Identification and Characterization of cis-Acting Elements Residing ...

JOURNAL OF VIROLOGY, July 2004, p. 7590–7601 Vol. 78, No. 140022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.14.7590–7601.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of cis-Acting Elements Residingin the Walleye Dermal Sarcoma Virus Promoter

Brett W. Hronek, Ashley Meagher, Joel Rovnak, and Sandra L. Quackenbush*Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Received 5 November 2003/Accepted 17 March 2004

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with tumors that appearand regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virusexpression between developing and regressing tumors. To understand the role of host cell factors in WDSVexpression, DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assayswere employed. DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclearextract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites.Additionally, three regions that contained no putative transcription factor binding sites were protected. EMSAconfirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa,not protected in DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context ofa luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed totranscription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase inluciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription.Anti-Jun and anti-Fos antiserum specifically inhibited protein-DNA complex formation, indicating the pres-ence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site.Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in DNase Ifootprint analysis by the walleye cell line nuclear extract and regressing-tumor nuclear extract. EMSA utilizingthe 15-bp repeat probe revealed that there are similarities of binding with W12 cell and developing-tumornuclear extracts and that the binding differs from that observed with regressing-tumor nuclear extract.

Walleye dermal sarcoma virus (WDSV) is a complex retro-virus found etiologically associated with dermal sarcomas inwalleye fish (Stizostedion vitreum) (14, 15, 31, 33, 34). There isa seasonal prevalence of this disease; tumors develop duringthe fall, regress in the spring, and are rarely seen in the summer(3). Dermal sarcoma can be experimentally transmitted towalleye fingerlings with cell-free tumor homogenates preparedfrom regressing spring tumors; however, homogenates pre-pared from developing fall tumors are unable to transmit thedisease (4, 13). This result is due to the apparently low levels ofonly subgenomic transcripts during tumor growth, while re-gressing tumors contain 1,000-fold-greater levels of full-lengthviral RNA, subgenomic viral transcripts, and infectious virus(4, 22). Thus, control of WDSV gene expression is particularlyimportant to tumor regression, because it correlates with aswitch to high levels of virus expression.

Complex retroviruses encode viral accessory proteins thatare necessary to upregulate transcription, while simple retro-viruses have inherently active promoters. The protein productof WDSV open reading frame a, OrfA or retroviral cyclin, isinvolved in the regulation of transcription (26, 36). Transient-expression assays determined that OrfA decreased basal activ-ity from the WDSV promoter in a walleye cell line (26, 36).OrfA was found to colocalize and copurify with RNA polymer-ase II and to interact with components of the mediator com-plex (26). These data suggest that OrfA may function to inhibit

virus expression during tumor development, although themechanism of transcription repression is not fully understood.

To elucidate the mechanisms responsible for the apparentshift in WDSV gene expression from a low level in growingtumors to overt virus expression in the spring, identification ofcis-acting regulatory elements within the viral promoter is nec-essary. A number of putative cis-acting regulatory elements,including an AP1, AP3, LVa, and an NF1 motif, have beenidentified in the U3 region of the WDSV long terminal repeat(LTR) (11, 35). Other sequences previously identified includea pentanucleotide direct repeat, TRTGT, that occurs five timesin the U3 region and a degenerate 15-bp repeat that occursthree times (11). Zhang et al. (35) used WDSV LTR deletionmutants to identify three regions within U3 that modulatedLTR activity. Two of these regions positively regulated tran-scription from the WDSV LTR, and the third region was foundto contain an apparent negative response element; however,individual sites were not conclusively identified.

In this report, we used DNase I footprint analysis and elec-trophoretic mobility shift assays (EMSA) to identify regions inthe WDSV U3 bound by a walleye cell line nuclear extract.Site-directed mutagenesis of a WDSV U3 reporter gene con-struct was used to determine the contribution of individualsites to transcription activation. Further analysis of an AP1 sitedemonstrated specific binding of walleye cell nuclear extracts,and anti-Jun and anti-Fos antiserum disrupted this binding. Inaddition, the 15-bp repeats are differentially protected inDNase I footprint analysis by the walleye cell line nuclearextracts and regressing-tumor extracts. EMSA revealed similarpatterns of binding to the 15-bp repeats with W12 cell and

* Corresponding author. Mailing address: Department of MolecularBiosciences, 7047 Haworth Hall, 1200 Sunnyside Ave., The Universityof Kansas, Lawrence, KS 66045. Phone: (785) 864-4022. Fax: (785)864-5294. E-mail: [email protected].

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developing-tumor nuclear extracts, which differ from that ob-served with regressing-tumor nuclear extract.

MATERIALS AND METHODS

Reporter-gene constructs and luciferase assays. The pGL3-U3 reporter vectorcontains the WDSV U3 region cloned upstream of the luciferase gene (26).Deletion mutant versions of the U3 region were generated by PCR with a fixed3� primer that incorporates a BglII restriction site (5�-GAAGATCTGAGACCCCGTTCTT-3�) and the following series of 5� primers, which each incorporatean XbaI restriction site: U3�1 (5�-TGCTCTAGATTCTTAAATTGTTAGTAAGGT-3�), U3�2 (5�-TGCTCTAGATTTCTATGTTGTGTTAAACTA-3�), U3�3(5�-TGCTCTAGATGTATACTGACTCATATGTAA-3�), U3�4 (5�-TGCTCTAGACCCAGATCAGCATGGTGCCAGA-3�), U3�5 (5�-TGCTCTAGATAAACCCATCTGTTTGTC-3�), U3�6 (5�-TGCTCTAGAGGCCTAAAGTAGAAATAACAA-3�), and U3�7 (5�-TGCTCTAGAGCATGTTGCCTTCAAACAGTGT-3�). The amplified products were digested with XbaI and BglII and ligatedinto the NheI and BglII sites in pGL3-Basic (Promega).

Mutations were made in the pGL3-U3 plasmid with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instruc-tions. The resulting mutated pGL3-U3 plasmids were sequenced for confirma-tion.

W12 cells, a walleye fibroblast cell line, were seeded into 24-well plates in 1 mlof minimal essential medium supplemented with 10% fetal bovine serum(Gibco), 4 mM glutamine, 100 U of penicillin ml�1, and 100 �g of streptomycinml�1. W12 cells were derived from walleye dermal sarcomas in the laboratory ofPaul Bowser, Cornell University. They do not contain viral sequences (25);however, they are susceptible to infection with WDSV (S. L. Quackenbush,unpublished data). Cells were transfected with 0.2 �g of a luciferase reportervector and 0.05 �g of pRL-TK (Promega) by using FuGENE6 (Roche) accordingto the manufacturer’s suggestions. Experiments were performed using the dual-luciferase reporter assay system (Promega), which sequentially measures fireflyand Renilla luciferase activities from a single sample. Cell lysates were harvested72 h after transfection with passive lysis buffer and then centrifuged at 20,000 �g for 5 min at 4°C. Luciferase activities were obtained with a TD-20/20 lumi-nometer (Turner Designs) according to the manufacturer’s instructions. Lucif-erase activity from the reporter vector was normalized for transfection efficiencyby using values obtained from the cotransfected pRL-TK vector. All transfec-tions were performed in quadruplicate. Student’s t test and 95% confidenceintervals based on a t distribution were used for statistical analyses. A P value lessthan 0.05 was considered significant.

