SCREENING ASSAY

Abstract

Disclosed are methods, kits and cells for screening for test compounds that are capable of inhibiting DNA-binding activity of a DNA-binding protein. The disclosed methods, kits and cells may include a reporter expression cassette that encodes a reporter expression product, wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the reporter expression product. Also disclosed are methods for producing a helix-constrained peptide that may be used in the screening methods disclosed herein. The methods, kits and cells find application, for example, in the identification of antagonists that may be useful in the treatment of cancers involving the DNA-binding protein.

Claims

1. A method for screening for an antagonist of a DNA-binding protein, the method comprising: i) providing a cell, wherein the cell comprises a test compound, a DNA-binding protein, and a reporter expression cassette that encodes a reporter expression product, wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the reporter expression product; and ii) determining expression of the reporter expression product in the presence of the test compound; wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting DNA-binding activity of the DNA-binding protein, and wherein some or all of the binding site(s) are located in the transcribed sequence of the reporter expression cassette.

2. The method of claim 1, wherein the reporter expression product is a reporter protein, optionally wherein the reporter protein is a cell survival protein, a cell reproduction protein a fluorescent protein, a bioluminescent protein, a protease, an enzyme that acts on a substrate to produce a colorimetric signal, a protein kinase, a transcriptional activator, or a regulatory protein such as ubiquitin.

3. The method of claim 2, wherein the reporter protein is a cell survival protein, optionally wherein the cell survival protein is an enzyme involved in synthesising compounds that are required for cell survival, or a protein that is able to inhibit action of a toxic agent.

4. The method of claim 3, wherein the cell survival protein is an exogenous cell survival protein that is able to compensate for a deficiency in an endogenous cell survival protein; and wherein the method is performed under selection conditions such that survival of the cell is dependent upon activity of the exogenous cell survival protein.

5. The method of claim 3, wherein the cell survival protein is dihydrofolate reductase (DHFR), optionally wherein the DHFR has an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1.

6. The method of claim 1, wherein the reporter expression cassette comprises between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 15, between 10 and 20, between 10 and 18 or between 12 and 16 binding sites.

7. The method of claim 2, wherein some or all of the binding site(s) are located in the protein coding sequence of the reporter expression cassette.

8. The method of claim 1, wherein the DNA-binding protein is a transcription factor or a DNA-binding fragment thereof, optionally wherein the DNA-binding protein is a eukaryotic transcription factor or a DNA-binding fragment thereof.

9. The method of claim 1, wherein the DNA-binding protein is a basic leucine zipper (bZIP) transcription factor, a basic helix-loop helix (bHLH) transcription factor, a bHLH leucine zipper (bHLH-ZIP) transcription factor, or a DNA-binding fragment thereof, and optionally wherein: a) the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6); b) the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8); c) the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9); d) the at least one binding site is a cAMP response element (CRE) having the nucleotide sequence TGACGTCA (SEQ ID NO: 10); e) the at least one binding site is a Maf recognition element (MARE) having the nucleotide sequence TGCTGA.sup.G/.sub.CTCAGCA (SEQ ID NO: 32) or TGCTGA.sup.GC/.sub.CGTCAGCA (SEQ ID NO: 33); or f) the at least one binding site is a PAP/CREB-2/PAR binding site having the nucleotide sequence TTACGTAA (SEQ ID NO: 34).

10. The method of claim 1, wherein the cell is a bacterial cell, such as an Escherichia coli cell.

11. The method of claim 1, wherein the cell is a eukaryotic cell, e.g. a mammalian cell, optionally a human cell.

12. (canceled)

13. The method of claim 11, wherein a mammalian cell was isolated from a human patient and wherein the DNA-binding protein is naturally produced by the cell, and optionally wherein the DNA-binding protein is suspected of being or known to be dysregulated in the cell.

14. The method of claim 1, wherein the method comprises administering a DNA-binding protein expression cassette that encodes the DNA-binding protein in order to provide the cell comprising the DNA-binding protein.

15. The method of claim 1, wherein the test compound is a peptidic test compound or a small molecule test compound.

16. (canceled)

17. The method of claim 15, wherein the test compound is a peptidic test compound expressed intracellularly from a test compound expression cassette, optionally wherein the method comprises providing the test compound expression cassette to the cell.

18. The method of claim 15, wherein the method comprises administering a cross-linking agent into the cell in order to introduce a cross-link between two amino acid residues in an alpha helix of the peptidic test compound to produce a helix-constrained peptidic compound.

19. The method of claim 15, wherein the method comprises administering the test compound extracellularly in order to provide the cell comprising the test compound, optionally wherein an increase in expression of the reporter expression product indicates that the test compound is capable of entering the cell as well as being capable of inhibiting DNA-binding activity of the DNA-binding protein.

20. The method of claim 19, wherein the test compound is a peptidic test compound, wherein the peptidic test compound comprises a helix-constrained peptide, and wherein the helix-constrained peptide comprises a cross-link between two amino acid residues.

21. (canceled)

22. A cell-free method for screening for an antagonist of a DNA-binding protein, the method comprising: i) contacting a test compound with a DNA-binding protein and a reporter expression cassette that encodes a reporter expression product, wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the reporter expression product; and ii) determining expression of the reporter expression product; wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting DNA-binding activity of the DNA-binding protein, wherein the method is carried out outside a cell in an in vitro system that comprises the components required for expression of the reporter expression product, and wherein some or all of the binding site(s) are located in the transcribed sequence of the reporter expression cassette.

23. (canceled)

24. A kit comprising: i) a reporter expression cassette that encodes a reporter expression product; and ii) a DNA-binding protein expression cassette that encodes a DNA-binding protein, wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the expression product, and wherein some or all of the binding site(s) are located in the transcribed sequence of the reporter expression cassette.

25. (canceled)

Description

SUMMARY OF THE FIGURES

[0216] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0217] FIG. 1. Transcription-Block Survival (TBS) Assay to derive functionally active AP-1 inhibitors. Schematic illustrating the principles of the TBS assay utilising mDHFR as a reporter expression cassette and TPA response elements (TREs) as DNA-binding sites. A) An mDHFR gene was generated that contains fifteen TREs introduced into its transcribed region, operably linked to a promoter (e.g. the lac operon) . . . B) Basic-cJun can form DNA-bound homodimers that bind to the TREs and prevent mDHFR transcription, inhibiting colony formation under selective conditions where mDHFR expression is necessary for cell survival. C) Peptides that bind to the coiled-coil region of basic-cJun (e.g. cFos) but do not dissociate the DNA-bound complex are not “functional antagonists” and will not rescue transcription of the mDHFR gene and therefore will not rescue colony formation under selective conditions. D) Expression of a functionally active inhibitor results in basic-cJun dissociating from TRE sites on the mDHFR gene leading to the restoration of mDHFR transcription-translation and colony formation.

[0218] FIG. 2. Luciferase reporter assay.

[0219] AP-1 driven Luciferase gene reporter assay with no transfection (control), transfected dummy vector (dummy-helix), acidic c-Jun, acidic c-Fos and library-derived peptide (acidic-FosW) treated. The acidic AP-1 proteins reduce the AP-1 driven luciferase activity where the peptide inhibitor from FIG. 1D has a dramatic reduction in the signal. The asterisk (*) indicates the results obtained when the peptide inhibitor from FIG. 1D was used. This data suggests that peptides which can dissociate DNA-bound AP-1 proteins in the bacterial TBS assay results in a strong phenotypic effect in eukaryotic cells.

[0220] FIG. 3. Quantification of results from TBS assay using TRE DHFR.

[0221] This figure provides quantification of colony formation in a TBS assay utilising the DHFR gene engineered to contain 15 TRE binding sites (TRE mDHFR) as a reporter and AP-1 as a DNA-binding protein. Bacterial cells transfected with Bacterial cells transfected with TRE mDHFR, the leucine zipper part of cJun (cJun LZ) and the leucine zipper part of cFos (cFos LZ) resulted in a large number (>300) of surviving bacterial colonies. Cells transfected with TRE mDHFR, cJun containing the basic DNA-binding domain (cJun bZIP) and cFos LZ resulted in a very low number of colonies (<20). This demonstrates that the basic DNA-binding domain of cJun is able to bind to the TRE sites in TRE mDHFR and inhibit expression of the DHFR protein. Cells transfected with TRE mDHFR, cJun bZIP and a peptide (Acidic FosW) that is able to dissociate the cJun bZIP protein from the TRE sites in TRE mDHFR results in substantial increase in bacterial colonies (>200).

EXAMPLES

Example 1—Development of a Generalised Approach to Derive Functionally Active Peptide Inhibitors of Transcription Factor Activity

[0222] Many rational design approaches, randomised screening approaches, and selection systems result in the successful identification of compounds capable of binding to given protein targets. However, what is much more difficult to ensure, is that binding to said target will result in ablating target protein function. There are many instances where formation of a protein-protein interaction (PPI) has not ensured loss of function. To address this major bottleneck in antagonist screening and design, and to accelerate the design of functionally active antagonists, we have taken inspiration from the transcription factor DNA-binding system and reversed their role in transcription.

Introducing DNA-Binding Sites into the DHFR Gene

[0223] It can be difficult to predict whether a compound that is derived to bind to given protein target will antagonise its function. To tackle this we have taken the gene corresponding to the essential enzyme, dihydrofolate reductase (DHFR), and introduced 15 TPA response elements (TREs) into the gene. This has been achieved using a combination of both silent and conserved mutations, such that the activity of the enzyme is preserved.

