the cells were rinsed twice with cold20mM-Tris pH7.5, 137mM-NaCl, 5mM-KCl, lmM-Na2HPO4buffer, then scraped into lml of cold 100 mM-potassiumphosphate (pH7.8), 1 mM-dithiothreitol followed by a 2mincentrifugation at 4000 g at 4°C. The pellet was resuspended in100 [A of the same buffer and immediately analyzed forluciferase activity or frozen and stored at —70 C.

Staining cells for fi-galactosidase activityOocytes and embryos were harvested at 24 to 72 h post-injection and rinsed through six 100 il drops of PBS+0.4%PVP-40 under oil. This was followed by a 20min fixation incold 2% formaldehyde, 0.2% glutaraldehyde, 0.4% PVP-40in 15mM-NaPO4 (pH7.4), 150mM-NaCl (phosphate-bufferedsaline; PBS). They were rinsed as before, then transferred to0.5 ml of reaction mix containing 5 mM-potassium ferro-cyanide, 5 mM-potassium ferricyanide, 25mM-MgCl2,2mgml~1 X-gal (Bethesda Research Labs) in PBS. The fixedembryos were incubated in the dark for 16 to 24 h at 37 °C in ahumidified chamber to prevent evaporation (Sanes et al.1986).

Luciferase assayIndividual embryos were harvested in 50/tl of reaction mix(25 mM-glycylglycine (pH7.8), 15mM-MgSO4, 5mM-ATP(pH7), 100^gml"1 BSA, 1 mM-dithiothreitol), subjected tothree cycles of freeze-thawing (-70cC to 37°C) and thenassayed in the presence of 1 mM-luciferin. Light emission fromextracts of microinjected embryos or transfected cells wereintegrated for the initial 10 s of emission at 25 °C in aluminometer (Monolight 2001, Analytical Luminiscence).Each group of assays was standardized using aliquots ofpurified luciferase (Sigma) stored at -70°C, and light unitdata were corrected for small variations from one experimentto the next (1506±46 light units/pg luciferase). This assay waslinear from 10 fg to 200 pg of luciferase per assay.

c-myc immuno-blotting analysisDifferentiated cells (2.4X106 HL60 and 4.4xlO6 NIH/3T3)were washed twice in cold PBS by centrifuging for 5 min at300g (4CC) and discarding the supernatants. Laemmli samplebuffer was added to the pellet to give a final concentration of2X104cellsiA~l. Since the cell suspension was too viscous topipette, it was sonicated for 60s while bathed in cool waterand then incubated for 5 min at 100°C. Oocytes and embryoswere rinsed twice in PBS containing 0.4% PVP-40. Embryoswere collected in this buffer (100 embryos/5 jil) and trans-

Transactivation of the adenovirus Ella promoter 947

ferred to a 1.5 ml centrifuge tube containing an equal volumeof 2X Laemmli sample buffer. These were then incubated for5 min at 100°C. Lysates were fractionated by electrophoresisin a 10% polyacrylamide gel containing SDS. Polypeptideswere electrophoretically transferred onto a nitrocellulosemembrane in a buffer containing 12.5mM-Tris (pH8.3),96mM-glycine, 20% methanol, and 0.01% SDS. The mem-brane was incubated for 90 min at 37°C in PBS containing 5 %non-fat milk, followed by lh at room temperature inlO/igml"1 of affinity-purified rabbit-anti-c-myc (Miyamoto etal. 1985) diluted in PBS containing 5 % non-fat milk and0.05% Tween 20. This was followed by six 5 min washes inPBS and 0.05% Tween 20, and then a lh incubation in0.2/iCiml"1 125I-Protein-A diluted as above. The membranewas then washed eight times for 5 min in PBS and 0.05 %Tween 20, air-dried and subjected to autoradiography usingKodak X-Omat AR film and a Dupont Cronex Lightning Plusintensifying screen.

Transfection of pEII-CAT into differentiated cell linesand CAT assaysApproximately 30-50% confluent 9 cm plates of HeLa,NIH/3T3, BHK hprt-, Rat 1, and Ltk- cell lines, grown inDMEM+10% fetal calf serum, were transfected by thecalcium phosphate technique of Graham and Van der Eb(1973). These cells received precipitates that contained 3^gpEll-CAT or pEII65-C4T with or without 5/xg of the Ad5ElA-expressing plasmid, pCE. About 16-24 h later the trans-fected cells were shocked with 20% glycerol in PBS forapproximately 2-3 min, followed by a PBS rinse, and werethen refed with DMEM+10% FCS. Two days later thesesamples were harvested by scraping in 0.25 M-Tris, pH8.0 andeither freeze thawing or mildly sonicating. Soluble extractswere obtained by centrifugation and CAT assays were per-formed using 100/xl of extract, 2.5 JX\ of 33.3 mgml"1 acetylCoA (Sigma), and 1.5 (A of [14C]chloramphenicol (Amer-sham; SSmCimmol"1, O^^Ci^l"1) for 3h at 37°C. Sampleswere extracted in ethyl acetate, and thin layer chromatograms(TLC) were resolved in 95% chloroform:5% methanol. TheTLCs were exposed to Kodak XAR film overnight.

