Proviral insertions within the int-2 gene can generate multiple anomalous transcripts but leave the protein-coding domain intact.

(1)

Proviral Insertions

within the int-2 Gene Can Generate

Multiple

Anomalous

Transcripts but

Leave the

Protein-Coding

Domain

Intact

CLIVE

DICKSON,'*

ROSALINDSMITH,' SHARON BROOKES,2AND GORDON PETERS2

Imperial Cancer Research Fund Laboratories, Lincoln'sInnFields, London WC2A

3PX,l

andImperialCancerResearch FundLaboratories, St. Bartholomews Hospital, DominionHouse, LondonECJA 7BE,2 UnitedKingdom

Received 10 August 1989/Accepted 13 October 1989

We examined the effects ofmouse mammary tumor virus integration on the multiple RNA transcripts expressedfrom the int-2 proto-oncogene invirally inducedbreast tumors. Proviral insertion eitherupstream ordownstream of thegenecouldsimultaneously activatetranscriptionfrom threedissimilar int-2 promoters.

In some tumors,theactivating provirus lies within thetranscription unitand disruptsthe structures ofthe variousRNAs. Insertions in the 5' region of thegene hadcomplexeffects depending onthe orientationand position of the provirus relative to the three promoters and intron-exon boundaries. RNase protection experimentsidentifiedtranscriptsinitiated in the virallongterminalrepeat,at normal andcrypticsites inthe

int-2sequences,and fromcrypticpromoters inaninvertedprovirus.At the3' end, insertions occurredwithin

the untranslated trailer and provided alternative terminationsignals that substituted for one orbothofthe normal thepoly(A) additionsites.However,innoinstance,of the20tumorsanalyzedindetail,didaprovirus

perturbthepresumedopenreadingframe of thegene.These datastrongly implicatethe normalproductofthe

int-2gene, whichisrelated to the fibroblast growthfactorfamily, as acontributoryfactor invirallyinduced mammary tumors.

The int-2 proto-oncogene is now known to encode a

memberofthe fibroblast growth factor family ofproteins,

and its expression, at least in the mouse, appears to be

restricted to specificembryonic cell typesat variousstages

ofdevelopment (2, 7, 26, 27). However, the initial

identifi-cation and structural characterization of the gene resulted

from its transcriptional activation in tumors induced by

mouse mammary tumor virus (MMTV) (3, 11, 16, 17). For

example, in the BR6 strain of mice, around 70% of the

spontaneously arising mammary tumors areclonal

prolifer-ationsof cells that havesustained a proviral insertion within

about 15 kilobase pairs (kb) on either side of the int-2 gene

(16, 18, 19).

Typically,these tumors express multiple int-2 transcripts

that are superficially analogous to RNAs detected in the

embryoand in culturedembryonal carcinoma (EC) cell lines,

suchasF9 andPCC4 (10, 24, 27). In EC lines, the expression of int-2increases when the cellsdifferentiate in response to

retinoic acid and dibutyryl cyclic AMP, and different cell

lines contain different patterns of transcripts. They have

therefore provided a convenient system in which to

charac-terizethevarious species of RNA (24). By a combination of

RNaseprotection analyses and blot hybridization with spe-cificprobes, six different classes of int-2 mRNA have been

distinguished that initiate atmultiple cap sites within three

separate promoter domains and terminate at either of two

polyadenylationsites (10, 24).

The reasons behind this complexity are unclear, since all transcripts are predicted to encode the same protein (11, 24).

One possibility would be to facilitate control of

tissue-specific expression, or there could be subtle differences in

the stability or translational efficiency of the various

tran-scripts. It was therefore essential to establish whether the

RNAs seeninMMTV-induced tumors are indeed analogous

to their embryonic counterparts or whether they represent

tumor-specific variants. Moreover, a subset of mammary

*Corresponding author.

tumors expressanomalous int-2 RNAs as aconsequenceof proviral integration within the transcription unit (19). Here wehave examined thestructuresof these int-2transcriptsin

a series oftumors and established that proviral insertions

caninfluence all three promoters and alter the structures of

transcriptsat either the 5' or3' end, but innocase do they

disrupt the protein-codingcapacity ofthe gene.

MATERIALSANDMETHODS

Analysis of tumorDNAand RNA.Theprocedures used for theextraction ofhigh-molecular-weightDNA andtotal RNA

fromMMTV-inducedmammary tumorshave beendescribed

previously (3, 17), and the examples analyzed here were

included in theseries of BR6 mammary tumors described in

an earlier report (19). Poly(A)+ RNA was enriched by

chromatography onpoly(rU)-Sepharose (3). Samples(5 ,ug)

werefractionated by electrophoresis in agarose gels

contain-ing formaldehyde (9) and transferred onto nylon filters by

blottingin 20x SSC (1x SSCis 0.15 M NaClplus0.015 M

sodium citrate). Hybridizations were performed with DNA

probes thatwere labeled with 32P by either nick translation

orprimingwith randomoligonucleotides.Theconditions for

hybridizingandwashingoffiltersandautoradiography were

asdescribed previously (3). In some experiments, 20 ,ug of

F9 orPCC4cell RNAs wasincluded as a control, and RNAs

weredenatured inglyoxal rather than formaldehyde (24). Exon-specific probes. Inaddition to the int-2 genomic DNA probe, designated int-2 f (11), which is specific for exon III of the gene, three furtherprobes were prepared that contained sequences present exclusively in exons la, I, and lb (24). These corresponded to nucleotides (nt) 590 to 1219, 1342 to 1681, and 1681 to 1815, respectively, in the genomic se-quence(11).

