Recruitment of transcription factors to the target site by triplex-forming oligonucleotides
Recruitment of transcription factors to the target site by triplex-forming oligonucleotidesFedor Svinarchuk1,2,*, Irina Nagibneva3, Dmitry Cherny4,5, Slimane Ait-Si-Ali3, Linda L. Pritchard3, Philippe Robin3, Claude Malvy1 and Annick Harel-Bellan3
1Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France, 2Department of Biochemistry, Novosibirsk Institute of Bioorganic Chemistry, 8 Prospect Lavrenteva, Novosibirsk 630090, Russia, 3Laboratoire `Oncogénése, Différenciation et Transduction du Signal', CNRS UPR 9079, CNRS, Institut Fédératif sur le Cancer (IFC-01), 7 rue Guy Moquet, 94801 Villejuif Cedex, France, 4Laboratoire de Microscopie cellulaire et moléculaire, CNRS URA 147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France and 5Institute of Molecular Genetics, RAS, Kurchatov's Square, Moscow 123182, Russia
Received May 19, 1997;Revised and Accepted July 18, 1997
ABSTRACT
Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.
INTRODUCTION
Triplex-forming oligonucleotides (TFOs) represent a new approach to artificially regulate gene expression through direct interaction with DNA. Indeed, TFOs targeted against gene promoters can modulate transcription of the targeted gene (1 ). Two types of TFOs have been described. Pyrimidine oligonucleotides are composed of thymidines and cytosines and bind in parallel orientation to runs of purine acceptors of a duplex DNA through Hoogsteen base pairing. In the second type of TFOs, the oligonucleotides are composed of guanosines and adenosines or thymidines, and bind in an antiparallel orientation to the purine acceptor strand via reverse Hoogsteen base pairing (reviewed in 2 ,3 ).
TFOs can bind specifically to their target sequences even if these targets are present as a single copy in a DNA molecule as long as a yeast chromosome (4 ), a human chromosome (5 ) or even in the whole human genome (6 ), which makes them very attractive compounds for gene-targeted therapy. They have been designed mainly to bind in the vicinity of transcription factor target sites and to act as competitors for these proteins. Alternatively, they can introduce a lesion in a sequence-specific manner which will prevent DNA/RNA polymerase movement through the target template. In all these cases, TFOs play a negative role, inhibiting a biochemical process by preventing normal interactions between the targeted DNA and protein factors (reviewed in 7 ). However, TFOs potentially could be used for more diverse purposes. For example, TFO-based DNA bending ligands have been designed (8 ). In the present work we have designed an oligonucleotide able to recruit transcription factors to the target DNA sequence.
These molecules (hairpin-TFOs) are bi-functional oligonucleotides, which contain a hairpin able to bind transcription factors, and a sequence forming a triple helix on a target DNA (see Fig. 1 A). The specific oligonucleotide that we have used in this study contains a homopurine triplex-forming sequence which is targeted to the vpx gene of HIV-2 (and SIV) and forms highly stable triple helices (9 ). This TFO portion is linked, through a nucleotide loop, to a double-stranded hairpin including transcription factor binding sites from the SRE enhancer of the c-fos gene promoter. The SRE enhancer (10 ) is present in the upstream regulatory sequence of a number of immediate early genes (11 ,12 ). The SRE is constitutively occupied by a complex of two proteins, p67SRF (13 ) and p62TCF (14 ). P67SRF recognizes a CArG box in the SRE (15 ). P62TCF, either the ELK-1 or the SAP-1 protein, does not bind autonomously to the element but requires the presence of SRF in order to efficiently contact DNA (16 ,17 ), thus forming a ternary complex.
MATERIALS AND METHODS
Plasmids
The plasmid pVPX1 containing the polypurine stretch of the SIV vpx gene was constructed by inserting the oligonucleotides 5"-CTAGACCTGGAGGGGGAGGAGGAGGAGGTCCG-3" and 5"-GATCCGGACCTCCTCCTCCTCCCCCTCCAGGT-3" into the XbaI-BamHI sites of the vector pBluescript II. All plasmids were purified on Qiagen columns, following the manufacturer's recommendations.
