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Previous Article | Next Article 
Journal of Virology, November 2004, p. 12120-12128, Vol. 78, No. 22
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.22.12120-12128.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
and
Thomas Hohn*
Friedrich Miescher Institute, Basel, Switzerland
Received 25 March 2004/ Accepted 24 June 2004
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ABSTRACT |
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INTRODUCTION |
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Previous work in our laboratory reported enhancement of expression by inclusion of the first 60 nucleotides (nt) of the 35S RNA leader sequence (stimulatory region 1 [S1]). At that time, we ascribed this solely to an effect on translation (20). More recently, we characterized an element in the equivalent position in the leader of the pregenomic RNA of rice tungro bacilliform virus (RTBV) that also enhances the expression of reporter genes in protoplast expression systems. A DNA-based element within this region was found to contribute to promoter activity and to be the target for the binding of several proteins from rice nuclear extracts (12, 28). These findings led us to reassess the CaMV leader S1 region, asking whether, in this case also, there could be elements in the transcribed region that contribute to promoter activity.
Here we describe the effect of the S1 sequence on reporter gene expression in protoplast transient expression systems. We find that repeats of a CT-rich motif positively affect promoter activity, both from their usual location within the leader and when translocated to a position upstream of the transcription start site. Plant nuclear proteins bind specifically to these motifs.
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MATERIALS AND METHODS |
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Replacement of the promoter fragment BamHI-BglII (–270 to –46) in pMTF-GUS with the –402-to-–130 region of the potato gst-1 promoter (26, 47) created pgst-MTF-GUS.
Mutations in the S1 leader were constructed by replacing the KpnI-NcoI S1 leader fragment of either p35S-GUS or p–90MTF-GUS with synthetic oligonucleotides carrying the desired mutations (sequences are given in Fig. 5A and B). The same strategy was used to clone leaderless constructs (nL) by using a short synthetic oligonucleotide with the sequence GTACCACCAC.
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GUS activities in protoplast extracts prepared after overnight incubation were determined as described previously (21). CAT protein levels were determined by using a CAT-ELISA kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. GUS activities were calculated relative to the CAT internal control in all cases. Activities cited are averages of at least three independent transformations, unless otherwise stated.
RNA analysis.
Total RNA was isolated from protoplasts 6 h after transfection and subjected to RNase protection analysis according to published protocols (23). A radioactively labeled probe for S1 or mutant constructs was synthesized by in vitro transcription (in the presence of [
-32P]UTP) using SP6 RNA polymerase from a plasmid constructed by inserting the NcoI-SnaBI fragment from the 5' end of the GUS gene into the NcoI-EcoRV polylinker sites of pGEM5 linearized at the NcoI site. An antisense probe for the internal control (CAT) was derived from a pGEM1 plasmid carrying the PvuII-EcoRI fragment from near the 5' end of the CAT gene cloned into HincII-EcoRI polylinker sites. This plasmid was then linearized with HindIII and transcribed with T7 RNA polymerase. Protected fragments were resolved on 6% polyacrylamide denaturing gels and visualized by autoradiography or by PhosphorImager analysis (Molecular Dynamics). Fragments corresponding to GUS and CAT mRNAs were identified based on their sizes. The different protected fragments were quantified by PhosphorImager analysis, and the relative abundance of transcripts produced from each tested mutant was calculated relative to the internal control. Variations were within 10% of the mean.
In vitro translation. To generate plasmids driven by the T7 promoter, the KpnI-EcoRI cassette containing the leader, the GUS gene, and the poly(A) site of the appropriate clones was cloned into the T7 expression vector pBluescript SK(–) (Stratagene).
T7-directed transcripts were transcribed in the presence of the cap analogue m7GpppG and translated to wheat germ extract (WGE) as described by Ryabova and Hohn (54). Globin RNA and WGE were from Roche Molecular Biochemicals.
