JOURNAL OF VIROLOGY, Feb. 1993, p. 813-8210022-538X/93/020813-09$02.00/0Copyright X) 1993, American Society for Microbiology

Regulation of Expression of Mouse Mammary Tumor Virus throughSequences Located in the Hormone Response Element: Involvement

of Cell-Cell Contact and a Negative Regulatory FactorELISABETH HARTIG, BETTINA NIERLICH, SIGRUN MINK,t GABRIELE NEBL,

AND ANDREW C. B. CATO*

Institute of Genetics and Toaxicology, Kernforschungszentrum Karlsruhe,P.O. Box 3640, W-7500 Karlsruhe 1, Germany

Received 7 July 1992/Accepted 19 October 1992

Mouse mammary tumor virus (MMTV) is a latently oncogenic retrovirus responsible for the neoplastictransformation ofmammary epithelial cells. Its expression is regulated by steroids, polypeptide growth factors,and cell-type-specific factors. Using GR mouse mammary cells and NIH 3T3 fibroblasts stably transfected withchimeric constructs of the long terminal repeat region ofMMTV, we have demonstrated a novel mechanism ofcell-type-specific expression of this virus. In confluent monolayer cultures that permit cell-cell interaction,MMTV long terminal repeat expression is positively regulated by sequences within the hormone response

element (HRE) that bind the transcription factors CTF/NFI and OTFI. Although these factors are present inNIH 3T3 cells, MMTV expression in these cells is not regulated by cell density. This is partially due to a

negative regulatory factor that binds sequences between -164 and -151 in the HRE. Mutations that destroythe binding site for this factor restored in part the cell density-regulated expression of MMTV to NIH 3T3fibroblasts. The HRE is thus a central coordinator of regulatory pathways that positively or negatively influencethe expression of MMTV.

Mouse mammary tumor virus (MMTV) is a slow trans-forming retrovirus that is causally associated with mammarycarcinomas in females of susceptible mice. During preg-

nancy and lactation, this virus is highly expressed in mam-mary glands of infected animals, and it is transmitted in milkto the newborn offspring during feeding (for reviews, see

references 21 and 31). High expression of MMTV results inreinfection and reintegration of MMTV copies into the hostgenome. During the process of reintegration of this virus,certain proto-oncogenes are inadvertently activated throughinsertional mutagenesis (12, 16, 33, 34). The increased ex-

pression of these proto-oncogenes marks the initiation pro-

cess of mouse mammary tumorigenesis.MMTV expression is closely linked with factors that

regulate proliferation and overt differentiation of the mam-

mary gland. Steroid hormones such as progesterone andglucocorticoids that are involved in these processes enhanceMMTV expression. This occurs through the binding of theprogesterone and glucocorticoid receptors to defined se-

quences located between positions -202 and -59 upstreamfrom the start of transcription in the hormone responseelement (HRE) of the viral long terminal repeat (LTR) region(7, 35). Epidermal growth factor, required for the prolifera-tion of the mammary epithelium (39, 40, 42), enhancesMMTV expression in a process that requires the progester-one receptor and the tyrosine kinase activity of the epider-mal growth factor receptor (24). MMTV expression is alsoinduced by lactogenic hormones. However, this regulationhas so far only been demonstrated in explant cultures ofmammary glands from parous mice (32). The sequences or

mechanisms involved are yet to be determined.Functional differentiation of mammary epithelial cells

* Corresponding author.t Present address: Max-Planck-Institut fur Immunbiologie,

W-7800 Freiburg, Germany.

depends not only on the presence of specific hormones andgrowth factors but on local environmental signals. These are

produced by extracellular matrix and by communicationwith adjacent cells which together generate a cascade ofevents leading to milk production. For instance, in CID 9cells, a subpopulation of the mouse mammary epithelial cellstrain COMMA-1D, an extracellular matrix-dependent tran-scriptional regulation of the milk protein gene, f-casein, hasbeen demonstrated (36). This regulation requires specificsequences in the 5' flanking region of the 0-casein gene

promoter (36). In another example, laminin, a component ofthe basement membrane of rat mammary epithelial cells,enhances the expression of ot-casein at the posttranscrip-tional level (45).

Cell-cell contact is also required for full differentiation ofsecretory epithelial cells. Mammary epithelial cells culturedon collagen I gels, for example, synthesize milk proteinsonly when the gels are floated into the medium (15). Thisevent triggers profound changes in cytostructure and in-duces a high degree of cell-cell interaction (15). In this study,we have investigated whether mammary epithelial cell-cellcontact increases MMTV expression. We stably transfectedMMTV LTR constructs into GR mouse mammary epithelialcells and mouse NIH 3T3 fibroblasts and cultured themunder conditions that promote cell-cell contact. A study ofMMTV expression under these culture conditions is thoughtto bring out specific differences in expression of this virus inmammary and nonmammary cells. The GR cells used for thisstudy maintain some mammary cell characteristics in cul-ture. They mediate the action of a recently identified mam-mary cell-specific enhancer element that functions exclu-sively in mammary cells (28, 29). In addition, the GR cellsconstitutively express the gene for the milk protein, ,B-casein(27a).Our experiments show that MMTV expression is regu-

lated by cell density in GR mouse mammary cells but not in

813

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814 HARTIG ET AL.

