Functional Knockout of the Corepressor CtBP by the Second Exon of Adenovirus E1A Relieves Repression of Transcription

Experimental Cell Research 268, 284–293 (2001)doi:10.1006/excr.2001.5280, available online at http://www.idealibrary.com on

Functional Knockout of the Corepressor CtBP by the Second Exonof Adenovirus E1A Relieves Repression of Transcription

Anders Sundqvist,* Edyta Bajak,* Sindhulakshmi D. Kurup,* Kerstin Sollerbrant,†and Catharina Svensson*,1

*Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden; and
†Ludwig Institute for Cancer Research, Karolinska Institute, Stockholm Branch, Box 240, SE-171 77 Stockholm, Sweden
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The C-terminal binding protein (CtBP) acts as atranscriptional corepressor upon recruitment to tran-scriptional regulators. In contrast, interaction be-tween CtBP and the adenovirus E1A protein is re-quired for efficient activation of E1A-responsivegenes, suggesting that E1A might block CtBP-medi-ated repression. Recruitment of CtBP to a promoter,either as a Gal4CtBP fusion or through an interactionwith a Gal4 fusion protein expressing the CtBP inter-acting domain (CID) of E1A, resulted in transcrip-tional repression. The second exon of E1A, containingthe CID, alleviated repression by Gal4E1ACID-re-cruited CtBP, but not Gal4CtBP-mediated repression,suggesting that E1A prevented repression by blockingpromoter recruitment of CtBP. E1ACID was also suf-ficient to derepress transcription from several co-transfected promoter constructs. Furthermore, induc-ible expression of E1ACID in established cell linesresulted in significant changes of endogenous geneexpression, possibly by sequestration of CtBP. To-gether, these data indicated that CtBP might act as awide-range regulator of transcription. Although CtBPwas shown to interact with histone deacetylases(HDACs), transcriptional repression by a Gal4CtBPfusion protein was not sensitive to inhibition ofHDACs by trichostatin A (TSA). In contrast, TSA elim-inated E1ACID derepression of E1A second exon-re-sponsive promoters. Although the reason for this dif-ference remains to be experimentally verified, it ispossible that the requirement for HDACs might differdepending on the mechanism by which CtBP becomespromoter recruited. © 2001 Academic Press

INTRODUCTION

The C-terminal binding protein (CtBP) was origi-nally identified as a protein interacting with the C-terminus of the transforming adenovirus E1A protein[1, 2]. CtBP binding to E1A requires a short amino acid

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motif, PLDLS (the CtBP-interacting domain or CID).CIDs also mediate the interaction between CtBP anddifferent transcription regulators, in particular thoseinvolved in repression of transcription. Several mem-bers of the CtBP family of proteins have been clonedfrom mammalian sources, as well as from Xenopus[3–5], supporting the notion that CtBP may function asan important corepressor of transcription in differentorganisms.

The CtBP homologue in Drosophila interacts with,t least, three short-range repressor proteins, Knirp,ruppel, and Snail [6, 7], and the long-range repressorairy [8]. These interactions are important for the

unction of the repressor proteins and consequently forormal embryonic development [6]. In mammalianells, CtBP has been shown to interact with a variety ofinc-finger-containing transcription regulators such asOG-2 [9], KLF8 [10], Ikaros [11], and ZEB [12]. Al-hough the exact functions of these factors remain un-lear, a common denominator seems to be their in-olvement in cellular development.The binding of CtBP to the second exon of E1A cor-

elates with inhibition of E1A 1 H-ras cotransforma-ion, tumorigenesis, and metastasis [1, 2]. This indi-ates that CtBP could be involved in normal cellrowth control, which is also supported by the obser-ation that CtBP interacts with members of the Poly-omb proteins [13], the ternary complex factor, Net14], and the wnt signaling mediator LEF-1/TCF [3].urthermore, the first identified CtBP-interacting pro-ein (CtIP) [15] binds, probably together with CtBP, tohe BRCT repeats of the tumor suppressor BRCA1,hereby blocking its ability to activate transcriptionrom the p21 promoter [16–18]. The CtIP–CtBP com-lex can also be recruited by the tumor suppressorrotein pRb, as well as by the pRb-related p130 pro-ein, and it is suggested that this recruitment of CtIP/tBP offers an additional mechanism for pRb/p130 toepress E2F-mediated transcription [19].In summary, available data suggest that the core-

ressor activity of CtBP requires interaction with a
ranscriptional regulator that mediates contact with

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285THE CtBP–E1A INTERACTION RELIEVES REPRESSION

DNA. In agreement with this, a Gal4CtBP proteinfusion is capable of repressing transcription from apromoter carrying Gal4 binding sites [11, 14, 19].Moreover, the E1ACID acts as a repressor domainwhen present in a Gal4E1A fusion protein [20].

