Two Independent Regions of Simian Virus 40 T Antigen IncreaseCBP/p300 Levels, Alter Patterns of Cellular Histone Acetylation, andImmortalize Primary Cells

Maria Teresa Sáenz Robles, Chikdu Shivalila,* Jeremy Wano,* Shelly Sorrells,* Alison Roos,* James M. Pipas

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cel-lular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transforma-tion requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expressionof SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Fur-thermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to in-creased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increasedin SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain arise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alterstheir overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT,one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain isnecessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. Thesestudies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms areused by T antigens to induce cell immortalization and transformation.

The large T antigen from simian virus 40 (SV40) (SVT) has beenshown to induce cell proliferation, immortalize primary cells,

and mediate tumorigenesis in numerous in vivo and in vitro sys-tems (1–3). SVT induces transformation by binding to specificcellular target proteins, thus altering pathways that regulate cellproliferation, death, and tissue homeostasis. For example, do-mains located in the carboxy-terminal half of SVT bind and inac-tivate the tumor suppressor p53, thereby blocking its ability toinduce cell cycle arrest and apoptosis. In addition, SVT binding tothe retinoblastoma (Rb) proteins (pRb, p107, and p130) throughan LXCXE motif and to hsc70 via its J domain antagonizes cellcycle exit (4–6). Strikingly, other DNA tumor viruses, such ashuman papillomaviruses and adenoviruses, also encode proteinsthat target Rb and p53, suggesting that these viruses converge on acommon set of cellular targets that are important for transforma-tion.

Disruption of additional cellular targets contributes to the tu-morigenic phenotype induced by SVT (7–12). In particular, SVThas been shown to bind CBP/p300 (13–15), closely related pro-teins that are also common targets of many viruses, including theDNA tumor viruses (16). For instance, the interaction of the ade-novirus E1A protein with CBP/p300 is necessary for transforma-tion (17). The E1A protein alters chromatin acetylation and geneexpression by removing CBP/p300 from the promoters of differ-entiation-specific and antiviral defense genes and positioningthem to activate genes involved in cell proliferation (18). Simi-larly, SVT binding to CBP/p300 is necessary for transformation(1). The association of SVT with CBP/p300 requires p53 and thusis thought to involve contacts of CBP/p300 with both p53 and SVT(1, 19, 20). The papillomavirus E6 protein also interacts with CBP/p300. In this case, E6 blocks the ability of CBP/p300 to acetylatep53 and thereby induces cell cycle arrest and senescence (21).

The CBP and p300 proteins are closely related and act as mo-lecular adaptors, associating with multiple transcriptional regula-tors and signaling molecules and thereby integrating many cellu-lar pathways. In fact, CBP/p300 bind more than 400 cellularproteins (22). In addition, CBP/p300 possess histone acetyltrans-ferase (HAT) activity and can alter gene expression through bothmodification and relaxation of chromatin and recruitment ofbasal and other transcriptional components to promoters (23).CBP and p300 are expressed in almost identical patterns in themouse (24), and homozygous p300 or CBP knockout mice dieduring embryogenesis (25–27). However, CBP and p300 func-tions are not completely redundant. For instance, p300 — but notCBP—is essential for the formation of the heart, small intestine,and lungs during embryonic development (28), and the two pro-teins play different roles in transcription regulation (29). Simi-larly, p300 and CBP play different roles during myogenesis, hema-topoietic differentiation, and stem cell renewal in mice (30, 31). Inaddition, p300 and CBP play different roles during differentiation,cell cycle exit, and apoptosis of embryonal carcinoma cells in cul-

Received 16 September 2013 Accepted 27 September 2013

Published ahead of print 2 October 2013

Address correspondence to James M. Pipas, pipas@pitt.edu.

* Present address: Chikdu Shivalila, Whitehead Institute for Biomedical Research,Cambridge, Massachusetts, USA; Jeremy Wano, The Robert Schattner Center,University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania,USA; Shelly Sorrells, Department of Oncological Sciences, Huntsman CancerInstitute, University of Utah, Salt Lake City, Utah, USA; Alison Roos, Department ofMolecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ture (32), and each protein seems to preferentially acetylate spe-cific lysine residues in certain histones, thus differentially regulat-ing chromatin activity (33).

To date, the effects of SVT on the activity of CBP/p300 arecontroversial: SVT has been shown to repress the transcriptionalactivity of CBP/p300 (34), but the overall HAT activity associatedwith those adaptors seems to increase in the presence of SVT (35).These differences could be attributed to genetic or epigenetic al-terations acquired during the establishment of cell lines used inthe different experiments. We have therefore used primary cells toevaluate the effects of SVT and SVT mutants on the CBP/p300pathway and to explore the molecular level at which SVT affectsthese epigenetic regulators.

MATERIALS AND METHODSIsolation of primary fibroblasts and cell culture conditions. Mouse em-bryo fibroblasts (MEFs) were harvested from 13.5-day-old FVB embryosas previously described (36) and grown at 37°C in 5% CO2 in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10% heat-inacti-vated fetal bovine serum, 100 U/ml penicillin, and 100 �g/ml streptomy-cin. MEF lines expressing different SVT mutants were previously gener-ated (37, 38).