[0224] All changes have been made in solvent exposed regions of the molecule to minimise the structural perturbations, with several proposed changes removed via close inspection of the accessible surface area (ASA) within the pdb file (PDBid=2FZJ (Cody et al. (2006)). This was done by inputting the pdb file into the ASA calculator at http://cib.cf.ocha.ac.jp/bitool/ASA/. A cut-off value of 20 was used—residues that had an ASA value lower than this were considered to be buried and not modified; residues that had an ASA value greater than this are considered exposed.

[0225] No changes have been made in residues deemed important for catalysis or NADPH binding. Methods of identifying the solvent exposed regions of the reporter protein are known. For example, it is possible to take the coordinate files for the reporter protein, e.g. a protein databank (PDB) file and use a program that calculates the accessible surface area (ASA) which informs the user how exposed/buried residues are within a structure. An exemplary ASA program can be found at http://cib.cf.ocha.ac.jp/bitool/ASA/. An exemplary cut-off value of 20 can be used, such that residues that are lower than this are considered to be buried and greater than this are considered exposed. In this way, the locations of solvent exposed residues can be identified and codons modified accordingly.

[0226] Shown below is the sequence of the mDHFR gene (SEQ ID NO: 11) with DNA mutations bold and underlined and changes within the translated protein sequence (SEQ ID NO: 31) shown. Shown in bold italics are the NheI and HindIII sites used for subcloning the gene into the pES300d vector. Mutations were made by inspection of the desired consensus sequences (TGACTCA or TGAGTCA) and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. For example, either of the two desired sequences above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00006 i) Frame 1: TGA CTC Axx 1 = stop 2 = LV 3 = I/M/T/N/K/S/R ii) Frame 2: xTG ACT CAx 1 = LMV 2 = TS 3 = HQ Iii) Frame 3: xxT GAC TCA 1 = FSYCLPHRITNVADG 2 = DE 3 = S

[0227] This gives rise to a number of codons to be identified for silent mutation and consequently a number of options for conserved or semi-conserved mutations that would permit the introduction of TREs into the mDHFR gene: [0228] i) No options [0229] ii) LSH, LSQ, LTH, LTQ, MSH, MSQ, MTH, MTQ, VSH, VSQ, VTH, VTQ [0230] iii) ADS, AES, CDS, CES, DDS, DES, FDS, FES, GDS, GES, HDS, HES, IDS, IES, LDS, LES, NDS, NES, PDS, PES, RDS, RES, SDS, SES, TDS, TES, VDS, VES, YDS, YES

[0231] From this we were able to implement the following changes into the mDHFR gene to give minimum perturbation to the overall sequence. Where possible mutations were silent or conservative. All mutations were also placed at solvent exposed sites and away from the catalytic centre (E116) and away from residues required for NADPH/substrate binding (A10/R71). This resulted in the introduction of 15 TREs into the mDHFR gene:

TABLE-US-00007  1. VSQ     (silent) = GTG AGT CAG  2. NEF.fwdarw.NES (F32S)   = AAT GAG TCA  3. MTT.fwdarw.MTQ (T40Q)   = ATG ACT CAG  4. TSS.fwdarw.TDS (S42D)   = ACT GAC TCA  5. VEG.fwdarw.VES (G46S)   = GTT GAG TCA  6. PEK.fwdarw.PES (K64S)   = CCT GAG TCA  7. ISR.fwdarw.LSQ (R78Q)   = CTG AGT CAA  8. IEQ.fwdarw.IES (Q103S)  = ATT GAG TCA  9. VDM.fwdarw.VDS (M112S)  = GTT GAC TCA 10. MNQ.fwdarw.MTQ (N127T)  = ATG ACT CAA 11. VTR.fwdarw.VTQ (R138Q)  = GTG ACT CAG 12. FES     (silent) = TTT GAG TCA 13. IDL.fwdarw.IDS (L154S)  = ATT GAC TCA 14. PEY.fwdarw.PES (Y163S)  = CCT GAG TCA 15. LSE.fwdarw.LSQ (E169Q)  = CTG AGT CAG

[0232] This design process gave rise to the following sequence:

TABLE-US-00008 A   S   V   R   P   L   N   C   I   V   A   V   S   Q   N   M   G  GTT CGA CCA TTG AAC TGC ATC GTC GCC   AAT ATG GGG I   G   K   N   G   D   L   P   W   P   P   L   R   N   E   S   K ATT GGC AAG AAC GGA GAC CTA CCC TGG CCT CCG CTC AGG   AAG Y   F   Q   R   M   T   Q   T   D   S   V   E   S   K   Q   N   L TAC TTC CAA AGA     AAA CAG AAT CTG V   I   M   G   R   K   T   W   F   S   I   P   E   S   N   R   P GTG ATT ATG GGT AGG AAA ACC TGG TTC TCC ATT   AAT CGA CCT L   K   D   R   I   N   I   V   L   S   Q   E   L   K   E   P   P TTA AAG GAC AGA ATT AAT ATA GTT   GAA CTC AAA GAA CCA CCA R   G   A   H   F   L   A   K   S   L   D   D   A   L   R   L   I CGA GGA GCT CAT TTT CTT GCC AAA AGT TTG GAT GAT GCC TTA AGA CTT  E   S   P   E   L   A   S   K   V   D   S   V   W   I   V   G   G  CCG GAA TTG GCG AGC AAA   GTT TGG ATC GTC GGA GGC S   S   V   Y   Q   E   A   M   T   Q   P   G   H   L   R   L   F AGT TCT GTT TAC CAG GAA GCC   CCA GGC CAC CTT AGA CTC TTT V   T   Q   I   M   Q   E   F   E   S   D   T   F   F   P   E   I  ATC ATG CAG GAA   GAC ACG TTT TTC CCA GAA  D   S   G   K   Y   K   L   L   P   E   S   P   G   V   L   S   Q  GGG AAA TAT AAA CTT CTC   CCA GGC GTC  V   Q   E   E   K   G   I   K   Y   K   F   E   V   Y   E   K   K GTC CAG GAG GAA AAA GGC ATC AAG TAT AAG TTT GAA GTC TAC GAG AAG AAA D   *   A   * GAC T AA

[0233] We have introduced 15 TREs via silent and conserved mutations into solvent exposed positions within the gene coding for the essential enzyme dihydofolate reductase (DHFR; FIG. 1A). We demonstrate that these changes result in a functional enzyme. Under selective conditions introduction of AP-1 prevents DHFR expression by binding to TRE sites within the gene, blocking transcription, and preventing colony formation under selective conditions. In contrast, attenuated versions of AP-1 that lack a basic DNA-binding region fail to prevent colony formation. Significantly, introducing peptides that can both bind AP-1 and antagonise function can be used to reverse this effect, leading to the formation of bacterial colonies. This represents a powerful approach for the selection of functionally active peptides that can bind to AP-1 components and abolish their function, leading to rapid library screening and identification of therapeutically interesting sequences.

Testing Functionality of DHFR Protein

[0234] The selection system is based on the fact that bacterial DHFR can be specifically inhibited using trimethoprim, rendering cells dependent upon murine DHFR (mDHFR) activity for their survival. The first test of the system was to establish that mDHFR protein refolds and is active. SDS-PAGE analysis was used to confirm that the protein is highly expressed upon addition of IPTG. Further evidence that the protein is expressed, folds, and is functionally active was verified by transformation of bacterial cells and confirmed by the presence of multiple colonies in minimal media containing trimethoprim (data not shown).

Establishing the TBS Assay

[0235] It was next necessary to establish that introduction of an AP-1 component (in this case basic-cJun) would result in binding to the 15 TRE's introduced within the mDHFR gene and therefore failure of the gene to be transcribed.

[0236] Three plasmids were used for the TBS Assay. These are i) p300-mDHFR (Cm; SEQ ID NO: 42) to express the 15×consensus sequence containing mDHFR, which is under control of the lac-operon; ii) p230d-basic-cJun (Amp; SEQ ID NO: 43) which is also under control of the lac-operon; iii) pREP4 (Kan; SEQ ID NO: 44) to express the lac repressor.

[0237] Cells were grown under non-selective conditions (i.e. LB/LB agar) containing Cm/Amp/Kan up until the time of the Assay. During TBS Selection Cells are grown in M9 minimal media (Agar or Broth) in the presence of Cm/Amp/Kan, as well as Tmp (to inhibit the bacterial copies of DHFR) and IPTG (to induce expression of mDHFR and bZIP proteins). During Assay selection, media-lacking ITPG is used to serve as a negative control to ensure that cell survival is exclusively driven by the loss on interaction between bZIP target protein and the consensus sequences located within the mDHFR gene. M9 agar plates with Tmp in the absence of IPTG do not form colonies (data not shown). Using IPTG to express mDHFR comprising the 15 TREs confers survival in M9 minimal medium containing Tmp to inhibit bacterial DHFR (data not shown).

[0238] As expected, overexpression of basic-cJun on the second plasmid resulted in a complete loss of bacterial colonies in minimal media (data not shown). Without wishing to be bound by theory, it is believed that this works because AP-1 binds to the multiple TREs found within the mDHFR gene and therefore works in the opposite way to its natural function. Rather it works by blocking transcription and preventing the machinery from moving along the DNA (FIG. 1B). As a control a version of cJun containing the leucine zipper, but lacking in the DNA-binding basic region (SEQ ID NO: 45), was tested. As expected this version did not prevent bacterial colony formation in minimal media (data not shown).

