Copyright © 1989,American SocietyforMicrobiology
Mapping of Functional Regions of Murine Retrovirus
Long
Terminal
Repeat Enhancers:
Enhancer Domains Interact and Are
Not
Independent in Their Contributions
to
Enhancer
Activity
TOMHOLLON't AND FAYTH K. YOSHIMURA2*
Department of Microbiology' andDepartment of Biological Structure,2 University of Washington, Seattle, Washington 98195
Received 22November 1988/Accepted 8 April 1989
We have used deletion andrecombinant longterminalrepeat(LTR)mutantstoexamineenhanceractivity differences between LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 murine
retroviruses.Deletionmutantanalysisrevealed that major control regions for MCF13 and Akv LTR enhancer
activity were similar but not identical. For both LTRs, major control regionswere distinctly different ina murine T-cellandafibroblastcell line.Recombinantenhancer analysis showed that LTRs could be divided into three regionscapableofaltering the levelofenhanceractivitythroughcooperativeorantagonisticinteraction.
The contribution ofeach regiontoenhancer activity was dependenton itscontext withrespect to the other
regions. LTRenhancer function indifferentcelltypesappearstobe the resultofthe interaction of enhancer modular elements.
Among the murine retroviruses which do not contain an oncogene within theirgenomes are viruses witha range of pathogenic potential, including those which replicate
with-out harming their hosts. Those murine leukemia viruses (MLVs) which are pathogenic are able to generate diseases which generally involve specific tissue types. To understand which viral genome sequences control disease specificity, recombinant retroviruses have been made by combining different regions of the genomes of retroviruses with dif-ferent diseaseproperties. Bycomparingdiseases inducedby the recombinants and their parental viruses, the major determinant of histological disease specificity has been mapped to DNA sequences within the U3regions of retro-viral LTRs (6-8, 13-15, 22, 23). Within these U3regions are tandemdirect repeat sequences which have been shown to betranscriptional enhancers (19, 20). Thisfinding has sug-gested thatenhancers are important determinants of
retro-viral disease specificity.
The implied linkage between enhancers and disease has been strengthened bythe observation that the exchange of tandem repeat sequences with a small adjacent DNA
se-quence in Moloney and Friend MLVs will change the diseasesthevirusescause(23).Moloneyviruscausesthymic lymphomas, and Friend virus causes erythroleukemias. A recombinant virus with Friend tandem repeat sequences and Moloney structural genes caused erythroleukemias. The
converse viral construction containing Moloney tandem repeat sequencesand Friend structural genes causedthymic
lymphomas.
Li and colleagues (23) also observed that mu-tant MLVs with one instead of two tandem repeats take muchlonger to induce disease than wild-type viruses. This observation has been supported by work from several groupswhich have shown with gene expression assaysthat long terminal repeats (LTRs) that have deleted onetandem repeatlose about half of their enhanceractivity(11,19, 32). Furthermore, stable and transient gene expression assays* Correspondingauthor.
tPresentaddress:InstitutPasteur,75724Paris Cedex15,France.
haveshown thatstimulationof geneactivityby enhancersof leukemogenic murine retroviruses is strongest in the cell type in which they induce disease (2, 3, 38).
Previous work in our laboratory has shown by means of stable gene expression assays that enhancer activity of the thymus lymphomagenic MCF13 MLV LTR is greatest in murine T-cell lines (38). That work also showed that the LTR of the nonpathogenic Akv virus does not have this T-cell-specific expression property, despite its 81% DNA
se-quencehomology withthe MCF13 LTR.
Studies of enhancersofDNAand RNA tumor viruses and the immunoglobulin heavy chain gene have shown that enhancersarecomposedof modules 15 to 20 basepairs(bp) in length which can further be subdivided into sequence motifs that are binding sites for trans-acting transcription-regulatoryproteins(16, 21,28, 33, 35). Avarietyof sequence motifs forbinding trans-acting proteins have beenfound in the Moloney MLV LTR(35). MCF13 and Akv LTRs have sequences similartotheMoloney motifswithin their tandem repeats, including those for nuclearfactor 1-CCAAT
tran-scription factor binding (26), the glucocorticoid-responsive element (5), the simian virus 40 (SV40) core consensus
sequence (17), and several other DNA-binding proteins which, to date, have only been described for murine
retro-viral LTRs. Akv and MCF13 enhancer mechanisms, then,
arealso likelyto depend oninteractions with transcription-regulatory proteins. Whichproteins may be involved in the T-cell preferential expressionof MCF13 is unknown. Nor is there any knowledge of modular enhancer elements within these LTRs.
Inthis report, we have made enhancer deletion mutants
and recombinant enhancers composed of both MCF13 and Akv LTR sequences in order to begin mapping sequences responsible for MCF13 and Akv enhancer differences. We
tested these constructions with transient gene expression assays in a murine T-cell and a fibroblast cell line. The results of theseexperiments suggest that enhancerfunction
of murine leukemia viruses in different cell types is theresult
of the interaction of enhancer modular elements. 3353
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MATERIALS ANDMETHODS
Cell lines. NIH3T3 murinefibroblastsweregrownin10% fetal bovine serum (FBS) (Hyclone
Laboratories,
Logan, Utah)-minimal essential medium (GIBCOLaboratories,
GrandIsland,N.Y.). L691 cellsare aC57/Leaden radiation-induced thymic lymphoma murine cell line (24); they weregrownin5% FBS-RPMI 1640(GIBCO).
