Oncogene (2007) 26, 7467–7479 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE C-terminal-binding interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3

RK Bruton1, M Rasti1, KL Mapp1, N Young1, RZ Carter1, IA Abramowicz1, GG Sedgwick1, DF Onion1, M Shuen2, JS Mymryk2, AS Turnell1 andRJA Grand 1

1Cancer Research UK Institute for Cancer Studies, The Medical School, University of Birmingham, Birmingham, UK and 2Departments of Oncology and Microbiology and Immunology, University of Western Ontario, Ontario, Canada

C-terminal-binding protein interacting protein (CtIP) was Introduction first isolated as a binding partner of C-terminal-binding protein (CtBP). It is considered to contribute to the Adenovirus early region 1A (AdE1A) is the first protein transcriptional repression and cell cycle regulatory to be expressedfollowing adenovirus infection andis properties of the retinoblastoma (Rb) family of essential for adenovirus E1-mediated transformation and to have a role in the cellular response to DNA (reviewedby Gallimore andTurnell, 2001; Berk, 2005). damage. Here, we have shown that CtIP is a novel target Two major E1A proteins are translatedfrom 13S and for the adenovirus oncoprotein early region 1A (AdE1A). 12S messenger RNAs differing only by the presence of AdE1A associates with CtIP in both Ad5E1-transformed a short amino-acidsequence locatedtowardsthe C cells and Ad5-infected cells and binds directly in terminus of the larger molecule. Comparison of the glutathione-S-transferase pull-down assays. Two binding amino-acid sequences of AdE1As from different viral sites have been mapped on Ad5E1A – the N-terminal serotypes indicates the presence of four highly conserved a-helical region (residues 1–30) and conserved region regions (CRs) locatedthroughout the molecule (Avva- 3(CR3)– the transcriptional activation domain. CtIP kumov et al., 2002, 2004). CR3 co-incides with the region can bind AdE1A and CtBP independently, raising the unique to the 13S mRNA product. AdE1A exerts its possibility that ternary complexes exist in Ad-transformed influence on infectedandtransformedcells through a and -infected cells. Significantly, reduction of CtIP complex series of protein–protein interactions (Galli- expression with small interfering RNAs results in reduc- more andTurnell, 2001). Most of the bindingsites for tion of the ability of a Gal4 DNA-binding domain-CR3 the cellular targets of AdE1A correspond to either the construct to transactivate a Gal 4-responsive luciferase CRs or to the N-terminal a-helical region. For example, reporter and this effect is reversed by reduction of CtBP the retinoblastoma (Rb) family of proteins bindto CR1 expression. Therefore, in this model, CtIP acts as a andCR2 (Dyson et al., 1992), p300 andCREB-binding transcriptional co-activator of AdE1A when dissociated protein (CBP) interact with the N-terminal region and from CtBP, through the action of AdE1A. These data CR1 (Eckner et al., 1994; Arany et al., 1995), while CtBP are consistent with observations that CtIP expression interacts with CR4 (Boyd et al., 1993). The modular is induced by AdE1A during viral infection and that nature of AdE1A has facilitated the mapping of protein- reduction of CtIP expression with RNA interference can binding sites and linking of biological functions to retard virus replication. In addition, AdE1A causes particular interactions andtherefore to certain regions of disruption of the CtIP/Rb complex during viral infection E1A. Binding of the Rb family or CBP/p300 is necessary by its interaction with CtIP, possibly contributing to for AdE1A to promote S phase entry, but binding of transcriptional derepression. both sets of proteins is required for AdE1A-mediated Oncogene (2007) 26, 7467–7479; doi:10.1038/sj.onc.1210551; transformation (Egan et al., 1988; Jelmsa et al., 1989; publishedonline 4 June 2007 Howe et al., 1990; Wang et al., 1993). CR3 contains a zinc (Zn2 þ )-finger motif andis the site of interaction with Keywords: adenovirus E1A; C-terminal-binding protein a number of proteins involvedin transcriptional regula- interacting protein; CtIP; C-terminal-binding protein; tion (Culp et al., 1988; Geisberg et al., 1994, 1995; Rasti CtBP et al., 2006). These interactions with proteins such as TATA-binding protein (TBP), associated transcription factor (ATFs), suppressor of ras (Sur2) 23 (MED23), proteasomal components andtrans-acting factors are necessary for transcriptional activation and Correspondence: Dr R Grand, Cancer Research UK Institute for for the expression of other viral early region proteins Cancer Studies, The Medical School, University of Birmingham, (reviewedby Jones, 1995; Avvakumov et al., 2004). Edgbaston, Birmingham, West Midlands, B15 2TT, UK. E-mail: [email protected] CR1, CR2 andCR3 are encodedby exon 1 of AdE1A Received15 June 2006; revised24 April 2007; accepted26 April 2007; andthis area of the protein is responsible for most of the publishedonline 4 June 2007 observedinteractions. However, the bindingsite for the CtIP binds directly to adenovirus early region 1A RK Bruton et al 7468 ubiquitous CtBP has been mappedto a CtBP, we investigatedthe relationship between CtIP and highly conservedPXDLS motif in CR4 in exon 2 (Boyd AdE1A and examined the possibility of direct interac- et al., 1993; Schaeper et al., 1995). Loss of binding of tion between the two proteins. When Ad5E1A was CtBP by AdE1A results in retarded viral replication immunoprecipitatedfrom Ad5E1-transformedcells (Grand et al., 2006). Significantly, the effect of binding (293) or from MCF7 cells infectedwith wt Ad5, CtIP of CtBP on AdE1A-mediated transformation is context was identified by western blotting as a co-precipitating dependent. Deletion of the C-terminal region of AdE1A protein (Figure 1a andc). In complementary experi- reduces the frequency of transformation by AdE1A ments, CtIP was immunoprecipitatedfrom Ad5E1- together with AdE1B, but increases the frequency of transformedandAd5-infectedcell lysates and transformation by AdE1A and activated ras (Douglas co-precipitating Ad5E1A was identified by Western et al., 1991; Subramanian et al., 1989, 1991). CtBP has blotting following electrophoresis on ‘urea gels’ in the been shown to interact with a large number of absence of sodium dodecylsulphate (SDS) (Figure 1b mammalian transcriptional repressors, such as BKLF, and d). The observation that Ad5E1A with a deletion Ikaros, Net andthe repressors Snail, Hairy encompassing the CtBP-binding site (dl 1135) co- andKnirps as well as histone deacetylases (HDACs) immunoprecipitates with CtIP suggests that the inter- (reviewedby Turner andCrossley, 2001; Chinnadurai, action is not mediated through CtBP (Figure 1d). CtIP 2002, 2004, 2006a, 2007). andAd12E1A were also co-immunoprecipitatedfrom The first mammalian CtBP-binding protein to be Ad12E1-transformed cells (Figure 1e). It is also notable isolatedwas C-terminal-bindingprotein interacting pro- that CtIP bound to Ad126f10E1A, which does not tein (CtIP). Like AdE1A, CtIP interacts with CtBP via a interact with CtBP (Grand et al., 2006) (Figure 1e). PXDLS motif andE1A may compete with CtIP for (Rather less CtIP appearedto be co-immunoprecipi- interaction with CtBP (Schaeper et al., 1998). The role of tatedfrom the rat cells than the human, possibly dueto CtIP is not clear – it binds to the Rb family and it has been reduced expression or the inability of the antibody to suggestedthat this is responsible for some of the recognize rat CtIP). In a further set of experiments, transcriptional repression properties of Rb through [35S]-labelledCtIP or CtBP1 were incubatedwith recruitment of CtBP andassociatedHDACs (Meloni glutathione-S-transferase (GST)-Ad512S and 13SE1A et al., 1999). CtIP also regulates Rb-dependent cell cycle or GST-Ad5E1A exons 1 and 2 (Figure 2). CtIP bound arrest in G1 through the modulation of Rb phosphoryla- directly to full-length Ad5E1A and Ad5E1A exon 1 tion (Chen et al., 2005). CtIP binds to breast cancer (expressedas GST-fusions), whereas CtBP1 interacted associated1 (BRCA1) andis involvedin the cellular with exon 2, confirming that the AdE1A/CtIP interac- response to DNA double strand breaks, being phosphory- tion is not mediated through CtBP. latedby ataxia telangiectasia mutated(ATM) (Wong et al., 1998; Yu et al., 1998; Li et al., 1999, 2000; Wu-Baer Dissociation constant for CtIP and Ad12E1A andBaer, 2001; Yu andChen, 2004). Significantly, Using enzyme-linkedimmunosorbant assays (ELISAs), BRCA1 catalyses CtIP ubiquitination andco-localizes the apparent K for AdE1A binding to CtIP has been with CtIP in DNA damaged-induced foci (Yu et al., 2006). d comparedwith values obtainedfor CtBP1 andCtBP2. Although the precise role of CtIP has yet to be determined PurifiedAd1213SE1A was coatedonto an ELISA plate in detail the protein must be of considerable importance to andGST-CtBP1, GST-CtBP2, GST-CtIP amino acids the functioning of the normal cell since CtIP knock-out 324–897 andGST-CtIP amino acids371–620 were mice die at an early stage in development (E4) largely serially diluted across the plate. Bound protein was through cell cycle arrest in G (Chen et al., 2005). 1 determined using an anti-GST antibody (Figure 3). Because of its close relationship to the Rb family of Using the Origin 7.5 software package apparent K s proteins andCtBP, we have consideredthe possibility d were determined. They were found to be 6, 2, 5 and that CtIP itself might be a novel target for AdE1A. Here, 79 nM for CtBP1, CtBP2, CtIP (324–897) andCtIP we have shown that these proteins interact independently (371–620), respectively. Thus, Ad12E1A has a similar of CtBP or Rb. CtIP binds to the N-terminal region and affinity for CtIP (324–897) as for CtBP 1 and2, CR3 of AdE1A regulating the ability of a Gal4 DNA- although binding to the shorter CtIP polypeptide is binding domain (DBD)-CR3 construct to transactivate a appreciably weaker. Gal-4-responsive luciferase reporter. This is consistent with our observation that knock down of CtIP expres- sion can retardthe rate of viral replication. Furthermore, The determination of the CtIP-binding site on AdE1A AdE1A disrupts interaction between CtIP and Rb and To determine the CtIP-binding site(s) on AdE1A modulates CtIP phosphorylation. [35S]methionine-labelledCtIP was incubatedwith various GST-AdE1A polypeptides (Table 1). It was observedfrom the autoradiograph shown in Figure 2 that CtIP binds to both GST-12S and GST-13S Results Ad5E1A as well as to GST-Ad5 E1A exon 1. Further analysis was undertaken using four GST-AdE1A poly- Ad5E1A binds CtIP peptides, which encompass all of exon 1 (Figure 4). CtIP As both CtIP andAdE1Acan associate with the Rb didnotbindto the region between amino acids41 and family of pocket proteins andwith the corepressor 140, which includes CR1, CR2 and the intervening