In this report we have used the second exon of ade-novirus E1A as a molecular trap for CtBP to demon-strate that sequestration of CtBP resulted in transcrip-tional derepression of several unrelated promoters.Furthermore, gene array analysis demonstrated that afunctional knockout of CtBP, using cell lines express-ing the E1ACID from an inducible promoter, resultedin a significant modulation of cellular gene activity.The mechanism by which CtBP repressed transcrip-tion is still unclear. It is possible that CtBP couldparticipate in a multiprotein complex with the capacityto modify chromatin. This is supported by observationsthat CtBP interacted with histone deacetylase(HDAC)-1 [21], HDAC-2 and -3 (this paper), andHDAC-4 and -5 [22]. The functional significance ofHDAC is, however, under discussion since Gal4CtBPrepression was insensitive to the histone deacetylaseinhibitor trichostatin A (TSA) [11, 23] (and this study).In contrast, the E1A second exon-sensitive promotersused in this study were found to be TSA inducible.However, derepression by E1ACID rendered transcrip-tion relatively insensitive to TSA. Possible explana-tions for this discrepancy will be discussed.

MATERIALS AND METHODS

Plasmids. The adenovirus major late promoter (MLP) frompTripCAT [24], the adenovirus E4 promoter from pE4CAT [25], andthe proliferating cell nuclear antigen (PCNA) promoter from PCNA-CAT [26] were subcloned into the luciferase reporter constructpUHC13-3 [27]. Hsp70Luc contains a fragment spanning nucleotide2175 to 1100 of the human hsp70 promoter. In pSVG5E1BLuc, theluciferase gene is under control of the SV40 enhancer from pSVE-CAT [24] upstream of the G5E1B cassette from G5E1BCAT [25].5xmycRELuc contains five copies of the consensus E-box sequence(CCACCACGTGGTGCCTC) upstream of the HSV thymidine kinase(tk) minimal TATA promoter. G5tkLuc contains five Gal4 DNA bind-ing sites followed by the tk promoter (2105 to 151) and the lucif-erase reporter gene.

Gal4ctE1A [20] express the last 44 amino acids of the second exonof E1A. Gal4ctE1ADCID is a derivative thereof, lacking amino acids225–238 of E1A243R. Gal4CtBP expresses the full-length CtBP pro-tein. GSTCtBP has been described [15]. FL-pcDNA3CtBP expressesan amino-terminal, FLAG-tagged, full-length CtBP protein.E1Aexon2 (pc-dl1119) and E1Aexon2DCID (pc-dl1135) have beendescribed [1, 28]. GSTctE1Aexon2 has been described [20]. Plasmidsfor expression of HDAC-1 (pING14HDAC1), HDAC-2, and HDAC-3(pCMV-HDAC2 and pCMV-HDAC3) have been described [29, 30].Plasmids pTRE-E1Aexon2 and pTRE-E1Aexon2DCID were gener-ated by replacement of the luciferase reporter gene in pTRELuc(Clontech) with the sequence from the second exon of E1A. Briefly,PCR fragments, carrying an initiator ATG inserted immediatelyupstream of the first amino acid in the second exon of E1A, weregenerated from the E1A-encoding plasmids pML00512S and
pML00512D225-238 [21].
Transfections and reporter gene analysis. The U2OS, Cos7,HeLa, and A549 cell lines were maintained in DMEM GLUTAMAX-1(Gibco) supplemented with 10% FCS, 100 U/ml penicillin, and 100mg/ml streptomycin. Transfections were done using the FuGENE-6transfection system (Roche) according to the manufacturer’s instruc-tion. Where indicated, TSA was added at 300 nM 22 h before har-vesting the cells. Luciferase assays were performed using the Lucif-erase Assay System (Promega) according to the manufacturer’sinstruction.

Establishment of cell lines. U2OS cell lines expressing the induc-ible TRE-E1Aexon2 and TRE-E1Aexon2DCID genes were estab-lished using the Tet-On system (Clontech). Briefly, the pTet-Ontransactivator protein was transfected into U2OS cells. StableU2OS-Tet-On clones were isolated following Gentamycin (G418) se-lection. In the subsequent step, U2OS-Tet-On cells were transfectedwith either pTRE-E1Aexon2 or pTRE-E1Aexon2DCID. Double-sta-ble transfected clones were isolated following hygromycin selection.

Immunoprecipitations and GST pull-down analyses. Cos7 cellswere transfected with 10 mg of FL-pcDNA3CtBP and 10 mg of eitherE1Aexon2 or E1Aexon2DCID. Cells were lysed in L buffer (10 mMTris, pH 7.0, 50 mM NaCl, 30 mM sodium pyrophosphate, 25 mMNaF, 2 mM EDTA, 1% Triton X-100 supplemented with CompleteProtease Inhibitor Mix; Roche). The cell extract was precleared withprotein A or G Sepharose (Pharmacia Biotech) and immunoprecipi-tated with either anti-E1A (sc-430; Santa Cruz Biotechnology) oranti-FLAG (Sigma) antibodies. The precipitates were washed fourtimes in L buffer and separated on a 12% SDS–PAGE gel followed byWestern blotting using the sc-430 (Santa Cruz Biotechnology) anti-body.