Nuclear extract preparation. Extracts were prepared by the method ofMayeda and Krainer (16), which is a modification of the method described byDignam et al. (9). Briefly, W12 cells were grown in minimal essential mediumsupplemented with 10% fetal bovine serum (Gibco), 4 mM glutamine, 100 U ofpenicillin ml�1, and 100 �g of streptomycin ml�1 at 20°C to 80% confluence in150-cm2 flasks. The cells were harvested and washed three times with coldDulbecco’s phosphate-buffered saline (D-PBS; 2.7 mM KCl, 1.5 mM KH2PO4,0.137 M NaCl, 8.1 mM Na2HPO4 · 7H2O). The packed-cell volume (PCV) wasmeasured, and the cell pellet was gently resuspended with 5 PCVs of buffer A (10mM HEPES-KOH [pH 8.0], 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol[DTT], and EDTA-free protease inhibitor cocktail [Roche]). The cells wereincubated on ice for 10 min and pelleted by centrifugation at 1,800 � g for 10min. Buffer A was added to 2 PCVs, and the cell suspension was homogenizedwith 30 strokes of pestle B in a Dounce glass homogenizer (Kontes GlassCompany) until the cells were �90% lysed, as determined by microscopy. Thelysate was centrifuged at 20,000 � g for 30 min at 4°C. The supernatant wasremoved, and the pellet, or packed nuclear volume (PNV), was measured. Then0.4 ml of buffer C (20 mM HEPES-KOH [pH 8.0], 0.6 M KCl, 1.5 mM MgCl2,0.2 mM EDTA, 25% [vol/vol] glycerol, 1 mM DTT, EDTA-free protease inhib-itor cocktail [Roche]) per ml of PNV was added. Cell nuclei were homogenizedwith 30 strokes of pestle A in a Dounce glass homogenizer. The suspension wasrocked at 4°C for 45 min and centrifuged for 45 min at 20,000 � g. Thesupernatant (nuclear extract) was dialyzed against buffer D (20 mM HEPES-KOH [pH 8.0], 100 mM KCl, 0.2 mM EDTA, 20% [vol/vol] glycerol, 0.5 mMphenylmethylsulfonyl fluoride, 1 mM DTT) overnight. The sample was centri-fuged at 20,000 � g for 30 min, and the supernatant (nuclear extract) wasaliquoted, frozen in liquid nitrogen, and stored at �80°C. Protein concentrationswere determined by the Bradford method (Bio-Rad).

Regressing and developing tumors were provided by James W. Casey and PaulR. Bowser, Cornell University. Nuclear extracts from regressing and developingtumors were prepared from a modification of the method described by Braun etal. (5). Briefly, the solid tumors were processed with a Tissue Tearor (Biospec

Products, Inc.) and washed in cold D-PBS. The pellet was resuspended in 0.5%NP-40 in PBS and rocked at 4°C for 30 min. The suspension was centrifugedbriefly, and the supernatant was removed. The pellet was resuspended in asolution containing 20 mM Tris-HCl (pH 8.0), 420 mM NaCl, 1.5 mM MgCl2, 0.2mM EDTA, 0.5 mM DTT, 25% glycerol, and EDTA-free protease inhibitorcocktail (Roche) and rocked at 4°C for 1 h. The suspension was centrifuged athigh speed for 30 min, and the supernatant (nuclear extract) was dialyzed againstbuffer D overnight. The nuclear extract was centrifuged for 30 min at 20,000 �g, and the supernatant was frozen and quantified as described above.

The W12 nuclear extract used to footprint the 15-bp repeats was processedidentically to that from the regressing and developing tumors, except treatmentwith the Tissue Tearor was omitted.

DNase I footprinting. The DNA probe used in the DNase I footprinting wasgenerated by PCR using PCR High Fidelity Supermix (Invitrogen). The primersused to amplify the U3 region of the WDSV promoter were RVprimer3 (5�-CTAGCAAAATAGGCTGTCCC-3�) and GLprimer2 (5�-CTTTATGTTTTTGGCGTCTTCCA-3�). The RVprimer3 was labeled with [�-32P]ATP by using T4polynucleotide kinase (PNK; New England Biolabs) according to the manufac-turer’s instructions. Briefly, the labeling reaction mixture contained 25 pmol of[�-32P]ATP (6,000 Ci/mmol, 10 mCi/ml), 20 pmol of RVprimer3, kinase reactionbuffer (70 mM Tris-HCl [pH 7.6], 10 mM MgCl2, 5 mM DTT) (New EnglandBiolabs), and 10 U of T4 PNK in a volume of 10 �l. The reaction mixture wasincubated at 37°C for 1 h and then heated to 95°C for 2 min. The PCR mixtureconsisted of PCR High Fidelity Supermix (Invitrogen), 0.2 �M 32P-labeledRVprimer3, 0.2 �M GLprimer2, and 50 ng of pGL3-U3 plasmid, in a finalvolume of 50 �l. The labeled U3 probe was purified with the QIAquick PCRpurification kit (QIAGEN) and resuspended in elution buffer (10 mM Tris-HCl,pH 8.5). The concentration of the labeled U3 was determined by measuring theabsorbance at 260 nm.

DNase I footprinting reaction mixtures consisted of 0.5 �g of lambda DNA(New England Biolabs), 15 fmol of labeled U3, and nuclear extract in bindingbuffer (25 mM Tris-HCl [pH 8.0], 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA,0.5 mM DTT, 10% [vol/vol] glycerol). Fifty microliters of a solution containing5 mM CaCl2 and 10 mM MgCl2 was added to the reaction mixtures, and themixtures were incubated at room temperature for 60 s. The reaction mixtureswere treated with 0.01 U of DNase I (Roche) for 30 to 90 s, as determined foreach probe, and the reaction was stopped by adding 90 �l of a stop solution (200mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, 100 �g of yeast tRNA/ml). The reaction mixtures were extracted with phenol-chloroform and precipi-tated with ethanol at �20°C. Samples were centrifuged at 20,000 � g for 10 minat room temperature. The supernatant was removed, and the DNA was washedonce with 300 �l of 70% ethanol and allowed to air dry. The pellets weresuspended in 11 �l of loading buffer (1:2 [vol/vol] 0.1 M NaOH-formamide, 0.1%xylene cyanol, 0.1% bromophenol blue). The samples were quantified by aPackard liquid scintillation counter, and equal counts (2 � 104 cpm) were loadedonto a 6% polyacrylamide sequencing gel (6% acrylamide-bisacrylamide [19:1],7 M urea, 1� TBE [89 mM Tris base, 110 mM boric acid, 2 mM EDTA]). Thegels were run at 1,500 V in 1� TBE, dried, and exposed to X-ray film at �80°C.Sequencing was performed with the Thermo Sequenase cycle sequencing kit(USB) according to manufacturer’s instructions, using the 32P-labeledRVprimer3, and run adjacent to the DNase I footprinting lanes to identify thesequences of the protected regions.

