Transcription factors are proteins that regulate cellular processes by activating and/or repressing the expression of specific genes and thereby the levels of the proteins encoded by regulated genes. Many transcription factors are implicated in human disease, including cancer. Often these diseases are characterised by gain or loss of the activity of a given transcription factor. Examples of gain/loss of transcription factor activities include mutation of the p53 gene (Nigro et al. 1989 Nature 342: 705-8) and Rb in retinoblastoma (Zheng L, Lee WH. Exp Cell Res. 264: 2-18).

Transcription factors are involved in hormone signalling. Breast cancer is a classic example of a hormone dependent cancer and the hormone estrogen (E2) regulates the growth of the majority of breast cancers. Other examples of hormone dependent cancer include endometrial cancer, ovarian cancer, thyroid cancer and prostate cancer as discussed in the following references: Sherman et al. The Management of Metastatic Differentiated Thyroid Carcinoma. Rev Endocr Metab Disord 2000.1 (3): 165-71; Makar et al.

Hormone Therapy in Epithelial Ovarian Cancer. Endocr Relat Cancer.

7 (2): 85-93 ; Schroder et al. Endocrine Treatment of Prostate Cancer. BJU Int. 1999.83 : 161-70; Chen et al. Endometrial Cancer. Oncology 1999.

13 (12): 1665-70; Feldman and Feldman. The development of androgen- independent prostate cancer (2001) Nature Reviews Cancer 1: 34-45.

Transcription factors regulate gene expression by binding to specific DNA sequences in the promoters of regulated genes. Their binding stimulates recruitment of the basal transcription factors including RNA polymerase II, resulting in messenger RNA synthesis. Transcription factors stimulate

recruitment of the basal transcription factors including RNA polymerase II through direct interaction with the basal transcription factors and/or by recruitment of transcriptional co-regulator complexes, which in turn recruit the RNA polymerase II complexes through direct interaction. In addition, co-regulator complexes facilitate gene expression by chromatin remodelling and include complexes that remodel chromatin by modification of core histones, for example by acetylation, arginine methylation. Transcription repression is brought about by several mechanisms. In the case of nuclear receptors interaction with N-CoR/SMRT results in recruitment of histone deacetylase complexes and consequent chromatin remodelling (Lemon and Tjian (2000) Genes Dev. 14: 2551-2569; Maldonado et al. (1999) Cell 99: 455-458).

Transcription factor activities are routinely studied in established cell lines using reporter genes. These are comprised of a synthetic promoter containing sequences required for basal transcription factor binding (e. g.

TATA box and transcription start site), the specific DNA sequences to which the transcription factor of interest binds and sequences encoding a protein, often of bacterial origin, whose activity can be readily assayed.

Examples include bacterial chloramphenicol acetyl transferase (CAT) that converts chloramphenicol to acetyl-chloramphenicol, B-galactosidase that can be used in simple colorimetric assays, firefly luciferase and green fluorescent protein (see, for example, Bronstein et al (1994) Chemiluminescent and bioluminescent reporter gene assays Ahal Biochem 219, 169-181 ; Tsien (1998) The green fluorescent protein Ann Rev Biochem 67, 509-544).

The nuclear receptor DNA binding protein superfamily includes oestrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR), retinoic acid receptor (RAR) and the like (see Mangelsdorf et al (1995) Cell 83, 835-839 for a review and nomenclature).

In the case of breast cancer the hormone oestrogen (E2) regulates the growth of the majority of breast cancers. E2 action is mediated by the transcription factors oestrogen receptor a (ERa) and estrogen receptor B (ERF) (Ali and Coombes (2002) Nature Reviews Cancer 2: 101-112).

The majority of patients present with localised disease, also known as primary breast cancer. The usual treatment is surgical excision of the tumour, followed by adjuvant therapy. Adjuvant therapies for ERa-positive disease are designed to reduce oestrogen levels or block its activity by binding to the receptor, as exemplified by tamoxifen. Tamoxifen is now the first line adjuvant treatment for ERa-positive disease in pre-and post- menopausal women and is beneficial in the treatment of metastatic disease, as well as localized disease. However, approximately 30%, of patients with ERa-positive disease do not respond to tamoxifen. Moreover, a substantial proportion of patients presenting with localized disease and all patients presenting with metastatic disease that initially respond to tamoxifen treatment become resistant. In the case of patients who initially respond but become resistant to tamoxifen ERa expression is lost in only-10% of cases. Moreover, one-third of resistant patients show a clinical response to treatment with a different anti-estrogen such as Faslodex (ICI 182, 780) or the use of aromatase inhibitors, drugs that inhibit estrogen synthesis.

Nevertheless and despite issues of de novo or acquired resistance to tamoxifen, it has become the adjuvant agent of first choice (Ali and Coombes (2002) Nature Rev. Cancer 2: 101-112).

ERa is a member of the nuclear receptor superfamily of transcription factors that activates gene expression upon binding as homodimers to small palindromic sequences known as estrogen response elements (EREs) in promoters of estrogen responsive genes. EREs conform to the general sequence GGTCAnnnTGACC, with some diversity from this sequence still providing a functional ERE (Klinge (2001) Nucleic Acids Res. 29: 2905- 2919). Transcription activation by ERa requires estrogen binding to the ligand or hormone binding domain (Shiau et al. (1998) Cell 95: 927-937)

and is mediated by co-regulator recruitment upon ligand binding (Glass & Rosenfeld (2000) Gene & Dev. 14: 121-141).

Resistance to endocrine therapy could theoretically arise through mutation of the ERa gene resulting in ERa protein that is inactive, is active in a ligand-independent manner or is activated by tamoxifen. Some ERa mutations have been described (Hopp and Fuqua SA. (1998) J Mammary Gland Biol Neoplasia. 3: 73-83). Resistance could also arise by growth factor-stimulated post-translational modification of ERa that results in an increase in ERa activity such that ERa is activated in the absence of ligand, or is activated by suboptimal concentrations of E2 and/or is activated, rather than inhibited by tamoxifen (Chen et al. (2001) Mol. Cell. 6,127-137).