Bacterially expressed GST, GSTctE1A, and GSTCtBP were pre-pared as described [21]. GST pull-down experiments using cell ex-tracts were essentially as follows: Doxycyclin-induced (2 mg/ml for20 h) TRE-E1Aexon2- or TRE-E1Aexon2DCID-expressing cells or[35S]methionine-labeled HeLa cells were disrupted in L buffer. Therude protein extracts were mixed with the GST fusion proteins andncubated at 14°C. The beads were washed four times in L buffer.ound proteins were separated on a 10% SDS–PAGE gel and visu-lized by immunoblotting using the polyclonal antibody against E1Asc-430; Santa Cruz Biotechnology) or by direct autoradiography.

In vitro translation of the HDAC proteins was performed using theommercially available TNT T7 or SP6 coupled reticulocyte systemPromega). [35S]Methionine-labeled proteins were incubated for 1 h

with the GST-fusion proteins in binding buffer (10 mM Tris, pH 7.5,150 mM NaCl, 1.5 mM MgCl2, 100 mM ZnCl2, 20 mM b-mercapto-thanol, and 0.1% NP-40). The beads were washed three times ininding buffer. Bound proteins were separated by 12% SDS–PAGEnd visualized by autoradiography.Gene array analysis. The TRE-E1Aexon2, TRE-E1Aexon2DCID,

nd the parental Tet-On-expressing cell lines were induced by doxy-yclin (2 mg/ml). Poly(A)1 RNA was extracted at 24 h postinduction

and cDNA probe synthesis, using array-specific primers, was doneusing the Atlas Pure Total RNA Labelling system (Clontech). Thethree different populations of 32P-labeled cDNA preparations (RNAisolated from TRE-E1Aexon2, TRE-E1Aexon2DCID, and Tet-On pa-rental cell lines) were hybridized to the Atlas Human Cancer 1.2Array membranes (Clontech). Signals were detected using a phos-phoimager (Fuji) and the results were quantified using the AtlasIm-age 1.01a software (Clontech).

RESULTS

Promoter Recruitment of CtBP Results in Repressionof Transcription

The E1A C-terminal binding protein acts as a tran-
scriptional repressor when recruited to a target pro-

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286 SUNDQVIST ET AL.

moter either directly as a Gal4CtBP fusion protein [11,14, 19] or indirectly through interaction with aGal4E1A fusion protein [20]. A Gal4E1A fusion ex-pressing only the last 44 amino acids of E1A, includingthe CID (Gal4ctE1A, Fig. 1A), repressed approxi-mately 10-fold transcription from a luciferase reportergene, driven by the SV40 enhancer and five bindingsites for Gal4 (SVG5E1Bluc, Fig. 1B). A similar level ofrepression was also observed on reporters containingGal4 binding sites upstream of the collagenase pro-moter, G5collLuc (data not shown) and the tk pro-moter, G5tkLuc (Fig. 1C). Elimination of the CtBPbinding capacity of Gal4ctE1A, by deletion of aminoacids 225–238 (Gal4ctE1ADCID, Fig. 1A), precludedrepression of both reporters (Figs. 1B and 1C) and, infact, resulted in an unexplained enhancement of lucif-erase expression from SVG5E1BLuc (Fig. 1B). Directpromoter recruitment of CtBP, using a Gal4CtBP fu-sion, resulted in a similar level of repression (Figs. 1Band 1C). To test whether repression by Gal4ctE1A

FIG. 1. A CtBP binding-competent competitor relieves transcripdifferent E1A constructs is shown in (A). U2OS cells were cotransfect(B and D) or G5tkLuc (C and D). The luciferase activity seen in thewas obtained by cotransfection of 0.25 mg of E1Aexon2 (expressing th

omain (E1Aexon2DCID). (D) A549 cells were cotransfected with 1 mThe data represent mean values obtained from at least three indepe

involved recruitment of CtBP, a competition experi-

ment was performed. Cotransfection of a plasmid en-coding a CtBP-binding-competent, 100-amino-acidfragment of the E1A protein (E1Aexon2, Fig. 1A) alle-viated Gal4ctE1A-mediated repression of theSVG5E1BLuc reporter (Fig. 1B). No effect was de-tected following coexpression of a plasmid encoding anE1A protein fragment lacking the CtBP binding site(E1Aexon2DCID, Figs. 1A and 1B). When the competi-tion strategy was applied on the Gal4CtBP-inducedrepression, no recovery of transcription was observed(Fig. 1B). This was in agreement with the hypothesisthat the E1A protein fragment would be unable todisplace CtBP from a promoter when it was covalentlytrapped in a Gal4 fusion.

