Proc. Natl. Acad. Sci. USAVol. 90, pp. 282-286, January 1993Biochemistry

Characterization of cis-acting elements regulating transcription ofthe human DF3 breast carcinoma-associated antigen (MUCI) geneMIYAKO ABE AND DONALD KUFELaboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115

Communicated by Christopher T. Walsh, October IS, 1992

ABSTRACT The present studies have examined the se-quences responsible for regulating transcription of the humanDF3 breast carcinoma-associted antigen (MUCI) gene. Aregion 1656 base pairs upstream to the DF3 transcriptioninitiation site was fused to the chloramphenical acetyltrans-ferase gene. Transient expression assays using a series ofdeleted constructs demonstrated that the region frown position-618 contains the regulatory sequences necessary for DF3transcription in human MCF-7 breast cancer cells. Furtheranalysis with internal deletion vectors and heterologous pro-moter constructs indicated the involvement of cis-acting ele-ments in the fragment extending from positions -598 to -485.By gel retardation and DNA footprinting, we have identified aprotein in MCF-7 cells that recognizes sequences betweenpositions -505 and -485. The results of Southwestern studiesdemonstrate that this protein has an apparent molecular massof 45 kDa. Taken together, these results suggest that DF3 genetranscription is regulated by a previously undescribed trans-acting factor.

The human DF3 (MUCI) gene encodes a high molecularmass glycoprotein that is aberrantly expressed by malignantmammary epithelium. The DF3 glycoprotein is a member ofa family of related carcinoma-associated antigens with coreproteins ranging from 160 to 230 kDa (1, 2). This antigen isexpressed on the apical borders of secretory mammaryepithelial cells and at high levels in the cytosol of lessdifferentiated malignant cells (3). DF3 antigen expressioncorrelates with the degree of breast tumor differentiation (4).Moreover, the detection of this antigen in human milk (5) hassuggested that its expression represents a differentiated func-tion of normal mammary epithelium. Other work has dem-onstrated that this family of glycoproteins regulates immunerecognition and cellular adhesion (6, 7).Sequence analysis of cDNAs coding for the DF3 protein

has demonstrated variable numbers of highly conserved(G+C)-rich 60-base-pair (bp) tandem repeats that contributeto allelic variation and expression of a polymorphic DF3glycoprotein (8, 9). Similar findings have been reported forthe related PEM, H23 and pancreatic glycoproteins (10-14).The N-terminal region contains hydrophobic signal se-quences that vary as a result of alternate splicing, whereas theC-terminal region includes a transmembrane domain andcytoplasmic tail (14-17). The gene contains seven exons,spans 4-7 kilobases (kb) depending on the number oftandemrepeats, and has been mapped to chromosome 1q21-24 (18,19). Other findings have demonstrated that the chromosomalregion around this locus is affected by mutations at highfrequency (9).

Previous studies have demonstrated that the DF3 gene ishighly overexpressed in human breast carcinomas and thatexpression ofthis gene is regulated at the transcriptional level(3, 20). The present work has examined the mechanisms that

control DF3 gene transcription. The results demonstrate thatthe regulatory sequences are located in the 5' flanking regionof this gene and that a previously undescribed cis element isfunctional in controlling transcription.*

MATERIALS AND METHODSCell Culture. Human MCF-7 breast carcinoma cells (Mich-

igan Cancer Foundation, Detroit) were grown as a monolayerin Dulbecco's modified Eagle's medium (DMEM) with 10oheat-inactivated fetal bovine serum, 100 units ofpenicillin perml, 100 ,Ag of streptomycin per ml, 2 mM L-glutamine, and0.25 unit insulin per ml.

Plasmid Construction. A pA vector was generated byexcising the simian virus 40 promoter and enhancer regionsfrom pSVT7 (21). Chloramphenicol acetyltransferase (CAT)expression vectors pSVT7CAT and pACAT were producedby inserting the CAT gene in the multicloning HindIll site ofpSVT7 and pA, respectively. A p-1656CAT vector wasconstructed by inserting theXmn I fragment (positions -1656to +31) of the DF3 gene into pACAT at the Pst I site byblunt-ended DNA ligation. The p-725CAT vector was pre-pared by inserting the blunt-ended Sst I/Xmn I fragment(positions -725 to +31) into pACAT. A series of deletionvectors were generated from p-725CAT after treatment withSst I/Xba I and subsequent digestion with exonuclease IIIand S1 nuclease. Sequencing ofthe constructs was performedby dideoxy termination (22).