Pools of MEFs expressing the early region of different viruses (SVT,BKVT [BK virus large T antigen], JCVT [JC virus large T antigen], andLPVT [lymphotropic papovavirus large T antigen]), truncated SVT mu-tants (SVTN136 and SVTC257-708), and SVT small or point mutations(N136D44N, N136dl89-97, N136F98A, N136E9K, N136K53R, N136D2K, andN136E107,108K) were obtained by infection with retroviral (pBabePuro) orlentiviral (pL6.3; Invitrogen) constructs encoding the different mutantsunder the control of the CMV promoter. In particular, mutations disrupt-ing the J domain and binding to hsc70 (D44N and K53R), unable to bindpRb proteins (E107 and 108K), unable to interact with Cul7 (F98A), orimpaired in binding Bub1 (dl89-97) and new mutations within the ami-no-terminal region of SVT (D2K and E9K) were used in this study. Uponinfection, cells were selected with the appropriate antibiotic (puromycin,2 �g/ml; blasticidin, 2.5 �g/ml) and tested for T antigen expression andsurvival after passage in culture.

To test stress conditions, cells in culture were subjected to DNA dam-age by treatment with doxorubicin (adryamicin; 0.5 �g/ml; 6 h), starva-tion in serum-free medium (6 h), or exposure to heat (42°C; 2 h). Aftereach treatment, the cells were washed with phosphate-buffered saline(PBS) and collected to obtain protein or RNA samples by standard meth-ods. When indicated, the inhibitor MG132 (10 �M) was added to the cellculture media for 6 h to block proteasome-mediated degradation, andcycloheximide (50 �g/ml) was used to block de novo protein synthesis.

Preparation and infection of hepatocytes. Hepatocyte preparationwas adapted from the protocol described by Gibco Invitrogen for theirhepatocyte product line in order to use embryonic tissue. Briefly, liversfrom embryonic day 18.5 (E18.5) stage mouse embryos were isolated anddigested with Liver Digest Medium (Invitrogen) for 30 min at 37°C, fil-tered through a 100-mm mesh screen, and washed three times with hepa-tocyte wash medium (Invitrogen). The resulting hepatocytes were platedonto collagen-coated plates (BD Biosciences) in Williams E medium sup-plemented with penicillin/streptomycin and 10% fetal bovine serum andkept at 37°C with 5% CO2. After 48 h, 5 � 106 cells were infected with aretroviral construct encoding the SVT early region in the presence of 8�g/ml of Polybrene, selected with puromycin (2 �g/ml), and collected foranalysis.

Protein analysis and immunofluorescence experiments. Protein ex-traction from and Western blot assays of cells in culture or murine tissueswere performed using standard procedures. Each Western blot experi-ment was performed at least 2 times and in most cases 3 to 6 times. Toanalyze protein expression under the microscope, MEFs were grown toconfluence on glass micro-cover slides (VWR Scientific), washed three

times with cold PBS, and fixed with 4% formaldehyde solution for 30 min.Every subsequent step was followed by 3 washes with cold PBS: (i) block-ing for 1 h in PBS supplemented with 3% bovine serum albumin (BSA)and 0.2% NP-40, (ii) incubation with appropriate dilution of primaryantibody in PBS for 24 h at 4°C, and (iii) incubation with the appropriateAlexa Fluor 488- or Alexa Fluor 568 (Invitrogen)-conjugated secondaryantibody (1:500) in PBS for 1 h at room temperature. The slides werecounterstained with 4=,6-diamidino-2-phenylindole (DAPI) andmounted for analysis by fluorescence microscopy. Each immunofluores-cence experiment was performed at least twice, and 3 to 4 times in mostcases.

Primary antibodies used. Monoclonal antibodies recognizing T anti-gen (416 and 419) and p53 (421) have been described previously (39). Thefollowing rabbit polyclonal antibodies were from Santa Cruz Biotechnol-ogy, Inc.: p107 C-18 (sc-318), p130 C-20 (sc-317), hsp70 K-20 (sc-1060),CBP A-22 (sc-369), �-tubulin D-10 (sc-5274), and p53 FL-393 (sc-6243).GAPDH-mouse monoclonal G8140-11 was from United States Biologi-cal, Swampscott, MA, USA. Anti-acetyl-histone H3 (Lys56) cloneEPR996Y and p300 mouse monoclonal clone RW128 were purchasedfrom Millipore. The following rabbit polyclonal antibodies were obtainedfrom Cell Signaling Technology Inc.: acetyl-CBP (Lys1535)/p300(Lys1499), number 4771; acetyl-histone H2A (Lys5), number 2576; his-tone H2B (V119), number 8135; acetyl-histone H2B (Lys5), number2574; histone H3 (D1H2) XP, number 4499; acetyl-histone H3 (Lys9)(C5B11), number 9649; acetyl-histone H4 (Lys5), number 9672; acetyl-histone H4 (Lys8), number 2594; and acetyl-histone H4 (Lys12) antibody,number 2591. Anti-acetyl-histone H3 (Lys18) was a generous gift fromRoberto Ferrari and has been described previously (40).