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7469 sequence indicating that the interaction was not radiography (Supplementary Figure 1). Densitometric mediated through the Rb family of proteins scanning shows that p107 boundequally well to 13S and (Figure 4b). However, strong binding of CtIP to the 12S E1A, but hardly at all to CR3, as expected N-terminal region (amino acids 1–40) and to CR3 was (Figure 4c). CtIP boundalmost as well to 13SE1A as observed(Figure 4b). did p107 and rather better than TBP. Binding of To give some indication of the significance of the Ad512SE1A to CtIP and TBP was reduced compared binding of CtIP to Ad5E1A, we compared this interac- to the 13S protein, but was largely comparable. tion with that seen for the well-characterizedAdE1A- Similarly, both CtIP andTBP boundto GST-Ad5CR3 binding proteins p107 and TBP. [35S]methionine-labelled (Figure 4c) CtIP, p107 andTBP were incubatedwith GST-Ad513- SE1A, GST-Ad512SE1A and GST-Ad5CR3. Bound Definition of the N-terminal-region-binding site protein was fractionatedby SDS–polyacrylamidegel A large number of cellular proteins bindto AdE1A electrophoresis (SDS–PAGE) andvisualizedby auto- through its N-terminal region – for example CBP, p300, YY1, TBP andthe proteasomal components S4 andS8. Although there are differences in the specific sites of interaction (Rasti et al., 2005), certain point mutations will disrupt most interactions. [35S]methionine-labelled CtIP was incubatedwith a panel of GST-Ad512SE1A proteins with point mutations in the N-terminal 30 amino acids as described previously (Rasti et al., 2005 andTable 1). Substitutions in a number of residues reduce binding appreciably. For example, replacement of I5 andH7, L19 andL20 andL23 andI24 with alanine all reduce binding to a very low level (Figure 5a). In addition,mutation of I11 andT12 andL28 andD30 reduced the interaction.