Recombinant DNA techniques. DNA from tumors W26, E115, and D157B was digested to completion with EcoRI

and ligated into the separated arms of XgtWES.XB, or X

L47.1,asdescribedelsewhere (3, 17). After in vitro

packag-ing, approximately 5 x 105 bacteriophage were plated on

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ANOMALOUS int-2 RNAs AFTER PROVIRAL INSERTION

Qm U) CU

W~ 0

(0 co

V-llJ

a

la

1 lb

Ill

.t. -2 .9

U-2.7

*

I!

q " -1.8

-1.6

Is:9

,IjiI

-2.9

-2.7

-1.6

FIG. 1. int-2 RNAs in mammary tumors. Samples (5 ,ug) of poly(A)+ tumor RNA were fractionated in denaturing agarose gels, transferred tonylonmembranes, and hybridized with32P-labeledprobes specific for mouse int-2 sequences. (a) RNA from tumorE155Awas hybridized sequentiallywith probesspecific for exonsIa,I, Ib,andIII as indicated (see Materials and Methods). (b) RNAs from the indicated mammary tumors werehybridized with the int-2f probe specific for exon III. The positions of the major classes of int-2 RNA and their sizes in kilobase pairsareindicatedon the right ofeach panel.

LE392 cells and screened with both MMTV long terminal repeat(LTR) and int-2 probes. Phage containing inserts that werepositive for both were plaque purified, and the

respec-tiveEcoRIfragments were transferred into plasmid vectors

for further analysis.

DNAsequence analysis. After the clonedEcoRIfragments

werecharacterized by restriction enzyme mapping, selected

fragments that spanned the virus-host junctions were

sub-cloned into M13mp vectors. DNA sequences were

deter-mined inboth orientations by the dideoxy-chain termination

procedure. For W26, the sequence of the entire LTR was determined toverifythat noadditional viral information was present. A 6-base-pair (bp) duplication was noted at each endofthe LTR,indicatingthat thesite of integration was at

nucleotide 1089 in thegenomic sequence (11). For E115 and

D157B, only the 5'junctions wererecovered and

character-ized, but these established both the position and the

orien-tation of the respective proviruses (indicated in Fig. 2).

RNase protection. Samples (generally 2

pRg)

ofpoly(A)+ tumor RNA or 20 ,ug of total RNA from EC cells were

subjectedtoRNaseprotection analysisexactlyasdescribed

by Smith etal. (24). Theuniformly labeled antisense RNA

probes were prepared by transcribing cloned fragments of

genomicDNAwithphageSP6 RNApolymerase.Theprobes

are numbered as in the previous report (24) (with the

addition of probes 1.1, 1.2, and 1.3), and the restriction

enzymesitesthat mark the 5' and 3' endsofthe

int-2-specific

sequencesin eachprobeareindicatedin the relevantfigures.

The sizes of eachproberefertotheint-2-specific sequences

only (occasionally, enzyme sites in the

polylinker

of the

transcribed

plasmid

weremore

convenient)

andwere

calcu-lated withoutconsideringexactcleavagepositionswithinthe

recognition sites. However, when measuring the sizes of

protected fragments, the exact ends were taken into

ac-count. Even so, we estimate that

fragment

sizes deduced

experimentallyare only accurateto +5bp.

RESULTS

Typicalandatypicalint-2 RNAs in MMTV-inducedtumors. Most tumors in which the int-2 gene is

transcriptionally

activated carry an MMTV provirus in adjacent cellular

DNA, rather than within the gene, in an orientation

appro-priate for enhancement of the normal int-2 promoters by

elements in the viral LTR (3, 18,19). These tumors typically

contain four sizes ofint-2 RNA, ofapproximately 2.9, 2.7,

1.8, and 1.6 kb, all of which are detectable with a probe for exonIIIofthe gene(Fig. 1). In the present study, the tumor

designated E155A was used to represent the typical case,

sincetheactivating MMTVprovirus is located about 10 kb

upstream ofint-2 (19). Results obtained with other typical

tumors were qualitatively indistinguishable. However, by analogy with ECcells,thefour bands detected on RNA blots were assumed to reflect six distinct types of RNA (10, 24). Thus, the2.9-kbbandisnowunderstood tocompriseRNAs that initiate at either the P1 or the P2 promoter. Tumor

RNAs ofthis sizecontained sequences derived from either

exon Ia(initiatedatP1)orexonI (initiatedatP2),while the

2.7-kbtranscriptwasnotdetectedby probes fortheseexons

(Fig. la). This latter RNA presumably initiates at a third

promoter,designatedP3(Fig.2),since itwasdetectablewith

aprobespecific forexon

Tb

(Fig. la) (24).Arationalization

of thesefindingsispresentedinFig. 2. Thesmaller 1.8- and

1.6-kb forms of int-2 RNA that are barely visible in the

reproductionofFig. la have been showntobeanalogsof the

2.9- and 2.7-kb classes, respectively, that terminate at the upstream poly(A) addition site (24). These smaller

tran-scripts are consistently underrepresented in tumors

com-paredwith EC cells.