Figure 1B furthermore shows that neither basal ex-pression from the SVG5E1BLuc reporter (in the pres-ence of Gal4 DBD) nor expression activated byGal4ctE1ADCID was significantly affected by coex-pression of E1Aexon2. In contrast, coexpression ofE1Aexon2—but not E1Aexon2DCID—and Gal4DBD,

al repression by promoter-recruited CtBP. A schematic view of thewith 0.1 mg of either Gal4 fusion construct and 1 mg of SVG5E1BLucsence of Gal4DBD alone was set to 1. Competition for CtBP bindingst 100 amino acids of E1A) or a derivative lacking the CtBP bindingf indicated reporter constructs and 0.25 or 0.5 mg of pcDNA3CtBP.ent experiments.

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Gal4ctE1ADCID, or Gal4CtBP resulted in a four- to


eightfold derepression of expression from the G5tkLucreporter (Fig. 1C). It has previously been shown thatthe SV40 promoter and the tk promoter demonstratedifferent sensitivity to HDAC-mediated repression,since the SV40 promoter was insensitive to treatmentwith the HDAC inhibitor TSA, whereas the tk pro-moter was derepressed by TSA treatment [31]. Simi-larly, Fig. 1D shows that the tk promoter, but not theSV40 promoter, was repressed by overexpression ofCtBP. Since we have previously shown that CtBP in-teracts with HDAC (see also Fig. 4A), it is possible thata complex containing CtBP and HDAC regulates ex-pression from the tk promoter but not from the SV40promoter.

Importantly, coexpression of E1Aexon2 andGal4ctE1A enhanced expression from G5tkLuc morethan 60-fold, compared to Gal4ctE1A alone (Fig. 1C),indicating that E1Aexon2 might have a dual effect inthis context, first to relieve CtBP repression of the“basal” G5tkLuc reporter and second to titrateGal4ctE1A-recruited CtBP.

The E1ACID Is Sufficient to Induce Transcriptionfrom Different Model Promoters

We have previously shown that the CtBP bindingactivity of E1A is essential for efficient activation oftranscription by full-length E1A [21]. The CID-ex-pressing second exon of E1A was furthermore suffi-cient to derepress transcription of the PCNA promoter[21], suggesting that the capacity of E1A to interactwith CtBP was sufficient to dampen the CtBP-medi-ated repression. As shown in Fig. 1C, the tk promoteralso responded to E1ACID and it was therefore impor-tant to investigate the specificity of the E1A-sensitiveand CtBP-dependent repression. A panel of reporterconstructs (Fig. 2A) driven by unrelated promoters wasanalyzed with respect to their response to the E1ACID. The promoters were derived from two adenovirusgenes (the major late promoter and the E4 promoter),two human genes (the proliferating cell nuclear anti-gen promoter and the hsp70 promoter), and an artifi-cial promoter containing five binding sites derived froma c-myc-responsive element. Cotransfection ofE1Aexon2 resulted in a 4- and 11-fold derepression ofall promoters (Fig. 2B). No effect was observed follow-ing coexpression of E1Aexon2DCID, supporting the hy-pothesis that derepression required the sequestrationof CtBP (Fig. 2B). Similar to the CtBP-mediated re-pression of the tk promoter (Fig. 1D), the E1A secondexon-inducible E4 (Fig. 2C), MLP, and Hsp70 promoter(data not shown) were also repressed by CtBP overex-pression. In addition, E1Aexon 2, but notE1Aexon2DCID, was able to block repression by over-expressed CtBP (Fig. 2C). In summary, we propose
that E1Aexon2 can derepress transcription by binding
to and interfering with the repressing capacity ofCtBP. The interaction between the E1ACID and CtBPwas verified by coimmunoprecipitation of CtBP andE1Aexon2—but not CtBP and E1Aexon2DCID—fromextracts of transfected cells (Fig. 2D). Moreover, in apull-down experiment using whole-cell extracts, onlyone predominant protein interacted specifically withGSTctE1A (Fig. 2E). This protein was, like CtBP, aphosphoprotein [20] and had the same size of 48 kDa(Fig. 2E). Although it cannot be completely excluded,these results do not support the existence of an addi-tional CID-binding factor that could be responsible forthe observed E1ACID derepression.