Generation of E1165-CAT transgenic miceThe no. 42-5 transgenic mouse line was produced by micro-injection of C57/CBA fertilized egg pronuclei (obtained atday.0.5 post-coitus) with the 1.95kb Xhol-Xbal pEIII65-CATfragment at a concentration of 2/igmP1. Six offspringwere obtained from surrogate mothers that received micro-injected 1-cell embryos. DNA samples were prepared fromlcm tail biopsies of these offspring, by mincing in 0.7 ml50mM-Tris pH8.0, lOOmM-EDTA, 0.5% SDS, 35^1lOmgrnl"1 proteinase K and incubating at 55°C overnight.These samples were phenol/chloroform-extracted, ethanol/sodium acetate-precipitated, and resuspended in 100/A Tris-EDTA buffer at 65 °C. BgM restriction digests of 16 /xlsamples were electrophoresed on 1 % agarose gels in Tris/borate/EDTA gels. Then southern blots were performedusing nitrocellulose and a random-primed CAT-specificprobe, which was ^P-labelled. The southern blots (andsubsequent DNA slot blots) confirmed that the offspring ofthe transgenic mouse no. 42-5 (female) contained the trans-gene at low copy number (data not shown). This transgenicline was then propagated in a heterozygous state, by matingwith non-transgenic C57/CBA mice.

CAT assays on transgenic tissuesAdult and embryonic tissues from transgenic mice wereexamined for expression by CAT assays. Freshly dissected

tissues isolated from numerous transgenic offspring weresonicated in cold 0.25 M-Tris pH8.0, heated at 65CC for 5 min(to inactivate potential deacetylases), centrifuged to collectsoluble extracts, and protein concentrations were determinedusing the Bradford protein assay (Biorad). Then 200 /ig ofeach sample was subjected to the CAT assay using 100 JJI totalof extract+Tris buffer, 2.5/A 33.3mgmP1 acetyl CoA, 1.5iA[14C]chloramphenicol (Amersham; 54mCi mmor' , 0.2 Cifd~l), and to incubation at 37°C for 90min. Purified CAT(Boehringer) was used as a positive control in these assays.Ethyl acetate-extracted samples were run on TLC plates in95% chloroform: 5% methanol and visualized by autoradi-ography. For the embryo assays, pooled embryo extracts(200fig and 1000 g) contained 12, 10, and 6 embryos from5.5, 7.5, and 9.5 days, respectively.

Immunohistochemical localization of CAT in ovariesThe ovary from a transgenic adult mouse was formalin-fixed,paraffin-embedded, sectioned, and dewaxed in xylene/ethanol. The mouse monoclonal antibody, CAT1, raisedagainst purified chloramphenicol acetyltransferase (gift fromJ. Gannon and D. Lane) was used as the primary antibody atdilutions ranging from 1:1 to 1:5000 in PBS containing 5%FCS. A secondary goat anti-mouse antibody that was conju-gated to colloidal gold was applied and subsequently silver-enhanced (Jannsen). Following a light toluidine blue staining,coverslips were mounted, and the sections were photo-graphed with an Olympus BH-2 compound light microscopesystem. Two primary antibody controls (PBS alone andmouse anti-actin) were included for comparison. The PBScontrol yielded no staining, whereas abundant staining wasobtained throughout the ovary section with anti-actin anti-body.

Results

Expression of Ella-promoter in mouse oocytes andpreimplantation embryosThe ability of mouse oocytes and preimplantationembryos to express the Ad5 Ella promoter was re-vealed by injecting plasmids that express the lacZ genewhen fused to the Ella promoter. Supercoiled plasmidDNA was injected into the oocyte germinal vesicle, themale pronucleus of 1-cell embryos, and one of thezygotic nuclei in 2-cell embryos. The amount of DNAinjected per nucleus and the time of incubation inculture after injection were those previously shown togive the optimum levels of gene expression using theHSV thymidine kinase (tk) promoter (Martinez-Salaset al. 1989). DNA injected into the nucleus was stablefor at least 3 days, while DNA injected into thecytoplasm was rapidly degraded (Wirak et al. 1985;Martinez-Salas et al. 1988). Maturation of oocytes wasprevented by culturing them in dbcAMP (Chalifouret al. 1986, 1987), and 1-cell embryos were preventedfrom entering S phase by incubation with aphidicolin(Martinez-Salas et al. 1989). These embryos retainedtheir two pronuclei and did not cleave into 2-cellembryos. Injected 2-cell embryos continued develop-ment to the morula and blastocyst stages. Previousstudies using these conditions revealed that promoterssuch as the tk-promoter respond to the same controlsthat regulate endogenous gene expression during pre-

948 T. P. Dooley and others

2 - 4 Cells

Fig. 1. Expression of pEU-lacZ inmicroinjected oocytes and earlyembryos. /S-galactosidase-positive cellsexhibit dark X-gal staining (refer toMaterials and methods for stainingprotocol).

implantation development, and that the level of geneexpression and the need for enhancers to activatepromoters or origins of DNA replication depends uponthe type of nucleus in which the DNA exists (Martinez-Salas et al. 1988, 1989).