Althoughthemajority of int-2-positivetumorsconformto

this typical pattern, our

examples

in whichthe

patterns

ofint-2transcriptswereunusual(19).Some of these

atypicalcases are shown in

Fig.

lb. From

relatively

detailed

mapping of the integrated

proviruses

in these tumors, it

becameclear that the anomalous

transcripts

arose

through

physical disruption of the int-2

transcription

unit and that

thiscouldoccur ateither the 5'or3'end.

Thus,

tumors

W26,

E115, and D157B

expressed

unusual RNAs as a result of

proviral insertion in the5'

region

of the gene, while several

others, suchasD124,

C46, V97A,

and

W106,

had

proviruses

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W26

D1

57B

1089 1195

E115

14

1503

P1

i1

::=la

lizi:il

1175 1671

P2

P3

B Pv S Sm St N Pv

0 ₂₅₀₀

1225

1681

-

Pvull-Sacl

Nael-BamHI

184

-~

Stul-Smal

991

Stui-Pvull

9

11

12

13

FIG. 2. Proviralinsertionsin thepromoterregion ofint-2. Intheupper part of thefigure,thestructuresofthreeclassesofint-2 RNAare

drawn to scale(relativetotherestrictionmapbelow),withexonsIa,I,andIbrepresentedasboxes and numbersreferringtocoordinates in thegenomicsequenceofMoore et al.(11).Shadedareascorrespondtoprotein-codingsequences,andthemultiplestartsites associated with promotersP1 and P2 aredepictedbythevertical lines. Thepositions andorientationsofintegrated provirusesin thenumbered mammary tumors are shown as arrowheads, with their positions in thegenomic sequenceindicated below. Therestriction map showsthe sites of cleavage for theenzymesBamHI (B), NaeI (N), PvuII (P),Sacl(S),SmaI(Sm),andStul(St)thatarerelevanttothegenerationofspecific antisense RNA probes. Not all sitesare included. The lines below themap define theextents and positions of the genomic sequences transcribed intoRNA probes, numberedas inreference 24. Thelength ofeachprobe and therestriction sitesthatdefine eachendareas

indicated.

within the presumed 3' untranslated region, leading to

ex-tendedforms of int-2 RNA (Fig. lb).

Locating proviruses by Southern blotting oftumor DNA

wasnotsufficientlyaccurate to drawfirmconclusionsabout

their effects on the structure and coding capacity of int-2 RNAs. Two approaches were therefore adopted to map these proviruses more precisely. First, recombinant DNA clones were isolated representing the junctions between

MMTVproviral DNA and int-2 genomic sequences. Inthe

second approach, tumor RNAs were subjected to RNase

protection analysis with a series of antisense probes derived

from specific regions of the genomic DNA. Since this

technique detects discontinuity between the RNA and

ge-nomicsequences, the mapping of hybrid RNAs generated by

viralinsertion canbe used to locate the site ofintegration.

Characterization ofvirus-hostjunctions. Bacteriophage K

libraries were constructed containing EcoRI-digested DNA

from tumors W26, E115, and D157B and screened with both int-2- and MMTV-specificprobes. Tumor W26 contained a

singleMMTV LTRin a 5' to 3' orientation relative to int-2,

resulting in an 11.3-kb EcoRI genomic DNA fragment as

opposed to the 10-kb normal allele (11). With E115 and

D157,only the respective5'junctionfragments were

recov-ered.Whenthesewerecharacterizedand the DNA sequence

was determined across the virus-cell boundaries, it was evident that the provirus in D157B was also in the same transcriptional orientationasthe int-2gene, but apparently

intact. In tumor E115, on the other hand, a

complete

proviruswas present inan invertedorientation. These data

also located the precise sites ofintegration in the

genomic

sequencedescribedbyMooreetal.(11)at nt1089, 1195,and

1503, respectively, in tumors W26, D157B, and E115

(Fig.

2). No common features were evident in the sequences

immediately adjacenttotheintegration sites.

The three proviruses in question lie within the promoter domains mapped in EC cells in previous studies (Fig.

2).

Thus, the LTR in tumor W26 had integrated in exon

la,

betweenP1 andP2,theprovirus in D157B lay just upstream of P2, while that in E115 split P2 and P3. We therefore attempted to reconcile these findings with the RNA struc-turespresent in therespectivetumorsby RNaseprotection.

Tumor RNAs containing exon Ia. int-2 transcripts that initiate atpromoter P1 and contain an exon Ta to Tb splice

have beenidentified in EC cells by both RNase mapping and

cDNAcloning (10, 24).However, since these transcriptsare

atrelativelylowabundance in F9 cells and are undetectable

in PCC4 cells, itwas important to establish whether

coun-terparts existed in typical and atypical mammary tumors. I

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NV,

"....V.M1.1.

