©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Repression by Human Adenovirus E1A N Terminus/Conserved Domain 1 Polypeptides in Vivo and in Vitro in the Absence of Protein Synthesis (*)

(Received for publication, January 4, 1995; and in revised form, July 26, 1995)

Chao-Zhong Song Christopher J. Tierney Paul M. Loewenstein Rozalia Pusztai (§) Janey S. Symington Qing-quan Tang Karoly Toth Akira Nishikawa Stanley T. Bayley (1) Maurice Green (¶)

From the Institute for Molecular Virology, Saint Louis University School of Medicine, St. Louis, Missouri Department of Biology, McMaster University, Hamilton, Ontario L85 4K1, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40-80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.


INTRODUCTION

Group C adenovirus (Ad) (^1)E1A encodes two multifunctional regulatory proteins of 243 and 289 amino acid residues (243R and 289R). The E1A proteins are involved in diverse cellular functions, including transcriptional activation, transcriptional repression, induction of cellular DNA synthesis, cell immortalization, cell transformation, as well as inhibition of metastasis and of cell differentiation (for reviews, see (1, 2, 3, 4, 5) ). E1A 243R differs from E1A 289R only by conserved region 3 (CR3), a 46-amino acid domain unique to 289R. E1A is the first viral gene expressed during productive infection of permissive human cells and is required to activate transcriptionally early viral genes. CR3 is essential (6, 7, 8, 9, 10) and sufficient (11, 12) for transactivation of early viral genes.

The 243R protein encodes domains required for the growth regulatory functions of E1A. An intriguing function of 243R is its ability to repress transcriptionally a set of cellular genes involved in growth regulation and differentiation(13, 14, 15, 16, 17) , as well as several viral promoters including those of SV40, polyoma virus, and human immunodeficiency virus type 1(18, 19, 20) . How the E1A repression function interfaces with its growth regulatory properties is not known. Furthermore, the molecular mechanism of transcriptional repression is not understood. Elucidation of mechanism would inform our understanding of the biological roles of E1A transcriptional repression.

One can imagine several mechanisms by which E1A might repress the activity of a target gene (for review, see (21) ). For example, E1A could (i) bind to promoter DNA and block access by a transcriptional activator; (ii) sequester an essential transcriptional factor through direct protein-protein interaction; or (iii) induce the de novo synthesis of a cellular repressor. In this report, we show by cell microinjection experiments that E1A repression does not require the de novo synthesis of cellular proteins. This result and the fact that E1A does not bind specifically promoter DNA(22, 23, 24) suggest that repression is mediated by direct interaction with a preexisting cellular factor(s) and predict that E1A repression is possible in a cell-free transcription reaction. In support of this prediction, we were able to establish in vitro transcription of E1A-repressible promoters and to demonstrate repression by purified E1A polypeptides. By use of the in vitro repression assay and a series of E1A deletion polypeptides, the sequence requirements for transcriptional repression of the human interstitial collagenase and rat insulin II promoters were mapped to two small E1A domains.


EXPERIMENTAL PROCEDURES

Plasmids

Plasmids used as templates for in vitro transcription include the adenovirus type 2 major late promoter-chloramphenicol acetyltransferase (CAT) (pMLPCAT)(25) , human interstitial collagenase-CAT (pCL-CAT3)(26) , rat insulin II-CAT (pICE-CAT)(27) , Rous sarcoma virus long terminal repeat-CAT (pRSVCAT), and human histone 4-CAT (pH4WTCAT)(28) . p1-11 which expresses the SV40 T antigen(29) , pSV2CAT, which contains the SV40 early promoter fused to the CAT gene, pSP6dl1500 and pSP6pm975, which express the E1A 12S and 13S gene products, respectively(11) , were used in microinjection assays.