Gel mobility shift assays. To prepare double-stranded DNA probes for gelmobility shifts, the DNA oligonucleotides (Invitrogen) were annealed by mixing1 nmol of each primer in 1� TEN buffer (10 mM Tris [pH 8.0], 1 mM EDTA,100 mM NaCl) and incubating the oligonucleotides at 95°C for 5 min and thenslowly cooling them until reaching room temperature. The double-strandedoligonucleotides (probes) contained the specified binding site surrounded byirrelevant bases. The double-stranded DNA probes were end labeled with[�-32P]ATP and T4 polynucleotide kinase (New England Biolabs). Ten micro-grams of W12 nuclear extract and 1 �g of poly(dI-dC) (Amersham PharmaciaBiotech) were added to EMSA reaction buffer (10 mM Tris [pH 7.5], 50 mMNaCl, 1 mM DTT, 1 mM EDTA, 5% [vol/vol] glycerol) in a total volume of 20�l and incubated for 10 min at room temperature. The labeled oligonucleotideprobe (0.3 ng) was added, and the mixture was further incubated for 20 min atroom temperature. Different concentrations of nuclear extracts ranging from 2.5to 20 �g prepared from the W12 cell line were initially tested in EMSA. Tenmicrograms of nuclear extracts was found to bind several different probes opti-mally and was subsequently used in all assays. When antibodies were included(c-Jun [Upstate Biotechnology] and c-Fos and sc-253X [Santa Cruz Biotechnol-ogy]), they were added following the 20-min incubation and the reaction mixturewas incubated at 4°C overnight. The following day, the samples were placed atroom temperature for 20 min and then separated on a 6% polyacrylamide gel

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(6% acrylamide, 37.5:1 acrylamide-bisacrylamide, 2.5% glycerol, 0.5� TBE [45mM Tris base {pH 8.3}, 45 mM boric acid, 1 mM EDTA]) in 0.5 M TBE. Thegels were run at 200 V for 45 min, dried, imaged and analyzed with the Cyclonestorage phosphor system and OptiQuant image analysis software (Packard).

Western blotting. Fifty nanograms of recombinant human Jun (rhAP1; Pro-mega), 20 �g of A431 cell lysate (Upstate Biotechnology), 10 �g of HeLa cellnuclear extract, and 10 �g of W12 cell nuclear extract were separated on a 12%NuPAGE gel (Invitrogen) in MOPS (3-[N-morpholino]propanesulfonic acid)running buffer (50 mM MOPS, 50 mM Tris base, 3.5 mM sodium dodecyl sulfate,1 mM EDTA) at 200 V for 70 min. The proteins were then transferred to anImmobilon-P membrane (Millipore) at 150 mA for 2 h. The membrane was thenblocked for 30 min in BLOTTO (5% nonfat dry milk and 0.5% Tween 20 in PBS)and incubated overnight with a 1:1,000 dilution of anti c-Jun rabbit antiserum(Upstate Biotechnology) in BLOTTO. The blot was then washed and incubatedfor 1 h with a 1:5,000 dilution of affinity-purified goat anti-rabbit immunoglobulinG conjugated with horseradish peroxidase (Kirkegaard & Perry Laboratories)and developed with the substrate 3,3�,5,5�-tetramethylbenzidine (Kirkegaard &Perry Laboratories).

RESULTS

Analysis of sequential 5� U3 deletion mutants. To identifythe cis-acting regulatory sites that contribute to WDSV U3-driven transcription, luciferase reporter assays were employed.Progressive 50-bp deletions from the 5� end of the U3 regionwere constructed (Fig. 1), and luciferase activity was measuredafter transfection into the walleye cell line W12, which is sus-ceptible to infection with WDSV (Fig. 1). Cells were cotrans-fected with reporter plasmid pRL-TK to control for transfec-tion efficiency, and the activities of the deletions are expressedas percent activity relative to that of the full-length WDSV U3.Deletion of the first 50 bases of the U3 region (U3�1; nucle-otides �440 to �391 based on the start of transcription atposition �1) resulted in a significant decrease in luciferaseactivity to 50% of that expressed from the full-length U3 re-gion (Fig. 1). Further deletion to nucleotide �341 (U3�2) didnot result in significant changes in luciferase activity comparedto that for U3�1. A significant increase in luciferase activity,above that of U3�2, was seen after removal of the next 50bases (U3�3; P 0.01), suggesting the presence of a negative

regulator of transcription. There was a decrease in luciferaseactivity back to the levels of U3�1 and U3�2 when nucleotides�440 to �191 (U3�4 and U3�5) were deleted, and expressionof luciferase activity from U3�6 and U3�7 (deletion of nucle-otides �440 to �91) was 1 to 2% of wild type. These trunca-tions of upstream elements of the U3 region of the WDSVpromoter delineate the enhancer region (nucleotides �440 to�141) and the basal promoter element (�140 to �1).

Walleye cell nuclear extracts protect sites within the WDSVU3 region. DNase I footprint experiments were conductedusing nuclear extracts from the walleye cell line W12, in orderto identify specific protein-DNA interactions in the WDSVLTR. The W12 walleye fibroblast cell line was originally de-rived from a dermal sarcoma but does not harbor WDSV (25).When W12 cells are infected with WDSV, only low levels ofthe accessory viral transcripts orf a and orf b are expressed,similar to the expression pattern seen in developing tumors(22; S. L. Quackenbush, unpublished data.). The U3 region ofthe WDSV LTR was 5� end labeled on the noncoding strandand incubated with increasing concentrations of nuclear ex-tracts. The reaction products were treated with DNase I, sep-arated on a 6% sequencing gel, and visualized by autoradiog-raphy.

The presence of a putative Oct1 binding site in the U3region (position �366 to �359) was predicted by analysis withthe MatInspector database (www.genomatix.de) (23). This sitewas protected with nuclear extracts from W12 cells (Fig. 2) andwas accompanied by a DNase hypersensitive site as the amountof nuclear extracts increased.

Two footprints which do not contain consensus binding sitespresent in the MatInspector database were identified in the U3region of the WDSV LTR. One of these regions, Novel #1,maps to �350 to �345 in the U3 region (Fig. 2), and the otherprotected region, referred to as Novel #2, maps to �147 to�141 in the U3 region (Fig. 2).