Preparation of nuclear extracts. Crude nuclear extracts were prepared from cell suspension cultures of O. violaceus or O. sativa line Oc as follows. About 4 x 108 suspension cells or protoplasts were disrupted in 30 ml of homogenization buffer (20 mM morpholineethanesulfonic acid [MES] [pH 6.0], 5 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 10 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 µg of leupeptin/ml, 1 µg of antipain/ml) with about 10 strokes in a Dounce homogenizer. The slurry was filtered sequentially through 60-, 40-, and 10-µm nylon mesh filters. The nuclei were pelleted by centrifugation at 1,200 x g for 5 min and resuspended in 50 ml of homogenization buffer. The solution was filtered again through 10-µm nylon mesh, and the nuclei were collected by centrifugation at 1,500 x g for 5 min and taken up in 20 ml of lysis buffer (20 mM HEPES [pH 7.5], 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, 10% [vol/vol] glycerol, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg of leupeptin/ml, 1 µg of antipain/ml). The nuclei were broken by a few strokes in a Dounce homogenizer and diluted to an optical density at 260 nm of 10. Saturated ammonium sulfate (one-ninth of the volume) was added and shaken to mix, and the solution was left to stand for 30 min. After centrifugation at 100,000 x g for 90 min, 0.33 g of ammonium sulfate/ml was added to the supernatant. After gentle shaking for 15 min to dissolve the ammonium sulfate completely, the solution was left to stand for 30 min and then centrifuged for 30 min at 20,000 x g. The pellet was resuspended in 1 ml of dialysis buffer (same as lysis buffer but without leupeptin and antipain) and dialyzed for 4 h against 200-ml batches of dialysis buffer (buffer exchanged hourly). The dialyzed solution was spun for 10 min at 10,000 x g, and the supernatant was collected and stored in aliquots at –80°C. All of these steps were performed at 4°C.
Crude nuclear extracts from O. sativa plant seedlings were prepared according to the work of He et al. (29).
Gel retardation assays. The DNA probe for gel shift analysis was produced by annealing two synthetic oligonucleotides corresponding to the first 60 nt of the 5' end of the CaMV leader sequence flanked by KpnI and NcoI sites. Note that, in the case of the S1 probe, the GTA present at position 53-55 (which derives from a Bst11071 cloning site present in the expression constructs) is replaced with the wild-type sequence TAA. The annealed products were digested with NcoI and labeled with 32P. Competitor DNA fragments were prepared by directly annealing two synthetic complementary oligonucleotides.
Binding reactions contained crude nuclear extracts (3 µl) and 5,000 cpm of labeled DNA (about 0.05 to 0.1 pmol) in a final volume of 15 µl of buffer containing 10 mM HEPES (pH 7.6), 8 mM MgCl2, 1 mM dithiothreitol, 4 mM spermidine, and 5% (vol/vol) glycerol. Ten micrograms of poly(dI-dC) · poly(dI-dC) (Pharmacia) was included as a nonspecific competitor. For the competition assays, variable amounts of competitor as indicated in the figure legends were included. Binding reactions were begun by addition of nuclear extract to the buffer. This reaction mixture was preincubated for 10 min at room temperature and incubated for a further 20 min at room temperature after addition of the probe and the competitors. Samples were then loaded onto a 5% native polyacrylamide gel (polyacrylamide to bisacrylamide, 40:1) in 1x Tris-borate-EDTA buffer. Following electrophoresis at 30 mA for 2 to 3 h at 4°C, gels were dried and autoradiographed.
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RESULTS |
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S1 sequences can enhance reporter gene expression from an upstream position. The results shown in Fig. 1 gave the first hint that the S1 sequences might be affecting transcriptional as well as posttranscriptional processes. Since the S1 region is transcribed and is thus present on the resulting mRNA, an obvious potential explanation for its stimulatory effects is that it acts as an enhancer of translation. Indeed, the S1 sequence is known to be relatively unstructured, a feature known to facilitate translation of a downstream open reading frame. However, if this were the only effect of the S1 region, the extent of stimulation would be expected to be independent of the promoter used, but this is not what was observed. Different promoters were affected to different extents by deletion of the S1 region. This led us to investigate whether S1 contains enhancer-like promoter elements.