CtIP binding to AdE1A CR3 It can be seen from Figure 4b andc that CtIP interacts with a GST polypeptide encompassing CR3. Further- more, CtIP has a higher affinity for 13SE1A than for the 12S protein (Figures 2 and4c). To confirm this, AdE1A CR3 regions from a variety of adenovirus serotypes were expressedas GST fusions. CtIP interactedwith each of these, although it appearedto have a higher affinity for the Ad5 and Ad9 polypeptides (Figure 5b). CR3 is considered to comprise an N-terminal region, a central Zn2 þ -finger domain (amino acids 147–177 in Ad5) and a short C-terminal sequence (amino acids 183– 188), which is essential for promoter targeting. To determine the site of interaction of CtIP on CR3,

Figure 1 Co-immunoprecipitation of CtIP andAdE1A.( a) Lysates from 293 cells were immunoprecipitatedwith antibodies against Ad5E1A(M58) or CtBP1 (E12) as shown. Co-immunopre- cipitating CtIP was detected by western blotting. (b) Lysates from 911 and293 cells were immunoprecipitatedwith two CtIP rabbit polyclonal antibodies. Immunoprecipitates were fractionated on ‘urea gels’ and co-immunoprecipitating Ad5E1A detected by western blotting. (c) Lysates from MCF7 cells infected(andmock infected) with Ad5wt were immunoprecipitatedfor Ad5E1A.Co- immunoprecipitating proteins were fractionatedon SDS gels and CtIP identified by western blotting. (d) Lysates from MCF7 cells infectedwith dl520 or dl1135 were immunoprecipitatedwith rabbit antibodies against CtIP. Co-immunoprecipitating proteins were fractionatedon ‘urea gels’ in the absence of SDS andwestern blottedfor Ad5E1A.( e) Lysates from Ad12E1-transformed human andrat cells were immunoprecipitatedwith an antibodyagainst Ad12E1A. Co-immunoprecipitating proteins were fractionated on gels run in the presence of SDS andblottedfor CtIP. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein; SDS, sodium dodecylsulphate.

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7470

Figure 3 Apparent dissociation constants of Ad1213SE1A for CtIP or CtBP. ELISA plates were coatedwith purifiedAd1213- SE1A (0.025 mg/well). Decreasing concentrations of GST-CtIP polypeptides 324–897 and 371–620, GST-CtBP1 or GST-CtBP2 were added and the concentration of bound protein determined using an antibody against GST. Apparent Kds were calculated from the data shown. CtBP1, C-terminal-binding protein; CtIP, C-terminal-binding protein interacting protein; GST, glutathione- S-transferase.

Table 1 Summary of GST-Ad5 proteins and polypeptides used in this study Nomenclature Dimensions (amino CtIP binding acids) of protein/ polypeptide

Ad512SE1A 1–243 O Ad513SE1A 1–289a O Ad5 exon 1 1–191a O Ad5 exon 2 192–289a — Ad5 1–40 1–40 O Ad5 41–80 41–80 — Ad5 81–140 81–140 — Ad5 141–185 141–185 O

Ad5 N-terminal mutants with var- 1–243 Dependent ious substitutions in the N-terminal on substitu- 30 amino acids of Ad512SE1A tion (usedin Figure 4) a 35 CR3 139–204 O Figure 2 CtIP binds directly to Ad5E1A. (i) [ S]methionine- a labelledCtIP or (ii) [ 35S]methionine-labelledCtBP1 were incubated CR3dl 139–160 160–204 O CR3dl 161–168 139–204 D 161–168a O with GST-Ad5E1A proteins and polypeptides (25 mg) as shown a by Coomassie Blue staining (iii). BoundCtIP or CtBP1 were CR3dl 169–177 139–204 D 169–177 — CR3dl 180–184 139–204 D 180–184a O fractioned by SDS–polyacrylamide gel electrophoresis and detected a by fluorography andautoradiography. Input represents 5% of CR3dl 188–204 139–188 O the amount added in the pull downs. AdE1A, adenovirus onco- protein early region 1A; CtBP1, C-terminal-binding protein; CtIP, Abbreviations: AdE1A, adenovirus oncoprotein early region 1A; CR, C-terminal-binding protein interacting protein; GST, glutathione- conservedregion; CtIP, C-terminal-bindingprotein interacting S-transferase. protein; GST, glutathione-S-transferase. aBasedon the Ad513SE1A protein.