NIH 3T3 mouse fibroblasts. This effect is mediated bybinding sites in the HRE for the transcription factors OTFIand CGT/NFI. Although these transcription factors arepresent in nuclear extracts of both GR and NIH 3T3 cells, inthe NIH 3T3 cells, the cell density-regulated expression ofMMTV is repressed. We have identified in these cells afactor that binds sequences between -164 and -151 in theHRE. This factor is in part responsible for repressing the celldensity effect in NIH 3T3 cells. Substitution mutations thatdestroyed the binding site for this factor restored part of thecell density-regulated expression of MMTV to NIH 3T3cells. The HRE therefore mediates regulation through cell-cell contact, but this effect is under negative control in NIH3T3 cells.

MATERIALS AND METHODS

Cell culture and transfection. Mouse mammary tumor cellline GR and mouse fibroblastic cell line NIH 3T3 werecultured as previously described in Dulbecco's modifiedEagle's medium supplemented with 10% fetal calf serum at37°C and in 5% CO2 (29). Stable transfections were per-formed with 13 ,g of the MMTV LTR chloramphenicolacetyltransferase (CAT) constructs, 10 p,g of calf thymusDNA, and 1 p,g of pSV2neo by using the calcium phosphatemethod as previously described (9). Transfectants wereselected in 400 ,g of G418 per ml and thereafter werecultured in the absence of this antibiotic. Unless otherwisestated, pooled clones of over 500 transfectants were seededat 1 x 105 and 3 x 106 cells per 9-cm culture dish. The cellswere grown for 2 days after seeding and then harvested forCAT assay.

Plasmid constructs. The chimeric MMTV LTR constructspLTR CAT9 and pHCwt have been previously described(29). The substitution mutants pHC6, pHC3, and pHC8 havealso been described (10), as has been the construct Oct T (4).The construct pHCmneg was constructed by overlap exten-sion by using the polymerase chain reaction method of Ho etal. (19). The template and primers used were pHCwt cleavedwith PvuII and the universal sequencing primer 5'-CAGCACTGACCCTIT'TG and the CAT primer 5'-AGGAGCTAAGGAAGCTAAAA-3'. The introduced mutated sequenceswere 5'-GTTCTfAAAACGGTIACCTGAGACAAGTG-3'and 5'-CAC1TGTCTCAGGTAACCGT1-TTAAGAAC-3'.

Northern (RNA) blots and S1 nuclease mapping experiment.Five micrograms of polyadenylated RNA isolated from GRcells at different cell densities was denatured and fraction-ated on 1% agarose and thereafter transferred onto HybondN+ membranes (Amersham) as previously described (9).The filters were hybridized at 65°C in 0.5 M phosphate buffer(pH 7.2), 1 mM EDTA, 7% sodium dodecyl sulfate (SDS),and 50 ,g of denatured calf thymus DNA per ml with a32P-labeled 0.5-kb BamHI fragment from the envelope re-gion of the MMTV construct pL2.6 (22) or an a-actin cDNA(30). After hybridization, the filters were washed once for 15min at 65°C with a 40 mM phosphate buffer containing 1 mMEDTA-5% SDS, 3 times with a 40 mM phosphate buffercontaining 1 mM EDTA-1% SDS, and once with 4x SSC(lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) atroom temperature before exposure for autoradiography. S1nuclease mapping was carried out with 10 ,g of total cellularRNA by using a 2.1-kb ClaI-HpaII fragment from constructpL2.6 (22) labeled at the 5' end at the HpaII site and a 3.0-kbEcoRI-BglII fragment labeled at the 5' end at the BglII site aspreviously described (11).

Immunoprecipitation. GR cells seeded at 1 x 105 and 3 x

106 cells per 9-cm culture plate were grown for 24 h.Thereafter, the cells were preincubated for 1 h in methi-onine-free Dulbecco's modified Eagle's medium withoutfetal calf serum. The cells were then incubated overnightwith fresh methionine-free medium and 100 p,Ci of [35S]me-thionine (500 ,uCi/mmol, Du Pont-New England Nuclear) perml. The radioactively labeled cells were lysed in radioimmu-noprecipitation assay buffer (50 mM Tris-HCl [pH 7.8], 1%Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 250mM NaCl, 5 mM EDTA). After centrifugation (60,000 rpm,2°C, 30 min; Beckman TL 100.2 rotor), equal amounts ofradioactively labeled lysate from the sparse and dense cul-tured cells were incubated on ice for 1 h with a monospecificantiserum directed against the rat glucocorticoid receptor(20). Thereafter, Pansorbin (Calbiochem) presaturated withnuclear extracts from GR cells was added for 30 min on iceand then pelleted. The pellets were washed four times withradioimmunoprecipitation assay buffer. Bound proteinswere eluted by boiling in SDS buffer (40% [vol/vol] glycerol,0.19 M Tris HCl [pH 8.8], 9% SDS, 15% [vol/vol] 3-mercap-toethanol, 0.01% [wt/voll bromophenol blue) for 10 min andseparated on an 7.5% SDS-polyacrylamide gel.