Reporter Assays. All vectors used in the reporter assayswere purified twice by cesium chloride gradient centrifuga-tion and phenol/chloroform extraction. Twenty microgramsof each plasmid was transfected into 3 x 105 MCF-7 cells bythe Ca2PO4 procedure (23). The cells were incubated incomplete medium for 48 hr after transfection, harvested, andlysed in 250 ,uM Tris-HCl (pH 8.0) by three cycles offreeze-thawing. CAT activity was assayed in 125-Al reactionmixtures containing 10-50 1ul of cell extract, 125 nCi of[14C]chloramphenicol (DuPont; 57 mCi/mmol; 1 Ci = 37GBq), 250 mM Tris'HCl (pH 8.0), and 25 ,ug of n-butyrylcoenzyme A (Sigma) for 1 hr at 370C. The reaction wasterminated by adding 300 jul of xylene. The nonbutyratedchloramphenicol was removed by washing twice with 100 y.of 0.25 M Tris HCl (pH 8.0). The xylene layer containing thebutyrated chloramphenicol was assayed by scintillation spec-troscopy (24).

Electrophoretlc Mobility Shift Assays. Nuclear proteinswere prepared according to previously described methods(25). Synthetic oligonucleotides were end-labeled with theappropriate a-32P-labeled dNTPs using DNA polymerase I(Klenow fragment) and purified by Nuc Trap 0ohltinns (Stra-tagene). Binding assays were performed as described (26).Ten micrograms of nuclear proteins was incubated with 10 ,ugof poly(dI-dC) in 25 Al of 10 mM Tris-HCl buffer (pH 7.5)

Abbreviations: CAT, chloramphenicol acetyltransferase.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. L06162).

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containing 50 or 150 mM KCI, 5 mM MgCI2, 1 mM dithio-threitol, 1 mM EDTA, 12.5% glycerol, and 0.1% Triton X-100for 30 min at room temperature. After adding the labeledprobe (1 x 105 cpm, 1 ng) and incubating for an additional 30min, the samples were separated in a low ionic strength 4.5%polyacrylamide gel (acrylamide/bisacrylamide, 30:0.8) con-taining 1 mM EDTA, 3.3 mM sodium acetate, and 6.7 mMTris-HCl (pH 7.5) or in a high ionic strength gel containing 50mM Tris-HCl (pH 8.5), 380mM glycine, and 2 mM EDTA (26,27). Unlabeled oligonucleotides as competitors, including theconsensus sequences of SP1, NF-1, AP-1, AP-2, and AP-3(Stratagene), were added at the same time as the labeledfragment.DNase I Footprint Analysis. Fragment f(-618/-410) was

ligated into pGEM3 at the Sma I site. The vector was digestedwith EcoRI and Acc I, end-labeled with [32P]dATP and dTTPat the EcoRI site, and purified in a 5% native polyacrylamidegel. The specific activity ofthe labeled fragment was -3 x 105cpm/ng. The assay was performed as described (28). Thereaction products were analyzed by 12% polyacrylamide/urea gel electrophoresis and subjected to autoradiography.Sequences were monitored by the Maxam-Gilbert procedure(29).Southwestern Analysis. Nuclear protein (200 ,Ag) was sep-

arated by 3-15% SDS/polyacrylamide gel electrophoresis

Proc. Natl. Acad. Sci. USA 90 (1993) 283

and transferred to a nitrocellulose sheet (30, 31). The sheetwas incubated in 10 mM Tris'HCI (pH 7.5) containing 5%skimmed milk and then incubated in 10 mM Tris HCl (pH 7.9)containing 32P-labeled oligonucleotide probe (106 cpm/ml),50 mM NaCl, 10 mM MgCI2, 0.1 mM EDTA, 1 mM dithio-threitol, and 10 ,ug of poly(dI-dC) per ml for 60 min at roomtemperature. The sheet was washed with the same buffercontaining 200 mM NaCl and exposed to x-ray film (32, 33).