polypeptide fragments were expressed as GST fusions as substitutions anddeletions cause some perturbation of described previously (Rasti et al., 2006 andSupplemen- the binding site, thus compromising, the interaction. tary Figure 2a). These were expressedin Escherichia coli, purifiedandincubatedwith [ 35S]-CtIP. CtIP boundto all CR3 polypeptides, except for 169–177 from which the The binding sites for AdE1A on CtIP C-terminal half of the Zn2 þ -finger is deleted (Supple- To map the reciprocal AdE1A-binding site(s) on CtIP, a mentary Figure 2b). This region is also requiredfor panel of CtIP fragments linkedto GST were expressed AdE1A’s binding to TBP and Sur2 (Geisberg et al., in E. coli andpurified.These were incubatedwith 1994; Boyer et al., 1999; Wang andBerk, 2002). [35S]methionine-labelledAd513SE1A. Interacting poly- However, it is interesting to note that the general level peptides were identified by SDS–PAGE and autoradio- of binding to the mutant CR3s was appreciably reduced graphy (Supplementary Figure 3). Multiple polypeptides comparedto wt except for CR3YF175 (Supplementary boundto AdE1A, covering much of the CtIP sequence. Figure 2b). It is possible, therefore, that all of these No polypeptide was available covering the first 45

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7471 amino acids of CtIP, but a comparison of the binding by CtBP does not displace CtIP from AdE1A fusions encompassing the N-terminal region suggests Although we have shown that the regions of AdE1A one binding site between amino acids 134 and 282. requiredfor CtIP bindingare quite distinct from the Another site of interaction was apparent between amino CtBP-binding site, we wished to confirm that the inter- acids 371 and 620, which may extend into the amino- action was not dependent on, or regulated by, CtBP. acidsequence between residues620 and690. [35S]methionine-labelledCtIP was incubatedwith either GST-Ad5E1A or GST-CtBP2 (Figure 6). Increasing concentrations of a synthetic peptide (EPGQPL- DLSCKRPRP) identical to the CtBP-binding site on Ad5E1A were added. After incubation, glutathione agarose beads were added and after washing, bound proteins were elutedwith glutathione andanalysedby SDS–PAGE. The CtBP2/CtIP complex was disrupted by the peptide, but the AdE1A/CtIP complex was unaffected(Figure 6).

CtIP enhances AdE1A CR3-dependent transactivation In view of the interaction of CtIP with CR3, we considered the possibility that it is involved in regulation of the transactivation properties of AdE1A. When tetheredto the DBD of Gal4, CR3 will stimulate transcription from Gal4-responsive promoters in the absence of Gal4 activation domain. Therefore, con- structs encoding Gal4 DBD or Gal4 DBD-CR3, and a Gal4 responsive luciferase reporter were transfectedinto cells in which CtIP expression was reduced using small interfering RNA (siRNA). Luciferase reporter activity was measuredafter 24 h. Reduction of CtIP expression reduced the ability of DBD-CR3 to transactivate the Gal4-responsive luciferase reporter by about 70% (Figure 7). Knock down of CtIP expression resulted in little reduction of activity of the control DBD or VP16- DBD, suggesting a specific effect of CtIP on CR3- mediated transcription. When CtBP expression was reduced, however, there was an increase in transactiva- tion of the reporter gene (Figure 7). Additionally, knock down of CtBP largely compensates for CtIP knock- down, giving activity intermediate between the low level observedwith CtIP siRNAs andthe high level with CtBP siRNAs (Figure 7).

CtIP and adenovirus infection In an attempt to understand the significance of the AdE1A/CtIP interaction for Ad infection, the level of expression of CtIP following AdE1A expression was examinedby western blotting. It was observedthat there

Figure 4 Mapping the CtIP-binding site in Ad5E1A. (a) Dimen- sions of GST-Ad5E1A polypeptides used in the pull-down assays. (b)[35S]methionine-labelledCtIP was incubatedwith GST-Ad5E1A polypeptides (25 mg) as shown. A Coomassie Blue stainedgel of the GST fragments is shown in the lower panel. (c)[35S]methionine CtIP, p107 andTBP were incubatedwith GST-Ad512SE1A, GST- Ad513SE1A and GST-Ad5CR3. Bound proteins were fractioned by SDS–polyacrylamide gel electrophoresis and visualized by fluorography andautoradiography. Densitometric scanning was usedto quantify the proportion of each of the proteins bound. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein; GST, glutathione- S-transferase.

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7472

Figure 5 Analysis of the binding site for CtIP in the N-terminal region of Ad5E1A. (a)[35S]methionine-labelledCtIP was incubated with GST-Ad512SE1A proteins carrying the amino-acid substitutions shown (20 mg). Boundproteins were isolatedwith glutathione agarose, eluted, fractionated by SDS–PAGE and detected by fluorography and autoradiography. Binding to wt Ad512S and Ad513S E1A is also shown. Input represents 5% of the total added in the pull-downs. Coomassie Blue-stained gels of the GST-AdE1As are shown. (b) CtIP binding to E1A CR3 from different viral serotypes. [35S]methionine-labelledCtIP was incubatedwith GST fusion proteins containing the CR3 regions of AdE1As, from different viral serotypes (25 mg). Boundproteins were fractionedby SDS–PAGE and detected by fluorography and autoradiography. Input represents 10% of the total added. A Coomassie Blue-stained gel of the GST-CR3 proteins is shown in the lower panel. AdE1A, adenovirus oncoprotein early region 1A; CR, conserved region; CtIP, C-terminal-binding protein interacting protein; GST, glutathione-S-transferase; SDS–PAGE, sodium dodecylsulphate–polyacrylamide gel electrophoresis.

