This promoter has its own MPE but shares the MLP UPE, suggesting the possibility ... with a model in which USF can stably interact with only one transcription.
Vol. 12, No. 4
MOLECULAR AND CELLULAR BIOLOGY, Apr. 1992, p. 1630-1638
0270-7306/92/041630-09$02.00/0 Copyright C) 1992, American Society for Microbiology
Evidence that USF Can Interact with Only a Single General Transcription Complex at One Time GUY ADAMI
AND
LEE E. BABISSt*
Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021 Received 16 September 1991/Accepted 27 January 1992
By in vitro analysis, the major late promoter (MLP) of adenovirus has been shown to be a simple promoter requiring two elements for efficient transcription: a minimal promoter element (MPE), where the general transcription factor-polymerase II complex binds, and a single functional upstream promoter element (UPE) which interacts with USF. Two hundred bases upstream of the MLP cap site and divergently oriented is the IVa2 promoter. This promoter has its own MPE but shares the MLP UPE, suggesting the possibility that these promoters are coordinately regulated. To determine mechanistically how this might function, we replaced the weak IVa2 minimal promoter with a strong MPE (from the viral ElA gene) and observed mutual inhibition of both promoters and unstable transcription factor binding. Only by duplication of the UPE could this inhibition be relieved. When tested independently, both promoters were shown to require the USF site for maximal activity. These results are compatible with a model in which USF can stably interact with only one transcription complex at a time. When two divergently oriented general transcription complexes compete efficiently for binding of USF, transcription is reduced to the same levels as if the USF site were absent. A typical mammalian RNA polymerase II (Pol II) gene contains a minimal promoter element (MPE), upstream promoter elements (UPEs), and an enhancer (28). The MPE is defined as a position-dependent element required for basal levels of transcription. It is located in the region immediately around the transcriptional start site and often contains an element at the cap site and a TATA consensus element just upstream (39). Enhancers and UPEs appear to play the major role in regulating both the specificity and rate of tissue-specific and developmentally regulated gene transcription, most likely by promoting general transcription factor-Pol II complex formation at the MPE (18, 28, 29, 43). The adenovirus major late promoter (MLP) has been used as a model system to gain insights into how the gene-specific transcription factors that bind to the upstream elements function and interact with the basic transcription machinery to promote transcription. In addition to Pol II, there are five known factors that make up the basic transcriptional machinery. They bind in an ordered reaction at the MLP MPE and are required for the initiation of transcription (3, 37, 38, 48). Among these, TFIID (as isolated from HeLa cells as a 120-kDa complex) has been shown to specifically recognize and to stably interact with the TATA consensus sequence present 25 bases upstream of the MLP cap site (3, 37, 38, 52). By using cell-free transcription systems prepared from uninfected HeLa cells, the MLP has been shown to contain a single UPE, the USF binding site (8, 30, 41). The interaction between the USF protein and the USF site has been shown to increase the rate of MLP transcription 10-fold (17, 41). USF has also been shown to increase the stability of the TFIID complex's binding to the MPE (see reference 42 for a review). These findings suggest that one way USF can increase the rate of MLP transcription is by promoting stable TFIID complex binding to the TATA containing MPE,
Corresponding author. t Present address: Department of Cell Biology, Glaxo Inc., Moore Drive, Research Triangle Park, NC 27709.