Oligonucleotide probes. The oligonucleotides used in thiswork were synthesized on a Pharmacia gene assembler(plus) DNA synthesizer. The double-stranded oligonucle-otides were obtained by annealing the following single-stranded oligonucleotides: CTF/NFI, 5'-AGCTITlGGAATCTATCCAAGTCG-3' and 5'-GATCCGACTTGGATAGATTCCAAA-3'; Spl, 5'-GATCGATCGGGGCGGGGCGATC-3' and 5'-GATCGCCCCGCCCCGATCGATC-3'; F3, 5'-AGCTITIrGGAATCTATCCAAGTCG-3' and 5'-GATCCGACTTGGATAGATfCCAAA-3'; neg (oligonucleotide N),5'-AGCTTAAAACAAGGATGTGAGACAAGTGG-3' and5'-GATCCCACTTGTCTCACATCCTTGTTTTA-3'; andmneg, 5'-GTTCTlTAAAACGGTTACCTGAGACAAGTG-3'and 5'-CACTTGTCTCAGGTAACCGTTl1TAAGAAC-3'.For gel mobility shift assay, the double-stranded oligonucle-otides were phosphorylated by using T4 polynucleotidekinase (Pharmacia) in the presence of [-y-32P]ATP (>5,000Ci/mmol; Amersham). Unincorporated probes were re-moved by passing the labeled oligonucleotide through NACsPrepas convertible columns (Bethesda Research Laborato-ries). In gel mobility assay identical results were obtained byusing the oligonucleotide neg end labeled at the 5' end or atthe 3' end with fill-in reaction with T7 polymerase.

Preparation of nuclear extracts and DNA-protein interac-tion. Nuclear extracts for DNA binding studies were pre-pared according to the method of Dignam et al. (13), exceptthat all the buffers used contained the following proteaseinhibitors: 1 mM phenylmethylsulfonyl fluoride, 2 mM ben-zamidine, 2.5 Kallikrein inhibitor units of aprotinin per ml,0.6 ,ug of pepstatin per ml, 1 p,g of leupeptin per ml, and 1 p,gof antipain per ml. Protein-DNA binding reactions werecarried out essentially as described by Barberis et al. (1).The assay mixture contained 10 fmol of labeled oligonucle-otide (10,000 cpm) and 10 ,ug of nuclear protein in a totalvolume of 20 p,l. Incubation for gel retardation assay with thenegative regulatory element (neg) was performed on ice, andthe gels were run at 4°C because of the particularly labilenature of the negative regulatory factor. The nuclear extractsfor this experiment were stored in liquid nitrogen. Nuclearextracts stored for 2 to 3 months at -70°C lose their bindingactivities.DNase I footprinting assay was carried out as previously

described (3) by using a 362-bp MMTV LTR fragment(-237/+125) of pHCwt (10) labeled at the HindIII and XhoI

J. VIROL.

REGULATION OF MMTV EXPRESSION BY SEQUENCES IN THE HRE 815

sites at the 5' and 3' ends, respectively. The incubation ofthe labeled DNA with protein for the identification of thebinding site for the negative regulatory factor was carried outon ice. DNase I digestion was carried out at room tempera-ture.CAT assay. GR and NIH 3T3 cells stably transfected with

MMTV LTR CAT constructs were disrupted by freezing andthawing five times in a dry ice-ethanol 37°C water bath. CATassays were performed with cellular extracts from these cellsas previously described (8).

RESULTS

MMTV is highly expressed in GR cells at a high cell density.We have already shown in transient transfection experi-ments that at a density of 2 x 106 cells per 9-cm culture dish,chimeric MMTV LTR constructs are more highly expressedin GR mouse mammary tumor cells than in NIH 313fibroblasts (29). Stable transfection of these two cell lineswith a CAT construct containing MMTV LTR sequencesfrom -1232 to +125 again resulted in an increased CATactivity in GR compared with that in NIH 313 cells afterseeding the cells at a high cell density (Fig. 1, striped bars).Such a difference in expression of the chimeric MMTV LTRconstruct in the two cell lines was not observed when thecells were seeded at a density of 105/9-cm culture dish (Fig.1A, filled bars).The cell density-induced expression ofMMTV in GR cells

is due to accumulation of RNA transcripts at the correctMMTV LTR start site of transcription as shown by S1nuclease mapping experiments (Fig. 1B, compare lane 2 withlane 1). The correctly transcribed mRNAs come from thetransfected and endogenous MMTV promoters. No otherinitiation sites were detected in the LTR in this assay,indicating that transcription at the correct start site is re-sponsible for the increased MMTV expression in the high-density cultures of GR cells. Transcripts initiated at thesimian virus 40 (SV40) promoter of a cotransfected pSV2neoconstruct showed only a small increase with cell density(Fig. 1B, compare lane 2 with lane 1).The contribution of active endogenous MMTV proviral

copies to the increased accumulation of RNA in the high-density GR cultures can be demonstrated in Northern blotexperiments. In these experiments polyadenylated RNAsisolated from GR cultures at two different densities werehybridized with a DNA fragment derived from the enveloperegion of the proviral MMTV DNA. The results showed ahigher level of expression of both the 8.9-kb primary and the3.8-kb processed MMTV transcripts in high- compared withlow-density cultures (Fig. 1C, compare lane 2 with lane 1). Acontrol a-actin transcript did not show this cell density effect(Fig. 1C). Thus, endogenous MMTV proviral DNA as wellas transfected chimeric MMTV LTR constructs is particu-larly well expressed in dense cultures of GR cells.That transfected MMTV in NIH 313 cells did not show

this cell density-induced expression indicates either thatMMTV expression in NIH 3T3 cells is under negativecontrol or that MMTV expression in GR cells is positivelyregulated by cell density.