Oligonucleotides
Oligodeoxynucleotides were synthesised using an Applied Biosystems 391A DNA synthesiser and purified by electrophoresis on denaturing 20% polyacrylamide gels. The different oligonucleotides used are presented in Figure 1 B.
DMS footprinting
To prepare a DNA fragment for modification by DMS the pVPX1 plasmid was cut with the ClaI restriction enzyme, 3" labelled with the Klenow fragment of DNA polymerase I, and digested with the XhoI restriction enzyme. A larger labelled fragment (1.0 pmol) was dissolved in a 20 [mu]l volume containing 50 mM MOPS, pH 7.2, 10 mM MgAc2 and 50 mM NaAc. After addition of the TFO (10 pmol), the mixture was incubated at 37oC for 2 h. Then 2 [mu]l of 5% DMS was added and the methylation reaction was performed for 2 min at 25oC. The reaction was stopped by the addition of 5 [mu]l of a solution containing 10% mercaptoethanol, 1 mM EDTA and 0.1 M NaAc. After two precipitations in ethanol, the samples were treated with 50 [mu]l of 10% piperidine at 95oC for 15 min, and the cleavage products were separated on a denaturing 6% polyacrylamide gel.
Electrophoretic mobility shift assay (EMSA)
SRF and ELK-1 were produced by in vitro translation as described (18 ). In some experiments, SRF was produced in bacteria as GST-SRF and prepared as described (19 ).
The SRE, SRE-TFO, SRE-mTFO and mSRE-TFO oligonucleotides, described in Figure 1 , were 5" end labelled (1000 Ci/mmol) using T4 polynucleotide kinase and [[gamma]-32P]ATP, purified on denaturing acrylamide gels, and annealed. Interaction between hairpin-TFOs and transcription factors was assayed in the following manner: 2 [mu]l in vitro translated SRF or ELK proteins were preincubated in 20 [mu]l of EMSA buffer (188 mM NaCl, 50 mM HEPES pH 7.9, 2.5 mM EDTA, 2.5 mM DTT, 12% glycerol) with 1-2 [mu]g salmon sperm, together with an excess of unlabeled competitor oligonucleotide (where indicated). After 15 min on ice, 32P-labelled oligonucleotide probes (0.5 pmol) were added, and the incubation was allowed to continue for an additional 10 min at room temperature. Samples were electrophoresed in 5% polyacrylamide gel in 0.5*TBE at 10oC in a Hoefer Mighty Small II apparatus. Gels were dried and analysed using a Phosphoimager (BAS 1000, Fuji), and by conventional autoradiography.
Formation of the multimolecular complex including the target DNA, hairpin-TFO, and the transcription factors was assayed in the following manner: 1 pmol of double-stranded DNA was first incubated with 1 pmol of the third-strand oligonucleotide in a 10 [mu]l solution containing 10 mM MgAc2, 20 mM TrisAc, pH 7.5, and 50 mM NaAc for 1 h to form the triple helix. Two [mu]l of this mixture were used for incubation with the transcription factors in 20 [mu]l of 0.5*TBE supplemented with 2 mM MgAc2 and 1 [mu]g of salmon sperm DNA, as described above. Samples were electrophoresed at 10oC in 0.5*TBE supplemented with 2 mM MgAc2.
Electron microscopy
After formation of the triple helix between pVPX1 linearized by KpnI and the SRE-TFO as described (20 ), a 5 [mu]l aliquot containing 1-3 mg/ml DNA (in 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, pH 7.4) was applied to a carbon film glow- discharged in the presence of pentylamine vapours using the method of (21 ). Samples were stained with 0.5-1% aqueous uranyl acetate for 10 s and blotted with filter paper; they were then observed with a Zeiss/LEO CEM-902 electron microscope in the annular dark-field mode as described by (22 ,23 ). Image recording and measurements of DNA molecules were performed with the built-in Kontron image analyser system and software.