Oocytes and embryos were stained for /3-galactosi-dase activity at various times post-injection. The resultsrevealed that the Ella promoter was very active at allstages of preimplantation development from the oocyteto the blastocyst (Fig. 1). No /?-galactosidase activitywas detected under these conditions when the sameplasmid without a promoter was injected. Furthermore,/3-galactosidase activity was restricted to those cellsreceiving the injected gene and their progeny; in 2 to 8cell embryos where individual blastomeres were readilyidentified, only half were stained. In all of the blasto-cysts derived from injected 2-cell embryos, staining wasrestricted to one portion of the embryo consistent withpEU-lacZ expression in the inner cell mass but not inthe trophectoderm. Attempts to produce blastocystsfrom either 2-cell embryos injected in both nuclei orfrom injected 1-cell embryos that were allowed todevelop in the absence of aphidicolin were unsuccess-ful. Therefore, we could not conclusively demonstrate

that this restricted pattern of pEU-lacZ expression wascell type-specific.

The levels of Ella-promoter activity in oocytes andpreimplantation embryos were quantified by injecting aplasmid in which the Ella promoter was fused to thefirefly luciferase gene. This assay allowed quantitativemeasurements of luciferase activity in individualoocytes and embryos (Martinez-Salas et al. 1989).Analogous data from Martinez-Salas et al. (1989) usingthe HSV tk-promoter was included for comparison.The HSV tk-promoter represents a promoter that doesnot respond to E1A, but does respond to enhancers andhas been well characterized in a variety of cell lines aswell as mouse and Xenopus oocytes and embryos(Weeks and Jones, 1985; Martinez-Salas, 1988;McKnight and Kingsbury, 1982).

At least 90% of the oocytes and embryos injectedwith pEII-/uc produced levels of luciferase substan-tially greater than the level observed with the sameexpression vector without a promoter (Fig. 2A and B).In general, Ella promoter activity paralleled that of thetk-promoter; it was lowest in oocytes and highest in 1-cell embryos arrested in S phase. In oocytes anddeveloping 2-cell embryos, the Ella-promoter was

Transactivation of the adenovirus Ella promoter 949

EII+

Oocytes 1-Cells 2-CellsFig. 2. Activity of adenovirus Ella-luciferase fusion gene inmouse oocytes and preimplantation embryos. Plasmid DNAwas injected into the germinal vesicle (GV) of mouseoocytes, the male pronucleus (MP) of 1-cell embryos, andone of the zygotic nuclei (ZN) of 2-cell embryos. Survivingova were cultured as described in Materials and methods,lysed and then luciferase activity measured in individualova. (A) 'EIF represents injection of lpgpEIIa-/Mc/GV,0.6pg/MP and 0.6pg/ZN. 'Low EIF represents injection of0.5 pg pEIIa-/wc/GV, 0.2pg/MP, and 0.3pg/ZN. 'LowEII+Ela' represents the same experiment as 'low EH' butwith an equal amount of pCE. The standard error of themean (error bars) for the groups of oocytes and embryosvaried from 15 % to 25 % of the mean, 'tk' and 'FlOltk'represent the same experiment as 'EH', but using ptk-/wcand pF101tk-/i<c. The promoterless control plasmid, pluc,yielded activities from 0.03 to 0.05 xl0~4 luciferase unitsper oocyte or embryo. These data were reproduced fromMartinez-Salas et al. (1989). (B) the 'EH' level of luciferaseactivity in each group of oocytes and embryos was definedas 100 % and the other data in that group were expressedrelative to this value.

8-fold more active than the tk-promoter. In 2-cellembryos, the polyomavirus (PyV) F101 enhancer stimu-lated tk-promoter activity to a level comparable to thatof the Ella-promoter alone. However, in oocytes, thisenhancer further reduced tk-activity 2-fold. The PyVenhancer mutation F101, which was selected for itsability to support virus growth in mouse embryonalcarcinoma F9 cells, was particularly effective in stimu-lating tk-promoter activity in developing 2-cell embryos(Martinez-Salas et al. 1989). Therefore, this mutation isperhaps recognized by a novel enhancer-activatingprotein expressed from 2-cell embryos through to theinner cell mass, which compensates for the presumedrepressive effects of the 'ElA-like' environment ofundifferentiated cells. And in 1-cell embryos, Ella

0 10 20Luciferase Activity (relative)