Polysome preparation and quantitative real-time PCR analysis. Pre-ribosomes, ribosomes, and polyribosomes were obtained according topreviously published protocols (41) with some modifications. MEFsgrowing in complete medium were allowed to become postconfluent,treated for 10 min with cold PBS-EDTA buffer containing cycloheximide(100 mg/ml), trypsinized, and collected. After disruption in lysis buffer(10 mM Tris, pH 7.5, 100 mM KCl, 10 mM MgCl2, 5 mM dithiothreitol[DTT], 100 mg/ml cycloheximide, 0.05% NP-40), the extract was shearedand debris was removed by centrifugation. Ribosomal components in thesupernatant were separated on 10 to 45% sucrose gradients by centrifu-gation at 27,000 rpm for 4 h at 13°C in an SW28 rotor (Beckman Instru-ments, Inc., Fullerton, CA). A Teledyne ISCO Foxy R1 density gradientfractionator was used to fractionate and analyze the gradients. All frac-tions containing polysomes were collected and pooled. Total RNA wasextracted from the pools by standard techniques, using Invitrogen puri-fication columns. Obtention of RNA and cDNA and transcript quantifi-cation by real-time PCR analysis were done as described previously (42)with primers specific for CBP (pair 5=-ACCCCAAACGAGCCAAACTC and5=-ACGCAGCATCTGGAACAAGG and pair 5=-TCCTTACGCCGCTCCAAATG and 5=-GCCCCCACTTCACCATCTTATG), p300 (pair 5=-TTTCTGTTGAGTCCGCATCCC and 5=-CAAAATCTGTGCCATCGCTGG and pair5=-CCAGCAAAAACAAGAGCAGCC and 5=-ATTGGGAGCAGGACAAGCGATG), mcm3 (5=-CGCAGGAAGAATGAAAAGAGG and 5=-CTGAGGAAGCAGGAAGTGAGA), p21 (5=-AGCCTGAAGACTGTGATGGG and5=-AAAGTTCCACCGTTCTCGG), thymidine kinase (TK) (5=-GAGAAAGAGGTGGAGGTGATT and 5=-CAAGAAGGGAACTGAAAACGG), Adh(5=-ATGACAGATGGGGGCGTGGATTAC and 5=-TGGAATGGACGAGTGGAGATTTC), and Rpl5 (5=-CCAAACGATTCCCTGGTAATGAC and 5=-GACGATTCCACCTCTTCTTCTTCAC). The results were normalizedagainst endogenous controls (Adh and/or Rpl5). Five different polysomepreparations—biological replicates—were obtained from control or SVT-expressing cells and one from SVTN136- and SVTC257-708-expressing cells.Each preparation was tested by real-time PCR at least once, and on threeoccasions twice. Within each real-time experiment, each experimental pointwas tested with four technical replicates. The results were analyzed with the7300 System SDS RQ Study Software (Applied Biosystems), which provided

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the RQ (mean expression level) and RQ min/RQ max (calculated standarderror from RQ) values.

RESULTSExpression of SV40 T antigen causes increased steady-state lev-els of CBP/p300. We have previously observed that several estab-lished cell lines express high levels of CBP/p300. However, severaltypes of nontransformed or primary cells contain low levels ofCBP and p300 proteins, as determined by Western blot analysis(Fig. 1A, B, D, and E). They include an established line of ratembryo fibroblasts (REF52), as well as cells obtained directly frommurine embryonic (MEFs and hepatocytes) or adult (enterocytesfrom intestinal villi) tissues. In contrast, expression of SVT inthose cells results in a marked increase of the CBP/p300 proteinlevels (Fig. 1A, B, D, and E). The increase of CBP/p300 in responseto SVT is not simply due to a higher proliferative status of the cellsor their level of confluence, as highly proliferative, underconfluentcontrol MEFs present much lower levels of CBP/p300 than similarcells expressing SVT (Fig. 1A). Furthermore, either subconfluent,confluent, or postconfluent cells expressing SVT show high levelsof CBP/p300, in contrast with control counterparts (Fig. 1A), sug-gesting that contact with other cells— or lack of it— does not playa role in controlling CBP/p300 protein levels. The increase inCBP/p300 levels is not due to the method used to introduce theSVT into cells, as cells expressing SVT after transfection (Fig. 1A

and B), viral infection (Fig. 1C), DNA integration in transgenicmice (Fig. 1E), or retroviral transduction (Fig. 1D and data notshown) show high levels of CBP/p300 in comparison to nonex-pressing counterpart cells.

The manipulations used to express ectopic products in cellscan potentially induce abnormal stresses that might influencethe status of cellular components. Thus, we studied the effectsof different types of stress on levels of CBP/p300 in controlMEFs or those expressing SVT. Neither inducing DNA damagewith doxorubicin, starving the cells (Fig. 2A), nor increasingthe temperature of incubation (Fig. 2B) raised the levels ofCBP/p300 in control MEFs. Similarly, cells expressing SVTshow high levels of CBP/p300 under all conditions tested. In-duction of DNA damage was confirmed by monitoring the in-crease of p53 in cells treated with doxorubicin, and the effec-tiveness of the heat shock treatment was confirmed byobserving the rise of hsp70 levels in heat-treated cells. In addi-tion, the method used to collect cells for protein analysis, byeither trypsinization, direct lysis in cell culture plates, or use ofcell scrapers, did not have any noticeable effect on the steady-state levels of CBP/p300 (data not shown).

