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© 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 12 3221 Cooperation between upstream and downstream elements of the adenovirus major late promoter for maximal late phase-specific transcription Guillaume Monctesert and Claude K6dinger* Laboratoire de Gene"tique Moleculaire des Eucaryotes du CNRS, Unite 184 de Biologie Moleculaire et de Genie Gen&ique de I'lNSERM, Faculty de M§decine, 11 rue Humann, 67085 Strasbourg Cedex, France Received April 12, 1991; Accepted May 13, 1991 ABSTRACT Transcription from the adenovirus major late promoter (MLP) is greatly stimulated during lytic infection, after replication of the viral DNA has started. This replicationdependent activation has previously been shown to be mediated by a positive regulatory cellular proteln(s). Binding of this factors) to sequence elements (DE1 and DE2), located between positions +76 and +124, with respect to the MLP transcriptlonal startsite, is detected only after the onset of DNA replication. Using a cellfree transcription system which mimics the late phase induction of the MLP and DNA binding assays, we now present evidence showing that maximal stimulation also depends on the MLP upstream element (UE), without Involving increased DNA binding activity of the corresponding factor (UEF) during the lytic cycle. Our results Indicate that the upstream and downstream elements act cooperatively on transcription efficiency, although no direct interactions between the cognate factors could be demonstrated. These observations strongly suggest that the elevated rate of transcription originating at the MLP startsite, late in infection, results from the simultaneous action of factors bound at the upstream and downstream elements onto a common target within the basal transcription machinery. INTRODUCTION Analysis of the molecular mechanisms underlying eukaryotic gene control has largely relied on the development of in vitro transcription systems using combinations of wild type and mutated exogenous DNA templates. Such systems have led to the identification of several protein factors which, together with RNA polymerase, contribute to the setting up of active initiation complexes and thereby contribute to the basal level of transcription (1-3). In addition to these general transcription factors, an increasing number of trans-acting factors have been identified, which bind to specific DNA sequence elements located at various positions with respect to the transcriptional startsite • To whom correspondence should be addressed (3—7). How these DNA-binding factors modulate basal promoter activity is still unknown. Accumulating evidence indicates however that, once bound to DNA, these factors achieve their transcriptional effects by establishing protein-protein contacts with the basal transcription apparatus, either directly or via intermediary connections with adaptor proteins (see 8, 9 for reviews). Temporal and tissue-specific transcriptional activation or repression of a given promoter will occur only if a particular combination of factors and cofactors has built up on it. The understanding of the molecular mechanisms implicated in this process clearly requires the identification and characterization of the factors involved, as well as their relationships with the other components of the transcription machinery. The major late promoter (MLP) of human adenovirus 2 or 5 is one of the eukaryotic promoters that have been most extensively studied (see 10, 11 for reviews). Efficient constitutive transcription from this promoter has been found to essentially depend on an intact TATA box centered at position - 2 8 , an upstream sequence element (UE) between —67 and - 4 9 and an initiator element encompassing the transcription startsite (12—18). Additional elements, located further downstream, have also been shown to contribute to basal promoter function (19, 20). The specific trans-acting factors recognizing these elements have subsequently been identified and their binding properties studied. Thus, binding of the TATA box recognition factor, BTF1 (21) or TFHD (22), appeared to be the prerequisite for die assembly of the initiation complex, which is further stabilized by interactions between ihllD and the factor bound to the nearby upstream element, MLTF, UEF or USF (12, 16, 22). TFUD also represents, in this promoter, the main target for direct activation by the adenovirus E la or the pseudorabies IE proteins (23, 24). Although the MLP is active at early times in infection, a strong stimulation occurs after the onset of viral DNA synthesis (10 for review). This activation cannot just be ascribed to an increase