Plasmids encoding E1A 1-80, E1A 1-80Delta4-25, E1A 1-80Delta26-35, E1A 1-80Delta30-49, E1A 1-80Delta70-80, and E1A 1-80Delta61-69 were constructed by polymerase chain reaction using as templates pLE2, pdl1101, pdl1102, pdl1103, pdl1105, and pdl1141, respectively(10) . Primer 1 and primer 2 were used for E1A 1-80, E1A 1-80Delta26-35, E1A 1-80Delta30-49, and E1A 1-80 Delta61-69; primer 3 and primer 2 were used for E1A 1-80Delta4-25; and primer 1 and primer 4 were used for E1A 1-80Delta70-80. The E1A 243R plasmid was polymerase chain reaction-synthesized using as template pUC18/12S (a gift of G. Chinnadurai) with primer 1 and primer 5. Primer sequences are as follows: primer 1, 5`-CCCGGATCCATGAGACATATTATC-3`; primer 2, 5`-CCCAGATCTTAAGTCAATCCCTTC-3`; primer 3, 5`-CCCGGATCCATGAGACATGAGGTA-3`; primer 4, 5`-CCCAGATCTAGAGTCGGGAAAAATC-3`; primer 5, 5`-ACTAGATCTTGGCCTGGGGCG-3`. Polymerase chain reaction products were digested with BamHI and BglII and then cloned into the BamHI and BglII sites of pQE-12 (QIAGEN). All E1A expression plasmids were sequenced to confirm primary structure. These constructs express the E1A polypeptides fused to six histidine residues at the C terminus.

Purification of Recombinant E1A Proteins

Biologically active six-His-tagged E1A 243R and E1A 1-80 deletion polypeptides used for cell microinjection and for in vitro transcription repression were purified by nickel-nitrilotriacetic acid affinity chromatography as recommended by QIAGEN. E1A proteins were eluted with 6 M guanidine HCl/PBS/10 mM dithiothreitol, followed by stepwise renaturation in a 3,000 molecular weight cut-off dialysis bag (Spectra/Por) by halving the guanidine HCl concentration at 2-3-h intervals until below 250 mM. Dialysis was continued against PBS/5 mM dithiothreitol, and proteins were concentrated with a Centriprep-3 (Amicon). The E1A 243R and 289R proteins, used only for microinjection studies, were prepared by expression from pAS1-E1A410 (289R) and pAS1-E1A412 (243R) and purified by ion-exchange chromatography on DEAE-Sephacel(22) .

Cell Microinjection

Microinjection was performed essentially as described previously(11) . A549 and HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum on scored 22-mm square coverslips in 35-mm plastic Petri dishes. For measurements of transcription repression, the SV40 T antigen expressing plasmid, p1-11, was injected at 10 µg/ml with or without E1A protein or plasmid into the nuclei of 100-200 cells. Injection mixtures contained 1% tetramethylrhodamine dextran (M(r) 70,000, Molecular Probes Inc.), which served as marker for injected cells. Cells were fixed in 3.8% paraformaldehyde/PBS for 10 min, permeabilized in methanol (-20 °C) for 5 min, and rehydrated in PBS. SV40 T antigen synthesis was measured by indirect immunofluorescence using monoclonal antibody to T antigen (1:200, Ab-2, Oncogene Science) as primary antibody and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Cappel) as secondary antibody. Coverslips were scored microscopically by double label immunofluorescence to identify injected cells (rhodamine) and cells expressing T antigen (FITC). To determine whether protein synthesis was necessary for E1A repression, pSV2CAT was injected with or without E1A 243R protein or plasmid (pSP6dl1500) (11) as described in the legend to Table 1. Cells were incubated in the presence or absence of cycloheximide or anisomycin for 6 h after injection(12) . The synthesis of CAT RNA was measured by in situ hybridization (12, 30) , using as probe CAT antisense RNA labeled with [S]CTP. After post-hybridization washes, cells on coverslips were dehydrated in ethanol, dried, mounted on microscope slides, and dipped in Kodak NTB-2 emulsion. Slides were developed after 2 days of exposure in the dark, and the percentage of microinjected cells that hybridized with the CAT DNA probe was scored by phase microscopy. Positive cells are those with at least twice as many photographic grains (usually too many to count) as the background (generally 30-50 grains).



In Vitro Transcription Repression

HeLa cell nuclear extracts were prepared essentially as described previously (31) except that nuclei were extracted with a final KCl concentration of 300 mM(32, 33) . Transcription reaction mixtures (25 µl) contained 12 mM HEPES, pH 7.9, 4 mM creatine phosphate, 0.1 mM EDTA, 10 mM MgCl(2), 0.7 mM dithiothreitol, 60-70 mM KCl, 15 mM NaCl, 8% glycerol, 500 µM ATP, CTP, GTP, and UTP, 20 units of placental RNase inhibitor (Boehringer Mannheim), 500 ng of template DNA, and 40-80 µg of nuclear extract protein. Template DNA was isolated using a QIAGEN kit and further purified by phenol extraction. E1A proteins and deletion polypeptides in PBS were added to reaction mixtures as indicated. For reactions in which E1A was omitted, an equal volume of PBS was added. Incubation was for 60 min at 30 °C. RNA transcripts were isolated, and primer extension analysis was performed using a 5`-end labeled 31-mer CAT primer(33) . Primer-extension products were analyzed by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography after overnight (12-h) exposure.