RESULTS

Sequence of the DF3 5' Flanking Region. We previouslyused a genomic library from MCF-7 cells to isolate a clonecontaining >2 kb of sequences upstream to the transcriptionstart site (15). The digestion of this clone with Xmn I resultedin a fragment extending from positions -1656 to +31. Pre-vious studies from this laboratory included sequences toposition -430 (15), and the remainder to position -1656 areincluded in Fig. 1A. A potential TATA box (TATAAA) wasidentified 25 bases upstream to the start site (15). The 5'flanking region also includes potential binding sites withhomology to the consensus sequences for (i) SP1 (GGGCGG)at positions -52, -56, -60, -102, -405, -582, -739, -806,and -1063; (ii) AP-1 (GAGAGGA) at positions -812 and-1046; (iii) AP-2 at positions -214 (GGGAGGCC) and -488

A5,-

-1656 GCTTCCGTGC GCCTAGAGCG CAGCCTGCGA CTGCGGGACC CAACAACCAC GTGCTGCCGC GGCCTGGGAT AGCTTCCTCC CCTCTGGCAC TGCTGCCGCA

-1556 CACACCTCTT GGCTGTCGCG CATTACGCAC CTCACGTGTG CTTTTGCCCC CGCCTACGTG CCTACCTGTC CCCAATACCA CTCTGCTCCC CAAAGGATAG

-1456 TTCTGTGTCC GTAAATCCCA TTCTGTCACC CCACCTACTC TCTGCCCCCC CCTTTGT TGAGACGG AGTCTTGCTC TGTCGCCCAG GCTGGAGTGC

-1356 AATGGCGCGA TCTCGGCTCA CTGCAACCTC CGCCTCCCGG GTTCAAGCGA TTCTCCTGCC TCAGCCTCCT GAGTAGCTGG GGTTACAGCG CCCGCCACCANF- I ER