A Cell Line, with Inducible E1A Second ExonExpression, Supports Derepression of theTransfected Reporter Genes

Cotransfection of transcription factors and re-porter genes into cells is a commonly used method tostudy regulation of transcription. A major disadvan-tage with this approach is that not all of the trans-fected cells will express all transfected genes. Toovercome this problem, we used the Tet-On system[27] to establish clones carrying inducible genes forE1Aexon2 and E1Aexon2DCID. Western blot analy-sis of selected U2OS-derived clones demonstrated astrong doxycyclin induction of TRE-E1Aexon2 andTRE-E1Aexon2DCID (Fig. 3A). The reporter con-structs used in the cotransfection study (Fig. 2A)were transfected into TRE-E1Aexon2-or TRE-E1Aexon2DCID-expressing cell lines. Doxycyclin in-duction of TRE-E1Aexon2 increased the reporter ac-tivity from PCNALuc approximately 11-fold andfrom Hsp70Luc approximately 7-fold (Fig. 3B). Incontrast, induction of TRE-E1Aexon2DCID failed toderepress transcription from the reporters (Fig. 3B).Similar results were obtained using the remainingluciferase reporter listed in Fig. 2A (data not shown).The capacity of CtBP to interact with the doxycyclin-induced TRE-E1Aexon2, but not with TRE-E1Aexon2DCID, was verified by pull-down experi-ment using GSTCtBP (Fig. 3C).

Derepression of Transcription by E1ACID Relieves theEffect of TSA

We have previously shown that CtBP has the ca-pacity to associate with HDAC-1 both in vitro and invivo [21]. Here, GSTCtBP was shown to also interactwith two additional members of the Class I histonedeacetylases, HDAC-2 and HDAC-3 (Fig. 4A). Tofurther investigate the involvement of HDACs incorepression by CtBP we analyzed the effect of thehistone deacetylase inhibitor TSA on the E1ACID-inducible promoters. If E1A blocked CtBP-mediated
repression of transcription by preventing the func-

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FIG. 2. The ability of the second exon of E1A to derepress transcription requires a functional CtBP binding domain. A schematicview of the different luciferase reporter constructs is shown in (A). (B) U2OS cells were cotransfected with 1 mg of either luciferasereporter and 0.25 mg E1Aexon2 or E1Aexon2DCID (see also Fig. 1A). Basal luciferase activity (set to 1) represents the activity from eachreporter obtained following cotransfection with the empty CMV vector (0.25 mg pcDNA3). Mean values obtained from at least threeindependent experiments are also shown. (C) A549 cells were cotransfected with 1 mg of E4Luc and increasing amounts of pcDNA3CtBP(0.25 and 0.75 mg). Where indicated 0.35 mg E1Aexon2 or E1Aexon2DCID was included in the transfection cocktail. (D) The interaction

etween the E1A second exon-encoded polypeptides and CtBP was demonstrated using immunoprecipitation and Western blot analysis.xtract from Cos7 cells, cotransfected with FL-pcDNA3CtBP and E1Aexon2 or E1Aexon2DCID, was immunoprecipitated with

antibodies recognizing FLAG-tagged CtBP (a-Flag) or E1Aexon2 (a-E1A). The recovered material was detected by Western blot analysisusing the polyclonal E1A (a-E1A). (E) The predominant interaction between GSTctE1A expressing the CID and a 48-kDa protein from[35S]methionine-labeled HeLa cells was demonstrated in a pull-down experiment. B and C show mean values obtained from at least
three independent experiments.

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tion or formation of a CtBP–HDAC-containing com-plex, it was assumed that the E1ACID-inducible pro-moters should also be induced by TSA. The effect ofTSA on expression from the PCNALuc reporter wasmeasured in the E1A-inducible cell lines. In the ab-sence of doxycyclin, TSA treatment resulted in a six-to eightfold induction of PCNALuc expression inboth inducible cell lines (Fig. 4B). Induction of TRE-E1Aexon2 gave the expected derepression of PC-NALuc expression, but simultaneous treatment withboth doxycyclin and TSA did not give a significantadditional increase in luciferase activity (Fig. 4B). Incells harboring the inducible TRE-E1Aexon2DCIDgene, TSA-enhanced expression from PCNALuc wassimilar, irrespective of the absence or presence ofdoxycyclin (Fig. 4B). Collectively, inactivation ofHDACs by TSA eliminated the need for E1A CID asa competitor for CtBP and therefore supports the

FIG. 3. Established Tet-On cell lines, harboring inducible genesfor TRE-E1Aexon2 or TRE-E1Aexon2DCID, display the capacity toderepress transcription in a CtBP binding-dependent manner. (A)Doxycyclin (Dox)-inducible expression of TRE-E1Aexon2 and TRE-E1Aexon2DCID was demonstrated by Western blot analysis using a

olyclonal E1A antibody (sc-430, a-E1A). (B) PCNALuc or Hsp70Luc0.5 mg) was transfected into the TRE-E1Aexon2- or TRE-1Aexon2DCID-expressing cells. Where indicated, 2 mg/ml doxycy-

clin was added 1 h posttransfection. The graph shows the averageresults from at least three independent experiments. (C) The capac-ity of doxycyclin-induced TRE-E1Aexon2, but not TRE-E1Aexon2DCID, to bind to CtBP was verified using a GST pull-downxperiment.

hypothesis suggesting that CtBP-mediated repres- i

sion involves HDACs. TSA was, however, not com-pletely without effect in cells in which E1Aexon2 wasinduced. This may indicate that suboptimal amountsof E1Aexon2 were expressed, that CtBP repressionoccurred also through non-E1A-targeted mecha-nisms, or perhaps, most likely, that TSA treatmenthas additional effects unrelated to the activity ofboth E1ACID and CtBP.

