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Journal of Virology logoLink to Journal of Virology
. 2013 Dec;87(24):13499–13509. doi: 10.1128/JVI.02658-13

Two Independent Regions of Simian Virus 40 T Antigen Increase CBP/p300 Levels, Alter Patterns of Cellular Histone Acetylation, and Immortalize Primary Cells

Maria Teresa Sáenz Robles 1, Chikdu Shivalila 1,*, Jeremy Wano 1,*, Shelly Sorrells 1,*, Alison Roos 1,*, James M Pipas 1,✉
PMCID: PMC3838271  PMID: 24089570

Abstract

Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their 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/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation.

INTRODUCTION

The large T antigen from simian virus 40 (SV40) (SVT) has been shown to induce cell proliferation, immortalize primary cells, and mediate tumorigenesis in numerous in vivo and in vitro systems (1–3). SVT induces transformation by binding to specific cellular target proteins, thus altering pathways that regulate cell proliferation, death, and tissue homeostasis. For example, domains located in the carboxy-terminal half of SVT bind and inactivate the tumor suppressor p53, thereby blocking its ability to induce cell cycle arrest and apoptosis. In addition, SVT binding to the retinoblastoma (Rb) proteins (pRb, p107, and p130) through an LXCXE motif and to hsc70 via its J domain antagonizes cell cycle exit (4–6). Strikingly, other DNA tumor viruses, such as human papillomaviruses and adenoviruses, also encode proteins that target Rb and p53, suggesting that these viruses converge on a common set of cellular targets that are important for transformation.

Disruption of additional cellular targets contributes to the tumorigenic phenotype induced by SVT (7–12). In particular, SVT has been shown to bind CBP/p300 (13–15), closely related proteins that are also common targets of many viruses, including the DNA tumor viruses (16). For instance, the interaction of the adenovirus E1A protein with CBP/p300 is necessary for transformation (17). The E1A protein alters chromatin acetylation and gene expression by removing CBP/p300 from the promoters of differentiation-specific and antiviral defense genes and positioning them to activate genes involved in cell proliferation (18). Similarly, SVT binding to CBP/p300 is necessary for transformation (1). The association of SVT with CBP/p300 requires p53 and thus is 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 acetylate p53 and thereby induces cell cycle arrest and senescence (21).

The CBP and p300 proteins are closely related and act as molecular adaptors, associating with multiple transcriptional regulators and signaling molecules and thereby integrating many cellular pathways. In fact, CBP/p300 bind more than 400 cellular proteins (22). In addition, CBP/p300 possess histone acetyltransferase (HAT) activity and can alter gene expression through both modification and relaxation of chromatin and recruitment of basal and other transcriptional components to promoters (23). CBP and p300 are expressed in almost identical patterns in the mouse (24), and homozygous p300 or CBP knockout mice die during embryogenesis (25–27). However, CBP and p300 functions are not completely redundant. For instance, p300—but not CBP—is essential for the formation of the heart, small intestine, and lungs during embryonic development (28), and the two proteins play different roles in transcription regulation (29). Similarly, p300 and CBP play different roles during myogenesis, hematopoietic differentiation, and stem cell renewal in mice (30, 31). In addition, p300 and CBP play different roles during differentiation, cell cycle exit, and apoptosis of embryonal carcinoma cells in culture (32), and each protein seems to preferentially acetylate specific lysine residues in certain histones, thus differentially regulating chromatin activity (33).

To date, the effects of SVT on the activity of CBP/p300 are controversial: SVT has been shown to repress the transcriptional activity of CBP/p300 (34), but the overall HAT activity associated with those adaptors seems to increase in the presence of SVT (35). These differences could be attributed to genetic or epigenetic alterations acquired during the establishment of cell lines used in the different experiments. We have therefore used primary cells to evaluate the effects of SVT and SVT mutants on the CBP/p300 pathway and to explore the molecular level at which SVT affects these epigenetic regulators.

MATERIALS AND METHODS

Isolation of primary fibroblasts and cell culture conditions.