RESULTS

Our initial goal was to determine whether protein synthesis is required for E1A repression. The experimental approach is based on a cell microinjection assay showing that transcriptional activation by E1A 289R occurs in the presence of inhibitors of protein synthesis (12) . We first developed a cell microinjection assay for transcriptional repression by E1A proteins. The reporter plasmid p1-11, encoding the SV40 T antigen, was coinjected into the nuclei of cells with an E1A protein or expression plasmid. SV40 T antigen synthesis was scored by immunofluorescence. To study E1A sequence requirements for repression, we expressed in Escherichia coli E1A 243R, E1A 1-80, and the E1A 1-80 deletion polypeptides shown in Fig. 1A. These recombinant E1A polypeptides were purified to near homogeneity as shown by SDS-polyacrylamide gel electrophoresis (Fig. 1B).


Figure 1: a, structure of the E1A 243R, E1A 1-80, and E1A 1-80 deletion polypeptides used for microinjection and for in vitro transcriptional repression. Shown in black are CR1 (residues 40-80) and CR2 (residues 120-139). b, SDS-polyacrylamide gel electrophoresis of recombinant E1A polypeptides stained by Coomassie Blue. Marker proteins with apparent molecular masses in kDa are shown on the left.



Transcription Repression by Microinjected E1A 243R and 289R Proteins

Various levels of E1A proteins were coinjected with p1-11 into human A549 cells and T antigen-synthesizing cells identified 18 h later by immunofluorescence (Fig. 2A). E1A 243R repressed T antigen synthesis, whereas E1A 289R was much less efficient. Similar results were obtained by coinjection with plasmids encoding the 243R protein (pSP6dl1500) and the 289R protein (pSP6pm975) (data not shown). Thus, the activity of each recombinant E1A protein in the cell microinjection assay reflects the repression function of the corresponding gene. The higher degree of repression by the E1A 12S gene product compared with that of the 13S product is in agreement with studies by Ad infection(11) . Presumably the activation region, CR3, in the E1A 13S product interferes with the function of the repression domain(s)(11) .


Figure 2: a, dose-response of recombinant E1A 243R and 289R proteins in repression of SV40 T antigen synthesis by microinjection assay. HeLa cells were coinjected into the cell nucleus with the SV40 T antigen expressing plasmid p1-11 (10 µg/ml) and the indicated amounts of E1A protein. From 150 to 200 cells were injected for each time point. SV40 T antigen synthesis was analyzed 18 h afterwards by indirect immunofluorescence. From 80-90% of cells injected with p1-11 alone expressed T antigen. The percent repression of T antigen producing cells is plotted against the concentration of coinjected E1A protein. b, time course of T antigen repression in cells coinjected with p1-11 (10 µg/ml) and E1A 243R protein (400 µg/ml) or E1A 243R gene (pSP6dl1500, 250 µg/ml). The percentage of SV40 T antigen positive cells is plotted against the time after microinjection. c, repression of SV40 T antigen expression in cells injected with p1-11 (10 µg/ml) alone (panelsA and B), p1-11 and E1A 1-80 polypeptide (100 µg/ml) (panelsC and D), or p1-11 and E1A 1-80Delta4-25 polypeptide (100 µg/ml) (panelsE and F). Included in each microinjection mixture was 1% tetramethylrhodamine dextran, which serves as marker for microinjected cells. The left-handpanels show cells staining positively for T antigen by FITC-conjugated second antibody. The right-handpanels show the same field viewed through filters allowing rhodamine containing cells to be observed.