The effect of TSA on Gal4CtBP-mediated repressionis controversial [11, 14, 19]. In our hands, TSA wasunable to relieve Gal4CtBP repression (Fig. 4C), sug-gesting that when CtBP is experimentally tethered to apromoter, the HDACs are not essential.

FIG. 4. TRE-E1Aexon2 expression and TSA inhibition of his-tone deacetylases result in similar levels of derepression of tran-scription and do not display additative effects. (A) GST pull-downassay demonstrating the ability of CtBP to interact with in vitro-synthesized HDAC-1,-2, and -3. (B) The TRE-E1Aexon2- or TRE-E1Aexon2DCID-inducible cells were transfected with 0.5 mg of

CNALuc. Where indicated, the transfected cells were inducedith 2 mg/ml doxycyclin (1 h posttransfection) and/or 300 nM TSA

(7 h posttransfection). Cells were harvested 24 h posttransfection.(C) U2OS cells were cotransfected with 1 mg of SVG5E1Bluc and

.1 mg of Gal4(1–147) or Gal4CtBP in the presence or absence ofSA. The graph shows the average results from at least three

ndependent experiments.

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Functional Knockout of CtBP by Induction ofE1ACID Resulted in a Wide-Range Modulation ofthe Cellular Gene Expression

A cell line allowing inducible expression of the CtBPbinding domain of E1A offers the opportunity to mea-sure the effect of functional inactivation of CtBP—through physical interaction with E1A—on total hostcell gene expression. Using macroarray filters we ana-lyzed the gene expression profile in TRE-E1Aexon2-and TRE-E1Aexon2DCID-expressing cell lines after24 h of doxycyclin induction—a time point when E1Aexpression had reached a steady-state level (data notshown). Induction of TRE-E1Aexon2 resulted in a pro-nounced modulation of cellular gene expression. Ex-pression from more than 4% of the 1176 analyzed genesincreased 2.5-fold or more, whereas more than 3% ofthe genes showed a greater than 2.5-fold reduction(Table 1). Induction of TRE-E1Aexon2DCID had signif-icantly less effect and caused up- or down-regulation ofonly 1.5% of the genes on the array (Table 1). Only0.5% of all genes showed the same pattern of regula-tion following induction of TRE-E1Aexon2 and TRE-E1Aexon2DCID. In summary, approximately 7% of theanalyzed genes were regulated in a CID-dependentmanner.

Although a wide variety of different genes were reg-ulated following induction of TRE-E1Aexon2 and TRE-E1Aexon2DCID, the most pronounced CtBP-depen-dent effects were observed on genes involved in cellcycle regulation, apoptosis, and intracellular commu-nications. More than one-third of the genes thatshowed an altered expression pattern after doxycyclin

TABLE 1

Number of Genes Regulated after Doxycyclin Induction ofE1Aexon2 and E1Aexon2DCID (%)a

E1Aexon2 E1Aexon2DCID

Up-regulated genes 4.2 0.8Down-regulated genes 3.4 0.7Regulated genes (total) 7.6 1.5Specifically up-regulated genes 4.1b 0.7c

Specifically down-regulated genes 3.0b 0.3c

Specifically regulated genes (total) 7.1 1.0

a Numbers indicate the percentage of genes showing altered ex-ression pattern following doxycyclin induction of TRE-E1Aexon2 orRE-E1Aexon2DCID. The changes in gene expression profile wereetermined by calculating the ratio between expression in the TRE-1Aexon2- or TRE-E1Aexon2DCID-expressing cells and expression

n the parental U2OS-Tet-On cell line. Only values exceeding 2.5-old up- or down-regulation were included.

b Percentage of genes up- or down-regulated exclusively in TRE-E1Aexon2-expressing cells.

c Percentage of genes up- or down-regulated exclusively in TRE-E1Aexon2DCID expressing cells.

induction fell into either of these categories (Table 2).

DISCUSSION

CtBP has been described as a transcriptional core-pressor that is recruited to promoters through interac-tion with a number of transcriptional regulators. Inthis report we have addressed the specificity of CtBPrepression by using its noncellular interaction partneradenovirus E1A. Our results demonstrated a wide-range capacity of the second exon of adenovirus E1A toregulate gene expression, a capacity that was depen-dent on its CID. Furthermore, competition experi-ments indicated that the observed effects were in fact aderepression, caused by the physical titration of CtBP(Figs. 1C and 2D).