in the intracellular viral template copy number, since, as previously suggested (25—27), replication is required to render the template competent for transcription. Similar conclusions have 3222 Nucleic Acids Research, Vol. 19, No. 12 also been reached in the case of the activation by DN A replication of SV40 (28), polyoma (29) and vaccinia virus late genes (30), or of the Xenopus jS-globin gene (31), for example. Besides these cis-acting alterations, the nature of which is still unknown, sequences downstream of the MLP transcription startsite have been shown by in vitro experiments to be essential for promoter activation (32, 33). DNAse I footprinting experiments combined with an in vitro transcriptional analysis of MLP deletion mutants, have established that MLP activation depends on sequence elements (DEI and DE2) located between +76 and +120 and correlates with the increased binding of a virus-induced 40 kD cellular factor to DEI (34, 35). Using a series of nondefective adenovirus recombinants expressing MLP—globin fusions, Leong et al. (36) have clearly established the role of the MLP downstream elements in the late phasespecific induction of this promoter during the lytic cycle of infection. In this study, we further examined the function of the neighbouring DEI (+86/+96) and DE2 (+113/ + 124) elements. We show that these elements are functionally redundant and most likely bind common proteins. In addition, our experiments reveal that the late phase-specific stimulation of the MLP results from a cooperative action of the upstream and downstream promoter elements, although no synergistic DNA-binding activity of the cognate proteins could be detected. MATERIALS AND METHODS Preparation of whole cell extracts HeLa cells, grown in Eagle medium supplemented with 5% calf serum, were infected with 10 PFU of adenovirus type 5 (wt) or its Ela-defective dl312 derivative (dl) per cell. Cells were harvested 20 h postinfection and extracts were prepared in parallel from wt and dl-infected cells, to minimize extract to extract variations. Experimental conditions were as previously described (35), except that the final dialysis was against buffer B containing 20 mM HEPES-NaOH (pH 7.9), 5 mM MgCl2, 100 mM KC1, 1 mM EDTA, 1 mM dithiothreitol and 17% glycerol (37). Recombinant plasmids The BamHI fragment of pML553 (34) comprising the MLP sequences between positions —259 and +553 was inserted into the BamHI site of M13mp9. The resulting single-stranded recombinant was used for oligonucleotide-directed mutagenesis. The mutated MLP fragments were recloned into the BamHI site of pBR322. The pG recombinant (34) contains the rabbit /3-globin gene, between positions -425 and +1700. These MLP and globin plasmids were used as templates for polymerase chain reactions (see below). In vitro runoff transcription Transcription reactions were carried out as previously described (35), with the following exceptions: i) final reaction volume was 32 y.\; ii) cell extracts (16 /tl) were preincubated for 15 min at 25 °C with sonicated salmon sperm DNA (200 ng); iii) the DNA templates were obtained by polymerase-chain-reaction (PCR) amplification of the wild type or mutated MLP sequences, between positions -137 and +314 (with respect to the MLP startsite), or the rabbit /3-globin gene sequences, between positions —290 and +225 (relative the corresponding startsite). PCR amplification was performed by incubating each plasmid (125 ng) with appropriate pairs of 25-nucleotide primers (1 fig) complementary to the borders indicated above, in a medium (100 fi\ final volume) containing 2 U Taq polymerase, 0.2 mM each dNTP, 100 mM Tris-HCl (pH 7.8), 10 jig/ml gelatin, 1.5 mM MgCl2, and subjecting the mixture to 30 cycles of (1 min at 92°C, 2 min at 55°C and 3 min at 72°C) in a Cetus PCR apparatus. After amplification, total DNA was purified by phenol—chloroform extraction and used as template in the in vitro transcription reactions. Transcripts were analyzed by electrophoresis on 5% polyacrylamide-urea gels which were vacuum-dried before exposure for autoradiography in the presence of an intensifying screen. All transcription assays were repeated at least three times, with independent template DNA and extract preparations. Electrophoretk band-shift assays Gel retardation assays were performed essentially as previously described (35). Briefly, about 0.3 ng (15,000 cpm) of 32 P-5'-end-labelled, double-stranded oligonucleotide probe (DEI or UE, see Figure 1) were incubated with 2 y\ (10 ng protein) of wt or dl extract, in the presence of 10 /tg of poly (dl-dC) as nonspecific competitor, in a medium (10 /il final volume) containing 50 mM KC1, 2 mM MgCl2, 