Departments of Microbiology and Immunology1, Biochemistry2, Pharmacology and Toxicology3 and Oncology4, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CanadaN6A 4L6
Author for correspondence: Joe Mymryk. Fax +1 519 685 8616. e-mail jmymryk{at}uwo.ca
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Abstract |
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Introduction |
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In addition to its many interactions with cellular proteins, the 289 residue E1A protein binds non-specifically to double-stranded (ds) DNA in vitro (Chatterjee et al., 1988 ) and a C-terminal fragment of E1A spanning amino acids 201289 is sufficient to bind dsDNA in vitro (Zu et al., 1992
). Substitution of aspartic acid for arginine at amino acids 205 and 206 or deletion of residues 201216 blocks binding of this C-terminal fragment of E1A to DNA (Zu et al., 1992
). In vivo, both of these mutants function as well as the wild-type protein to activate and repress transcription, suggesting that the interaction of E1A with DNA is not required for either of these activities (Zu et al., 1992
).
In this study, we tested a number of fragments of E1A for DNA binding and determined that only the C-terminal region of E1A bound DNA in vitro. This region of E1A bound both single-stranded (ss) and dsDNA and binding required a basic region spanning amino acids 201220. In addition, a minimal fragment of E1A encompassing amino acids 201218 was sufficient to bind DNA and several cation-exchange resins. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain (AD) inhibited growth when expressed in Saccharomyces cerevisiae, this did not require the region we identified as necessary for DNA binding in vitro. Our results suggest that E1A may not function as a DNA-binding protein in vivo.
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Methods |
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To express E1A fused to glutathione S-transferase (GST), DNA sequences encoding amino acids 182, 93138, 139204, 201289, 201218 and 221289 of E1A were excised from the corresponding yeast expression vectors described above with EcoRI and XhoI and ligated into the same sites of pGEX-4T1 (Amersham Pharmacia). pGEX-4T1 vectors encoding amino acids 201289 of E1A with deletions of residues 227239, 239254, 255270, 271284 and 285289 were constructed by replacing the wild-type XbaIXhoI fragment with the corresponding mutant fragments derived from a series of vectors provided by G. Chinnadurai, St Louis University Health Sciences Center, St Louis, MO, USA (Schaeper et al., 1995 ).
Expression and purification of GSTE1A fusion proteins.
GSTE1A fusion proteins were expressed and purified as described by the pGEX-4T1 supplier (Amersham Pharmacia), with minor modifications. An overnight culture of Escherichia coli BL21 cells transformed with either pGEX-4T1 or one of the pGEX-4T1 recombinants described above was diluted 1:10 in LB medium containing 50 µg/ml ampicillin and grown at 37 °C. After 1 h, expression of the fusion protein was induced by the addition of IPTG to a final concentration of 0·2 mM and cultures were grown for a further 3 h at 37 °C. Cells were harvested, resuspended in 1/200 vol. PBS and lysed by mild sonication. DNaseI was added to 20 µg/ml and lysates were incubated for 30 min at room temperature. Subsequently, 10% Triton X-100 was added to a final concentration of 1% and the lysates were centrifuged at 21000 g for 5 min at room temperature. Then, 1/10 vol. 50% glutathione bead slurry was added to the supernatant and mixed gently at room temperature for 5 min. Beads were washed twice with 100 vols PBS, once with TEK buffer (10 mM TrisHCl pH 7·4, 1 mM EDTA and 50 mM KCL) containing 1·0 M NaCl and twice with TEK buffer alone. The GSTE1A fusion proteins were then eluted from the beads with 50 mM TrisHCl pH 8·0 containing 5 mM glutathione and dialysed against TEK buffer.
Assay for binding to nucleic acid cellulose, cellulose phosphate and carboxymethyl Sepharose.
GSTE1A fusion proteins were assayed for binding to ds and ss calf thymus DNA cellulose (Sigma), cellulose phosphate (Whatman Nuclepore) and carboxymethyl Sepharose (Amersham Pharmacia). dsDNA cellulose, ssDNA cellulose and carboxymethyl Sepharose were allowed to swell in TEK buffer overnight at 4 °C, washed with TEK buffer twice and then resuspended in an equal volume of buffer. Weighed cellulose phosphate was stirred gently into 25 vols 0·5 M NaOH, left for 5 min at room temperature and washed with TEK buffer until the pH of the flow-through dropped to below 11·0. The resin was then mixed with 25 vols 0·5 M HCl, left for 5 min at room temperature and washed with TEK buffer until the pH of the flow-through reached 3·0. The cellulose phosphate resin was then resuspended in an equal volume of TEK buffer. Prior to binding assays, 1 ml of the slurry was washed twice with 25 ml TEK buffer and resuspended in 500 µl TEK buffer.