Fig. 3. Activity of the Ella-luciferase gene in mouseNIH/3T3 cells and human 293 cells. 3T3 and HeLa cellswere transfected with pEIIa-/uc±pEla (pCE) at the molarratios indicated (Materials and methods). Levels ofluciferase activity are presented relative to the controlplasmid, pluc, lacking the Ella promoter within the samecell type. Note that direct comparisons of actual levels ofexpression cannot be made from this relative data, becausethe actual expression levels of pEIIa-/wc are significantlyhigher in 293 cells than 3T3 cells.

promoter activity was equivalent to tk-promoter ac-tivity with or without an enhancer (Fig. 2). Previousstudies have shown that replication origins and pro-moters that normally require an enhancer for activity inmouse differentiated cells function efficiently withoutan enhancer when present in the pronuclei of 1-cellembryos arrested in S phase (Martinez-Salas et al. 1988,1989). Thus, in all three developmental stages (oocytes,1-cell embryos and 2-cell embryos) the Ella promoteractivity was equal to or greater than the activity of thereference tk promoter either with or without anenhancer element.

Coinjection of lower amounts of pEII-/«c with anequal amount of pCE, a plasmid encoding the Ad5 E1Agene, did not stimulate Ella-promoter activity, but, infact, reduced luciferase expression about 3-fold inoocytes and developing 2-cell embryos (Fig. 2B). Sincecotransfection of pEII-/«c with pCE stimulated lucifer-ase expression in mouse 3T3 cells up to 28-fold (Fig. 3),the Ella-promoter was already fully active in mouseoocytes and early embryos and could not be super-stimulated by E1A. Furthermore, the response ofmouse oocytes and early embryos to over expression ofE l A protein was similar to 293 cells, a transformedhuman cell line that expresses Ad5 E1A from anintegrated viral genome (Fig. 3). All three cell typesexpress the Ella promoter at full activity and all threecell types reduced the level of expression in the pres-ence of additional E1A, for some yet unknown reason.

pEII-65 CAT transgenic miceHaving established that Ella promoter activity was highin the undifferentiated stages of oogenesis and earlyembryogenesis, we then created a transgenic mouse lineto further characterize the pattern of Ella expression.

950 T. P. Dooley and others

NH3T3 HELA BHK HPRT-

AcCM

CM -

65 65 WT WT*

65 65 WT WTI I

65 WT WTE1A

Fig. 4. CAT assays on transfected differentiated cell lines. Calcium phosphate transient transfections of pEIIa-C4r(WT) orpEII65-C47"(65) either with (+) or without the Ad5 ElA-expressing plasmid, pCE, were analyzed by CAT assays. NIH3T3, HeLa, and BHK hprt- represent three of the differentiated mammalian cell lines which were transfected. Thepositions of the unmodified and acetylated forms, of chloramphenicol on the TLC are indicated by 'CM' and 'Ac CM',respectively.

In order to demonstrate the extent to which the Ella-reporter gene used in the following transgenic studiescould be transactivated by El A, a variety of differen-tiated cell lines were transfected with pElI-CATeitherin the presence or absence of an ElA-expressingplasmid. Transient expression of the pEII-C4Tgene intransfected cell lines, derived from four differentspecies, was assessed by CAT assays (NIH/3T3, Hela,and BHK hprt-, in Fig. 4; Ltk- and Rat 1, data notshown). This assay monitors the enzymatic conversionof [14C]chloramphenicol to its acetylated forms. Boththe wild type and -65/-75 linker-scanning mutant Ellapromoters (WT and 65, respectively) were transcribedin all cell lines at low basal rates in the absence of anElA-expression plasmid (Fig. 4). However, the level ofElla expression was elevated dramatically in the pres-ence of E1A, consistent with previously reported re-sults. The linker-scanning mutant form of this promoterwas also ElA-inducible, albeit both the basal andinduced levels of transcription in several cell types(NIH 3T3, Hela, and BHK hprt-) were less than thoseobtained with the wild-type promoter. pEII-C4rgeneexpression was also observed in stably-transformedPCC4 undifferentiated EC cells, but was drasticallyreduced upon differentiation (data not shown).

To determine the tissue specificity of an Ella pro-moter activity, a transgenic mouse line was establishedby microinjecting a pEII65-C4T restriction fragmentinto fertilized mouse eggs. This promoter was chosenbecause it exhibited a lower basal level of transcriptionin the absence of E1A in some cell types, yet was stilltransactivated by El A (Murthy etal. 1985; see pEII65-C4Tdata above). Thus the EII65 promoter should be asensitive tool for identifying the primary sites of high-

level transactivation. By microinjection, we obtained atransgenic line of mice (derived from the no. 42-5female) that contained this reporter gene integrated atlow copy number (data not shown). The pEII65-C4.rtransgenic line transmitted this allelle as a single auto-somal 'dominant' trait lacking any noticeable terato-genic or dysplastic abnormalities.