10 mM EDTA and 2.5% Ficoll. After 10 min at 25°C, the complexes were separated by non-denaturing polyacrylamide (4.5%, acrylamideibisacrylamide 80:1) gel electrophoresis. After the run, the gel was transferred onto Whatman 3MM paper and vacuum-dried before autoradiography. DNAse I footprinting assays About 1 ng (10,000 cpm) of the BamHI (-259) -HindHI (+200) MLP fragment of pML553,32P-3'-end-labelled at the HindHI site (non-transcribed strand) was incubated in buffer B (40 fd final volume), in the presence of 2 /tl wt extract and 1 /ig of poly (dldC), for 15 min at 25°C. Where indicated, the extract was preincubated for 15 min at 25 °C with specific competitor doublestranded oligonucleotides, before addition of the probe DNA. After digestion by DNAse I (15 min at 25° C with 100 Kunitz units per assay), the DNA fragments were phenol—chloroform extracted and separated on a 6% polyacrylamide sequencing gel, next to DNAse I-treated (10 units per reaction) naked probe DNA. After the run, the gel was vacuum-dried and exposed for autoradiography. RESULTS DEI and DE2 are involved in the late phase-specific activation of the MLP In vitro transcription analysis and DNA binding studies of the adenovirus MLP have established the requirement of a sequence element spanning positions +86 to +96 (DEI), for the replication-dependent activation of this promoter. The contribution to this regulation of the nearby downstream element (DE2, +113/+124) and the upstream promoter element (UE, —67/-49) was examined by introducing site-directed alterations into the corresponding MLP sequences (see Figure 1). To analyze die effect of these mutations on the late phase-specific activation of the MLP, the template efficiencies of the resulting mutants were tested in the presence of extracts prepared from cells infected for 20 h widi wild type adenovirus-5 (wt extracts). The transcriptional activity directed by these late-infected cell extracts was compared to that of extracts prepared 20 h after infection with dl312 (dl extracts), an adenovirus-5 derivative which is Nucleic Acids Research, Vol. 19, No. 12 3223 I Ttmplam | DEI DE2 1 WT V//1 +86 + 9 6 + 1 1 3 + 124 + 3 1 4 iyy i v//x A2 m! A2 ItfV I mUm1 X//A +85 +96 _/\_rzza_ +85 +96 mUA12 Footprint proba | C€1 (BamHO DE2 (Hlndlll) •259 OBgonudeotfdM W/A DE12 + 124 +75 062 +104 (GTAGGCCACG) (TTQTCAGTTT) DEI +75 (GTAGACTACG) OEImi +132 +104 - I XX I +75 +104 (TTTTCACTTT) ~ ~ Figure 1. Diagrams of the adenovirus MLP DNA fragments used for in vitro transcription and protein binding assays. The PCR-amplified DNA fragments used as templates for run-off transcription are depicted, with promoter elements shown as boxes, and coordinates given relative to the startsite ( + 1 , arrow). The WT template corresponds to the natural adenovirus type 2 sequence between positions -137 and +314. Point mutations (xx) correspond to G-to-A and C-to-T transitions at positions - 6 2 and - 6 0 (in mU) and to G-to-T and G-to-C transversions at positions +88 and +92 (in ml), respectively (see below). Deletions spanning DEI (Al), DE2 (A2) or both elements (A12) arc shown with corresponding coordinates. The BamHI-Hindlll fragment, excised from pML553 (see Materials and Methods) was used as probe in the DNAse I footprinting experiments. The chemically synthesized double-stranded oligonucleotides used as probes or competitors in the bandshift and footprinting experiments are schematized at the bottom. Relevant nucleotide sequences are given next to corresponding wild type and mutated elements (alterations relative to the natural sequence are underlined). defective for Ela expression and whose DNA replication is delayed compared to that of wt-infected cells. We have in fact previously shown that the transcriptional activity of these dl extracts was identical to that of extracts prepared from wt-infected cells at 6 h post-infection (early phase) or at 20 h post-infection from cells which were grown in the presence of cytosinearabinoside to prevent DNA replication (34). Typical transcription assays, run in the presence of various DNA templates, are shown in Figure 2. Since some of the specific signals generated by the wt extracts were nearly saturated after a 1.5 h exposure time (middle row of lanes), the corresponding lanes were also exposed for a shorter time (0.5 h, upper row). Under our incubation conditions, a DNA fragment spanning the wild type MLP sequence between positions -137 and +314 ('WT' template, see Figure 1) was transcribed about 30-fold more efficiently in wt extracts than in dl extracts (Figure 2A, lane 1), while a control rabbit /3-globin fragment (glob) was transcribed at roughly equal efficiencies in