FIG. 1. Luciferase activity of WDSV U3 deletion mutants. WDSV wild-type and U3 deletion mutant reporter constructs were transfected intoW12 cells. Relative luciferase values were corrected for transfection efficiency with pRL-Tk. The luciferase activity of each deletion mutant isexpressed as percent activity relative to that for the full-length U3 region. Putative cis-acting sites previously identified by others are shown (11,36). All experiments were performed in quadruplicate, and the means standard deviations of luciferase expression from three independentexperiments are shown. �, statistically significant value (P 0.05).

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Holzschu et al. (11) previously identified a 5-bp direct re-peat, TRTGT, that is present in the WDSV U3 region fivetimes. No known transcription factors are predicted to bind tothis sequence. However, two of these 5-bp repeats are part oftwo E4BP4 sites. All five repeats were protected by DNase Ifootprinting, and four of the five 5-bp repeats are shown in Fig.2. One region of protection overlaps the first two 5-bp repeatsand maps to �335 to �328. The second region of protection,maps to �319 to �311 in U3 (Fig. 2). This second regioncontains the third and fourth 5-bp repeats, and the first of twoE4BP4 sites, E4BP4 #1, that are present in the U3 region.

Using nuclear extracts prepared from W12 cells, a largefootprint was found to map to �293 to �270 in the U3 region(Fig. 2). The footprint overlaps a predicted Winged helix nude(Whn) binding site, an AP1 transcription factor binding site,and the second E4BP4 binding site, E4BP4 #2. The fifth 5-bp

direct repeat, is contained within E4BP4 #2. The AP1 andE4BP4 #2 binding sites are directly adjacent to each other andoverlap by 1 bp (Fig. 2).

These results demonstrate that proteins present in walleyecell nuclear extracts bind to specific sequences in the U3 regionof the WDSV promoter. These proteins may include homologsof Oct1, Whn, E4BP4, and components of the AP1 bindingcomplex. Also included in the walleye cell nuclear extracts areproteins capable of binding DNA sequences that do not con-tain known transcription factor binding sites.

Protected sites are specifically bound by nuclear proteins.EMSA were performed to confirm the specific binding of acellular factor(s) to sites protected by DNase I footprinting andalso to identify additional sites that were not protected.

A probe containing the predicted Oct1 binding site from theWDSV U3 region was incubated with W12 cell nuclear extracts

FIG. 2. DNase I footprint of the WDSV U3 region with W12 nuclear extracts. DNA probes were 5� end labeled on the noncoding strand andincubated with increasing amounts of W12 nuclear extracts. Dideoxy sequence reactions were run in adjacent lanes for reference. �, hypersensitivesite. The locations of the protected sequences are indicated on the right.



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and separated on a nondenaturing polyacrylamide gel. Theformation of a protein-DNA complex was observed as a singleshifted band (Fig. 3A, lane 2). The specificity of protein bind-ing to the Oct1 probe was demonstrated by competition with a100-fold molar excess of the unlabeled Oct1 probe (Fig. 3A,lane 3). Competition of nuclear extract binding was not possi-ble with a 100-fold molar excess of an unlabeled mutated Oct1probe or with a nonspecific competitor probe, further demon-strating specific binding (Fig. 3A, lanes 4 and 5).

Three regions that were protected in DNase I footprintanalysis but that do not contain known transcription factorbinding sites were subjected to EMSA with walleye cell nuclearextracts. Two shifted bands were observed when the Novel #1probe was used in EMSA (Fig. 3B, lane 2). The upper bandwas competed with excess unlabeled probe, but not with thenonspecific competitor probe (Fig. 3B). There appears to be alittle competition with the mutated Novel #1 probe for bindingto nuclear extracts (Fig. 3B, lane 4). The lower band was notcompeted with any of the probes (Fig. 3B) and likely repre-sents a nonspecific band. When a probe containing the first two5-bp repeats was incubated with W12 nuclear extracts, a pre-dominant shifted band was observed (Fig. 3C, lane 2), themajority of which could be competed with excess unlabeled

probe (Fig. 3C, lane 3). A nonspecific competitor did notcompete for binding of nuclear extracts; however, the mutated5-bp-repeat probe seemed to compete for some binding (Fig.3C, lanes 4 and 5). A minor, slower-migrating band was alsopresent; however, it was not competed with excess unlabeledprobe, suggesting that this is a nonspecific band. Incubation ofnuclear extracts with the Novel #2 probe resulted in the for-mation of a protein-DNA complex that appears to contain twobands (Fig. 3H, lane 2). A 100-fold molar excess of unlabeledprobe competed for binding of the nuclear extracts, as did themutated Novel #2 probe (Fig. 3H, lanes 3 and 4), whereas anonspecific competitor was unable to compete for binding(Fig. 3H, lane 5).

EMSA revealed the formation of three protein-DNA com-plexes when W12 nuclear extracts were incubated with a probethat contained the E4BP4 #1 site (Fig. 3D, lane 2). Band 1 wassuccessfully competed by 100-fold molar excess of unlabeledprobe (Fig. 3D, lane 3), and the majorities of bands 2 and 3were competed. The nonspecific competitor did not competefor binding of nuclear extracts (Fig. 3D, lane 5). The mutatedE4BP4 #1 probe competed for binding to nuclear extracts(Fig. 3D, lane 4). The mutation made in the E4BP4 probe did

FIG. 3. W12 nuclear extracts specifically bind to sites found protected by DNase I footprint analysis. Oligonucleotides containing individualbinding sites (Oct1, Novel #1, 5-bp repeats, E4BP4 #1, Whn, AP1, E4BP4 #2, and Novel #2) were end-labeled with 32P and incubated with 10�g of W12 nuclear extract. Lane 1, oligonucleotide alone; lane 2, oligonucleotide and 10 �g of W12 nuclear extract; lane 3, oligonucleotide, 10�g of W12 nuclear extract, and unlabeled oligonucleotide; lane 4, oligonucleotide, 10 �g of W12 nuclear extract, and unlabeled mutatedoligonucleotide; lane 5, oligonucleotide, 10 �g of W12 nuclear extract, and unlabeled nonspecific competitor oligonucleotide. In panels D and Gthe three bands are indicated.



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not change the 5-bp repeat sequence, which may still bindnuclear extract.