Transfection and analysis of gene expression. L691 cells were washed and suspended in RPMI without serum at
107
cells in 0.4 ml pertransfectionexperiment. Then,
10jig
each ofchloramphenicol acetyltransferase(CAT)
construct plas-mid and3-galactosidase
plasmid containing the SV40early
promoter (pCH110) (12) was mixed with cells to betrans-fected. Transfection procedures were performed by means ofelectroporation, essentially as described by Potter et al. (29), except that a cylindrical electroporation chamber de-sign was used which improved transfection
efficiency
about fivefold over cuvette chamberdesigns (T. Hollon and F. K.Yoshimura, submitted for publication).
Trypsinized
NIH 3T3 cells were washed andsuspended
in RPMI without serum at3 x106
cells in 0.4 ml pertransfectionexperiment.
Then4 ,ug eachof CATconstructplasmid andpCH110wasmixed withNIH3T3
cells.
tobe transfected.Electroporation
conditions were the same as for L691 cells. Allplasmids
transfected were in supercoiled form. Electroporated L691 cellswere placed in RPMI pluspenicillin-streptomycin plus
5% FBS for 2 days before cellprotein
extracts were har-vested. Electroporated NIH 3T3 cells were placed in mini-mal essential mediumplus 10% FBS plus penicillin-strepto-mycin and were alsoharvested after 2 days.Protein extract harvests and
nonchromatographic
CAT assays were done essentially as describedby Sleigh
(34), except that protein extracts for CAT assays were heat treatedat65°Cfor 10 min in the presence of 5 mM EDTA(4)
in order to denature cellular enzymes interfering with the CATassay. The averagecountsperminute of14C-acetylated
chloramphenicolextractedfor MCF13 LTR
(clone M2)
CAT assayswas29,800cpmin L691 cellsand13,700
cpmin NIH 3T3 cells.3-galactosidase
activity measurements wereusedto correctCAT activitymeasurementsfor variationsdueto
transfection efficiencies. Each
3-galactosidase
assay wasdone by using 100 jig of
protein
extractin 0.25 M Tris(pH
7.8)plus 10V1
50 mMCPRG(chlorophenol
red P-D-galacto-pyranoside; Boehringer MannheimBiochemicals,
Indianap-olis, Ind.)-70VI
Z buffer(25)-deionized
water to a total volume of 100VI.
Zbuffer is 0.06 M Na2HPO47H20-0.04
MNaH2PO4.
H20-0.01 M KCl-1 mMP-mercaptoethanol
(pH 7.0). The assaywas done at 37°C for 150 min, and the i-esultingP-galactosidase
activitywas read at 574nm. Plas-mids were transfected an average of 12times each in L691 cells and 9 times each in NIH 3T3 cells.Plasmid constructs. The LTR+CAT constructions were made in several steps according to standard
procedures.
Constructions were verified by analysis with appropriate restriction endonuclease digestion procedures. The SphI-BamHI DNA fragment from pSV2CAT (9) containing the SV40 promoter without enhancer and the CAT gene wasligated into the SphI and HindIll sites ofpUC18after first making BamHI and HindIII ends blunt with Klenow
frag-ment treatment. This plasmid was called pUCCAT. PstI-DraIfragments ofMCF13 and Akv LTRs were ligated into theXbaI site ofthepoly-cloning site of pUCCATto create
M2PrS andA2PrS;PstI andXbaI sites wererendered blunt in orderto achieve ligation. To make clones A2 and M2, a
PstI-HindIII fragment was first removed from pUCCATin
orderto removethe SV40promoter.
Next,
theHindlll site at the 5' end of the CAT gene was made blunt endedby
Klenow
fragment
treatment.PstI-SmaI LTRfragments
werethen
ligated
intotheCATvector toreplace
thedeletedSV40
promoter; thiscreated A2and M2.Deletion mutants. A2 and M2 were eachcut with EcoRV and
religated
in the absence of the EcoRV-EcoRVfragment
withinthe tandem repeats.This
generated
AAAandMMM,
which each haveonetandem repeatdeleted. BamHI-EcoRV
fragments
weredeletedfromAAAand MMMtocreateOAA andOMM;
BamHI andApaI
fragments
were deleted to createOOAandOOM(BamHI
wasin thepUC18
poly-cloning
site).
AOA and MOMweremadeby
deleting
theEcoRV-ApaI
fragments
from AAA andMMM.PrMwasmadeby
deleting
theBamHI-DraI
fragment
from M2.Recombinant enhancers. PstI-EcoRV sequences were
ex-changed
between MMM and AAA to create AMM and MAA.EcoRV-ApaI
sequences wereexchanged
betweenMMMand AAAtomake MAM and AMA. AAM and MMA resulted from
exchanging
ApaI
toSmaIsequences between AAA and MMM.RESULTS
Experimental
strategyand constructionofmutants.Figure
1 shows a
comparison
of the Akv and MCF13 LTRse-quences involved in the DNA constructions used in this
study.
Sequences
present as tandem direct repeats in the LTRs are enclosed in brackets. MCF13 and Akv tandem repeats are 69 and 99bp,
respectively.