either extract (Figure 2A, lane 3224 Nucleic Acids Research, Vol. 19, No. 12 probe Template. $<E% rue • • • • • • Bamsma rttainod DE1 • - - * - - • LDE2 • • • . - - • • DE1 UE extract wt dl - : - wt dl + - + - . + -UEF wt (0.5h expo) I* L (1^h expo) 1 2 12 3 4 9 6 7 12 3 4 3 Free 1 2 3 4 5 6 7 4 5 6 Figure 2. Comparative mutational analysis of MLP activity in wt and dl extracts. In vitro transcription was performed as described in Materials and Methods with wt extracts (wt, top and middle series of lanes) or dl extracts (dl, bottom lanes), in the presence of the PCR-amplified MLP templates (60 ng) or rabbit /3-globin template (glob, 400 ng), as indicated. Intact (+) or altered ( - ) promoter elements present in each MLP template are noted. The specific run-off transcripts were separated by gel electrophoresis and visualized by autoradiography: reactions carried out in the presence of wt extracts were exposed for 0.5 h (upper series of lanes) and 1.5 h (middle), those run in parallel, but in me presence of dl extracts, were exposed for 1.5 h (bottom). The longer exposure time (wt and dl, 1.5 h expo) was chosen to visualize the overall extent of late phase-dependent stimulation of the MLP activity. The non-saturating exposure time (wt, 0.5 h expo) allows comparison of relative MLP activities in wt extracts. The major band in each lane corresponds to the specific transcript, with the expected length (globin-specific transcripts are indicated by arrow-heads). Panels A, B and C correspond to independent experiments and illustrate the functional redundancy of DEI and DE2 and the role of UE, the cooperativity between the DE and UE elements, and the ability of DEI and DE2 to separately cooperate with UE, respectively. Figure 3. Comparative analysis of DEF and UEF binding activities in wt and dl extracts. Standard band-shift assays were performed with the DEI (lanes 1 - 3 ) or UE (lanes 4 - 6 ) oligonucleotide probes (see Figure 1). The specificity of the DEF and UEF complexes (arrows) was determined by experiments using appropriate competitor oligonucleotides (not shown). The slower migrating bands in lanes 1 and 2 correspond to ubiquitous complexes previously described (35) UE TATA DE1 D£2 I dl extract Z rrr~i m I 1 8). Introduction of a double-point mutation into DEI or deletion of the whole element (as in ml and Al, respectively; see Figure 1) did not significantly affect stimulation of the corresponding templates by the wt extracts, under these in vitro transcription conditions (Figure 2A, lanes 2 and 3; Figure 2C, lane 2). Similarly, deletion of the DE2 element (as in A2) had no effect on MLP activity (Figure 2A, lane 4 and Figure 2C, lane 7). By contrast, simultaneous alteration or deletion of both elements (as in mlA2 and A12) reduced about 4-fold MLP activity in wt extracts, without affecting basal MLP activity as measured in dl extracts (Figure 2A, lanes 5 and 6, Figure 2B, lane 2 and Figure 2C, lane 4). These results clearly suggest that both DEI and DE2 are involved in the late phase-specific activation of the MLP. Furthermore, since either one of these elements mediates by itself most of this activation, we conclude that DEI and DE2 are functionally redundant elements. The MLP upstream element cooperates with the downstream elements Preliminary experiments (not shown) revealed that the late phasespecific stimulation of the MLP was substantially reduced if binding of the upstream element factor (UEF) was titrated with competitor UE oligonucleotides, prior to the transcription reaction. To confirm the involvement of the UE element in the late phase-specific MLP activation suggested by this observation, we first investigated the effect of UE-directed mutations on this phenomenon. In agreement with earlier transcription analyses which used partially purified transcription factors from uninfected HeLa cells (16), a double-point mutation of the UE element (mU, see Figure 1), that abolished UEF binding (see below), reduced about 3-fold J wt extract mUA12 1 WT (mU+A12)-mU412 0 20 40 60 BO 100 Relative activity {%) Figure 4. Cooperativity between the UE and DE elements for late phase-specific stimulation of MLP activity. MLP mutants were schematically depicted on the left (see Figure 1). with altered promoter elements crossed (X)- Non-saturated exposures of autoradiographs of the experiment in Figure 2 (and others not shown) were scanned with a densitometer and the areas corresponding to the specific transcripts generated in the presence of wt or dl extracts were diagrammed next to the corresponding template, relative to the transcriptional activity of the WT template in wt extracts (100%). The bottom line represents