RESULTS AND DISCUSSION
Hairpin-TFOs form stable triplexes with target DNA
SRF and ELK-1 bind to the hairpin-TFO
The interaction between the transcription factors and the hairpin-TFO was analysed by EMSA. The SRE-TFO and SRE-mTFO hairpins both contain a CArG box, specifically recognized by SRF. Both oligonucleotides bound SRF as efficiently as a classic double-stranded probe used as a control (Fig. 5 A); the ratio of the retarded band to the total radiolabelled oligonucleotide was the same for all three probes (SRE, SRE-TFO and SRE-mTFO). The SRE sequence of the c-fos promoter contains, in addition to the CArG box, an Ets box (Fig. 1 ). The Ets box is recognised by ELK-1 but only when the adjacent CArG box is occupied by SRF (16 ,17 ), thus forming a ternary complex which can be observed in EMSA as a supershifted band as compared with that of the SRE/SRF complex (18 ). As seen in Figure 5 B, lanes 4 and 7, SRE-TFO and SRE-mTFO have the ability to bind SRF and ELK-1 simultaneously. The formation of the ternary complex was specific, since no retarded bands were detected when similar experiments were performed with mSRE-TFO, in which the CArG and Ets boxes are mutated (Fig. 5 A, lane 5). Thus, the SRE-TFO is able to specifically interact with the transcription factors.
Formation of a multimolecular complex: target DNA/hairpin-TFO/transcription factors
The formation of a multimolecular complex including the target DNA, the hairpin-TFO, and transcription factors was demonstrated by EMSA. In these studies, an end-labelled ClaI-Ecl136I fragment (102 bp) from the pVPX1 plasmid which contains the target sequence was prepared. When this probe was incubated with the SRE-TFO or the mSRE-TFO, but not with the SRE-mTFO, a retarded band corresponding to the triplex structure could be observed (Fig. 6 A, lanes 2, 3, 6, 7). Addition of the SRF protein resulted in the appearance of a further retarded band migrating with a mobility similar to that of the SRE-TFO/SRF (Fig. 6 A, lane 3). It is not surprising that the mobility of the multimolecular complex (duplex DNA/hairpin-TFO/SRF) was similar to that of the hairpin-TFO/SRF complex; indeed, in EMSA the mobility of protein-DNA complexes is mainly dictated by the protein. The target DNA/hairpin TFO/SRF complex was not observed when one of the two functional sites of the bi-functional oligonucleotide was mutated, i.e., in the case of the SRE-mTFO or the mSRE-TFO (Fig. 6 A, lanes 1, 2). Furthermore, no complexes were observed in the absence of TFOs, indicating that the protein could not bind directly to the target DNA probe. Thus the appearance of the complex requires an interaction between the hairpin-TFO and both the target DNA and the transcription factors. The specificity of the interaction was further confirmed by competition experiments: an excess of unlabelled SRE resulted in a decrease in the intensity of the corresponding band (Fig. 6 B, lanes 1-3). In addition, after binding to the target DNA, the SRE-TFO retains its ability to form a ternary complex with SRF and ELK-1 proteins, as demonstrated by the `super-super shift' observed in the presence of ELK-1 (Fig. 7 ).
Current models for transcription activation (27 ,28 ) suggest that the recruitment of transcription factors to a gene promoter region plays a pivotal role in gene regulation. In addition, double hybrid experiments demonstrate that transactivation domains are independent of the DNA binding domains and seem to be functional whatever the way in which they are recruited to the promoter region. We suggest that hairpin-TFOs able to recruit transcription factors to the target DNA may prove useful in the design of new tools for artificial regulation of gene expression.
ACKNOWLEDGEMENTS
We thank Dr E. Lescot for oligonucleotide synthesis. We are very thankful to Drs D.Trouche and A.Debin for fruitful discussions.This work was supported by a SIDACTION research fellowship to F.S.
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