Acetylation levels of CBP/p300 and specific histone residuesare increased in cells expressing SV40 T antigen. CBP/p300 pos-sesses acetyltransferase activity (43, 44) and can add acetyl resi-

FIG 1 CBP and p300 steady-state protein levels increase in cells expressing SV40 T antigen. (A) Steady-state levels of CBP and p300 in control orSVT-expressing MEFs grown to different levels of confluence as shown by Western blotting with specific antibodies. The results were confirmed in at leasttwo cell lines/pools of each kind. The increase in CBP/p300 levels is even more noticeable between control and SVT-expressing cells in 2-day-postcon-fluent MEFs. (B) Steady-state levels of CBP and p300 in REF52 cells, monitored by Western blotting. Expression of SVT results in increased levels of bothproteins in 2-day-postconfluent cells. (C) Protein extracts were obtained from cells 4 days postinfection with SV40 viruses and studied by Westernblotting. Levels of CBP and p300 increase in cells infected with SV40 particles. (D) Embryonic hepatocytes show increased levels of CBP/p300 upon stableSVT expression. (E) The effect of SVT on CBP/p300 is not restricted to cell culture. Shown are steady-state levels of CBP and p300 in villus-enriched cellpopulations from control or transgenic mice expressing SVT in the intestinal epithelium. Expression of SVT correlates with a significant increase in CBP,p300, and CBP/p300 autoacetylation levels. The levels of p130 and p107 were monitored to ensure functionality of the T antigen. Ac, acetyl. The black linesindicate composite images.

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dues to multiple proteins, including transcription factors and hi-stones (43, 45–49), thus possibly controlling gene activityepigenetically. We hypothesized that the SVT-induced increase inCBP/p300 levels might change gene regulation through modifica-tion of the epigenetic code. We first used an antibody that detectsendogenous levels of CBP or p300 only when acetylated at lysine1535 or lysine 1499, respectively. Posttranslational acetylation ofp300 in specific lysine residues within an activation loop motif,including Lys1499, has been linked to an increase in its acetyl-transferase activity (50). Consistent with this hypothesis, theincreased levels of CBP/p300 in cells expressing SVT correlatewith increased CBP/p300 acetylation levels, as determined byimmunofluorescence (Fig. 3A) and Western blot analysis (Fig.3B), suggesting increased activity of these proteins in the pres-ence of SV40 T antigen. Acetylation of histones by CBP/p300has been associated with transcriptional activation, histone de-position, and DNA repair (43, 51, 52), and thus, we used apanel of antibodies recognizing specific acetylated residues indifferent histones. In contrast with other viral proteins, likeadenovirus E1A, which have been shown to induce a drasticreduction in the acetylation of cellular histone H3 lysine 18(H3K18ac) (40), SVT does not alter the acetyl levels of H3K18or other specific histone residues, including H4K8 (Fig. 3C andD) and H2AK5, H2BK5, H3K9, and H4K5 (Fig. 3D). However,cells expressing SVT show a significant increase in the levels ofH3K56 (Fig. 3D and 4B) and H4K12 (Fig. 3D and 4C).

Polyomavirus large T antigens increase the acetylation levelsof CBP/p300 and specific histone residues in primary MEFs.While SV40 is one of the best-characterized polyomaviruses, all

members of this group encode large T antigens. Therefore, wenext examined if the T antigens encoded by other members of thePolyomaviridae induce high CBP/p300 levels. We observed thatMEFs expressing BKVT, JCVT, or LPVT also increase the steady-state levels of CBP/p300 (Fig. 4A and C). Also, similar to that ofSV40, these T antigens induce increased levels of H3K56 (Fig. 4B)and H4K12 (Fig. 4C). Interestingly, the murine polyomaviruslarge T antigen does not show this effect (data not shown). Theseresults suggest that polyomavirus T antigens increase protein lev-els and autoacetylation of CBP/p300 and regulate chromatin acet-ylation.

Two independent functions encoded in separate regions ofSVT affect CBP/p300. Next, we examined a series of SVT mutantsfor the ability to increase CBP/p300 levels and autoacetylation, aswell as for changes in histone acetylation. Previously describedmutants, as well as newly generated truncation mutants, were usedto explore the effects on CBP/p300 levels (Fig. 5D) (see Materialsand Methods). Several characterized amino acid substitution mu-tants generated in the context of full-length SVT (ptc1, ptc2,E107,108K, and D44N) retained the ability to increase CBP/p300(Fig. 5A). Furthermore, MEFs stably expressing different trun-cated versions of SVT encompassing either the N terminus (N136and N625) or the C terminus (C257 to C708) of SVT show in-creased levels (Fig. 5A and B) and autoacetylation (Fig. 5C) of CBPand p300. We conclude that two independent functions encodedin separate regions of SVT affect CBP/p300 levels and autoacety-lation.