The 60-nt S1 leader sequence was transferred from its downstream position to a location upstream of the weakened –90MTF promoter (S1–90MTF-nL-GUS, S1rev–90MTF-nL-GUS). Compared to that of the leaderless construct (–90MTF-nL-GUS), reporter gene expression was roughly twofold higher regardless of the orientation of the S1 sequence (Fig. 2). A duplicated S1 in the upstream position (2xS1rev–90MTF-nL-GUS) stimulated expression more than a single copy. These results indicate that S1 contains a DNA-based enhancer element(s).
The drop in the expression level upon deletion of the 5' untranslated region can be partially ascribed to reduced translatability due to the short leader remaining (20 bp in contrast to 63 bp in S1). By replacing the S1 leader with a random sequence (Linker) of the same length and with a similar secondary structure (with a free energy of –1.7 compared to –1.2 for S1), we were able to distinguish translational effects deriving from shortening of the leader from sequence-specific effects influencing promoter strength by comparing results from in vivo expression in protoplasts (Fig. 2B) with results from in vitro translation assays carried out in WGE (Fig. 2C). Replacing the S1 leader region with the Linker did not cause any changes in translatability (Fig. 2C), although there was a striking drop in GUS expression in vivo (Fig. 2B). In contrast, deletion of S1 (nL) negatively influenced the translational process. RNA protection assays performed with protoplasts transiently transfected with various leader mutants also demonstrated a clear correlation between reporter gene expression and mRNA amount (data not shown). This latter correlation, together with the results shown in Fig. 1A, which clearly show differing GUS expression levels from identical mRNAs, lead us to consider it unlikely that changes in mRNA stability and half-life contribute to the differences in expression seen in Fig. 2B.
Nuclear proteins bind to CT stretches within S1. DNA gel mobility shift experiments were performed to search for binding of potential transcription factors to the S1 element. Radioactively labeled double-stranded S1 DNA was used as a probe in experiments with nuclear extracts from protoplasts of O. violaceus and O. sativa. Two complexes were observed with all extract preparations tested (see Fig. 3A, complexes I and II, for O. sativa nuclear extracts; results with O. violaceus extracts were similar [data not shown]). Both shifts are specific: they can be competed by unlabeled S1 but not by unrelated DNA fragments from either the promoter or parts of an unrelated reporter gene (CAT) (Fig. 3A). A third complex (complex III) appeared with some protoplast extract preparations tested, but the corresponding protein factor(s) is either more abundant or more stable in extracts prepared from seedlings (see below).
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A competitor fragment corresponding to part of the hsp26-1 gene sequence containing the GAGA element (45) was able to compete with S1 for binding of these proteins (Fig. 3D). However, an artificial GAGA fragment competed much less efficiently, suggesting an influence either of the number of GAGA motifs (hsp26-1 contains three, while S1 and artificial GAGA have only two each) or of context and flanking sequences on protein binding.
A nuclear extract from O. sativa seedlings was used for the latter experiment. In this extract, an additional, higher-molecular-weight complex was also observed. Addition of competitors revealed that this complex (Fig. 3D, complex III) is specific for S1 but is not significantly competed by the hsp26-1 GAGA element.
Competition gel shift assays were then performed to further characterize the binding sites and the types of complexes formed between the S1 region and nuclear proteins. An oligonucleotide in which the CT motifs were mutated to GA motifs (MUT GA) competed only weakly with wild-type S1 sequences for the formation of DNA-protein complexes, confirming the importance of the flanking sequences for protein binding. Interestingly, the MUT GA oligonucleotide appears to compete slightly with S1 for the formation of complex I but hardly at all for complex II, suggesting that there might be an additional protein(s) involved in the formation of the latter that can stabilize the proteins that bind to CT motifs.