To examine the large protected region from position �293to �270, which contains the Whn, AP1, and E4BP4 #2 sites,individual probes for each of the sites were tested by EMSA. Apredominant protein-DNA complex formed with the Whnprobe (Fig. 3E, lane 2). A 100-fold molar excess of unlabeledprobe efficiently competed for binding of the complex (Fig. 3E,lane 3). The mutated Whn and the nonspecific competitorprobes did not compete for binding to nuclear extracts (Fig.3E, lanes 4 and 5). A probe containing only the AP1 site alsoyielded a predominant shifted band (Fig. 3F, lane 2), andspecificity of binding was demonstrated by competition withexcess unlabeled probe and lack of competition with the mu-tated AP1 and nonspecific competitor probes (Fig. 3F, lanes 3to 5). Mobility shift assays of the E4BP4 #2 site revealed theformation of three protein-DNA complexes, similar to thatobserved with the E4BP4 #1 probe (Fig. 3G and D). Again,the majorities of bands 2 and 3 were competed with excessunlabeled probe, whereas band 1 was completely eliminated(Fig. 3G, lane 3). Interestingly, analysis by densitometry deter-mined that bands 2 and 3 consistently bound 40% more of thelabeled E4BP4 #2 probe than of the E4BP4 #1 probe (Fig. 3Gand D). The two sites differ by 1 bp (Table 1), although thecontribution of this base in binding E4BP4 has not been pre-viously determined. The nonspecific competitor probe did notcompete for binding of nuclear extracts (Fig. 3G, lane 5). Themutated E4BP4 #2 probe competed somewhat for binding(Fig. 3G, lane 4) and again, like the E4BP4 #1 probe, con-tained the 5-bp repeat sequence, suggesting that a protein(s)other than E4BP4 may bind to this probe.

Three sites not protected from DNase I in footprinting as-says were evaluated with EMSA. When a probe containing apredicted NF1 site was incubated with W12 nuclear extract, ashifted band was observed, which was competed by an excess ofunlabeled probe and not by mutated NF1 or nonspecific com-petitor probes (Fig. 4A). Two sites identified by Holzschu et al.

(11), LVa and AP3, were also investigated. A large, predom-inant protein-DNA complex was observed with both probes(Fig. 4B and C, respectively), and both complexes could becompeted by a 100-fold molar excess of unlabeled probe (Fig.4B and C). Neither a mutated AP3 probe nor a nonspecificcompetitor probe competed for binding of nuclear extract,indicating the specificity of binding (Fig. 4C). The mutatedLVa probe was not successful at eliminating binding of nuclearextracts (Fig. 4B, lane 4).

These results confirm specific binding of proteins present inW12 nuclear extracts to sites found protected by DNase Ifootprinting and two sites that were not protected by footprintanalysis.

TABLE 1. cis-acting sites in the WDSV U3 region

SiteSequence (reference)

Consensus WDSVa Mutantb

NF1 ATTGGCT (27) ATTCGCT AccCcCTOct1 ATTTGCAT (30) ATATGCAT CaATGCAc (32)Novel #1 ?c ATACAT ATAggg5-bp repeats ? TATGTTGTGT TaaaaaaaGTE4BP4 #1 TYAYGTAA (7) TTATGTAA TTATGTcA (6)Whn NNNNACGCNNN (28) CCAGACGCTGT CCAGACaCTGT (28)AP1 TGACTCA (2) TGACTCA TacCTCA (24)E4BP4 #2 TYAYGTAA (7) ATATGTAA ATATGTcA (6)AP3 TAACCACA (17) TAACCACA TAAaacCA (17)Novel #2 ? GCTGTAT GCccccTLVa GAACAGT (29) GAACAGT TAACAtT (29)CHR1 TTCAARY (39) TTCAAAC ccCAACAFREAC4 CWNTGTTTACWTWRG (21) CAGTGTTTTCTTCAA CAGTGggTTCTTCAACHR2 TTCAARY (39) TTCAAC TTgggACHNF3� VAWTRTTKRYTY (20) CAGTATTTACTT CAGTcgaTcCTTSTAT5 TTCYNRGAAR (10) TTCTGAGAAT TTtTGAGAATPax 2/5/8 KNANRCTTWA (8) GAATCCATGA GAATCgggGACDP ATCGATTA (1) ATCCATGA ATCgggGA

a Bases in boldface deviate from the consensus sequence.b Bases in lowercase were mutated in the luciferase constructs.c ?, sequence unknown.

FIG. 4. W12 nuclear extracts specifically bind to sites not foundprotected by DNase I footprint analysis. Oligonucleotides containingindividual binding sites (NF1, LVa, and AP3) were end labeled with32P and incubated with 10 �g of W12 nuclear extract. Lanes 1, oligo-nucleotide alone; lanes 2, oligonucleotide and 10 �g of W12 nuclearextract; lanes 3, oligonucleotide, 10 �g of W12 nuclear extract, andunlabeled oligonucleotide; lanes 4, oligonucleotide, 10 �g of W12nuclear extract, and unlabeled mutated oligonucleotide; lanes 5, oli-gonucleotide, 10 �g of W12 nuclear extract, and unlabeled nonspecificcompetitor oligonucleotide.



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AP1 binds to the WDSV LTR. Characterization of the pro-teins that bind to the AP1 site in the WDSV U3 region re-quired an antibody that recognizes components of AP1 fromwalleye cells. An amino acid alignment was performed withc-Jun from humans (Homo sapiens), mice (Mus musculus),chickens (Gallus domesticus), carp (Cyprinus carpio), and fugu(Fugu rubripes) (data not shown). Based on the conservation ofthe DNA-binding domain of Jun among the species examined,an antibody that recognized the DNA-binding domain wasselected for the detection of c-Jun from walleye. Nuclear ex-tracts from W12 cells were subjected to Western blot analysiswith an antibody that was generated against the avian DNA-binding domain of c-Jun (Fig. 5). The anti-Jun antibody rec-ognizes recombinant human Jun (rhAP1), Jun from two hu-man cell lines, A431 and HeLa, and a protein that migrates atthe correct molecular mass from walleye cell (W12) nuclearextracts (Fig. 5), which suggests that this protein is the Junequivalent in W12 nuclear extracts.

EMSA were used to further investigate the binding of W12nuclear extracts to the AP1 site alone and also to the region ofWDSV U3 that encompasses the Whn, AP1, and E4BP4 #2sites. Anti-Jun antiserum could specifically disrupt the protein-DNA complex formed with W12 nuclear extracts and a probecontaining only the AP1 site (Fig. 6A, lane 3), whereas normal

rabbit serum did not affect complex formation (Fig. 6A, lane4). A distinct protein-DNA complex was observed when anoligonucleotide containing the Whn, AP1, and E4BP4 #2 siteswas incubated with W12 nuclear extracts (Fig. 6B, lane 2).Inclusion of anti-Jun antiserum in the reaction inhibited theformation of the complex (Fig. 6B, lane 3). The anti-Jun anti-serum was generated against the DNA-binding domain of Jun,suggesting that the anti-Jun antibody prevents the binding ofwalleye Jun to DNA, which results in disruption of the com-plex, instead of formation of a supershift. As illustrated in Fig.6B, lane 4, normal rabbit serum was unable to disrupt complexformation, indicating that inhibition of complex formation withanti-Jun antiserum was specific and that walleye Jun waspresent in the complex. To further assess the composition ofthe AP1 complex, EMSA was performed with an antibody thatrecognizes all of the Fos family members (c-Fos, FosB, Fra1,and Fra2). The anti-Fos antibody was also able to disruptcomplex formation (Fig. 6A), suggesting that the AP1 complexis a c-Jun/Fos heterodimer.