The SVT effect on the steady-state levels of CBP/p300 is me-diated through preferential association of CBP/p300 transcriptswith polysomes. Gene expression profiling experiments withMEFs indicated no significant changes in the transcriptional levelsof CBP or p300 mRNAs either after SV40 infection or upon ex-pression of SVT (53). We confirmed this result using CBP- andp300-specific primers by RT-PCR analysis of cDNAs from SVT-expressing MEFs and REF52 cells (Fig. 6A) or by real-time PCRanalysis of MEFs (not shown). Thus, changes in CBP/p300 levelsare not due to increased transcription triggered by SVT expres-sion. Next, we determined the effect of SVT on CBP/p300 steady-state levels in the presence or absence of MG132, a potent inhibi-tor of the proteasome. Neither control (DMSO) nor MG132treatment altered the levels of CBP/p300 with or without SVTpresent in 2-day-postconfluent cells (Fig. 6B), while increased lev-els of hsp70 in the presence of MG132 indicated the effectivenessof the treatment. Proteasome inhibition of underconfluent, ac-tively growing cells produced a similar outcome (not shown).These results indicate that SVT does not increase CBP/p300 levelsby preventing proteasome-mediated degradation. Furthermore,SVT does not alter the cytoplasmic/nuclear distribution of CBP/p300 transcripts (not shown); thus, differences in the transport ofspecific RNAs do not account for the observed increases in CBP/p300 protein levels in the presence of SV40 T antigen. In addition,treatment with cycloheximide does not significantly alter theamounts of CBP/p300 proteins either in control or in SVT-ex-pressing MEFs (Fig. 6C). Our data indicate that protein degrada-tion does not account for the SVT-mediated increase of CBP/p300steady-state levels.

Finally, we examined the association of CBP/p300 transcriptswith actively translating polysomes. Although CBP/p300 tran-scriptional levels remain similar in control and SVT-expressingcells, the amount of CBP/p300 RNAs present in polysomes is sig-

FIG 2 Different types of stress do not affect the steady-state levels of CBP/p300. Mouse embryo fibroblasts were treated with different stress inducers 2days after reaching confluence. The corresponding protein extracts were re-solved in SDS-PAGE gels and analyzed with different specific antibodies. Nei-ther DNA damage by treatment with doxorubicin, starvation in serum-freemedium (A), nor exposure to heat shock (B) affects the levels of CBP/p300with or without SVT present in the cells.

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nificantly increased in SVT-transformed cells (Fig. 6D). Remark-ably, a higher association of CBP and p300 transcripts with poly-some populations is similarly observed in both SVTN136- andSVTC257-708-expressing cells (Fig. 6D), both of which also expressconsiderably higher levels of CBP/p300 proteins. In contrast, wefound p21 transcripts associated with polysomes, as expected, ac-cording to their abundance in different cells: p21 is a p53 targetand thus is upregulated in response to disruption of the RB/E2Fpathway by SVTN136, but p21 transcripts remain low in controlcells or those expressing SVT or SVTC257-708 (Fig. 6D), as bothproteins can bind and prevent p53-mediated upregulation of p21.In addition, we examined transcripts normally unchanged (glyc-eraldehyde-3-phosphate dehydrogenase [GAPDH]) or overex-pressed (mcm3 and TK) in the presence of SVT, and in each case,we found that they associate with polysomes, as expected (data notshown). Our results indicate that SV40 T antigen controls CBP/p300 levels through an increase in their overall translation.

A functional J domain within the amino-terminal region ofSVT is required to raise CBP/p300- and histone-specific acety-lation levels and to immortalize MEFs in culture. As two inde-pendent SVT truncations are capable of raising CBP/p300 levels ina similar way, we initiated the characterization and mutagenesis ofthe amino-terminal region and assessed the abilities of different

mutants to raise the levels of CBP/p300. In the context of theSVTN136 truncation, most of the individual mutations tested donot preclude SVT from increasing the steady-state levels of CBP/p300, even though the mutations render proteins unable to inter-act with Bub1 (N136dl89-97), Cul7 (N136F98A), or the pRB proteins(N136E107,108K). In contrast, mutations precluding interactionwith hsc70 (N136D44N and N136K53R) completely abolish the abil-ity of T antigen to raise the steady-state levels of CBP/p300 (Fig.7A). This phenomenon is not due to a general block or disruptionof the J domain, as only mutations between helix II and helix III(N136D44N) or in helix III (N136K53R) of the J domain, but not inother parts of the structure (N136D2K and N136E9K), prevent the Tantigen-mediated increase in the steady-state levels of CBP/p300(Fig. 7A). Moreover, different mutations within the amino termi-nal region of SVT are still capable of immortalizing cells in culture,but mutations inactivating the J domain of the protein and pre-cluding interaction with hsc70 completely abolish the capacity ofSVTN136 to immortalize MEFs, and the cells die after 4 or 5 pas-sages (Fig. 7B). Mutations in the J domain that do not abolishinteraction with hsc70 (N136D2K and N136E9K) retain the abilityto immortalize primary cells and to increase CBP/p300 levels. Fi-nally, we investigated if a possible connection exists between theSVTN136-mediated increases in p300 levels and the acetylation lev-

FIG 3 Cells expressing SVT show increased acetylation levels of CBP (Lys1535) and/or p300 (Lys1499) and correlate with increased acetylation of specifichistone residues. (A and B) Immunofluorescence with specific antibodies indicates an increase in the steady-state levels of CBP and p300 and the CBP(Lys1535)/p300 (Lys1499) acetylation in postconfluent MEFs expressing SVT (A), a result confirmed by Western blot analysis of postconfluent MEFs (B).(C and D) Concomitantly, SVT-expressing cells show increased levels of specific histone acetylation residues (H3K36 and H4K12) with respect to controlcells, as determined by immunofluorescence (C) and Western blotting (D). Acetylation of other specific histone residues (H2AK5, H2BK5, H3K9, H3K18,H4K5, and H4K8) and histones overall (H2B, H3, and H4 general acetylation) remains unaltered in the presence of SVT. wt, wild type. The black linesindicate composite images.