Two additional competitor variants were tested. In MUT GT, every C in the CT motifs of the oligonucleotide was changed to G (Fig. 4A); in MUT CA (partial), only the three internal T residues of each motif were changed to A. Both these oligonucleotides competed as well as wild-type S1 for complex II while losing completely the ability to compete complex I (Fig. 4B). Again, this is consistent with a requirement for another protein component in complex II that recognizes either the sequence between the two CT stretches or sites overlapping the CT motifs. Different mutations were introduced in the context of the full-length S1 sequence and used as labeled probes in gel shift assays. The MUT GT mutation supported the formation of complexes II and III but not that of complex I (Fig. 4C). The latter cannot be formed on this sequence, because the CT motifs are lacking. However, we interpret complex II as being composed of an additional protein interacting with the sequence between the CT stretches on the probe and recruiting the CT-binding factor(s) through protein-protein interactions. Consistent with this interpretation, mutation of this region resulted in loss of complex II formation (Fig. 4A, MUT AAA-TTT; Fig. 4C, S1 AAA-TTT).
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As evidence supporting this suggestion, when hsp26-1 DNA was used as a competitor, the formation of complex III was stimulated while complexes I and II were completely competed, suggesting competition for overlapping binding sites (see Fig. 3D). Further studies of complex III should be carried out with seedling extracts, where the abundance of the corresponding proteins is apparently much greater.
CT-rich motifs in the S1 region enhance expression in vivo. To investigate the importance of the CT motifs in vivo, S1 mutants were created in which the CT motifs were changed to CA motifs. In the three protoplast types tested and in the context of the weak –90MTF promoter, this led to a substantial decrease in expression (Fig. 5A, mutant CA). As expected, the decrease was only minor in the context of the full-strength wild-type 35S promoter with dicot protoplasts, but still substantial with rice protoplasts. When the CT motifs were mutated to GA motifs to restore GAGA-like elements, activity in the context of weak upstream promoter was partially recovered in O. violaceus and N. plumbaginifolia protoplasts and completely restored in rice protoplasts (Fig. 5A, mutant GA). Mutant GT (Fig. 5A), in which CT motifs were replaced with GT motifs, also led to a drastic decrease in activity. These results correlate well with the results of the gel shift assays, i.e., mutants unable to compete to form complex I are also unable to direct full-strength expression. Consistently, the AAA-TTT mutant, which in gel shift assays still allowed formation of complex I, had no detrimental effect on expression in vivo.
We next tested the effect of removing only one of the CT stretches, or altering the distance between the two motifs. Mutation of one CT motif to an unrelated sequence reduced reporter gene activity by roughly half, with a further reduction if both CT motifs were changed. Increasing the distance between the two CTmotifs did not affect expression (Fig. 5B).
Since all of these mutations are downstream of the transcription start site, they might potentially exert their effects at a posttranscriptional level. To check if any of these mutations affected translation, a series of mRNAs corresponding to the S1 leader (wild type or mutated) plus the GUS coding region was prepared by in vitro transcription. Production of GUS protein from these RNAs was monitored after in vitro translation in WGE, both by direct visualization of the translation product and by quantification of GUS activity in the extract. The results demonstrated that the mutations introduced into the leader sequence had only slight effects on translatability (Fig. 5C).
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DISCUSSION |
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To reveal these effects of the S1 region, it was necessary to use the relatively weak –90MTF promoter. A promoter can be weak due to reduced TFIID stability on the promoter (27, 38, 52). Many activators or enhancers influence promoter strength by facilitating TFIID association with the cognate promoter and/or stabilization of the preinitiation complex. In a strong promoter such as the wild-type 35S promoter, the redundant and combinatorial effects of the powerful upstream enhancers make it likely that the subtle effects of S1 sequences are normally overshadowed.