which results in an increase in the rate of Pol II loading. The USF site can stimulate MLP transcription in an orientationindependent manner (24) and can also stimulate transcription from the divergent IVa2 promoter, located 210 bases upstream (35). The IVa2 promoter lacks a conventional TATA consensus element, although it requires TFIID for transcription. The IVa2 promoter binds the TFIID complex weakly (compared with the MLP) and just downstream of the cap site (6). Earlier work has shown that the MLP and the IVa2 promoter compete, with the strong MLP dominating in transcription because of its canonical MPE (34, 35). As a result, IVa2 transcription is extremely low and is difficult to detect unless the MLP sequences are deleted (6, 35). Hypothetically, an ideal way to link the expression of two genes would be to have them arranged divergently sharing the same UPE. Although there is some debate on this issue, this seems to be the case for the IVa2 promoter and MLP (31, 34) at early times after virus infection of HeLa cells (25, 32, 36). In mammalian genomes divergent transcription has been observed for a number of different cellular gene pairs (4, 11, 16, 44, 51). Curiously, only rarely do these promoter pairs have conventional MPEs containing the TATA consensus. We created this situation artificially in an attempt to understand whether there were mechanistic constrains on the type of MPEs seen in the MLP and IVa2 promoter pair. Our findings allow some conclusions to be made concerning how the basic transcription machinery and gene-specific transcription factors interact. While others have shown that multiple transcription factors can interact simultaneously with one general transcription complex, the converse does not seem to be true (7). MATERUILS AND METHODS Constructs containing the MLP paired with different divergent MPEs. The MLP plasmid contains the IVa2 promoter and MLP with a G-less cassette of 400 bases fused at nucleotide (nt) +11, relative to the MLP cap site. The MLP/+TATA and MLP/-TATA plasmids contain the adenovirus ElA gene MPE up to the cap site at +1, with or
*
5
1630
VOL. 12, 1992
INTERACTION OF USF AND GENERAL TRANSCRIPTION COMPLEXES
without a TATA element inserted at -202 bp relative to the MLP cap site. For both constructs, the IVa2 gene and cap site have been deleted. The ElA MPE is oriented to drive transcription divergently in relation to the MLP. To make these constructs plasmid pML(C2AT), which contains the MLP fused to a G-less cassette (40), was digested with SalI (3' to the G-less cassette) and HgaI (nt -202 relative to the MLP cap site). Two oligonucleotide duplexes extending from -32 and +1, relative to the ElA cap site, were ligated to the HgaI site in the following fragments: 5'-TTCGTCCC CGGTGAGTTCCTCAAGAGGCCAAACTGCA-3' (MLP/ -TATA) and 5'-TTCGTCGGTA'TTTATACCCGGTGTTCC TCAAGAGGCCAAACTGCA (MLP/+TATA); the lower strand is not shown. This created a PstI site, and these fragments were cloned into the Bluescript plasmid vector between the Sall and PstI sites, to create MLP/-TATA and
MLP/+TATA. Single-promoter constructs with G-less cassettes fused to the E1A/lVa2 chimeric promoter. (i) Ela+TATA+USF. The MLP/+TATA construct was digested with PmII (nt -58, in relation to MLP cap site) and with SacI, which cuts inside the vector sequence, thus deleting the MLP and its G-less cassette. To these sites was ligated an oligonucleotide duplex, the top strand of which was 5'-GTGACCGGGTGTTC GAATTCG-3'. This restored the USF site and created an EcoRI site. (ii) E1A+TATA-USF. The MLP/+TATA construct was digested with PmIl (nt -59) and HincII, which cuts inside the vector. The PmlI site was blunted with T4 polymerase, and then the plasmid was allowed to self ligate, resulting in the loss of the major late (ML) MPE and G-less cassette. The resulting plasmid was digested with KpnI, which cuts inside the vector. This site was blunted with T4 polymerase, and then an EcoRI linker was inserted. A similar pair of constructs containing the ElA promoter with the TATA deletion (ElA-TATA+USF and ElA-TATA-USF) were also made. For all of these constructs, a G-less cassette was joined to the E1A/IVa2 promoter. This was done by digesting each plasmid with PstI (which cuts at +3 relative to the ElA cap site) and then removing the 3' overhang with T4 polymerase. This was followed by digestion with BamHI, which cuts inside the vector. Into this site was inserted the G-less cassette as isolated from pC2AT. The G-less cassette was inserted as a SacI-to-BamHI fragment where the SacI site was blunted with T4 polymerase. Versions of MLP/+TATA and MLP/-TATA