gcr1-dependent transcriptional activation of yeast retrotransposon ty2-917

. 13: 917–930 (1997)

GCR1-Dependent Transcriptional Activation of YeastRetrotransposon Ty2-917

SEZAI TU}RKEL1*, XIAO-BEI LIAO2 AND PHILIP J. FARABAUGH3

1Department of Biology, Faculty of Arts & Sciences, Abant izzet Baysal University, 14280 Bolu, Turkey2Laboratory of Immunopathology, NIAID, Bldg 7, Room 301, Bethesda, MD 20892-0705, U.S.A.3Department of Biological Sciences and Program in Molecular and Cellular Biology, University of Maryland,Baltimore, MD 21228, U.S.A.

Received 2 October 1996; accepted 15 February 1997

Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstreamand downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstreamenhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on theGCR1 product, a transcription factor which also regulates the genes encoding yeast glycolytic enzymes. EliminatingGCR1 causes a 100-fold decrease in transcription of Ty2-917. Activation by the isolated Ty2-917 UAS also stronglydepends on GCR1. Unexpectedly, GCR1-dependent activation by the Ty2-917 enhancer is strongly position-dependent. Activation by the enhancer in its normal position within the transcription unit depended strongly onGCR1, but eliminating GCR1 reduced activation only three-fold when the enhancer was moved upstream of thetranscribed region. Gel mobility shift and DNaseI protection assays indicated that GCR1 binds specifically tomultiple sites within the Ty2-917 UAS and enhancer regions. ? 1997 by John Wiley & Sons, Ltd.

Yeast 13: 917–930, 1997.No. of Figures: 5. No of Tables: 3. No. of References: 61.

— yeast retrotransposon; Gcr1; transcription

INTRODUCTION

Ty elements are a dispersed, repetitive gene familyof the yeast Saccharomyces cerevisiae (Cameron etal., 1979) with structural and functional similari-ties to vertebrate retroviruses (Boeke et al., 1985;Garfinkel et al., 1985). Five families of Ty elementshave been characterized in yeast (reviewed inFarabaugh, 1995; Sandmeyer, 1992). Transcrip-tional control of Ty elements is complex, with thebest-studied examples being the closely related Ty1and Ty2 elements (reviewed in Garfinkel, 1992)and the distantly related Ty3 element (Bilanchoneet al., 1993). Transcription of Ty1 and Ty2 ele-ments is controlled by multiple sites both upstreamand downstream of the transcriptional initiationsite. The 5* non-transcribed region of Ty1 and Ty2elements includes an upstream activation site(UAS) and TATA-site (Liao et al., 1987). Unlike

UASs of other yeast genes, this UAS does notautonomously induce transcription at high levels(Fulton et al., 1988; Liao et al., 1987). Instead itstimulates transcription dependent on activatingsequences located within the transcribed portion ofthe element. Both Ty1 (Errede et al., 1984, 1985,1987; Fulton et al., 1988; Yu and Elder, 1989) andTy2 elements (Farabaugh et al., 1989; Liao et al.,1987) include downstream transcriptional acti-vation regions which extend over several hundrednucleotides starting immediately downstream ofthe transcription initiation site. Ty2 elementsinclude a second nearly 200 bp region which nega-tively regulates transcription (Farabaugh et al.,1989, 1993; Turkel and Farabaugh, 1993). Tran-scriptional control of Ty3 more closely resemblesnormal yeast genes in that the control sites, bothfor activation and repression, reside in the 5*non-transcribed region (Bilanchone et al., 1993).The size of the Ty1 and Ty2 transcriptional

control regions imply that they bind multiple*Correspondence to: Sezai Turkel.Contract grant sponsor: US Public Health Service

CCC 0749–503X/97/100917–14 $17.50? 1997 by John Wiley & Sons, Ltd.

transcription factors. Genetic and biochemicalexperiments have identified several gene-specificregulators which recognize Ty1 or Ty2, includingSTE12 (Company et al., 1988; Errede andAmmerer, 1989), MATa1·MATá2 (Errede et al.,1985), RAP1 (Gray and Fassler, 1993), MCM1(Errede, 1993; Gray and Fassler, 1993) andTYE5/GCR1 (Ciriacy et al., 1991; Lohning et al.,1993). Transcriptional activation probably de-pends on cooperative interactions among thesemany DNA-bound factors. The involvement ofmany activation proteins also suggests that acti-vation may be redundant. Consistent with thisidea, the removal of the binding site for any oneof these proteins usually has little effect on ex-pression of the element. The lone exception ismutation of the STE12 site which causes a five-fold decrease in activation (Company et al.,1988). Probably because of the complexity of thetranscriptional control signals, many trans-actingmutations affecting Ty transcription target ele-ments of the general transcriptional machinery,e.g. the TATA-binding factor (Eisenmann et al.,1989), histones (Clark-Adams et al., 1988), orfactors which are proposed to remodel chromatinto regulate transcription (reviewed in Carlsonand Laurent, 1994; Peterson and Tamkun, 1995;Winston and Carlson, 1992).Previously we have reported functional analysis

of transcriptional regulatory regions of Ty2-917(Farabaugh et al., 1993). In the present study, weanalyse the effects of GCR1 (TYE5), showing thatGCR1 binds to at least two sites within the Ty2-917 enhancer region and one site in the Ty2-917 5*non-transcribed region. Furthermore, GCR1 isessential for Ty2-917 transcription since loss ofGCR1 causes a 100-fold reduction in Ty2-917transcription. Unexpectedly, when the GCR1-dependent enhancer region is placed outside thetranscription unit, the normal location for tran-scriptional activation sites, it becomes largelyGCR1-independent. The positional specificityof the GCR1 effect suggests a model in whichGCR1 plays a partly structural role, counteractingthe disruptive effect of transcription through theTy2-917 enhancer.