-1256 CGCTCGGCTA AT1TTTGTAG TTrTTAGTAG AGACGAGGTT TCACCAT~r GCTG GTCTTGAACC C ~rg TGATCCACTC GCCTCGGCCT

-1156 TCCAAAGTGT TGGGATTACG GGCGTGACGA CCGTGCCACG CCCGATCTGC CTCTTAAGTA CATAACGGCC CACACAGAAC GTGTCCAACT CC CAP- 1

-1056 CGTTCCAACG :TCA CATACCTCGG TGCCCCTTCC ACATACCTCA GGACCCCACC CGCTTAGCTC CATTTCCTCC AGACGCCACC ACCACGCGTCER

- 956 CCGGAGTGCC CCCTCCTAAA GCTCCCAGCC GTCCACCATG CTGTGGT CTCCCTCCCT GGCCACGGCA t2ITC TCTCCCGGGC CCTGCTTCCCA-1 SPi

- 856 TCTCGCGGGC TCTCGCTGCC TCACTTAAGC AGCGCTGCCC TrAcE G C CGAGCGGCCC CTCAGCTTGC GCGGCCCAGC CCCGCAAGGCER SPI

- 756 TCCC CTAGA CGAGC TCCTGGCCAG TGGTGGAGAG TGGCAAGGAA GGACCCTAGG GTTCATCGGA GCCCAGGTTT ACTCCCTTAAAP-3 SPI

- 656 GTGGAAATTT CTTCCCCCAC TCCCTCCTTG GCTTTCTCCA AGGAGGGAAC CCAGGCTGCT ,;AA=C GCTt2 3j GGACTGTGGG TTTCAGGGTAAP-2

- 556 GAACTGCGTG TGGAACGGGA CAGGGAGCGG TTAGAAGGGT GGGGCTATTC CGGGAAGTGG TGGGGGGA CA CTAGCACCTA GTCCACTCATSPI

- 456 TATCCAGCCC TCTTATT'CT CGGCCCCGCT CTGCTTCAGT GGACCCGGGG A222GA AGTGGAGTGG GAGACCTAGG GGTGGGCTTC CCGACCTTGC

- 356 TGTACAGGAC CTCGACCTAG CTGGCTC TCCCCATCCC CACGTTAGTT GTTGCCCTGA GGCTAAAACT AGAGCCCAGG GGCCCCAAGT TCCAGACTGCAP-2

- 256 CCCTCCCCCC TCCCCCGGAG CCAGGGAGTG GTTGGTGAAA AGCTGGAAA CAACGGGTA GTCAGGGGGT TGAGCGATTA GAGCCCTTGTSPi SPI

- 156 ACCCTACCCA GGAATGGTTG GGGAGGAGGA GGAAGAGGTA GGAGGTAGGG GAGcv=G GGTTTTGTCA CCTGTCACCT GCTCCGGCTG TGCCTAfi- 56 3 GGAGTGGGGG GACCGGTATA AAGCGGTAGG CGCCTGTGCC CGCTCCACCT CTCAAGCAGC CAGCGCCTGC CTGAATCTGT TCTGCCCCCT

+ 45 CCCCACCCAT TTCACCACCA CCXM

Bn

;56 +31 -

-725 +31CA5

-686 +31 ,_-ICATI 1n

-618 +314C~A 5

-549 +3137

-448 +31E35

5

-167 +31i-l 7

-19.- +31____CEA 5

CA~ 14

11/////////////7/Ag/X////////////////////EmE///////////W |I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

@e~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Ie le~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ID~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

l'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

m~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IE~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

1, , 1,, 1,20 40 60 80 100 120

Relative CAT Activity

FIG. 1. Transient expression anal-ysis of DF3 promoter deletion con-structs. (A) Nucleotide sequence ofthe 5' flanking region ofthe DF3 gene.Sequences from positions +1 (tran-scription start site) to -430 are in-cluded in ref. 15. (B) MCF-7 cellswere transfected with deleted pro-moter fragments linked to CAT. Rel-ative CAT activities are expressed asvalues compared to that' obtainedwith p-686CAT (assigned a value of100). The results represent the mean+ SD of the indicated number (n) ofexperiments.

-16

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284 Biochemistry: Abe and Kufe

(GGGAGCCC); (iv) AP-3 (GGAAAGTCC) at position -5%;(v) NF-1 (GGCCAA) at position -1208; and (vi) the estrogenreceptor half-site (GGTCA) at positions -750, -885, and-1184 (Fig. 1A and ref. 15).Functional Analysis of the Putative DF3 Promoter Region.

Previous studies have demonstrated that DF3 gene expres-

sion is regulated at the transcriptional level in MCF-7 cells(20). A series of 5' deleted DF3 promoter-CAT constructswere transfected into these cells to identify the functional ciselement(s). Transfection of plasmid p-1656CAT resulted inCAT activities that were -25-fold higher than that obtainedwith pACAT (Fig. 1B). Similar levels of CAT activity were

obtained with p-725CAT, p-686CAT, and p-618CAT (Fig.1B). In contrast, transfection of p-549CAT was associatedwith a decrease in transcriptional activity that was '15% ofthat found with p-686CAT. Moreover, transfection of themore extensively deleted constructs (p-448CAT, p-324CAT,p-167CAT) yielded levels of relative activity that were similarto that obtained with constructs containing a minimal pro-

moter region (p-19CAT) (Fig. 1B). Taken together, thesefindings indicated that the sequences between positions -618and -549 include elements involved in the control of DF3gene transcription.To further define the functional element(s), we prepared a

series of internal deletion vectors using p-686CAT. Deletionof the region from positions -603 to -410 decreased (>80%)transcription of p-686CAT to a level similar to that obtainedwith p-549CAT (Fig. 2A). Similar findings were obtained withreinsertion of f(-505/-410) or f(-481/-410) (Fig. 2A).These results were thus in concert with the likelihood that thefunctional sequences were upstream to position -549 (Fig.1B). However, ligation of f(-588/-505) into this cassette hadlittle if any effect, whereas reinsertion of f(-600/-505)resulted in only partial (54%) recovery of CAT activity (Fig.2A). In contrast, f(-598/-485) conferred complete restitu-tion of the activity obtained with p-686CAT (Fig. 2A). Theseresults indicated that sequences within f(-598/-485) were

responsible for control of DF3 gene transcription.A heterologous promoter was used to determine the en-