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a

1840

10CrU)(

Cm LLUJ

3 I

M

*

-218

-p -1 76

138',

'S1

b

MLL

LO L()

w-

517-

398-

296-

221-I

4 (P1)

-41

Um

154-

145-91'>

_

-75

FIG. 3. RNaseprotection analysisofexonIa. Approximately2 ,ug ofpoly(A)+ tumor RNA from the indicated tumors or 20 p.goftotal RNAfrom eitherF9 orPCC4cells that had been treated withretinoic acid anddibutyrylcyclic AMP wassubjectedtoRNase protection analysisasdescribed previously(24). (a) Results obtained with the probedesignated 12 in Fig. 2. Numbersto theleft of the panel identify thepositionsandsizes(nucleotides)of theRNase-resistant products.Thesizes wereestimated relativeto aseries ofplasmidDNAfragments of known length (HinfI-digested pAT153)and astandard DNAsequence ladder, indicated as lane M. Numbers on the rightidentify the positions andsizesofthestandardmarkers. (b) Probe 13 was used todelineatethe multiple start sitesassociatedwithinitiationatpromoter

P1.The arrowheadsontherightidentifyproductscorrespondingtothe major cap sitesobserved in F9 cells. Numbers on the left identify the sizes(in nucleotides) and positions ofDNAmarkerscalibrated witha sequenceladder.

Theexpression of exon

Ta

wasfirstexamined by protection

of the 184-nucleotide antisense RNA transcribed from the

genomic DNA segment designated as probe 12 (Fig. 2).

Assumingthat the structureofthetranscript wasasdepicted

in Fig. 2(subsequently confirmedwithmultiple probe

com-binations), probe 12 should have detected discontinuity at

thesplice donor siteatthe 3' end ofexon Ta.TumorE155A,

in which the provirus lies distant from the int-2 gene,

contained RNAs that include exon la and splice at the

predicted position, yielding a protected fragment of 138 nt

(Fig. 3a). The 184-nt fragment corresponded to full

protec-tion of theprobeandmay havebeenindicative ofunspliced

forms in thepreparationof total RNA used in thesestudies;

thecontrol RNA fromrat208Fcells and F9 cell RNA didnot

yield thisfullyprotectedproduct, indicating thatconditions

were adequatefor completedigestionof the probe.

A different result was obtained with RNA from tumor

W26, in which the solo LTR lies within exon

Ta.

Two

protected fragmentswereobservedthatwereeachabout50

ntsmaller than thoseseenin thetypicaltumorsample.Other data

(Fig.

4) confirmthatthelargerof thetwo(-135 nt)was

derivedfrom unspliced RNAand represented the distance

between the LTRandtheend oftheprobe

(calculated

tobe

137 nucleotides), while the -90-nt fragment reflected the

distance between theLTRand the end ofexon

Ta

(calculated

as 91 nucleotides). It is not clear why this latterfragment

resolves intomultiple species, 1base apart,but the effect is

consistently seen with probes that span this

discontinuity

(Fig. 4aand other datanot shown). TumorE115, in which

theproviruslies downstreamof the

splice

donor

site,

didnot

show evidenceoftranscriptsthatcontainexon Ia

(data

not shown).

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a

( U

(P2)j

321 0'

(Pi)

U')

I

M

--1 244

co

CJQ-WW

C\C L

ll LL

4..

wt

*

-520

'

-396

a

-354

496 'o

(P2),J

'

-218

I

.. ..

a"

a

-176

(SA)

147 (P3)

141 o

188

Po

(SD)

91

0 I

4

-75

FIG. 4. RNase protection analysis ofexons I andIb. (a) RNase protection was performed on theindicated RNA samples with the

1,225-nucleotide PvuII-SacI probe designated9in Fig. 2. Numbers onthe left of the figure identify the positions andsizes ofrelevant RNase-resistant fragments, calculated relative to the standard markers and sequence ladder on the right (lane M). Discontinuities

characteristic of the multiplestartsin P2 and P1 (24)areindicated by the lines, and other discontinuitiesareidentifiedthatcorrespondtothe

splice donor inexonIa(SD), the spliceacceptorinexonlb (SA), and initiationatP3 (10).The 321-ntfragmentmapsthepositionofprovirus

integration intumorE115. (b) A similar analysiswasperformedwith probe11.Since this probe extends fromthe NaeIsiteatnucleotide1681,

shorteranalogsareproduced for each ofthediscontinuitiesshown in panela.Thepositions of the multiplestartsites associatedwith theP2 promoterareindicated by arrowheads, while the 496- and 188-nt fragments correspondtothesites of proviral integration intumorsD157B andE115,respectively. The arrowhead adjacenttothe -500-nt fragment in the W26 laneidentifies the novelP2initiationsitecharacteristic ofthistumor,while the larger bandinthistrackderives from unspliced RNA initiated in theLTR. Thesizesof fragments (innucleotides)

werededuced directly fromthesequence ladder (laneM).

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Although these results implied splicing from the 3' end of exon Ia, in at least a proportion of the tumor RNAs, it was

of interest to examine the 5' end of such transcripts, to

ascertain whether they initiated at the same cap sites as in F9 cells. The analyses were therefore repeated with probe 13, which extends upstream of promoter P1. InE15SA, aseries of protected fragments were observed that were indicative of multiple start sites withinP1, each fragment being equivalent to acomplete exon Ta(Fig. 3b). Most of these 5' ends were analogous to those seen in F9 cells, but there were also additional discontinuities reflected in fragments of between 280 and 450 nt. Itis not clear whether these represent novel, tumor-specific initiation sites or simply an increase in the efficiencies with which normal cap sites are employed. Neither the W26 nor E11S tumor RNAs gave signals for initiation at P1 (data not shown).