Mouse embryo fibroblasts (MEFs) were harvested from 13.5-day-old FVB embryos as previously described (36) and grown at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. MEF lines expressing different SVT mutants were previously generated (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], and LPVT [lymphotropic papovavirus large T antigen]), truncated SVT mutants (SVTN136 and SVTC257-708), and SVT small or point mutations (N136D44N, N136dl89-97, N136F98A, N136E9K, N136K53R, N136D2K, and N136E107,108K) were obtained by infection with retroviral (pBabePuro) or lentiviral (pL6.3; Invitrogen) constructs encoding the different mutants under the control of the CMV promoter. In particular, mutations disrupting the J domain and binding to hsc70 (D44N and K53R), unable to bind pRb proteins (E107 and 108K), unable to interact with Cul7 (F98A), or impaired in binding Bub1 (dl89-97) and new mutations within the amino-terminal region of SVT (D2K and E9K) were used in this study. Upon infection, cells were selected with the appropriate antibiotic (puromycin, 2 μg/ml; blasticidin, 2.5 μg/ml) and tested for T antigen expression and survival after passage in culture.

To test stress conditions, cells in culture were subjected to DNA damage by treatment with doxorubicin (adryamicin; 0.5 μg/ml; 6 h), starvation in serum-free medium (6 h), or exposure to heat (42°C; 2 h). After each treatment, the cells were washed with phosphate-buffered saline (PBS) and collected to obtain protein or RNA samples by standard methods. When indicated, the inhibitor MG132 (10 μM) was added to the cell culture media for 6 h to block proteasome-mediated degradation, and cycloheximide (50 μg/ml) was used to block de novo protein synthesis.

Preparation and infection of hepatocytes.

Hepatocyte preparation was adapted from the protocol described by Gibco Invitrogen for their hepatocyte product line in order to use embryonic tissue. Briefly, livers from embryonic day 18.5 (E18.5) stage mouse embryos were isolated and digested with Liver Digest Medium (Invitrogen) for 30 min at 37°C, filtered through a 100-mm mesh screen, and washed three times with hepatocyte wash medium (Invitrogen). The resulting hepatocytes were plated onto collagen-coated plates (BD Biosciences) in Williams E medium supplemented with penicillin/streptomycin and 10% fetal bovine serum and kept at 37°C with 5% CO2. After 48 h, 5 × 106 cells were infected with a retroviral construct encoding the SVT early region in the presence of 8 μg/ml of Polybrene, selected with puromycin (2 μg/ml), and collected for analysis.

Protein analysis and immunofluorescence experiments.

Protein extraction from and Western blot assays of cells in culture or murine tissues were performed using standard procedures. Each Western blot experiment was performed at least 2 times and in most cases 3 to 6 times. To analyze protein expression under the microscope, MEFs were grown to confluence 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) blocking for 1 h in PBS supplemented with 3% bovine serum albumin (BSA) and 0.2% NP-40, (ii) incubation with appropriate dilution of primary antibody in PBS for 24 h at 4°C, and (iii) incubation with the appropriate Alexa Fluor 488- or Alexa Fluor 568 (Invitrogen)-conjugated secondary antibody (1:500) in PBS for 1 h at room temperature. The slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted for analysis by fluorescence microscopy. Each immunofluorescence experiment was performed at least twice, and 3 to 4 times in most cases.

Primary antibodies used.

Monoclonal antibodies recognizing T antigen (416 and 419) and p53 (421) have been described previously (39). The following rabbit polyclonal antibodies were from Santa Cruz Biotechnology, Inc.: p107 C-18 (sc-318), p130 C-20 (sc-317), hsp70 K-20 (sc-1060), CBP A-22 (sc-369), β-tubulin D-10 (sc-5274), and p53 FL-393 (sc-6243). GAPDH-mouse monoclonal G8140-11 was from United States Biological, Swampscott, MA, USA. Anti-acetyl-histone H3 (Lys56) clone EPR996Y and p300 mouse monoclonal clone RW128 were purchased from Millipore. The following rabbit polyclonal antibodies were obtained from Cell Signaling Technology Inc.: acetyl-CBP (Lys1535)/p300 (Lys1499), number 4771; acetyl-histone H2A (Lys5), number 2576; histone H2B (V119), number 8135; acetyl-histone H2B (Lys5), number 2574; 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 from Roberto Ferrari and has been described previously (40).