MATERIALS AND METHODS

Strains and growth conditionsThe S. cerevisiae strains used in this study were

the congenic pair of strains S150-2B (MATa leu2-3

leu2-112 Ähis3 trp1-289 ura3-52) and HBY4(MATa Ägcr1::HIS3 leu2-3 leu2-112 his3 trp1-289ura3-52; Scott et al., 1990). Yeast strains weretransformed with 2ì-based plasmids carrying theURA3+ gene as a selectable marker, as describedby Ito et al. (1983). Transformants were grown inliquid YNB media containing 2% glycerol, 2%lactate and supplemented with 20 mg of histidineand tryptophan, and 30 mg of leucine per litre, asrequired. â-Galactosidase assays were done in trip-licate, as described previously (Farabaugh et al.,1989). Standard error in these assays was lessthan 10%.

Plasmids and oligonucleotidesAll plasmids used in this study are listed in Table

1. To construct the plasmid pST1-Enh, the Ty2-917 enhancer region extending from 240 to 559 bpwas isolated from YEP917-559 (Farabaugh et al.,1989), which contains the entire upstream untran-scribed region of Ty2-917, as a 319 bp XhoI/BamHI fragment, and ligated into XhoI/BamHIsite of the previously described expression vectorpST1 (Turkel and Farabaugh, 1993). In pST1 theactivation sites in the HIS4 promoter have beenreplaced by a polylinker which includes a uniqueXhoI site. A 36 nucleotide long Ty2-UAS oligo-nucleotide, corresponding to positions 95 to131 bp of the element, was synthesized with a XhoIhalf-site at the 5* end, and a SalI half-site at the 3*end; immediately interior to the SalI half-site is acomplete SalI site. Inserting the oligonucleotideinto the unique XhoI site of pST1 generates uniqueXhoI and SalI sites flanking the oligonucleotide.The single UAS oligonucleotide was reiterated toconstruct pST1-4U using the XhoI and SalI sites asdescribed (Turkel and Farabaugh, 1993). Con-struction of YEP917-1072 and YEP917-1072-NUwere described previously (Farabaugh et al., 1989).YEP917-1072 contains the first 1072 bp of Ty2-917fused to the Escherichia coli lacZ gene in a 2ì-URA3-based vector. YEP917-1072-NU containsthe region of Ty2-917 extending from position 240to 1072, and therefore entirely lacks the upstreamnon-transcribed portion of the element, includingthe UAS. The structure of each constructed plas-mid was confirmed by restriction mapping andDNA sequencing. We have shown that these plas-mids do not vary in their copy number in varioustransformants (Farabaugh et al., 1989).pRS416-UAS and pRS416-Enh435 were con-

structed to prepare gel mobility shift and DNaseI

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. 13: 917–930 (1997) ? 1997 by John Wiley & Sons, Ltd.

Table 1. List of plasmids.

Plasmid Description Source

YEP917-555 Ty2-917::lacZ fusion at position 555 of Ty2-917, includes entire enhancer (2ì-URA3) Farabaugh et al. (1993)YEP917-754 Ty2-917::lacZ fusion at position 754, includes enhancer and downstream repression

region (2ì-URA3) Farabaugh et al. (1993)YEP917-1072 Ty2-917::lacZ fusion at position 1072, includes enhancer, downstream repression region

and further downstream region up to position 1072 (2ì-URA3) Farabaugh et al. (1989)YEP917-1072-NU Ty2-917::lacZ fusion at position 1072. First 240 bp of YEP917-1072 deleted and

replaced with UAS-less HIS4 TATA box region. It includes enhancer, downstreamrepression region and further downstream region up to position 1072 (2ì-URA3) Farabaugh et al. (1989)

YEP912-613 Ty1-912::lacZ fusion at position 613 of Ty1-912; includes entire enhancer (2ì-URA3) P. Farabaugh (unpublished results)pST1-Enh HIS4::lacZ fusions to codon 32 of HIS4. The HIS4 transcriptional activators are

replaced by a polylinker into which the Ty2-917 enhancer (240–559) was inserted(2ì-URA3) This study

pST1-1U, 4U As pST1-Enh, except into the polylinker one or four UAS oligonucleotides wereinserted into the polylinker (2ì-URA3) This study

pRS416-UAS Non-transcribed portion of Ty2-917 (1–240) inserted into polylinker of pRS416 (54)(CEN-ARS-URA3) This study

pRS416-Enh435 Ty2-917 enhancer (240–435) inserted into polylinker of pRS416 (CEN-ARS-URA3) This studypFN8X-n HIS4::lacZ fusion (2ì-URA3) Nagawa and Fink (1985)pMBP-GCR(690-844) malE::GRC1 fusion; for purification of MalE-GCR1 fusion protein Huie et al. (1992)

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protection probes of Ty2-917 UAS and enhancerregion. A HindIII/XhoI fragment of YEP917-559was isolated and ligated into XhoI/HindIII-cleavedpRS416 (Sikorski and Hieter, 1989) to givepRS416-UAS. The Ty2-917 enhancer region, ex-tending from 240 to 435 bp, was amplified byPCR. One primer used introduced a unique BglIIsite immediately downstream of position 435. ThePCR product was digested with XhoI/BglII, andthe purified 195 bp fragment was ligated into theXhoI/BamHI site of pRS416 to obtain plasmidpRS416-Enh435.