Ourprevious
workusing
stable geneexpression
assayssuggested
that sequence differencesbetween these LTRswereresponsible
fora5-to10-foldincrease of MCF13 LTR enhancer
activity
overAkv in murine T-cell lines(38).
Although
tandemrepeats in the U3region
of other MLVs have been shown to function asenhancer elements
(20),
Laimins et al.(19)
have demon-strated that tandemrepeats maynotbe the sole locations of enhanceractivity.
Therefore,
ouroperational
definition of LTR enhancers has allowed for thepossibility
that not all enhanceractivity
isprovided by
theMCF13 andAkvtandem repeats.Figure
2 shows ourenhancer constructions. For ease ofmanipulations
involving
cloning,
all deletion andrecombi-nant enhancer mutants were
generated
from constructscontaining only
oneofthetandem repeats from eachLTR.To
begin
to mapregions
responsible
for LTR enhanceractivity,
we made use ofrestriction enzyme sites found at similarpositions
in thetwoLTRs. EcoRVandApaI
restric-tion sites(Fig.
1)
within each tandemrepeatare23bp
apartand straddle
SV40-like
enhancercore consensus sequences(17).
We therefore refer to this DNA segment as the coreregion.
EliminationofDNA segmentsterminating
inone or theotherof theserestriction sitescreatesdeletionmutantsin whichcomparable
regions
have been lost from Akv and MCF13 LTRs. We reasoned thatby
comparing
enhancer activities of deletion mutants with theoriginal LTRs,
wewould be able to map LTR
regions controlling
enhanceractivity
in different cell types andtoidentify regions
respon-sible for the differencesbetween thetwoenhancers.To create recombinant
enhancers,
weagain
took advan-tageof the EcoRV andApaI
sites,
whicheffectively
allowedus todivide eachLTR into threeparts: sequences 5' tothe core
region
(PstI-EcoRV),
coreregion (EcoRV-ApaI),
and sequences 3' to the coreregion, including
the promoters(ApaI-SmaI).
For convenience we call these the5',
core, and 3' LTRregions.
MCF13 and Akv DNA segments cuton November 10, 2019 by guest
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PstI
CTGCAGTAACGCCATTTTGCAAGGCATGGAAAAGTACCAGAGCTGA.GTTCTCAAAAGTC...ACAAG
G A T G AAACAAGA
EcoR V Core
GAAGTTTAGTTAAAGAATAAGGCTGAA*AAAACTGGGACAGGGGCCAAACAGGATATC4iTGGTCGAIGCA
A AG ... G A GT C TA A |
ApaC
CCTGGGCCCCGGCTCAGGGCCAAGAgAGATGGTACTCAGATAAAGCGAAACTAGCAGCAGTTTCTGGAA
TA C C C A]T T AC A A
DraI
MCF13 AGTCCCACCTCAGTTTCAAGTTCCCCAAAAGACCGGGAAA.
AACCCCAAGCCTTATTTAAACTAA~~
Akv CC AG AACTGTC CA G G T G TC C
MCF13 CAGCTCGCTTCTCGCTTCTGTAACCGCGCTTTTTGCTCCCCAGCCC ATAAGGTAAAAACCCCACA
Akv C A G T G
SmaI
CTCGGCGCGCCAGTCATCCGATAGACTGAGTCGCCCGGG C
B. MCF13 or Akv LTR
TATA CCAAT Cap Site
I
I
11_ _
I~II I~I 'I"t tr
I
Il~~~cl
Pr1
p
I I
E A
I I
E A
U3
I I
D S
U5 R
FIG. 1. Comparison of Akv and MCF13 LTR nucleotide sequences and structure of LTRs. (A) Akv and MCF13 LTR nucleotide
sequencesbetweenthe PstI andSmaI sitesarealigned for comparison. Sequence differencesareshown for the AkvLTR,withblankspaces foridentical bases and dots for deletions. The sequencesarefrom thepublished data of Van Beverenetal.(37) and Yoshimuraetal.(38). Nucleotidespresentastandemdirectrepeatsin the LTRsareshown withinbrackets, but onlyasinglerepeatfor each LTR isshownhere. Boxes surround theSV40-likecoresequencesandCCAAT and TATA motifs in thepromoter.(B) Generalizedstructureof the MCF13 and AkvLTRs.Restrictionendonuclease sitescommontothe Akvand MCF13 LTRsarePstI(P), EcoRV (E), ApaI (A), DraI (D), and SmaI(S).
LTRs usedinconstructionstodirectCATgenetranscriptionwerePstI-SmaI LTR fragments. The PstI sitestarts atbase36atthe 5' end of eachLTR; the SmaI sitestarts atabout33bases 3'toeachcapsite. Thehorizontalarrowsshow thetandem directrepeats;boxes marked Carethe23-bpcoreregion between the EcoRV and ApaI sites. The DraI site is5bp5'toeachCCAAT box. The DraI-SmaIregion includes
thepromoter(Pr). Arrows marking R and US LTR regionsaretoscale with the U3 region.
withoneortheother restrictionenzyme wererecombinedto create LTRs containing enhancers that contained both
MCF13 and Akv sequences. Comparing recombinant en-hancers would let us ask what effect each region had on enhancerfunctionwhenlinkedtodifferentsequencesofAkv
or MCF13 origin. We also made constructions which ex-changedLTR enhancers and promoters, enablingusto ask whether these enhancers work differently with different
promoters. Enhancers and promoters were exchanged at a DraIsite 5bp5'tothe CCAATboxof each promoter.