Polysome preparation and quantitative real-time PCR analysis.

Preribosomes, ribosomes, and polyribosomes were obtained according to previously published protocols (41) with some modifications. MEFs growing 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 sheared and debris was removed by centrifugation. Ribosomal components in the supernatant were separated on 10 to 45% sucrose gradients by centrifugation at 27,000 rpm for 4 h at 13°C in an SW28 rotor (Beckman Instruments, Inc., Fullerton, CA). A Teledyne ISCO Foxy R1 density gradient fractionator was used to fractionate and analyze the gradients. All fractions containing polysomes were collected and pooled. Total RNA was extracted from the pools by standard techniques, using Invitrogen purification columns. Obtention of RNA and cDNA and transcript quantification by real-time PCR analysis were done as described previously (42) with primers specific for CBP (pair 5′-ACCCCAAACGAGCCAAACTC and 5′-ACGCAGCATCTGGAACAAGG and pair 5′-TCCTTACGCCGCTCCAAATG and 5′-GCCCCCACTTCACCATCTTATG), p300 (pair 5′-TTTCTGTTGAGTCCGCATCCC and 5′-CAAAATCTGTGCCATCGCTGG and pair 5′-CCAGCAAAAACAAGAGCAGCC and 5′-ATTGGGAGCAGGACAAGCGATG), mcm3 (5′-CGCAGGAAGAATGAAAAGAGG and 5′-CTGAGGAAGCAGGAAGTGAGA), p21 (5′-AGCCTGAAGACTGTGATGGG and 5′-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 normalized against endogenous controls (Adh and/or Rpl5). Five different polysome preparations—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 three occasions twice. Within each real-time experiment, each experimental point was tested with four technical replicates. The results were analyzed with the 7300 System SDS RQ Study Software (Applied Biosystems), which provided the RQ (mean expression level) and RQ min/RQ max (calculated standard error from RQ) values.

RESULTS

Expression of SV40 T antigen causes increased steady-state levels of CBP/p300.

We have previously observed that several established cell lines express high levels of CBP/p300. However, several types of nontransformed or primary cells contain low levels of CBP and p300 proteins, as determined by Western blot analysis (Fig. 1A, B, D, and E). They include an established line of rat embryo fibroblasts (REF52), as well as cells obtained directly from murine embryonic (MEFs and hepatocytes) or adult (enterocytes from intestinal villi) tissues. In contrast, expression of SVT in those cells results in a marked increase of the CBP/p300 protein levels (Fig. 1A, B, D, and E). The increase of CBP/p300 in response to SVT is not simply due to a higher proliferative status of the cells or their level of confluence, as highly proliferative, underconfluent control MEFs present much lower levels of CBP/p300 than similar cells expressing SVT (Fig. 1A). Furthermore, either subconfluent, confluent, or postconfluent cells expressing SVT show high levels of CBP/p300, in contrast with control counterparts (Fig. 1A), suggesting that contact with other cells—or lack of it—does not play a role in controlling CBP/p300 protein levels. The increase in CBP/p300 levels is not due to the method used to introduce the SVT into cells, as cells expressing SVT after transfection (Fig. 1A and B), viral infection (Fig. 1C), DNA integration in transgenic mice (Fig. 1E), or retroviral transduction (Fig. 1D and data not shown) show high levels of CBP/p300 in comparison to nonexpressing counterpart cells.

Fig 1.

Fig 1

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CBP and p300 steady-state protein levels increase in cells expressing SV40 T antigen. (A) Steady-state levels of CBP and p300 in control or SVT-expressing MEFs grown to different levels of confluence as shown by Western blotting with specific antibodies. The results were confirmed in at least two 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-postconfluent MEFs. (B) Steady-state levels of CBP and p300 in REF52 cells, monitored by Western blotting. Expression of SVT results in increased levels of both proteins in 2-day-postconfluent cells. (C) Protein extracts were obtained from cells 4 days postinfection with SV40 viruses and studied by Western blotting. Levels of CBP and p300 increase in cells infected with SV40 particles. (D) Embryonic hepatocytes show increased levels of CBP/p300 upon stable SVT 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 cell populations 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 lines indicate composite images.