DNA mobility shift assayspMBP-GCR1(690-844) was used to prepare

MalE-GCR1 fusion protein as described pre-viously (Huie et al., 1992). The MalE-GCR1protein was expressed in E. coli using a vectorwhich expresses the fusion as about 10% of totalcell protein, and purified by amylose affinitychromatography, a procedure which yields over90% pure protein (Kellermann and Ferenci, 1982).To prepare a radiolabelled Ty2-917 fragment, the277 bp HindIII/XhoI fragment of pRS416-UAS,containing the entire untranscribed region of Ty2-917, was purified and labelled by filling in the3* recessed ends of the fragment with Klenowfragment of DNA polymerase I as described(Sambrook et al., 1989). A labelled enhancer frag-ment was prepared by similar labelling of a 195 bpXhoI/XbaI fragment of pRS416-Enh435.Gel mobility shift reactions were carried out in a

20 ìl reaction which contains 20 000 cpm radio-labelled DNA, 3 ìg polydI-polydC, 0·5 ìg BSA,and 100 ng denatured herring sperm DNA inreaction buffer A (12 m-HEPES–NaOH pH 7·5,60 m-KCl, 5 m-MgCl2, 4 m-Tris–HCl pH 7·4,0·6 m-EDTA pH 8, 0·6 m-DTT, 0·5 m-CaCl2,and 10% (vol/vol) glycerol). The MalE-GCR1 fu-sion protein (10–500 ng) was added as indicated inthe Figure legends. Binding reactions were incu-bated at 25)C for 20 min, then immediately loadedon a 4% low ionic strength polyacrylamide gel(29% acrylamide, 1% bisacrylamide) and run inlow ionic strength buffer at 4)C for 4–5 h (Ausubelet al., 1991). The gels were dried under vacuumand exposed to Kodak XAR-5 film with anintensifying screen for 6–10 h at "80)C.To calculate the approximate equilibrium dis-

sociation constant (Kd) of the MalE-GCR1 foreach of its binding sites within the Ty2-917 ele-ment, we assumed that the protein was essentially

pure. The protein used was actually at least 90%pure based on visualizing preparations separatedby SDS–polyacrylamide gel electrophoresis (datanot shown), therefore the calculated Kd valueshave an error based on this assumption of no morethan 10%.

DNaseI protection assaysTy2-917 UAS and enhancer region probes end-

labelled at either end were prepared by digestingpRS416-UAS with either XhoI or HindIII. Linear-ized plasmids were end-labelled by end filling withKlenow fragment using 50 ìCi of [á-32P]dATP anddCTP, then digested either with HindIII or withXhoI to obtain each singly-labelled fragment.Singly end-labelled Ty2-917 enhancer probes wereprepared similarly by end-labelling unique XhoIand XbaI sites immediately adjacent to theenhancer-fragment of pRS416-Enh435.Binding reactions for DNaseI protection assay

were performed essentially as for gel mobility shiftassays, except 1–2 ng (50 000 cpm) of end-labelledTy2-917 UAS or enhancer probes were used, andpoly dI:poly dC was omitted. DNaseI treatmentof samples was carried out with a commercial‘Core Footprinting’ kit (Promega) according tothe manufacturer’s specifications. Single-strandednicks were introduced with 0·2 unit DNaseI perreaction for 1–1·5 min. DNaseI-treated sampleswere analysed in a DNA sequencing gel (5·7%acrylamide, 0·3% bisacrylamide, 50% urea).

RESULTS

Transcription of Ty1 and Ty2 is GCR1-dependentTy elements can insert upstream of other genes

and activate their transcription. Insertions into theADH2 gene, encoding the ADHII isozyme of alco-hol dehydrogenase, relieve its glucose repression,resulting in constitutive expression (Williamsonet al., 1981). Insertion of a Ty1 element into theADH2 promoter was used to isolate trans-actingmutations which interfere with Ty-mediated tran-scriptional activation (Ciriacy et al., 1991). Thesemutations defined five genes, TYE1–TYE5. Mostof these genes had been identified previously inother genetic screens. TYE2, TYE3 and TYE4 cor-respond to SWI3, SNF2 and SNF5, respectively(Ciriacy et al., 1991; Lohning et al., 1993), threegenes which have a pleiotropic effect on transcrip-tion apparently by antagonizing the repressiveeffects of chromatin (Carlson and Laurent, 1994;