was a markedincrease in CtIP expression co-incident infection (Figure 8a). Interestingly, the pattern of with the expression of Ad5E1A (Supplementary Figure expression of AdE1A is rather different. Up to about 4). It is unlikely that E1B55K expression plays a part in 30 h expression was similar in CtIP-knockdown cells and this as infection with Ad5dl1520 produced a similar untransfectedcells. At later times, however, AdE1A effect to dl520 and wt. Similarly, binding of CtBP by continuedto be expressedwhen CtIP expression had AdE1A does not contribute to this effect as Ad5dl1135 been reduced, although in the untransfected cells (in which there is a deletion of the CtBP-binding site of expression of AdE1A was diminished as expected. These Ad5E1A) again produces the same effect on CtIP data are consistent with results of a quantitative PCR expression nor does the interaction of AdE1A with study in which it was shown that adenovirus genomes CtIP as Ad5dl1101 causes an increase in CtIP expression (measuredas a function of hexon production)were even though deletion at the N terminus and the lack of more abundant in control, infected A549 cells than in CR3 mean that no interaction couldtake place. those treatedwith CtIP siRNA (Figure 8b). Because the sites for CtIP interaction on Ad5E1A overlap those for a number of other well-characterized binding partners, it is not feasible to use mutational The impact of AdE1A on properties of CtIP analysis as a means of determining the specific role of The experiments described above outline certain effects CtIP during viral infection or Ad-mediated transforma- of CtIP on AdE1A attributable to the interaction. In tion. Therefore, the effect of reduction in CtIP expres- complementary experiments, the effects of Ad5E1A on sion, using siRNAs, on Adinfection was assessed two aspects of CtIP biology were examined– the (Figure 8). It was observedthat knock downof CtIP interaction with Rb family proteins andphosphoryla- in A549 cells reduced the rate of expression of AdE1B, tion in response to ionizing radiation (IR). It has E4orf6, L4 andviral structural proteins, following Ad5 previously been shown that CtIP interacts with Rb and

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7473

Figure 6 Complex formation of Ad5E1A, CtBP1 and CtIP. GST- Ad5E1A or GST-CtBP2 was incubated with [35S]-labelledCtIP in the presence of increasing concentrations of a peptide containing the PLDLS motif. Protein mixtures were incubatedwith glu- tathione agarose beads. The level of bound GST-Ad5E1A or GST- CtBP2 is shown by SDS–PAGE andstaining for total protein (i). 35 Bound[ S]-CtIP was detected by fluorography and autoradio- Figure 7 The effect of CtIP on Ad5E1A transactivation. CtIP or graphy (ii). AdE1A, adenovirus oncoprotein early region 1A; CtBP expression was reduced in A549 cells using appropriate CtBP2, C-terminal-binding protein; CtIP, C-terminal-binding siRNAs. Control cells were transfectedwith a nonspecific control protein interacting protein; GST, glutathione-S-transferase. siRNA. After 3 days, cells were transfected with pcDNA 3-Gal4 DBD anda Gal4-responsive luciferase reporter, pcDNA-Gal4 DBD-CR3 anda Gal4-responsive luciferase reporter or VP16 DBD p130 (Meloni et al., 1999). It can be seen from the anda Gal4-responsive luciferase reporter. ( a) At 24 h post- western blots in Figure 9a that immunoprecipitation of transfection luciferase activity was determined. (b) Western blot CtIP results in co-immunoprecipitation of Rb andp130. showing reduction of CtIP expression (upper panel) and CtBP expression (lower panel) by siRNA. AdE1A, adenovirus oncopro- No interaction with p107 was detected (data not shown). tein early region 1A; CtBP2, C-terminal-binding protein; CtIP, C- However, viral infection andsubsequent expression of terminal-binding protein interacting protein; DBD, DNA-binding AdE1A disrupts the Rb/CtIP interaction (Figure 9a). To domain; GST, glutathione-S-transferase; siRNA, small interfering determine whether this effect was due to the binding of RNA. AdE1A to CtIP or Rb MCF7 cells were infected with Ad5dl1108, which has a deletion in CR2 and does not bindto the Rb family proteins. It can be seen that there Discussion is little difference between samples infected with dl1108 and dl520 (Figure 9b). This strongly suggests that CtIP was originally isolatedas a bindingpartner for targeting of Rb by AdE1A has relatively little effect CtBP1 (Schaeper et al., 1998). Since then it has been on the level of CtIP/Rb complex but that it is mainly shown to be involvedin the cellular response to DNA disrupted by binding to CtIP. In the case of these double-strand breaks through its association with viruses, this interaction is through sequences in the N- BRCA1 andits phosphorylation by ATM. It is also terminal region, as they both express Ad512SE1A. The involvedin the transcriptional repression by the Rb difference between the effect of viruses expressing 12S family possibly by recruitment of CtBP andassociated and13SE1A (Figure 9a), which disrupts virtually all of HDACs (reviewedby Chinnadurai,2006b). It has the complex and dl520 (Figure 9b) is presumably due to recently been shown that knockout mice, which do not the additional CtIP-binding site in CR3. dl1520 was express CtIP die at an early stage of development, usedin the experiment shown in Figure 9a to obviate probably due to aberrant cell cycle regulation (Chen any possible AdE1B effects and reduce viral replication. et al., 2005). It has been surmisedthat CtIP expression is In a further experiment, the effect of AdE1A on CtIP necessary for correct phosphorylation of Rb and phosphorylation following IR was examined. A549 cells subsequent cell cycle progression. andcomparable cells stably expressing either 12S or 13S The association of AdE1A with CtBP has been long Ad5E1A were exposed to IR (20 Gy) and then harvested established(Boyd et al., 1993). We now present evidence at the times shown (Figure 10). It can be seen that CtIP to demonstrate a direct interaction between AdE1A and is hyperphosphorylatedin A549 cells in the first hour CtIP. This has been confirmedby a number of after irradiation (upper panel). However, both 12S and approaches. The two proteins co-immunoprecipitate 13SE1A inhibit substantially the phosphorylation reac- (Figure 1) andinteract after in vitro transcription – tion probably through direct interaction with CtIP translation confirming direct binding (Figure 2). Inter- (lower panels). estingly, the proteins make two contacts – binding

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7474

Figure 8 The effect of CtIP knockdown of Ad5 infection. A549 cells were transfected with appropriate small interfering RNAs to reduce expression of CtIP. After 72 h cells were infected with Ad5 wt (2 pfu/cell). Cells were harvestedat the times shown. ( a) Western blotting was carriedout for expression of viral proteins, CtIP and b-actin as shown. (b) Quantitative PCR was carriedout as described in the Materials andmethodssection. Virus particles/cell in control andCtIP knockdowncells are plottedagainst time of infection. CtIP, C-terminal-binding protein interacting protein.