In searching for specific targets for CtBP-mediatedrepression, we deliberately selected unrelated promot-ers, both of native origin (Hsp70Luc, E4Luc, G5tkLuc,and MLPLuc) and designed to carry defined bindingsites for known transcription factors (5xmycRELuc andSVG5E1BLuc). From these and previously publishedexperiments [21] no single factor responsible for thepromoter recruitment of CtBP could be identified. Al-though the consensus CID sequence, PXDLS, wasfound in the amino acid sequence of some of the pro-moter-specific transcription factors (e.g., ATF-2 recog-nizing the PCNA and E4 promoters), other factorslacked close homologues to this motif. Therefore, wefind it is possible that, in addition to its interactionwith DNA-binding factors carrying the PXDLS motif,CtBP might also become promoter recruited throughadditional bridging factors.

A potential candidate for a bridging factor is theCtBP interaction protein CtIP [15]. CtIP has beenshown to mediate the contact between CtBP and theBRCT domain of BRCA1 [16, 17] and pRB/p130 [19,32]. Significantly, these interactions have been impli-cated in BRCA1- and pRb/p130-mediated regulation oftranscription [16, 19].

CtBP also interacts with several members of theHDAC family of proteins ([21, 22] and this paper).HDACs are found in association with chromatin-mod-ulating complexes such as Sin3 (reviewed in [33]) butalso in ATP-dependent nucleosome mobilization com-plexes such as Mi2/NuRD (reviewed in [34]). It remainsto be determined whether the deacetylase activity ofthe HDACs is always an essential component of thechromatin-modulating complexes or if the HDAC pro-teins may also act as molecular bridges through bind-ing to corepressors (reviewed in [35]). In a recent re-port, it was shown that MITR (a transcriptionalrepressor with homology to Class II HDACs but lack-ing the deacetylase domain), similar to HDACs, couldrecruit CtBP to the MEF2 transcription factor [22].This suggests that HDACs, just like CtIP, may act asan additional bridging factor and, as such, be required
for CtBP repression. In agreement with this model,

n 2


transcriptional derepression by E1ACID was lost incells treated with the HDAC inhibitor TSA (Fig. 4B).Although it remains to be experimentally verified, it ispossible that the TSA inactivation of HDACs obstructsthe interaction between HDAC and CtBP, thereby in-terfering with the promoter recruitment of CtBP.

Alternatively, CtBP-mediated repression was alsoobtained using a Gal4CtBP fusion (Fig. 1) [11, 14, 19]or a Gal4E1A fusion expressing the CID (Fig. 1). Thissuggested that CtBP (by itself or by serving as a plat-form for additional factors) directly participated in re-pression of transcription. Importantly, Gal4CtBP-me-diated repression was found to be insensitive to TSAtreatment [11, 19] (Fig. 4C), suggesting that HDACswere not essential when CtBP was given promoterbinding capacity through the Gal4 DNA binding do-main. This model is supported by the observation thata mutant of Gal4CtBP, with diminished capacity tointeract with HDAC2, was still able to repress tran-scription [11]. In summary, the differential TSA sensi-

TAB

Specific Genes Regulated after Indu

Gene

Cell cycle regulatorsCyclin ICdk2 (cyclin-dependent kinase 2)ERK5 (extracellular signal-regulated kinase 5)Wee1/p87; cdc2 tyrosine 15-kinaseSTK-1 growth factor receptor tyrosine kinaseCyclin-dependent kinase 5 activatorGrowth inhibitory factor; metallothionein-III (MT-III)CDC37 homolog

Intracellular signal transduction modulators and effectorsMAPKK1 (MAP kinase kinase 1)MEKK5 (MAP/ERK kinase kinase 5)cAMP-dependent protein kinase; PKA C-gMitogen-activated protein kinase p38bSerine/threonine protein kinaseProtein tyrosine phosphataseSerine/threonine protein phosphatase 6 (PP6)b-Adrenergic receptor kinase 2Rho6 proteinGDI-dissociation inhibitor RhoGDIgInhibitor of the RNA-activated protein kinase, 58-kDaGRB2-associated binder 2

Apoptosis-related proteinsInsulin-like growth factor I receptorCytotoxic ligand TRAIL receptorRAIDD; death domain-containing protein CRADDCaspase-9 precursorCaspase-7 precursorNbk apoptosis inducer; Bcl-2 interacting killerhIAP3 apoptosis inhibitorDAPK1 (death-associated protein kinase 1)RATS1

Note. Grouping into families was according to the gene list for thindicate that the named genes were up or down-regulated more tha

tivity of CtBP repression of native promoters compared

to Gal4CtBP repression of promoters carrying Gal4binding sites supports the idea that HDACs may not berequired to mediate the contact between Gal4CtBP andthe target promoter nor for the actual CtBP repressoractivity. Instead HDAC—like MITR [22]—might be re-quired to establish a CtBP-containing, promoter-boundcomplex on a native promoter.