with the ElAI lVa2 promoter fused to a G-less cassette. pSmaF (40), containing the MLP (nt 4120 to 6583 of the adenovirus 2 genome), was digested with PmlI (nt -58 relative to MLP cap site) and SmaI, which cuts at nt +535. This fragment was ligated into ElA+TATA+USF to create a construct with an intact MLP and a chimeric E1A/IVa2 promoter. Only the E1A/IVa2 promoter was followed by a G-less cassette in this construct (+TATA ElA/MLP). To make the version of this with the ElA-TATA G-less cassette (-TATA ElA/MLP), the starting vector was E1ATATA-USF. Constructs with the duplication of the USF site and controls. Constructs with the duplication of the USF site and controls were made starting with the two versions of the ElA MPE with and without a TATA element, coupled and not coupled to a USF site. The starting vector was synthesized by partially digesting MLP/+TATA with AflIl (nt -110), then blunting it with T4 polymerase, and then ligating an EcoRI linker. The ElA promoter in this plasmid upstream of the
1631
unique EcoRI site at -110, relative to the MLP cap site, was then replaced with four different inserts from the various versions of the E1AIIVa2 promoter. An EcoRI-SstI fragment was inserted into the starting vector between the EcoRI (nt -110 relative to the MLP) and SstI (located in the vector) sites. This added approximately 70 bases to the IVa2 ML intergenic region and duplicated nt -110 to -44 in the 2x USF construct and nt -110 to -58 in the 1 x USF construct. Additional vector sequence is contained in the lx USF constructs to make the insertions of similar lengths (within several bases). These constructs are diagrammed in Fig. 5. Reference plasmids. The reference plasmids Alb400 and Alb286 were used as internal controls, with Alb286 also used as a challenge template. Alb400 has been previously described (14). It contains positions -650 to +25 of the mouse albumin promoter fused to a G-less cassette of about 400 bases. Alb286 contains the same promoter, except it is fused to a shorter G-less cassette. It was constructed by Bal 31 digestion of Alb400 at the HindIII site to give a G-less cassette of 300 bases. Alb300 was synthesized from this plasmid by cloning a HaeIII-XbaI fragment containing nt -650 through the G-less cassette into the HincII-XbaI sites of Bluescript+. The AMLP construct is identical to the MLP plasmid, except the G-less cassette is shortened on the 3' end. All of the plasmid constructs described contain at most a single G-less cassette. Clones were verified by sequencing across the promoters and/or restriction enzyme digestion. Extract preparation and in vitro transcription reactions. Transcriptionally active adult rat liver nuclear extracts were prepared as previously described (19). In vitro transcription reactions were as described by Gorski et al. (14), with approximately 60 to 80 ,ug of total protein. While the total amount of functional template in any reaction was variable, the total DNA concentration was brought to the same level (1.0 ,ug) by the addition of the promoterless pC2AT plasmid DNA. When reference plasmids (0.1 ,ug) were included, they were preincubated with the extract for 15 to 20 min prior to the addition of the test template. This was necessary so that the reference plasmid transcription level would be independent of the level of the test template. The transcription products were analyzed on a 5% denaturing acrylamide gel. Molecular weight standards were 3'-end-labelled fragments of pBR322 cut with HpaII endonuclease. Template challenge experiments. Liver extract was incubated for 20 min at 300C with the test template without added nucleoside triphosphates (NTPs). While the level of test template varied, the total DNA concentration was adjusted to the same level for each reaction. To start transcription, 0.2 ,ug of the Alb300 plasmid and NTPs was added and the mixture was then incubated for an additional 30 min at 30°C. In vitro transcription in the presence of USF oligonucleotide duplex. Liver extract was incubated for 20 min at 300C as described earlier with the addition of 75 ng of an oligonucleotide duplex. The USF oligonucleotide duplex is 5'-GGTG TAGGCCACGTGACCGGG-3' hybridized to the complementary strand. The USF mutant oligonucleotide duplex is 5'-GGTGTATGCTACATGGCCGGG-3' hybridized to the complementary strand. RESULTS Under conditions of limiting levels of specific DNA template, the presence of a divergent EIA MPE can inhibit MLP transcription. Using a rat liver hepatocyte in vitro transcription system (14) and the natural divergent promoter pair
1632
ADAMI AND BABISS
MOL. CELL. BIOL.