hancing activities of sequences upstream to position -485.Fragments f(-753/-505), f(-614/-505), f(-588/-505), andf(-598/-485) were inserted upstream to the thymidine ki-nase promoter in pBLCAT2 (ref. 34, Fig. 2B). CAT activityof pBLCAT2 was normalized to 100. Insertion of f(-753/-505) into this vector had little if any effect, whereasf(-598/-485) enhanced TK promoter activity by >10-fold(Fig. 2B). These results provided further support for thepresence of functional elements between positions -598 and-485. Moreover, the finding thatf(-614/-505) and f(-588/-505) decreased the activity of pBLCAT2 (Fig. 2B) empha-sized the importance of sequences between -505 and -485.

Interaction of Nuclear Proteins with the DF3 Promoter. Theresults of the reporter assays prompted further studies on theinteraction of nuclear proteins with the putative regulatoryregions of the DF3 promoter. Electrophoretic mobility shiftassays were performed with f(-507/-483). Incubation ofend-labeled f(-507/-483) with nuclear proteins from MCF-7cells resulted in a clearly detectable retarded fragment (Fig.3A). To determine whether this complex reflected specificDNA-protein binding, unlabeled f(-507/-483) was added tothe reaction mixture. In these experiments, a 50-fold excess

of unlabeled probe partially inhibited formation of the com-

plex, whereas a 250-fold excess resulted in complete inhibi-tion (Fig. 3A). In contrast, use of unlabeledf(-611/-579) as

a competitor had no detectable effect (Fig. 3A). There was

also no detectable inhibition of this complex when usingunrelated oligonucleotides containing SP1, AP-2, NF-1,

AP-1, or AP-3 sequences (Fig. 3A and data not shown). Thesefindings, taken together with the absence of a detectable bandwhen using nuclear proteins from HL-60 (DF3 antigen-

A-M -103 -410 +31

CAT-410 505

I-481 -410

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4-505

1-505 -600

-549P1~~~~~A

I00XX05N ~ CA

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0 100Relative CAT Activity

200

0 500 1000Relative CAT Activty

FIG. 2. Transient expression of p-686CAT internal deletion vec-tors and heterologous promoter constructs. (A) p-686CAT wasdigested with BstXI and Sma I to create a construct with deletion ofthe region from positions -603 to -410. Smaller fragments off(-603/-410) were religated into the deleted region. Relative CATactivities are expressed as values compared to that obtained withp-686CAT. The results represent the mean ± SD of the indicatednumber (n) of independent experiments. (B) Fragments of the DF3promoter were inserted upstream to thethymidine kinase (TK)promoter in pBLCAT2. MCF-7 cells were transfected with theindicated constructs. The results (mean ± SD) are expressed asrelative CAT activity compared to that obtained with TK-CAT(assigned a value of 100).

negative cells; data not shown and ref. 8), supported theformation of a specific f(-507/-483)-MCF-7 nuclear proteincomplex. There was also no detectable complex formationwhen incubating labeled f(-515/-496) orf(-529/-510) withthe MCF-7 nuclear proteins (data not shown). Finally, alter-ations in the sequence of f(-507/-483) at position -496/-495 resulted in nearly complete inhibition of complexformation (Fig. 3B).The interaction of nuclear proteins with the DF3 promoter

was further addressed by DNA footprinting. End-labeledf(-618/-410) was incubated with increasing amounts ofMCF-7 nuclear proteins and then subjected to digestion withDNase I. The demonstration that one protected region ex-tended from positions -485 to -505 (Fig. 4) was in concertwith the sequences identified by reporter and gel retardationassays. Although another region extending from positions-513 to -525 was also protected by these nuclear proteins,there was no evidence for interaction at other sites (Fig. 4).Moreover, there was enhancement of sequences adjacent tothe protected regions (at positions -505 to -510 and up-stream to position -530) (Fig. 4).

6 -

VANIV1111111111/.Z//// II

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Proc. Natl. Acad. Sci. USA 90 (1993)

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Proc. Natl. Acad. Sci. USA 90 (1993) 285

A_ I

0

ooU) ED

C O O %o

° to Ul) Ul) LOoj 04C

Bcv0

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0LO)

o

oo E

CLCU,

-485-

tit, 4U.