[0239] Lastly, we added compounds known to bind to the cJun coiled coil to establish if they are also functionally active peptides—i.e. capable of sequestering basic-cJun as a non-functional heterodimer and therefore prevent DNA-binding and rescue transcription. The c-Jun homodimer/DNA complex forms a very tight interaction, therefore a bonafide c-Jun inhibitor is required not only to bind to the c-Jun homodimer in solution but also to disassociate the DNA bound complex to prevent AP-1 transcriptional activity. Peptides which bind to the coiled coil domain (the dimerization motif of c-Jun homodimer) do not necessarily dissociate the DNA-bound complex (FIG. 1C). Those that do not will therefore not rescue bacterial growth of TMP/IPTG treated cells (data not shown). However, peptides which lead to dissociation of the c-Jun homodimer from the DNA will enable the transcription of the mDHFR gene and rescue TMP/IPTG bacterial cells and grow. This is demonstrated by using the FosW peptide that is known to bind to cJun with high affinity (K.sub.D=90 nM; Mason et al. 2006). Expression of the FosW peptide resulted in the formation of colonies (data not shown), demonstrating that the FosW peptide was able to rescue transcription of the mDHFR gene and therefore is a functionally active antagonist (FIG. 1D).

[0240] This assay allows for the selection of specific peptides capable not only of binding to the coiled coil region predicted to be required for driving AP-1 dimerization, but importantly that function by shutting down the DNA-binding activity of the protein. Thus the peptides must be functionally active to be selected and can be readily isolated from binders that are not of use by using cell survival as a marker for success.

[0241] In Vitro Activity in Mammalian Cells

[0242] Wild-type AP-1 proteins have a long stretch of basic amino acids at the N-terminal of the protein which enables DNA binding. Acidic versions of AP-1 proteins can be generated with a long stretch of negatively charged amino acids (in place of the positive) which can serve as a DNA mimic. Acidic AP-1 peptide variants can therefore form heterodimers with wild type AP-1 proteins driven by both the dimerization motif and the negative/positive charge interaction, resulting in their sequestration from the DNA.

[0243] In order to test whether the peptides identified as being functional antagonists in the TBS assay are also likely to have the same activity in eukaryotic cells, a AP-1 driven luciferase gene reporter assay was carried out. This assay was carried out using no transfection (control), transfected dummy vector, acidic c-Jun, acidic c-Fos and library-derived peptide treated (FosW). The results are shown in FIG. 2. The acidic AP-1 proteins reduce the AP-1 driven luciferase activity where the peptide inhibitor from Example 1 (FosW) has a dramatic reduction in the signal. This data suggests that peptides which can dissociate DNA-bound AP-1 proteins in the bacterial TBS assay results in a strong phenotypic effect in eukaryotic cells.

Discussion

[0244] There are many assays to derive high affinity PPI's using library based approaches, but very few that guarantee loss of function within the target protein. Using AP-1 as our exemplar system, we have developed an assay to derive functionally active sequences capable of shutting down transcription factor activity. We have shown using the essential enzyme mDHFR that i) enzymatic activity is preserved upon introduction of 15 TREs into the gene under selective conditions activity becomes lost when basic-cJun is introduced the basic region within basic-cJun is an absolute requirement for this loss of mDHFR activity, and iv) peptides derived to bind to cJun can be separated into those that result in loss of AP-1 DNA binding activity (and therefore function) and those that do not. This assay therefore uses cell survival as a marker to allow rapid screening of peptide libraries and consequently the derivation of functionally active antagonists of transcription factor function.

Example 2—Library Creation

TBS Assay—Genetically Encoded Library Construction:

[0245] As described above, three plasmids were used for the TBS Assay. These are i) p300-mDHFR (Cm) to express the 15×consensus sequence containing mDHFR, which is under control of the lac-operon; ii) p230d-basic-cJun (Amp) which is also under control of the lac-operon; iii) pREP4 (Kan) to express the lac repressor.

[0246] Genetically encoded libraries are created using overlap extension PCR, subcloned into the p410d vector (Tet) and plated out. Each colony then represents a member of the library. We typically collect 2-5× the library size in colony numbers to gain approx. 95% total coverage. The maximum library size screenable using the approach is 10.sup.6. Once the library is complete colonies are pooled and mini-preparation of DNA performed. Finally the plasmid library is transformed into cells containing p300/p230/pREP4. During single step selection cells are plated onto LB agar (to demonstrate successful transformation), M9 agar lacking IPTG (as a negative control where no bZIP or mDHFR is expressed) and finally onto M9 agar containing Cm/Amp/Kan/Tet/Tmp/IPTG to drive production of bZIP/mDHFR/Library such that cell viability is only restored if a given library member can prevent the bZIP target from interacting with the cognate sequences within the mDHFR gene. Surviving colonies can next be pooled, grown and serially diluted in liquid cultures under selective conditions (M9 minimal medium with 1 μg/ml trimethoprim). Fastest growth, and hence the highest affinity interacting partners dominated the pool. Library pools as well as colonies from individual clones were sequenced to verify the arrival at one sequence. To assess library quality we sequence pools and single clones to find approximately equal distributions of varied amino acids. Pooled colonies exceeded the library size 5-10 fold. Using more recent ligation methods (Topo/Gibson/Gateway) it may be possible to move into TBS directly from ligation, giving the significant advantage of being able to screen larger libraries (possibly up to 10.sup.10 or 10.sup.11), however processes will need to be put into place (e.g. next gen sequencing) to ensure that library size and quality is fully represented prior to transformation into the TBS assay.

[0247] Another possibility is to use pET24a as an alternative to the pREP4 vector used to express the lac repressor. This would allow the expression of both the lac repressor and library member/antagonist off a single plasmid, i.e. avoiding the need for another antibiotic.

TBS Assay—Extracellular Compound Addition:

[0248] For extracellular libraries, cells containing p300-mDHFR plasmid are grown in the presence of p230d-bZIP and pREP4 plasmids under non-selective conditions (LB agar/media). Once ready for assay overnights can then be placed into each well of microtitre plates (96, 384, 1536) at A.sub.600=0.05 and compound libraries screened by direct addition to each well. Plates are incubated at 37° C. and with shaking and successful compounds identified by monitoring of the absorbance signal at 600 nm. This extracellular compound addition method has the advantage of allowing the user to move away from standard peptide libraries (e.g. one can profile for helix constrained peptides, peptidomimetics, non-natural amino acids etc, or even small molecule libraries) and importantly allows the user to profile for cell penetrance concomitantly with functional antagonism of the bZIP target. Once again, all proteins are under control of a lac promoter, and expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG).

Selection of Winner Peptides

[0249] Briefly, during TBS only peptides (intracellular) or compounds (extracellular) that can interact with the bZIP target and dissociate it from the consensus sequences within the mDHFR gene will result in colony formation/cell growth on M9 minimal medium plates/media with 1 μg/mItrimethoprim to inhibit bacterial DHFR.

Example 3—Further TBS Assay Experiment

[0250] A further experiment was carried out to establish the TBS assay as a suitable assay for identifying functional antagonists.

[0251] This experiment used similar plasmids to those set out in Example 1, with the exception that pET24a was used to express both the lac repressor and test compound using a single plasmid. Specifically, three plasmids were used: i) p300d expressing the mDHFR gene modified to include 15 TREs (TRE mDHFR, generated as described in Example 1); ii) p230d expressing either the leucine zipper part of cJun (cJun LZ) or cJun with the basic region and leucine zipper (cJun bZip); iii) pET24a expressing the lac repressor and either the leucine zipper part of cFos (cFos LZ) or the acidic FosW peptide (acidic FosW).

[0252] Plates were generated as set out in the following table:

TABLE-US-00009 Plate p300d p230d pET24a 1 TRE mDHFR cJun LZ cFos LZ 2 TRE mDHFR cJun bZIP cFos LZ 3 TRE mDHFR cJun bZIP Acidic FosW

[0253] The number of colonies produced in each plate were counted and this quantification is illustrated in FIG. 3. The cJun LZ expressed in control plate 1 is unable to bind and inhibit expression of TRE DHFR, therefore resulting in a large number (>300) bacterial colonies. cJun with the basic DNA-binding domain (cJun bZip) is able to bind and inhibit expression of DHFR and therefore when expressed in plate 2 resulted in a substantial reduction in the number of bacterial colonies (<20) compared to plate 1. Co-expression with a peptide that binds to the coiled-coil region of cJun (cFos LZ) does not dissociate the DNA-bound complex and therefore is not able to rescue expression of the DHFR protein in the TBS assay. However, co-expression with a peptide (acidic FosW) that is able to dissociate the DNA-bound AP-1 transcription factor from its binding sites in the TRE DHFR gene results in a substantial increase in the number of colonies compared to plate 2

[0254] Overall, these results provide further evidence that the TBS assay can be used to identify antagonists that are able to inhibit DNA-binding activity of a transcription factor such as AP-1.

Example 4—Introducing the CRE, CCAAT and Ebox Binding Sites into the Dhfr Gene

[0255] Constructs were designed whereby the CRE, CCAAT and Ebox binding sites, respectively, were inserted into the DHFR gene. These constructs can be tested in the TBS assay as described in Examples 1 and 3.