Tumor RNAs containing exonlb. Previous studies on EC cells established that int-2 transcripts that include exon Ia

are spliced such that the donor site (nucleotide 1175) is linked to the consensus acceptor site (nucleotide 1661) that marks the 5' end of exon lb (10, 24) (Fig. 2). However, we now recognize that other transcripts initiate at a unique site very close to the 5' end of exon Ib, at what was originally

interpreted as an alternative splice acceptor (nucleotide

1667). Although separated by only 6 bases, the two

discon-tinuities, attributabletoeitherspliced RNA initiated at P1 or

de novo initiation at promoter P3, can be readily

distin-guished. For example, when probe 9 (Fig. 2) was used in

RNaseprotection experimentswith tumorE1S5ARNA, two protected fragments of 147 and 141 nt were observed at

approximately equivalent intensities (Fig. 4a). This would

imply that transcription initiation at promoters P1 and P3

occurredwith roughlyequalefficiency in typical mammary

tumors, in some contrastto the situation in F9 cells where P3, reflected in the 141-nt product, was clearly dominant (24). It also contrasts with the result obtained from tumor W26 in which the converse was true (Fig. 4a). Here, the

majorRNA species initiate in the MMTV LTR, and about

70% ofthesetranscripts undergoaspliceattheendofexon

Ta(91-ntfragment in Fig. 3a). The recipient ofthis splice is

presumablythe 5' end ofexon

Tb,

asreflected in the 147-nt

protected fragment, providing an explanation for the

strengthof thissignal relative tothefragmentgenerated by

initiation atP3 (Fig. 4a).

With tumor

E115,

the signal indicative of the

splice

acceptor sitewasextremely faint(Fig. 4a). Since there was

noevidence forRNAsplicing from exonIain this tumor, it

is possible that minor transcripts initiating at a

cryptic

promoter(s) within the MMTV provirus become

spliced

to

this site. However, the signal

corresponding

to initiationat

P3 was also unexpectedly weak,

despite

the fact that the

MMTV

provirus

inEllSis

presumably

inanideal

position

to enhance

transcription

from thispromoter.Either the levelof

transcription from P3 is inherently low in this tumor, for

reasons that are not clear, or the signals derived from P3

were masked by more abundant

transcripts

initiated

else-where. The most likely

origin

of these RNAs would be initiation from cryptic promoters on the

negative

strand of the MMTVprovirus,but this hasnotbeen

formally

proved.

Tumor RNAs containing exon I. The

major protected

fragmentgenerated fromEllSRNAwas

approximately

321

ntand corresponded to

discontinuity

atthe siteof

proviral

integration.Thismappedabout 100nucleotides downstream

ofthe P2

domain,

but

proviral

insertion

appeared

toprevent initiation at this promoter. However, in the

typical

tumor,

E1SSA, initiation within P2 could also be visualized with

probe

9, as evidenced

by

a

heterogeneous

set ofbands of

between 450 and 600nt. These are very similar in

distribu-tion to the analogous start sites observed at much lower

levels in PCC4 cells (Fig. 4a). The dataare complicated by

the fact that

probe

9 can also detect heterogeneous starts within P1

(inactive

in PCC4 cells), as represented

by

the characteristic series of

protected

fragments

between 215and 265nt. These latter

fragments

werenotobservedwith RNA from W26 and

E115, confirming

thatP1doesnotfunction in thesetumors

(Fig. 4a). However,

withW26

RNA,

therewas

evidence for novel initiation sitesin the upstream

region

of

P2

(fragments

of-650to750

nt)

anda

protected

fragment

of

around 91 nt was

again

observed

(Fig. 4a),

representing

the distance between the MMTV LTR and the

splice

donor site in exon

Ta,

as in

Fig.

3a.

Theseconclusionswere

supported

and clearerindications

for initiation in P2wereobtained

by

using probe

11

(Fig.

4b).

This

probe

extendedfrom the NaeI siteat1681

(Fig. 2)

and

therefore

yielded

shorter

analogs

of each of the

fragments

generated

with

probe

9.

Only

thoseoverabout 150ntin size are shown in the

gel depicted

in

Fig.

4b.

Transcription

initiation at P2 was reflected in

protected

fragments

of

between 308 and 443 nt, with the

major

start site

(361-nt

product)

centered on nucleotide 1320.

Thus,

typical

mam-mary tumors such as E1SSA expressRNAs that arealmost exact counterparts ofthose initiatedat P2inPCC4 cells. In

W26,

onthe other

hand,

a different set of

presumptive

cap siteswere

again

evident with

probe 11,

themost

prominent

of which

(-500-nt

fragment)

mapped

about 150 nucleotides

upstream of the

major

start

normally

associated with P2.

Since a

fragment

of similar size was also observed with

E1SSA

RNA,

albeitatmuch lower

intensity (Fig.

4b),

it may

correspond

toa

normally

rarecapsitewhose usage is

greatly

enhanced

by

the

nearby

insertionofthe MMTV LTR. The

larger product

from W26 RNA would be consistent with

unspliced

RNA sinceit represents thedistancebetween the LTR

(1089)

and the NaeI site

(1681)

that marks the end of

probe

11.

Probe 11 also revealed discontinuities that confirmed the sitesof

proviral

integration

intumorsEllS

(188-nt

fragment)

and D157B

(496-nt

fragment)

relative to the NaeI site. For

D157B,

there was insufficient tumor RNA to

perform

a

comprehensive analysis,

but the results shown in

Fig.