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Peterson and Tamkun, 1995; Winston andCarlson, 1992). Why mutations in these genes in-terfere with Ty1 activation of ADH2 is not clear,though it may suggest that gene-specific activatorsof Ty1 must compete with repressive chromatinstructures to activate the gene. The TYE5 genecorresponds to the GCR1 gene (Lohning andCiriacy, 1994), a transcriptional activator of glyco-lytic genes (Baker, 1986; Holland et al., 1987). Thetye5 mutant effect suggests that GCR1 may be agene-specific activator of Ty1.We wanted to test this hypothesis by assaying

the effect of eliminating GCR1 activity on expres-sion of Ty1::lacZ and Ty2::lacZ fusion reporterconstructs. Previously we constructed a collectionof lacZ fusions to the Ty2-917 (Farabaugh et al.,1989, 1993) and Ty1-912 elements (unpublisheddata). These constructs contain various portions ofthe downstream transcriptional activation regionsof the two elements. In the plasmid YEP917-555,the Ty2-917 element is translationally fused to lacZat position 555 of the element. The plasmid in-cludes the downstream transcriptional activationregion of the element, responsible for maximaltranscriptional activation (Farabaugh et al., 1993).A second plasmid, YEP917-754, includes the first754 bp of the element, including a further down-stream transcriptional repression site which re-duces Ty2-917 transcription four-fold (Farabaughet al., 1993). A third plasmid, YEP912-613, has thefirst 613 bp of Ty1-912, including the Ty1-912 en-hancer, fused to lacZ, and also stimulates maximaltranscription (data not shown).The three lacZ reporter constructs, YEP917-555,

YEP917-754 and YEP912-613, and an irrelevantcontrol, pFN8X-n, a HIS4::lacZ fusion plasmid(Nagawa and Fink, 1985) whose expression is ex-pected to be independent of GCR1, were trans-formed into a GCR1+ and a congenic Ägcr1 yeaststrain. As shown in Table 2, the transformants ofeach plasmid express high levels of â-galactosidasein the wild-type strain. However, when the plas-mids were introduced into a congenic strain inwhich the GCR1 gene had been deleted (Ägcr1),each of the Ty reporter plasmids expressed signifi-cantly less â-galactosidase activity. Elimination ofGCR1 reduced YEP917-555 expression 92-fold,and YEP917-754 expression decreased 30-fold.By contrast, the effect of Ägcr1 on YEP912-613expression was much weaker, decreasing it by only11-fold. At the same time the deletion had littleeffect on expression of theHIS4 reporter construct,as expected. Because glycolytic gene transcription

is activated by GCR1, Ägcr1 strains do not growwell on glucose as sole carbon source. Thereforefor these assays cells were grown on glyceroland lactate as carbon source. Exposing cells toglucose for short periods had no significant effecton transcription of any of the constructs (data notshown). Clearly, GCR1 has a very significant rolein activation of Ty1 and Ty2 transcription, thoughfrom these data we cannot distinguish between adirect and indirect effect.

The Ty UAS is a GCR1-dependent activatorBoth Ty1 and Ty2 elements include a UAS

located about 110 bp upstream of the transcrip-tional initiation site (Liao, 1989; Liao et al., 1987).The existence of the site was controversial sinceinsertion of an oligonucleotide encompassing thesite stimulated little transcription (Fulton et al.,1988). However, reiteration of the site revealed ahighly synergistic effect so that as few as fourcopies of the UAS strongly stimulate transcription(Liao, 1989). This type of synergism is recognizedas a common feature of transcription activationsites (Ptashne, 1988).Since transcription of a Ty1 and a Ty2 element

strongly depended on GCR1 we wished to testwhether the UAS is a GCR1-dependent activator.In order to test GCR1 dependence we used con-structs in which the Ty UAS activates transcriptionof a heterologous gene, HIS4. The constructs weremade by inserting one or more copies of the UASinto a polylinker upstream of a HIS4 promoterfrom which all of the normal transcriptional acti-vator binding sites (GCN4, BAS1, BAS2 andRAP1) had been deleted (Turkel and Farabaugh,1993). One copy of the Ty UAS (pST1-1U) only

Table 2. Transcription of Ty2-917 is highly dependenton GCR1.

Plasmids

â-Galactosidase activities (U)a

GCR1b Ägcr1b

YEP912-613 9400 870YEP917-555 3450 37YEP917-754 750 25pFN8X-n 1600 1000

aUnits are expressed in micromoles of ONPG cleaved perminute per mg of protein.bGCR1 and Ägcr1 indicate S150-2B and HBY4 strain trans-formants respectively.

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? 1997 by John Wiley & Sons, Ltd. . 13: 917–930 (1997)

slightly increases transcription of a HIS4-lacZreporter fusion gene, while four copies (pST1-4U)stimulates expression 126-fold (see column 1 ofTable 3, lines 1–3). When these constructs wereintroduced into a Ägcr1 strain, expression wasreduced to near background levels, a four-foldreduction for pST1-1U and a 27-fold decrease forpST1-4U. The Ty UAS, by this criterion, appearsto be strongly dependent on GCR1 to stimulatetranscription.

The Ty2-917 enhancer shows an unexpected loss ofGCR1 dependence when moved outside thetranscription unitIn the intact element, most of the transcriptional

activation potential is located downstream ofthe transcription initiation point (reviewed inGarfinkel, 1992). For Ty2-917, deletion of theUAS only causes a ten-fold decrease in transcrip-tion (Liao et al., 1987), but complete deletionof the downstream enhancer causes a muchmore severe 1000-fold decrease in transcription(Farabaugh et al., 1989, 1993). Therefore, if tran-scription of the Ty2-917 element is strongly depen-dent on GCR1, as shown in Table 2, the enhanceris likely also to be GCR1-dependent. The Ty2-917enhancer can stimulate transcription when insertedalone into the same truncated HIS4 promoter usedto assay UAS activation to generate pST1-Enh (asshown in Table 3). The Ty2-917 enhancer stimu-lates expression of the HIS4-lacZ reporter 367-fold. However, whereas loss of GCR1 reducedtranscription of a Ty2-917-lacZ fusion up to 93-fold (Table 2, line 2), the expression of pST1-Enhwas reduced only 2·7-fold in the Ägcr1 strain