WeusednonchromatographicCAT transientgene expres-sion assays (34) to test enhancer activity. Results of CAT
assaysofwild-typeLTRenhancersin themurineL691 T-cell
andNIH3T3fibroblastcell linesareshown inFig. 3. In the T-cell line, CAT activity of the MCF13 LTR with two tandem repeats (M2) is 30 times greater than that of the construct withonlytheMCF13 promoter(PrM).The
differ-encebetweenthesesameclones in NIH 3T3 cellsis50-fold. The Akv enhancer attached to the MCF13 promoter (A2PrM)is20 timesasactiveasPrM in the T-cell line and 75
times as active in fibroblasts. This confirms our previous
report (38) that both LTRs contain sequences which are
capable ofenhancing transcriptional activityfrom a murine leukemia viruspromoter.That thesesequencesare function-ingasenhancers issupported by previouswork in whichwe haveshown that thisactivityisorientationindependent (38), ahallmark of enhancers (17).
When we compared the relative strengths of the two LTRs, we found that M2produced2.4timesasmuch CAT
activity as theAkv LTR (A2) in L691 cells. In fibroblasts,
their strengths were reversed; Akv was 2.7 times more active than MCF13. Between celllines,the LTRs differedin relative strength by about sixfold, as can be seen by com-paringM2 and A2 activities in NIH 3T3 and L691 cells(Fig. 3).
LTR enhancers show promoter preference. We created A2PrM and M2PrA (the MCF13 enhancer and the Akv promoter),which exchangeenhancer and promoter regions (Fig. 2), totest whetherthe enhancers functioned indepen-dently of their promoters. Berg et al. (1) reported that MCF13
A.
AkvMCF13 Akv
MCF13
Akv AA G
MCF13 Akv
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[image:3.612.101.507.76.430.2]M2
I
IH
iHI[
IPi
I
A2
IU2PrA I IIIII
:
AmpC
LTI CAT
A2PrM
M2PrS
I
III
M
A2PrS
PrM
DG
DELETfON MUTANTS
OAA
AOA
OOA
P EAO D S
ff'_C4-' -b
~cowe
AAA-MAM AMA
MAA 11
AMM ~ :
MMA
I I I
P EA D S
[image:4.612.70.306.66.398.2]44-PC4-3' -*
FIG. 2. MCF13 and Akv LTR constructions used for transient expression CAT assays. All LTRs were cloned into a
pUC18-derivedplasmid containing the CATgene.Theplasmid designatthe
upperright illustrates the general form of these constructions. At the
upperleftareLTRs consisting of PstI-Smal fragments. Open boxes represent MCF13 regions; Akv regionsare stippled. Boxes below
thosemarked C and PrepresenttheEcoRV-ApaI coreregion and
thepromoter,respectively. M2 and A2arethewild-type LTRs; the 2denotes thepresenceof both tandemrepeats,shown witharrows.
PrM, PrA, and PrSarethe MCF13 and Akv LTRpromotersand the SV40earlypromoter(hatched), respectively. M2PrA is the MCF13 enhancer withAkvpromoter;asimilar notation describes the other
enhancer-promoter recombinations. MMM and AAA correspondto
LTRs withonetandemrepeatremovedfrom M2 and A2. Each letter M or A stands fora 5' (PstI-EcoRV), core (EcoRV-Apal), or 3'
(ApaI-SmaI) LTR region. For the deletion mutants, a0is usedto
denote a deleted region. Restriction enzyme sites important to
deletion mutant and recombinant enhancer construction are PstI
(P), EcoRV (E), ApaI (A), DraI (D), and SmaI (S). Arrows below therestriction sites indicate the 5',core,and 3'regions.
enhancers can sometimesfunction differently with different promoters. Akv and MCF13 promoters differ by 5 bp be-tween the CCAAT box andthe transcription initiation site (38), although their CCAAT and TATAbox sequences are identical.
Thedifference between M2 and M2PrAwaslessthan17% in both cell lines (Fig. 3), which we judged not to be
significant. The A2versusA2PrMcomparison showed larger differences. A2PrM had 40%moreactivitythanA2in Tcells and 45% less activity than A2 in fibroblasts. These differ-ences also correlate with ourdata showingthat the MCF13 LTR has greater activity than Akv in T cells but less in
fibroblasts. For these reasons, it appears that the effect of the MCF13 promoter on the enhancers may be of
signifi-'~- 200
|
1000.