Binding assays of GSTE1A fusion proteins were performed as follows: 100 µl DNA cellulose, cellulose phosphate or carboxymethyl Sepharose resin slurry was centrifuged briefly and the supernatant was removed. Then, 30 µg recombinant protein was diluted to a final volume of 100 µl in TEK buffer, mixed gently with the resin in a microcentrifuge tube and allowed to bind for 30 min on ice. After centrifuging briefly, the supernatant (flow-through) was withdrawn into a fresh tube and the resin was washed twice with 100 µl TEK buffer. The wash fractions were also collected. Fusion protein was eluted sequentially with 100 µl TEK buffer containing 0·1 M NaCl, 0·2 M NaCl, 0·3 M NaCl, 0·4 M NaCl, 0·6 M NaCl and 1·0 M NaCl. Aliquots of 24 µl from each fraction were mixed with 8 µl 4x sample buffer (0·0625 M TrisHCl pH 6·8, 10% glycerol, 2% SDS, 0·05% b-mercaptoethanol and 0·0005% bromophenol blue), boiled for 4 min and separated on 12% SDSpolyacrylamide gels. Proteins were visualized by staining with Coomassie brilliant blue.
Analysis of growth inhibition in yeast.
To determine the effect of each fragment of E1A on yeast growth, yeast strain w3031a (obtained from M. Smith, University of Virginia, Charlottesville, VA, USA) was transformed with pJG4-5+ or corresponding derivatives expressing portions of E1A using the lithium acetate method (Adams et al., 1998 ). Cells were plated on synthetic complete (SC) selective medium containing galactose to induce recombinant protein expression. After 4872 h of growth at 30 °C, pictures of plates were taken using a Pharmacia Biotech ImageMaster VDS image capture system. Toxicity of E1A fragments was assessed by visually examining the size of yeast colonies and comparing them to those that received the empty pJG4-5+ plasmid. Similar tests were carried out in yeast strains FY86 (GCN5) and FY1370 (
gcn5) (Roberts & Winston, 1997
) (obtained from C. Brandl, University of Western Ontario, London, Ontario, Canada). Yeast doubling time analysis was performed by diluting overnight cultures of transformed yeast to 0·1 A600 in SC selective medium containing galactose. Triplicate cultures were grown with agitation at 30 °C and A600 was measured at various times post-inoculation.
Western blot analysis of recombinant protein expression in yeast.
Expression levels of the C-terminal fragments of E1A in yeast were determined by Western blot analysis. Yeast were transformed with pJG4-5+ or pJG4-5+ derivatives expressing the C-terminal portion of E1A and plated on SC selective medium containing glucose. Colonies of transformed yeast were picked from each plate and used to inoculate 5 ml liquid SC selective medium containing glucose. These cultures were then grown in a cell culture rotator at 30 °C for 24 h. Cells from 1·5 ml aliquots of each culture were harvested by centrifugation, washed with sterile distilled H2O to remove residual glucose and grown for a further 24 h in 5 ml SC selective medium containing galactose to induce E1A expression. Cells were harvested from 1·5 ml aliquots of each culture, washed twice in 1 ml HSE buffer (20 mM HEPES pH 7·5, 150 mM NaCl and 1 mM EDTA), resuspended in 200 µl HSE buffer containing Complete Protease Inhibitor Cocktail (Roche) and transferred into fresh tubes containing 0·3 g acid-washed glass beads (425600 µm; Sigma). Cells were lysed by 15 cycles of vortexing for 30 s followed by 30 s of incubation on ice. After lysis, samples were centrifuged at 21000 g for 10 min. A sample of 200 µl of each supernatant was transferred to fresh microcentrifuge tubes, recentrifuged as above and the protein concentration of the supernatant was measured using the Bio-Rad DC Protein assay (Bio-Rad Laboratories). From each extract, 30 µg of protein was separated on 12% SDSpolyacrylamide gels and transferred to a PVDF membrane. Western blot analyses were performed with the ECL Plus system (Amersham Pharmacia) using the 3F10 anti-HA rat monoclonal antibody (Roche) and goat anti-rat IgG horseradish peroxidase-conjugated antibody (Pierce). Blots were visualized using a Molecular Dynamics Storm 860 system.
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Results |
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To precisely identify the region(s) of E1A required for interaction with DNA, we constructed a series of mutants with deletions spanning the entire C-terminal region of E1A and tested the ability of these recombinant proteins to bind to dsDNA in vitro (Fig. 2). Only the mutant lacking amino acids 201220 failed to interact with dsDNA cellulose, suggesting that the dsDNA-binding domain of E1A resides entirely within this region (Fig. 2B
). To determine if this portion of E1A was sufficient for DNA binding, we expressed amino acids 201218 of E1A as a GST fusion protein and tested it for binding to dsDNA cellulose. This fragment alone was sufficient to bind dsDNA cellulose at least as efficiently as the fragment spanning amino acids 201289 and, thus, represents the minimal portion of E1A necessary and sufficient to bind dsDNA (Fig. 2B
).