4b

suggested

that the

major

transcripts

in this tumor were

initiated in the viral

LTR,

upstream ofand

substituting

for P2.

Tumor RNAs with abnormal 3' ends.

Although

proviral

insertionstendtobemore common5'tothe int-2 gene, there

are nevertheless several tumors in which

proviruses

have been

mapped

at the 3' end of the gene. In S of the 20

int-2-positive

BR6 tumors, the

provirus

was inserted within

the

transcription

unit,

in the same

transcriptional

orienta-tion.

Figure

lbshowed that thesetumors contained

atypical

int-2

RNAs,

presumably

owing

to

perturbation

of the normal termination

signals

for

transcription. Indeed,

some ofthese RNAswere shown

by

sandwich

blotting

to be

hybrid

tran-scripts

comprising

both int-2 and MMTV LTR sequences (22; data not

shown),

suggesting

that

they

terminate in the

viralLTR.

However,

since the int-2 gene hastwoalternative

polyadenylation

signals,

it was of interestto determine the

precise

location ofthevarious

integrated

proviruses

andto assess whether any of the insertions affected the

protein-coding

domain.

Rather than construct recombinant

phage

librariesfor all thesetumors,welocated the

proviruses by

RNase

mapping.

Although

this method is less

precise

than

cloning

and

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Z79 Fm 6260 D124

6203

5641

V97P

6789

C46D.

6781

El46

-7725 W106 .

7505

7508

6404

H

p Ac Pv

5000

954

1367

E

8000

500

EcoRl-Hindill

HindlIl-Pvull Hindlil-Acci

1.3 1468 Hindill-Psti

FIG. 5. Proviralinsertions in the 3' untranslated regionofint-2. The3' end of the int-2geneis drawntoscale relativetotherestriction

map,withexonIIIdepictedasboxesinwhichprotein-codingsequencesareshaded. Theexonterminates at twoalternativepolyadenylation signalsatnucleotides7508 and 6404 in the genomicsequence(11).Arrowheadsatthetop of thefigure identifythepositionsandorientations

ofproviruses in the indicatedtumors.The restrictionmapshows thecleavage sites forthe enzymesAccI(Ac),EcoRI(E), Hindlll(H),PstI

(P), and PvuII (Pv) thatwereused ingenerating thenumbered antisenseprobesshown beneaththe map.Onlyrelevant sitesareshown.

quencing of virus-host junctions, we estimated that the

positions deduced from the sizes of protected fragments wereaccurate to withinabout 5 bases.The results of these

analyses aresummarized inFig. 5, anddatasupportingthe conclusions are presented in Fig. 6. Briefly, a series of antisense probes wereprepared, designated 1, 1.1, 1.2,and 1.3(24)(Fig. 5), thatwerecapable ofdetecting discontinuity between tumor-specific RNA and the genomic sequences

encompassing exon III. Wherever possible, each

disconti-nuity and therefore site of integration was mapped with at leasttwospecific probes. For example,theprovirusintumor D124waslocatedatnucleotide 6203 (+5) by using probes1.2 and1.3. Withtheformer, extendingto the AccI site

(nucle-otide 5997), a protected fragment of 206 nt was obtained (datanotshown), whilethelatter, extendingtothePstIsite

(5896), yielded a 302-nt fragment (Fig. 6a), suggesting that integration had occurredatthese distances from the respec-tive restriction sites. By similar reasoning, the provirus in tumor Z79was mapped to nucleotide 6260, approximately

359 ntfromthe PstI site(Fig. 6a). Representative data for

theother tumors and therespective probes used are

illus-trated inFig. 6b andc.

DISCUSSION

Multiple int-2 promoters respond to proviral activation.

Previousstudiesonthe expression of theint-2geneinmouse

embryos and EC cells have indicated aninherent transcrip-tionalcomplexity for which there isasyetnoclear explana-tion (7, 10, 24, 26, 27). Much of the complexity arises through the differential usage of three distinct promoter

regions in which RNA transcripts initiate at multiple sites (10, 24). However, all the transcripts characterizedthus far would be predicted to encode the same 27-kilodalton

pro-tein, now recognized as a member of the fibroblast growth

factor family (2, 4, 11). One possibility is that differential promoterusagereflectsaneedtorespondtofactors govern-inggeneexpression invery specific celltypes andathighly

restricted times duringdevelopment.

InMMTV-induced mammarytumors, expression ofint-2 is activated inacelltypeinwhichthegeneisnormallysilent, atleastasjudged by steady-statelevels of RNA(3,7).Itwas

therefore important to establish whether the transcription

patterns intumors reflected those seenin EC cells, suchas

F9 and PCC4, and whether the different int-2 promoters

responded differently to activation by MMTV. The three

promoter domains that have been distinguished, designated P1, P2, and P3 (10, 24) (Fig. 2), havenoobvious features in

common;theP1 region hasanaveragebasecomposition, P2 isveryG+C-rich, while the regionnowassociated with P3is unusually C+T-rich, analogous to some ribosomal gene

promoters(5). Northern (RNA) blotting analyses with DNA probes specific for exons Ia, I, and Ib indicated that in a

typicaltumor,suchasE155A, transcriptsaredetectable that correspondtoinitiationateachof the threepromoters(Fig. la). Thiswas subsequently confirmed by RNase protection withaseriesofantisenseprobes, aspresentedinFig. 3 and 4. Qualitatively, the RNAs initiated at each promoter are

very similar to those seen in EC cells, although there are minor variations in the distribution ofcap sites.