(Table 3, line 4). This reduction is enough toconfirm that GCR1 has a role in transcriptionalactivation by the Ty2-917 enhancer, since theGCR1-independent HIS4 control showed littleeffect of the loss of GCR1 (Table 2, line 4).However, the reduced dependence of GCR1 issurprising. In the Ägcr1 strain the enhancerinduces much higher expression when presentupstream of the HIS4 promoter than expected.Note that in this case the enhancer alone stimu-lates 11-times more expression (410 u; Table 3,line 4) than the combination of the UAS and en-hancer stimulates when the enhancer is withinthe transcribed region (37 u; Table 2, line 2).The difference in GCR1-dependence of pST1-

Enh and the YEP917-plasmids could be causedeither by the position of the enhancer, or by thefact that in the YEP917 plasmids the UAS andenhancer interact synergistically to stimulate tran-scription. To determine which effect causes thedifference in GCR1-dependence we measured theeffect of GCR1 on a YEP917 construct with andwithout the UAS. As shown in Table 3 (line 5),transcription of the plasmid YEP917-1072 stronglydepends on GCR1; transcription is reduced 12-foldin the Ägcr1 background. The lower level ofexpression of YEP917-1072 is caused by thepresence of downstream repression sites in Ty2-917 (Farabaugh et al., 1989, 1993). Eliminating theUAS in this construct, YEP917-1072-NU, causedan almost 2·5-fold decrease in transcription in theGCR1 strain (Table 3, line 5). This level of tran-scription was still strongly dependent on GCR1since in the Ägcr1 strain expression was reducedsix-fold, to almost the same level of transcriptionthat we observed in all YEP917 constructs trans-formed into the Ägcr1 strain. Thus, deleting theUAS from YEP917-1072 did not eliminate GCR1-dependence of enhancer-driven transcription. Thisresult indicates that the response of the down-stream enhancer to GCR1 depends on its positionwithin the transcribed region, and not on aninteraction between the UAS and enhancer.

GCR1 binds to multiple sequences within the Ty2enhancer element and the UASThe GCR1 dependence of the Ty1 and Ty2

enhancers and the Ty UAS could result fromGCR1 binding directly and activating transcrip-tion of the gene. Alternatively, GCR1 mightactivate the expression of some other gene(s) nec-essary for Ty expression, and indirectly regulate its

Table 3. The Ty2-917 UAS is a GCR1-dependentactivator.

Plasmids

â-Galactosidase activities (U)a

GCR1b Ägcr1b

pST1 3 5pST1-1UAS 35 8pST1-4UAS 380 14pST1-Enh 1100 410YEP917-1072 245 20YEP917-1072-NU 100 17pFN8X-n 1600 1000

aUnits are expressed as in Table 2, footnote a.bYeast strains are as defined in Table 2, footnote b.

922 . .


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transcription. GCR1 activates the transcription ofcontrolled genes by binding to multiple CTTCCsequences (Baker, 1991). Inspection of the se-quence of Ty2-917 reveals three consensus bindingsites with the Ty2-917 UAS and enhancer, one inthe UAS at position 115 and two in the enhancerat positions 363 and 401.To determine if GCR1 actually binds to these

sites we first performed gel mobility shift assaysusing a MalE-GCR1 fusion protein (Huie et al.,1992). This protein consists of the DNA bindingdomain of GCR1, the 155 C-terminal residues ofthe protein, from 690 to 844, fused to the maltose-binding protein of E. coli, malE. The advantage ofthis protein is that it binds to DNA exactly asGCR1 does, but it can be purified essentially tohomogeneity on an amylose affinity column (Huieet al., 1992).Figure 1 presents the result of binding increasing

amounts of affinity-purified MalE-GCR1 fusion tothe Ty2-917 UAS region (as described in Materialsand Methods). With increasing concentrations ofthe MalE-GCR1 fusion, we find evidence for threeGCR1 complexes with the UAS. The first complex

(labelled CI in Figure 1) shows half-maximal binding at about 38 n. The equilibrium dissociatioconstant (Kd) for a DNA binding proteinequivalent to the reciprocal of the concentration awhich half of the DNA binds in the reactionprovided the concentration of protein is mucgreater than the concentration of DNA (Care1991; Riggs et al., 1968). In our reactions the DNconcentration is less than 10"13 . Thereforhalf-maximal binding of complex CI at 38 nindicates an apparent Kd of 2·6#107 "1. Increasing the amount of MalE-GCR1 results in thformation of two further complexes, CII and CIIwith apparent Kd values of about 3·2#106 "

and 8·3#105 "1.A similar analysis was performed using a frag

ment of the enhancer including the region from thinitiation site to position 435. The fully functionenhancer fragment includes sequences to positio555 (Farabaugh et al., 1993); however, we founno MalE-GCR1 binding sites distal to position 43(data not shown).Figure 2 shows that in increasing amounts, th