The manipulations used to express ectopic products in cells can potentially induce abnormal stresses that might influence the status of cellular components. Thus, we studied the effects of different types of stress on levels of CBP/p300 in control MEFs or those expressing SVT. Neither inducing DNA damage with doxorubicin, starving the cells (Fig. 2A), nor increasing the temperature of incubation (Fig. 2B) raised the levels of CBP/p300 in control MEFs. Similarly, cells expressing SVT show high levels of CBP/p300 under all conditions tested. Induction of DNA damage was confirmed by monitoring the increase of p53 in cells treated with doxorubicin, and the effectiveness of the heat shock treatment was confirmed by observing the rise of hsp70 levels in heat-treated cells. In addition, the method used to collect cells for protein analysis, by either trypsinization, direct lysis in cell culture plates, or use of cell scrapers, did not have any noticeable effect on the steady-state levels of CBP/p300 (data not shown).

Fig 2.

Fig 2

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Different types of stress do not affect the steady-state levels of CBP/p300. Mouse embryo fibroblasts were treated with different stress inducers 2 days after reaching confluence. The corresponding protein extracts were resolved in SDS-PAGE gels and analyzed with different specific antibodies. Neither DNA damage by treatment with doxorubicin, starvation in serum-free medium (A), nor exposure to heat shock (B) affects the levels of CBP/p300 with or without SVT present in the cells.

Acetylation levels of CBP/p300 and specific histone residues are increased in cells expressing SV40 T antigen.

CBP/p300 possesses acetyltransferase activity (43, 44) and can add acetyl residues to multiple proteins, including transcription factors and histones (43, 45–49), thus possibly controlling gene activity epigenetically. We hypothesized that the SVT-induced increase in CBP/p300 levels might change gene regulation through modification of the epigenetic code. We first used an antibody that detects endogenous levels of CBP or p300 only when acetylated at lysine 1535 or lysine 1499, respectively. Posttranslational acetylation of p300 in specific lysine residues within an activation loop motif, including Lys1499, has been linked to an increase in its acetyltransferase activity (50). Consistent with this hypothesis, the increased levels of CBP/p300 in cells expressing SVT correlate with increased CBP/p300 acetylation levels, as determined by immunofluorescence (Fig. 3A) and Western blot analysis (Fig. 3B), suggesting increased activity of these proteins in the presence of SV40 T antigen. Acetylation of histones by CBP/p300 has been associated with transcriptional activation, histone deposition, and DNA repair (43, 51, 52), and thus, we used a panel of antibodies recognizing specific acetylated residues in different histones. In contrast with other viral proteins, like adenovirus E1A, which have been shown to induce a drastic reduction in the acetylation of cellular histone H3 lysine 18 (H3K18ac) (40), SVT does not alter the acetyl levels of H3K18 or other specific histone residues, including H4K8 (Fig. 3C and D) and H2AK5, H2BK5, H3K9, and H4K5 (Fig. 3D). However, cells expressing SVT show a significant increase in the levels of H3K56 (Fig. 3D and 4B) and H4K12 (Fig. 3D and 4C).

Fig 3.

Fig 3

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Cells expressing SVT show increased acetylation levels of CBP (Lys1535) and/or p300 (Lys1499) and correlate with increased acetylation of specific histone 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 control cells, 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 lines indicate composite images.

Fig 4.

Fig 4

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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 antibodies recognizing specific acetylated lysine residues. Expression of each of these polyomavirus T antigens produced a significant increase in both p300 and the autoacetylated 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 cells expressing different polyomavirus T antigens was detected with antibodies against SVT, and the exposure time was adjusted according to the intensity of the signal observed. None of the available antibodies to detect T antigen cross-react with LPVT. The black lines indicate composite images.