through the N-terminal region of AdE1A and CR3 at the N terminus of AdE1A so far examined (Rasti (Figures 2, 4, 5 andSupplementary Figure 2; summar- et al., 2005 andFigure 5a). When consideredagainst this izedin Table 1). Although a large number of proteins background, the binding pattern of CtIP to mutated bindclose to the N terminus of AdE1A, there appear to Ad5E1A in vitro resembles that seen with S8 andTBP be subtle differences in the binding sites. For example, it (Rasti et al., 2005). In particular, mutations in amino has previously been shown that mutation of I5 to glycine acids 5–7, 11–12, 19–20, 23–24 and 28–30 all interfere has relatively little effect on CBP/p300 binding in vitro, with binding even though none of these is predicted to but completely disrupts interaction with P/CAF (Rasti disrupt the a-helix which is considered to extend from et al., 2005). Similarly, mutation of D21 to alanine residue 10 to 30. disrupts AdE1A’s interaction with Ran, but not with P/ The second major binding site on AdE1A for CtIP is CAF, CBP/p300, hGCN5, TBP andS8. However, located in the CR3 transactivation domain. It is difficult substitution of L19 and/or L20 negates all interactions to account for the rather markeddifferences seen in the

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7475

Figure 10 Ad5E1A inhibits phosphorylation of CtIP. A549 cells and A549 cells stably expressing Ad512SE1A or Ad513SE1A were subjectedto IR (20 Gy). Cells were harvestedat the times shown andwestern blottedfor CtIP and b-actin. PhosphorylatedCtIP can be seen as a slower migrating band. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein.

Figure 9 Ad5E1A disrupts complexes between CtIP and Rb and constitute part of the binding site in CR3 for MED23, a p130. (a) MCF7 cells were mock infectedor infectedwith dl1520 (20 pfu/cell) for 24 h. CtIP was immunoprecipitatedusing a goat component of Mediator complex (Boyer et al., 1999; antibody. After fractionation by SDS–polyacrylamide gel electro- Wang andBerk, 2002). Interestingly, the site of phoresis samples were western blottedfor Rb or p130 as shown, ( b) interaction with MED23 largely overlaps that observed as for (a) except cells were infectedwith dl520 or dl1108 andblotted for CtIP (169–177, Supplementary Figure 2). Mutation for Rb. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C- of Ad5 E1A amino acids 154, 160, 171 and 173–176 terminal-binding protein interacting protein; Rb, retinoblastoma. negates MED23 binding; however, CtIP binds to Y175F, whereas MED23 does not. binding of CR3 regions from different viral serotypes as It is now widely accepted that AdE1A adopts a they are highly homologous; although it is presumed modular structure, such that small deletions introduced that our results are attributable to subtle structural in one region have little or no effect on other regions of differences (Figure 5b). The observation that CR3 the protein andsmall polypeptidescan have similar regions from all AdE1As examined bind to CtIP binding properties to the whole molecule. This is not the indicates a conservation of function, presumably neces- case for CtIP, which probably has extensive secondary sary for optimal activity of AdE1A. Almost all of the andtertiary structure. Therefore, it is not a straightfor- deletions and substitutions introduced into CR3 reduce wardundertaking to map precisely sites of interaction the interaction with CtIP, suggesting that integrity of the for AdE1A. However, it does appear that an N-terminal whole region is requiredfor optimal binding(Supple- region (amino acids 134–282) and a central region mentary Figure 2). However, the major site of interac- (amino acids 371–690) when expressed as GST fusion tion on Ad5E1A CR3 appears to be located between proteins will bindto Ad513SE1A (Supplementary Figure amino acids 169–177, which encompasses the two C- 3). This latter site encompasses the PXDLS CtBP- terminal cysteines involvedin the Zn 2 þ -finger domain binding motif at residues 490–494, while the former (Culp et al., 1988; Webster andRicciardi, 1991). It is includes the LXCXE Rb-binding site (amino acids 153– notable that mutation of the two N-terminal cysteines of 157) andmuch of the proposedsite of interaction with the Zn2 þ -finger (for example, in dl 139–160) do not LM04 (Schaeper et al., 1998; Sum et al., 2002). negate CtIP interaction – therefore it seems likely that From the mutational analysis of AdE1A, it is clear binding of the metal ion per se is not requiredfor the that the interaction with CtIP occurs irrespective of interaction. TBP binds to CR3 through amino acids 147, CtBP binding. In a more detailed study, it has been 150, 157 and171–174 (Geisberg et al., 1994) – the latter demonstrated that the addition of an appreciable molar three residues falling in the site necessary for CtIP excess of CtBP1 does not displace CtIP from GST- interaction. Similarly, mutation of several amino acids Ad5E1A (data not shown). As this is the case, it is within CR3, which do not interfere with TBP binding, possible that ternary complexes comprising AdE1A, blocks transcriptional activation. These amino acids CtBP andCtIP couldexist in vivo. Similarly, the addition