MalE-GCR1 fusion binds to form four complexewith this shortened enhancer fragment. The appaent Kd of the first complex (labelled CI in Figure 2is between 5·2#107 "1 and 1·2#107 "

approximately equivalent to the first UAS complex. The other complexes form with decreasinKd values, CII between 1·2#107 "1 an6·5#106 "1, CIII about 6·5#106 "1, and CIabout 3·2#106 "1.Determining exact Kd values from this analys

is not possible, partly because of the lack oa sufficient number of data points, and partbecause the complexes form in overlapping concentration intervals, making it difficult to detemine the concentration showing half-maximbinding. However, the data can be used to estimathe affinity of GCR1 for the several sites presenindicating a range of affinities from less than 50 nto over 1 ì. We presumed that the range oaffinities reflects the deviation among the seversites from a consensus GCR1 binding site (Huand Baker, 1996; Huie et al., 1992). Since inspetion of the sequence only identified three consensu5*-CTTCC-3* binding sites, it was likely that abouhalf of the seven complexes reflect binding tnon-consensus binding sites.DNaseI protection assays were performed t

identify the sites bound by the MalE-GCR1 fusioprotein. Figure 3 presents the result of performinsuch an experiment on a fragment encompassin

Figure 1. GCR1 forms multiple complexes with the non-transcribed region of Ty2-917. DNA mobility shift assay wasdone with bacterially expressed, purified MalE-GCR1 fusionprotein using Ty2 UAS region as a probe. Lanes 1 through 7represent reactions with increasing amounts of MalE-GCR1 (0,25, 50, 100, 400, 800 ng and 1·5 ìg respectively). Specificprotein–DNA complexes formed are labelled CI, CII and CIII.F indicates the position of free probe.

? 1997 by John Wiley & Sons, Ltd. . 13: 917–930 (199

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the entire 5* non-transcribed region of Ty2-917; thefastest migrating band shown corresponds to pos-ition 168, the second base in the TATA box. Thefragment that was end-labelled on the non-codingstrand of the element also gave similar DNaseIprotection results (data not shown). The dataclearly show that there is one major binding sitefor GCR1 which maps to within the geneticallydefined UAS, roughly mapped in the interval from100 to 129 (Liao et al., 1987). Clear protection isevident at the lowest concentration of GCR1 used,78 n. The protected region on the coding strandextends from 106 to 125; on the non-coding strandit extends from position 105 to 129 (data notshown).These results are summarized in Figure 4, which

shows that the protected region includes theconsensus GCR1 binding site, CTTCC. GCR1protects most if not all of the Ty UAS fromDNaseI cleavage (from 100 to 129). There were noother binding sites apparent in the non-transcribedportion of the element, though at the highest

concentration the gel mobility shift analysis woulhave predicted at least one more complex.DNaseI protection analysis identified tw

major, and one minor binding site for GCRwithin the transcribed portion of the elemenFigure 5 presents the results using a fragment othe non-coding strand including the region fromposition 240 to 435, though the fastest migratinband in the Figure corresponds to position 41The results are again summarized in Figure 4. W

Figure 2. GCR1 binds to the Ty2 enhancer element and formsfour distinct protein–DNA complexes. Results of mobility shiftexperiments with increasing amounts of MalE-GCR1 areshown in lanes 1 through 6 (0, 10, 25, 100, 200 and 400 ngprotein respectively). CI, CII, CIII and CIV identify theprotein–DNA complexes formed between the MalE::GCR1and enhancer region of Ty2-917.

Figure 3. DNaseI protection of Ty2-917 non-transcribed rgion by MalE-GCR1. Results of DNaseI protection analysusing the top strand of the Ty2-917 5* non-transcribed regioDNaseI protection was performed with increasing amountsMalE-GCR1. Lanes 1 to 4 correspond to reactions with 0 n250 ng, 500 ng and 1 ìg of protein. G and Y are MaxamGilbert DNA sequencing reactions; G represents cleavageguanines, and Y cleavage at cytosine and thymine. The numbers adjacent to the G lane indicate positions within Ty2-91

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found no evidence for a binding site downstreamof position 416 (data not shown). The Figureshows two obvious protected regions, one extend-ing from 289 to 305, and one from 395 to 413.Each region is clearly protected in the presenceof as little as 125 n-GCR1. The second ofthese protected sites overlaps a consensus GCR1binding site (CTTCC) while the first includes anon-consensus site (ATTCC). Protection of thesesites is clear on the coding strand as well, from 289to 303, and from 395 to 411 (data not shown), assummarized in Figure 4. Interestingly, the secondconsensus GCR1 binding site, from 363 to 367,shows no apparent protection, even at the highestconcentration of GCR1 used (250 n).The fact that DNaseI protection analysis

showed only two strong binding sites contrastswith gel mobility shift experiments which suggestthat at least three molecules of GCR1 would haveassociated with the region at the highest concen-tration of GCR1. The reason for the discrepancy isnot known, though it may be that insufficientconcentrations of GCR1 to protect this weaker sitewere present in the experiment. The results ofthe mobility shift experiment suggest otherwise;however, the apparent Kd derived from the twomethods could be quite different.