the calculated sum of the transcription activities separately elicited by the mU and A12 templates. The final value was adjusted for the basal promoter activity by deducting one times the activity of the minimal promoter retained within the mUA12 template. basal MLP activity, as measured in dl extracts. In addition, this mutation reduced about 6-fold the activity measured in wt extracts, thus decreasing the overall activation by about 2-fold (compare the ratios of the signals generated by wt and dl extracts in lanes 1 and 7, Figure 2A and see Figure 4). These results support the conclusion that the UE element contributes to the late phase-specific stimulation of the MLP. This could be achieved either directly through the activation of the cognate transcription factor (UEF) itself or through the cooperative interaction between UEF, the DE-recognizing factor (DEF) and the transcription machinery. It has previously been shown by Leong et al. (23) that the intrinsic in vitro transcriptional and DNA binding activities of UEF were not affected by infection with pm975, an adenovirus-2 derivative expressing mainly the large Ela protein. In agreement with this conclusion, identical amounts of UEF-specific retarded Nucleic Acids Research, Vol. 19, No. 12 3225 B Competitor (molar excess) UEm UE DE12 DE2 -- --• 8 --8888 DE1m1 DE1 88 8 3 8 8 ! | | | 8888 ; • : ' • > • • Extract -67- UEC UEm tmn. DE1 I +86- OE2 +124- DE2 12 3 4 5 6 7 8 It 9 10 H 11 12 •MM -« 12 3 4 5 6 7 8 910111213 14151617181920212223 2425 Figure 5. DNAse I footprinting of the MLP region. A) A comparative DNAse I protection analysis was carried out (see Materials and Methods) with the WT MLP and the indicated mutant probes, in the presence of wt extract. The corresponding naked probe digestion pattern ( - ) is shown next to each protection assay (+). The UE, DEI, and DE2 elements discussed in the text are positioned. Landmark coordinates are given, relative to the MLP startsite. B) Competition analysis of the footprints on the WT MLP probe was performed by preincubating the wt extract with increasing amounts (molar excesses as mentioned) of the indicated doublestranded competitor oligonucleotides (depicted in Figure 1), before addition of the labelled probe DNA and DNAse I treatment. The uncompeted digestion pattern is shown in lane 25, and naked probe patterns in lanes 1 and 24. complexes were also observed by comparative gel-shift analysis of dl and wt extracts (Figure 3, compare lanes 5 and 6). By contrast, under the same probe-excess conditions, the level of DEF DNA-binding aictivity was dramatically increased in wt compared to dl extracts (Figure 3, compare lanes 1 and 2). Thus, while activation of DEF clearly correlates with the late-specific stimulation of MLP (35), no such a direct activation of UEF could be detected. We next examined the combined effect of alterations of the UE and both the DEI and DE2 elements on basal MLP activity (as tested in dl extracts) and on the extent of MLP activation by the wt extracts. As shown in Figure 2B and depicted in Figure 4, mUA12, a mutant promoter simultaneously lacking functional UE, DEI and DE2 elements (see Figure 1), displayed a 2 to 3-fold reduced basal activity compared to the WT template. This reduction was in fact exclusively caused by the mutation of the UE element, consistent with the low levels of DEF binding activity in dl extracts. In agreement with this conclusion, an alteration of the UE element alone (as in mil) produced the same effect as the mUA12 mutation (Figure 2B, compare lanes 3 and 4), while a deletion of the whole DE region had no effect on basal activity (Figure 2B, lane 2). By contrast, when assayed in wt extracts, the mUA12 mutation had more dramatic effects on template efficiency than mutations which separately destroy either the UE (as in mU) or the DE elements (as in A12) (Figure 2B, compare lanes 2 and 3 with lane 4). As previously suggested (23, 34), the residual activation of mUA12 transcription by the wt extract most likely reflects the stimulatory effect of the Ela gene products mediated by the intact TATA box retained in this mutant. A quantitative analysis (Figure 4) of the results presented in Figures 2A and B (and others not shown), reveals in fact that the UE and DEI +DE2 elements cooperate for maximal promoter activation: the effect of the DE and UE elements is about 3 times more pronounced when these elements are present together (activity of the WT template), than when present separately (cumulative activity of the MU and A12 templates). That the template efficiencies of mutants lacking either the DEI (ml or Al templates) or the DE2 element (A2 template) were nearly