Mutation and/or reduction in levels of co-repressor complexes and/or increased levels of co-activator proteins could also lead to resistance. In vitro resistance to tamoxifen has been correlated with reduction in amounts of the co-repressor N-CoR (Lavinsky et al. PNAS 95: 2920-2925), whilst overexpression and/or amplification of the AIB1 coactivator is observed in almost 5% of breast cancers (Anzick et al (1997) Science 277: 965-968 ; Bautista et al. (1998) Clin. Cancer Res. 4: 2925-2929). See also Kothari (2003) Br J Cancer 88, 1071-1076.

Methods for assessing transcription factor activity in a patient's cells are described in PCT/GB03/00878 and Kothari (2003) Br J Cancer 88, 1071- 1076.

Genbank Accession No BK000210, generated by comparison of the Takifugu rubripes genome with the human genome sequence, (Gilligan et al. (2002) Gene 294: 35-44) describes a putative polypeptide coding sequence similar to zinc finger protein 91 (HPF7, HTF10), which has been named ZNF366. This has subsequently been confirmed by the NEDO human cDNA sequencing project (Genbank Accession No Nom152625).

GenBank Accession No AAC60294 describes a Takifugu rubripes polypeptide with significant homology to NM_152625. GenBank Accession

No XP_138810 describes a Mus musculus polypeptide with significant homology to NM_152625. GenBank Accession No BI184476 describes a Sus scrofa EST polynucleotide sequence which, when translated in antiparallel, gives homology to Nom 152625.

We have identified a polypeptide (termed ERZFP) which is capable of modulating the activity of a transcription factor, for example a nuclear receptor DNA binding protein, for example an oestrogen receptor polypeptide, for example ERa. The polypeptide is capable of inhibiting transcription factor activity in a reporter gene assay. Accordingly, the polypeptide is useful in medicine, for example in relation to the treatment of honnone-dependent cancers, uterine hyperplasia, endometriosis, and post- menopausal symptoms eg bone loss.

A first aspect of the invention provides a polypeptide (ERZFP) having the amino acid sequence of Figure 1A (Seq ID No 1) or an amino acid sequence (variant) having at least 45 % identity with the amino acid sequence of Figure 1A (Seq ID No 1) for use in medicine.

In an embodiment of the first aspect of the invention the amino acid sequence has at least 50% identity with the amino acid sequence of Figure 1A (Seq ID No 1).

A second aspect of the invention provides a fragment or fusion or derivative of the polypeptide of the first aspect of the invention for use in medicine.

The polypeptide may be recombinant or naturally occurring. The polypeptide may be substantially pure.

By"substantially pure"we mean that the said polypeptide is substantially free of other proteins. Thus, the pharmaceutical composition may be or may be formed from any composition that includes at least 30% of the protein content by weight as the said polypeptide, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said polypeptide.

Thus, the invention also includes compositions comprising, or formed from compositions comprising, the said polypeptide and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.

Thus, the substantially pure said polypeptide may be combined with other components ex vivo, said other components not being all of the components found in the cell in which said polypeptide is found. It will be appreciated that the said substantially pure polypeptide may be obtained by expression from a recombinant nucleic acid, for example in a prokaryotic or eukaryotic cell, as discussed further below.

Variants of the polypeptide having the amino acid sequence of SEQ ID No 1; Figure 1A (whether naturally-occurring or otherwise) may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below.

By"fragment of said polypeptide"we include any fragment which retains activity, for example the ability to interact with or bind to a transcription factor, for example a nuclear receptor DNA binding activity, or which has the effect of modulating, for example inhibiting, the transcription activity of

the transcription factor, for example a nuclear receptor DNA binding activity; or which is useful in some other way, for example, for use in raising antibodies or in a binding assay. The fragment may bind to the API region of a nuclear receptor DNA binding protein, for example of the estrogen receptor, for example of ERa. Such binding may be dependent on the nuclear receptor DNA binding protein being phosphorylated or mutated to mimic phosphorylation, for example on/in the API region, for example on the residue equivalent to Serl 18 of ERa.

Nuclear Receptors have a modular structure having at least three distinct domains, defined by the fact that these domains can be isolated and their activities assayed independently of the others. There is a near central DNA binding domain, comprised of two zinc fingers. C-terminal to the DNA binding domain is the ligand binding domain and the tightly associated transcription activation function AF2. Upon binding ligand this domain recruits co-activators or corepressors leading to gene regulation. N-terminal to the DNA binding domain is another region required for transcription activation, called AF1. The activity of this region is often regulated by phosphorylation, which in the case of ERalpha, can lead to activation of estrogen-regulated genes in the absence of ligand.

By"fusion of said polypeptide"we include said polypeptide fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope.

Fusions to any variant, fragment or derivative of said polypeptide are also included in the scope of the invention.

By"variants"of the polypeptide having the amino acid sequence of SEQ ID No 1; Figure 1A we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide (for example, the ability to bind to, or the ability to inhibit transcription activity of, a transcription factor, for example a nuclear receptor DNA binding protein or fragment thereof, for example ER, for example ERa).

Examples of variants are the pufferfish sequence reported in Gilligan et al (supra) which has 58% identity with the polypeptide having the amino acid sequence of SEQ ID No 1; Figure 1A (88% in the ZF domain), the murine polypeptide described in GenBank Accession No XP_138810 (a Mus musculus polypeptide) which has 74% identity with the polypeptide having the amino acid sequence of SEQ ID No 1; Figure 1A, and the porcine polypeptide, which is a translation of the Sus scrofa EST polynucleotide sequence described in GenBank Accession No BI184476. The partial porcine polypeptide has 47% identity with the polypeptide having the amino acid sequence of SEQ ID No 1; Figure 1A, however, as the porcine polypeptide is not full length, the complete polypeptide is likely to have a higher percent identity to SEQ ID No 1.