Effect of individual cis-acting sites on WDSV U3-driventranscription. To evaluate the functional role of the cis-actingelements protected in DNase I footprint analysis and identifiedwith EMSA, transcriptional activity of the WDSV U3 wasanalyzed with a luciferase reporter gene assay. Protein-bindingregions identified in both footprint analysis and EMSA weresubjected to site-directed mutation (Table 1). Mutations ofNovel #1 and Novel #2 were analyzed to confirm that muta-tions did not result in the generation of known sites. WalleyeW12 cells were transfected with each luciferase reporter con-struct and pRL-TK, a Renilla reporter plasmid, to control fortransfection efficiency, and the ratio of relative luciferase ac-tivity to Renilla activity for the wild-type WDSV U3 was set at100%.

The Oct1 site that was protected by DNase I footprint anal-

FIG. 5. Detection of Jun in walleye cell nuclear extracts. Recom-binant human Jun (rhAP1), A431 cell lysate, HeLa cell nuclear extract(NE), and W12 cell nuclear extract were separated on a NuPage 12%Bis-Tris gel and transferred to an Immobilon membrane. The blot wasprobed with rabbit anti-Jun antiserum that recognizes the DNA-bind-ing domain of avian Jun.

FIG. 6. c-Jun in W12 nuclear extracts binds to the AP1 consensus site in the WDSV LTR. (A) W12 nuclear extracts were incubated with a5�-end-labeled oligonucleotide containing the AP1 binding site. (B) W12 nuclear extracts were incubated with a 5�-end-labeled oligonucleotidecontaining the Whn, AP1, and E4BP4 #2 binding sites. Anti-Jun antiserum (Jun), normal rabbit serum (N), anti-Fos antiserum (F), or irrelevantantiserum (I) was added to the specified reaction mixture. The reaction products were separated on a 6% nondenaturing polyacrylamide gel andvisualized with a phosphorimager.



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ysis (Fig. 2A) was mutated, and expression levels were com-pared to wild-type U3 expression. Expression from the WDSVOct1 mutant reporter construct resulted in significantly lessactivity than that observed with the wild-type U3 construct(69.2% 9.3%; Fig. 7B; P 0.03).

The Novel #1 site protected in DNase I footprint analysiswas subjected to mutation, and luciferase activity was mea-sured (Fig. 7B). Three bases were mutated in the Novel #1 site(Table 1), and mutation of this site did not significantly affectluciferase expression (104.5% 5.6%; Fig. 7A).

The first two of the 5-bp repeats present in the U3 regionmapping to �336 to �326 (Fig. 2D) were mutated, and lucif-erase expression was measured. Mutation of six of the pro-tected bases (Table 1) did not result in a measurable change inluciferase activity compared to that for the wild type (99.9% 10.3%; Fig. 7B). Mutation of the predicted E4BP4 binding site,E4BP4 #1, which overlaps the fourth 5-bp repeat at position�317 to �310, did not result in a significant change in lucif-erase activity compared to that for the wild type, (94.4% 4.7%; Fig. 7B).

Mutation of the AP1 site in the WDSV U3 reporter con-struct resulted in a significant decrease in luciferase expressioncompared to that of the wild-type U3 construct (73.0% 4.4%; Fig. 7B; P 0.009). The predicted E4BP4 site adjacentto the AP1 binding site, E4BP4 #2, was mutated and luciferaseactivity was measured. In contrast to the activity from themutated E4BP4 #1 site, luciferase activity resulting from mu-tation of the second E4BP4 site was significantly reduced com-pared to wild type (58.5% 8.9%; Fig. 7B; P 0.02). Also,mutation of the Whn site resulted in significantly reducedluciferase activity (70.2% 3.0%; Fig. 7B, P 0.003).

A third site, Novel #2, found to be protected by DNase I

footprinting (Fig. 2C), was mutated, and luciferase activity wasmeasured. Transfection of this mutated U3 reporter constructinto W12 cells resulted in a significant increase in luciferaseactivity (137.8% 1.7%; Fig. 7B; P 0.001). These datasuggest that the Novel #2 region may bind a transcriptionalrepressor.

Zhang et al. (35) conducted a functional analysis of theWDSV LTR using a luciferase reporter construct by introduc-ing progressive deletions from the 5� end of the WDSV LTRand measuring the luciferase activity of these deletion mutants.When a region of the WDSV U3 containing an NF1 site wasdeleted, there was a decrease in luciferase activity compared tothat for the wild-type LTR (35). This site was not found to beprotected by DNase I footprinting but did bind W12 nuclearextract in EMSA (Fig. 4A). Mutation of the NF1 site resultedin a significant decrease in expression compared to that for thewild-type WDSV U3 (86.2% 1.8%; Fig. 7C; P 0.006).

Two putative transcription factor binding sites in the WDSVU3, AP3, and LVa, were previously identified by Holzschu etal. (11). Again, these sites were not protected by DNase Ifootprint analysis (data not shown); however, they were foundto bind nuclear extracts in mobility shift assays (Fig. 4B and C).Transfection of the WDSV U3 reporter constructs containingeither an AP3 or LVa mutation into W12 cells resulted insignificantly lower levels of luciferase activity: 76.1% 9.7%(Fig. 7C; P 0.05) or 56.5% 8.7% (Fig. 7C, P 0.01),respectively.

These data demonstrate that several cis-acting sites presentin the U3 region of the WDSV LTR functionally contribute totranscription activation.

Regressing-tumor nuclear extracts protect the 15-bp re-peats. Nuclear extracts prepared from regressing tumors ob-

FIG. 7. Activity of mutant WDSV U3 reporter constructs. (A) Diagram of the transcription factor binding sites characterized by DNase Ifootprint analysis, EMSA, and luciferase assays. (B) Relative luciferase activity of the pGL3-U3 luciferase plasmid with mutations in the indicatedsites (Table 1) found protected by DNase I footprint analysis. (C) Relative luciferase activity of the pGL3-U3 luciferase plasmid with mutationsin the indicated sites (Table 1) that were not found protected by DNase I footprint analysis. The luciferase activity of the mutants is expressed aspercent activity relative to that for the full-length U3 region. All experiments were performed in quadruplicate, and the means standarddeviations of luciferase expression from three independent experiments are shown. �, statistically significant (P 0.05).



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tained from naturally infected walleye fish were used in DNaseI footprint analysis. The sites found protected with W12 cellnuclear extracts (Fig. 2) were also protected with the tumorcell nuclear extracts (data not shown). In addition, a verystrong footprint was observed overlapping the degenerative15-bp repeats that map to �82 to �36 in the U3 region (Fig.8A, C, and D). Immediately apparent is a very predominantDNase I hypersensitive site (Fig. 8A and C), which corre-sponds to the A in the middle of the first 15-bp repeat. Thecorresponding A in the middle of the second 15-bp repeat isalso present as a hypersensitive site, although it is not as in-tense as that in the first 15-bp repeat (Fig. 8B and C). Thereare four regions of protection which are interspersed betweenfour additional hypersensitive sites (Fig. 8A, C, and D).