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FIG. 1. Transcription conditions in which the presence of a divergent ElA MPE can inhibit ML transcription. (A) In the schematic representation of the promoter region of the AMLP construct, arrows start at the cap sites and indicate the orientations of the IVa2 and ML transcripts. The positions of the MLP MPE and the USF binding site are indicated. Nucleotide numbers are in relation to the MLP cap site (nt + 1). The G-less cassette fused to the MLP contains 286 bases and is represented by a box. The autoradiogram shows the polyacrylamide gel electrophoresis-separated transcription products made from in vitro transcription of the AMLP plasmid. At the top of each lane is the quantity in micrograms of this plasmid. For all assays prior to each reaction 0.1 pg of a construct containing the Alb promoter fused to a 400-base G-less cassette was added as an internal control. The bands are identified according to size (in bases) and are labelled according to the promoter from which they were synthesized. (B) Constructs are schematized as described above. The ElA MPE is diagrammed as a hollow box with or without the TATA consensus sequence. In vitro transcription is done with three plasmids at high (1-,ug) and low (0.1 ,ug) levels of specific template. The autoradiogram is of the gel electrophoresisseparated RNA products synthesized in vitro by using the template marked above each lane. The autoradiogram is overexposed, and so the signals for the transcription products with 1.0 ,ug of template are
MLP/IVa2 (34), we examined promoter activity as a function of the presence of divergent TATA-containing MPEs. A 38-nt oligonucleotide duplex containing just the ElA MPE (12) and no other binding sites was inserted into the IVa2 promoter at +2 (-212 relative to the MLP start site) to make the MLP/+TATA plasmid (see Fig. 1 line drawings). The ElA MPE sequence that was cloned was sufficient for transcription in this context, since it contains the TATA consensus element and all but 1 base of the initiator consensus sequence element (46). In this construct the MLP was intact. A control plasmid, MLP/-TATA, was created by inserting in the same place and orientation a similar oligonucleotide duplex except that the TATA sequence was deleted. Transcription from the MLP and all subsequent promoters was monitored by using the G-less cassette system, as previously described (40). The titration of MLP transcriptional activity as a function of template levels is shown in Fig. 1A. Unless otherwise stated, a low level of reference plasmid (Alb400 or Alb286) was preincubated with the extract as an internal control in this and all subsequent assays. This facilitates comparisons between different experiments. Total DNA levels in each reaction were equalized by the addition of a promoterless vector plasmid. The plasmid used for the titration shown in Fig. 1A was AMLP, although the plasmid MLP/-TATA gave identical results (data not shown). When 0.5, 1.0, or 1.5 p,g of specific template was used, no increase in the rate of MLP transcription was scored. The system could no longer support an increase in transcription above 0.5 ,ug of specific DNA template. An increase in template levels from 0.1 to 0.5 p,g of DNA resulted in a marked increase in transcription levels. (A longer exposure of the film did reveal a signal in the lane labelled 0.1 ,ug [data not shown].) We thus operationally define levels of 1.0 ,ug of promoter-containing plasmid as DNA template excess and 0.1 p,g as template limiting. In vitro transcription is often performed under conditions of template excess. We wanted to look at the interaction of two promoters on the same DNA template. Therefore, we did our assays under conditions that favored the assembly of transcription complexes on all promoters (large amounts of transcription factors and limiting amounts of the promotercontaining template). This would create a situation in which there was sufficient extract so that all promoters could interact with the available transcription factors. We examined transcription from the MLP in two different constructs (with and without an intact, divergent ElA MPE: MLP/ +TATA and MLP/-TATA, respectively). As a control we also examined transcription from the MLP with an intact IVa2 promoter (MLP). The autoradiogram in Fig. 1B is overexposed to facilitate the comparison of transcription at the low template levels. When 1.0 pg of all of the MLPcontaining templates was used for transcription analysis, equally high levels of ML transcription products were observed. We speculate that at this high promoter-containing plasmid concentration, the majority of active templates have only a single promoter functioning, since some or all transcription factors are limiting. On the basis of the data in Fig.
not in the linear range. Products are marked according to the promoter and the size of the G-less cassette. All reaction mixtures were preincubated with 0.1 ,ug of a construct containing the mouse Alb promoter fused to a 286-base G-less cassette as an internal control. The markers shown are HpaII-digested pBR322 3' end labelled with Klenow fragment and [ 2P]dCrP.