For example, it is considered that the pufferfish sequence reported in Gilligan et al (supra) will have similar functional properties to the polypeptide having the amino acid sequence of SEQ ID No 1; Figure 1A as the zinc finger DNA binding domain, the potential CtBP binding motif and the potential nuclear receptor LBD (ligand binding domain) binding LXXLL motifs (see Figure 4) are present in the pufferfish polypeptide.

By"conservative substitutions"is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln ; Ser, Thr; Lys, Arg; and Phe, Tyr.

It is particularly preferred if the polypeptide variant has an amino acid sequence overall or in at least the zinc finger domain which has at least 50% identity with the amino acid sequence of sequence of SEQ ID No 1; Figure 1A, preferably at least 65%, more preferably at least 75%, still more preferably at least 80%, yet more preferably at least 90%, and most preferably at least 95% or 99% identity with the amino acid sequence of SEQ ID No 1 ; Figure 1 A.

It is also preferred that the CtBP binding motif and the potential nuclear receptor LBD (Ligand Binding Domain) motif (LXXLL) are also present.

The LXXLL motif is an helical region in coactivators that has been shown to interact with the LBD. Some corepressors, like RIP140, TIFlalpha also use LXXLL motif to interact with the estrogen-bound ER to repress ER activity (see, for example, Glass & Rosenfeld 2000).

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (Thompson et al (1994) Nucl Acid Res 22,4673-4680). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

Scoring matrix: BLOSUM.

A particular embodiment of the invention makes use of a substantially pure human ERZFP polypeptide or naturally occurring allelic variants thereof.

An amino acid sequence is shown in Figure 1A and in Accession No Nom 152625.

It is particularly preferred, although not essential, that the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 30% of the activity of ERZFP with respect to the binding or modulation of the transcription factor activity of transcription factor, for example a human ERa, or other nuclear receptor DNA binding protein, or other transcription factors, as discussed in Example 1 and shown in Figure 10. It is more preferred if the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the activity of ERZFP with respect to the modulation of ERa transcription factor activity or any one of the alternatives described above. However, it will be appreciated that variants or fusions or derivatives or fragments which are devoid of transcription factor modulatory activity may nevertheless be useful, for example by interacting with another polypeptide, or as antigens in raising antibodies.

A third aspect of the invention provides a polynucleotide encoding the polypeptide of the first or second aspect of the invention for use in medicine.

A fourth aspect of the invention provides a polynucleotide having the nucleic acid sequence of Figure 1B ; Seq ID No 2 or a nucleic acid sequence having at least 50 % identity with the nucleic acid sequence of Seq ID No 2; or a fragment thereof; or a polynucleotide complementary any thereto, for use in medicine.

We include the polynucleotides described in GenBank Accession No AAC60294 (a Takifugu rubripes polynucleotide), GenBank Accession No XP-138810 (a Mus musculus polynucleotide) and GenBank Accession No BI184476 (a Sus scrofa polynucleotide) in the third and fourth aspects of the invention.

Preferences and exclusions for a polynucleotide variant are the same as given above in relation to a polypeptide variant.

The polynucleotide may be recombinant or naturally occurring. The polynucleotide may be substantially pure.

A polynucleotide comprising a fragment of the recombinant polynucleotide encoding the polypeptide or a variant, fragment, fusion or derivative may also be useful. Preferably, the polynucleotide comprises a fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length. Such polynucleotides are useful as PCR primers, which may be useful in, for example, diagnostic methods. A polynucleotide complementary to the polynucleotide (or a fragment thereof) encoding the polypeptide or a variant, fragment, fusion or derivative may also be useful. Such complementary polynucleotides are well known to those skilled in the art as antisense polynucleotides. Such molecules may be useful therapeutically.

The polynucleotide or recombinant polynucleotide used in the invention may be DNA or RNA, preferably DNA. The polynucleotide may or may not contain introns in the coding sequence; preferably the polynucleotide is a cDNA.

A"variation"of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to the gene or to a variation of type (i) as just defined. For example, different codons can be substituted which code for the same amino acid (s) as the original codons.

Alternatively, the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis"Science, 229: 193- 210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.

Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) encoding a polypeptide of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency. Such polynucleotides includes any genomic DNA. Accordingly, the polynucleotide used in the invention includes polynucleotide that shows at least 60%, preferably 70%, and more preferably at least 80% and most preferably at least 90%

homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful. Such a polypeptide may be a functional homologue of the polypeptide of the invention. The polypeptide may, for example, have a similar transcription regulatory activity to the ERZFP polypeptide or may be able to substitute for the ERZFP polypeptide in a cell.

It will be appreciated that such a method may be used in the identification of a functional homologue of ERZFP.

It is preferred that the polynucleotide is derivable from a mammal, for example a human, or a domesticated animal, for example a companion animal such as a cat or dog.

Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group.

DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0. 1XSSC and 6XSSC and at temperatures of between 55°C and 70°C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By"high stringency"we mean 2XSSC and 65°C. 1XSSC is 0. 15M NaCl/0. 015M sodium citrate.

Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.

A fifth aspect of the invention provides a pharmaceutical composition comprising a polypeptide or polynucleotide as defined in any one of the

preceding aspects of the invention and a pharmaceutically acceptable carrier.

Whilst it is possible for a polypeptide or polynucleotide of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier (s) must be"acceptable"in the sense of being compatible with the polypeptide or polynucleotide of the invention and not deleterious to the recipients thereof.