M2 A2 M2PrA A2PrM M2PrS A2PrS PrM FIG. 3. CAT activities of wild type LTRs and enhancer-pro-moterrecombinants. L691 T- and NIH 3T3fibroblast murine cell linesweretransiently cotransfected with LTR+CAT plasmids and 3-galactosidase (pCH110) plasmids.Nonchromatographic CAT ac-tivity measurements (34) are counts per minute of 14C-acetylated chloramphenicol extracted from CAT assays with ethyl acetate. CATactivities measured for these transfections werecorrected for transfectionefficiency variation with ,3-galactosidaseactivity mea-surements. Theresults ofmultipleindependent cotransfections are presented as averages. Error bars show standard errors of the means.The levelofMCF13 wild-type LTR (M2)activity has been set at100for each cellline,and all other CATactivityaverages are expressed relativetothe M2 levels. ForFig.3, 4,and 5, theaverage countsperminute of14C-acetylatedchloramphenicol extractedfor M2 CATassays was 29,800 cpm inL691 cells and 13,700cpm in NIH 3T3 cells.
cance.Whenwerecombined enhancers with theSV40 early
promoter (M2PrS and A2PrS), we found that in both cell
lines MCF13 and Akv had at least 10 times more activity
with their own promoters than with theSV40promoter.
DifferentLTRregionscontrol enhanceractivity in aT-cell
and a fibroblast cell line. To map regions important for enhanceractivity in each cell line, we made deletion mutants whichprogressivelydeleted similarregionsfrom the MCF13 and Akv LTRs. We first deleted one of the two tandem repeatsequences from each LTRto createMMM and AAA (Fig. 2). Inournomenclature, each letter MorA stands for
a5', core, or3'region. In thedeletion mutants, 0 is usedto
denotea deleted region. The 5' region from PstI to EcoRV wasdeleted to create OMM and OAA. Deleting5' and core
regionsfrom PstIthroughtheApaI sites resulted in OOM and OOA. The core region included between EcoRV andApaI was deleted in MOM and AOA. By comparing M2 with
MMM,OMM,MOM, andOOM in both cell lines andsimilarly for Akv mutants, contributions of the deleted regions to
enhanceractivitywere determined.
CAT activities of M2 and its deletion mutants are
com-paredinFig.4. InL691 T cells(Fig.4A),neither theloss of
oneof the tandem repeats(MMM)northefurther loss of the 5'region (OMM) causedmorethana15%decline in enhancer activity from the M2 level. Loss ofthe coreregion(MOM), however, caused a70% declineinactivity fromthe levelof the M2clone.Surprisingly, deleting both5' andcoreregions inOOMdidnotlower enhanceractivitytothe MOMlevel,but rather, the activity of OOM was twice the activity of MOM. This suggested that the MCF13 5' region is suppressing enhancer activity in MOM. On the other hand, this result
couldbe duetoantagonistic interactionsof 5' and 3'regions
no longer separated by the core region. Comparison of deletion mutants with MCF13 promoterPrM showed that the major control regions ofM2 enhanceractivity are the
core region and sequences between it and the promoter. A
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[image:4.612.325.565.81.216.2]L691
L~~~-MMM AAA OMM OAA MOM AOA OOM OOA PrM
M2 A2 MMM MA OMM OM MOM AOA OOM OOA PrM
FIG. 4. CAT activities of deletion mutants in L691 T cells and NIH 3T3fibroblasts. Barsshowaverage CAT activities of MCF13 and AkvdeletionmutantsinL691 cells(A)andNIH3T3cells(B).
MCF13-derived mutantsare
represented by
stippled
barsand mu-tantsof Akvorigin
areshownby
open bars.second enhancer tandem
repeat
seemedtocontribute littletoMCF13 enhancer
activity
in L691cells.In NIH3T3
fibroblasts,
MCF13 deletionmutantsshowed adifferentpattern
(Fig.
4B).
The lossofonetandemrepeat
inMMMlowered enhanceractivity by
onethirdfromthe M2level. OMM had
only
halftheactivity
ofM2. MOM hadonly
20% of theactivity
ofM2. Whereas OOM had 60% ofM2activity
in L691cells,
it hadonly
10% ofthat in NIH 3T3cells. In the
fibroblast
cellline,
themajor
controlregions
of MCF13 enhanceractivity
reside ina second tandemrepeat
and in the 5' and core
regions.
A
comparison
ofA2andits deletionmutantsin L691cells(Fig.
4A)
showed that the deletion of one tandemrepeat
(AAA)
or5'(OAA)
or coreregion
(AOA)
is withoutsignifi-cant effect.
OOA,
in which 5' and coreregions
have beendeleted,
hadnearly
70% more enhanceractivity
thanA2,
whichmayindicatethataregionrepressingenhanceractiv-ity
has been removed fromOOA. In L691cells, then,
mostof theenhanceractivity
ofA2is controlledby
sequencesinthe3'
region.
Itisinteresting
that inL691cells,
OOA hasgreater
activity
than AOA andOOM hasgreater
activity
than MOM.These
parallel
results with different LTRssupport
oursug-gestion
that sequences in the 5'region
may beinhibiting
enhancer
activity.
In NIH3T3
cells,
AAAhadonly
60% ofA2activity
dueto the deletion of one tandemrepeat
(Fig.
4B).
Additional deletions ofthe 5'region
(OAA),
coreregion
(AOA),
and both5' and core
regions
(OOA)
showedlarge
declines inactivity
fromthe AAA and A2 levels. In this cellline,
thecontrolling
-A
regions
ofenhanceractivity
are a second copy ofarepeat
sequence and 5' and core
regions.
Regions responsible
for differences in MCF13 and Akv enhancer activities. We have also derivedinformationabout theregions
ofthe LTRwhichaccountforthedifferences in enhancer activities between MCF13 and Akv in the L691 T-celllineand in NIH 3T3fibroblasts. In L691cells,
M2had 2.4 times the CATactivity
of A2(Fig.