FIG. 3. Gel retardation analysis of nucleoprotein complexesfound with f(-507/-483). (A) f(-507/-483) (CCGGGAAGTG-GTGGGGGGAGGGAGC) was end-labeled and incubated with 10,gof MCF-7 nuclear proteins. Unlabeled f(-507/-483) and f(-611/-579) were added at a 50- or 250-fold molar excess compared to thelabeled probe. The unlabeled oligonucleotide corresponding to theconsensus sequence for SP1 was added at a 50-fold molar excess. (B)f(-507/-483) and a mutated fragment at positions -4% and -495(CCGGGAAGTGGCAGGGGGAGGGAGC) were end-labeled (both1 x 105 cpm, 5 x 104 cpmlng) and incubated with MCF-7 nuclearproteins.

Southwestern analysis was performed to identify the sizeof the protein(s) that interact with f(-507/-483). This frag-ment interacted with a species with an apparent molecularmass of 45 kDa (Fig. 5). This interaction was inhibited by a100-fold excess of unlabeled to labeled fragment. In contrast,there was no detectable interaction of f(-507/-483) withnuclear proteins from HL-60 cells (Fig. 5). Moreover, incu-bation of labeled f(-611/-579) with the MCF-7 nuclearproteins failed to demonstrate DNA-protein interaction (datanot shown). Taken together, these results indicated thatf(-507/-483) specifically interacts with a 45-kDa protein.

DISCUSSIONPrevious work has demonstrated that DF3 gene expression isregulated at the transcriptional level (20). In this context,although treatment of MCF-7 cells with phorbol 12-myristate13-acetate increases DF3 mRNA levels and the rate of DF3transcription, there is little if any effect of this agent onstability of DF3 transcripts (20). Moreover, the finding thatDF3 protein production correlates directly with mRNA levelshas suggested that transcriptional control is the major deter-minant of DF3 antigen expression (20). On the basis of thesefindings and the demonstration that this gene is overex-pressed in breast carcinoma cells, we cloned the 5' flankingregion of the DF3 gene and found sequences correspondingto various putative regulatory elements. The results demon-strate that transcription of the DF3 gene is regulated bysequences between positions -598 and -485. This regioncontains an AP-2-like site at position -587 (CCCCAGCC)

MCFM-,'I e-

0 e

C Z - N U0

Eii~l"I

* ..::: "

-505- 40ato_ww*

--513

-525--530- 0

_il

I'd S._ . '

aA

IGGnAG

(r3G

T'

G

---485 X A

--505 --__ T

--530 G

G

AA

G

A

5'

-505 G~~5

Fic. 4. DNase I analysis of protein binding to f(-618/-410).f(-618/-410) was end-labeled (1 x 104 cpm/0.1 ng) and incubatedwith the indicated amounts of MCF-7 nuclear proteins. The sampleswere subjected to DNase I digestion and analyzed in 12% polyacryl-amide gels. C represents analysis of cytosines as determined byMaxam-Gilbert sequencing. The hatched boxes reflect regions pro-tected from DNase I digestion.

and potential SP1 binding sites at positions -582(GGGCGG), -532 (GAGCGC), and -492 (GGGAGG). Thefinding that deletions at either end of this region are associ-ated with decreased or complete loss of transcriptional ac-tivity suggested regulation by at least two distinct elements.

Reporter assays with the DF3 promoter demonstrated thatsequences functional for transcription were located upstreamto position -549, in particular between positions -598 and-588. However, the finding that f(-600/-505) only partiallyreconstitutes transcriptional activity of internal deletion vec-tors suggested the involvement of another downstream ele-ment. Indeed, the heterologous promoter studies confirmedthe functional importance of sequences between positions-505 and -485. Moreover, the demonstration that thesesequences confer transcriptional activation in the reverseorientation provided support for their role as an enhancer.The upstream region of interest between positions -598 and-588 (TGGAAAGTCC) contains the consensus sequence forAP-3 binding. However, there was no detectable interactionof MCF-7 nuclear proteins with this putative element by gelretardation or protection assays. One possible explanationfor these findings is that protein binding to this regionrequires the formation of a complex involving downstreamsequences within f(-598/-485). Whereas further studies areneeded to resolve this issue and the nature of the functionalupstream sequences, the present work has focused on theputative downstream element between -505 and -485.Taken together with reporter assays, the results of gel

retardation and footprinting studies demonstrate a functional

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32p-f(-507/-483)N 0

(DLL0 _

- +

sequence between positions -505 to -485. Characterizationof this DNA binding protein will provide an opportunity todetermine whether increased activation of DF3 gene tran-scription in breast tumors relates to increased expression ofthat factor.