Typically, the carriers will be water or saline which will be sterile and pyrogen free.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (polypeptide or polynucleotide of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by

compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

A sixth aspect of the invention provides an expression vector comprising a polynucleotide as defined in relation to the preceding aspects of the invention, or a recombinant polynucleotide suitable for expressing a polypeptide (or variant or fragment or derivative or fusion) as defined in relation to the preceding aspects of the invention. Preferences and exclusions for the said polynucleotide variant are the same as indicated in relation to preceding aspects of the invention.

By"suitable for expressing"is meant that the polynucleotide is a polynucleotide that may be translated to form the polypeptide, for example RNA, or that the polynucleotide (which is preferably DNA) encoding the polypeptide of the invention is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.

The polynucleotide may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by any desired host; such controls may be incorporated in the expression vector.

Thus a further aspect of the invention is a replicable vector suitable for expressing a polypeptide used in the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative. Preferences and exclusions for the

said polynucleotide variant are the same as for the polypeptide used in the invention. For example, the replicable vector may be suitable for expressing a fusion of the polypeptide, in particular a GST fusion. It will be appreciated that a construct is not a recombinant polynucleotide as defined above if it lacks sequences necessary for the translation and therefore expression of the encoded polypeptide used in the invention.

The present invention also relates to a host cell transformed with a polynucleotide expression vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc. , Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.

Transformation of appropriate cell hosts with a DNA expression construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.

Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A

Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275,104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.

For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2,637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2. 5X PEB using 6250V per cm at 25: FD.

Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) MethodsE7zzy7nol. 194,182.

Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98,503 or Berent et al (1985)

Biotech. 3,208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity.

Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.

Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.

A method of making the polypeptide used in the invention or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative comprises culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said polypeptide, and isolating said polypeptide or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative from said host cell. Methods of cultivating host cells and isolating recombinant proteins are well known in the art.

A seventh aspect of the invention provides a gene therapy vector comprising a polynucleotide as defined in any one of the preceding aspects of the invention, for example a polypeptide encoding ERZFP or a fragment or fusion or variant or derivative thereof ; or a polypeptide capable of acting as an antisense or other agent for reducing the amount of ERZFP in a cell.

Gene therapy vectors known in the art may be useful in the practise of this aspect of the invention. Examples of suitable vectors, gene therapy constructs and delivery systems which may be adapted in relation to the present invention, including systems in which expression of the encoded polypeptide is under the control of an inducible promoter, are described in, for example WO01/18038.

Vectors, constructs, tissue-specific promoters and routes of administration for gene therapy aspects of this invention are known to a person of skill in the art, and are described for example in WO 01/18038, which is incorporated herein by reference.

A number of viruses have been used as gene transfer vectors, including papovaviruses, eg SV40 (Madzak et al (1992) J. Gen. Virol. 73,1533- 1536), adenovirus (Berkner (1992) Curr. Top. Microbiol. Immunol. 158,39- 61; Berkner et al (1988) BioTechniques 6,616-629 ; Gorziglia and Kapikian (1992) J. Virol. 66,4407-4412 ; Quantin et al (1992) Proc. Natl. Acad. Sci. USA 89,2581-2584 ; Rosenfeld et al (1992) Cell 68, 143-155; Wilkinson et al (1992) Nucleic Acids Res. 20,2233-2239 ; Stratford-Perricaudet et al (1990) Hum. Gene Ther. 1,241-256), vaccinia virus (Moss (1992) Curr. Top. Microbiol. Immunol. 158, 25-38), adeno-associated virus (Muzyczka (1992) Curr. Top. Microbiol. Immunol. 158,97-123 ; Ohi et al (1990) Gene 89, 279-282), herpes viruses including HSV and EBV (Margolskee (1992) Curr. Top. Microbiol. Immunol. 158,67-90 ; Johnson et al (1992) J. Virol. 66,2952-2965 ; Fink et al (1992) Hum. Gene Ther. 3,11-19 ; Breakfield and Geller (1987) Mol. Neurobiol. 1,337-371 ; Freese et al (1990) Biochem. Pharmacol. 40,2189-2199), and retroviruses of avian (Brandyopadhyay and Temin (1984) Mol. Cell. Biol. 4,749-754 ; Petropoulos et al (1992) J.

Virol. 66,3391-3397), murine (Miller (1992) Curr. Top. Microbiol.

Immunol. 158,1-24 ; Miller et al (1985) Mol. Cell. Biol. 5,431-437 ; Sorge et al (1984) Mol. Cell. Biol. 4,1730-1737 ; Mann and Baltimore (1985) J.

Virol. 54,401-407 ; Miller et al (1988) J. Virol. 62,4337-4345), and human origin (Shimada et al (1991) J. Cliva. Invest. 88,1043-1047 ; Helseth et al (1990) J. Virol. 64,2416-2420 ; Page et al (1990) J. Virol. 64,5370-5276 ; Buchschacher and Panganiban (1992) J. Virol. 66,2731-2739). To date most human gene therapy protocols have been based on disabled murine retroviruses.

An eighth aspect of the invention provides the use of a compound for modulating, for example inhibiting, the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein, wherein the compound is (a) the polypeptide ERZFP (SEQ ID No 1 ; Figure 1A) ; or, (b) a fragment of the polypeptide ERZFP which binds to the transcription factor, for example a nuclear receptor DNA binding protein; or, (c) a variant of the polypeptide or fragment as defined in (a) or (b) which binds to the transcription factor, for example a nuclear receptor DNA binding protein; or, (d) a fusion or derivative of the polypeptide or fragment as defined in (a), (b) or (c) which binds to the transcription factor, for example a nuclear receptor DNA binding protein; or, (e) a peptidomimetic of the polypeptide, fragment, variant, fusion or derivative as defined in (a), (b), (c) or (d) which binds to the transcription factor, for example a nuclear receptor DNA binding protein; or, (f) a compound, for example an antibody or antibody fragment which mimics the binding of the polypeptide, fragment, variant, fusion or derivative as defined in (a), (b), (c) or (d) to the transcription factor, for example a nuclear receptor DNA binding protein

Examples of variants, fusions, derivatives and fragments are discussed above. The term"peptidomimetic"refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent, but that avoids the undesirable features. For example, morphine is a compound which can be orally administered, and which is a peptidomimetic of the peptide endorphin.