Soluble extracts from various transgenic adult so-matic tissues were subjected to CAT assays (seeFig. 5A). The transgenic line did not express CAT inany of the adult somatic tissues that were analyzed. Thetissues chosen for examination contained a large varietyof endo-, ecto-, and mesodermally-derived cell typesand yet none of these somatic tissues was positive in theCAT assays. These results suggest that this promoterwas transcribed at non-detectable levels under theconditions chosen, in non-germ line tissues. Of moresignificant interest was the observation that all (8/8)females were CAT-positive in this assay in ovariantissue (Fig. 5B). The amount of CAT activity in theovaries was estimated to be at least 20x higher than inthe 'non-expressing' somatic tissues of the same mice.This activity was not due to a natural endogenous CAT-like activity in non-transgenics, since CAT activity waspresent only in the transgenic females. The absence ofthis activity in the testis of male transgenics (seeFig. 5B) demonstrates that this ability to form activetranscription complexes was not germ line-specific, butrather ovary-specific. Transactivation of this EII-reporter gene in adult ovaries indicates that at least onetype of cell within the ovary is capable of making activetranscription complexes, presumably via the cw-actingtranscription factor binding sites within this E1A-activitable promoter. Alternatively, the site of inte-

Transactivation of the adenovirus Ella promoter 951

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Fig. 5. CAT assays on transgenic mice tissues.CAT assays were performed on 200 ng of solubleprotein from individual EII65-C47"transgenicmice tissues. (A) Lack of activity in somatictissues: lane 1, 0.05 units CAT(Boehringer); lane2, brain; lane 3, kidney; lane 4, lung; lane 5,heart; lane 6, liver; lane 7, spleen; lane 8,intestine; lane 9, skin; lane 10, uterus; lane 11,eye; lane 12, mammary gland; lane 13, legmuscle; lane 14, abdominal muscle; lane 15, tail.(B) Germ-line and embryonic tissues: lanes 1-3,ovaries from three different adults; lane 4, testis;lane 5, no protein-negative control; lane 6,0.15 units CAT; lanes 7 and 8, pooled 9.5-dayembryo extracts at 200 ^g and lmg total protein,respectively; lanes 9 and 10, pooled 7.5-dayembryo extracts at 200 j/g and 1 mg total protein,respectively; lane 11, 0.2 units CAT. Thenumbers adjacent to each lane refers to thenumber of times that result was obtained fromdifferent transgenic offspring.

gration of this transgene might cause it to be aberrantlyhormonally-activated and thus expressed only in thefemale ovaries. This alternative appears unlikely, be-cause the phenotype of these transgenic mice wasobtained by screening rather than direct phenotypicselection. Pedigree analysis of this transgenic line re-vealed that the pattern of pEII65-C4T expression wasidentical, regardless of the sex of the transgenic parentmouse. Thus, the ovaries from offspring of both trans-genic males or females were CAT-positive, demon-strating that transmission of the transgene via the'CAT-negative' testis of the male parent does notirreversibly inactivate this transgene. These resultsindicate that only the adult ovary contains the necessary'activated' transcription factors to mediate the transac-tivation of an ElA-dependent promoter.

The fact that the Ella promoter appeared to be fullyactive when injected into mouse oocytes suggested thatthe expression of CAT in the transgenic ovaries was dueprimarily to transactivation of the transgene in develop-ing oocytes. To test this hypothesis, histological sec-tions of transgenic ovaries were treated with an anti-

CAT antibody preparation and were visualized by silverenhancement of immunogold-bound complexes(Fig. 6A). The specific staining of oocytes was due tothe primary anti-CAT antibody, as controls lacking aprimary antibody exhibited no staining (Fig. 6B), andan anti-actin primary antibody demonstrated strongimmunostaining throughout the ovary sections (datanot shown). Significantly lower levels of diffuse non-nuclear, cytoplasmic staining was also seen in somecorpus lutea (data not shown). The presence ofimmuno-reactive cytoplasmic CAT enzyme in theoocytes of adult female ovaries confirmed our hypoth-esis that the pEU65-CAT transgene was expressedprimarily in the oocytes.

To determine whether or not CAT activity waspresent in the post-implantation embryos as well asoocytes, embryos at days 5.5, 7.5, and 9.5 post-coitusfrom female heterozygous transgenics were examined.Presuming that only 50 % of the embryos contained thetransgene, a large number of embryos were pooled toprepare mixed extracts for CAT assays. These extractsexhibited no CAT activity even when 5-fold higher than

952 T. P. Dooley and others

Fig. 6. Immunohistochemical detection of CAT in transgenic ovary. Photomicrographs are presented of sections(488xmagnification) from the ovary of a transgenic female that were treated with (A) mouse anti-CAT antibody, CAT 1, or(B) no primary antibody. Samples were visualized by binding with gold conjugated goat anti-mouse IgG, and silverenhancement. Note the localization of the anti-CAT antibody within the cytoplasm of the developing oocytes (A).

normal amounts of extract were used (Fig. 5B; day 5.5,data not shown). This result is consistent with thefindings of Suemori et al. (1988). By infecting E1A-deficient adenovirus into embryos and scoring thetransactivation of Ella, they also found evidence of anElA-like activity that was restricted to preimplantationembryos, i.e. no activity was found after day 4.5.