DISCUSSION

The data presented here identify the GCR1 proteinas an important activator of Ty transcription.GCR1 activates transcription of both Ty1 and Ty2elements, but its role in Ty2 transcription appears

more crucial. Both elements include an identicUAS, about 110 bp upstream of the transcriptioinitiation site. Though this site does not stimulasignificant transcription by itself, multiple tandemcopies of the site strongly stimulate transcriptionIn addition, the UAS synergizes with the downstream enhancer region (Liao et al., 1987One model for synergism suggests that multipactivators must contact the basal transcriptiomachinery simultaneously in the process of initiation (Choy and Green, 1993). The necessity foreiteration of the Ty UAS may indicate that thsite binds few transcriptional activators, perhaponly one. Synergism between the UAS and thdownstream enhancer may mean that factobound downstream cooperate with UAS-bounfactors in stimulating transcription. A seconmodel proposes that synergism results from cooperativity among multiple activators binding icompetition with repressive chromatin (Chang anGralla, 1994). Under this model, reiterating the TUAS, or combining the UAS with the downstreamenhancer, allows the UAS binding factors tcompete effectively against repressive chromatin.Both the strong enhancers and the weaker UAS

depend on GCR1 for their activity. In fact, loss oGCR1 caused an almost 100-fold decrease ienhancer-dependent transcription of Ty2-917,much stronger effect than has been reported foany other activator of Ty1 and Ty2 transcriptionBut the role GCR1 plays in transcription actvation is surprisingly sensitive to the placement oits binding site within the transcribed portioof the element. The enhancer becomes largely

Figure 4. Location of GCR1 binding sites with the transcription regulatory regions ofTy2-917. DNaseI-protected regions in both strands are indicated by solid lines. Partiallyprotected regions are indicated by dotted lines. Consensus (CTTCC) and near-consensus(ATTCC) GCR1 binding site sequences are shown in bold. The first 420 bp of the elementare represented in the Figure.


92 Ty2-917

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GCR1-independent activator when it is placedoutside the transcription unit, in the 5* non-transcribed position normally occupied by yeastactivation sites. In this position the enhancer con-tinues to strongly activate transcription, yet loss ofGCR1 causes only a less than three-fold decreasein transcription. What is the origin of thisunexpected loss of GCR1 dependence?It has long been recognized that moving a UAS

from its normal position in the 5*-non-transcribedregion to within the transcription unit eliminatesits ability to activate transcription (Guarente and

Hoar, 1984; Struhl, 1984). The simplest interpreation of this transcriptional interference is that thelongating RNA polymerase passing throughtranscriptional control region disrupts its ability tstimulate transcription by physically displacinactivator proteins from the DNA. Transcriptionknown to displace nucleosomes during elongatio(Kornberg and Lorch, 1991) and transcriptiothrough a centromere can interfere with its funtioning (Snyder et al., 1988). It is therefore perhapsurprising that the Ty1 and Ty2 enhancers funtion at all. Clearly they must have the ability tovercome the effect of passage of elongatinpolymerases. This unusual ability may depend othe presence of GCR1 protein.GCR1 binding sites form part of what appea

to be a complex transcriptional control region iboth Ty1 and Ty2 elements. There is strong evdence for only one transcriptional control site ithe 5* non-transcribed region, the UAS (Liao et al1987); however, in both elements, the first severhundred nucleotides of the transcribed regiostrongly stimulate transcription. The UAS noappears to function as a GCR1-dependent actvator. We show here that GCR1 binds to a consensus recognition sequence located within thgenetically defined UAS, and that activation by thUAS depends on the presence of the GCR1 protein. We had previously found that it synergizewith the downstream enhancer to promomaximal transcription (Liao et al., 1987). Here wshow that reiteration of the UAS converts it to aefficient autonomous activator. Consistent witthese data, GCR1 does not bind efficiently ta single consensus site; rather, efficient bindinrequires interaction with a second nearby proteineither a second GCR1 or another protein such athe ubiquitous transcriptional activator, RAP(Henry et al., 1994; Scott and Baker, 1993Reiteration of the UAS then probably allowefficient in vivo binding of GCR1, and thus transcriptional activation, rather than simply increaing the efficiency of transcriptional activation bDNA-bound factors.GCR1 also binds to two sites within the fir

173 bp of the transcribed region—a canonicbinding site, CTTCC, at position 401–405, anddivergent site, ATTCC, at position 298–294. Itsomewhat surprising that the divergent site bounGCR1 while a consensus site at position 367–36did not. Apparently sequence elements from ouside the core site must strongly affect affinity (Huand Baker, 1996). The relative importance o

Figure 5. DNaseI protection of the Ty2-917 enhancer regionby MalE-GCR1. DNaseI protection analysis of the codingstrand of the Ty2-917 enhancer. Two protected regions areindicated by the brackets at the right of lane 5. Lanes 1 to 5represent reactions with 0, 50, 100, 400 and 800 ng of MalE-GCR1. The numbers adjacent to lane 1 indicate positionswithin Ty2-917.


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these sites should not be inferred from these data,however, since given the propensity for GCR1 tosynergize, the affinity of GCR1 for each site in vivomay respond sensitively to the presence or absenceof adjacent bound factors. Our in vitro bindingassays may also fail to reveal possible synergisticinteractions between DNA-bound GCR1 proteinssince they involve a MalE-GCR1 fusion whichincludes only the characterized C-terminal 155amino acid DNA-binding domain lacking 690N-terminal amino acids.Preliminary data suggest that one of the three