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els of specific histone residues. In fact, MEFs expressing amino-terminal truncation mutants able to increase p300 steady-statelevels (N625 and N136E9K) are still able to raise the levels of H3K56and H4K12 acetylation (Fig. 8). However, expression of N136 mu-tants lacking a functional J domain (N136D44N and N136K53R) is

not sufficient to raise the levels of those specific histone acetyla-tions (Fig. 8). We conclude that the ability of the amino-terminal136 amino acids of SVT to immortalize cells, to increase CBP/p300 levels, and to control acetylation of specific histone residuesrequires a fully functional J domain.

FIG 4 Expression of different polyomavirus large T antigens results in increased specific CBP/p300 acetylation and raised levels of H3K56 and H4K12.Postconfluent MEFs expressing large T antigens from SV40, BKV, JCV, or LPV were analyzed by Western blotting and immunofluorescence with antibodiesrecognizing specific acetylated lysine residues. Expression of each of these polyomavirus T antigens produced a significant increase in both p300 and theautoacetylated form of CBP/p300 (A and C), as well as a rise in the acetylation of the specific histone residues H3K56 (B) and H4K12 (C). Staining of cellsexpressing different polyomavirus T antigens was detected with antibodies against SVT, and the exposure time was adjusted according to the intensity of thesignal observed. None of the available antibodies to detect T antigen cross-react with LPVT. The black lines indicate composite images.

FIG 5 Mutational analysis of SVT effects on CBP/p300. (A) Mutations within full-length T antigen do not abolish its ability to alter the levels of CBP/p300, asMEF mutants affecting the J domain (D44N), binding to Rb proteins (E107 and 108K), or binding to p53 or CBP/p300 (ptc1 and ptc2) or lacking expression ofsmall t (dl1140) raise the levels of CBP/p300 to similar extents as full-length SVT. (B and C) Two truncations of T antigen, SVTN136 and SVTC257-708, are able toincrease the steady-state levels of CBP/p300 (B) and autoacetylation (C). �-Tubulin was used as a loading control; levels of p107 and p130 were monitored toensure functionality of the tested T antigens. (D) Summary of SV40 T antigen mutants. Functional domains of SV40 T antigen are indicated above the depictionsof amino- and carboxy-terminal truncations, as well as the locations (�) of small and point mutations used in the study. OBD, origin binding domain; HR, hostrange domain. The black lines indicate composite images.

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DISCUSSION

DNA tumor viruses encode oncoproteins that act by binding keycellular regulatory proteins and thereby activating, inhibiting, orredirecting their functions. SVT-mediated transformation re-quires binding to both the retinoblastoma family of tumor sup-pressors (pRb, p130, and p107) and p53. The first interaction an-tagonizes Rb proteins’ ability to act as transcriptional repressors,thus increasing the expression of cell proliferation-related genes,and binding to p53 blocks their ability to bind to promoters andstimulate expression of growth arrest and cell death genes. Simi-larly, the E1A and E1B proteins of adenoviruses and the E7 and E6proteins encoded by papillomaviruses bind and inactivate Rb pro-teins and p53. In addition, adenoviruses and papillomavirusestarget the transcriptional adapter proteins CBP/p300, and in bothcases, this interaction is required for transformation.

CBP/p300 are highly similar multifunctional proteins that in-teract with numerous cell components to control multiple cellularpathways. The proteins are both transcriptional coactivators andcontain intrinsic cytoplasmic and HAT activities (43, 44, 54–56).CBP/p300 are used as scaffolds, coupling chromatin remodelingto the transcriptional machinery and playing essential roles ingrowth control and regulation, development, and homeostasis(23, 43, 52). Furthermore, CBP/p300 might play a role in tumor

suppression (27, 57, 58), and the fact that mutations of p300 re-sulting in loss of acetyltransferase activity have been found in pri-mary tumors, as well as in cancer cell lines (57), suggests thatacetylation of cellular proteins is a critical signal in cell growth.CBP/p300 mediate the effects of numerous cellular proteins, in-cluding transcription factors like c-myb (59) and E2Fs (60, 61),and several viral proteins target CBP/p300 to manipulate the hostmachinery in their favor (62–65).

Previous studies have shown that SVT binds CBP/p300 andthat SVT complements adenovirus E1A mutants defective forCBP/p300 association in transformation assays (66). The associa-tion of SVT with CBP/p300 requires SVT binding to p53, andfurthermore, the formation of the SVT-p53-CBP/p300 complexresults in the acetylation of p53 on K373 and of SVT on K697 (19,20). The effect of SVT on CBP/p300 activity has been the subject ofcontroversy. One study reports that SVT represses CBP/p300transcriptional activities, while another concludes that SVT en-hances the histone acetyltransferase activities of these proteins(34, 35). We have previously shown that interaction with CBP/p300 plays an essential role in SVT-mediated transformation (38).Here, we show that cells expressing SVT accumulate high levels ofhyperacetylated CBP/p300 and that SVT-expressing cells also ex-hibit increased acetylation of histones H3K56 and H4K12, modi-