The use of a compound as defined in this aspect of the invention may be in vivo or in vitro.

By'inhibiting'we include where the compound reduces the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein by at least 25%, preferably 50%, 60%, 70%, 80% or 90%.

Most preferably the compound reduces the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein by 95%.

Compounds that may be of use in this aspect of the invention include an antibody or antibody fragment that mimics the binding of the polypeptide, fragment, variant, fusion or derivative as defined in (a), (b), (c) or (d) to the transcription factor, for example a nuclear receptor DNA binding protein.

Methods of producing antibodies that recognise polypeptides are well known to those skilled in the art and can be considered routine.

Further compounds included in this aspect of the invention include a polypeptide that mimics the binding of the polypeptide, fragment, variant, fusion or derivative as defined in (a), (b), (c) or (d) to the transcription factor, for example a nuclear receptor DNA binding protein. One method of identifying such a compound is by using a derivative of a two-hybrid library screening system. In this screen, the transcription factor activity of the

transcription factor, for example a nuclear receptor DNA binding protein is monitored by the expression of a reporter gene under control of the transcription factor, for example a nuclear receptor DNA binding protein. A library of polynucleotides is transformed into the yeast cells and the reporter gene expression monitored. Any polynucleotides which encode polypeptides that mimic the binding of the polypeptide, fragment, variant, fusion or derivative as defined in (a), (b), (c) or (d) to the transcription factor, for example a nuclear receptor DNA binding protein can be identified by measuring the expression of a reporter gene, ie a compounds that may be of use in this aspect of the invention would result in a decrease in reporter gene expression. Such a system and the controls required to identify a polypeptide of use in this aspect of the invention can be performed by a person skilled in the art.

Alternatively, test compounds may be supplied to the yeast cells described above and the expression of a reporter gene monitored. Compounds that may be of use in this aspect of the invention would result an decrease in reporter gene expression, ie the compound inhibits the transcription factor activity of the transcription factor, for example a nuclear receptor DNA binding protein. Again, this screen and the necessary controls can be performed by a person skilled in the art.

A ninth aspect of the invention provides the use of a compound for modulating, for example inhibiting, the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein in a cell, wherein the compound is: (a) a compound as defined in relation to the preceding aspect of the invention; or,

(b) a polynucleotide encoding the polypeptide, fragment, variant, fusion, derivative or compound as defined in (a), (b), (c), (d) or (f) of the previous aspect of the invention.

Preferences as to the inhibition of the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein are those provided in the previous aspect of the invention.

By'cell'we include both prokaryotic or eukaryotic cells. Eukaryotic cells may be unicellular, such as yeast, or it may be comprised in a multicellular organism such as a human or animal body or a plant. Hence this aspect of the invention includes where the compound modulates the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein in a cell, in which the cell is part of a human body.

A tenth aspect of the invention provides a method for modulating, for example inhibiting, the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein, the method comprising the steps of: (a) providing a compound as defined in the eighth aspect of the invention; and, (b) exposing the transcription factor, for example a nuclear receptor DNA binding protein to the compound.

Preferences as to the inhibition of the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein are those provided in the eighth aspect of the invention.

By'providing'we include where the endogenous gene encoding ERZFP is upregulated hence providing a larger quantity of the ERZFP polypeptide.

This includes increasing the copy number of the endogenous ERZFP gene.

An embodiment of this aspect of the invention is where the compound is provided by providing a polynucleotide encoding the polypeptide, fragment, variant, fusion, derivative or compound as defined in (a), (b), (c), (d) or (f) of the eighth aspect of the invention.

A further embodiment of this aspect of the invention is where the transcription factor, for example a nuclear receptor DNA binding protein is in a cell. By'cell'we include the types of cells listed in the previous aspect of the invention.

A further embodiment of the eighth, ninth or tenth aspects of the invention is where the use or method is performed in vitro.

An eleventh aspect of the invention provides the use of a compound for modulating, for example promoting, the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein in a cell, wherein the compound is: (a) a compound which inhibits the binding of the polypeptide ERZFP (SEQ ID No 1; Figure 1A) to the transcription factor, for example a nuclear receptor DNA binding protein; or, (b) a compound which reduces the amount of ERZFP in the cell.

This aspect of the invention may be useful in, for example, the stimulation of peroxisome proliferation-activated receptor gamma (PPARy), retinoic acid receptor a and vitamin D3 receptor. Stimulation of these proteins has benefits in the treatment of breast cancer. Activation of PPARy has also

been linked to benefits in the treatment of colon and prostate cancers, as well as a role in some forms of diabetes. Hence compounds that are suitable for use in this aspect of the invention may be beneficial for the treatment of a number of different disorders.

By'promoting'we include where the compound increases the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein by at least two-fold, preferably three-fold, five-fold, ten- fold. For the avoidance of doubt, two-fold means a doubling of transcription factor activity. Most preferably the compound promotes the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein by twenty five-fold.

Compounds that may be of use in this aspect of the invention include an antibody or antibody fragment that recognises ERZFP and prevents binding of ERZFP to the transcription factor, for example a nuclear receptor DNA binding protein. Methods of producing antibodies or antibody fragments that recognise polypeptides are well known to those skilled in the art and can be considered routine.