Sequences that mediate high level of MMTIV expression inGR cells are coincident with the HRE. MMTflV LTR se-quences from -237 to +125 when stably transfected into GRcells also showed a cell density effect. This region of theMMTV contains the HRE that mediates glucocorticoidinducibility of expression at the MMTV LTR promoter. Theinduction of MMTV expression occurs by a prior binding of

A -

-E8

0

2 7-

E 6 -

- _

0

=3-aes 2-co

O

T

B (DOU,0 X

3 9 2 -392-

1 2

106--

CD0C

k b

8. 9

3.8

NIH3T3

SV40

MMTV

MMTV

1 2

2. 1 ."Wt | Actin

FIG. 1. MMTV LTR expression in mouse mammary GR cellsbut not in mouse fibroblastic NIH 3T3 cells is enhanced by celldensity. Mouse mammary tumor GR cells and NIH 3T3 fibroblastsstably cotransfected with MMTV LTR (-1232/+125) and pSV2neoconstructs were seeded at 1 x 105 and 3 x 106 cells per 9-cm culturedish and grown for 2 days. Thereafter, the cells were harvested andCAT activity was determined. (A) The bar diagram represents theaverage CAT activity of four independent determinations + stan-dard deviation in cells seeded at 1 x 105 (filled bars) or at 3 x 106(striped bars). (B) Ten micrograms of total cellular RNA from thelow- and high-density-cultured GR cells were used in S1 nucleasemapping experiments in which MMTV LTR and SV40 probes wereused simultaneously to detect the correct start of transcription at theMMTV LTR and SV40 promoters. The correct SV40 and MMTVLTR start sites of transcription are identified by a 392- and a 106-bpfragment, respectively. (C) Five micrograms of poly(A)+ RNA fromthe low- and high-density GR cultures was hybridized in Northernblot experiments with an MMTV envelope fragment as well as ana-actin cDNA fragment. The full-length 8.9-kb MMTV transcript aswell as the 3.8-kb spliced transcript is indicated. The 2.1-kb a-actintranscript is also shown.

VOL. 67, 1993

816 HARTIG ET AL.

A

35

3x10 105 3x 105 3x10 6

B~ ~ ~ ~ L

O X

12

FIG. 2. The glucocorticoid receptor is not responsible for theincreased MMTV expression mediated by the HRE in high-densitycultures of GR cells. GR mouse mammary tumor cells stablytransfected with the MMTV LTR-CAT construct (pHCwt) were

seeded at 1 x 105 and 3 x 106 cells per 9-cm culture dish and treatedwithout or with hormone and antihormone. Additionally, the cells atboth densities were metabolically labeled with [35S]methionine andthe glucocorticoid receptor was immunoprecipitated. (A) Immedi-ately after seeding, the GR cells were treated without or with 10-7 Mof the glucocorticoid dexamethasone (Dex) or the glucocorticoidantagonist RU486 and grown for 2 days. The cells were thenharvested, and CAT activity was determined with 50 p1g of protein.Similar results were obtained in three independent expenments. (B)After seeding the GR cells at the two indicated cell densities, theywere grown for 24 h and thereafter labeled overnight with [35S]me-thionine. The cells were then disrupted, and equal amounts ofradioactively labeled proteins were incubated with a monospecificanti-glucocorticoid receptor antibody to immunoprecipitate the glu-cocorticoid receptor. The arrow indicates the position of the glu-cocorticoid receptor on SDS-polyacrylamide gel electrophoresis.

glucocorticoid to its receptor followed by the interaction ofthe hormone-receptor complex with specific sequences

within the HRE (for a review, see reference 2). Conceivablyin vivo conditions, other than the steroid, that activate thereceptor could also enhance expression at the MMTV LTRpromoter through the HRE. To investigate whether this is

the case, MMTV LTR expression in low- and high-densitycultures of GR cells was studied in the presence or absenceof the glucocorticoid antagonist RU486 that traps the glu-cocorticoid receptor in an inactive state (18) or as a control,in the presence of the synthetic glucocorticoid dexametha-sone.

In cultures containing RU486, the increased level ofMMTV expression at high cell density was not reduced (Fig.2A, compare lanes 3 and 4). Instead, the levels of CATactivity determined in cultures containing RU486 were

slightly higher than in the absence of this antagonist, indi-cating a partial agonistic action of RU486 (Fig. 2A, compare

lanes 3 and 4 with 1 and 2). In the presence of dexametha-sone, the CAT activity of the transfected cells in both low-and high-density cultures was further elevated (Fig. 2A,lanes 5 and 6). CAT activity induced by dexamethasone inthe GR cells is reduced by an equimolar concentration of

RU486 to the level mediated by this antagonist alone (resultsnot shown).As RU486 did not inhibit the increased expression of

MMTV and dexamethasone rather enhanced the level ofexpression, it appears that the increased MMTV expressionat a high cell density in the absence of hormone is differentfrom the glucocorticoid receptor-mediated expression at theMMTV LTR promoter. This conclusion is further corrobo-rated by the finding that equal amounts of the glucocorticoidreceptor are expressed in GR cells at both low and high celldensities (Fig. 2B).