Quantita-tively, eachpromoter is moreactive in tumors thanin EC cells, and althoughP2 and P3aredominant,it is clear thatall three promoters function in mammary tumors. This con-trastswith the situation in F9 andPCC4cells,inwhichonly twoof thethreepromotersfunctionineach celltype:P1and P3in F9 cells, and P2 and P3 in PCC4 cells (10, 24). Since the

sequences involved in directing initiation from each

pro-1.1 1.2

OME'S'N

m

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a

PROBE1.3

Z79

-Dl124'o

~XX'359

sxwow'302

cxxxx30

04 0)

o N

b

PROBE 1.1

V971

C46

r

1468 _ 954

isssssss 376 ssssssss 368

et CY

0>

C

PROBE

1 El46m"

Wl

061m-500

<X'&%ss 360

'xx% 140/141

CD

_3O

Co0

6

* 4 360

* 4 359 368 - *

4376

302'0

140 , 4*141

FIG. 6. RNasemapping of proviruses in exon III ofint-2.Poly(A)+ RNA from the indicated tumors was subjected toRNase mapping with probes thatcrossed the sitesofintegration. The location of the provirus (shown by the arrowhead) was deduced from the length of the RNase-resistant fragment(striped line) compared with theinput probe (solid line). Each insertion was mapped relative to restriction sites that marked theendsoftwoindependentprobes (Fig. 5). Theautoradiographs in the lower part of the figure give examples of the data obtained with probes1.3(a), 1.1 (b), and 1 (c) on the indicated tumors, with the respective probe and product depicted graphically above each panel. Thesizes of the protected fragments (innucleotides), indicated to the left and right of the panel, were calculated relative to standard markers andaDNAsequenceladder.

motermay overlap (D. Grinberg and J. Thurlow, personal

communication), itis possible that MMTV influencesgene

expressionthrough a common element.

MMTV insertions that perturb int-2 promoter usage. In

mostint-2-expressing mammary tumors, the MMTV

provi-rusliesoutside the transcription unit and the patterns of int-2

transcription are very similar if not indistinguishable from

those describedherefortumorE155A(3, 16, 19).However,

thereare also examples that expressanomalous transcripts

resulting from proviral insertions in the 5' and 3'

untrans-latedregions of the gene.Inthreeofthese,W26,E115, and D157B, theprovirushas been accurately located within the int-2 promoterregions by sequencingacross the virus-host

junctions. By combining this information with detailed

RNaseprotection analyses,wehave accountedfor themajor

int-2transcripts in thesetumors.

Inthe firstexample, W26, int-2expressionis activatedby

a single MMTV LTR in exonla. We previously concluded

that themajor site for int-2 transcription in this tumor was

likelytobe theviral promoter in the LTR, basedon primer

extensionanalysis(11; datanotshown). Herewe confirmed

this conclusion but showed that transcripts initiated in the

LTR may havetwo structures, depending on whetherthey

undergo splicing between exons la and Ib. The calculated

size of the major spliced transcript wouldbe 2.75 kb, very

similar to that estimated from Northern blotanalysis (Fig.

lb), whilethelessabundantunspliced form wouldbe around

3.2 kb. However, there is also evidence for transcription

initiation outside theLTR,mostlyfromasite in themost5'

region ofP2, yielding an approximately 3.0-kb RNA. The

reason for initiation at this unusual cap site is unclear but

mayreflect influences of the MMTV enhanceron the most

proximal cap site in the P2 promoter. A bidirectional

en-hancermightalso have been

expected

toactivate

transcrip-tion from thenearbyP1promoter, butnoinitiationatP1was

discernibleintumorW26.

Nevertheless,

it is also clear that

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W26 contains minor int-2-specific transcripts for which we cannot as yet provide a full explanation, other than the possibility of unspliced orpartially spliced forms.

WithtumorD157B,therewasinsufficient RNAtoconduct

a comprehensive analysis, but Northern blots indicated a

transcription pattern similar to that in W26 (Fig.

lb).

This

would be consistent with transcription initiation from the viral promoter, presumably in the 3' LTR of thefull-length

MMTV provirus present in this tumor. Since the provirus liesintheupstream partof the P2domain, it has thepotential to subvert transcription from both P1 and P2 and may also

override P3 by promoter occlusion effects (1, 6).

In tumorE115, on the other hand, the ostensibly intact

provirus is in the inverse orientation and cannot program transcription from the normalviralpromoter. Nevertheless,

the RNase protection experiments we present here suggest thatthemajor transcriptinthistumormustinitiatewithin the provirus, presumably at some cryptic promoter on the

antisense strand of viral DNA. The position of the provirus wouldpreventinitiation inP2,and therewasnoevidencefor

transcription fromP1 cap sites. Thus, we conclude that the major2.8-kbRNA species consistently seenin thistumoris

initiated inthe provirus, while the less abundant2.7-kb RNA isanormal P3-derived transcript.