identical to those of the WT template (Figure 2A, compare lane 1 with lanes 2—4) strongly suggests that each of these downstream elements may separately cooperate with the UE element to achieve maximal promoter activation. In agreement with this conclusion, mutants either retaining the DEI or the DE2 element, but lacking the UE element (mUA2 and mUAl templates, respectively) were as poorly responsive to the latespecific stimulation as a mutant (mU template) lacking only the UE element (Figure 2, compare panels B and C). DNAse I protection studies suggest that the DEI and DE2 elements bind the same factor To examine the effect of the forementioned individual and combined mutations on the protein binding activity of the 3226 Nucleic Acids Research, Vol. 19, No. 12 respective MLP elements, we performed DNAse I footprinting experiments. A typical protection pattern of the WT MLP probe by the wt extract is presented in Figure 5 A Qane 2), next to the DNAse I digestion profile of the naked DNA (lane 1). The strong protections spanning the UE, DEI and DE2 elements are indicated. Additional, weaker protections, spanning the TATA box region (between UE and the startsite) or the region directly upstream of DEI, are observed. These protections, also found with dl extracts (34), have not been further analyzed. Deletion of either one of the DE elements Qanes 3 - 6 ) did not affect the protection over the UE element, consistent with the UEF footprint being detected in dl extracts which contain only very low DEF binding activity (35). Similarly, an alteration of the UE element which abolishes UEF binding (lanes 7-12) had no effect on protein binding to the downstream elements. These results suggest that efficient binding of either the UE or DE-specific factors can occur independently from each other, in agreement with earlier gel-shift or protection assays with crude or purified protein fractions (12, 16, 17, 35). We also performed competition experiments in which specific footprints on the WT MLP template were competed with increasing concentrations of selected synthetic oligonucleotides. As shown in Figure 5B, when the reaction was challenged with oligonucleotides spanning only the DEI or only the DE2 element (DEI or DE2, see Figure 1), protections over both DEI and DE2 elements were simultaneously abolished in each case Qanes 2 - 5 and 10-13). The resulting DNAse I digestion patterns, within the MLP downstream area, were indistinguishable from that of naked DNA (lanes 1 and 24) or after competition of wt extracts with an oligonucleotide (DE12, see Figure 1) spanning the whole DE region (Figure 5B, lanes 14-17). Under the same conditions, a mutated oligonucleotide (DElml, see Figure 1), used as nonspecific competitor, did not alter the digestion pattern (lanes 6 - 9 ) . These results suggest that the DEI and DE2 elements are binding sites for the same factor (see Discussion). Strikingly, the DE2 oligonucleotide also competed for the footprint which spans the UE element (lanes 10—13), whereas no such a competition could be obtained, under similar conditions, with the DEI or the DEI2 oligonucleotides (see lanes 2—5 and 14—17). Sequence comparisons of the oligonucleotides used in these experiments revealed in fact significant homologies between part of the UE element ( - 6 3 / - 5 4 , non-transcribed strand) and a region partially overlapping the DE2 element (+120/+129, transcribed strand), which may explain these results. As expected, the DE12 oligonucleotide which lacks most of the conserved sequences because it only extends to position +124 (instead of +132, as the DE2 oligonucleotide, see Figure 1), did not compete for the UE protection. Similarly, the UE oligonucleotide, while readily competing for UEF-specific binding (lanes 18—23), had no detectable effect on proteins binding to the DEI or DE2 elements, whether used at the concentrations shown in this experiment (lanes 18-23) or at higher concentrations (not shown). DISCUSSION Previous studies have delineated sequence elements located downstream of the MLP transcription startsite which are critical for the late phase-specific activation of this promoter (34-36). A protein (DEF), with an apparent size of 40 kDa, has previously been identified, whose binding to at least one of these downstream elements (DE) correlated with this transcriptional activation (35), pointing to DEF as a potential positive transcription factor. In this report, we demonstrate the participation of the MLP upstream element (UE) in this replication-dependent stimulation. We show that this contribution was not due to elevated binding activities of the cognate UEF (or MLTF or USF) factor late in