Specific sequences in the HRE contribute to the high level ofexpression of MMTV in GR cells in the absence of steroidhormone. To identify sequences in the HRE involved in theincreased expression in GR cells, we stably transfected intothe GR cells constructs with 6-bp substitution mutations invarious regions of the HRE. Pools of GR cells transfectedwith MMTV LTR constructs with mutations in the steroidhormone receptor binding sites showed no differences inexpression in sparsely and densely cultured cells (results notshown). Some mutations outside the steroid receptor bindingsites shown schematically in Fig. 3A produced clear func-tional differences with cell density.

In low-density cultures, CAT activity was almost identicalin all the stable transfectants. In high-density cultures,elevated expression was observed in clones containing thewild-type construct pHCwt and mutant pHC3, and a mod-erate level was seen in mutant pHC6 (Fig. 3B). In contrast,CAT activity in cells containing mutants pHC8 and Oct Twas drastically reduced (Fig. 3B). Thus, in the HRE, se-quences at the regions mutated in pHC8 and Oct T areimportant for the high-level expression of MMTV in thehigh-density cultures of GR cells.The HRE of the MMTV LTR mediates negative cell-type-

specific regulation. The sequences mutated in constructspHC8 and Oct T in the wild-type HRE are binding sites forCTF/NFI and OTFI (4, 10, 27). Gel retardation experimentscarried out with oligonucleotides corresponding to se-quences in these regions and extracts from low- and high-density cultures of GR cells failed to show any differencesthat could explain the cell density effect. An example of thegel retardation experiment with CT7F/NFI oligonucleotideshowing no difference in the retarded band with extractsfrom low- and high-density GR cells is shown in Fig. 4A,lanes 3 and 4. Competition experiments with GR and NIH3T3 nuclear extracts showed that homologous (Fig. 4B,lanes 2 and 5) but not unrelated (Fig. 4B, lanes 3 and 4)oligonucleotides competed for the formation of the boundcomplex, indicating the specificity of binding to the CTF/NFI oligonucleotide. The nuclear extracts from the various-density cultures of GR cells, when incubated with a labeledbinding site for the transcription factor Spl, showed only atiny difference in DNA binding activity (Fig. 4A, lanes 5 and6). The DNA binding activity of Spl is not known to beaffected by cell density. Correction for this difference inbinding for the CTF/NFI binding activity was still notenough to account for the cell density effect at the MMTVLTR promoter mediated through the CTF/NFI binding site.Nuclear extracts from low- and high-density cultures of NIH3T3 cells also showed identical patterns of DNA bindingactivity for CTF/NFI (Fig. 4) and for OTF1 (results notshown), as was obtained with nuclear extracts from GRcells. This poses problems in understanding the lack of celldensity-regulated expression of MMTV in NIH 3T3 cells asopposed to GR cells. In a search for mechanisms that couldaccount for the absence of cell density-regulated expression

J. VIROL.

REGULATION OF MMTV EXPRESSION BY SEQUENCES IN THE HRE 817

A -200 -150 -100 -50, HRE,

BSI BSII BSIII BSIVTATA

- - _I NFIRlflEIiF/

A NIH3T3 GR

pHC, tOCTD OCTP

pHC6

_GGTT _ _ _

- /ATCGA1C

4GGAAT/ i

to1o\ -T7GTAAATGCTTATGTA

1' §cAGCTTGGiTA

bound

pHC3

pHC8

OctT

B 20-

o 10E

e sopHCwt pHC6 pHC3 pHC8 OctT

FIG. 3. Specific sequences in the HRE mediate increased ex-pression of MMTV in dense cultures of GR cells in the absence ofsteroid hormones. (A) MMTV LTR-CAT chimeric constructs con-taining substitution mutation in the HRE in sequences outside thereceptor binding sites were stably transfected into GR cells. Theshaded bar refers to the area defined as the HRE. BSI to IV are theglucocorticoid receptor binding sites (35). The DNA sequenceindicated above the mutated site is the sequence as it occurs in theMMTV LTR (e.g., GGGTIT in pHC6), whereas the sequencebelow (e.g., CCCGGG in pHC6) is the mutated version. The TATAbox and start of transcription (CAP) are indicated. Also indicatedare positions for the NFI (10) and the degenerate octamer motifs OctD and Oct P (4). (B) Pooled clones of a couple of hundredtransfectants seeded at 1 x 105 and 3 x 106/9-cm culture dish weregrown for 2 days. Thereafter, the cells were harvested and CATactivity was determined. The bar diagrams represent the averageCAT activity standard deviation obtained from four independentdeterminations in cells seeded at 1 x 105 (filled bars) and 3 x 106(striped bars).

of MMTV in NIH 3T3 cells, we examined the DNA bindingactivities of nuclear extracts from confluent cultures of thiscell line in DNase I footprinting experiments over the HRE.