Expression of c-myc during oogenesis andembryogenesisOnclercq et al. (1988) have reported that c-myc protein,like El A protein, is capable of transactivating theadenovirus EIV and Ella promoters in HeLa andmouse EC cells. If c-myc were present at sufficientlyhigh levels in mouse oocytes and embryos, it might playa role in the transactivation of ElA-inducible pro-moters'. Therefore, the steady-state levels of c-myc inmouse oocytes, embryos and differentiated cell lineswere determined by Western blot analysis (Fig. 7).Mouse oocytes contained 150 to 1000 pg of c-myc,depending on their age. Since mouse oocytes consist of

24 ng of protein, this was equivalent to 0.6 % to 4 % oftotal oocyte protein. The levels of c-myc in mousepreimplantation embryos were reduced by 3 to 20-foldrelative to mouse oocytes. In comparison, cell linessuch as HL60 that have amplified the c-myc genecontained only about 1.1 pg of c-myc per cell, anddifferentiated cells with a single copy of the c-myc genesuch as mouse 3T3 fibroblasts contained only 0.03 pg/cell of c-myc. Since the level of c-myc mRNA in 3T3cells is equal to or greater than the level of c-myc RNAfound in a wide variety of tissues from newborn andadult mice (Zimmerman et al. 1986), the levels of c-mycprotein in mouse embryos and oocytes are from 3000 to30000-fold higher per cell (or approximately 75-foldhigher in concentration) than most mouse differen-tiated cells. Similar high levels of c-myc expression inXenopus oocytes have been reported (Taylor et al.1986; Godeau et al. 1986; King et al. 1986; Hourdry et al.1988). The correlation between high c-myc levels,expression of the Ella promoter, and the report thatc-myc can substitute for E1A to transactivate Ella

Transactivation of the adenovirus Ella promoter 953

BIk Oocytes Embryos Diff. Cells c-myc BIki5d 25d 2C M / B " H L 6 0 3T3"20 4o

Table 1. Summary of various cell types which arecapable of Ella transactivation

Fig. 7. Detection of c-myc protein in extracts from mouse oocytes, embryos and differentiated cells. Oocytes (100 per lane)were isolated from CD-I females at 15 and 25 days after birth. Embryos were prepared from CD-I females 6-8 weeks old:1-cell embryos (1C, 20 per lane), 2-cell embryos (2C, 60 per lane), and morula and blastocysts (M/B, 60 per lane). Extractswere also prepared from mouse NIH/3T3 cells (3T3, ~5xlO4 per lane) and human HL60 cells containing amplified copies ofthe c-myc gene (HL60, ~ lx l0 4 per lane). Proteins were fractionated by gel electrophoresis, transferred to a nitrocellulosemembrane, incubated with affinity-purified rabbit anti-c-myc antibody, and reacted with 125I-protein-A. Blank reactionswithout cell extract (BIk) and standard aliquots of 20 ng and 40 ng of purified c-myc protein (Watt et al. 1985) were includedas controls. The amounts of c-myc was calculated from these and similar data at 15Opg/l5-day old oocyte (15d),>l000pg/25-day old oocyte (25d), 300pg/one-cell embryo (lc), 100pg/two-cell embryo (2c), 50pg/early morula, 300pg/latemorula and blastocyst (M/B). HL60 cells contained about 1.1 pg/cell and NIH/3T3 cells contained about 0.03pg/cell.(Diff., differentiated).

(Onclereq et al. 1988), suggests that c-myc mightaccount for the efficient use of this promoter duringmammalian oogenesis and embryogenesis.

Discussion

The data presented above clearly demonstrate that theadenovirus Ella early promoter is transcriptionallyactivated in mouse oocytes and preimplantation em-bryos. These findings were confirmed by differentapproaches: microinjection of Ella promoter-reportergenes into ova and cleavage-stage embryos and con-struction of transgenic mice containing an integratedEII promoter-reporter gene (summarized in Table 1.)Microinjection of pEH-lacZ or pEll-luciferase re-vealed high levels of gene expression in all of the earlycleavage stages of preimplantation embryos. Our re-sults are in agreement with the findings of Suemori et al.(1988), who also demonstrated the presence of Ellatransactivation in 2.5- to 4.5-day-old mouse embryosfollowing infection by an ElA-defective adenovirus.We have extended these studies to include mouseoocytes, fertilized ova, and early cleavage-stage em-bryos. The presence of the oocyte-specific activity wasconfirmed in the ovaries of adult female transgenics.The ability to transactivate Ella was lost during thegeneration of post-implantation embryonic and adult