Time Course of Repression by Microinjected E1A 243R Protein or Plasmid

HeLa cells on coverslips were coinjected with p1-11 and E1A 243R protein or E1A 243R expression plasmid, pSP6dl1500. Coverslips were fixed at different times after microinjection, and the number of cells expressing T antigen was determined by indirect immunofluorescence. When p1-11 alone was injected, T antigen was detected in about 30% of cells at 3 h, and reached a plateau of 80-90% at about 10 h (Fig. 2B). When pSP6dl1500 was coinjected with p1-11, little or no T antigen was detected at 6-7 h, and less than 15% of cells expressed T antigen at 10 h, corresponding to about 80% repression (Fig. 2B). Coinjection with the E1A 243R protein repressed T antigen synthesis in 80-100% of cells up to 7 h postinjection, but only in 50-60% thereafter, probably reflecting intracellular degradation of injected 243R protein (Fig. 2B). Based on these results, subsequent microinjection experiments were assayed at 6 h after injection of E1A polypeptide.

Microinjected E1A 1-80 Polypeptide Strongly Represses SV40 T Antigen Synthesis

Several studies have implicated a requirement for the E1A N-terminal region and CR1 for transcriptional repression (for review, see (5) ). We therefore tested the ability of the E1A 1-80 polypeptide to repress transcription. Cells were coinjected with p1-11, E1A 1-80 polypeptide, and rhodamine-conjugated dextran (to serve as a marker for microinjected cells) and analyzed by double-label immunofluorescence (Fig. 2C). Fields in the left-handpanels were visualized through FITC filters, which display cells that synthesize T Ag, whereas identical fields in the right-handpanels were visualized with rhodamine filters, which show cells that are microinjected (panels under Marker). The majority of cells (80-90%) injected with p1-11 alone synthesized T Ag (panelA), whereas those coinjected with E1A 1-80 were blocked in T antigen synthesis (80-90% repression in several experiments) (panelC). The sequence specificity of E1A 1-80 in transcriptional repression is demonstrated by the inability of the deletion polypeptide, E1A 1-80Delta4-25, to repress T antigen synthesis (panelE).

Repression by E1A 243R Protein Does Not Require de Novo Cellular Protein Synthesis

The ability of the E1A 243R protein to repress transcription in microinjected cells permits the experimental determination of whether cellular protein synthesis is required for E1A repression. Cells were pretreated for 30 min with the protein synthesis inhibitor cycloheximide or anisomycin, coinjected with pSV2CAT and E1A 243R protein, and incubated in the presence of inhibitor for 6 h. Under these conditions, greater than 90% of protein synthesis is blocked (12) . Expression from the SV40 early promoter was measured by in situ hybridization for CAT-specific RNA using as probe S-labeled CAT antisense RNA. As control for the efficacy of protein synthesis inhibition, the E1A 12S plasmid, pSP6dl1500, was coinjected with pSV2CAT into drug treated cells. Repression by pSP6dl1500 should be substantially reduced in the presence of a protein synthesis inhibitor because E1A protein synthesis would be substantially reduced.

When pSP6dl1500 and pSV2CAT were coinjected, between 77 and 84% repression of CAT expression was observed in the absence of inhibitor, but only 20% inhibition when cells were treated with cycloheximide (Table 1, part A) and 0% when cells were treated with the more stringent inhibitor, anisomycin (Table 1, part B). Thus protein synthesis appears to be substantially blocked by these agents. By contrast, repression by E1A 243R protein was only slightly reduced by cycloheximide in HeLa cells (78-63%, Table 1, part A) and even less by the more effective inhibitor, anisomycin (69-64%, Table 1, part B); the small decrease in E1A repression in the presence of inhibitor may reflect some cell toxicity of these drugs. Our results using two different inhibitors and two cell lines support the conclusion that repression by E1A does not require de novo protein synthesis.

E1A Polypeptides Can Repress Expression of E1A-repressible Promoters in an in Vitro Transcription System

The results of cell microinjection predict that E1A repression should be possible in a cell-free transcription extract. To test this prediction, we optimized conditions for in vitro transcription of the collagenase and insulin II promoters that have been reported to be repressible by E1A in vivo(14, 26, 27, 34) and then determined whether purified E1A proteins and polypeptides would repress transcription in vitro. The plasmid, pCL-CAT3, contains a 3.8-kilobase fragment from the 5`-flanking portion of the collagenase gene to nucleotide +37 fused to the CAT gene(26) . Transcription was measured by primer extension analysis with an oligomer complementary to the CAT mRNA sequence and yielded a correctly initiated product. Clear repression was seen with 500 ng of E1A 243R protein, and the signal was reduced to background levels with 1000 ng (data not shown). Because E1A 1-80 polypeptide strongly inhibited expression of the SV40 promoter in the cell microinjection assay (Fig. 2C), we determined whether it is sufficient to repress transcription of the collagenase promoter in vitro. Different levels of polypeptide were tested, and as shown in Fig. 3A, strong repression was seen with 50 ng of E1A 1-80.