VOL. 12, 1992
1633
INTERACTION OF USF AND GENERAL TRANSCRIPTION COMPLEXES
1A, we expected that using 0.1,ug of DNA template would result in conditions of factor excess and the possibility of expression of both promoters on a single template. As expected, total transcription was low for all templates at the lower (0.1-,ug) DNA level. However, transcription from the divergent MLP contained in the MLP/+TATA plasmid was inhibited about 10-fold versus that from the MLPs in the MLP/-TATA and wild-type MLP construct (Fig.1B). This result was observed with three different pairs of MLP/TATA constructs and with more than three different nuclear extract preparations (although the extent of inhibition varied from 5to 10-fold). In conclusion, the divergent ElA MPE inhibited transcription from the MLP, and this occurred only at low specific template levels (template-limiting conditions). The promoter construct with the divergent ElA MPE competes poorly in a template challenge assay. Template challenge experiments were done to examine more closely the levels of excess transcription factors as a function of template concentrations. Several of the general transcription factors have been shown to bind stably enough to DNA to resist competition for transcription by a secondarily added template (10, 49). In this assay an initial template was incubated in a transcription extract without the addition of NTPs to allow stable interactions with transcription factors to occur. This was followed by the addition of a constant amount of the Alb286 challenge (second) template (containing the Alb promoter fused to a 286-bp G-less cassette). The same experiments using a second template with the MLP fused to a 286-bp G-less cassette gave similar results (data not shown). Levels of transcription from the second template reflect the amounts of accessible transcription factors that remain following incubation with the first template. Included is a negative control in which the first template (pC2AT) does not contain a promoter and thus does not compete efficiently. When 1,ug of the MLP plasmid was used in the preincubation reaction, there was no detectable transcription from the second (Alb286) template, as shown in Fig. 2. As a control, when the order of addition is reversed by adding 0.1 Fig of the challenge template first and then 1.0 Fig of the second template, the challenge template was transcribed at 100% (Fig. 1). When 0.2 Fig of the first template was used, transcription from the challenge template was about 40% of the control level, indicating that transcription factors are accessible to the second template, although not at 100%. Results with MLP/+TATA and MLP/-TATA, shown in Fig. 2, gave additional information. When 1.0 Fig of either template was used, transcription from the challenge template was similarly undetectable. Surprisingly, under conditions of limiting template (0.2 Fig of template), the MLP/+TATA construct failed to compete efficiently for transcription factors. The challenge template was transcribed as well as if the first template were not present. Therefore, these findings suggest that at 0.2-,ug template conditions, in which there is some excess of transcription factors, the divergent MLP/ +TATA construct is not binding some transcription factor(s) stably. This effect is directly attributable to the ElA MPE. It has been shown for the MLP that USF and the TFIID complex are the only factors or groups of factors that bind stably through the course of the template challenge assay using a HeLa cell extract (49). If we assume that the liver and HeLa cell extract behave similarly, then the TFIID complex must not be stably binding to the MLP in the MLP/+TATA construct. We would not be able to monitor USF binding in this assay, since the mouse albumin and ML UPEs do not share specific factor requirements (reference 26
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FIG. 2. Template challenge assays show that the MLP/+TATA construct competes poorly for transcription factors. The constructs are described in the legend to Fig. 1B, with the addition of the plasmid which contains a G-less cassette but no PC2AT control and the mouse albumin promoter-containing plasmid. The promoter albumin promoter construct is similar to the internal control used for the experiments described in the legend to Fig. 1B. All possible albumin transcription factor binding sites are schematized (27). Each lane of the autoradiogram represents the separated products from in vitro transcription; the products are identified according to size. The lanes are labelled according to the identity of the first (at 0.2 or 1 Fig) which is preincubated with the extract. template After addition of NTPs, the second template was added, as described in Materials and Methods.
and data not shown). The general transcription machinery
are presumably the requirements (i.e., the TFIIDthatcomplex) at least one entity binding same. Our findings suggest less stably in the divergent promoter construct is part of the basic transcription machinery (shared by the albumin promoter and MLP).
The ElA chimeric promoter is an efficient promoter and conrequires the USF site for maximal transcription.the Four USF site structs (Fig. 3) were created to test whether
contributed to transcription from the ElA/IVa2 hybrid MPE. All of these constructs lacked the ML MPE. Two contained the +TATA and -TATA versions of the ElA MPE, fused to a G-less cassette extending to nt -160 relative to the wild-type IVa2 start site. They contained the wild-type USF site. As a control, a pair of constructs that lacked the USF
site was made. These constructs were tested for transcription at levels of 1 FLg of template. The results shown in Fig. 3A indicate that only the E1A+TATA constructs (lanes 1 and 2) gave efficient transcription. The USF site in the promoter increased ElA MPE transcription about threefold
1634
ADAMI AND BABISS
MOL. CELL. BIOL. ElA #TATA +USF E1A MPF
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FIG. 3. The ElA chimeric promoter is an efficient promoter that requires the USF site for maximal transcription. (A) ElA/IVa2 chimeric promoter constructs are shown. The USF site is either intact or partially deleted as schematized. Symbols are as described for Fig. 1B. Lanes of the autoradiogram are numbered according to the identity of the promoter construct shown in the diagram. All reaction mixtures contained 1.0 ,ug of each promoter construct. (B) Only at high template levels does the ElA chimeric promoter work efficiently when an intact MLP is present on the same template. Shown are comparisons of ElA+TATA+USF promoters with or without an intact MLP. Lanes of the autoradiogram are numbered according to the identity of the specific promoter construct which was tested at the level indicated. Otherwise, see the legend to Fig. 1B for details.