It is also possible to screen for polypeptides that inhibit the binding of the polypeptide ERZFP to the transcription factor, for example a nuclear receptor DNA binding protein. Such a screen may be conducted in yeast cells using a derivative of a two-hybrid library screening system. In this screen, the binding of ERZFP to the transcription factor, for example a nuclear receptor DNA binding protein is monitored by the expression of a reporter gene under control of the transcription factor, for example a nuclear receptor DNA binding protein. A library of polynucleotides is transformed into the yeast cells and the reporter gene expression monitored. Any polynucleotides which encode polypeptides that inhibit the binding of

ERZFP to the transcription factor, for example a nuclear receptor DNA binding protein can be identified by measuring the expression of a reporter gene, ie compounds of use in this aspect of the invention would result in an increase of reporter gene expression. Such a system and the controls required to identify a polypeptide of use in this aspect of the invention can be performed by a person skilled in the art.

Alternatively, test compounds may be supplied to the yeast cells described above and the expression of a reporter gene monitored. Compounds that may be of use in this aspect of the invention would result an increase of reporter gene expression, ie the compound inhibits the binding of ERZFP to the transcription factor, for example a nuclear receptor DNA binding protein. Again, this screen and the necessary controls can be performed by a person skilled in the art.

As mentioned above, other compounds that may be of use in this aspect of the invention include those compounds that reduces the amount of ERZFP in the cell.

An embodiment of the eleventh aspect of the invention is wherein the compound which reduces the amount of ERZFP in the cell is an antisense or triplex-forming oligonucleotide or peptide nucleic acid (PNA) which binds to a polynucleotide encoding ERZFP.

Antisense oligonucleotides are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA- DNA duplex is formed. These nucleic acids are often termed"antisense" because they are complementary to the sense or coding strand of the gene.

Recently, formation of a triple helix has proven possible where the

oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise a sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.

By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly (A) addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene. Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts. Inhibition of viral function by antisense oligonucleotides has been demonstrated in vitro using oligonucleotides which were complementary to the AIDS HIV retrovirus RNA (Goodchild, J. 1988"Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides", Proc. Natl. Acad. Sci.

(USA) 85 (15), 5507-11). The Goodchild study showed that oligonucleotides that were most effective were complementary to the poly (A) signal; also effective were those targeted at the 5'end of the RNA,

particularly the cap and 5'untranslated region, next to the primer binding site and at the primer binding site. The cap, 5'untranslated region, and poly (A) signal lie within the sequence repeated at the ends of retrovirus RNA (R region) and the oligonucleotides complementary to these may bind twice to the RNA.

Typically, antisense oligonucleotides are 15 to 35 bases in length. For example, 20-mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et al, Breast Cancer Res Treat 53: 41-50 (1999) ) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, J Neurosurg 91: 261-7 (1999)).

However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10,11, 12,13, or 14 bases, or 36, 37,38, 39 or 40 bases.

Also included in this aspect of the invention is the reduction of ERZFP by the method of RNA interference, a form of antisense.

A twelfth aspect of the invention is a method for modulating, for example promoting, the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein in a cell, the method comprising the steps of : (a) providing a compound as defined in the eleventh aspect of the invention or a polynucleotide encoding the compound; and, (b) exposing the cell to the compound or to the polynucleotide encoding the compound.

Preferences as to the promotion of the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein are those provided in the eleventh aspect of the invention.

A further embodiment of the eighth, ninth, tenth, eleventh or twelfth aspects of the invention is wherein the nuclear receptor DNA binding protein is a steroid hormone receptor protein, for example an estrogen receptor (ER), for example ERa Steroid hormone receptor proteins, including estrogen receptors, have been discussed above (see Mangelsdorf et al (1995) Cell 83, 835-839 for a review and nomenclature). The polynucleotide sequence encoding these proteins is well known (for example, the coding sequence of a human estrogen receptor a is provided in GenBank accession X03635.

A thirteenth aspect of the invention is a method for identifying a compound which modulates, for example promotes the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein, comprising the steps of : (a) providing the transcription factor, for example a nuclear receptor DNA binding protein or a fragment thereof comprising the AF1 domain or fragment thereof, and a compound as defined in the eighth aspect of the invention; (b) exposing a test compound to the transcription factor, for example a nuclear receptor DNA binding protein or fragment and/or a compound as defined in the eighth aspect of the invention; (c) determining whether the test compound modulates, for example inhibits, the ability of the transcription factor, for example a nuclear receptor DNA binding protein or fragment to bind to the compound as defined in the eighth aspect of the invention;

(d) selecting a compound which modulates, for example inhibits, the ability of the transcription factor, for example a nuclear receptor DNA binding protein or fragment to bind to the compound as defined in the eighth aspect of the invention.

An example of this method of the invention was provided in the eleventh aspect of the invention, ie a derivative of the yeast two-hybrid library screening system.

Alternatively, the method of this aspect of the invention may be performed in vitro. For example, the ability of the transcription factor, for example a nuclear receptor DNA binding protein or fragment to bind to the compound as defined in the eighth aspect of the invention may be monitored by measuring any change in amount of the transcription factor, for example a nuclear receptor DNA binding protein/compound complex using non- denaturing polyacrylamide gel electrophoresis. Other methods of monitoring in vitro protein/compound binding are well known to those skilled in the art.