infection, in contrast to DEF. Even though these factors bind independently to their respective sites, our results suggest that they cooperate to elicit the observed transcriptional effect, since the activation by both elements together is greater than the sum of the effects of each alone. DNA-binding competition experiments indicate that the two major downstream elements (DEI and DE2) most likely bind the same factor(s), a conclusion also supported by the observation that these elements are redundant in their ability to separately cooperate with UE and achieve maximal (or nearly maximal) stimulation of the MLP, in vitro. Synergistic promoter activation has previously been observed under conditions where no cooperative DNA binding of the activators occurred (38—41). From their results the authors suggested that activators may cooperate not by directly interacting with each other, but by simultaneously contacting a particular component(s) of the transcription machinery. In this respect, it may be relevant that UEF and BTF1 (or TFIID) stimulate MLP activity by cooperatively binding to their recognition sites. Whether DEF-mediated activation also involves BTF1, remains to be established. While the UE recognition factor (UEF), a protein which belongs to the helix-loop-helix family of regulatory proteins and binds as a dimer to its recognition site, is now well characterized (42), still little is known about the protein(s) which bind to the DE elements. Comparative band-shift, UV cross-linking and south-western analyses (35) have indicated that a host-cell protein of about 40 kDa (DEF) interacts with DEI. The observation that DEI and DE2 may bind the same protein (36 and present study), suggests that DE2, which shares no obvious sequence homology with DEI, must contact a distinct domain of DEF. Thus, both DE elements may interact with the same DEF molecule or alternatively, each element may bind its own copy of the same factor. Whereas in our present in vitro transcription system DEI and DE2 appeared as interchangeable and redundant elements, they behaved as distinct elements, each one contributing to part of the transcriptional effect, when assayed in vivo, after recombinant adenovirus infection (36). The reason for the discrepancy between the results of these in vitro and in vivo experiments is not clear, but could reflect the involvement of additional factors, whose effects would not be detected in the in vitro transcription system. Such a possibility is in fact supported by our earlier observation that a protein fraction, purified by chromatography on a DEI-affinity column, produced footprints over both the DEI and DE2 elements, but with a pattern over the DE2 element, different from that generated by the starting material (35). Despite a strong sequence homology (9/10) between the UE element ( - 6 3 to -54) and a segment overlapping the 3' portion of DE2 (+120 to +129), the UEF protein did not appear to bind to this downstream region, since competition by the upstream element had no visible effect on the protection pattern of the DE2 region, under our footprinting conditions (see Figure 5B). However, we cannot exclude the interesting possibility that such an interaction of UEF with the DE2 neighbourhood might occur in vivo, which could at least partially account for the in vivo transcriptional phenotype observed by Leong et al. (36). In this respect, it is worth mentioning that a functional equivalent of Nucleic Acids Research, Vol. 19, No. 12 3227 UEF, partially purified from duck erythrocytes, has been shown to transactivate expression from the histone H5 gene by interacting with an intragenic element (43). Leong et al. (36) have detected an additional, but much weaker protein binding site (Rl), located closer to the startsite, between +37 and +68. It appears however from their results that deletion of this element affects MLP activity, both at early and late times after infection. If true, this would suggest that the Rl region is not primarily involved in late phase-dependent events. We have notrepeatedlyobserved this protection in our binding experiments and have not analyzed it further. Interestingly however, there exists a striking sequence homology (7/8) between segments in Rl (+41 to +48) and DE2 (+117 to +124). The significance of this homology remains to be clarified. Another adenovirus gene whose activation depends, at least in part, on viral DNA replication is the peptide IX gene. The mechanisms proposed for this promoter activation imply the synthesis of a sufficient amount of new template molecules which would result in the dilution of inhibitory DNA-binding proteins and allow the redistribution