Polyomavirus large T antigens increase the acetylation levels of 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, we next examined if the T antigens encoded by other members of the Polyomaviridae induce high CBP/p300 levels. We observed that MEFs expressing BKVT, JCVT, or LPVT also increase the steady-state levels of CBP/p300 (Fig. 4A and C). Also, similar to that of SV40, these T antigens induce increased levels of H3K56 (Fig. 4B) and H4K12 (Fig. 4C). Interestingly, the murine polyomavirus large T antigen does not show this effect (data not shown). These results suggest that polyomavirus T antigens increase protein levels and autoacetylation of CBP/p300 and regulate chromatin acetylation.

Two independent functions encoded in separate regions of SVT affect CBP/p300.

Next, we examined a series of SVT mutants for the ability to increase CBP/p300 levels and autoacetylation, as well as for changes in histone acetylation. Previously described mutants, as well as newly generated truncation mutants, were used to explore the effects on CBP/p300 levels (Fig. 5D) (see Materials and Methods). Several characterized amino acid substitution mutants 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 truncated versions of SVT encompassing either the N terminus (N136 and N625) or the C terminus (C257 to C708) of SVT show increased levels (Fig. 5A and B) and autoacetylation (Fig. 5C) of CBP and p300. We conclude that two independent functions encoded in separate regions of SVT affect CBP/p300 levels and autoacetylation.

Fig 5.

Fig 5

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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, as MEF 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 of small 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 to increase the steady-state levels of CBP/p300 (B) and autoacetylation (C). β-Tubulin was used as a loading control; levels of p107 and p130 were monitored to ensure functionality of the tested T antigens. (D) Summary of SV40 T antigen mutants. Functional domains of SV40 T antigen are indicated above the depictions of amino- and carboxy-terminal truncations, as well as the locations (×) of small and point mutations used in the study. OBD, origin binding domain; HR, host range domain. The black lines indicate composite images.

The SVT effect on the steady-state levels of CBP/p300 is mediated through preferential association of CBP/p300 transcripts with polysomes.

Gene expression profiling experiments with MEFs indicated no significant changes in the transcriptional levels of CBP or p300 mRNAs either after SV40 infection or upon expression of SVT (53). We confirmed this result using CBP- and p300-specific primers by RT-PCR analysis of cDNAs from SVT-expressing MEFs and REF52 cells (Fig. 6A) or by real-time PCR analysis of MEFs (not shown). Thus, changes in CBP/p300 levels are not due to increased transcription triggered by SVT expression. Next, we determined the effect of SVT on CBP/p300 steady-state levels in the presence or absence of MG132, a potent inhibitor of the proteasome. Neither control (DMSO) nor MG132 treatment altered the levels of CBP/p300 with or without SVT present in 2-day-postconfluent cells (Fig. 6B), while increased levels of hsp70 in the presence of MG132 indicated the effectiveness of the treatment. Proteasome inhibition of underconfluent, actively growing cells produced a similar outcome (not shown). These results indicate that SVT does not increase CBP/p300 levels by preventing proteasome-mediated degradation. Furthermore, SVT does not alter the cytoplasmic/nuclear distribution of CBP/p300 transcripts (not shown); thus, differences in the transport of specific 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 the amounts of CBP/p300 proteins either in control or in SVT-expressing MEFs (Fig. 6C). Our data indicate that protein degradation does not account for the SVT-mediated increase of CBP/p300 steady-state levels.

Fig 6.

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Mechanism of action of SVT on CBP/p300. (A) CBP and p300 transcript levels are not increased upon expression of SVT. RNA was extracted from 2-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 affect CBP/p300 steady-state levels in either the absence or presence of SVT. Confluent control and SVT-expressing MEFs were treated with 10 μM MG132 to prevent proteasome-mediated degradation. Samples were collected at the indicated time points, and protein levels were analyzed by immunoblotting. Hsp70 was used as a control of treatment, and β-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 Western blotting. No significant changes in the steady-state levels of CBP/p300 were observed at any point. (D) Presence of specific transcripts in polysomal populations from control and SVT-expressing cells. Polysomes were isolated, and total RNA was extracted from control and SVT-expressing cells. The levels of specific transcripts were determined by real-time PCR and normalized against the levels of control, nonchanging transcripts (e.g., Adh and Rpl5) present in the same samples (RQ values). The error bars indicate four replicates from a single representative experiment. Despite showing similar transcriptional levels in all cells tested, the association of CBP and p300 transcripts with polysome populations is increased in cells containing full-length SVT, SVTN136, or SVTC257-708. See Results for a description of the association of other transcripts with polysomes.