DNase I footprint analysis of the 15-bp repeats with W12

nuclear extracts revealed a striking difference from that ob-served with the tumor nuclear extract (Fig. 8B to D). Tworegions of protection are found with W12 nuclear extracts, aswell as five faint hypersensitive sites (Fig. 8B to D). Two of thehypersensitive sites are located at the A in the middle of thefirst two 15-bp repeats, similar to that found with the regress-ing-tumor extracts (Fig. 8). The additional three hypersensitivesites do not correspond to the hypersensitive sites found byusing regressing-tumor nuclear extract.

EMSA was used to confirm binding of nuclear extracts to the15-bp repeats. An oligonucleotide containing the 15-bp repeatregion was incubated with W12 cell and regressing- and devel-oping-tumor extracts and separated on a nondenaturing poly-acrylamide gel (Fig. 9). Two predominant protein-DNA com-plexes were observed with all three of the nuclear extracts. A

FIG. 8. Differential protection of the degenerate 15-bp repeats in WDSV U3. DNase I footprint analysis of the degenerate 15-bp repeats afterincubation with regressing-tumor nuclear extracts (NE) (A), W12 nuclear extracts (B), and both W12 and regressing-tumor nuclear extracts (C).DNA probes were 5� end labeled on the noncoding strand and incubated with the indicated quantities of nuclear extracts. Dideoxy sequencereactions were run in adjacent lanes for reference. �, hypersensitive sites. (D) Representative diagram showing the sequence of the 15-bp repeats(boxed). The regions of protection (black bars) and hypersensitive sites (�) found with the W12 nuclear extracts and the regressing-tumor nuclearextracts are shown on the top and bottom of the sequence, respectively.



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slower-migrating protein-DNA complex was detected with theW12 cell and developing-tumor nuclear extracts but was notpresent with the regressing-tumor extracts. An additional pro-tein-DNA complex formed with the developing-tumor andW12 cell nuclear extracts; however, the intensity of the bandwas stronger with the developing-tumor extracts.

These data demonstrate a significant difference in the foot-printing patterns of the 15-bp repeats between the W12 nu-clear extracts and regressing-tumor nuclear extracts. Also, astriking similarity in binding of nuclear extracts from W12 cellsand developing tumors to the 15-bp repeat probe was demon-strated by EMSA.

The 15-bp repeat region contributes to negative regulationof WDSV U3 transcription. Analysis of the 15-bp repeat regionwith the MatInspector database identified two cell cycle genehomology region sites (CHR) and one each of forkhead-re-lated activator 4 (FREAC4), hepatocyte nuclear factor 3�(HNF-3�), STAT5, CCAAT displacement protein (CDP), andPAX 2/5/8 sites (Fig. 10B). These sites were mutated by site-directed mutagenesis in the context of the pGL3-U3 luciferaseplasmid and assayed for luciferase expression as describedabove. The CDP and Pax sites overlap; therefore, it was notpossible to mutate one without disrupting the other site. As aresult, both sites were disrupted in the luciferase reporter con-struct. Mutation of STAT5, CHR#1, and FREAC4 did notresult in a significant difference in luciferase activity (86.7% 7.5%, 79.1% 12.7%, and 105.1% 14.3%, respectively)compared to that of the wild type (100%) (Fig. 10A). However,mutation of the HNF-3�, CHR#2, and CDP/Pax sites resultedin an increase in luciferase activity: 163.8% 20.8% (P 0.04), 151.4% 10.3% (P 0.02), and 148.5% 17.5% (P 0.05), respectively (Fig. 10A). These data demonstrate thatthree sites within the 15-bp repeat region of the WDSV pro-moter contribute to negatively regulate transcription in W12cells.

DISCUSSION

There are large differences in WDSV gene expression be-tween developing and regressing tumors collected from natu-rally infected walleye (4, 22). The WDSV protein OrfA func-tions to inhibit expression from the WDSV promoter intransient-transfection experiments conducted with a walleyecell line. However, very little is known regarding what host cellfactors are necessary for transcription from the WDSV pro-moter or whether OrfA functions through a DNA-bindingprotein. In this study, specific host cell protein-DNA interac-tions in the WDSV U3 region were identified and the func-tional contribution of each site was evaluated.

Initially, mutants with 5� progressive 50-bp deletions of theU3 region were constructed and luciferase activity was mea-sured. Based on these data, the enhancer region of the WDSVU3 was mapped to nucleotides �440 to �141 and the basalpromoter was mapped to �141 to �1. DNase I footprint anal-ysis was performed using walleye cell nuclear extracts, and sixregions of protection were found. Individual sites included inthese protected regions were an Oct1 site, an AP1 site, a Whnsite, two E4BP4 sites, a series of 5-bp repeats, and two novelsites. Specific binding of nuclear extracts to these sites wasconfirmed by EMSA. There was a little competition with themutated Novel #1 and the 5-bp repeat probes. Currently we donot know what proteins bind to these sites; therefore, it ispossible that the mutations did not completely disrupt binding.A region of protection, including a prominent hypersensitivityband, of the Novel #2 site was clearly identified in DNase Ifootprint analysis. EMSA confirmed binding of nuclear ex-tracts to the Novel #2 probe. The unlabeled probe competed

FIG. 9. Binding of W12 and developing- and regressing-tumor nu-clear extract to the 15-bp repeats. An oligonucleotide containing the15-bp repeats was end labeled with 32P and incubated with the indi-cated nuclear extract. Lane 1, oligonucleotide alone; lane 2, oligonu-cleotide and 10 �g of W12 nuclear extract (W12); lane 3, oligonucle-otide and 10 �g of developing-tumor nuclear extract (Dev); lane 4,oligonucleotide and 10 �g of regressing-tumor nuclear extract (Reg).

FIG. 10. Activity of mutant WDSV U3 reporter constructs.(A) Relative luciferase activity of the pGL3-U3 luciferase plasmid withmutations in the indicated sites found in the 15-bp repeats. The lucif-erase activity of the mutants is expressed as percent activity relative tothat for the full-length U3 region. All experiments were performed inquadruplicate, and the means standard deviations of luciferase ex-pression from three independent experiments are shown. �, statisticallysignificant (P 0.05). (B) Representative diagram showing the se-quence of the 15-bp repeats (boxed). The transcription factor-bindingconsensus sites are underlined.



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for binding, as did the mutated probe, indicating that themutation did not disrupt binding. Interestingly, the mutation ofthe Novel #2 site resulted in an increase in luciferase activity,suggesting the presence of a negative element; however, thissite does not contain consensus binding sites for any knowntranscription factors. To further address the importance of theNovel #2 site in transcription will require identification of thehost proteins that bind to this region.