Inserting the CRE Binding Site into the DHFR Gene

[0256] CRE is usually defined as TGACGTCA (SEQ ID NO: 10). Mutations in the DHFR gene can be made by inspection of the desired consensus sequence and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. The CRE is 8 bp as so can span four codons. For example, the sequence defined above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00010 A: Frame 1: TGA CGT CAx 1 = stop 2:R 3:H/Q B: Frame 2: xTG ACG TCA 1:L4V 2:T 3:S C: Frame 3: xxT GAC GTC Axx 1:FLIVSPTAYHNDCRG 2:D 3:V 4:IMTNKSR

[0257] From this it is possible to implement changes into the mDHFR gene to give minimal perturbation to the overall sequence. Mutations should be placed at solvent exposed sites and away from the catalytic centre and where possible mutations should be silent or conservative.

[0258] An example of an mDHFR gene that is modified to contain CRE binding sites is shown as follows:

TABLE-US-00011 ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGG GATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGT TCAAGTACTTCCAAAGAATGACGTCAACCTCTTCAGTGGAAGGTAAA CAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGA GAAGAATCGACCTTTAAAGGACAGAATTAATATAGTGACGTCAAGAG AACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTG GATGATGCCTTAAGACTTATTGAACAACCGGAATTGACGTCAAAAGT AGACATGGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCA TGAATCAACCAGGCCACCTTAGACTCTTTGTGACGTCAATCATGCAG GAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATA TAAACTTCTCCCAGAATACCCAGGCGTGACGTCAGAGGTCCAGGAGG AAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAA GCTTAA

[0259] Nucleotide residues in bold underline indicate consensus CRE binding sites.

[0260] Nucleotide residues in lowercase and italics correspond to the restriction enzyme sites for AscI and HindIII at the 5′ and 3′ ends of the sequence, respectively.

[0261] The resulting amino acid sequence is shows as follows:

TABLE-US-00012 M V R P L N C I V A V S Q N M G I G K N G D L P W P P L R N E F K Y F Q R M T  T S S V E G K Q N L V I M G R K T W F S I P E K N R P L K D R I N I V   S R E L K E P P R G A H F L A K S L D D A L R L I E Q P E L   S K V D M V W I V G G S S V Y Q E A M N Q P G H L R L F V T   I M Q E F E S D T F F P E I D L G K Y K L L P E Y P G V   S E V Q E E K G I K Y K F E V Y E K K D

[0262] Amino acid residues in italics are solvent exposed residues. The other residues are classed as buried residues.

[0263] Amino acid residues in bold underline are residues that have been altered as a result of the insertion of CRE into the nucleotide sequence.

[0264] A summary of the amino acid changes is provided as follows:

TABLE-US-00013 1. MTT.fwdarw.MTS (T40S)  = ATG ACG TCA ASA at posn = 36 2. VLS.fwdarw.VTS (L76T)  = GTG ACG TCA ASA at posn = 21 3. LAS.fwdarw.LTS (A107T) = TTG ACG TCA ASA at posn = 37 4. VTR.fwdarw.VTS (R138S) = GTG ACG TCA ASA at posn = 57 5. VIS.fwdarw.VTS (L167T) = GTG ACG TCA ASA at posn = 99
Inserting the CCAAT Binding Site into the DHFR Gene

[0265] CCAAT is usually defined as ATTGCGCAAT (SEQ ID NO: 9). Mutations in the DHFR gene can be made by inspection of the desired consensus sequence and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. The CCAAT is 10 bp and so can span five codons. For example, the sequence defined above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00014 A:Frame 1: ATT GCG CAA Txx 1:I 2:A 3:Q 4:FLSYCW* B:Frame 2: KAT TGC GCA ATx 1:YHND 2:C 3:A 4:IM C:Frame 3: xxA TTG CGC AAT 1:LIVSPTAQKERG* 2:L 3:R 4:N

[0266] From this it is possible to implement changes into the mDHFR gene to give minimal perturbation to the overall sequence. Mutations should be placed at solvent exposed sites and away from the catalytic centre and where possible mutations should silent or conservative.

[0267] An example of an mDHFR gene that is modified to contain CCAAT is shown as follows:

TABLE-US-00015 ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGG ATTGGCAAGAACGGAGACCTACCCTGGCCTCCATTGCGCAATGAGTTC AAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAG AATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAG AATCGACCATTGCGCAATAGAATTAATATAGTTCTCAGTAGAGAATTG CGCAATCCACCACGAGGAGCTCATTTTATTGCGCAATCCTTGGATGAT GCATTGCGCAATATTGAACAACCGGAATTGGCGAGCAAAGTAGACATG GTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAA CCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTGAA AGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTC CCAGAATACCCAGGCGTCCTCTCTGAATTGCGCAATGAAAAAGGCATC AAGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAAGCTTAA

[0268] Nucleotide residues in bold underline indicate CCAAT consensus binding sites.

[0269] Nucleotide residues in lowercase and italics correspond to the restriction enzyme sites for AscI and HindIII at the 5′ and 3′ ends of the sequence, respectively.

[0270] The resulting amino acid sequence is shows as follows:

TABLE-US-00016 M V R P L N C I V A V S Q N M G I G K N G D L P W P P L R N E F K Y F Q R M T T T S S V E G K Q N L V I M G R K T W F S I P E K N R P L R N R I N I V L S R E L R N P P R G A H F I A Q S L D D A L R N I E Q P E L A S K V D M V W I V G G S S V Y Q E A M N Q P G H L R L F V T R I M Q E F E S D T F F P E I D L G K Y K L L P E Y P G V L S E V Q E E K G I K Y K F E V Y E K K D

[0271] Amino acid residues in italics are solvent exposed residues. The other residues are classed as buried residues.

[0272] Amino acid residues in bold underline are residues that have been altered as a result of the insertion of CCAAT into the nucleotide sequence.

[0273] A summary of the amino acid changes is provided as follows:

TABLE-US-00017 1. PL        RN (silent) 2. PLKD.fwdarw.PLRN (K69R, D70N) = ASA at posns = 90, 97 3. ELKE.fwdarw.ELRN (K81R, E82N) = ASA at posns = 175, 131 4. LAKS.fwdarw.IAQS (L90I, K92Q) = ASA at posns = 47, 139 5. ALRL.fwdarw.ALRN (L100N)      = ASA at posn  = 46

[0274] A further CCAAT site could be inserted to make the following mutation:

TABLE-US-00018 6. EVQE.fwdarw.ELRN (V170L, Q171R, E172N)
Inserting the Ebox Binding Site into the DHFR Gene

[0275] In the context of c-Myc, Ebox is usually defined as CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8). Mutations in the DHFR gene can be made by inspection of the desired consensus sequence and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. For example, the sequences defined above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00019 A:Frame 1: CAC GTG xxx = 1:H 2:V 3:Anything B:Frame 2: xCA CGT Gxx = 1:S/P/T/A 2:R 3:V/A/D/E/G C:Frame 3: xxC ACG TGx = 1:FLIVSPTAYHNDCRSG 2:T 3:C/W/* A:Frame 1: CAC ATG xxx = 1:H 2:M 3:anything B:Frame 2: xCA CAT Gxx = 1:S/P/T/A 2:H 3:V/A/D/E/G C:Frame 3: xxC ACA TGx = 1:FLIVSPTAYHNDCRSG 2:T 3:C/W/*

[0276] From this it is possible to implement changes into the mDHFR gene to give minimal perturbation to the overall sequence. Mutations should be placed at solvent exposed sites and away from the catalytic centre and where possible mutations should silent or conservative.

[0277] An example of an mDHFR gene that is modified to contain Eboxes is shown as follows:

TABLE-US-00020 ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGG ATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTC AAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAG AATCTGGTGATTATGGGTAGGCGCACGTGGTTCTCCATTCCTGAGAAG AATCGACCTTTAAAGGACAGAATTAATATAGTTCTCTCACGTGAACTC AAAGAACCACCACGTGGAGCTCACGTGCTTGCCAAATCACTGGATGAT GCATTAAGACTTATTGAACAACCGGAATTGGCGTCACGTGTAGACATG GTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAA CCAGGCCACGTGAGACTCTTTGTGACACGTGTCATGCAGGAATTTGAA AGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTC CCAGAATACCCAGGCGTCCTCTCACGTGTCCAGGAGGAAAAAGGCATC AAGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAAGCTTAA

[0278] Nucleotide residues in bold underline indicate Ebox consensus binding sites.

[0279] Nucleotide residues in lowercase and italics correspond to the restriction enzyme sites for AscI and HindIII at the 5′ and 3′ ends of the sequence, respectively.

[0280] The resulting amino acid sequence is shows as follows:

TABLE-US-00021 M V R P L N C I V A V S Q N M G I G K N G D L P W P P L R N E F K Y F Q R M T T T S S V E G K Q N L V I M G R   T W F S I P E K N R P L K D R I N I V L S R E L K E P P R G A H   L A K S L D D A L R L I E Q P E L A S   V D M V W I V G G S S V Y Q E A M N Q P G H V R L F V T R V M Q E F E S D T F F P E I D L G K Y K L L P E Y P G V L S   V Q E E K G I K Y K F E V Y E K K D

[0281] Amino acid residues in italics are solvent exposed residues. The other residues are classed as buried residues.

[0282] Amino acid residues in bold underline are residues that have been altered as a result of the insertion of Ebox into the nucleotide sequence.