(lane 1 versus lane 2), demonstrating its contribution to efficient transcription from the hybrid ElA promoter. The divergent MLP and ElA promoter are mutually inhibitory. Next we determined the level of transcription from the E1A/IVa2 fusion promoter in a situation in which the MLP was intact. A version of the MLP/+TATA plasmid containing the ElA MPE fused to a G-less cassette was made (Fig. 3B). This plasmid contained an intact MLP, although its transcription was not scored since it was not fused to a G-less cassette. Transcription from the ElA chimeric promoter in the MLP/+TATA construct was tested at two template levels (1.0 and 0.10 ,ug) and was compared with transcription from the same promoter in a construct with the MLP deleted. As in earlier experiments to control for transcription product recovery, a small amount of the Alb286 template is preincubated in the transcription extract. At the high template levels the E1A/IVa2+TATA promoter with the MLP deleted was about twofold more active than that in the construct without the MLP deletion (Fig. 3B, lane 1 versus lane 2). This discrepancy was expected, since the MLP/+TATA construct contains the MLP competing for transcription factors. At low template levels, no transcription from the ElA promoter in the MLP/+TATA construct was detected, while the same promoter without a divergent MLP was active. We note that the ML MPE inhibited ElA MPE-driven transcription more than three- or fourfold in the plasmid MLP/+TATA. Since the MLP in this construct was
also inhibited (Fig. 1B), it seems that the two TATAcontaining MPEs (the ML MPE and the ElA MPE) are mutually inhibitory. At low template levels the sum of transcription from both promoters is much less than that from the construct containing a single intact MLP. Finally, note that the ElA chimeric promoter works quite efficiently at high levels of promoter-containing DNA templates even when the MLP is intact on the same plasmid. When assayed at similar template levels, transcription from the IVa2 promoter is undetectable (34). The USF site is the source of inhibition in the divergent promoters. It has been shown that when the USF site is deleted from the MLP, transcription is reduced 10- to 20-fold and the general transcription factors bind less efficiently (8, 41). Because this level of inhibition was similar to the amount observed in the divergent TATA-containing promoters, an experiment was done to examine whether the USF site was playing a role in the inhibition. USF was specifically competed out of the in vitro transcription reaction by preincubation with a oligonucleotide duplex containing the USF site or with a control oligonucleotide duplex containing a mutant USF (15). Transcription of the same three constructs diagrammed in Fig. 1B was compared at low template levels. The left three and right three lanes in Fig. 4 are control experiments with no oligonucleotide and a control mutant oligonucleotide duplex, while the middle three lanes are from the actual experiment. The presence of the USF-
INTERACTION OF USF AND GENERAL TRANSCRIPTION COMPLEXES
VOL. 12, 1992
taneously with the bound USF. It seemed reasonable that the duplication of this cis element might relieve this inhibition. Sequences extending from nt -112 to -45 (relative to the MLP start site) were duplicated in the MLP, so that two USF sites were now present in both the MLP/-TATA and MLP/+TATA constructs (see Fig. 5). A pair of analogous plasmids was constructed in which a similar sequence minus the USF site was duplicated in order to control for nonspecific spacing effects (bp 112 to -59 and vector sequence). For all four of these constructs only MLP transcription was monitored with the G-less cassette. The results of these studies are shown in Fig. 5. At high template levels, all MLP-containing plasmids showed similar levels of transcription after normalization to the control level (lanes 1 to 6). Note that the presence of two USF sites did not increase transcription efficiencies appreciably. This lack of synergy may be due to the large separation (>70 bases) of the sites (see reference 18 for a review). At low template concentrations (conditions of factor excess), duplication of the USF site relieved the E1A+TATA MPE inhibition of the MLP (lane 7 versus lane 12). In contrast, the control plasmid with similar ElA and ML MPE spacing, but lacking the USF site duplication, showed inhibition (lane 8 versus lane 12). Note that the duplication of the USF site had no effect on transcription from the ElA-TATA construct (lane 10 versus lane 12). These findings can be explained only by a relief of interference by the E1A+TATA promoter. Apparently, when there are enough general transcription complexes to potentially bind at the two MPEs, they compete for a stable interaction with a single USF, which seems to result in a loss cooperativity between the general and gene-specific factors. We have repeated many of these experiments using a construct with the mouse albumin promoter from nt -325 to +25 fused to a G-less cassette. The polyomavirus early transcription unit MPE (nt 118 to 145) with Sall sticky ends
-C