We also examined three additional putative binding sitesidentified by others (11, 35) for their contribution to transcrip-tion of WDSV. Two sites, AP3 and LVa, were previouslyidentified in the U3 region (11). Mobility shift assays wereperformed with these sites using walleye cell nuclear extract,and specific binding was observed. Mutation of both the AP3and LVa sites resulted in decreased luciferase activities, sug-gesting the importance of these two sites in the transcriptionactivation driven by the WDSV U3 region. The reason we didnot observe binding in the DNase I footprinting experiments isnot known; however, difficulties in DNase I footprinting usingcrude nuclear extracts have been previously reported (19).

An additional putative site, NF1, was first identified in da-tabase analysis performed by Zhang et al. (35). In their dele-tion analysis study, removal of the first 60 bp of the U3 region,including the putative NF1 site mapping to �403 to �397,resulted in decreased luciferase activity (35). In our deletionanalysis, we also observe a decrease in luciferase activity afterremoval of the first 50 bp (U3�1), thus supporting Zhang et al.(35). Based on this information, we further investigated theNF1 site by performing mobility shift analysis and site-directedmutagenesis on the NF1 site in the context of the luciferasereporter plasmid. Specific binding was observed in mobilityshift analysis, and mutation of NF1 resulted in a significantdecrease in luciferase activity.

Previous work by Zhang et al. (35) using deletion mutantsdemonstrated that deletion of nucleotides �440 to �325,which removed the first four 5-bp repeats, resulted in an in-crease in luciferase activity, suggesting that a negative regula-tor of transcription functions through these repeats. In thisreport, deletion of �440 to �291 also resulted in an increase ofluciferase activity. However, site-directed mutation of the 5-bprepeats did not result in a significant change in luciferaseactivity. Therefore, we cannot unequivocally define this regionas binding a negative regulator of transcription.

The Whn, AP1, and E4BP4 #2 sites reside in close proximityto each other (nucleotides �294 to �269). When Zhang et al.deleted the portion of the U3 region from �440 to �275,which removes the Whn binding site, the entire AP1 site, andthe E4BP4 #2 site, a reduction in luciferase activity was ob-served (35). In our deletion analysis, removal of the Whn, AP1,and E4BP4 #2 sites (U3�4) resulted in a decrease in luciferaseactivity (Fig. 1, compare U3�3 to U3�4), confirming the ob-servation by Zhang et al. (35). We further extend these studieswith site-directed mutations of the Whn, AP1, and E4BP4 #2sites, which all resulted in significant decreases in activity com-pared to that observed with the wild-type U3 construct.

The AP1 site present in the U3 region was further analyzedwith antiserum generated against the DNA-binding domain ofavian Jun. Western blot analysis demonstrated the presence ofwalleye Jun in W12 nuclear extracts, and this antiserum couldspecifically disrupt the protein-DNA complex formed in mo-

bility shift assays. This demonstrates that Jun is present inwalleye cell nuclear extracts and participates in binding to theAP1 site. Antiserum that recognizes all of the Fos family mem-bers was also able to disrupt complex formation, suggestingthat a c-Jun/Fos heterodimer binds to the AP1 site. The iden-tification of the Fos family member will require antibodies thatrecognize individual Fos proteins. Interestingly, the anti-Junantiserum disrupted the majority of the protein-DNA com-plexes formed with W12 nuclear extracts and a probe contain-ing the Whn, AP1, and E4BP4 #2 sites. This suggests that AP1is the predominant factor binding to this region or that AP1binding is required for Whn and E4BP4 to bind. However,footprint analysis revealed protection over all three sites, andsite-directed mutagenesis of all three sites resulted in de-creased levels of luciferase activity. Therefore, further analysisof this region using additional antibodies and/or purified pro-teins is necessary.

Experiments addressing the functional role of the twoE4BP4 sites revealed that mutation of only E4BP4 #2 resultedin a decrease in luciferase activity. Mobility shift analysis ofeach site revealed similar patterns of binding, although bands2 and 3 of E4BP4 #2 consistently bound more nuclear extractthan those of E4BP4 #1. The binding pattern observed isconsistent with a previously published report investigating therole of E4BP4 regulation by interleukin-3 in Baf-3 pro-B lym-phocytes (12). E4BP4 #1 matches the E4BP4 consensus se-quence determined by Cowell et al. (7) exactly, whereas E4BP4#2 differs from the consensus sequence by one base. The basethat differs from the consensus in E4BP4 #2 is the last base ofthe AP1 site. We currently don’t know why mutation of E4BP4#2 results in decreased activity from the WDSV reporter con-struct, while no difference is apparent with mutation of E4BP4#1. However, the difference could be attributed to the se-quence difference between the two sites, which leads to aweaker binding of E4BP4 to E4BP4 #1, as suggested by themobility shift assays, or perhaps E4BP4 needs to interact withother transcription factors, such as AP1. With respect to thisidea, a recent report has indicated that the purified dimeriza-tion domains of Jun and Fos family members do not interactwith the purified dimerization domain of E4BP4; however,these experiments do not rule out an interaction of the full-length proteins (18).

Perhaps the most interesting observation resulting fromthese experiments concerns the 15-bp repeats in the U3 region.The footprint patterns of the 15-bp repeats for the W12 andregressing-tumor nuclear extracts are clearly distinct. This dif-ference could be attributed to different cellular factors bindingto the 15-bp repeats, which is supported by the distinct foot-print patterns, and to the presence of different hypersensitivesites. Another possibility would be different levels of the samecellular factors, which is supported by two similar regions ofprotection and by the presence of two identical hypersensitivesites. This leads to the hypothesis that the 15-bp repeats con-tribute to the differential expression of WDSV. EMSA re-vealed a different pattern of protein-DNA complex formationwhen nuclear extracts from regressing tumors were comparedto those formed on the 15-bp repeat probe with W12 cell andextracts from developing tumors. These results suggest that thetranscription factor compositions of the developing tumors andW12 cells are similar. Interestingly, when W12 cells are in-



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fected with WDSV, only low levels of accessory viral tran-scripts, like those seen with developing tumors, are detected(Quackenbush, unpublished). Six different transcription factor-binding sites were identified within the 15-bp repeat region.Mutation of three of these sites, HNF3�, CHR2, and CDP/Pax, resulted in an increase in luciferase activity when activityin W12 cells was assayed, suggesting that these proteins maycontribute to the low level of viral gene expression observedearly in infection with WDSV. CDP is a repressor of mousemammary tumor virus (MMTV) expression (37, 38). Expres-sion of CDP decreases as the mammary gland differentiates,which correlates with increased MMTV expression. Furtherstudies identifying the factor(s), either host or viral or a com-bination of both, that binds to the 15-bp repeats will provideinsight into the regulation of WDSV gene expression.

ACKNOWLEDGMENTS

We thank Paul R. Bowser and James W. Casey for providing tumorsamples.

This research was supported by research project grant RPG-00-313-01-MBC from the American Cancer Society. J.R. was supported byNational Research Service Award F32 CA88572-01.

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