FIG 6 Mechanism of action of SVT on CBP/p300. (A) CBP and p300 transcript levels are not increased upon expression of SVT. RNA was extracted from2-day-postconfluent MEFs, and following cDNA synthesis, specific primers were used to evaluate transcriptional activity by semiquantitative RT-PCRs (shown)and real-time PCR analysis (not shown). Ribosomal protein 5 (Rpl5) was used as an internal control. (B) Inhibition of the proteasome pathway does not affectCBP/p300 steady-state levels in either the absence or presence of SVT. Confluent control and SVT-expressing MEFs were treated with 10 �M MG132 to preventproteasome-mediated degradation. Samples were collected at the indicated time points, and protein levels were analyzed by immunoblotting. Hsp70 was used asa control of treatment, and �-tubulin was used as a loading control. (C) Treatment with cycloheximide does not alter the steady-state levels of CBP/p300.Subconfluent SVT-expressing or control MEFs were treated or not with cycloheximide for different time intervals, and protein extracts were analyzed by Westernblotting. No significant changes in the steady-state levels of CBP/p300 were observed at any point. (D) Presence of specific transcripts in polysomal populationsfrom control and SVT-expressing cells. Polysomes were isolated, and total RNA was extracted from control and SVT-expressing cells. The levels of specifictranscripts were determined by real-time PCR and normalized against the levels of control, nonchanging transcripts (e.g., Adh and Rpl5) present in the samesamples (RQ values). The error bars indicate four replicates from a single representative experiment. Despite showing similar transcriptional levels in all cellstested, the association of CBP and p300 transcripts with polysome populations is increased in cells containing full-length SVT, SVTN136, or SVTC257-708. SeeResults for a description of the association of other transcripts with polysomes.

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fications associated with CBP/p300 HAT activity. The T antigensencoded by the human polyomaviruses BKV and JCV or by themonkey virus LPV also induce high CBP/p300 levels and enhanceH3K56 and H4K12 acetylation, suggesting that these effects are acommon feature of polyomavirus T antigens.

To characterize the mechanism used by SVT to increase thelevels of CBP/p300, we examined several possibilities. For in-stance, we determined that SVT does not increase the levels ofCBP/p300 mRNAs, change the distribution of CBP/p300 RNAsbetween the nucleus and/or the cytoplasm, or prevent CBP/p300from being degraded by the proteasome. However, analysis ofpolysomes from SVT-expressing cells and control cells showed ahigher proportion of CBP/p300 RNAs associated with the activelytranslating polysomes in SVT-transformed MEFs, despite the factthat the two cell types show similar CBP/p300-specific transcrip-

tional levels. Furthermore, cells expressing the truncation mutantSVTN136 or SVTC257-708 also show increased association of CBP/p300 transcripts with polysome populations. Similar to full-lengthSVT, these mutants also raise the levels of CBP/p300 and theirlevels of acetylation. We conclude that SVT enhances the associa-tion of CBP/p300 mRNAs with polysomes, thus increasing theirtranslation and contributing to higher levels of protein accumu-lation. We speculate that T antigen may (i) induce a signalingpathway that regulates the translation of CBP/p300 mRNAs or (ii)bind to components of the translation machinery and directlyregulate translation.

Mutational analysis indicates that SVT contains two segmentsthat independently alter CBP/p300 levels and histone acetylation.Either an amino-terminal (SVTN136) or a carboxy-terminal(SVTC257-708) truncation of the protein increases the steady-state

FIG 7 Mutations inactivating the J domain preclude SVT from increasing the steady-state levels of CBP/p300 and from immortalizing primary cells in culture.The first 136 amino acids of SVT can bind/affect the function of multiple proteins, and thus, mutations preventing SVT from binding to Rb proteins (E107 and108K), to Bub1 (dl89-95), or to Cul7 (F98A) were generated in the context of the N136 amino-terminal fragment, as well as mutations along the structure, at thebeginning of the sequence (D2K), within the first helix (E9K), in the loop within helices II and III (D44N), or in the third helix of the J domain (K53R). (A)Mutations producing a defective J domain (D44N and K53R) impair the ability of the amino-terminal truncation to raise the steady-state levels of CBP/p300.Each mutation is labeled with a specific color dot below the Western blot. (B) An intact amino-terminal region per se is able to immortalize primary MEFs, exceptwhen lacking an intact J domain (mutations D44N and K53R). Images of cells expressing different N136 mutants after 4 or 5 passages in cell culture are shown.Cells expressing N136D44N or N136K53R are unable to proliferate and eventually die, while cells expressing mutations in other residues proliferate at different ratesand survive further cell passages. (C) The color scheme shown in panel A is maintained to identify the position of each mutant within the structure of the SVTN136

protein (Protein Data Bank [PDB] 1GH6 [77]). Mutant D2K affects the second amino acid in the protein and is not included in the structure. Pools of cellsexpressing each mutant were generated and evaluated on two separate occasions, and cells expressing N136, N136E9K, N136D44N, and N136K53R were producedone additional time for confirmation purposes. The black lines indicate composite images.