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oligo FIG. 4. USF is required for the inhibition of the MLP by a divergent EIA MPE. The experiment is similar to that used to produce the data shown in the right half of Fig. 1B. Each specific template is used at 0.1 pg. As marked for the middle set of assays, the transcription extract was preincubated for 20 min with 75 ng of an oligonucleotide duplex that contains the USF site (36). The third set of assays is in a transcription extract that is preincubated with 75 ng of a mutant USF oligonucleotide duplex that does not bind USF. See Materials and Methods for details. oligo
containing oligonucleotide produced a 5- to 10-fold decrease in transcription from the MLP/-TATA and MLP plasmids. MLP transcription from the MLP/+TATA construct was not affected. It is as if USF had no contribution to the transcription seen from this promoter. Interestingly, with USF competed away, all three promoter setups were transcribed at the same low level. The ElA MPE no longer was able to inhibit transcription from the divergent MLP. We conclude that USF is required for the inhibition of the divergent MLP, although these data do not indicate how direct this effect is. One model to explain the inhibition seen in the divergent TATA-containing promoter setup is that two efficient promoters surrounding a single USF site cannot interact simul-
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MLP400
I _1 |.TATA 2212 EIAAMPEl
iMLPi-TATA -
-
ALB286 _ U122 F1
100%
-TT
0.lug Template (24hr exposure)
FIG. 5. Duplication of the USF site relieves divergent TATA-containing MPE inhibition. Lanes of the autoradiogram of the separated transcription products are labelled according to the number of promoter constructs are tested in the assay. Otherwise, see the legend to Fig. 1B for details. Note that the autoradiogram from the assay with 1 p,g of the test plasmid is exposed for a shorter time than that of the assay with 0.1 pg of the specific plasmid.
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(5'-TCGACTTGATATAATTAAGCCCCAACCGCCTCT3') was inserted as a duplex in both orientations in a polylinker just past nt -325 relative to the albumin promoter. This insert contains the TATA consensus and transcription start site. A control construct had similar inserts of a polyomavirus MPE containing a 6-base deletion of the TATA consensus. Specific inhibition of the albumin promoter was observed only at low promoter template concentrations and only when a TATA consensus-containing MPE was inserted in the promoter region (data not shown). Furthermore, the inhibition required that the inserted MPE be arranged divergently. A codirectionally oriented MPE (wild type or mutant) did not inhibit transcription from the albumin promoter (data not shown). The suggestion is that divergent transcription driven by a single dominant UPE can be inhibitory for transcription factors besides USF. DISCUSSION The divergent arrangement of the adenovirus MLP and IVa2 promoter prompted us to ask questions concerning coordinate regulation of these two genes. The MPE of the MLP, but not that of the IVa2 promoter, contains a consensus TATA element. By changing the IVa2 promoter so it now contained an MPE with both a cap site and an upstream consensus TATA box, we originally sought to examine restraints on divergent transcription in a mammalian system. The experimental findings we describe have now allowed us to make several conclusions about the interactions of genespecific transcription factors, the general transcription machinery, and the different types of MPEs. We have identified a sequence (the ElA MPE) that can inhibit transcription in cis only under conditions in which transcription factors are at or approaching saturating levels (as measured by template challenge assays and by transcription levels). This MPE binds the TFIID transcription factor complex and the transcription machinery (33). Insertion of the ElA MPE upstream of the MLP in a divergent orientation resulted in inhibition of transcription from the MLP by the destabilization of transcription factor binding. MLP activity was inhibited to levels as if the USF site was not present. Inhibition was dependent on the ElA MPE containing only an intact TATA element and cap site. This was not unexpected, as the TATA element is an important part of an efficient promoter; in fact, its insertion upstream of a nonTATA-containing MPE resulted in increases in transcription and stabilization of TFIID binding (1, 23). The question is how can a sequence inserted 200 bases away from the MLP MPE have an effect on its ability to drive transcription? The DNA-bound transcription factors and the general transcription machinery interact directly or indirectly. When present together the general and upstream transcription factors can show qualitative changes in binding (22, 41), increased stability of DNA template binding (13, 41), cooperation in blocking nucleosome inhibition of transcription(53), and finally, association on affinity chromatography columns (2). We propose that USF bound at its site cannot stably interact with the two surrounding divergently arranged transcription complexes simultaneously. This model is supported by several facts: (i) only the efficient E1A+TATA promoter inhibits transcription, (ii) like the ML MPE, the E1A+TATA promoter requires the USF site for maximal function (Fig. 3A), (iii) when the promoters are arranged divergently, total transcription decreases (the two promoters are mutually inhibitory) (Fig. 3B), (iv) USF is required to observe the inhibition at the ML MPE (Fig. 4),
MOL. CELL. BIOL.