[0283] A summary of the amino acid changes is provided as follows:

TABLE-US-00022 1. KTW.fwdarw.RTW (K56R)   = CGC ACG TGG ASA at posn = 143 (exposed) 2. SPE     (silent) = TCA CGT GAA ASA at posn = N/A 3. PRG     (silent) = CCA CGT GGA ASA at posn = N/A 4. HFL.fwdarw.HVL (F89V)   = CAC GTG CTT ASA at posn = 71 (exposed) 5. SKV.fwdarw.SRV (K109R)  = ACA CGT GTA ASA at posn = 109 (exposed) 6. HLR.fwdarw.HVR (L132V)  = CAC GTG AGA ASA at posn = 1.6 7. TRI.fwdarw.TRV (I139V)  = ACA CGT GTC ASA at posn = 1.6 8. SEV.fwdarw.SRV (E151R)  = ACA CGT GTC ASA at posn = 141 (exposed)

[0284] Changes 6 and 7 are located at residues that are classed as buried. Accordingly, constructs could be made that contain all 8 Ebox sites, one that is lacking site ‘6’, one that is lacking site ‘7’ and one that is lacking both sites ‘6’ and ‘7’ in order to determine whether the mutation at these ‘buried’ sites affect the function of the resultant DHFR protein.

Example 5—Expanding the TBS Assay for Use with Additional Transcription Factor Targets

[0285] Experiments were carried out to establish the TBS assay for use in identifying functional inhibitors of transcription factors other than AP-1. The following modified DHFR genes were generated:

TABLE-US-00023 Nucleotide Total number Nucleotide mDHFR TBS sequence of of binding sequence of mutant binding site sites mDHFR mutant: Notes TRE mDHFR TGA(C/G)TCA 15 SEQ ID NO: 4 See Example 1 for construction details CCAAT mDHFR ATTGCGCAAT  6 SEQ ID NO: 38 See Example 4 for construction details EBOX mDHFR CAC(G/A)TG  8 SEQ ID NO: 40 See Example 4 for construction details CRE mDHFR TGACGTCA  5 SEQ ID NO: 36 See Example 4 for construction details

[0286] As set out in Example 1, expression of mDHFR TRE was able to restore bacterial colonies in the presence of TMP, indicating that the protein produced by this mDHFR mutant was functional. Similar experiments were carried out to determine if the other mDHFR mutants (mDHFR CCAAT, mDHFR EBOX, mDHFR CRE) were also active. These experiments revealed that expression of the mDHFR mutant in the presence of TMP resulted in an increased number of colonies compared to plates where TMP was present without the mDHFR mutant being expressed (data not shown). These experiments confirm that the DHFR proteins produced by the mDHFR mutants were functional and able to confer survival in the presence of TMP.

[0287] Experiments were also carried out to determine whether the TBS assay could be used to identify functional antagonists of C/EBP alpha and BZLF1. C/EBP alpha is a bZip transcription factor that binds CCAAT sites and upregulation in human cells is associated with colorectal cancer growth, metastasis and indicates poor survival outcome. BZLF1 is a bZip transcription factor that binds TRE and CCAAT sites and is associated with Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, nasopharyngeal carcinoma and lymphomas.

[0288] For C/EBP alpha, p230d plasmids were generated that encoded either C/EBP alpha with the DNA-binding basic region (C/EBPα incl. basic region) or without the basic region (C/EBPα minus basic region). Cells were then plated with p230d expressing either of these C/EBP alpha constructs together with p300d expressing mDHFR CCAAT and an empty pET24a plasmid. The experiment was carried out using 8 μM TMP and in the absence and presence of 1 mM IPTG (to induce expression of the C/EBP alpha and DHFR proteins).

[0289] Cells were first transfected with CCAAT mDHFR, a plasmid encoding C/EBPα incl. basic region, and an empty pET24a plasmid in the presence of 8 μM trimethoprim (TMP) but without IPTG. TMP inhibits bacterial DHFR, resulting in no bacterial colonies (data not shown). Addition of 1 mM IPTG induces transcription of mDHFR and C/EBPα proteins and resulted in a very low number of bacterial colonies (approx. 8; data not shown) being observed. This result suggests that the DNA-binding domain of C/EBPα was able to bind to the CCAAT binding sites and inhibit expression of mDHFR, as expected based on previous results using the TBS assay (see Example 1 and 3). Expression of mDHFR and C/EBPα minus basic region results in a large number (>100) of bacterial colonies. This suggests that the absence of a DNA-binding domain prevents the C/EBPα protein from binding the CCAAT sites in CCAAT mDHFR, permitting expression of the mDHFR protein and allowing bacterial cell survival.

[0290] The experiment revealed that co-expression of mDHFR CCAAT with the C/EBP alpha protein that contains the basic region resulted in substantially fewer colonies compared to the plate containing cells that express mDHFR CCAAT and C/EBP alpha lacking the basic region. This result indicates that the DNA-binding basic region of the C/EBP alpha protein was able to bind the CCAAT binding sites in mDHFR CCAAT and inhibit production of mDHFR, suggesting that these constructs could be used in the TBS assay to identify compounds that are capable of inhibiting DNA-binding activity of the C/EBP alpha protein.

[0291] A similar experiment was carried out to assess whether the constructs described above could be used to identify compounds capable of inhibiting the DNA-binding activity of BZLF1. p230d plasmids were generated that encoded either BZLF1 with the DNA-binding basic region (BZLF1 incl. basic region) or without the basic region (BZLF1 minus basic region). Cells were then plated with p230d expressing either of these BZLF1 constructs together with p300d expressing mDHFR TRE and an empty pET24a plasmid. The experiment was carried out using 8 μM TMP and in the absence and presence of 1 mM IPTG (to induce expression of the BZLF1 and DHFR proteins).

[0292] The results demonstrated that co-expression of mDHFR TRE with the BZLF1 protein that contains the basic region resulted in substantially fewer colonies compared to the plate containing mDHFR TRE and BZLF1 lacking the basic region (data not shown). This result indicates that the DNA-binding basic region of the BZLF1 protein was able to bind the TRE binding sites in mDHFR TRE and inhibit production of mDHFR, suggesting that these constructs could be used in the TBS assay to identify compounds that are capable of inhibiting DNA-binding activity of the BZLF1 protein.

[0293] In summary, these results provide evidence that the TBS assay can be used to identify compounds capable of inhibiting DNA-binding activity of the transcription factors other than AP-1, such as the C/EBP alpha and BZLF1 transcription factors. This further demonstrates that the TBS assay can be utilised as a general method for identifying functional antagonists of DNA-binding proteins.

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[0332] For standard molecular biology techniques, see Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