GCR1 binding sites, the strong binding site atposition 401–405, is especially important in vivo (S.Turkel and P. J. Farabaugh, unpublished results).A deletion removing the 24 bp from position 387 to410 virtually eliminates transcriptional activationby the Ty2 enhancer. Surprisingly, deletions ofeither half of this region, Ä399–410, which includesthe GCR1 site, or Ä387–398, which does not, havea much smaller effect on activation, reducing ex-pression less than three-fold. These data suggestthat a second transcriptional activation proteinbinds immediately upstream of GCR1 on a sitewhich at least partially overlaps the deleted region.We have been unable to identify a potential bind-ing protein by comparing the sequence of the bind-ing region to known DNA recognition sequencesof yeast transcriptional activators.GCR1-regulated promoters often include bind-

ing sites for one or more of the ubiquitoustranscription factors RAP1, ABF1 and REB1.For example, activation of the PGK1 and ENO2promoters requires a combination of GCR1,RAP1 and ABF1 (Henry et al., 1994; Willettet al., 1993), and the TPI1 promoter requiresGCR1, RAP1 and REB1 (Scott and Baker,1993). GCR1 interacts synergistically with RAP1,but not with ABF1 or REB1 (Henry et al., 1994;Scott and Baker, 1993; Willett et al., 1993). Thesynergy of GCR1 and RAP1 may suggest aphysical interaction between the factors. Thoughboth factors can bind independently to the DNA(Buchman et al., 1988; Huie et al., 1992), Huieet al. have suggested that RAP1 may facilitateGCR1 binding to DNA (Huie et al., 1992). Itis also possible that they collaborate in recruit-ing the putative co-activator proteins GCR2(Uemura and Fraenkel, 1990) and GAL11/SPT13(Nishizawa et al., 1990). GCR2 has been shownto physically interact with GCR1 (Uemura andJigami, 1992), and GAL11 stimulation of PGKtranscription requires the presence of RAP1

(Stanway et al., 1994), again suggesting a physcal interaction. Recruiting the combination othese two co-activators may produce synergy bincreasing the efficiency of forming a transcription pre-initiation complex. The transcriptioncontrol regions of Ty2-917 described here do noinclude RAP1, REB1 or ABF1 binding site; nconsensus site could be identified by computeanalysis, nor could we find any evidence ofRAP1 or an ABF1 complex in vitro (S. Turkand P. J. Farabaugh, unpublished results). Iparticular, the region immediately upstream othe GCR1 site at position 401–405, which includes the putative binding site for an interactinprotein, does not include binding sites for any othese proteins.What is the molecular role of GCR1 i

activating Ty2 transcription? There are severpossibilities. First, GCR1 may form a part oftranscriptional activation complex, interactinwith the basal transcription machinery to increasthe formation of RNA polymerase II pre-initiatiocomplexes. Alternatively, GCR1 may functionegatively as an anti-repressor by excluding nucleosomes from the UAS/enhancer region, aproposed in the TDH3 gene where it may excludtwo positioned nucleosomes from the promote(Pavlovic and Horz, 1988). Finally, its effect mabe to counteract the presumed disruptive effect orepeated passage of elongating polymerasethrough the enhancer region.The unusual position-dependence of GCR1 a

tivation argues against the first model, and makethe third hypothesis the most attractive. The concept is that GCR1 forms a part of a complex otranscription activators, though it has little direrole in stimulating transcription (consistent witthe lack of effect of Ägcr1 when the enhancerpositioned upstream of the transcription unitRather, GCR1 would provide a molecular ‘scafold’ so that when RNA polymerase II readthrough the enhancer, the interactions betweeGCR1 and the other transcription factors, aideperhaps by interactions among the other factorstabilizes the three-dimensional structure of thcomplex so that it can reassociate with the DNafter passage of the polymerase. An inch-wormtype mechanism might allow the polymerasto read through such a large complex withouactually dissociating it from the DNA. RNpolymerase II would only disrupt protein–DNcontacts over a short interval, perhaps a few helicturns of the DNA. As the enzyme passed throug

92 Ty2-917


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each region of the enhancer, the DNA bindingproteins held in the GCR1-stabilized complexwould simply reassociate with the DNA, and at notime would the entire complex dissociate.The ability of Ty elements to activate transcrip-

tion is modulated by elements of the SNF/SWIcomplex (Winston and Carlson, 1992), a 2 MDaprotein complex whose role is to antagonize therepressive effect of chromatin (reviewed in Carlsonand Laurent, 1994; Peterson and Tamkun, 1995;Winston and Carlson, 1992). The SNF/SWI com-plex appears to antagonize repressive chromatinby facilitating the binding of transcription factorsto nucleosomes, and perhaps by promoting thedissociation of histones from these ternary com-plexes (Cote et al., 1994). Given this connectionbetween repressive chromatin and Ty transcrip-tion, the function of GCR1 may not simply be tostabilize an enhancer-bound activation complex,but to facilitate the exclusion of chromatin. In thismodel, the passage of RNA polymerase II throughthe enhancer may both cause dissociation of theDNA-bound activation proteins, and allow thereplacement of these factors by nucleosomes.GCR1 could disrupt binding by the nucleosomes,allowing for rapid reformation of the activatingcomplex. In this model, the small effect of GCR1upstream of the transcription initiation site mayreflect the fact that once bound, the activationcomplex stably associates with the DNA. Increas-ing the rate of formation of the complex in thiscase might have little effect on transcription outputsince the dissociation constant (Kd) for the com-plex might be much more dependent on theslow rate of dissociation (k"1) than on association(k1). Downstream, where the half-life of boundcomplex would be determined by the frequency oftranscription through the enhancer, its effect onassociation might dominate the Kd.

ACKNOWLEDGEMENTS

We thank Dr Henry Baker (University of Florida)for yeast strains and the MalE-GCR1 expressingplasmid. This work was supported by grantGM-29480 from the US Public Health Service.

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