(Received for publication, January 4, 1995; and in revised form, July 26, 1995)
From the
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.
Group C adenovirus (Ad) ()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.
Plasmids encoding E1A 1-80, E1A
1-804-25, E1A 1-80
26-35, E1A
1-80
30-49, E1A 1-80
70-80, and E1A
1-80
61-69 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 1-80
26-35, E1A
1-80
30-49, and E1A 1-80
61-69; primer
3 and primer 2 were used for E1A 1-80
4-25; and primer
1 and primer 4 were used for E1A 1-80
70-80. 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.
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.
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 1-804-25
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.
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.
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.
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
1-8061-69 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 1-8061-69 polypeptide of in
vitro transcription using pCL-CAT3 as template. Transcripts were
analyzed by primer extension as described under ``Experimental
Procedures.''
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.''
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
-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.