TABLE-US-00024 Sequence Annex Amino acid sequence of wild-type murine dihydrofolate reductase (SEQ ID NO: 1) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNRPLKD RINIVLSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIM QEFESDTFFPEIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Amino acid sequence of murine dihydrofolate reductase engineered to contain TRE sites (SEQ ID NO: 2 MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNESKYFQRMTQTDSVESKQNLVIMGRKTWFSIPESNRPLKD RINIVLSQELKEPPRGAHFLAKSLDDALRLIESPELASKVDSVWIVGGSSVYQEAMTQPGHLRLFVTQIM QEFESDTFFPEIDSGKYKLLPESPGVLSQVQEEKGIKYKFEVYEKKD Amino acid sequence of wild-type human dihydrofolate reductase (SEQ ID NO: 3) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG RINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Nucleic acid sequence for the protein coding sequence of murine dihydrofolate reductase engineered to contain TRE sites (SEQ ID NO: 4) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGAGTCAGAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCC TCCGCTCAGGAATGAGTCAAAGTACTTCCAAAGAATGACTCAGACTGACTCAGTTGAGTCAAAACAGAATCTGGTGA TTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGTCAAATCGACCTTTAAAGGACAGAATTAATATAGTTCTGAGT CAAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAGTC ACCGGAATTGGCGAGCAAAGTTGACTCAGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGACTCAAC CAGGCCACCTTAGACTCTTTGTGACTCAGATCATGCAGGAATTTGAGTCAGACACGTTTTTCCCAGAAATTGACTCA GGGAAATATAAACTTCTCCCTGAGTCACCAGGCGTCCTGAGTCAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTT TGAAGTCTACGAGAAGAAAGACTAA Nucleic acid sequences of TPA response elements (TRE) TGACTCA (SEQ ID NO: 5) TGAGTCA (SEQ ID NO: 6) Nucleic acid sequences of Ebox response elements CACGTG (SEQ ID NO: 7) CACATG (SEQ ID NO: 8) Nucleic acid sequence of C/EBP protein response element ATTGCGCAAT (SEQ ID NO: 9) Nucleic acid sequence of cAMP response element (CRE) TGACGTCA (SEQ ID NO: 10) Nucleic acid sequences of Maf recognition elements (MAREs) TGCTGA.sup.G/.sub.CTCAGCA (SEQ ID NO: 32) TGCTGA.sup.GC/.sub.CGTCAGCA (SEQ ID NO: 33) Nucleic acid sequence of PAP/CREB-2/PAR binding site TTACGTAA (SEQ ID NO: 34) Nucleic acid sequence of polynucleotide encoding engineered murine dihydrofolate reductase including restriction enzyme sites (SEQ ID NO: 11) GCTAGCGTTCGACCATTGAACTGCATCGTCGCCGTGAGTCAGAATATGGGGATTGGCAAGAACGGAGACCTACCCTG GCCTCCGCTCAGGAATGAGTCAAAGTACTTCCAAAGAATGACTCAGACTGACTCAGTTGAGTCAAAACAGAATCTGG TGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGTCAAATCGACCTTTAAAGGACAGAATTAATATAGTTCTG AGTCAAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGA GTCACCGGAATTGGCGAGCAAAGTTGACTCAGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGACTC AACCAGGCCACCTTAGACTCTTTGTGACTCAGATCATGCAGGAATTTGAGTCAGACACGTTTTTCCCAGAAATTGAC TCAGGGAAATATAAACTTCTCCCTGAGTCACCAGGCGTCCTGAGTCAGGTCCAGGAGGAAAAAGGCATCAAGTATAA GTTTGAAGTCTACGAGAAGAAAGACTAA Nucleic acid sequences of example reading frames Example reading frame 1: TGA CTC Axx (SEQ ID NO: 12) Example reading frame 2: xTG ACT CAx (SEQ ID NO: 13) Example reading frame 3: xxT GAC TCA (SEQ ID NO: 14) Amino acid sequence of example reading frame 3 (SEQ ID NO: 15) FSYCLPHRITNVADG Amino acid sequence of example codon triplets containing TREs GTGAGTCAG (SEQ ID NO: 16) AATGAGTCA (SEQ ID NO: 17) ATGACTCAG (SEQ ID NO: 18) ACTGACTCA (SEQ ID NO: 19) GTTGAGTCA (SEQ ID NO: 20) CCTGAGTCA (SEQ ID NO: 21) CTGAGTCAA (SEQ ID NO: 22) ATTGAGTCA (SEQ ID NO: 23) GTTGACTCA (SEQ ID NO: 24) ATGACTCAA (SEQ ID NO: 25) GTGACTCAG (SEQ ID NO: 26) TTTGAGTCA (SEQ ID NO: 27) ATTGACTCA (SEQ ID NO: 28) CCTGAGTCA (SEQ ID NO: 29) CTGAGTCAG (SEQ ID NO: 30) Amino acid sequence of engineered murine dihydrofolate reductase used during design process (SEQ ID NO: 31) * = stop codon ASVRPLNCIVAVSQNMGIGKNGDLPWPPLRNESKYFQRMTQTDSVESKQNLVIMGRKTWFSIPESNRPLK DRINIVLSQELKEPPRGAHFLAKSLDDALRLIESPELASKVDSVWIVGGSSVYQEAMTQPGHLRLFVTQI MQEFESDTFFPEIDSGKYKLLPESPGVLSQVQEEKGIKYKFEVYEKKD*A* Nucleotide sequence of an exemplary murine dihydrofolate reductase gene engineered to include CRE binding sites (SEQ ID NO: 36) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACGTCAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGAC AGAATTAATATAGTGACGTCAAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTT TGGATGATGCCTTAAGACTTATTGAACAACCGGAATTGACGTCAAAAGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACGTCAATCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTGACGTCAGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA Amino acid sequence of an exemplary murine dihydrofolate reductase engineered to include CRE binding sites (SEQ ID NO: 37) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTSTSSVEGKQNLVIMGRKTWFSIPEKNRPLKDRINIVTS RELKEPPRGAHFLAKSLDDALRLIEQPELTSKVDMVWIVGGSSVYQEAMNQPGHLRLFVTSIMQEFESDTFFPEIDL GKYKLLPEYPGVTSEVQEEKGIKYKFEVYEKKD Nucleotide sequence of an exemplary murine dihydrofolate reductase gene engineered to include CCAAT binding sites (SEQ ID NO: 38) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCC TCCATTGCGCAATGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGA TTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCATTGCGCAATAGAATTAATATAGTTCTCAGT AGAGAATTGCGCAATCCACCACGAGGAGCTCATTTTATTGCGCAATCCTTGGATGATGCATTGCGCAATATTGAACA ACCGGAATTGGCGAGCAAAGTAGACATGGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAAC CAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTG GGGAAATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAATTGCGCAATGAAAAAGGCATCAAGTATAAGTT TGAAGTCTACGAGAAGAAAGACTAAGCTTAA Amino acid sequence of an exemplary murine dihydrofolate reductase engineered to include CCAAT binding sites (SEQ ID NO: 39) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNRPLRNRINIVLS RELRNPPRGAHFIAQSLDDALRNIEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDL GKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Nucleotide sequence of an exemplary murine dihydrofolate reductase gene engineered to include Eboxes (SEQ ID NO: 40) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGCGCACGTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGAC AGAATTAATATAGTTCTCTCACGTGAACTCAAAGAACCACCACGTGGAGCTCACGTGCTTGCCAAATCAC TGGATGATGCATTAAGACTTATTGAACAACCGGAATTGGCGTCACGTGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACGTGAGACTCTTTGTGACACGTGTCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTCCTCTCACGTGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA Amino acid sequence of an exemplary murine dihydrofolate reductase engineered to include Eboxes (SEQ ID NO: 41) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRRTWFSIPEKNRPLKDRINIVLS RELKEPPRGAHVLAKSLDDALRLIEQPELASRVDMVWIVGGSSVYQEAMNQPGHVRLFVTRVMQEFESDTFFPEIDL GKYKLLPEYPGVLSRVQEEKGIKYKFEVYEKKD Nucleotide sequence of p300-mDHFR plasmid used in Examples (SEQ ID NO: 42) CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACA ATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCACCATCACCATCACCATgctagcgttcgaccattga actgcatcgtcgccgtgagtcagaatatggggattggcaagaacggagacctaccctggcctccgctcaggaatgag tcaaagtacttccaaagaatgactcagactgactcagttgagtcaaaacagaatctggtgattatgggtaggaaaac ctggttctccattcctgagtcaaatcgacctttaaaggacagaattaatatagttctgagtcaagaactcaaagaac caccacgaggagctcattttcttgccaaaagtttggatgatgccttaagacttattgagtcaccggaattggcgagc aaagttgactcagtttggatcgtcggaggcagttctgtttaccaggaagccatgactcaaccaggccaccttagact ctttgtgactcagatcatgcaggaatttgagtcagacacgtttttcccagaaattgactcagggaaatataaacttc tccctgagtcaccaggcgtcctgagtcaggtccaggaggaaaaaggcatcaagtataagtttgaagtctacgagaag aaagactaagcttAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTT GTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAAGCTAGTTTGGGAGGTTCCAACTTTCAC CATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGA GAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAG TTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCAC AAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGA CGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGC TCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAAC CTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTT TGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACA AGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAA TTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGG GGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGT TGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCCTCTAGAGCTGCCTCGCGCGTTTCGGTGATGACGGT GAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATA CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCG TAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT GAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACC ATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG CCGCGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAA AGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGT TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT TTCGTCTTCAC Nucleotide sequence of p230d-basic-cJun plasmid used in Examples (SEQ ID NO: 43) CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACA ATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCGCATTAAAGCCGAACGCAAACGGATGCGCAACCGCA TCGCAGCCTCCAAGTGCCGCAAACGCAAATTGGAGCGCATCGCCCGCTTGGAAGAAAAGGTGAAAACCCTGAAAGCA CAGAACTATGAGCTGGCCTCCACCGCCAACATGTTGCGCGAACAGGTGGCCCAGCTCGGCGCGCCTCATCACCATCA CCATCACTGATAAAGCGCGCCTTGATAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCT CAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGGCGAGATT TTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTA AAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTT TTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCA TCCGGAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCC ATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCG CAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGC CAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCA TGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGC TTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGG CAGTTATTGGTGCCCTTAAACGCCTGGGGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCG AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCCTCTAGAGC TGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTA AGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCC AGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATA TGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGG ATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTC CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGC TACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGG ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCAC CAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAC Nucleotide sequence of pREP4 expressing the lac repressor used in Examples (SEQ ID NO: 44) AAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAG AAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCA GGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAG CTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGG CGCAGGGGATCAAGATCTGATCAAGAGACAGGATGACGGTCGTTTCGCATGCTTGAACAAGATGGATTGCACGCAGG TTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCG TGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAG GACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAG TATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAA CATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGG GCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCG ATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCG GACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCT CGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGG GACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCT ATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAG TTCTTCGCCCACCCCGGGCTCGATCCCCTCGCGAGTTGGTTCAGCTGCTGCCTGAGGCTGGACGACCTCGCGGAGTT CTACCGGCAGTGCAAATCCGTCGGCATCCAGGAAACCAGCAGCGGCTATCCGCGCATCCATGCCCCCGAACTGCAGG AGTGGGGAGGCACGATGGCCGCTTTGGTCGACAATTCGCGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCT TTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGG GCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGT TGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACA TGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATG GTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATA TTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGAC CCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGG TCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTC GTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGC GCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTT GGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCA CCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACC ACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCAACGTA AATGCATGCCGCTTCGCCTTCGCGCGCGAATTGTCGACCCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTA ACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCA GTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCG CTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGAT TTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGC CCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCC GCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCC CCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACC ACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGG ACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAA ACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATC TTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATA CGATATAAGTTGTTAATTCTCATGTTTGACAGCTTATCATCGAT Nucleotide sequence of control cJun plasmid lacking the DNA-binding basic region used in Examples (SEQ ID NO: 45) CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACA ATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCACCATCACCATCACCATGCTAGCATCGCCCGGCTGG AGGAAAAAGTGAAGACCTTGAAGGCCCAGAACTATGAGCTGGCGTCCACGGCCAACATGCTCCGGGAACAGGTGGCA CAGCTTGGCGCGCCTTAAGGTAGCTCTAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACC TCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGGCGAGAT TTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGT AAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTT TTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTT AATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAAC GCCTGGGGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT ATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCCTCTAGAGCTGCCTCGCGCGTTTCGGTGAT GACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACA AGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAG TGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACA GATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG TTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGG GCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCT ATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGG CTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCA AAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATG GCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC AGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTA TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAC Nucleotide sequence of pET24a plasmid containing FosW (SEQ ID NO: 46) ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAT TGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTG GTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCTTAAGGCGCGCCCAGTTTCTCCAGCTGT TTCTGGAGGTCTTCGATCTCTTTGCGCAAGGCATAGTTGCGTTCTTCCAGCTGTTCAATCTCGGCCTGCAGTTCATC GAGTTCCTGTTCGAGCTCTTCGGCCTCTTTTTCCAATTCTTCGTTCTCGCGGGCCAGTTCTTCGGCCCGCTGTTCCA GGCTAGCCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATT CCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCA CCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGG CTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCA TGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATA AGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGA GAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGA CCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAG TCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGG TGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGG CTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATT TCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGC ATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTG GCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTC CGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGG CGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGAT ACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGT GGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAA AAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAG GTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTC GACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTT ATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTT TCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCA CTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGATC GTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGA GCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTT CGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTA CCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCAT CCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCA TCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAG GAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGA CGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTT TCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGG AGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGA TAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAA ATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC GGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG CCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTC CAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATAC AAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATT TATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGAT GCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTG GCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTG CGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCA GTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCA GGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAAC AAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGAT AACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGA TCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTG ATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATA CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTG TAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC GGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACT ATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCAC CCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCT TGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAG TGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCA Nucleotide sequence of pET24a plasmid containing cFos (SEQ ID NO: 47) ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAT TGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTG GTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCTTAAGGCGCGCCGAGTTTTTCCTTCTCC TTCAGCAGGTTGGCAATTTCGGTCTGCAGGGCGTACTTCTCATCTTCCAGTTGGTCTGTCTCCGCTTGGAGTGTATC AGTGCTAGCCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAA TTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCAT CACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCG GGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTG CATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCA TAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAA GAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCA GACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGG AGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCC AGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGT GGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTG GGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTA TTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGA GCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTC TGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATG TCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGAT GGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACG ATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGC GTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAG AAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGAC AGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATC TCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCAC TTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGC TTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGT CACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGA TCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGC GAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGT TTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGC TACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGC ATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAG CATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAAC AGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTG GACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCG TTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCG GGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGC GATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTG AAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGC TCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAA CGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGC TGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAAACTGTCTGCTTACATAAACAGTAAT ACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGA TTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCG ATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAAC TGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCAC TGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGG CAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCT CAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGA ACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTG ATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAG GATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTAT TGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGA TACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAAT TGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAA TCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCA CTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATC ACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAG CTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCC A CAAT box GGCCAATCT (SEQ ID NO: 48) CArG box CC(A/T.sub.6GG (SEQ ID NO: 49) E2 box CAGGTG and CACCTG (SEQ ID NOs: 50 and 51) HY box TG(A/T)GGG (SEQ ID NO: 52) T box TCACACCT (SEQ ID NO: 53) TATA box TATAAA (SEQ ID NO: 54) X box GTTGGCATGGCAAC (SEQ ID NO: 55) Y box (A/G)CTAACC(A/G)(A/G)(C/T) (SEQ ID NO: 56) ATA box AAATAT (SEQ ID NO: 57) CGCG box (A/C/G)CGCG(C/G/T) (SEQ ID NO: 58) DREB box TACCGACAT (SEQ ID NO: 59) Fur box GATAATGATAATCATTATC (SEQ ID NO: 60) G box GCCACGTGGC (SEQ ID NO: 61) GCC box AGCCGCC (SEQ ID NO: 62) H box ACACCA (SEQ ID NO: 63) Prolamin box TGTAAAG (SEQ ID NO: 64) Pyrimidine box CCTTTT (SEQ ID NO: 65) TACTAAC box ATTTACTAAC (SEQ ID NO: 66)