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Growth inhibition is mediated by the C-terminal portion of E1A
Although E1A binds dsDNA in vitro, no evidence has been presented to suggest that E1A binds dsDNA in vivo. Studies in the simple eukaryote S. cerevisiae have demonstrated that expression of a chimeric protein composed of a strong transcriptional AD fused to a DNA-binding domain can inhibit yeast growth, probably by trapping general transcription factors at genomic sites (Berger et al., 1992 ; Pina et al., 1993
; Marcus et al., 1996
). We reasoned that fusion of a strong AD to the DNA-binding region of E1A might inhibit growth in a similar manner, providing evidence for an interaction of E1A with DNA in vivo. We constructed vectors expressing portions of E1A fused to a strong AD and tested their ability to inhibit yeast growth, as measured by colony size (Fig. 4A
). Yeast expressing fragments spanning amino acids 182, 93138 or 139204 of E1A fused to an AD did not strongly inhibit yeast growth, consistent with their inability to interact with dsDNA in vitro. However, the fragment encompassing amino acids 187289 of E1A had a strong negative effect on yeast growth, as demonstrated by the overall decrease in colony size, consistent with the presence of a DNA-binding domain within this fragment. In addition, growth inhibition by the C-terminal portion of E1A was only observed when this fragment was fused to an AD (Fig. 4A
). The inability of the C-terminal fragment to inhibit growth without the AD was not simply a result of a decreased level of protein expression, as determined by Western blot analysis (Fig. 4B
).
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Discussion |
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Although it is well established that E1A can bind dsDNA in vitro, we wished to determine whether this interaction occurred or was relevant in vivo. As E1A does not bind dsDNA in a sequence-specific fashion (Chatterjee et al., 1988 ; Zu et al., 1992
), we took advantage of previous studies in the simple eukaryote S. cerevisiae, which demonstrated that fusion of a strong transcriptional AD to a DNA-binding domain can inhibit yeast growth. This probably occurs by sequestering limiting general transcription components at genomic sites, as growth inhibition can be overcome by mutation of transcriptional co-activator proteins, such as Gcn5p (Berger et al., 1992
; Pina et al., 1993
; Marcus et al., 1996
). We reasoned that fusion of a strong AD to the DNA-binding region of E1A might inhibit growth in a similar manner, providing evidence for an interaction of E1A with DNA in vivo. We tested various fragments of E1A fused to a strong AD (Fig. 4A
) for their ability to inhibit yeast growth and observed that only a fragment encompassing the C terminus of E1A had an effect on yeast growth (Fig. 4B
; Table 1
). In addition, growth inhibition by this portion of E1A was only observed when this fragment was fused to an AD (Fig. 4B
; Table 1
). These results are fully consistent with the presence of a DNA-binding domain that functions in combination with a strong AD to inhibit growth by trapping limiting general transcription factors.
However, further tests rule out this interpretation of the data. Firstly, growth inhibition by the C terminus of E1A was not dependent on the presence of Gcn5p, which differs from previous results using the Gal4p DNA-binding domain fused to a strong AD (Fig. 5). Secondly, the minimal DNA-binding domain was not sufficient to inhibit yeast growth when fused to a strong transcriptional AD (Fig. 6B
; Table 1
). Thirdly, mutant
205221, which lacks the DNA-binding region, inhibited yeast growth as well as the wild-type fragment of E1A (Fig. 6B
; Table 1
). These results indicate that the C-terminal region of E1A does not bind DNA in vivo and suggest that growth inhibition by this portion of E1A occurs via an alternative mechanism. Interestingly, mutational analysis demonstrated that growth inhibition requires most of the C-terminal region of E1A, with the exception of residues 205221 and 255270 (Figs 6
and 7A
; Table 1
). This pattern of activity does not coincide with the binding to CtBP or Yak1p, the two known cellular proteins targeted by the C-terminal portion of E1A (Schaeper et al., 1995
; Zhang et al., 2001
), suggesting that interaction with additional, as yet unknown, cellular factors may be responsible for growth inhibition. However, the lower level of expression of mutants with deletions within residues 187221 makes it difficult to unequivocally determine if this region is required for growth inhibition (Fig. 7
).
Taken together, our results raise the distinct possibility that the observed binding of E1A to DNA is an artefact of in vitro analyses, resulting from a simple ionic interaction of a basic portion of E1A with the phosphodiester backbone of DNA and that additional cellular proteins are targeted by the C-terminal portion of E1A.
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Acknowledgments |
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References |
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Received 13 August 2001;
accepted 13 November 2001.