Finally, we examined the association of CBP/p300 transcripts with actively translating polysomes. Although CBP/p300 transcriptional levels remain similar in control and SVT-expressing cells, the amount of CBP/p300 RNAs present in polysomes is significantly increased in SVT-transformed cells (Fig. 6D). Remarkably, a higher association of CBP and p300 transcripts with polysome populations is similarly observed in both SVTN136- and SVTC257-708-expressing cells (Fig. 6D), both of which also express considerably higher levels of CBP/p300 proteins. In contrast, we found p21 transcripts associated with polysomes, as expected, according to their abundance in different cells: p21 is a p53 target and thus is upregulated in response to disruption of the RB/E2F pathway by SVTN136, but p21 transcripts remain low in control cells or those expressing SVT or SVTC257-708 (Fig. 6D), as both proteins can bind and prevent p53-mediated upregulation of p21. In addition, we examined transcripts normally unchanged (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) or overexpressed (mcm3 and TK) in the presence of SVT, and in each case, we found that they associate with polysomes, as expected (data not shown). 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 of SVT is required to raise CBP/p300- and histone-specific acetylation levels and to immortalize MEFs in culture.

As two independent SVT truncations are capable of raising CBP/p300 levels in a similar way, we initiated the characterization and mutagenesis of the amino-terminal region and assessed the abilities of different mutants to raise the levels of CBP/p300. In the context of the SVTN136 truncation, most of the individual mutations tested do not preclude SVT from increasing the steady-state levels of CBP/p300, even though the mutations render proteins unable to interact with Bub1 (N136dl89-97), Cul7 (N136F98A), or the pRB proteins (N136E107,108K). In contrast, mutations precluding interaction with hsc70 (N136D44N and N136K53R) completely abolish the ability of T antigen to raise the steady-state levels of CBP/p300 (Fig. 7A). This phenomenon is not due to a general block or disruption of the J domain, as only mutations between helix II and helix III (N136D44N) or in helix III (N136K53R) of the J domain, but not in other parts of the structure (N136D2K and N136E9K), prevent the T antigen-mediated increase in the steady-state levels of CBP/p300 (Fig. 7A). Moreover, different mutations within the amino terminal region of SVT are still capable of immortalizing cells in culture, but mutations inactivating the J domain of the protein and precluding interaction with hsc70 completely abolish the capacity of SVTN136 to immortalize MEFs, and the cells die after 4 or 5 passages (Fig. 7B). Mutations in the J domain that do not abolish interaction with hsc70 (N136D2K and N136E9K) retain the ability to immortalize primary cells and to increase CBP/p300 levels. Finally, we investigated if a possible connection exists between the SVTN136-mediated increases in p300 levels and the acetylation levels of specific histone residues. In fact, MEFs expressing amino-terminal truncation mutants able to increase p300 steady-state levels (N625 and N136E9K) are still able to raise the levels of H3K56 and H4K12 acetylation (Fig. 8). However, expression of N136 mutants lacking a functional J domain (N136D44N and N136K53R) is not sufficient to raise the levels of those specific histone acetylations (Fig. 8). We conclude that the ability of the amino-terminal 136 amino acids of SVT to immortalize cells, to increase CBP/p300 levels, and to control acetylation of specific histone residues requires a fully functional J domain.

Fig 7.

Fig 7

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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 and 108K), 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 the beginning 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, except when 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 rates and 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 cells expressing each mutant were generated and evaluated on two separate occasions, and cells expressing N136, N136E9K, N136D44N, and N136K53R were produced one additional time for confirmation purposes. The black lines indicate composite images.

Fig 8.

Fig 8

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Mutations inactivating the J domain remove the ability of truncated SVT proteins to increase H3K56 and H4K12 acetylation levels. MEFs were transduced with lentiviral constructs expressing different T antigen mutants and kept in culture for 6 days to allow SVT expression. Double immunofluorescence staining with 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 and N136K53R) failed to raise the acetylation levels of H3K56 and, to a lesser extent, those of H4K12.

DISCUSSION

DNA tumor viruses encode oncoproteins that act by binding key cellular regulatory proteins and thereby activating, inhibiting, or redirecting their functions. SVT-mediated transformation requires binding to both the retinoblastoma family of tumor suppressors (pRb, p130, and p107) and p53. The first interaction antagonizes 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 and stimulate expression of growth arrest and cell death genes. Similarly, the E1A and E1B proteins of adenoviruses and the E7 and E6 proteins encoded by papillomaviruses bind and inactivate Rb proteins and p53. In addition, adenoviruses and papillomaviruses target the transcriptional adapter proteins CBP/p300, and in both cases, this interaction is required for transformation.

CBP/p300 are highly similar multifunctional proteins that interact with numerous cell components to control multiple cellular pathways. The proteins are both transcriptional coactivators and contain intrinsic cytoplasmic and HAT activities (43, 44, 54–56). CBP/p300 are used as scaffolds, coupling chromatin remodeling to the transcriptional machinery and playing essential roles in growth 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 resulting in loss of acetyltransferase activity have been found in primary tumors, as well as in cancer cell lines (57), suggests that acetylation of cellular proteins is a critical signal in cell growth. CBP/p300 mediate the effects of numerous cellular proteins, including transcription factors like c-myb (59) and E2Fs (60, 61), and several viral proteins target CBP/p300 to manipulate the host machinery in their favor (62–65).

Previous studies have shown that SVT binds CBP/p300 and that SVT complements adenovirus E1A mutants defective for CBP/p300 association in transformation assays (66). The association of SVT with CBP/p300 requires SVT binding to p53, and furthermore, the formation of the SVT-p53-CBP/p300 complex results 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 of controversy. One study reports that SVT represses CBP/p300 transcriptional activities, while another concludes that SVT enhances 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 of hyperacetylated CBP/p300 and that SVT-expressing cells also exhibit increased acetylation of histones H3K56 and H4K12, modifications associated with CBP/p300 HAT activity. The T antigens encoded by the human polyomaviruses BKV and JCV or by the monkey virus LPV also induce high CBP/p300 levels and enhance H3K56 and H4K12 acetylation, suggesting that these effects are a common feature of polyomavirus T antigens.

To characterize the mechanism used by SVT to increase the levels of CBP/p300, we examined several possibilities. For instance, we determined that SVT does not increase the levels of CBP/p300 mRNAs, change the distribution of CBP/p300 RNAs between the nucleus and/or the cytoplasm, or prevent CBP/p300 from being degraded by the proteasome. However, analysis of polysomes from SVT-expressing cells and control cells showed a higher proportion of CBP/p300 RNAs associated with the actively translating polysomes in SVT-transformed MEFs, despite the fact that the two cell types show similar CBP/p300-specific transcriptional levels. Furthermore, cells expressing the truncation mutant SVTN136 or SVTC257-708 also show increased association of CBP/p300 transcripts with polysome populations. Similar to full-length SVT, these mutants also raise the levels of CBP/p300 and their levels of acetylation. We conclude that SVT enhances the association of CBP/p300 mRNAs with polysomes, thus increasing their translation and contributing to higher levels of protein accumulation. We speculate that T antigen may (i) induce a signaling pathway that regulates the translation of CBP/p300 mRNAs or (ii) bind to components of the translation machinery and directly regulate translation.

Mutational analysis indicates that SVT contains two segments that 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 levels of CBP/p300 and alters histone acetylation in a similar way to full-length SVT, indicating that two different regions of T antigen can affect CBP/p300 independently. This explains previous observations that single mutations within the context of full-length SVT, even ptc1 and ptc2, which prevent interaction between CBP/p300 and SVT, did not abolish the capacity of SVT to affect CBP/p300 levels (38). Furthermore, both SVT fragments control the levels of CBP/p300 by increasing the loading of CBP/p300 mRNAs onto polysomes. This is not the first time that SVT has been reported to have redundant functions in the amino- and carboxy-terminal regions. Truncation mutants similar to the ones shown in this study were previously shown to independently extend the life span of primary cells in culture and to cooperate with an activated ras oncogene to induce transformation (67). In fact, both SVTN136 and SVTC257-708 extend the life span of primary MEFs (data not shown), a fact that perhaps suggests a link between the ability to alter CBP/p300 and the capacity of oncoproteins to immortalize cells in culture.

The SVT region expressing the first 136 amino acids of the protein is sufficient to block pRb proteins and to stimulate E2F-dependent transcription, and it also contains binding sites for hsc70, Bub1, and Cul7. Mutations disrupting each one of these functions produce proteins that are still able to raise CBP/p300 levels, except those mutations affecting the loop between helices II and III or the third helix of the J domain (N136D44N and N136K53R), which disrupt interaction with the chaperone hsc70. In addition, amino-terminal truncations containing a functional J domain increase specific histone acetylation levels (H3K56 and H4K12), while J domain-defective truncations fail to affect those residues. Furthermore, both the intact and mutated amino-terminal truncations are capable of extending the life span of MEFs in vitro, with the exception of N136D44N or N136 K53R. Cells expressing either mutant behave like nonexpressing primary counterparts and survive only 4 or 5 passages in cell culture. These results also suggest a link between CBP/p300 protein levels, the acetylation of specific histone residues, and the ability of cells to become immortal in culture. Remarkably, N136E107,108K—a mutant unable to interact with the pRb proteins—is still able to increase CBP/p300 levels and extend the life span of cells in culture. To our knowledge, this is the first report to indicate a vital role of the J domain in SVT in virus-mediated immortalization and transformation that is not dependent on interaction with the retinoblastoma family of proteins.

We characterized the acetylation status of multiple histone residues in SVT-expressing cells. CBP/p300 have been shown to add acetyl groups to specific histone residues, both in vivo and in vitro (33, 49, 68, 69). Many histone residues are bona fide substrates of CBP/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 specific protein has been shown, such as the preferential acetylation of H4K12 in chromatin by CBP and the H4K8 preferential modification by p300 (49). We found that, in primary MEFs, the increase in CBP/p300 correlates with a similar rise in acetylation of H4K12 and H3K56, while other residues tested showed no significant alterations.

Acetylation of H4K12 has been linked to multiple cellular and multicellular processes, including transcriptional control of genes linked to cell viability and growth (70); modification of newly synthesized histones prior to chromatin assembly (71); consolidation of memory in mice (72); and changes during meiosis, aging, and fertility (73, 74). When acetylated in K12, the H4 protein recruits the bromodomain proteins Brd2 and Brd3 to genomic regions/promoters and allows active transcription (75). On the other hand, H3K56 acetylation is put onto newly synthesized H3 (76) and perhaps marks this histone for appropriate chromatin assembly. Thus, H3K56 acetylation increases in S phase, and it also increases after DNA damage, when the modification localizes to sites of DNA repair, together with (P)ATM, g-H2AX, CHK2, and p53 (68).

Our results indicate that, in addition to manipulating the cell machinery by controlling two of the main tumor suppressors, pRb and p53, large T antigens from polyomavirus tamper with the epigenetic markers in normal cells. A clear precedent has been established with the adenovirus E1A protein, which alters histone acetylation and increases H3K18 levels by a mechanism involving CBP/p300 (40). By redirecting CBP/p300 from the promoters of genes mediating growth arrest or differentiation to those of genes stimulating proliferation, E1A changes the epigenetic program and alters the cellular transcriptional pattern (18). In contrast to E1A, we do not observe changes in the acetylation of H3K18, but rather at H4K12 and H3K56, residues that are not altered by E1A. However, it is likely that SVT redirects CBP/p300 to modify cellular gene expression, and this possibility will be the subject of future studies.

ACKNOWLEDGMENTS

This work was supported by grant NIH R01CA098956 to J.M.P.

We thank Jelena Jacovlievic and John Wolford (Carnegie Mellon University, Pittsburgh, PA) for kindly helping with the preparation of polysome gradients, Ping An for critical reading of and suggestions on the manuscript, and Han Na Choi for excellent technical help.

Footnotes

Published ahead of print 2 October 2013

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