Numbered Clauses

[0333] The following numbered clauses, describing aspects and embodiments of the invention, are part of the description.

[0334] 1. A method for screening for an antagonist of a DNA-binding protein, the method comprising: [0335] i) providing a cell, wherein the cell comprises a test compound, a DNA-binding protein, and a reporter expression cassette that encodes a reporter expression product, [0336] wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the reporter expression product; and [0337] ii) determining expression of the reporter expression product in the presence of the test compound; [0338] wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting DNA-binding activity of the DNA-binding protein.

[0339] 2. The method of clause 1, wherein the reporter expression product is a reporter protein.

[0340] 3. The method of clause 2, wherein the reporter protein is a cell survival protein, a cell reproduction protein a fluorescent protein, a bioluminescent protein, a protease, an enzyme that acts on a substrate to produce a colorimetric signal, a protein kinase, a transcriptional activator, or a regulatory protein such as ubiquitin.

[0341] 4. The method of clause 3, wherein the reporter protein is a cell survival protein, optionally wherein the cell survival protein is an enzyme involved in synthesising compounds that are required for cell survival, or a protein that is able to inhibit action of a toxic agent.

[0342] 5. The method of clause 3, wherein the reporter protein is a cell reproduction protein, optionally wherein the cell reproduction protein is an enzyme involved in synthesising compounds that are required for cell proliferation.

[0343] 6. The method of clause 4, wherein the cell survival protein is an exogenous cell survival protein that is able to compensate for a deficiency in an endogenous cell survival protein; and [0344] wherein the method is performed under selection conditions such that survival of the cell is dependent upon activity of the exogenous cell survival protein.

[0345] 7. The method of clause 5, wherein the cell reproduction protein is an exogenous cell reproduction protein that is able to compensate for a deficiency in an endogenous cell reproduction protein; and [0346] wherein the method is performed under selection conditions such that proliferation of the cell is dependent upon activity of the exogenous cell reproduction protein.

[0347] 8. The method of clause 6 or clause 7, wherein the exogenous cell survival protein is an orthologue of the endogenous cell survival protein, or the exogenous cell reproduction protein is an orthologue of the endogenous cell reproduction protein.

[0348] 9. The method of any one of clauses 6 to 8, wherein the exogenous cell survival protein or exogenous cell reproduction protein is resistant to selection conditions that inhibit the function of the endogenous cell survival protein or endogenous cell reproduction protein.

[0349] 10. The method of any one of clauses 6 to 9, wherein the selection conditions comprise the addition of a selection agent that inhibits the function of the endogenous cell survival protein or endogenous cell reproduction protein.

[0350] 11. The method of any one of clauses 4, 6, and 8 to 10, wherein the cell survival protein is dihydrofolate reductase (DHFR), optionally wherein the DHFR has an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1.

[0351] 12. The method of any one of the preceding clauses, wherein the reporter expression cassette comprises between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 15, between 10 and 20, between 10 and 18 or between 12 and 16 binding sites.

[0352] 13. The method of any one of the clauses 1 to 11, wherein the reporter expression cassette comprises at least 2, at least 5, at least 10, at least 12, or at least 15 binding sites.

[0353] 14. The method of any one of clauses 2 to 13, wherein the reporter protein retains at least 50%, at least 70%, at least 90%, or at least 95% of the function of a parent reporter protein, and wherein the parent reporter protein is encoded by a parent reporter expression cassette that corresponds to the reporter expression cassette but does not comprise the binding site(s).

[0354] 15. The method of any of the preceding claims, wherein some or all of the binding site(s) are located in the transcribed sequence of the reporter expression cassette

[0355] 16. The method of any one of clauses 2 to 15, wherein some or all of the binding site(s) are located in the protein coding sequence of the reporter expression cassette, optionally wherein the majority or all of the binding sites located in the protein coding sequence of the reporter expression cassette were introduced as silent, semi-conservative and/or conservative mutations.

[0356] 17. The method of clause 16, wherein the majority or all of the binding sites located in the protein coding sequence of the reporter expression cassette are located at positions that encode a solvent exposed residue in the reporter protein.

[0357] 18. The method of any one of clauses 15 to 17, wherein the majority or all of the binding sites located in the protein coding sequence of the reporter expression cassette are not located at positions that encode a residue that forms part of the catalytic centre of the reporter protein.

[0358] 19. The method of any one of clauses 2 to 18, wherein the reporter protein has an amino acid sequence that is at least 80% identical to a parent reporter protein, wherein the parent reporter protein is encoded by a parent reporter expression cassette that corresponds to the reporter expression cassette but does not comprise the binding site(s).

[0359] 20. The method of any one the preceding clauses, wherein the method comprises administering the reporter expression cassette in order to provide the cell comprising the reporter expression cassette.

[0360] 21. The method of any one of the preceding clauses, wherein the DNA-binding protein is a transcription factor, or a DNA-binding fragment thereof.

[0361] 22. The method of clause 21, wherein the DNA-binding protein is a eukaryotic transcription factor, or a DNA-binding fragment thereof, optionally wherein the eukaryotic transcription factor is a human transcription factor.