4A).
When one tandemrepeat
was deleted from eachLTR,
MMMactivity
wasgreater
than theactivity
ofAAA,
but thedifferencewas about halfofthedifferencebetween M2 and A2.Comparing
5'
region
deletionmutants, OMMwasmoreactivethanOAA,
and thedifferencewas about75% ofthedifferencebetweenM2and A2. But when thecore
region
wasdeleted,
compar-ison ofMOM and AOA and
comparison
of OOM and OOA showed that thegreater
activity
associated with MCF13sequences had
disappeared.
Thus,
regions
mostimportant
for the MCF13 and Akv LTR difference seemed to be a second copy ofthe tandem
repeat
and the coreregion.
InNIH 3T3
cells,
A2 CATactivity
was2.7times asgreat
as M2activity
(Fig.
413).
When one tandemrepeat
was deleted from eachLTR,
theactivity
of AAA exceededMMM
activity,
butthis differencewasabout halfthediffer-encebetween A2 and M2. The OMMversusOAA
comparison
showed that theoriginal
difference between A2 and M2activity
waseliminatedwhen 5'regions
weredeleted.Com-parison
ofthecoreregion
deletionmutantsshowed that AOAhad more
activity
thanMOM,
whichsuggested
the coreregion
did notdeterminethe differencein Akv and MCF13 enhancer activities. OOA hadgreater
activity
thanOOM,
whichsuggests
that the 3'region
also contributed to the difference between Akv and MCF13 in NIH 3T3 cells.However,
the presence of a second tandemrepeat
and sequences within the 5'region
seemed to be themajor
determinant ofthe difference inLTR enhanceractivities in this cell line.Comparing
recombinant enhancers. With enhancer con-structsMAM, AMA, MMA,
AAM, MAA,
and AMM cre-atedfromparts
ofMMMandAAA,
wewishedtoassessthe relative contribution of eachregion
to enhanceractivity
when associated with sequences from another LTR.Com-parisons
ofcoreregion
deletion mutants MOM and AOA inFig.
4A indicated thatcoreregions
accountforsome ofthe differences between MMM and AAA in L691 cells. We wishedtoknowwhether eachrecombinantenhancer withan MCF13coreregion
would havegreater
activity
in L691cells than thecorresponding
Akv coreregion
recombinant. Fourcomparisons
ofcoreregion exchanges
arepossible:
MMMversus
MAM,
AMA versusAAA,
MMA versusMAA,
andAMM versus AAM. Evidence that an MCF13 core
region
recombinantalways
hasgreater
activity
than its Akv coreregion
counterpart
wouldsuggest
that thecontribution ofthecore
region
to enhanceractivity
isindependent
of thesequences
surrounding
it. On the otherhand,
if some en-hancers with Akv coreregions
are more thanorequally
as active as theircounterparts
with MCF13 coreregions,
thecore
region
contributionmaybedependent
onadjacent
5'or3'
region
sequences.Figure
5A shows the CAT results from four coreregion
exchanges
inL691 cells. MMMhas 47%moreactivity
thanMAM,
which iscomparable
to the difference between the activities ofMMM and AAA. The AMA versus AAAandMMA versus MAA
comparisons,
however,
showed a smalleror nochange
when Mand Acoreregions exchanged
places.
Inaddition,
the AMMcomparison
with AAM showed that the Akv coreregion
couldgreatly
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MMM MAM AMA AM MMA MM AMM MM PrM FIG. 5. CATactivities of recombinant LTR enhancers. Recom-binant enhancers arearranged toshow CATactivitychangesfrom
exchanging core regions in L691 cells (A) and NIH 3T3 cells (B).
Stippled bars represent recombinant enhancers with MCF13 core
regions;recombinant enhancers with Akvcore r-egionsareshownby
openbars. Fourcomparisons ofcoreregion exchangesare shown-,
lefttoright: MMMversus MAM, AMAversusAAA, MMAversus
MAA,and AMMversusAAM.Thepromoter PrM is shown insolid bars. The recombinant enhancers can also be compared for the effects of 5' or 3' region exchanges. The 5' region exchanges are
MMM versus AMM, MAA versus AAA. MAMversus AAM. and
MMA versus AMA. The 3' region exchanges are MMM versus
MMA. AAM versus AAA. MAM versus MAA. and AMM versus
AMA.
enhancer activity. Conversely, this comparison indicated that the MCF13 core regionwas depressingenhancer
activ-ity. This wasa surprisingresult notpredicted by analysis of the deletion mutants of Fig. 4A. Enhancers with MCF13
coreregions apparently did not always havegreater activity
in L691 cells than those with Akv core regions. These data suggest that whether an Akv or MCF13 core region
de-creases, increases, ormakes no change in enhancer activity
dependson the context of enhancer sequences with respect
toother regions.
Data from the AOA versus MOM comparison in Fig. 4B
indicated that the core region was not responsible forAAA being more active than MMM in fibroblasts. This informa-tion would predict that core region exchanges in NIH 3T3 cells would havelittle effectonenhanceractivity. Of the four
core region exchangesshown in Fig. SB,three showed little
change. But the AMM and AAM exchange showed that a core region canhave alarge effect on enhancer function in
NIH 3T3 cells.Aswith L691cells,the dataindicate thatcore
region exchanges did not always have the same effect on
enhanceractivity.Inboth celllines, thecoreregionseems to
be influenced by sequences in otherregions.