Sequences involved in transcriptional activation can function in a distance- and orientation-independent manner (5, 10), and the S1 leader sequence fits these criteria (Fig. 2). Consistent with the synergism of enhancer elements (13, 33, 43, 51), a copy number-dependent increase in reporter gene expression could be demonstrated.
A growing number of regulatory elements located downstream of transcription start sites have been reported, many of which affect gene expression on the transcriptional level. Two types of such elements can be distinguished. The first (designated DPE, for downstream promoter element) is located close to the transcription start site and interacts directly with subunits of the transcription factor TFIID to maximize the stability of the TFIID-DNA complex, thereby contributing to promoter strength. DPEs, apparently essential for full promoter activity, are associated either with TATA-less promoters (1, 8, 37, 49, 62) or with promoters endowed with a weak TATA box context and an Inr consensus (36, 41). The second type of element influences promoter activity more indirectly; it is the target of sequence-specific nuclear DNA binding proteins that function either as activators or repressors or by altering DNA conformation and even chromatin structure. Such proteins are often involved in temporal and spatial regulation of gene expression (14-16, 30, 53).
The involvement of CT-rich sequences (inverted: GAGA) in gene regulation has been reported for many years. (CT)/(GA) repeats were first described in Drosophila heat shock protein and histone gene promoters (22, 45) and were later assigned to diverse groups of genes, including inducible, homeotic, and housekeeping genes (24). In addition to Drosophila, such elements have been shown to play an important role in transcriptional regulation in many other organisms, including vertebrates (4, 35, 59), plants (2, 6, 7, 15, 16, 55), and viruses (28), suggesting the involvement of a universally valid mechanism of gene regulation.
GAGA elements have been most thoroughly examined in Drosophila melanogaster, where they are the binding targets of "GAGA transcription factor" (GAF) and the Pipsqueak (Psq) protein (56). A single trinucleotide sequence (GAG) represents a sufficient binding site for GAF (61), in contrast to Psq, which needs a longer GAGA element (39). The two proteins can bind directly to each other, thus establishing a protein complex bound to chromosomal target sites in vivo (56). Biochemical studies indicate that GAF activates RNA polymerase II transcription by counteracting chromatin repression (18). At the hsp70 and hsp26 promoters, GAF directs the formation of an open chromatin structure by nucleosome positioning that allows the association of additional factors necessary for transcriptional competence and creation of nucleosome-free DNase I-hypersensitive sites (44, 60). It appears that the chromatin remodeling complex NURF plays a key role in this process by altering the nucleosome structure in an ATP-dependent manner to allow GAGA factor binding (57). Recent studies on GAF showed that its binding on DNA can positively contribute to the recruitment of TFIID, confirming the notion that it is a widely used transcription factor (40).
GAGA-binding proteins (GBP) have also been identified in plants. Originally detected in soybeans, several GBP homologues have been found to date in several dicot plants and in the monocot rice (O. sativa). In soybeans, GBP binds to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element, and is implicated in the control of that gene (55).
Several lines of evidence support our speculation that the protein(s) forming complex I on the S1 leader region of CaMV belongs to the class of GBP found in plants. Like soybean GBP, our protein(s) seems to bind the GAGA motif in a very sequence-specific manner, and binding correlates with enhanced promoter activity. Deletion of one of the two CT motifs in the S1 leader region reduced activity almost as much as deletion of both motifs, suggesting a synergistic effect mediated by the bound proteins. On the other hand, insertion of a random 20-bp fragment between the CT motifs had no effect (Fig. 5B). Similar characteristics have been reported for Drosophila GAF, which was shown to oligomerize into higher-order complexes that could then bind to multiple sites spaced at variable distances to synergistically activate transcription (17, 34). The notion that our CT-binding protein has features similar to those of Drosophila GAF was further supported by the observation that an hsp26 gene fragment containing a known GAGA element could efficiently compete with S1 for binding of this protein(s) (Fig. 3C).