[0362] 23. The method of any one of the preceding clauses, wherein the DNA-binding protein is a basic leucine zipper (bZIP) transcription factor, a basic helix-loop helix (bHLH) transcription factor, or bHLH leucine zipper (bHLH-ZIP) transcription factor, or a DNA-binding fragment thereof, and optionally wherein [0363] a) the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6); [0364] b) the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8); [0365] c) the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9); [0366] d) the at least one binding site is a cAMP response element (CRE) having the nucleotide sequence TGACGTCA (SEQ ID NO: 10); [0367] e) the at least one binding site is a Maf recognition element (MARE) having the nucleotide sequence TGCTGA.sup.G/.sub.CTCAGCA (SEQ ID NO: 32) or TGCTGA.sup.GC/.sub.CGTCAGCA (SEQ ID NO: 33); or the at least one binding site is a PAP/CREB-2/PAR binding site having the nucleotide sequence TTACGTAA (SEQ ID NO: 34).

[0368] 24. The method of clause 23, wherein: [0369] a) the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6); [0370] b) the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8); or [0371] c) the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9).

[0372] 25. The method of any one of the preceding clauses, wherein [0373] a) the DNA-binding protein is AP-1 or a member of the Fos/Jun subfamily of transcription factors (such as c-Jun), or a DNA-binding fragment thereof, and the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6), optionally wherein the reporter expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 4; [0374] b) the DNA-binding protein is a bHLH transcription factor (such as c-Myc), or a DNA-binding fragment thereof, and the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8), optionally wherein the reporter expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 40; [0375] c) the DNA-binding protein is a member of the C/EBP subfamily of transcription factors (such as C/EBP alpha), or a DNA-binding fragment thereof, and the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9); optionally wherein the reporter expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 38; [0376] d) the DNA-binding protein is BZLF1, or a DNA-binding fragment thereof, and the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6), or a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9), optionally wherein the reporter expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 4 or sequence set forth in SEQ ID NO: 38.

[0377] 26. The method of any one of the preceding clauses, wherein the cell is a bacterial cell, such as an Escherichia coli cell.

[0378] 27. The method of any one of clauses 1 to 25, wherein the cell is a eukaryotic cell.

[0379] 28. The method of clause 27, wherein the eukaryotic cell is a mammalian cell, optionally a human cell.

[0380] 29. The method of clause 28, wherein the mammalian cell was isolated from a human patient and wherein the DNA-binding protein is naturally produced by the cell, and optionally wherein the DNA-binding protein is suspected of being or known to be dysregulated in the cell.

[0381] 30. The method of any one of clauses 1 to 28, wherein the method comprises administering a DNA-binding protein expression cassette that encodes the DNA-binding protein in order to provide the cell comprising the DNA-binding protein.

[0382] 31. The method of any one of the preceding clauses, wherein the test compound is a peptidic test compound or a small molecule test compound.

[0383] 32. The method of clause 31, wherein the test compound is a peptidic test compound.

[0384] 33. The method of clause 32, wherein the peptidic test compound is expressed intracellularly from a test compound expression cassette.

[0385] 34. The method of clause 33, wherein the method comprises providing the test compound expression cassette to the cell.

[0386] 35. The method of any one of clauses 32-34, wherein the method comprises administering a cross-linking agent into the cell in order to introduce a cross-link between two amino acid residues in an alpha helix of the peptidic test compound to produce a helix-constrained peptidic compound.

[0387] 36. The method of clause 35, wherein the method comprises determining expression of the reporter expression product both before and after the addition of the cross-linking agent.

[0388] 37. The method of clause 31 or clause 32, wherein the method comprises administering the test compound extracellularly in order to provide the cell comprising the test compound, optionally wherein an increase in expression of the reporter expression product indicates that the test compound is capable of entering the cell as well as being capable of inhibiting DNA-binding activity of the DNA-binding protein.

[0389] 38. The method of clause 37, wherein the test compound is a peptidic test compound, wherein the peptidic test compound comprises a helix-constrained peptide, and wherein the helix-constrained peptide comprises a cross-link between two amino acid residues.

[0390] 39. The method of clause 35 or clause 38, wherein the cross-link is formed between residues i and i+4 in the peptidic test compound.

[0391] 40. The method of any one of clause 35, clause 38 and clause 39, wherein the cross-link is formed between cysteine residues in the peptidic test compound.

[0392] 41. A method for producing a helix-constrained peptide in a cell, the method comprising: [0393] i) providing the cell containing a peptidic test compound comprising an alpha helix; and [0394] ii) adding a cross-linking agent to the cell, wherein the cross-linking agent chemically modifies the peptidic test compound to introduce a cross-link between two amino acid residues in the alpha-helix, thereby producing a helix-constrained peptide.

[0395] 42. The method of clause 41, wherein the cross-link is formed between residues i and i+4 in the helix-constrained peptide.

[0396] 43. The method of clause 41 or clause 42, wherein the cross-link is formed between cysteine residues.

[0397] 44. The method of any one of clauses 41-43, wherein the method further comprises determining expression of the reporter expression product both before and after the addition of the cross-linking agent.

[0398] 45. A cell-free method for screening for an antagonist of a DNA-binding protein, the method comprising: [0399] i) contacting a test compound with a DNA-binding protein and a reporter expression cassette that encodes a reporter expression product, [0400] wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the reporter expression product; and [0401] ii) determining expression of the reporter expression product; [0402] wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting DNA-binding activity of the DNA-binding protein, and [0403] wherein the method is carried out outside a cell in an in vitro system that comprises the components required for expression of the reporter expression product.

[0404] 46. A method of generating a reporter expression cassette for use in screening for an antagonist of a DNA-binding protein, the method comprising introducing at least one binding site into the reporter expression cassette that encodes a reporter expression product; [0405] wherein the at least one binding site is introduced into the reporter expression cassette such that binding of a DNA-binding protein to the at least one binding site in the reporter expression cassette inhibits expression of the reporter expression product.

[0406] 47. The method of clause 46, wherein the reporter expression product is a reporter protein.

[0407] 48. The method of clause 47, wherein the reporter protein is a cell survival protein, a fluorescence protein, a bioluminescence protein, a protease, an enzyme that acts on a substrate to produce a colorimetric signal, a protein kinases, a transcriptional activator, or a regulatory protein such as ubiquitin.

[0408] 49. The method of any one of clauses 46-48, wherein the reporter expression cassette comprises between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 15, between 10 and 20, between 10 and 18 or between 12 and 16 binding sites.

[0409] 50. The method of any one of clauses 46-49, wherein the reporter expression cassette comprises at least 2, at least 5, at least 10, at least 12, or at least 15 binding sites.

[0410] 51. The method of any one of clauses 46 to 50 some or all of the binding site(s) are located in the transcribed sequence of the reporter expression cassette.

[0411] 52. The method of any one of clauses 47 to 51, wherein the reporter protein retains at least 50%, at least 70%, at least 90%, or at least 95% of the function of a parent reporter protein, and wherein the parent reporter protein is encoded by a parent reporter expression cassette that corresponds to the reporter expression cassette but does not comprise the binding site(s).

[0412] 53. The method of any one of clauses 47 to 52, wherein the at least one binding site is introduced into the protein coding sequence of the reporter expression cassette, optionally wherein the majority or all of the binding sites are introduced as silent, semi-conservative and/or conservative mutations in the protein coding sequence of the reporter expression cassette.

[0413] 54. The method of clause 53, wherein the majority or all of the binding sites are introduced in the protein coding sequence of the reporter expression cassette at positions that encode a solvent exposed residue in the reporter protein.

[0414] 55. The method of any one of clauses 52 to 54, wherein the majority or all of the binding sites are introduced in the protein coding sequence of the reporter expression cassette at positions that encode a solvent exposed residue in the reporter protein.

[0415] 56. The method of any one of clauses 47 to 55, wherein the reporter protein has an amino acid sequence that is at least 80% identical to a parent reporter protein, wherein the parent reporter protein is encoded by a parent reporter expression cassette that corresponds to the reporter expression cassette but does not comprise the binding site(s).

[0416] 57. The method of any one of clauses 46 to 56, wherein the DNA-binding protein is a transcription factor or a DNA-binding fragment thereof.

[0417] 58. The method of clause 57, wherein the DNA-binding protein is a eukaryotic transcription factor or a DNA-binding fragment thereof, optionally wherein the eukaryotic transcription factor is a human transcription factor.

[0418] 59. The method of any one of clauses 46 to 58, wherein the transcription factor is a basic leucine zipper (bZIP) transcription factor, a basic helix-loop helix (bHLH) transcription factor, a bHLH leucine zipper (bHLH-ZIP) transcription factor or a DNA-binding fragment thereof.

[0419] 60. The method of any one of clauses 46-59, wherein: [0420] a) the DNA-binding protein is AP-1 or a member of the Fos/Jun subfamily of transcription factors (such as c-Jun), or a DNA-binding fragment thereof, and/or the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6); [0421] b) the DNA-binding protein is a bHLH transcription factor, such as c-Myc or Max, or a DNA-binding fragment thereof, and/or the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8); [0422] c) the DNA-binding protein is a member of the C/EBP subfamily of transcription factors (such as C/EBP alpha), or a DNA-binding fragment thereof, and/or the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9); [0423] d) the DNA-binding protein is BZLF1, or a DNA-binding fragment thereof, and/or the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6), or a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9).

[0424] 61. A kit comprising: [0425] i) a reporter expression cassette that encodes a reporter expression product; and [0426] ii) a DNA-binding protein expression cassette that encodes a DNA-binding protein [0427] wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the expression product.

[0428] 62. The kit of clause 61, wherein the kit further comprises a test compound.

[0429] 63. A cell comprising: [0430] i) a reporter expression cassette that encodes a reporter expression product; and [0431] ii) a DNA-binding protein expression cassette that encodes a DNA-binding protein; [0432] wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the expression product.