The recombinant enhancer measurements also allowed comparison ofthe effects ofexchanging5' regions. In L691 cells, comparisons of5'regionexchanges (Fig. 5A) showed resultssimilartothose inNIH 3T3cells (Fig. SB). InbothT
cells and fibroblasts, the MMM versus AMM comparison suggested that the 5' MCF13 region increases enhancer activity. In both cell lines, theMAM versus AAM compar-isons indicated a decline in activity due to the 5' MCF13 region. In both cell lines, two comparisons, MMA versus AMA and MAA versusAAA, did not show much change. The data suggest that in the right sequence context the
almost silent 5' enhancer domains in L691 cells can be activated. In eithercell line,a 5' region can cooperate with other regions to stimulate activity, interact antagonistically to suppress enhancer activity, or make no change. As with the core region, the data indicate that the 5' region
contri-bution to enhancer activity is not independent ofthe other
sequences.
When wecompared 3' regionexchangesin L691cells (Fig. SA), we found one large change in activity, i.e., the AAM
and AAA exchange. The other exchanges (MMM versus
MMA, MAM versus MAA, and AMM versus AMA) indi-catedonlysmallchangesinactivity.For NIH 3T3 cells(Fig. SB) exchanges, MMM versus MMA, MAM versus MAA, and AMM versus AMA indicated the 3' Akv region
stimu-lated enhancer activity. The fourth exchange, AAM versus
AAA, did not show an increase in activity by the 3' Akv
region.When3' enhancerregionsareexchanged, promoters
are exchangedaswell. Itispossiblethat the promoters have influenced these results. Experiments described earlier (Fig. 3) showed Akv enhancer sequences could be influenced by MCF13 promoters. In any case, our data show that a 3'
enhancer region plus promoter exchange does not always give the same result. It seems that the activity of the 3' region (perhaps with some promoter influence) can be
af-fectedby the 5' and core regions.
DISCUSSION
We have examined differences in enhancer activities
be-tween LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 retroviruses. These experimentsare afirst step toward pinpointing LTR sequences that control
enhancer activity and tissue-specific expression and that
may possibly contribute to viral pathogenicity. We have found thatwithin acellline, similar,butnotalwaysidentical,
LTR regions are the major control regions of enhancer activity. Despite the fact that this is not a fine mapping of
LTRenhancers, the regulatoryregions inthe twocell lines
were clearly different. This finding suggests that there are
modular functional divisions within each LTR. Regions controlling differences between Akv and MCF13 enhancer activities inthetwocell lineswerealsonotidentical,further
suggestingseparable functional elements in LTRenhancers. More refined mappingwill be neededto preciselylocate the modular elements.
Functional attributes of LTR regions. We observed dif-ferent contributions that a second copy of the enhancer tandem repeatcanmaketoenhanceractivityin different cell lines. Infibroblasts, deletion ofone tandem repeat in either the AkvorMCF13 LTR resultedinlossof activity similarto the50%activitylosspreviously reportedfor other retroviral
LTRs (11, 19, 32). In contrast, in the T-cell line loss ofone
tandem repeatdid not significantly reduce either MCF13 or
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[image:6.560.38.280.72.369.2]Akv enhancer function. Li et al. (23) demonstrated that deletion of an LTR tandem repeat substantially delays the onset of Moloney virus-induced thymic lymphoma and of Friend MLV-induced erythroleukemia. While the disease caused by Moloney viruses with one or two tandem repeats did not change, 20% of the Friend viruses with a deleted repeat caused myelogenous or megakaryocytic leukemia. The induction of these new leukemias by the mutant Friend virus suggests that in the Friend LTR the deletion of one tandem repeat may have affected the tissue-specific expres-sion of this enhancer. This result may have some bearing on ourfinding that in both cell lines a second copy of the repeat sequence contributed to the overall activity differences
between Akv and MCF13 LTRs.
Our findingthat the differences among LTRs in the T-cell line was partly attributable to the core region (Fig. 4A) was particularly interesting. The SV40 core consensus sequences of Akv (GTGGTCAA) and MCF13 (GTGGTCGA) differ only at the seventh base. Sequence changes in the core region of the SV40 enhancers have been shown to greatly change enhancer activity (39). Experiments by Schirm et al. (31) have suggested that core sequences differing by only one base could be used to assemble enhancers with very different patterns of tissue-specific expression. Tissue-preferential expression by Akv and MCF13 LTRs may be more compli-cated than differences in core motifs, however. The 23-bp
EcoRV-ApaI
segment we call the core region for conve-nience contains the site for at least one other DNA-bindingtr-ans-acting
protein. This DNA-binding protein has been identified by Speck and Baltimore (35) for the Moloney MLV LTR. They have shown that this protein, named leukemia virus factor C (LVc), binds to the sequence CCTGC. The MCF13 sequence CCTGG (a four of five match) and Akv sequence CTAGG (a two of five match) are at LTR positions corresponding to the location of the Moloney LVc motif. The LVc motifs in the core region near theApaI site contain the only sequence mismatches in this region besides theSV40 core motifs. The poor two of five match for Akv suggests that LVc does not bind to this Akv sequence. Thiesen and colleagues (36) have shown that nucleotide differences in the Moloney and Friend MLV LTR LVc regions contribute to tissue-specific differences in transcrip-tional activity of Moloney and Friend LTRs. Moloney and Friend core motif sequences are identical. It is
possible,
then, that sequence differences in either the core or LVc motifs contribute to the difference between MCF13 and Akv LTR function or, alternately, that both motifs do so to-gether.Our deletion mutant analysis indicated possible repressor activity in L691 T cells. The00A deletion mutant was more active than the intact Akv LTR, A2. Furthermore, 00A
activity was twice as great as AOA activity and, likewise, 00Mactivity was twice as great as the activity of MOM (Fig. 4A). These results suggest that each LTR has a repressor active in the 5' region in L691 cells. In view of our recom-binant enhancer results which showed that 5' regions in L691 cells could increase, decrease, or make no change in en-hancer activity, this suppression of activity may depend on interactions with other enhancer regions. Whether the re-pression we have observed has relevance for viral replica-tion or pathogenicity we do not yet know.