In the only other system in which comparable analyses have been reported, the activation of c-myc by avian and

murine leukemia viruses, there are close analogies to all threeof these scenarios,although thetranscriptionpattern is inherentlyless complex(8, 12, 20, 21, 23). Thus, proviruses havebeenfound within the firstexonorintron thatcandrive

expression from the viral promoter, enhance transcription fromthe c-myc promoters, orinduce initiation from cryptic

promoters. Although the generalities of the enhancer and promoterinsertion models stillapply (reviewed inreference 15), itis nowclear that the influences of retroviral LTRs on

proto-oncogene transcription can be manifold.

MMTV insertions thatperturb transcriptional termination.

Additional complexityin the transcription ofthemouse int-2 genearisesthroughusageoftwoalternativepolyadenylation signals, bothvariants oftheconsensus(10, 11, 24).Although

we cannot rigorously exclude some influences on RNA

stability or translational efficiency, it seems likely that this

complexity is largely irrelevant. InEC cells, up to 50% of transcripts terminate at the upstream poly(A) site (10, 24),

but in MMTV-induced mammary tumors, the majority of int-2 transcripts terminate at the downstream site, and the 1.8- and 1.6-kb RNA size classes are barely discernible on

Northern blots (Fig. 1). One possible explanation wouldbe

thatthe transcriptioncomplexes thattraverse thegenehave

different components in different cell types or in

MMTV-infected cells. Alternatively, the rate of transcription may

simply be higher in tumors than inEC cells, making it less

likelytoachieveterminationattheupstream signal. Insome

tumors, for example, E146, there is readthrough of both

polyadenylation signals, andthree sets of transcripts canbe

resolved (Fig. lb). Here the provirusis very close tothe 3'

end ofthe transcription unit (Fig. 5 and 6), allowing

termi-nation at the normal signals within the viral LTR and

extendingeach class ofRNAby the length ofthe U3region (-1.2 kb). In other tumors, a similar phenomenon occurs,

buttheproviruslieswithinthe transcription unit (Fig. 5 and

6).Depending onthelocation ofthe provirus relativetothe

two polyadenylation signals, this can result in extended

forms of the 2.9- and 2.7-kb RNAs but normal 1.8- and

1.6-kb transcripts,asfortumorsV97A,C46, and W106. The latter example is remarkable in that the integration has

occurred very close

(within

afew

bases)

tothe downstream

poly(A) addition site.

Alternatively,

asin D124and

Z79,

the

provirus lies upstream ofboth

signals,

resulting

in

only

one

setoftranscripts (Fig. 5 and

6).

The loss ofint-2 sequences

from the 3' end ofthese RNAs is

compensated

for

by

the addition of viral sequences,

making

their sizes very similar

to the normal 2.9- and 2.7-kb species

(Fig.

lb).

Proviral insertion does not perturb the

protein-coding

do-main. Retroviral activation of cellular proto-oncogenes, whether resulting fromtransductioninto the viral genome or

insertional mutagenesis by integrated proviral DNA, can often involve structural alterations to the gene

product

(reviewed in reference 15). However, there are also

exam-ples of insertionally activated oncogenes whose

coding

po-tential remains unaltered, implying that it is thenormalgene product that contributes to tumorigenesis. Obvious

exam-ples of the latter are the

int-i,

pim-J,and myc genes(12, 13,

20, 23, 25), and here weestablished that int-2 belongsin this category. Despite the complexity of

transcripts

and several

examplesofproviral insertions that disturb the

transcription

unit, all the proviruses mapped so far lie outside the open readingframethatspecifiesthe int-2protein. Atthe 5' endof

the gene, the most intrusive provirus (E115) is

approxi-mately 275 nt upstream of the presumed AUG initiation

codon. Although there is recent evidence for initiation at a

non-AUG codon, leading to amino-terminally extended formsof theprotein (P. Acland, M. Dixon, G. Peters, andC.

Dickson, submitted for publication), these would still be unaffected byinsertions upstream of P3. At 3' end, the most intrusive provirus that has been mapped to date, in tumor

D124, is -150 nucleotides downstream of the termination codon. Although we cannot rule out pointmutations orsmall alterations in thecoding sequences, we wouldconclude that the contribution of int-2 to oncogenesis is a function of elevated or inappropriate expression of the normal gene product. It has recently been shown that the product can function as a mitogen for cultured mammary epithelial cells and fibroblasts, in line with its classification as a member of the fibroblast growth factor family (4, 14). Similarly, under appropriate circumstances, the gene can induce morpholog-ical transformation of NIH 3T3 cells (R. Deed, W. Walther, C. Dickson, and G. Peters, unpublished data) and lead to epithelial hypertrophy in transgenic mice (W. J. Muller, F. S. Lee, C. Dickson, G. Peters, P. Pattengale, and P. Leder, submitted for publication). However, the normal role of int-2 is more likely to be as an inductive agent modulating the growth or differentiation of specific embryonic lineages (26, 27). The effect of MMTV integration, in

whatever

orientation and location relative to the int-2 transcription unit, is to reactivate the expression of a fetal gene in an adult tissue but not to alter the specificity of the gene product.

ACKNOWLEDGMENTS

We are grateful to Robert Moore, Mark Dixon, Marysia

Placzek,

and JanWitkowskifor contributing to some aspects of this work and to Roger Watson and Steve

Goodbourn

for helpful discussions on the manuscript.

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