A fourteenth aspect of the invention is a method for identifying a compound which modulates, for example inhibits, the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein, comprising the steps of : (a) providing the transcription factor, for example a nuclear receptor DNA binding protein or fragment thereof comprising the API domain or fragment thereof, and optionally a compound as defined in the eighth aspect of the invention; (b) exposing a test compound to the transcription factor, for example a nuclear receptor DNA binding protein or fragment, and/or compound as defined in the eighth aspect of the invention;

(c) determining whether the test compound modulates, for example promotes the ability of the transcription factor, for example a nuclear receptor DNA binding protein or fragment to bind to the compound as defined in the eighth aspect of the invention or whether the test compound mimics the effect of the binding of the compound as defined in the first aspect of the invention to the transcription factor, for example a nuclear receptor DNA binding protein or fragment; (d) selecting a compound which modulates, for example promotes the ability of the transcription factor, for example a nuclear receptor DNA binding protein or fragment to bind to the compound as defined in the eighth aspect of the invention or which mimics the effect of the binding of the compound as defined in the eighth aspect of the invention to the transcription factor, for example a nuclear receptor DNA binding protein or fragment.

Examples of this method of the invention may be performed are provided in the previous aspect of the invention, with the exception that the compounds selected would be those that promote the ability of the transcription factor, for example a nuclear receptor DNA binding protein or fragment to bind to the compound.

PCT/GB03/00878 describes a novel method for characterising the hormone dependency of a primary cancer cell or cells which comprises the steps of (1) exposing the cell or cells to a recombinant viral vector comprising a reporter gene which comprises a hormone-dependent promoter; and (2) assessing the expression of the reporter gene in the said cell or cells. The method is also presented in Kothari et al (2003) Br J Cancer 88 (7): 1071-6.

Such a method may be useful in compound screening methods of the thirteenth and fourteenth aspects of the invention in which the effect of a

test compound on the interaction between ERZFP and a transcription factor, for example a nuclear receptor DNA binding protein is assessed.

The screening methods of the thirteenth and fourteenth aspects of the invention could be conducted using, for example, African Green Kidney Monkey cells that are estrogen receptor-negative or Era positive breast cancer cell lines, e. g. MCF7 or T47D. The cell lines could be modified to include a stable integration of an estrogen-responsive reporter (e. g. luciferase).

A further embodiment of the thirteenth or fourteenth aspects of the invention is wherein step (c) comprises the step of measuring transcription or translation from a reporter gene under the control of the transcription factor, for example a nuclear receptor DNA binding protein. Methods of measuring transcription or translation from a reporter gene are provided below.

A fifteenth aspect of the invention is a compound identified or identifiable by the method of the thirteenth or fourteenth aspects of the invention.

An embodiment of the fifteenth aspect of the invention is wherein the compound promotes the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein.

A further embodiment of the fifteenth aspect of the invention is wherein the compound inhibits the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein.

A sixteenth aspect of the invention is a compound as defined in fifteenth aspect of the invention for use in medicine.

A seventeenth aspect of the invention is a method for treating a patient with a hormone-dependent type of cancer comprising administering to the patient a compound as defined in the ninth aspect of the invention or the sixteenth aspect of the invention where the compound inhibits the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein.

By'hormone-dependent type of cancer'we include breast cancer, endometrial cancer, ovarian cancer, thyroid cancer and prostate cancer as discussed above.

At least two different types of EC are thought to exist (see Emons et al (2000) Endocr Relat Cancer 7 (4), 227-242). Type I is associated with an estrogen-dominated hormonal environment, endometrioid histology and development from endometrial hyperplasia; this form is considered to be responsive to endocrine therapy. Type II is not associated with an estrogen- dominated hormonal environment, has a serous histology and develops from atrophic endometrium in elderly women; this form is not considered to be responsive to endocrine therapy.

An eighteenth aspect of the invention is a method for treating a patient with post-menopausal problems, for example bone loss, comprising administering to the patient a compound or polynucleotide as defined in the twelfth aspect of the invention or the sixteenth aspect of the invention where the compound promotes the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein.

A nineteenth aspect of the invention is the use of a compound as defined in ninth aspect of the invention or the sixteenth aspect of the invention where

the compound inhibits the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein in the manufacture of a medicament for treating a patient with a hormone- dependent type of cancer.

A twentieth aspect of the invention is the use of a compound or polynucleotide as defined in twelfth aspect of the invention or the sixteenth aspect of the invention where the compound promotes the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein in the manufacture of a medicament for treating a patient in need of promotion of the transcription factor activity.

An embodiment of the seventeenth or nineteenth aspects of the invention is wherein the cancer is breast cancer or an endometrial cancer (EC) or ovarian cancer. A further embodiment of these aspects of the invention is wherein the patient has an estrogen receptor positive form of breast cancer.

A further embodiment of these aspects of the invention is wherein the patient has estrogen-independent estrogen receptor transcription factor activity form of breast cancer.

The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.

Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier (s) must be"acceptable"in the sense of being compatible with the compound of the invention and not deleterious to

the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.

A twenty-first aspect of the invention is the method or use of the seventeenth or nineteenth aspects of the invention in which the patient is selected on the basis of a method for providing information for deciding on a therapeutic strategy for a patient, comprising the step of assessing the activity of the transcription factor, for example a nuclear receptor DNA binding protein transcription factor in a cell or cells from the patient, comprising the steps of : (1) exposing the cell or cells to a recombinant viral vector comprising a reporter gene which comprises a promoter under the control of the transcription factor, for example a nuclear receptor DNA binding protein transcription factor (transcription factor-dependent promoter); and, (2) assessing the expression of the reporter gene in the said cell or cells.

A twenty-second aspect of the invention is the method or use of the seventeenth or nineteenth aspects of the invention in which the patient is selected on the basis of a method for characterising the hormone dependency of a primary cancer cell or cells, comprising the steps of : (1) exposing the cell or cells to a recombinant viral vector comprising a reporter gene which comprises a hormone-dependent promoter; and, (2) assessing the expression of the reporter gene in the said cell or cells.

A method which can be of use in the twenty-first and twenty-second aspects of the invention is provided in PCT/GB03/00878 and is also presented in Kothari et al (2003) Br J Cancer 88 (7): 1071-6.