In comparing this work with our previously published work showing T-cell-specific activity of the MCF13
en-hancer, we observed several important differences. Our previous report used stable gene expression assaysbased on the G418 resistance
gene,
Neo'r, to show that the MCF13enhancer was 5- to 10-fold more active in T cells than the
Akvenhancer;infibroblasts, thetwo LTRshad nearlyequal activity. In this report, using transient expression CAT
assays,wehavefound asixfold difference between Akvand MCF13 enhancer strength, but this difference is distributed between the two cell lines we tested. In the T-cell line, MCF13 is2.4 timesas active as Akv, and in NIH 3T3 cells, Akv is approximately 2.7 times more activethan MCF13. It is possible that the difference between our stable and tran-sient assay resultsis duetodifferentdesignsofourNeo' and
CAT plasmid constructions. However, several laboratories have noted that stable and transient gene expression assays are not always comparable in results and that widely used reporter genes, such as tk, CAT, and Neor, apparently can interact with promoter sequences so that assays with dif-ferent reporter genes sometimes give different results (10, 18, 27). Nevertheless, our past and present data have dem-onstrated that the MCF13 LTR is more active than Akv in T-cell lines.
Cooperation of LTR regions. The deletion mutant data (Fig. 4) showed that the 5'LTRregions in L691 cells and the 3' regions in NIH 3T3 cells made no substantial
contribu-tions to enhancer activity. From this data, it could be predicted that exchanging5' regions in recombinant
enhanc-ers would not influence enhancer activity in L691 cells. Similarly, 3' region exchanges should have little effect in
NIH 3T3 cells. Unexpectedly, we found instead that each
Akv or MCF13 region was capable of stimulating or
sup-pressingenhancer activity orof making no significant
differ-ence upon exchange. Regions which the deletion mutant data indicated were functionally almost silent could in some recombinations substantially affect enhancer activity. We suggest that in the wild-type LTRs each region is relatively activeorinactivein partbecauseofthe influence oftheother regions. The three regions seem to act in concert to control enhanceractivity, asenhancer modules within them interact cooperatively or antagonistically to raise or lower enhancer
activity. This would mean that the magnitude of LTR enhanceractivity and possibly the tissue-specific expression
attributable to any one module would not necessarily
indi-cate its functions in combination with other modules from
either its cognate or other enhancers.
Our conclusions are supported by the experiments of Rochford et al. (30), demonstrating that recombining en-hancer sequences can have unpredictable effects on viral function. By deleting the B domain of the polyomavirus enhancer from the polyomavirus genome and recombining
the remaining A domain with the lymphotrophic Moloney
virus enhancer, they obtained polyomavirus expression in
the pancreas, an organ in which neither parental virus is expressed. An example such as this together with our own data suggests that interaction among LTR enhancer
mod-ules, rather than solely a sequence change within a binding
motif, may be responsible for the MCF13 LTR contribution to lymphomagenesis. The unexpected activities of the
recombinant enhancers in CATassays suggest that some of the enhancers might have interestingpathological properties in retroviruses.
In summary, our experiments are the beginning of a
definition ofthefunctionalproperties ofsequence motifs and
modules withina pathogenicand anonpathogenic LTR. Our deletion mutant analysis hasprovided evidenceoffunctional
divisions within LTR enhancers. The unexpected
interac-tions within the recombinant enhancers suggestthat further studies of how enhancer modules work together will be
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important for betterunderstanding of how enhancers control transcription.
ACKNOWLEDGMENTS
We thank Kurt Diem and Lisa Garbrick for expert technical
assistance. pCH110 was a kind gift from Frank Lee and Gordon
Ringold. We thank Richard Palmiter and Ron Reeder for critical reading of the manuscript.
T.H. waspartially supported by a Public Health Service
Molec-ular and Cellular Biology Predoctoral Training Grant from the National Institutes of Health. This work was supported by Public
Health ServicegrantCA44166from the National Institutesof Health
to F.K.Y. and by a Department of Energy grant (DE-FG 06086 ER60409). Duringasubstantialpartoftheperiod that this workwas
performed. F.K.Y. was a Scholar of the Leukemia Society of
America.
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