In agreement with the known low affinity of OTFI for thedegenerate octamer motifs in the MMTV LTR promoter (4),no clear footprints were observed at the positions -47 and-42 in the regions labeled Oct D and Oct P on the noncodingand coding MMTV DNA sequence (Fig. SA and C). How-ever, a clear footprint over the CTF/NFI binding site wasobtained on the noncoding and coding strands of the MMTVLTR (Fig. 5A and C, NFI). A strong hypersensitive site wasobserved at position -151. We designated this FP forfootprint (Fig. SA, FP). At a higher resolution, this hyper-sensitive site was found to correspond indeed to the 3'boundary of a weak DNase I footprint specifically generatedby nuclear extracts from NIH 3T3 cells that extends fromposition -164 to -151 (Fig. SB). Note the appearance of

B NIH3T3 GR

I 11 1 competitorNFI OligoN - NFI O;iqoN loox molar excess

bound

free

1 2 3 4 5 6FIG. 4. Nuclear extracts from confluent GR and NIH 3T3 cells

do not show differences in binding to C'F/NNFI oligonucleotide. (A)End-labeled CTF/NFI and Spl oligonucleotides (approximately 10fmol, 10,000 cpm) were incubated with 10 ,ug of nuclear extract fromlow- and high-density NIH 3T3 and GR cells. The bound and freelabeled oligonucleotides are indicated. (B) End-labeled CTF/NFIoligonucleotide (10 fmol, 10,000 cpm) and 12 Lg of nuclear extractfrom confluent NIH 3T3 and GR cells were used in gel retardationexperiments. Indicated are reactions containing a 100-fold molarexcess of specific (NFI) or nonspecific (OligoN) oligonucleotidesused as competitors.

another hypersensitive site at position -164 and the reducedintensities of the bands in the FP region in the lane contain-ing 70 pg of nuclear protein (Fig. 5B). The interaction ofnuclear proteins from NIH 3T3 cells with the HRE sequenceat the FP region on the coding strand is also clear from thereduction of the intensity of bands between positions -164and -151 at increasing protein concentrations (Fig. 5C, FP).To further characterize the protein binding to FP, an

oligonucleotide (neg) corresponding to this region was usedin a gel retardation experiment with nuclear extracts fromboth GR and NIH 3T3 cells. Both extracts formed specificcomplexes with the oligonucleotide. Complex A, which is adoublet, is inhibited with homologous double-stranded oli-gonucleotide (Fig. 6, oligonucleotide neg, lanes 2 and 6) butnot with an unrelated oligonucleotide F3 (Fig. 6, lanes 4 and8). An oligonucleotide, mneg, mutated at positions contactedby the protein in methylation interference analysis (resultsnot shown) failed to compete (Fig. 6, lanes 3 and 7).Complexes B and C (Fig. 6) are inhibited with the single-stranded neg oligonucleotide but not with an unrelatedsingle-stranded oligonucleotide (results not shown).

bound

f r e e

In _ S11 _ 1urCD x CD x Cl X

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~ W

I 1 2 3 4 15 6 1

NFI Spl

-. .,.

VOL. 67, 1993

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818 HARTIG ET AL.

B N i3T3 G C:Tpqpr1te8 n ' 7

_w:.i

864- 551

- 2

- fi'.- b 4

- 4 2

C NNI F13T3 G C I

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nF)::*NF. ..'

i Op*f*db_

::F..

Am

a'

*, ........ *I,

g...

as

FIG. 5. Nuclear extracts from NIH 3T3 cells bind specific se-

quences in the HRE of MMTV LTR in DNase I footprinting assay.

(A and B) A 362-bp HindIII-XhoI fragment from the MMTV LTR(-237/+125) labeled at the 3' end of the XAoI site was used in a

DNase I footprinting experiment as previously described (10) withdifferent concentrations of nuclear extracts from confluent NIH 3T3cells. The positions of interaction of different proteins with se-

quences in the HRE are indicated by the open boxes. Panel B showsa high-resolution electrophoretic separation of the DNase I foot-printing reaction products of panel A. (C) An autoradiogram of a

DNase I footprinting experiment carried out with the 362-bp Hin-dIII-XhoI fragment from the MMTV LTR (-237/+125) labeled at 3'end of the HindIII site.

NIH 3T3

I_ I

GR

I .. I-: L-

con rk,.Itr2n0x molnor .xxcess

4- B

C

rme

7 1 2 3 4 5 6 7 8

FIG. 6. Nuclear extracts from confluent GR and NIH 3T3 cellsbind specifically to an oligonucleotide encompassing the FP regionin the HRE of MMTV LTR. End-labeled oligonucleotide neg (10fmol, 10,000 cpm) was incubated with 10-p,g nuclear extracts fromconfluent NIH 3T3 and GR cells and used in gel retardationexperiments. Indicated are reactions containing 200-fold molarexcess of specific or nonspecific oligonucleotides used as competi-tors.

As mutations in the neg oligonucleotide could not inhibitthe formation of complex A, the mutations destroy thebinding site of the protein that generates this complex. Wetherefore investigated the functional activity bestowed ontothe MMTV LTR promoter by introducing these mutationsinto the MMTV LTR. GR and NIH 3T3 cells stably trans-fected with the mutant MMTV LTR construct pHCmneg(Fig. 7A) were seeded at low and high densities. Theincreased expression at the MMTV LTR promoter in high-density cultures of GR cells was unaffected by the mutation,indicating that the factor that binds this sequence in GR cellsdoes not play a major role in MMTV LTR expression(results not shown). Furthermore, glucocorticoid responseat the MMTV LTR promoter in GR cells in low- andhigh-density cultures was not affected by the mutation in theFP region (results not shown).