Figure 3: Transcription repression by purified recombinant E1A 1-80 polypeptide in vitro of (a) collagenase promoter and (b) insulin II promoter. pCL-CAT3 (a) and pICE-CAT (b) were used as templates for the in vitro transcription reaction. Transcripts were detected by primer extension analysis using a CAT primer as described under ``Experimental Procedures.'' Reaction mixtures contained from 0 to 1000 ng of E1A 1-80 as indicated.



The rat insulin II plasmid pICE-CAT, which contains the promoter sequence from -100 to +8 fused to the CAT gene(35) , was analyzed next. E1A 1-80 represses pICE-CAT in a dose-dependent manner (Fig. 3B). Striking repression was obtained with as little as 50 ng of E1A 1-80.

Two Small Domains within the E1A 1-80 Polypeptide Are Sufficient for in Vitro Repression

As shown above, the collagenase and insulin II promoters, which are repressed by E1A in vivo are repressed in vitro by the E1A 1-80 polypeptide. E1A 1-80 contains only the N-terminal nonconserved region and CR1. To map the regions within E1A 1-80 essential for its repression function, E1A 1-80 polypeptides containing deletions of amino acid residues 4-25, 26-35, 30-49, 48-60, and 70-80 (see Fig. 1A) were expressed in E. coli, purified, and tested for repression of transcription in vitro. E1A repression of pICE-CAT requires residues within 4-25 and 48-60 since only deletion of these sequences inactivated the E1A 1-80 repression function (Fig. 4). E1A sequences within residues 30-49 and 70-80 are not required; E1A 1-80 polypeptides deleted in these sequences repressed as well as did wild-type E1A 1-80. E1A 1-80Delta26-35 is not necessary for repression activity (Fig. 4), although deletion appears to reduce repression slightly. We conclude that residues 4-25 and and 48-60 are essential for repression, whereas residues 30-49 and 70-80 are dispensable. Similar findings were obtained when the same experiments were done with pCL-CAT3 as template (data not shown).


Figure 4: Sequences within E1A 1-80 required for repression of in vitro transcription. pICE-CAT template was transcribed in vitro in the presence of 1000 ng of wild-type (WT) E1A 1-80 polypeptide or the indicated E1A 1-80 deletion polypeptides. Transcripts were analyzed by primer extension as described under ``Experimental Procedures.''



The above results indicate that two distinct E1A regions are required for repression in vitro. The first includes residues 4-25 within the N-terminal region, and the second includes residues 48-60 within CR1. To delineate the C-terminal boundary of the CR1 sequence needed for E1A repression, E1A 1-80Delta61-69 was constructed, purified, and tested for the ability to repress transcription in vitro relative to wild-type E1A 1-80. Dose-response measurements with 100-1500 ng of these two polypeptides showed no difference in repression of pICE-CAT (Fig. 5). Similar results were obtained with pCL-CAT3 (data not shown). Thus E1A residues 61-69 are not required for E1A repression in vitro. To summarize our findings, there are two ``active sites'' for E1A repression in vitro; they are located within residues 1-25 and 48-60.


Figure 5: Repression by 100-1500 ng of E1A 1-80 or E1A 1-80Delta61-69 polypeptide of in vitro transcription using pCL-CAT3 as template. Transcripts were analyzed by primer extension as described under ``Experimental Procedures.''



E1A Repression in Vitro Is Promoter-specific

To investigate the promoter specificity of E1A repression in vitro, we determined whether E1A 1-80 can repress the transcription of the Ad major late promoter, the Rous sarcoma virus long terminal repeat promoter, and the human histone 4 (H4WT) promoter. As shown in Fig. 6, MLPCAT transcription was not repressed by E1A 1-80 or E1A 1-80 deletion polypeptides. Likewise, RSVCAT and H4WTCAT transcription were not repressed by 0.5-2 µg of E1A 1-80. Based on these results, we conclude that E1A repression in vitro is promoter specific.


Figure 6: Transcription in vitro of the major late promoter, Rous sarcoma virus, and human histone 4 promoters is not repressed by E1A 1-80 polypeptides. A, MLPCAT was transcribed in vitro in the presence of 1.0 µg of E1A 1-80 (WT) or various E1A 1-80 deletion polypeptides. RSVCAT (B) and H4WTCAT (C) were transcribed in vitro in the presence of the indicated amounts of E1A 1-80 polypeptide. Transcripts were analyzed by primer extension as described under ``Experimental Procedures.''