unlabeledf(-507/-483)

kDa

200L0-

92.5-

69.0-

46.0- r'

30.0-

2 1.5-14.3-

FIG. 5. f(-507/-483) interacts with a 45-kDa nuclear protein(arrow) in MCF-7 cells. Nuclear proteins (200 ,ug) from MCF-7 andHL-60 cells were separated by polyacrylamide gel electrophoresisand transferred to a nitrocellulose sheet. Samples were incubatedwith 32P-labeled f(-507/-483) in the absence (-) or presence (+) ofa 100-fold molar excess of unlabeled fragment.

interaction of protein(s) with the sequence GGGAAGTG-GTGGGGGGAGGGA (positions -505 to -485). To our

knowledge, this sequence has not been previously identifiedas a cis element, although similar sequences have beenidentified as being functional in regulating transcription of themouse immunoglobulin heavy chain gene (CCACCA) (35)and the adenine phosphoribosyltransferase gene (GC-CCCACC) (36). As such, it is not clear whether activation ofthis element is specific for regulating DF3 gene expression.Moreover, little is known about the elements responsible fortranscriptional control of genes coding for the mucin-like highmolecular mass glycoproteins. Indeed, although cDNAs en-

coding human intestinal (37) and porcine submaxillary gland(38) mucins have been cloned, the promoter region of thesegenes have yet to be identified. Other studies have demon-strated that the carcinoembryonic antigen (CEA) gene con-

tains conserved sequences that bind nuclear proteins (39).However, it is not known whether these sequences are

functional in regulating CEA expression. The present resultsdo not exclude the possibility that other elements withinf(-598/-485) may also contribute to transcriptional activa-tion of the DF3 gene. For example, DNA footprint analysisdemonstrated protection of the region extending from posi-tions -525 to -513. This sequence contains a potentialSP1-like site.

Previous studies have demonstrated that =75% of primaryhuman breast carcinomas express high levels of DF3 antigen(3). A similar percentage of patients with metastatic breastcarcinoma have elevated plasma levels of this glycoprotein(40, 41). Other studies have shown that human breast tumortissues overexpress the DF3 gene at the mRNA and proteinlevels compared to normal breast tissue and benign lesions(13). These findings suggest that aberrant regulation at thetranscriptional level is responsible for increased DF3 geneexpression in breast tumors. Although decreased expressionof a transcriptional repressor could contribute to induction ofthe DF3 gene, the present findings indicate the involvementat least in part of a positive regulatory factor(s) that interactswith elements in f(-598/-485). Southwestern studies havedemonstrated the interaction of a 45-kDa protein with the

This investigation was supported by Public Health Service GrantCA38879 awarded by the National Cancer Institute, the Departmentof Health and Human Services, and by a Biomedical ResearchSupport Grant from the Dana-Farber Cancer Institute.

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Schlom, J. (1984) Hvbridoma 3, 223-232.4. Lundy, J., Thor, A., Maenza. R., Schlom, J., Forouhar, F., Testa, M. &

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itriou, J. & Bramwell, M. (1987) Nature (London) 328, 82-84.11. Gendler, S., Burchell, J., Duhig, T., Lamport, D., White, R., Parker. M.

& Taylor-Papadimitriou, J. (1987) Proc. Nati. Acad. Sci. USA 84,6060-6064.

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13. Hareuveni, M., Tsarfaty, I., Zaretsky, J., Kotkes, P., Horev, J., Zrihan,S., Weiss, M., Green, S., Lathe, R., Keydar, I. & Wreschner, D. H.(1990) Eur. J. Biochem. 189, 475-486.

14. Lan, M. S., Batra, S. K., Qi, W.-N., Metzgar, R. S. & Hollingsworth,M. A. (1990) J. Biol. Chem. 265, 15294-15299.

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