In NIH 3T3 cells, the situation was different. An exampleof typical results obtained with NIH 3T3 cells transfectedwith the wild-type and mutant MMTV LTR constructs isshown in Fig. 7B. In non-hormone-treated, high-density-cultured cells transfected with pHCwt, no increased expres-sion at the MMTV LTR promoter was observed, as alreadyreported in Fig. 1 (Fig. 7B, compare lanes 1 and 2). Athreefold increase in MMTV LTR expression with celldensity was, however, observed in the absence of glucocor-ticoid in cells containing the mutation in the FP region (Fig.7B, lanes 5 and 6). This suggests that the factor binding tothe FP sequence represses the cell density-mediated expres-sion of MMTV. This effect was, however, still less than thecell density effect observed in GR cells (Fig. 2A). Thus, themutation at FP only overcomes part of the suppression of thecell density effect in NIH 3T3 cells. Even then, the factorbinding to the FP region is not specific for the reduction ofthe cell density effect. This idea stems from the fact that inlow-density cultures the mutation increased expression atthe MMTV LTR promoter (Fig. 7B, compare lanes 1 and 5).In the presence of dexamethasone, NIH 3T3 cells trans-fected with the wild-type construct pHCwt also showed a

cell density effect in expression at the MMTV LTR promoter

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REGULATION OF MMTV EXPRESSION BY SEQUENCES IN THE HRE 819

A 2 -i%D -' C -- transduction (26), it is not always clear which precise geneHRE targets are at the receiving end of the cascade of events. In

ES' B2ESIIES!BSM TA one case, extracellular matrix-integrin interaction was= _ 1 A / pHCV t shown to promote the expression of the P-casein gene (37).

OCT,/\TD A function-blocking anti-integrin antibody severely dimin-.nu-':ATG ished the expression of this gene in response to extracellularW<=// 117 pF1Crneg matrix signals (37).

GG=TACC In this work, we have shown that MMTV expression iselevated at a high cell density. This effect is observed in anumber of mouse mammary epithelial cells such as GR and

G92'.6 3-5-2 NMuMG or RAC 10P (results not shown) but not in mousefibroblastic NIH 3T3 cells. We do not know the factors or

* * * * * * mechanisms involved in transferring signals generated from**: the cell-cell interaction to changes in transcription at the

MMTV LTR promoter. From the above-described role ofintegrins in the regulation of 1-casein expression, it istempting to postulate the involvement of these receptors in

* * * * * * * * theincreasedexpressionofMMTVindenselyculturedcells.However, the lack of basement membrane and the reduced

2 3 level of expression of laminin and other components of theCn1-' 6%^1n'-C i5' i\>40'- Ah G basement membrane in monolayer culture on plastic (38)-l -make it unlikely that integrins are involved in the cell______________________ -- -_ ~ density-induced expression of MMTV. Nonetheless, we

have identified the DNA binding motifs that serve as targets7. Mutation in the HRE increases expression of a MMTV for the increased expression of MMTV in high-density-AT construct in densely cultured NIH 3T3 cells. (A) Sche- cultured cells. These are the binding sites for the transcrip-epresentation of the HRE of the MMTV LTR showing the tion factors CTF/NFI and OTFI.as of the glucocorticoid receptor binding sites BSI to IV, NFI In gel retardation experiments with extracts from sparselyF1 binding sites, TATA box, and the start site of transcrip- and densely cultured GR cells, no differences were observedhe mutation in between receptor binding sites I and II that in the factors binding to the CTF/NFI or OTFI binding sites.ts the sequence 5'-AAGGATG-3' to 5'-GGTTACC-3' has This indicates that the changes in functional activities ofen indicated. This mutant construct is denoted pHCmneg. (B))f NIH 3T3 cells stably transfected with pHCwt and pHCm- CTFINFI and OTFI with cell-cell contact are not reflected inre seeded at 1 x 105 and 6 x 106 cells per 9-cm culture dish changes in the DNA binding activities of these proteins. No)wn for 2 days. Eight hours before the cells were harvested, differences were also observed in a comparison of the DNAf of the cultures was treated with dexamethasone (Dex), and binding activities of these two transcription factors in nu-er halfwas left untreated. The cells were then harvested, and clear extracts from low-density and confluent cultures ofctivity was determined. Lanes 1 to 4, pHCwt transfectants; NIH 3T3 cells. This finding contrasts with the report ofto 8, pHCmneg transfectants. This is a typical example of Goyal et al. (17), which demonstrated different DNA bindingconsistently obtained in four different experiments. forms of CTF/NFI in exponentially growing and confluent

cultures of NIH 3T3 cells. While we cannot explain thereason for the difference in our results and those of Goyal et