Although most data on CtBP interactions support amodel in which CtBP repression requires a physicalassociation with the target promoter, through interac-tion with DNA-bound transcription factors, it cannotbe excluded that CtBP (also) physically interferes withthe formation of a basal transcription initiation com-plex. This possibility gets support from a low E1ACIDderepression of basal (TATA only) promoter activity(data not shown).

In our investigation, only the SVG5E1Bluc reportercontained a promoter that was insensitive to CtBPrepression/E1ACID derepression. Possibly, the natureof the SV40 enhancer, with multiple and tightly spaced

2

n of E1Aexon2 and E1Aexon2DCID

enBank Accession No. Exon2 Exon2DCID

D50310 2M68520 2 2U25278 1U10564 2U02687 2U34051 2D13365 1U63131 2

L05624 2D84476 1M34182 1U53442 1 1AJ000512 1D15049 2X92972 1X69117 1Y07923 1U82532 1U28424 1 1U43885 1 1

X04434 2 1U90875 1U84388 1U56390 1U37448 2U34584 2U45880 2X76104 1U37688 1

tlas Human Cancer 1.2 Array membranes (Clontech). “1” and “2”.5-fold, respectively, following induction of the second exon of E1A.

LE

ctio

G

e A

transcription factor binding sites, might preclude ac-

cct

frtpsdetuIca

1

1


cess of CtBP. However, the insensitivity to CtBP re-pression is most likely not caused by high transcrip-tional activity per se, since phorbol ester induction of acollagenase promoter containing Gal4 binding sites didnot prevent Gal4CtBP repression (data not shown).

The immortalizing activity of adenovirus E1A re-quires binding of a number of cellular proteins to theN-terminal half of the protein [36]. This enables E1A tocooperate with several dominant oncogenes to give fulltransformation (reviewed in [37]). In contrast, the sec-ond exon of E1A confers suppression of transformation,tumorigenicity, and metastatic potential of E1A1ras-transformed cells (reviewed in [28]). Gene array anal-ysis demonstrated a pronounced effect of E1ACID tomodulate gene expression (Table 1). Of the genes show-ing an altered expression pattern in the presence ofE1A exon 2, a number of interesting tendencies wereobserved. The capacity to bind CtBP correlated withaltered expression of genes involved in (i) cell cycleregulation, (ii) regulation of apoptosis, and (iii) intra-cellular cell signaling (Table 2). Taken together, theseregulatory events are consistent with the previous ob-servation that the second exon of E1A restricts thelevel of tumorigenicity of the E1A-transformed cells [1,38] possibly by reducing cell growth and promotingapoptosis. In contrast, very few of these genes wereaffected in the E1Aexon2DCID-expressing cells, indi-ating that a failure to regulate these genes couldontribute to the observed hypertransforming pheno-ype of E1A mutants lacking the CtBP binding domain.

Although there is a strong link between hypertrans-ormation and a lost ability to bind CtBP [1], otheregions in the second exon may actively contribute tohe observed phenoptype [1, 38]. In light of the unex-lained ability of Gal4ctE1ADCID to activate tran-cription from the SVG5E1BLuc reporter (Fig. 1B), aiscovery of additional activities residing in the secondxon of E1A might be expected. As a first indication,he gene array analysis identified genes that were reg-lated exclusively by E1Aexon2DCID (Tables 1 and 2).mportantly, E1Aexon2DCID demonstrated a specificapacity to induce expression of the RhoGTPase Rho6nd the dissociation inhibitor Rho GDIg. The family of

Rho GTPases is receiving increasing attention due totheir possible involvement in transformation [39] andit is noteworthy that studies of hypertransforming E1Asecond-exon mutants have implicated members of theRho family of GTPases as effectors during E1A1H-rashypertransformation [40]. Thus, it is possible that theregulation of certain members of the family of RhoGTPases by E1Aexon2DCID might be involved in es-tablishing the hypertransformed phenotype.

Despite the general assumption that CtBP is a core-pressor of transcription, approximately 3% of the 1176analyzed genes were repressed after induction of the
second exon of E1A. Since the analyses were performed
after a 24-h induction period, this might simply be theresult of the CID-dependent derepression of genes,which, in turn, causes repression of secondary targetgenes. Alternatively, the repression observed afterE1ACID induction—and sequestering of CtBP—mightreflect a capacity of CtBP to directly activate transcrip-tion. This interpretation is supported by the observa-tion that Drosophila CtBP may also act as a coactivatorof transcription [41].

The authors thank Drs. E. Seto, G. Akusjarvi, and T. Punga forkind gifts of plasmids. This work was supported by the SwedishCancer Society.

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Received April 5, 2001Revised version received May 16, 2001

Documents

Functional Knockout of the Corepressor CtBP by the Second Exon of Adenovirus E1A Relieves Repression of Transcription