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levels of CBP/p300 and alters histone acetylation in a similar wayto full-length SVT, indicating that two different regions of T an-tigen can affect CBP/p300 independently. This explains previousobservations that single mutations within the context of full-length SVT, even ptc1 and ptc2, which prevent interaction be-tween CBP/p300 and SVT, did not abolish the capacity of SVT toaffect CBP/p300 levels (38). Furthermore, both SVT fragmentscontrol the levels of CBP/p300 by increasing the loading of CBP/p300 mRNAs onto polysomes. This is not the first time that SVThas been reported to have redundant functions in the amino- andcarboxy-terminal regions. Truncation mutants similar to the onesshown in this study were previously shown to independently ex-tend the life span of primary cells in culture and to cooperate withan activated ras oncogene to induce transformation (67). In fact,both SVTN136 and SVTC257-708 extend the life span of primaryMEFs (data not shown), a fact that perhaps suggests a link betweenthe ability to alter CBP/p300 and the capacity of oncoproteins toimmortalize cells in culture.

The SVT region expressing the first 136 amino acids of theprotein is sufficient to block pRb proteins and to stimulate E2F-dependent transcription, and it also contains binding sites forhsc70, Bub1, and Cul7. Mutations disrupting each one of thesefunctions produce proteins that are still able to raise CBP/p300levels, except those mutations affecting the loop between helices IIand III or the third helix of the J domain (N136D44N and N136K53R),which disrupt interaction with the chaperone hsc70. In addi-tion, amino-terminal truncations containing a functional J do-main increase specific histone acetylation levels (H3K56 and

H4K12), while J domain-defective truncations fail to affectthose residues. Furthermore, both the intact and mutated ami-no-terminal truncations are capable of extending the life spanof MEFs in vitro, with the exception of N136D44N or N136 K53R.Cells expressing either mutant behave like nonexpressing pri-mary counterparts and survive only 4 or 5 passages in cell cul-ture. These results also suggest a link between CBP/p300 pro-tein levels, the acetylation of specific histone residues, and theability of cells to become immortal in culture. Remarkably,N136E107,108K—a mutant unable to interact with the pRb pro-teins—is still able to increase CBP/p300 levels and extend thelife span of cells in culture. To our knowledge, this is the firstreport to indicate a vital role of the J domain in SVT in virus-mediated immortalization and transformation that is not de-pendent on interaction with the retinoblastoma family of pro-teins.

We characterized the acetylation status of multiple histone res-idues in SVT-expressing cells. CBP/p300 have been shown to addacetyl groups to specific histone residues, both in vivo and in vitro(33, 49, 68, 69). Many histone residues are bona fide substrates ofCBP/p300, such as all sites in H2A and H2B; H3 residues K14,K18, and K56; and H4 residues K5, K8, K12, and K14 (33, 49, 68,69). In some cases, a link between a particular residue and a spe-cific protein has been shown, such as the preferential acetylation ofH4K12 in chromatin by CBP and the H4K8 preferential modifi-cation by p300 (49). We found that, in primary MEFs, the increasein CBP/p300 correlates with a similar rise in acetylation of H4K12

FIG 8 Mutations inactivating the J domain remove the ability of truncated SVT proteins to increase H3K56 and H4K12 acetylation levels. MEFs were transducedwith lentiviral constructs expressing different T antigen mutants and kept in culture for 6 days to allow SVT expression. Double immunofluorescence stainingwith antibodies against SVT and either H3K53 or H4K12 was performed in cells after fixation and permeabilization. Proteins maintaining an intact J domain(N625 and N136E9K) retained the ability to increase acetylation of H3K56 and H4K12. However, mutant proteins harboring a defective J domain (N136D44N andN136K53R) failed to raise the acetylation levels of H3K56 and, to a lesser extent, those of H4K12.

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and H3K56, while other residues tested showed no significant al-terations.

Acetylation of H4K12 has been linked to multiple cellular andmulticellular processes, including transcriptional control of geneslinked to cell viability and growth (70); modification of newlysynthesized histones prior to chromatin assembly (71); consolida-tion of memory in mice (72); and changes during meiosis, aging,and fertility (73, 74). When acetylated in K12, the H4 proteinrecruits the bromodomain proteins Brd2 and Brd3 to genomicregions/promoters and allows active transcription (75). On theother hand, H3K56 acetylation is put onto newly synthesized H3(76) and perhaps marks this histone for appropriate chromatinassembly. Thus, H3K56 acetylation increases in S phase, and it alsoincreases after DNA damage, when the modification localizes tosites of DNA repair, together with (P)ATM, g-H2AX, CHK2, andp53 (68).

Our results indicate that, in addition to manipulating the cellmachinery by controlling two of the main tumor suppressors, pRband p53, large T antigens from polyomavirus tamper with theepigenetic markers in normal cells. A clear precedent has beenestablished with the adenovirus E1A protein, which alters histoneacetylation and increases H3K18 levels by a mechanism involvingCBP/p300 (40). By redirecting CBP/p300 from the promoters ofgenes mediating growth arrest or differentiation to those of genesstimulating proliferation, E1A changes the epigenetic programand alters the cellular transcriptional pattern (18). In contrast toE1A, we do not observe changes in the acetylation of H3K18, butrather at H4K12 and H3K56, residues that are not altered by E1A.However, it is likely that SVT redirects CBP/p300 to modify cel-lular gene expression, and this possibility will be the subject offuture studies.

ACKNOWLEDGMENTS

This work was supported by grant NIH R01CA098956 to J.M.P.We thank Jelena Jacovlievic and John Wolford (Carnegie Mellon Uni-

versity, Pittsburgh, PA) for kindly helping with the preparation of poly-some gradients, Ping An for critical reading of and suggestions on themanuscript, and Han Na Choi for excellent technical help.

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