B, lanes 3 and 4). This effect may be different from the al. (17), it is not clear whether the reported differences inediated by the mutation at -164 and -151, as it is DNA binding activity of CTF/NFI have any functionalTed with both the wild-type and mutant MMTV LTR consequences.ucts (Fig. 7B, compare lanes 3 and 4 with 7 and 8). If the signal transducing factor in the cell density regula-a number of negative control mechanisms operate at tion of MMTV LTR expression is from a cell adhesion?,E of the MMTV LTR in NIH 3T3 cells. The negative molecule, then this type of regulation of gene expressiontory element at -164 to -151 that we have identified would also be expected in NIH 3T3 cells. Indeed, we doe only one of these negative regulatory elements. observe a somewhat reduced cell density effect at the

MMTV LTR promoter in these cells, but only after mutatingDISCUSSION a certain sequence in the HRE. This sequence, termed FP,

binds a factor that is present in both nuclear extracts of GRe expression in mammary epithelial cells is regulated and NIH 3T3 cells. However, in GR cells, the FP bindingious factors including steroids, polypeptide hormones, site seems to be functionally inactive. The isolation and1ll-cell interactions (43). Cells isolated from the mam- characterization of the factors binding to this region in GRgland usually lose their ability to express milk protein and NIH 3T3 cells will clarify their roles in the cell-type- andwhen cultured on plastic (44). This is overcome in cell density-regulated expression of the MMTV.cases, such as in the HC11 cells, by culturing the cells Recently, Tanaka et al. (41) showed that in vivo competi-[fluency. Confluent monolayer cell culture is an impor- tion of a factor binding to sequences at positions -163 torerequisite for the induction of expression of the milk -147 in the HRE increased the glucocorticoid hormonen ,B-casein by lactogenic hormones in the mouse mam- response at the MMTV LTR promoter. They explained theirHCll cells (14). finding as resulting from the competition of a single-strandedthelial cell-cell contacts promote the deposition of DNA binding protein that interacts with the HRE to represstent membrane (37) from within which integrins trans- the glucocorticoid response of the MMTV LTR (41). Our gelsignals resulting in various intracellular changes (37). retardation studies revealed that our negative regulatory FPagh a number of integrins are candidates for signal sequence is bound by a labile double-stranded DNA binding

VOL. 67, 1993

CAT acr

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FIG.LTR-C.matic rpositiorand OTtion. T1convertalso beePools oneg we]and greone halthe oth4CAT aslanes 5results

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820 HARTIG ET AL.

protein as well as single-stranded DNA binding proteins.Our substitution mutation that destroyed the negative regu-lation of MMTV expression in NIH 3T3 cells destroyed onlythe DNA binding activity of the double-stranded DNAbinding protein but not those of the single-stranded DNAbinding proteins. It is therefore very likely that the factor wehave reported in this work is different from that described byTanaka et al. (41). As our negative regulatory factor re-

presses expression of the MMTV without any obvious effecton the hormone response, we would postulate that it isresponsible for the low level of MMTV expression in manycell types in the absence of steroid hormones. In fact,Langer and Ostrowski (25) have shown that multiple copiesof our negative regulatory sequence repressed expression inNIH 3T3 cells when cloned in front of the thymidine kinasepromoter of a thymidine kinase-luciferase fusion construct.The proteins that bind the negative regulatory sequence in

the MMTV LTR that we have described in this work may beimportant in enhancing hormone inducibility at the MMTVLTR promoter as well as at other steroid hormone-regulatedpromoters by reducing the basal level of expression of thesegenes. In this connection it is of interest to note that thesequence 5'-AAGGATGT-3' that we have mutated in theHRE of the MMTV LTR also occurs as 5'-CAGGATGT-3'in the HRE of the tyrosine aminotransferase (TAT) gene

overlapping the principal glucocorticoid receptor bindingsite II at position -2500 upstream from the start of transcrip-tion of this gene (23). Although it is not yet known whetherthe FP sequence in the TAT gene binds a transcription factorin the absence of hormone, it is tempting to speculate a

similar function of this sequence in the TAT gene. Thisspeculation is even more appealing in view of similaritiesobserved in the arrangement of nucleosomes on the HREs ofthese two genes in the absence of hormone (6). Whatever thefunction of the sequence 5'-CAGGATGT-3' in the TATgene, our results clearly demonstrate that in the HRE of theMMTV LTR it mediates a negative regulation of expressionat the MMTV LTR promoter in NIH 3T3 cells in the absenceof hormone.Taken together, our results show that the HRE plays

multiple roles in MMTV LTR expression. It endows theMMTV LTR promoter with steroid hormone responsive-ness, it mediates a cell density-regulated expression, andnegatively mediates expression in a cell-type-specific man-

ner.

ACKNOWLEDGMENTS

We are grateful to W. Hoeck and B. Groner for making availableto us antisera to the rat glucocorticoid receptor. We thank J.Weinmann and B. Besenbeck for their excellent technical assistanceand I. Kammerer for typing the manuscript.

This work was supported by DGF grant Cal30/1-1 to A.C.B.C.

ADDENDUM IN PROOF

Our negative regulatory sequence 5'-AAGGATGT-3' oc-

curs as 5'-GAGGATGT-3' in other strains of MMTV. Re-cent experiments have shown that this point mutation has no

effect on the DNA binding activity of our negative regulatoryfactor (E. Hartig and A. C. B. Cato, unpublished data).

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