DISCUSSION

We describe here two lines of evidence that E1A repression does not require the induction of cellular protein synthesis. First, E1A 243R protein inhibits transcription of an E1A-repressible promoter in vivo in the presence of inhibitors of protein synthesis, as demonstrated by microinjection-in situ hybridization experiments. Second, the E1A 1-80 polypeptide strongly represses transcription in vitro of E1A-repressible promoters in a dose-dependent manner. These findings support the conclusion that E1A repression involves direct protein-protein interactions. Mutational analysis indicates that only two E1A amino acid sequence elements, the E1A N terminus (residues 1-25) and the middle portion of CR1 (residues 48-60), are required for transcriptional repression in vitro. Repression probably involves protein-protein interaction among these two putative E1A contact sites and a cellular factor(s). The N-terminal region is not conserved based upon primary sequence comparisons among different E1A serotypes(36) . However, conservation at a secondary or higher structural level may be important for the E1A repression function, as suggested by Gedrich et al.(37) , who noted a conserved three-amino-acid sequence termed ILE, which exists within a predicted alpha-helical structure.

On the basis of the results reported here and our studies with the human immunodeficiency virus type 1 long terminal repeat promoter(38) , we propose that E1A repression of the two cellular and two viral promoters studied is mediated through a common mechanism. First, protein synthesis is not required for repression of these promoters. Second, E1A 1-80 is sufficient for repression. Third, deletion of amino acids 4-25 inactivates repression. Specific cis-acting upstream promoter elements are unlikely to be targets for E1A repression, since E1A-repressible promoters do not possess a common sequence motif. Deletion analysis of the SV40 enhancer has failed to identify a specific sequence required for E1A repression of the SV40 early promoter(39) . Whereas the collagenase promoter contains a collTRE element (the consensus AP-1 binding site, TGAGTCA), which seems important for E1A repression(26, 34) , the rat insulin II promoter construct, pICE, contains neither a TRE element nor an SV40 enhancer element but possesses an enhancer element between nucleotides -100 and -91 (GCCATCTGC), which seems necessary to confer E1A repressibility (35) . These findings indicate that there are no specific upstream elements required for E1A repressibility.

Because E1A repression appears to be mediated by a common mechanism that does not require de novo protein synthesis, E1A repression domains must interact with a specific cellular factor(s). Several factors have been reported that can bind sequences within the E1A N terminus. First is the p300/CBP family of putative coactivators (40, 41, 42, 43, 44, 45, 46, 47) . p300 associates with E1A in extracts of Ad-infected and Ad-transformed cells (for review, see (4) ). p300 has recently been cloned, and a mutant lacking the E1A binding region was shown to overcome partially E1A repression in transient expression analysis (48) . Second, Dr1, a cellular transcriptional repressor that targets TFIID, can also bind the E1A N terminus(49) . Third, YY1, a multifunctional transcription factor that can either activate or repress transcription, depending upon its target, has recently been reported to bind E1A N-terminal sequences(50) . Most recently, it has been shown that the TBP component of TFIID can both bind an E1A N-terminal sequence and overcome E1A repression in vitro(51) . The finding that E1A 1-80 can target TBP should not be surprising since TFIID is central to transcriptional regulation (for review, see (52) ). The in vitro transcription-repression system described here should be valuable for further studies defining the molecular mechanism(s) of E1A repression and the interaction of the E1A N-terminal domain(s) with cellular transcription regulators.


FOOTNOTES

*
This work was supported by Research Career Award AI-04739 and Public Health service grant CA-29561 to M. G. from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Inst. of Microbiology, University Medical School of Szeged, Szeged, Hungary.

To whom correspondence should be addressed. Tel.: 314-577-8401; Fax: 314-577-8406.

(^1)
The abbreviations used are: Ad, adenovirus; CR3, conserved region 3; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.


ACKNOWLEDGEMENTS

We thank Roland Stein, Steven Frisch, Michael Mathews, Nat Heinz, Li Yu, Yakov Gluzman, Michael Green, and G. Chinnadurai for gifts of plasmids; Andrew Kravetz for technical assistance and laboratory humor; and Carolyn Mulhall for editorial assistance.


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