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7476 of a PVDLS-containing peptide, at a concentration directly affect CtIP’s ability to regulate Rb-dependent sufficient to dissociate CtIP and CtBP2 had no effect S-phase entry, perhaps by controlling its phosphoryla- on the Ad5E1A/CtIP interaction (Figure 6). Results of tion (Chen et al., 2005) (Figure 9). Moreover, CtIP’s the ELISA analysis show that the affinity of AdE1A for phosphorylation-dependent association with BRCA1 the larger CtIP polypeptide (324–897) is comparable regulates the G2/M transition checkpoint following to that for CtBP andcan therefore be considereda DNA damage (Yu and Chen, 2004). Until we under- strong interaction. The apparent Kd of the smaller CtIP stand how AdE1A binding impinges on each of these polypeptide (371–620) is significantly greater than that of (andperhaps other) activities, it will not be possible to the larger polypeptide probably due to the absence of the assess the full implications of the AdE1A/CtIP associa- binding site located towards the N terminus. tion, although it is very likely that it helps to optimize The precise biological consequences of the AdE1A/ viral replication through cell cycle control andtran- CtIP interaction are not clear at present. However, scriptional regulation. basedon the CR3-Gal4 study,it is apparent that CtIP binding enhances transcriptional activation by AdE1A CR3 (Figure 7). This is consistent with the observation Materials and methods that reduction in CtIP expression with siRNA retards the expression of viral proteins andthe rate of viral Cell lines and viruses replication (Figure 8). It seems, therefore, that the A549 cells are derived from a human nonsmall cell lung interaction of AdE1A with CtIP is necessary for optimal carcinoma andMCF7 cells derivefrom a human breast CR3-dependent transcriptional activity and ultimately carcinoma. 293 and911 cells are human embryo kidneycells viral infection. Reduction of CtBP levels results in an andembryo retinoblasts, respectively, transformedwith Ad5E1. HER2 and HER10 cells are human embryo retino- increase in CR3-dependent activation (Figure 7a); this blasts transformed with Ad12E1. Ad12E1HLBRK cells are couldbe attributable to the increasedconcentration of Hooded Lister baby rat kidney cells transformed with ‘free’ CtIP. Thus, it is possible to propose a model in Ad12E1DNA and 6f10 cells are similar except for a point which AdE1A dissociates CtBP and CtIP complexes by mutation in the PVDLS motif of Ad12E1A (P-S), such that interaction with CtBP andthen bindsto CtIP to activate it does not bind CtBP (Grand et al., 2006). MCF7 cells were transcription. It is assumedthat the interaction of the N- maintainedin RPM1 supplementedwith 2 m M glutamine and terminal region of AdE1A with CtIP probably fulfils a 8% fetal calf serum (FCS). All other cells were maintainedin different function, possibly the disruption of the CtIP/ Dulbecco’s modified Eagle’s medium supplemented with Rb complex (see below). The induction of CtIP glutamine andFCS. The following viruses were usedin this expression during viral infection (Supplementary Figure study: Ad5dl520, which expresses 12SE1A but not 13SE1A; Ad5dl1135 expresses 12SE1A with a deletion between amino 4) couldbe attributable to a directeffect of E1A on CtIP acids 225 and 238 (the CtBP binding site); Ad5dl1108 expresses transcription or stability, but is more likely due to 12SE1A with a deletion between amino acids 124 and 127 in AdE1A-induced progression of the infected cells into S- CR2; Ad5dl1520 expresses Ad512S and 13SE1A but not the phase. It has been previously reportedthat CtIP E1B55K protein. A549 cells were infectedwith virus at a expression is cell cycle dependent (Yu and Baer, 2000). multiplicity of infection (MOI) of 30 pfu/cell for the induction It has been shown here that AdE1A expression causes of CtIP expression and2 pfu/cell for CtIP-knockdownexpe- disruption of the CtIP/Rb and CtIP/p130 complexes riments andharvestedat appropriate times. MCF7 cells and this appears to be primarily due to binding to CtIP were infectedwith virus at an MOI of 30 pfu/cell for the rather than the pocket proteins themselves, since similar co-immunoprecipitation experiments. effects were observedafter infection with Ad5 dl520 and dl1108; this latter virus expresses an AdE1A, which does Protein expression not bindRb or p130 (Figure 9). It shouldbe notedthat CtIP, Ad513SE1A, TBP, p107, CtBP1 and CtBP2 were transcribedandtranslatedandlabelledwith [ 35S]methionine interaction with CR3 is probably requiredfor maximal using wheat germ or rabbit reticulocyte lysates following the dissociation of the Rb/CtIP complex. manufacturer’s instructions (Promega, Southampton, UK). A It is also apparent that AdE1A inhibits CtIP large panel of Ad5E1A wt and‘mutant’ polypeptideswere phosphorylation following IR (Figure 10). It is not clonedinto pGEX 4T-1 for expression as GST fusions certain whether this is a result of direct interaction of (Table 1). CtBP1, CtBP2 anda panel of CtIP polypeptide AdE1A with CtIP or an indirect action, possibly fragments were also expressedas GST fusion proteins. E. coli through action on ATM (the proposedkinase involved). (BL21) were transformedwith the appropriate pGEX vector Phosphorylation of CtIP regulates interaction with andgrown at 37 1. Protein expression was induced with ITPG BRCA1, andprobably other BRCT-containing proteins (0.5 mM) for 3 h at 301. GST-fusion proteins were purifiedwith (Yu andChen, 2004) andthe G2/M transition glutathione-agarose (Sigma, St Louis, MO, USA) essentially as described by Rasti et al. (2005) andBarral et al. (2005). checkpoint (Yu andChen, 2004). Ad12E1A was expressed in E. coli, purifiedandrenaturedas Because of the complexity of CtIP’s mode of action, it described previously (Grand et al., 1998) is difficult to determine which of its functions are primary targets for AdE1A. The CtIP/CtBP complex Reduction of gene expression by RNA interference plays a role in E2F/Rb-mediated repression and it has Purified, annealed, double-stranded 21-mer RNA oligonucleo- been suggestedthat AdE1Acouldalleviate this repres- tides with dTdT overhangs were purchased from Qiagen sion through dissociation of the complex (Meloni et al., (Crawley, West Sussex, UK). The targetedgene sequence was: 1999). Furthermore, it is possible that AdE1A can CtIP (nucleotides 2719–2739), 50-AAC TGC TTG GGC ACA