of RNA polymerase and activating transcription factors over the clean, newly synthesized DNA templates. While RNA polymerase transit, from the nearby upstream Elb unit, may by itself cause promoter occlusion by preventing the attachment of necessary factors at the pIX promoter (44), negative regulatory factors may also directly repress transcription from this promoter on non-replicated molecules. A binding site for such a repressor has recently been identified, between positions +33 and +122 of the pIX gene (45). In addition, this author has shown that the upstream promoter element of the pIX gene suppresses the transcriptional repression mediated by the downstream element. Whereas promoter occlusion may similarly be invoked to explain the replication-dependence of MLP activation in vivo, it is unlikely that such a phenomenon could account for the results observed in our in vitro system, since (i) the template fragments used in the present study do not contain other promoters besides the MLP, (ii) essentially no end-to-end transcription takes place on these templates, (iii) the downstream element binding proteins act as positive transcription factors and (iv) no specific repressor binding sites have so far been identified within the MLP. influence the binding of specific transcription factors to the corresponding MLP sequences. Transcription from the MLP in vivo has previously been shown to pause or terminate prematurely around position +190, at late but not early times after infection (49). It has recently been reported that this termination site was promoter-specific since it did not function efficiently when inserted downstream of a heterologous promoter (50), suggesting that pausing required interactions of the elongation complex with specific upstream sequences or proteins bound to them. The possibility that the DE elements identified here and the cognate factors may be involved in the control of polymerase stalling at this site seems however unlikely. Extended pausing in vitro was indeed not observed under the standard incubation conditions used in our study, but only in the presence of Sarkosyl (51 and our unpublished observation). In addition, the results of Wiest and Hawley (50) show that the MLP sequences between +33 and +133 were not required for the Sarkosyl-dependent termination. Transcriptional control from the long terminal repeat (LTR) of human immunodeficiency virus provides an alternative, intriguing example of cooperation between upstream and downstream promoter elements. A number of studies have shown that the virally encoded trans-activator Tat enhances transcription of its own gene by interacting with a Tat-responsive element (TAR), an RNA target located within the 5' region of the transcript. This Tat-TAR complex seems in turn to act on particular upstream promoter elements lwithin the LTR and thereby elicit efficient transactivation (52-54). While it is clear that DEF binds to DNA, it is not known at present whether it exhibits, in addition, RNA binding activity. The elucidation of the mechanism of action of DEF also implies the understanding of the process leading to its own activation: it will for instance be essential to determine whether the dramatic increase in DNA binding activity observed late in infection corresponds to increased DEF copy numbers or to posttranscriptional modifications of preexisting molecules, and to identify the events responsible for these alterations. Clearly answers to these questions await further characterization of the DEF protein(s). Reach et al. (46) have recently reported the construction and transcriptional analysis of adenovirus mutants harboring alterations within the natural MLP upstream region. Their results indicate that mutagenesis of the UE element only weakly affected (not more than 2 fold) MLP activity, late in infection. On the other hand, these authors observed that transcription from the MLP was markedly impaired when an additional mutation was introduced into the inverted CAAT box located directly upstream of UE, between positions - 7 6 and - 8 0 . We detected no protections over this region, neither with cell extracts (Figure 5A, lanes 7—12) nor by genomic footprinting (47). It may nevertheless be of interest to examine whether altering this CAAT element, which is retained in our template molecules, will further enhance the dependence on UE of the late phase-specific transcriptional stimulation seen in vitro. Chang and Shenk (48) recently demonstrated the contribution of the DNA-binding protein (DBP), encoded by the adenovirus E2a gene, to the transcriptional activation of the MLP. 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