Cell type

Differentiated non-ECDifferentiated non-EC+ElAUndifferentiated ECDifferentiated ECMurine oocytesMurine 1-cell embryosMurine 2-cell embryosMurine 4-8 cell embryosMurine morula embryosMurine blastocyst embryosMurine day 2.5—4.5 embryosMurine day 5.5 embryosMurine day 7.5 embryosMurine day 9.5 embryosMurine adult somatic tissuesMurine adult testisMurine adult ovary

Transactivation

—++—+++++++-—---+

References

1; this work1; this work

22

This workThis workThis workThis workThis workThis work

33; this work3; this workThis workThis workThis workThis work

(1) Numerous reports have demonstrated the transactivation ofElla by El A in various differentiated cell lines (Jones & Shenk,1979; Murthy et al. 1985; Strair et al. 1988; for review ontransactivation see Jones et al. 1988). (2) Transactivation of Ella inEC cells by an ElA-like activity (Imperiale etal. 1984). (3)Suemori et al. 1988.

954 T. P. Dooley and others

somatic tissues. This lack of expression in somatictissues of transgenic mice further strengthens our hy-pothesis that this activity is a marker of the undifferen-tiated state during murine development, i.e. the pres-ence of this marker is restricted to the relatively'undifferentiated' state of murine oogenesis and earlyembryogenesis.

Our data also correlate well with the reported be-havior of the Ella promoter in undifferentiated ECcells. Undifferentiated embryonal carcinoma cell linesexpress Ella in the absence of ElA, suggesting thatthey may contain an endogenous factor which substi-tutes for El A (Imperiale et al. 1984). The identity of theElA-like activity in EC cells is currently unknown. Ourmicroinjection and transgenic data lead us to believethat 'normal' undifferentiated oocytes and preimplan-tation embryos of mice also contain an 'ElA-like'environment resembling that found in undifferentiatedEC cells. Thus, EC cells are not unique in their abilityto transactivate the Ella promoter, because mouseoocytes to blastocyst also exhibit this property. Blasto-cysts are composed of two cell types; the totipotentinner cell mass that gives rise to the embryo, and thedifferentiated trophectoderm that gives rise to theplacenta. Mouse EC cell lines share many character-istics with primitive endoderm that arises from the innercell mass during the late stage of blastocyst develop-ment (Hogan etal. 1983). Therefore, it is not surprisingthat 'Ela-like' activity appears to be restricted toundifferentiated cells such as the inner cell mass, since itis only found in EC cell lines prior to their differen-tiation. This conclusion is further supported by the factthat Ella activity was barely detectable in expanded orhatched blastocysts (4.5-day embryos; Suemori et al.1988) and undetectable in extracts of 5.5-, 7.5- and 9.5-day embryos (Fig. 5B).

What might the role of this transactivation potentialbe in undifferentiated cells during oogenesis and em-bryogenesis? It seems quite Likely that this activity maybe responsible for the transcriptional regulation ofvarious cellular genes. ElA has been shown to becapable of transcriptionally regulating various endogen-ous genes, such as heat shock, globin, MHCI, andothers (Imperial et al. 1984; Kingston et al. 1985). Thestrikingly high degree of similarity of the human braincreatine kinase promoter to the Ella promoter suggeststhat both ElA and the ElA-like activity could transacti-vate this promoter and possibly others which sharesimilar transcription factor binding sites (Daouk et al.1988). Thus, the endogenous ElA-like activity we aremonitoring during oogenesis and embryogenesis mightbe responsible for transcriptional regulation of numer-ous cellular genes. This activity may even possibly act asa developmental switch mechanism for determining theexpression of a larger set of cellular genes, i.e. the lossof this activity during early development might signalthe switching 'off of genes which are ElA-activatibleand the switching 'on' by depression of genes which arenormally repressed by this presumed transregulator(assuming this ElA-like activity contains both thetransactivator and the repression functions of ElA). A

report by Montano and Lane (1987) addressed whetherover-expression of exogenous ElA in undifferentiatedEC cells would block their ability to undergo differen-tiation. They found that exogenous ElA is not capableof inhibiting EC cell differentiation and, surprisingly,ElA actually induced differentiation in their hands.This intriguing result suggests that the endogenouscellular ElA-like activity does not 'hold' EC cells intheir undifferentiated state, whereas one might havepredicted that release from the undifferentiated state isaccomplished by the loss of the cellular ElA-likeactivity. Their perplexing results do not addresswhether the ElA-like activity is dispensable in theundifferentiated state. Thus, the ability to transactivateElla in cells lacking ElA appears to be a marker for theundifferentiated state during early development. Ellarepresents an example of a promoter that is differen-tially regulated as mammalian cells undergo differen-tiation. As such, this information might result in theidentification of cellular genes which are regulated in asimilar manner, and which may play a role in theprocesses of differentiation.