Oncogene CtIP binds directly to adenovirus early region 1A RK Bruton et al 7477 CGT GTA, CtBP1 (nucleotides 913–933) 50-CTGGATGTG Analytical methods CACGAGTCGGAA andCtBP2 (nucleotides1765–1795) 5 0- For pull-down assays in vitro [35S]methionine-labelledproteins TCGGATGGTCTTTGTAACTGA. Typically, 5  105 A549 were incubatedwith GST fusion protein (20 mg) in PBS containing cells were transfectedwith siRNA by electroporation (960 mF 2mMEDTA,1%TritonX100,0.5MNaClfor2h.Glutathione- and220 V in 4-mm-deepcuvettes). A nonsilencing siRNA with agarose beads (70 ml) were addedandrotatedfor1 h. After no known homology to any human gene was usedas a washing, boundproteins were elutedwith 25 m M glutathione (pH negative control (QIAGEN). 8.0) andfractionatedby SDS–PAGE. [ 35S]methionine-labelled proteins were detected by fluorography using Amplify (Amersham, Gal 4 reporter assays Little Chalfont, Buckinghamshire, UK) andautoradiography. At 3 days after reduction of CtIP or CtBP expression by RNA Immunoprecipitation experiments were carriedout in 0.15 M interference cells were transfectedwith 1 mgoftheGal4- NaCl, 20 mM Tris–HCl (pH 7.2), 2 mM EDTA, 1% NP40. Cells responsive luciferase reporter together with either 100 ng Gal4- were lysedby homogenization, clarifiedby centrifugation (40 K DBD or 100 ng Gal4-DBD-Ad5 CR3 (all cloned into pcDNA3). r.p.m., 30 min) andincubatedwith antibodies overnight at 4 1. DBD-VP16 was usedas a control. DNA-Lipofectamine Antigen-antibody complexes were isolated using protein complexes were incubatedwith cells according to the manu- G-agarose (Sigma), washedandfractionatedby SDS–PAGE facturer’s instructions (Invitrogen, Paisley, UK). At 24 h post- or on acrylamide gels in the presence of 9 M urea but the transfection, cells were lysedandluciferase activities determined absence of SDS (GrandandGallimore, 1984). according to the manufacturer’s instructions (Promega). ELISAs were performedas describedpreviously (Molloy et al., 1998, 2001). Briefly, Ad12E1A was coated on the ELISA Quantitative PCR plate (0.025 mg/well). GST-CTIP (amino acids 324–896 or 371– Ad5 infected A549 cells (2 pfu/cell) were washed in cold 620), GST-CtBP1 or GST-CTBP2 was added and serially phosphate-bufferedsaline (PBS) andsolubilizedin 200 mlof diluted. Bound fusion protein was detected with goat polyclonal antibody against GST (Amersham). lysis buffer (0.45% NP-40, 0.45% Tween 20, 2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3)). Proteinase K (100 mg) 1 was added and incubated at 55 C overnight, before being Antibodies 1 inactivatedat 95 C for 10 min. The samples were vortexed CtIP was routinely immunoprecipitatedanddetectedin vigorously, and5 ml volumes usedin the PCR reactions as 7 western blots using a goat polyclonal antibody (T16; Santa described below. A549 cells (10 ) were extractedto serve as a Cruz, Santa Cruz, CA, USA). It was also immunoprecipitated quantitative control of cell number. Control Ad5 DNA was using two rabbit polyclonal antibodies (Ab1 and 2) raised isolatedfrom CsCl gradient-purifiedvirus andcopy number against GST-CtIP. Rb was detected with a mouse monoclonal determined with DNA-binding PicoGreen (Invitrogen) (Mura- antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) kami andMcCaman, 1999). andp107 (C18) andp130 (C20) with rabbit polyclonal Amplification of DNA was by real-time monitoring of antibodies (Santa Cruz) Ad5E1A was immunoprecipitated changes in fluorescence intensity using dual-labelled fluoro- anddetectedinwestern blots using the mouse monoclonal genic Taqman probes. Adenovirus genomes were detected antibody M58 (Becton-Dickinson). Ad12E1A was immuno- using hexon-specific forwardprimer 5 0-CCACCCTTCTTTA 0 0 precipitatedwith the mouse monoclonal Mab6A anddetected TGTTTTGTTTGA-3 , reverse primer 5 -GCAGGTACAC in western blots with a mouse monoclonal Mab19. CtBP1 was GGTCTCGATGA-30anddual-labelledfluorogenic probe 5 0- 0 immunoprecipitatedanddetectedin western blots with a (FAM)TCTTGACGTGGTCCGTGTGCACC(TAMRA)-3 . mouse monoclonal antibody (E12) (Santa Cruz). CtBP2 was Cellular genomes were detected using the b2-microglobulin- detected with a mouse monoclonal antibody (Becton-Dick- specific forwardprimer 5 0-GGAATTGATTTGGGAGAGC 0 0 inson). Ad5 structural proteins were detected with a rabbit ATC-3 , reverse primer 5 -CAGGTCCTGGCTCTACAATTT polyclonal antibody (R61/99), Ad5E1B55K with the mouse ACTAA-30 anddual-labelledfluorogenicprobe 5 0-(VIC)A 0 monoclonal 2A6 andL4 with a rat monoclonal antibody. GTGTGACTGGGCAGATCATCCAGCTTC(TAMRA)-3 . b-Actin was detected with a mouse monoclonal (AC74) PCR reactions were preparedin a final volume of 25 ml (Sigma). containing  1 Taqman Universal PCR Mastermix (Applied Biosystems, Foster City, CA, USA) with 300 nM primers and 200 nM for hexon amplification or 300 nM primers and50 n M probe for b2-microglobulin amplification. Thermal cycling Acknowledgements conditions comprised 2 min at 501C, 10 min at 951C and40 rounds of amplification (denaturation for 15 s at 951C and We are most grateful to Professor RichardBaer (Columbia annealing andextension for 1 min at 60 1C). All test samples University, USA) for the CtIP constructs andDr Richard were run in triplicate andtemplate-negative samples servedas Lundblad (Salk Institute, USA) for the gift of His-tagged controls. Fluorescent signals were detected by an ABI CtBP1. We thank Dr Vivien Mautner andDr Thomas Dobner 7500FAST system (AppliedBiosystems). for gifts of antibodies. We also thank Dr Mautner for help The number of hexon and b2-microglobulin copies in the test with the quantitative PCR experiments. We thank Cancer samples was calculatedfrom standardcurves generatedusing Research UK, the Breast Cancer Campaign andthe CIHR for standard DNA. Results were expressed as number of virus generous funding and the Iranian government for a PhD genomes/cell. studentship to MR.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene