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Originally published In Press as doi:10.1074/jbc.M414564200 on February 28, 2005

J. Biol. Chem., Vol. 280, Issue 23, 21915-21923, June 10, 2005
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p400 Is Required for E1A to Promote Apoptosis*

Andrew V. Samuelson{ddagger}§, Masako Narita{ddagger}, Ho-Man Chan¶||, Jianping Jin{ddagger}, Elisa de Stanchina{ddagger}**, Mila E. McCurrach{ddagger}, Masashi Narita{ddagger}{ddagger}{ddagger}, Miriam Fuchs§, David M. Livingston§, and Scott W. Lowe{ddagger}§§

From the {ddagger}Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, December 27, 2004 , and in revised form, February 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The adenovirus E1A oncoprotein promotes proliferation and transformation by binding cellular proteins, including members of the retinoblastoma protein family, the p300/CREB-binding protein transcriptional coactivators, and the p400-TRRAP chromatin-remodeling complex. E1A also promotes apoptosis, in part, by engaging the ARF-p53 tumor suppressor pathway. We show that E1A induces ARF and p53 and promotes apoptosis in normal fibroblasts by physically associating with the retinoblastoma protein and a p400-TRRAP complex and that its interaction with p300 is largely dispensable for these effects. We further show that E1A increases p400 expression and, conversely, that suppression of p400 using stable RNA interference reduces the levels of ARF, p53, and apoptosis in E1A-expressing cells. Therefore, whereas E1A inactivates the retinoblastoma protein, it requires p400 to efficiently promote cell death. These results identify p400 as a regulator of the ARF-p53 pathway and a component of the cellular machinery that couples proliferation to cell death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal cells possess innate tumor suppressor mechanisms that couple proliferation to apoptosis, thereby limiting the transforming potential of oncogenic mutations (reviewed in Ref. 1). For example, overexpression of the c-myc oncogene or loss of the retinoblastoma protein (Rb)1 tumor suppressor deregulates cell cycle control but also can promote apoptosis. Consequently, cells with these lesions proliferate inappropriately but also become hypersensitive to apoptosis in response to survival factor depletion and DNA-damaging agents. Subsequent mutations that disable apoptosis allow proliferation to continue unabated, leading to oncogenic transformation or tumor progression.

The ARF-p53 pathway is an important circuit in the signaling network that couples proliferation to cell death (2). ARF is the alternative reading frame product of the INK4a/ARF tumor suppressor locus, which also encodes the cyclin-dependent kinase inhibitor p16INK4a. Several oncogenes, including c-Myc and E1A, induce ARF message and protein, which in turn activates p53 by interfering with its negative regulator Mdm2 (2). p53, in turn, increases the transcription of genes that ultimately target different components of the apoptotic machinery (3). Although oncogenes can also promote apoptosis through ARF- and p53-independent mechanisms, apoptosis induced by Myc or E1A is severely compromised in cells lacking ARF or p53 (4, 5), and loss of ARF or p53 can cooperate with proapoptotic oncogenes to promote transformation in vitro and tumorigenesis in mice (reviewed in Refs. 1 and 6). How ARF senses hyperproliferative signals is poorly understood, but it can involve transcriptional control of the ARF promoter by E2F proteins as well as control of higher order chromatin structure by chromatin remodeling complexes (7–9).

The adenovirus E1A oncoprotein promotes uncontrolled cell cycle progression and apoptosis and has been widely used as a tool to identify cellular activities that function in both processes. E1A stimulates proliferation by physically associating with proteins that act at critical control points in the normal cell cycle (10). Perhaps the best understood of these involve the Rb family, which interacts with the E1A protein through an LXCXE motif located in a central domain known as conserved region 2. As a consequence of this interaction, E1A inactivates the Rb family proteins, thereby releasing E2Fs to constitutively activate S phase genes. Notably, Rb inactivation is not sufficient for E1A action, and, in fact, N-terminal regions of E1A also contribute to promoting S phase entry and oncogenic transformation. For example, the N terminus of E1A binds and interferes with the function of p300 and CBP, two highly related transcriptional co-activators that stimulate the expression of genes involved in growth inhibition and differentiation (11, 12). Also, E1A uses overlapping sequences to target p400 and TRRAP, two unrelated proteins that interact with each other and other additional proteins involved in chromatin remodeling (13). The nature of specific genes targeted by these complexes is not well understood, but the interactions appear important for transforming activity of both E1A and Myc (13–16).

The cellular proteins targeted by E1A to promote apoptosis are controversial, although many studies correlate apoptosis with the ability of E1A to bind p300/CBP (17–21). Indeed, consistent with the potential relevance of this interaction, p300 can directly control p53 stability. For example, p300-Mdm2 complexes can interact with p53, and contribute to its turnover, suggesting that E1A binding to p300 may lead to increased p53 stability by disrupting p53 degradation (22) and E1A may block polyubiquitination of p53 by p300 and Mdm2 (23). Alternatively, E1A may stabilize p53 by inhibiting Mdm2 transactivation through a p300-dependent mechanism (24). Additionally, inactivation of p300 increases p53 levels and activity (25), and expression of p300 can prevent apoptosis in some settings (26). However, other studies suggest that additional E1A targets are important, either instead of or in combination with p300/CBP. These include certain Id transcription factors, the p21 cyclin-dependent kinase inhibitor, C-terminal binding protein, p400-TRRAP (27), and Rb (17, 20, 28–31). Although it is formally possible that E1A promotes apoptosis through multiple mechanisms that depend on context, most studies have examined the proapoptotic activity of E1A in immortalized or tumor-derived lines that may have defects in signaling pathways relevant to E1A action, thereby complicating the results (20).



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  		FIG. 1.
Summary of E1A structure-function analysis. A, a graphic depiction of E1A 243R and the panel of mutants tested in this study. The conserved regions are indicated by light boxes (CR1 and CR2), deletions are indicated by gaps, and point mutations are indicated by asterisks. The specific amino acids removed in the deletion ({Delta}) mutations are listed on the left. {Delta}N and {Delta}CR2 have been previously described (20). The RG2 mutant has an arginine to glycine mutation at amino acid 2. The E55 mutant has mutations to alanine at amino acids 55 to 59 and a glutamic acid to glycine mutation at amino acid 60 (65). The M9 mutant has a glutamic acid to lysine, aspartic acid to asparagine, and glutamic acid to glycine mutations at amino acids 55, 56, and 60, respectively (66). The 143 mutant encodes the first 143 amino acids of E1A. B, IMR90 or U-2 OS cells expressing the indicated E1A mutants were evaluated for their ability to interact with p300/CBP, Rb, and p400 by co-immunoprecipitation and immunoblotting as described under "Experimental Procedures." E1A and its mutants were graded as having complete (+), reduced (±), or no ability (–) to interact with the indicated protein. Symbols in parenthesis indicate predicted results based on published literature, whereas nd indicates that binding has not been determined. C, IMR90 cells (Human) or MEFs (Murine) expressing the indicated E1A mutants were assessed for p53 levels, and their ability to undergo apoptosis was assessed following adriamycin treatment. Compared with full-length E1A, mutants either retained complete (+), reduced (±), or no ability (–) to increase p53 protein levels. Relative apoptosis for each mutant was normalized to full-length E1A as described under "Experimental Procedures." D, E1A-p400 and -Rb interaction is inseparable from the ability of E1A to promote apoptosis. Top, schematic diagram of full-length E1A with regions required for interacting with p300, Rb, and p400 indicated. Middle and bottom, relative apoptosis of each mutant to full-length E1A (y axis) versus amino acids deleted or altered for each mutant (x axis) in IMR90 (middle) or MEF (bottom) cells. The lengths of horizontal bars are scaled to reflect size and position of a given deletion. The dotted line illustrates boundaries within E1A that demarcate regions dispensable and required for promoting apoptosis.

 
Our laboratory has studied the effects of following retrovirus-mediated delivery of E1A into nonimmortal cells, thereby allowing E1A functions to be examined in genetically normal cells outside the context of adenovirus infection. Using this approach, we previously showed that E1A uses at least two functional domains that act in trans to induce ARF and p53 and promote apoptosis (5, 20). One domain includes conserved region 2, which promotes apoptosis by binding and inactivating Rb. Interestingly, whereas E1A targets all three Rb family members, the apoptotic defect of E1A mutants unable to bind Rb is reversed in cells lacking only Rb. A second region requires an N-terminal E1A domain that overlaps E1A sequences required to bind p300/CBP. However, the E1A mutants used in our study and in most others were not refined enough to exclude other targets. We therefore performed a more extensive analysis, which included RNA interference, to identify and validate N-terminal targets of E1A involved it apoptosis. Our results indicate that, in normal fibroblasts, the E1A-p300/CBP interaction is largely dispensable for E1A to induce ARF, p53, and apoptosis. Instead, E1A targets a p400-containing complex, which is essential for its proapoptotic activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
E1A Mutants and Retroviral Vectors—Ad5 E1A-12S mutants were cloned into both the pLPC and WZL vectors (32). The p400-EB mutant (which has a point mutation at residue 1086 that abolishes ATP binding) was subcloned into a Maloney murine leukemia virus-based retroviral vector (pLPC). To make a pSIN vector, a self-inactivating short hairpin vector with a puromycin selection marker, a BglII-ClaI fragment from pMSCV-puro-GW (33) was cloned into the BglII-EcoRV site in pQCXIX (Clontech), designated as pSIN-puro-GW. As SalI-EcoRI fragments, p400 short hairpin constructs with H1 promoter were subcloned from pSuper (34) into an XhoI-EcoRI site of pSIN-puro-GW, which flanks the GW cassette. As a control vector, the H1 promoter cassette was subcloned into the same site in the pSIN-puro-GW from the pSuper vector. All vectors were sequenced throughout the open reading frame or hairpin region.

Cell Culture and Gene Transfer—IMR90 fibroblasts and U2-OS cells were obtained from ATCC. Primary mouse embryo fibroblasts (MEFs) were derived from day 13.5 embryos and used between passages 2 and 4. All cells were maintained at 37 °C in 7.5% CO2 using Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin G/streptomycin sulfate (Sigma). Retroviruses were generated as described previously (35). IMR90s expressing an ecotropic receptor or MEFs were infected three times either with ecotropic or amphotropic retroviruses, and the infected populations were selected using either 1.5 µg/ml puromycin (Sigma) or 75 µg/ml hygromycin B (Roche Applied Science). Multiple constructs were introduced by simultaneous co-infection followed by sequential selection, with transduction efficiencies consistently >60% prior to selection and >95% after selection. For transfections, U-2OS cells were plated at 1 x 106 cells/10-cm plate, transfected with Fugene 6 (Roche Applied Science), and collected 48 h after transfection. For each transfection reaction, 10 µg of plasmid in 800 µl of serum-free Dulbecco's modified Eagle's medium with 25 µl of Fugene 6 was used. In all experiments, a pBABE lacZ control was included to measure transfection efficiency, which was ~40%.

Immunoprecipitations and Protein Expression—For E1A immunoprecipitations, retrovirally transduced IMR90s and transfected U2OS cells were collected for co-immunoprecipitation analysis 1 day postselection or 2 days after transfection, respectively. Cells were lysed in 20 mM Tris-HCl (pH 8.0), 170 mM KCl, 0.5% Nonidet P-40 containing Complete protease inhibitor mixture with EDTA (Roche Applied Science), rotating for 30 min at 4 °C. Lysates were centrifuged for 15 min at 13,000 rpm at 4 °C to remove nonsoluble debris. Protein levels were normalized using Bradford (Bio-Rad), antibodies were added, and lysates were incubated with rotation for 2 h at 4 °C. The antibodies used in various immunoprecipitations were as follows: for E1A, 50 µlofM73 (36); for T Ag, 50 µl of Pab416 (37); for p400, 20 µl of PO212 (13); for p300, 50 µl of NM11 (38); and for Rb, 25 µl of XZ55 and 25 µl of C36 (39). The equivalent of 25 µl of drained beads of Protein A-Sepharose was added, and lysates were incubated with rotation for an additional 1 h at 4 °C. Immunoprecipitations were washed four times with lysis buffer, resuspended in 2x SDS reducing buffer (1x SDS reducing buffer: 62.5 mM Tris (pH 6.8), 10% glycerol, 0.02% SDS, and 5% {beta}-mercaptoethanol), boiled for 5 min, and resolved by 6% SDS-PAGE. Gels were transferred to activated nitrocellulose in 48 mM Tris, 390 mM glycine, 0.1% (w/v) SDS, and 20% methanol for 13 h at 350 mA at 4 °C. Membranes were blocked in TBS with Tween 20 (0.2%) and milk (5%). The antibodies used to probe membranes of various immunoprecipitations were anti-p400 (RW144 1:3 (11)), anti-p300 (NM11 1:50 (38)), anti-CBP (A-22 1:1000; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and anti-Rb (XZ55 1:100, C36 1:100, H14001A 1:1000 (39) and Pharmingen).

For immunoblotting, cell pellets were lysed in 1x SDS reducing buffer (above) by boiling for 5 min. Debris was pelleted by centrifugation at 14,000 rpm for 5 min. Samples were normalized using a Bradford assay and was resolved by SDS-PAGE. Protein was transferred to either nitrocellulose or activated polyvinylidene difluoride (Immobilon-P) membranes using one of two conditions: either for 90 min at 100 V at room temperature using a 25 mM Tris (pH 8.3), 192 mM glycine, and 20% MeOH transfer buffer or for 16 h at 350 mA at 4 °C using a 50 mM Tris (pH 8.3), 380 mM glycine, 0.1% SDS, and 20% methanol transfer buffer. Membranes were blocked as above. The antibodies used to probe membranes of various immunoblots were anti-E1A (sc-430 (13 S-5) 1:1000; Santa Cruz Biotechnology), anti-p53 (DO-1 (human) or 505 (murine) 1:1000; Novocastra), anti-ARF (sc-8340 (human) 1:1000 (Santa Cruz Biotechnology); ab80 (murine) 1:1000 (Abcam); anti-p400 (above); anti-{beta}-actin (ac-15 1:3500; Sigma); and anti-tubulin (B-512 1:5000; Sigma).

Northern Blots—Northern blots were conducted using total RNA purified by Trizol (Invitrogen). Twenty µg of total RNA was fractionated on 1.0% formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham Biosciences) in 10x SSC (1x SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, 0.1% SDS). Hybridization was performed at 65 °C with 32P-labeled probes in 7% SDS, 0.5 M phosphate buffer (pH 7.2), 0.1 mg/ml tRNA, and 10 mM EDTA. The membranes were washed twice at 65 °C in 2x SSC for 10 min, twice at 65 °C in 1x SSC for 10 min, and twice at 65 °C in 0.1x SSC for 10 min. Probes for ARF and 18 S rRNA were labeled by random priming (T7 QuickPrime; Amersham Biosciences).

Immunofluorescence—Cells plated on coverslips were fixed in 4% paraformaldehyde at room temperature for 15 min and permeabilized in phosphate-buffered saline containing 0.2% Triton X-100 for 5 min at 4 °C. Slides were blocked in phosphate-buffered saline containing 0.5% normal goat serum (Sigma) for 30 min at room temperature and subsequently incubated for 1 h at room temperature with a primary antibody in blocking solution. The antibodies used were anti-p19ARF (1:100; Novus Biologicals NB200-106) and anti-E1A (1:50; M58 + M73). Slides were washed three times with phosphate-buffered saline and incubated for 1 h at room temperature with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594-conjugated secondary antibodies (1:1000; Molecular Probes, Inc., Eugene, OR). Slides were washed three times with phosphate-buffered saline, incubated for 1 min at room temperature in blocking solution containing 1 µg/ml 4',6'-diamidino-2-phenylindonle (Sigma), mounted with Vectashield (Vector Laboratories), and viewed under a Zeiss immunofluorescence microscope (Axioscop 50; Thornwood, NY).

Cell Viability—Cells (1 x 105) were plated into 12-well plates 24 h before treatment. Twenty-four h following treatment with adriamycin or serum withdrawal, adherent and nonadherent cells were pooled and analyzed for viability by trypan blue exclusion. At least 200 cells were counted for each data point. Cell death was confirmed to be apoptosis by visualizing chromatin condensation with 1 µg/ml 4',6'-diamidino-2-phenylindonle (Sigma). All values represent data from at least three separate experiments. Relative apoptosis (see Fig. 1) was determined by comparing cell death 24 h after treatment with increasing doses of adriamycin (0.1, 0.2, and 0.5 µg/ml). Values ((percentage of apoptosis mutant/percentage of full-length E1A) x 100) were averaged for all doses and represent the average of at least three independent trials. Values were internally controlled by calculating relative apoptosis based on the values for full-length E1A in the same trial.



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  		FIG. 2.
E1A associates with p300, CBP, Rb, and p400 through distinct regions. A, E1A-p300, -CBP, and -Rb interaction was examined in IMR90 cells expressing empty vector (V), full-length E1A, RG2, {Delta}2–11, {Delta}26–35, or {Delta}CR2 by immunoprecipitation with either an E1A antibody (E) or a nonspecific control antibody (T), with subsequent Western blotting for p300, CBP, or Rb. 293 cells express E1A and serve as a positive control. B, E1A-p400 interaction was examined in IMR90 or U-2 OS cells expressing empty vector (V), full-length E1A, {Delta}N, RG2, {Delta}26–35, or {Delta}CR2 by immunoprecipitation with either an E1A antibody (E), a nonspecific control antibody (T), a p400-specific control antibody (400), or a p300-specific control antibody (300) with subsequent Western blotting for p400 and p300 using a monoclonal antibody that crossreacts with both p400 and p300. Similar results have been found for the E1A-TRRAP interaction (13, 15). C, to confirm equal loading in B, E1A expression in IMR90 or U-2 OS cells expressing the indicated E1A mutants were examined by Western blotting. Similar expression levels were also found between samples in A (not shown).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In order to better understand the apoptotic activity of E1A, we conducted an extensive structure-function analysis to correlate the regions of E1A capable of binding specific cellular proteins with those required to engage the ARF-p53 pathway and to promote apoptosis. To do this, we cloned a panel of E1A mutants that altered or deleted various residues in the N-terminal 143 amino acids into a pLPC vector that also expressed a puromycin resistance gene. Since the 12 S form of E1A (which contains conserved regions 1 and 2 but not conserved region 3) is sufficient to induce p53 and apoptosis (40), all of the N-terminal mutants were studied in a 12 S E1A background. Each vector was then packaged into a retrovirus and introduced into IMR90 normal diploid human fibroblasts or early passage MEFs. The resulting populations were placed in puromycin (~2 days) to eliminate uninfected cells, and all assays were conducted shortly thereafter to minimize selection for or against individual E1A mutants. Using this approach, we produced a series of polyclonal cell populations harboring full-length or mutant E1A.

Interactions between E1A and Cellular Proteins in Normal Cells—Although the regions of E1A required for interacting with key transformation targets such as p300, CBP, and Rb have been previously identified, no study has examined these interactions in normal cells. Therefore, to rule out any differences between cell types, we examined the ability of key E1A mutants to interact with p300, CBP, and Rb when expressed in IMR90 fibroblasts. For p300/CBP and Rb, the results in normal human IMR90 fibroblasts were largely similar to what has been reported in other cell types, with only minor variations in requirements for binding p300 within the CR1 region (summarized in Fig. 1B and Refs. 41 and 42)). For example, the RG2 and {Delta}2–11 mutants fail to interact with p300 and CBP but retain Rb binding. In contrast, the E1A {Delta}CR2 mutant interacts with p300 and CBP but not Rb, whereas the E1A {Delta}26–35 mutant interacts with p300, CBP, and Rb (Fig. 2A).

The N-terminal region of E1A is also required for binding to p400-TRRAP chromatin-remodeling complexes, although this interaction has not been mapped comprehensively. Therefore, we tested the ability of our E1A mutants to interact with p400 in either IMR90 or U2OS cells by co-immunoprecipitation (summarized in Fig. 1B and examples shown in Fig. 2B; see also Ref. 13)). Significantly, E1A requires amino acids 16–36 to interact with p400, a region that is distinct from those required for p300 or Rb interaction (Fig. 1D, top). The {Delta}26–35 mutant, which has been previously described (13), is unique in that it only weakly binds p400-TRRAP yet retains its ability to interact with p300 and Rb (Fig. 2, A (compare lanes 5 and 8) and B (compare lanes 5 and 8)). Together, this analysis created a comprehensive map of the regions of E1A required to interact with p300, CBP, Rb, and p400 in normal cells that confirms and extends reports from immortal or tumor-derived lines (Fig. 1, A, B, and D, top).



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  		FIG. 3.
Sensitization to drug-induced apoptosis correlates with p400 and Rb binding. IMR90 (Human) or MEF (Murine) cell populations expressing empty vector, E1A, {Delta}N, RG2, {Delta}2–11, {Delta}26–35, or {Delta}CR2 were treated with the indicated doses of adriamycin, and viability was determined 24 h later by trypan blue exclusion. The percentage viability represents the actual percentage of surviving cells in the total population after treatment. Each value represents the mean ± S.D. of the data from at least three separate experiments.

 



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  		FIG. 4.
Sensitization to apoptosis following factor withdrawal correlates with p400 interaction. MEF cell populations expressing the indicated construct underwent serum withdrawal (0.1% FBS, black bars) or remained in high serum (10.0% FBS, gray bars), and viability was determined 24 h later by trypan blue exclusion. Each value represents the mean ± S.D. of the data from at least three separate experiments.

 
A p400-TRRAP Complex Is Important for E1A to Promote Apoptosis—We next identified the regions of E1A required to promote apoptosis. Whereas E1A can directly promote apoptosis in the context of adenovirus infection (43–45), retrovirally transduced cells can tolerate E1A but become highly sensitized to apoptosis following treatment with DNA-damaging agents or upon growth factor depletion (46, 47). Therefore, IMR90 and MEF cell populations expressing an empty vector, full-length E1A, or various E1A mutants were treated with adriamycin, a commonly used chemotherapeutic agent known to damage DNA, and analyzed for cell viability 24 h later by trypan blue exclusion (summarized in Fig. 1C, shown in Table I and Fig. 3). Importantly, we have previously shown that E1A-expressing cells undergo apoptosis under the conditions used in this study (adriamycin treatment and serum depletion) using DNA ladder analysis, annexin V staining, apoptotic chromatin condensation, caspase cleavage, poly(ADP-ribose) polymerase cleavage, and electron microscopy (20, 32, 47–49). We have also shown that death in these circumstances is blocked by loss of p53 (46, 47) or overexpression of the antiapoptotic proteins E1B19K or Bcl-2 (32). In this study, all cell death was confirmed to be apoptotic by assessing chromatin condensation using 4',6'-diamidino-2-phenylindonle (32).


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  		TABLE I
p400 and Rb binding by E1A is required for apoptosis

Human (IMR90) or murine (MEF) cell populations expressing the listed constructs were treated with the indicated doses of adriamycin, and viability was determined 24 h later by trypan blue exclusion. Values are the actual percentage of surviving cells in the total population after treatment. Each value represents the mean and the S.D. of the data from the listed number of trials.

 
Unexpectedly, the regions of E1A required to promote apoptosis following adriamycin treatment did not correlate with the ability to bind p300/CBP but, rather, with the ability to bind p400 and Rb (compare Fig. 1D, top and bottom). As an example, the E1A RG2 mutant retained its ability to interact with p400 (Fig. 2B, lane 7) but not p300/CBP, yet still promoted apoptosis in response to adriamycin (Fig. 3, B and G). Conversely, the E1A {Delta}26–35 mutant retains its ability to bind p300/CBP (Fig. 2A, lane 8) but not p400 (Fig. 2B, lane 8), and is defective at promoting apoptosis (Fig. 3, D and I). p400 and TRRAP form larger complexes in cells either lacking or expressing E1A, and E1A mutants unable to interact with p400 are also unable to associate with TRRAP (13, 15). Whereas most of our experiments use adriamycin to trigger apoptosis, similar results are observed in MEFs following serum depletion, although in MEFs the p300-defective mutants also show a partial apoptotic defect (Fig. 4). Therefore, a p400-TRRAP complex may be an important target of E1A in apoptosis.

Induction of ARF and p53 by E1A Requires Binding to p400-TRRAP—We also tested the same panel of E1A mutants for their ability to activate the ARF-p53 pathway. To examine p53 levels, IMR90 cells or MEFs expressing each E1A mutant were subjected to immunoblotting using a p53-specific antibody and compared with cells expressing full-length E1A. Not surprisingly, the regions of E1A required for p53 induction overlap with those required for apoptosis in both cell types (summarized in Fig. 1C). For example, RG2 fails to bind p300/CBP but retains interactions with p400-TRRAP and Rb and efficiently induces p53 (Fig. 5, A (IMR90s) and B (MEFs); compare lanes 1 and 4), whereas {Delta}26–35 and {Delta}CR2 retain p300/CBP binding but do not bind p400-TRRAP or Rb, respectively, and are defective at inducing p53 (Fig. 5A (IMR90s) and Fig. 5B (MEFs); compare lane 2 with lanes 6 and 7).

Cells expressing select E1A mutants were also examined for ARF expression by Northern blotting, immunoblotting, and immunofluorescence. Consistent with their failure to efficiently induce p53, cells expressing {Delta}CR2 or {Delta}26–35 were compromised in their ability to induce ARF mRNA in MEFs (Fig. 5C, compare lane 2 with lanes 4 and 6) and protein (Fig. 5, A and B, lanes 6 and 7) and did not contain an increase in nucleolar ARF (Fig. 5D). We did note, however, that increasing the levels of E1A {Delta}26–35 by increasing the multiplicity of infection induced more ARF protein, which is consistent with the known ability of this mutant to bind some p400 (data not shown; also see Ref. 13). Although this dosage sensitivity probably contributed to variability between experiments (e.g. compare ARF induction in Fig. 5 and 8), the E1A {Delta}26–35 mutant was always impaired in its ability to induce ARF, and increased E1A dosage never fully rescued ARF levels nor altered cellular sensitivity to proapoptotic stimuli. These results suggest that E1A induces ARF and p53 by interacting with Rb and a p400-TRRAP complex that, in turn, act cooperatively to control ARF mRNA expression.



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  		FIG. 5.
Induction of the ARF-p53 pathway by E1A correlates with p400 interaction. A and B, p53 and ARF protein levels in populations of IMR90 (Human) (A) or MEF (Murine) (B) cells expressing empty vector (lane 1), full-length E1A (lane 2), {Delta}N (lane 3), RG2 (lane 4), {Delta}2–11 (lane 5), {Delta}26–35 (lane 6), or {Delta}CR2 (lane 7) were examined by Western blotting. E1A protein levels were included to confirm equal expression. C, ARF message levels in populations of MEFs expressing the indicated E1A proteins were determined by Northern blot analysis. 18 S RNA served as a loading control. D, ARF localization in populations of MEF cells expressing the indicated E1A proteins was determined by immunofluorescence. E1A expression was also confirmed by immunofluorescence (not shown). Fibrillarin was used as a marker for nucleoli. Nuclear staining is shown using 4',6'-diamidino-2-phenylindonle. Cells shown are a representative set of the larger population.

 



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  		FIG. 6.
E1A modulation of p300 activity is dispensable for p53 induction and apoptosis. A, IMR90 (Human) or MEF (Murine) cell populations expressing empty vector, E1A, or E1A 143 were treated with the indicated doses of adriamycin, and viability was determined 24 h later by trypan blue exclusion. Each value represents the mean ± S.D. of the data from at least three separate experiments. B, ARF and p53 protein levels in populations of MEF cells expressing empty vector, full-length E1A, or E1A 143 were examined by Western blotting. E1A protein levels were examined by Western blotting to confirm equal expression, whereas tubulin serves as a general loading control.

 
N-terminal E1A Sequences Are Sufficient for Apoptosis— Previous studies demonstrate that the C-terminal domain of E1A can inhibit p300 histone deacetylase activity independently of the N-terminal domain and can promote anoikis (apoptosis of cells in suspension) of carcinoma cells by targeting the C-terminal binding protein (30). To determine whether the E1A C terminus contains domains required for apoptosis in normal cells, we examined the E1A 143 mutant for its ability to induce apoptosis, ARF, and p53 in MEFs and IMR90 cells. This fragment lacks the entire C-terminal domain and is unable to inhibit p300/CBP histone acetyltransferase activity or bind C-terminal binding protein but remains able to bind p300/CBP, p400-TRRAP, and the Rb family. Both IMR90 cells and MEFs expressing E1A 143 efficiently underwent apoptosis in response to adriamycin (Fig. 6A) and serum withdrawal (data not shown), as did cells expressing a double mutant, also lacking amino acids 2–11 ({Delta}2–11.143) and thus unable to stably bind p300 nor harbor C-terminal functions (data not shown). Moreover, cells expressing E1A 143 retained their ability to induce both ARF and p53 protein (Fig. 6B and data not shown). Hence, the N-terminal region of E1A (amino acid residues 12–143) is sufficient to promote apoptosis and activate p53, independently of its ability to physically associate with p300, modulate p300 activity, and bind C-terminal binding protein. These results indicate that E1A targets a limited set of proteins, perhaps only Rb and p400-TRRAP, to modulate apoptosis.

p400 Is Required for Apoptosis Associated with E1A—To determine whether the E1A-p400 interaction might be required for apoptosis, we tested whether a p400 fragment capable of binding E1A, but not TRRAP, could interfere with apoptosis. This fragment consists of a conserved SWI2/SNF2-like domain and is very similar to one that has been shown to bind both E1A and TIP48/49 (also known as TAF54{alpha}/{beta}) and can partially rescue the transformation defect of E1A {Delta}26–35 (13).2 A retrovirus expressing p400-EB (Fig. 7A) was introduced into IMR90 cells expressing E1A, and the resulting populations were examined for viability following adriamycin treatment. Compared with similar populations expressing a control vector, p400-EB-expressing cells showed substantially reduced apoptosis upon adriamycin treatment (Fig. 7B). Although only a correlation, this observation is consistent with the possibility that the E1A-p400-TRRAP interaction is important for apoptosis.



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  		FIG. 7.
A p400 fragment can inhibit E1A-mediated apoptosis. A, schematic diagram of p400. The top diagram depicts full-length p400. The Gal4-p400 fragment fusion construct is shown below. Note that p400-EB is able to bind E1A but unable to bind TRAAP (13) (D. Livingston, unpublished observations). B, IMR90 cell populations were retrovirally transduced with empty vector, full-length E1A, Gal4-p400-EB, or full-length E1A and Gal4-p400-EB. Cell populations were treated with the indicated dose of adriamycin for 24 h, and viability was determined by trypan blue exclusion. Each value represents the mean ± S.D. of the data from at least three separate experiments.

 
In principle, E1A might promote apoptosis by inactivating p400 or, conversely, by modulating p400 functions. If E1A inactivates p400, then suppressing p400 activity should increase the ability of E1A {Delta}26–35 (which binds p400 poorly) to promote apoptosis. In fact, such an approach revealed that the requirement for conserved region 2 in apoptosis involved its ability to inactivate Rb (20). Conversely, if E1A requires p400 to promote apoptosis, then suppression of p400 activity should reduce cell death in cells expressing full-length E1A. To distinguish between these possibilities and to directly link p400 to apoptosis, we generated two distinct short hairpin RNAs (shRNAs) capable of stably suppressing p400 protein using RNA interference. We then co-expressed each p400 shRNA (sh-p400.1 and sh-p400.2) with a control vector, full-length E1A, or {Delta}26–35 and analyzed the resulting populations for ARF and p53 expression and susceptibility to apoptosis. Since we noted some variability between experiments, data from multiple experiments are shown in the immunoblots and averaged in viability plots.

E1A induced p400 protein that is only partially dependent on the p400 binding domain (Fig. 8A, compare lane 4 with lane 7), suggesting that E1A does not promote apoptosis simply by inducing p400 levels. In normal IMR90 cells, both sh-p400.1 and sh-p400.2 efficiently suppressed p400 protein to nearly undetectable levels, and the cells underwent a senescent-like arrest as described in detail elsewhere (34). However, in cells expressing full-length E1A or {Delta}26–35, neither shRNA was able to completely repress p400, typically producing knockdowns only slightly less than normal cells, with sh-p400.1 being typically less effective than sh-p400.2 (Fig. 8A, compare lanes 1, 5, and 6). Nonetheless, both shRNAs were effective and could reduce p400 levels to or below normal levels.

Interestingly, despite E1A levels comparable with controls, cells expressing E1A and p400 shRNAs consistently showed a reduction in ARF and p53 protein, with cells co-expressing sh-p400.2 typically showing a more dramatic effect (Fig. 8A, Experiment 1, compare lane 4 with lane 6). Moreover, these cells showed a substantial reduction in apoptosis compared with E1A-expressing cells harboring a control vector (Fig. 8B). Similar results were observed in multiple experiments and required efficient knockdown of p400, since expression of the same p400 shRNAs in a different retroviral backbone produced only a modest suppression of p400 and had little effect on ARF or p53 levels or susceptibility to apoptosis (data not shown). As described above, cells expressing E1A {Delta}26–35 showed only a slight increase in ARF and p53 levels (Fig. 8A, compare lane 1 with lane 7). However, these proteins where further reduced by expression of p400 shRNAs, probably due to the residual p400 binding activity of this mutant (Fig. 8A, compare lane 7 with lanes 8 and 9). Importantly, neither p400 shRNA rescued the apoptotic defect of E1A {Delta}26–35 (Fig. 8B). Therefore, E1A does not inactivate p400 to promote apoptosis but, instead, requires p400 activity for efficient cell death.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we used the adenovirus E1A oncoprotein to probe the mechanisms whereby normal cells couple proliferation and apoptosis in response to oncogenic stress. We show that, in addition to binding the retinoblastoma gene product, E1A must also associate with a p400-containing complex to engage the ARF-p53 pathway and sensitize cells to apoptotic stimuli. Accordingly, the E1A regions required for binding p400 are also required for apoptosis, and disruption of the E1A-p400 interaction substantially attenuates cell death. However, we also show that, whereas E1A inactivates Rb, it requires p400 to effectively promote apoptosis. Thus, E1A induces p400 protein, and suppression of p400 expression attenuates the induction of ARF, p53, and apoptosis in E1A-expressing cells. It is noteworthy that the ability of E1A to bind p400 is also important for its transforming potential (13, 15, 16). Therefore, our results identify p400 as a novel component of the cellular machinery that couples proliferation to cell death.

Although the immediate consequences of the association between E1A and Rb or p400 are distinct, both interactions probably mediate E1A activities through their ability to alter transcription. For example, E1A disrupts the Rb-E2F interaction, freeing up E2F to constitutively activate target genes involved in cell cycle progression and apoptosis. In the case of p400, E1A perturbs the normal composition of the p400 complex (13). TRRAP is a part of five large multisubunit complexes implicated in chromatin remodeling including the P/CAF, TFTC, hSTAGA, TIP60, and p400 complexes (13, 50–54), and p400 associates with the TIP60 and p400 complexes but not the P/CAF complex (13). Although it remains to be determined whether E1A interacts with a subset of these complexes, the hSTAGA and TIP60 complexes have been implicated in DNA repair. Notably, however, ARF is not responsive to DNA damage (6), and TRRAP has an essential role in normal cellular proliferation; therefore, it is more likely that deregulation of p400-TRRAP remodels chromatin to promote hyperproliferation and, directly or indirectly, apoptosis. Consistent with this view, p400 complexes possess chromatin remodeling activity through their association with TIP60, by selective histone acetylation and subsequent nucleosomal exchange at sites of DNA lesions (55). Furthermore, TIP60 mutations affect DNA double strand break repair and inhibit {gamma}-irradiation-induced apoptosis. Conversely, ectopic expression of TIP60 increases apoptosis (53, 54).

p400 also acts as a regulator of cellular senescence by repressing the p21 cyclin-dependent kinase inhibitor (34), which, in turn, can attenuate oncogene-induced apoptosis (56). Thus, by increasing p400 levels, E1A may allow p400-containing complexes to more effectively repress certain antiapoptotic genes, thereby shifting the cellular default state from cell cycle arrest to apoptosis. Alternatively, this requirement may reflect the production of distinct, non-p400-containing chromatin-modifying complexes produced by a redistribution of p400 complex partners resulting from both the increase in p400 levels and the displacement of certain subunits by E1A. In principle, such changes may alter the biochemical properties of the p400 complex by displacing TIP48/49 in a manner that cannot be produced merely by inactivating p400.

Whatever the precise mechanism, it is clear that neither the E1A-Rb nor E1A-p400 interactions act in isolation; E1A must simultaneously target both proteins to efficiently promote cell death. Consistent with these findings, mutational analysis in Caenorhabditis elegans has recently demonstrated functional redundancy between p400-TRRAP and Rb homologs in cell fate determination (57). These interactions ultimately modulate distinct cellular activities, since E1A mutants defective in binding either p400 or Rb stabilize p53 and promote apoptosis when expressed in trans (20). Presumably, these downstream processes cooperate to promote apoptosis in multiple ways, perhaps controlling distinct activities required for apoptosis, or converging to regulate certain apoptotic targets.

Although E1A may target many processes involved in apoptosis, its ability to control ARF expression is clearly important for this activity (5). Although ARF is an E2F target gene, it differs from canonical E2F targets in that it is not cell cycle regulated, nor is it expressed in rapidly dividing tissues (1). Indeed, studies in mice confirm that ARF is almost completely buffered from normal proliferative signals but responds to aberrant proliferative stimuli (58). One component of this buffer involves E2F3b, a repressive E2F family member that is bound to the ARF promoter in normal cells and is exchanged with activator E2Fs upon oncogene expression (9). Another component involves the polycomb group protein Bmi-1, which acts at the level of higher order chromatin organization to repress ARF expression in vitro and in vivo (7).



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  		FIG. 8.
E1A requires the p400-TRRAP complex to promote apoptosis. A, two representative sets of experiments of p400, ARF, and p53 protein levels in populations of IMR90 cells expressing empty vector, full-length E1A, or {Delta}26–35 alone or in combination with either of two short hairpins to p400 (sh.1 and sh.2) were examined by Western blotting. E1A protein levels were examined by Western blotting to confirm equal expression and tubulin or {beta}-actin protein levels confirm equal loading. B, cell populations expressing the indicated construct underwent adriamycin treatment for 24 h, and viability was determined by trypan blue exclusion. Each value represents the mean ± S.D. of the data from at least three separate experiments.

 
Our data identify p400 and its associated complexes as an additional regulator of the ARF locus. One attractive explanation for these observations is that p400 complexes contribute to the repressive chromatin environment of the ARF locus, such that binding by E1A alleviates this repression and/or produces a new complex that contributes to ARF transactivation. Consequently, the E1A interaction with p400, although incapable of inducing ARF alone, may create a permissive chromatin environment that allows free activator E2Fs to stimulate ARF transcription. Whether such a mechanism acts directly or indirectly remains to be determined but may explain how p400 cooperates with Rb loss to mediate E1A effects.

Our results are intriguing in light of studies implicating the p300/CBP transcriptional co-activators as critical for E1A to stabilize p53 and promote apoptosis. These studies showed that certain E1A mutants defective at binding p300/CBP were unable to stabilize p53 or promote apoptosis (17–20, 59) and that overexpression of p300 in E1A-expressing cells could destabilize p53 and suppress apoptosis (24). However, the regions of E1A required for binding p300/CBP and p400 overlap, and most studies have not used mutants that distinguish between the two interactions. However, we see that an E1A mutant capable of binding p300 but not p400 is defective at inducing ARF, p53, and apoptosis (see also Ref. 27); conversely, we see mutants capable of binding p400 but not p300/CBP that retain these activities (e.g. RG2 and {Delta}2–11). Thus, whereas p300 can clearly regulate p53 levels and activity (22, 24), this is not the predominant mechanism through which E1A stabilizes p53 and promotes apoptosis in normal cells. Nonetheless, the E1A-p300/CBP interaction may contribute to apoptosis in some circumstances (see Fig. 4), perhaps due to its ability to act as a p53 transcriptional co-activator (60, 61).

DNA tumor virus oncoproteins have evolved to target key nodes in cellular signaling networks and, consequently, have been widely used to identify cellular activities involved in normal growth control. For example, studies using E1A identified Rb as an important tumor suppressor, E2F as a key component of cell cycle control, and p53 as a crucial mediator of apoptosis (e.g. see Refs. 20, 40, and 62–64). Although the contribution of p400 to the apoptotic programs that limit spontaneous tumorigenesis remains to be determined, we suspect that its role extends beyond adenovirus. In this regard, it is intriguing that c-myc, a cellular oncogene that also engages the ARF-p53 pathway to promote apoptosis, binds and induces p400 protein and requires a p400 complex to promote transformation (34). Nonetheless, in fibroblasts not expressing proapoptotic oncogenes, p400 may act to control cellular life span, in part, by directly suppressing the p21 cyclin-dependent kinase inhibitor (34). Together, these data suggest that p400-containing complexes act as a crucial node in networks that couple proliferation, apoptosis, and transformation and that their activity can be dramatically influenced by a transforming oncogene. A more complete understanding of the composition and activity of these complexes will provide further insights into the innate tumor suppressor mechanisms that couple proliferation to cell death and limit malignant transformation.


   FOOTNOTES
 
* This work was supported by a grant from the NCI, National Institutes of Health (to D. M. L.) and NCI, National Institutes of Health Project Grant CA13106 (to S. W. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Department of Defense Breast Cancer Research Program predoctoral fellowship. Back

|| Supported by a Human Frontier long term fellowship. Back

** Supported by a Tularik postdoctoral fellowship. Back

{ddagger}{ddagger} Supported by a Department of Defense Breast Cancer Research Program postdoctoral fellowship and an Uehara Memorial Foundation research fellowship. Back

§§ AACR-NFCR Research Professor. To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-8406; Fax: 516-367-8454; E-mail: lowe{at}cshl.org.

1 The abbreviations used are: Rb, retinoblastoma protein; CBP, CREB-binding protein; cAMP-response element-binding protein; MEF, mouse embryo fibroblast; shRNA, short hairpin RNA. Back

2 M. Fuchs and D. M. Livingston, unpublished observations. Back



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