The second complex (complex II) seems also to be formed by a protein(s) that binds in a sequence-specific manner to the region located between the two CT motifs (CAAAT), although adjacent sequences overlapping the CT motifs also seem to be involved (Fig. 3B and 4B and C). In contrast to the CT motifs, no functional influence on transcriptional efficiency could be detected by mutagenesis (Fig. 5A), precluding any speculation on biological relevance.
In addition to the CT motif interacting with the GAGA-binding factor, a search of plant databases for potential transcription factor binding sites (by using the Patch program, version 1.0-public, and the TRANSFAC 6.0-Public database; Biobase GmbH, Wolfenbüttel, Germany [http://www.gene-regulation.com/pub/databases.html#transfac]) using the S1 sequence revealed a CAAAT motif specifically bound by the maize high-mobility-group (HMG) protein (25). HMG proteins are relatively abundant chromatin-associated nonhistone nucleoproteins that are able to bend DNA, thus facilitating the binding of various transcription factors to their cognate DNA sequences (9, 42). Sequence-specific binding of HMG proteins involves not only the core binding sequence but also 5' and 3' flanking sequences (58), which would be consistent with our observations.
Interestingly, several high-affinity binding sites for HMG1 have been identified in the 5' long terminal repeat of human immunodeficiency virus type 1. One of these coincides with a transcriptional activator binding site located downstream of the transcriptional start site in the 5' untranslated region. It was therefore suggested that HMG1 might play a fundamental role in the expression of human immunodeficiency virus type 1 by determining the nature of transcription factor-promoter interactions (30). A similar case was found in plants at the phytochrome A (PHYA) gene promoter, where HMG-I/Y was found to stimulate the sequence-specific binding of the transcriptional activator GT-2 to the promoter (46).
Like NURF, which is required for efficient binding of GAF to GAGA motifs, the protein(s) forming complex II may function to modify DNA or chromatin structure to facilitate the binding of GAF-like factors to their cognate DNA sequences. The reason why no transcriptional effect is observed upon mutation of the binding site for complex II may lie in the experimental setup. Functional tests were performed by transiently transfecting DNA into protoplasts, followed by determination of reporter gene expression after 12 h of incubation. In contrast to the CaMV minichromosome, which has been shown to exist as a chromatin-like nucleoprotein complex with nucleosome subunits in the nuclei of infected leaves (48, 50), the transfected DNA is originally neither arranged in nucleosomes nor properly packed in chromatin. In this situation, the GAGA-binding protein could bind by itself without the help of the complex II-forming protein, which would, however, be absolutely required under natural conditions. Alternatively, the protein may simply be inactive in protoplasts.
Whether the positive effect of the S1 leader is restricted to specific cellular or environmental conditions in the context of the viral life cycle remains an open question. Taking into account the modular structure of the complete 35S promoter and the demonstrated tissue specificity of the different domains 5' of the TATA box (3), it is possible that the cis elements in the S1 region are responsible for guaranteeing a minimal basal activity of the promoter under every possible circumstance. This notion is supported by the occurrence of CT motifs in plant housekeeping genes, such as those encoding ribosomal proteins L12 and L13, suggesting involvement of these motifs in a universally valid mechanism of gene regulation (6). Transcription from the genomic promoter is of critical importance to a pararetrovirus, not only for production of viral proteins but also for viral replication. The use of a common, and thus abundant, transcription factor to ensure a minimal level of promoter activity could reflect a fundamental survival strategy in these viruses, as evidenced by the finding of a similar mechanism in the plant pararetrovirus RTBV (28).
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ACKNOWLEDGMENTS |
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H.M.R. was supported by a grant from the Swiss National Research Foundation.
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FOOTNOTES |
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Present address: Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111-1800.
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REFERENCES |
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gene promoter. Mol. Gen. Genet. 238:428-436.[CrossRef][Medline]
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