How might Ella transactivation occur inundifferentiated cells?Transactivation of the Ad5 Ella early promoter by ElAhas been extensively studied in vitro (Siva Raman et al.1986; Kovesdi et al. 1986; Murthy et al. 1985; andreferences therein). Thus far, ElA has not been shownto bind directly at the DNA level to the Ella promoter.Mutational and biochemical analysis of this promoterhas delineated the positions of a number of transcrip-tion factor binding sites, including the sites for the E2Fand ATF transcription factors (revised in Jones et al.1988). Two E2F binding sites are located within theregion from -32 to -71 of the transcriptional start site,adjacent to a single ATF site (Reichel et al. 1987; SivaRaman et al. 1986). These transcription factors arethought to play similar roles in transactivating the Ellapromoter in both ElA-containing differentiated cellsand undifferentiated EC cells. Adenovirus infection ofHeLa or differentiated EC cells results in increasedbinding of E2F to the Ella promoter (Reichel et al.1987; Kovesdi etal. 1986; Lathangue and Rigby, 1987).Furthermore, the binding of E2F to this promoteroccurs in undifferentiated EC cells, whereas this factordeclines upon differentiation. ATF levels, on the otherhand, are not affected by ElA. The precise mechanismby which ElA acts either directly or indirectly on theseand other factors is not yet known, although Lillie andGreen (1989) have proposed that ElA might directlyinteract with some DNA-bound transcription factor(s)resulting in complex formation and thus transactiv-ation. Alternatively, ElA might stimulate modificationof these factor(s) resulting in an increase in the in-itiation of transcription.

What might the identity of the 'ElA-like' activity bein oocytes, embryos and undifferentiated EC cells? Themechanism by which Ella transactivation occurs inundifferentiated cells is not currently known and itmight be accomplished in the absence of ElA by one of

Transactivation of the adenovirus Ella promoter 955

several different plausible mechanisms. Some evidenceprovided by Onclercq et al. (1988) suggests that overex-pressed c-myc protein can transactivate both the Ellaand EIV promoters. Our observation that c-myc levelsare very high in oocytes and embryos (in terms ofabsolute amounts and relative concentrations) would beconsistent with this interpretation, provided that the c-myc protein is available at sufficient levels in an activestate within the nucleus. However, this correlationbetween elevated c-myc levels and Ella expressionshould not be interpreted as a verification that c-myc isactually an ElA-like analog in oocytes and embryos,although this remains a valid possibility.

In addition to c-myc, numerous other non-ElA viralgene products have been demonstrated to transactivatethe adenovirus early gene products. The list includespseudorabies IE, HSVICP4, EBV early transactivator,human cytomegalovirus, SV40 small T, HTLV Itransactivator and HPV E7 (Feldman et al. 1982;Tevethia and Spector, 1984; Tremblay et al. 1985; Wongand Levine, 1986; Workman et al. 1988; Loeken et al.1988; Phelps et al. 1988). Possibly, a putative cellularhomologue to one of these genes exists, and its productis responsible for the promiscuous transactivation of theElla promoter during oogenesis and embryogenesis.Alternatively, one might also speculate that undifferen-tiated cells synthesize or activate a transcription factorwhich is capable of interacting with the promoter toinitiate transcription (i.e. E2F or ATF, etc.), in an E1A-independent manner. Currently, studies to address theroles of transcription factors are technically difficult toperform on such limited quantities of cells derived fromoocytes or early stage embryos. And, finally, undiffer-entiated cells might regulate the Ella promoter by yetanother mechanism independent of an E1A analog.Through this possible mechanism the Ella promoter ismerely expressed at a constitutive uninduced level inundifferentiated cells. And this promoter is repressed indifferentiated cells, although currently no evidenceexists in favor of this 'constitutive vs repressed' model,and if it were true one would expect exogenous E1A toenhance expression of Ella in oocytes and preimplan-tation embryos, and yet the opposite result occurred inour hands. These perplexing alternative interpretationsof the possible mechanism of apparent Ella transactiv-ation have not been fully addressed to date. Therefore,at this stage there is no direct evidence showing that theincreased expression of Ella in undifferentiated cells isbeing accomplished by a cellular El A analog, such asc-myc. However, if an ElA-like transactivation effectordoes exist, it may be possible to identify it by a geneticapproach. To this end, we have created ElA-dependentcell lines, which become drug-resistant when expressingE1A. These lines could be used as potential screening'traps' for the isolation of novel cDNA clones whichencode the putative ElA-like activity from undifferen-tiated cells (unpublished results, Dooley and Jones).

We wish to acknowledge Vyviene Attenburrow for assist-ance in generating transgenic mice, Sadhan Majumder for theconstruction of pEIIa-/uc and pluc, Rita Tilley and George

Elia for their help with the immunohistochemistry and PegKornacker for preparation of the manuscript. Plasmid con-structs, cell lines, and antibodies were graciously provided byB. Thimmapaya, J. Morganstern, P. Goodfellow, M. Friedand D. Lane. T.P.D. was supported by a fellowship from theHelen Hay Whitney Foundation.

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(Accepted 6 October 1989)