and (v) this inhibition is relieved by duplication of the USF site (Fig. 5). When surrounded by efficient divergent MPEs, the USF does not function. It is as if the two surrounding general transcription complexes compete for a stable interaction with a single USF complex. The situation would be roughly analogous to a receptor ligand interaction with interference by an inhibitor. As the interactions of the general transcription machinery and DNA-bound USF are controversial, it is difficult to speculate on the biochemical nature of this inhibition. Somehow, though, it functionally results in destabilization of both interactions and USF's contribution to transcription is compromised. Another explanation of our results is that efficient divergent elongation of two transcripts is inhibited because of topological constraints. It is possible that at low DNA template levels, the divergent promoters initiate transcription but the rapid unwinding of the DNA template that allows efficient elongation in both directions cannot occur. Since we used only functional assays that looked at completely transcribed products, this would appear as a transcriptional defect. Three experiments suggest that this explanation is incorrect. (i) All plasmids contain one single G-less cassette; transcription in the opposite orientation would be limited, since 0-methyl G (40) is present in the reaction mix. (ii) Inhibition of factor binding was observed in the template challenge assay prior to the addition of NTPs. (iii) Duplication of the USF site was shown to relieve the promoter interference at the MLP. This final observation indicates that if inhibition is due to divergent transcription and constraints on DNA unwinding, then one must assume that the addition of an extra USF site specifically prevents this unwinding from being a problem. It has been proposed that more than one gene-specific transcription factor can interact simultaneously with the basic transcription factor-Pol II complex (9, 18, 45). Using an artificial promoter containing multiple GALA binding sites, Carey et al. (7) demonstrated that during transcriptional activation as many as five factors can simultaneously interact with the Pol II transcription complex. Our results now suggest that the converse is not true. We find that only one transcriptional complex can interact with a single USF. This is not contradictory, since the transcriptional complex is larger than a gene-specific transcriptional factor, such as the USF dimer (15). To further support our hypothesis, we have inserted MPEs in both orientations approximately 325 bases upstream of the mouse albumin transcriptional start site and have observed similar kinds of transcriptional inhibition only when the MPE is intact and arranged divergently. The two sites responsible for the bulk of transcriptional activation of the albumin promoter in liver extract (HNF-1 and C/EBP) are within 30 bases of each other and likely work cooperatively in their interaction with the general transcription machinery (27). These results suggest that in the albumin promoter there are similar restraints on interactions of UPE-bound factors and surrounding general transcription complexes. The USF complex is likely not the only transcription factor (or transcription factor pair) that can interact with only one general transcription complex at a time. The data here demonstrated a large reproducible mutual inhibition of in vitro transcription when divergent conventional TATA-driven promoters share a UPE. Using two experimental approaches, we could relieve this TATAdependent inhibition of divergent promoter function (i) by duplicating the UPE (and so losing coordinate control) and (ii) by removal of the TATA from one of the MPEs. Although our results are remarkably consistent with what is
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INTERACTION OF USF AND GENERAL TRANSCRIPTION COMPLEXES
naturally occurring mammalian bidirectional transcription units, we are cautious to extrapolate from our findings. The phenomenon of TATA-less promoters driving divergent transcription is the rule only in mammalian genomes; many exceptions exist in Drosophila and yeast genomes (21, 50). One would have to postulate that there is something about the basic mammalian transcription system that is quantitatively or qualitatively different from the Drosophila or yeast system. Furthermore, the inhibition we see depends on a single efficient UPE being surrounded by divergent MPEs. We should note that divergent transcription in the polyomavirus uses multiple blocks of UPEs (5, 47). One would not expect insertion of an efficient MPE in the late orientation to inhibit total divergent transcription, and that is what is seen (data not shown). Finally, it should be noted, it would be unlikely for transient transfection studies to show divergent promoter inhibition, because in transfections DNA is present in the nucleus at high levels probably analogous to those in the in vitro experiments done at high template levels (11, 20). In these types of experiments transcription factors would likely be at limiting concentraseen in
tions.
2. 3.
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