PCT/GB03/00878 and Kothari et al (2003) Br J Cancer 88 (7): 1071- 6describe a novel functional assay based on a transcription factor reporter

gene (for example a reporter gene under the control of a hormone response element) in which purifiedprima7y cells from a patient, for example cancer cells, maintained in short term cultures, can be infected with the reporter gene. Using this assay, transcription factor, for example hormone receptor, activity can be assessed reliably, in vitro, in primary cells, for example primary cancer cells. The information obtained can be used in guiding treatment of the patient. This may be useful in relation to determining which patients may be particularly suitable for treatment using the compositions or methods of the present invention.

The information regarding the activity of the transcription factor is used in deciding on a therapeutic strategy for the patient. For example, the information may be used in deciding whether the patient has a particular disease or condition, or in further characterising the form of the disease or condition that the patient has. The therapeutic strategy may be a prophylactic strategy, for example if the patient has no (for example if there is a family history of a particular disease or condition) or preliminary symptoms. By therapeutic strategy or regime is included a strategy concerning, for example, surgery, administration of pharmaceutical compounds and/or diet.

In a preferred embodiment the transcription factor is a nuclear hormone receptor protein and the reporter gene comprises a response element for the nuclear hormone receptor protein. Thus, the reporter gene preferably comprises a hormone-dependent promoter.

Preferably the hormone-dependent promoter is an estrogen-dependent promoter. Thus, the promoter preferably comprises one or more Estrogen Response Elements (EREs), for example two EREs. EREs are found in estrogen-regulated genes. Still more preferably, the Estrogen Response Element is the Estrogen Response Element derived from the progesterone

receptor (PR) gene or the PS2 (trefoil related protein) gene. The ERE has several different sequences that may be used in the construct (Klinge et al.

Estrogen Receptor Interaction with estrogen response elements. Nucleic Acids Res 2001.29 (14): 2905-19).

Hence an embodiment of the twenty-first or twenty-second aspects of the invention is wherein the transcription factor-dependent promoter or hormone-dependent promoter is an estrogen-dependent promoter. A further embodiment of these aspects of the invention is wherein the promoter comprises one or more Estrogen Response Elements (EREs).

Alternatively, the hormone-dependent promoter may be an androgen- dependent promoter (ie comprising one or more Androgen Response Elements (AREs) ) or a thyroid hormone dependent promoter (ie comprising a Thyroid hormone Response Element (TRE) or a progesterone receptor dependent promoter or other steroid hormone dependent promoter or retinoic acid receptor dependent promoter or PPAR (peroxisome proliferator receptor) or VDR (vitamin D receptor) dependent promoter.

Thus, the hormone-dependent promoter comprises a binding site for a nuclear receptor DNA binding protein such as a steroid hormone receptor protein, as well known to those skilled in the art. The nuclear receptor DNA binding protein superfamily includes estrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR), retinoic acid receptor (RAR), peroxisome-proliferator activated receptors (PPAR), thyroid hormone receptor and the like (see Mangelsdorf et al (1995) Cell 83, 835- 839 for a review and nomenclature; also Chawla et al (2001) Nuclear receptors and lipid physiology: opening the X-files Science 294 (5548), 1866-1870).

Hence a further embodiment of the twenty-second aspect of the invention is wherein the hormone-dependent promoter is an androgen-dependent promoter or a thyroid hormone dependent promoter or a retinoic acid dependent promoter or a progesterone-dependent promoter or a PPAR (peroxisome proliferator receptor) or VDR (vitamin D receptor) dependent promoter.

As noted above, the reporter gene comprises a transcription factor- dependent promoter or a hormone-dependent promoter. Thus, transcription of the reporter gene is considered to be under the control of the promoter.

The transcribed nucleic acid may be any nucleic acid whose transcription is detectable, for example leads to the production of a detectable product.

The detectable product may be the transcribed RNA (including a portion or processed version thereof) ; this may be detected by techniques well known to those skilled in the art, for example a technique involving hybridisation of the transcribed RNA (or portion thereof) to one or more complementary nucleic acids. For example, the transcribed RNA may be detected by a technique involving the polymerase chain reaction (PCR) or other amplification reaction; preferably the technique involves quantitative reverse-transcriptase PCR (RT-PCR). Suitable techniques may be described in, for example, Van Trappen PO et al. Molecular quantification and mapping of lymph-node micrometastases in cervical cancer. Lancet 2001 Jan 6; 357 (9249): 15-20, Aerts J et al. A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood. Ann Oncol. 2001 Jan; 12 (1) : 39-46.

Alternatively or additionally, the transcribed RNA may encode a polypeptide which may be detected immunologically or as a result of its enzymatic or other biological activity. For example, the transcribed RNA

may encode an enzyme such as (3-galactosidase which is capable of producing a colour change (or fluorescence change) in a reagent (for example IPTG or CRPG (chloro-phenol-red-guanothiocyanate)).

The transcribed RNA may encode a luciferase enzyme (for example from firefly (Photinus pyralis) or seapansy (Renilla reiformis)). Luciferase catalyses a reaction involving D-luciferin which results in light emission, as well known to those skilled in the art. Alternatively, the transcribed RNA may encode a fluorescent protein, for example a protein belonging to the Green Fluorescent Protein (GFP) family (for example the Green Fluorescent protein from Aqueorea victoria). GFPs are intrinsically fluorescent proteins, which may emit blue, yellow light or green light. Miyawaki et al (1997) Nature 388, 882-887 describes a GFP-based Ca2+ sensing system; Mitra et al (1996) Gene 173,13-17 describes a two-GFP-based system for use in identifying protease inhibitors; WO 97/28261 discloses a two-GFP system in which the GFP donor and GFP acceptor are linked by a peptide containing a protease cleavage site. WO 95/07463 describes uses of GFPs ; WO 96/23898 relates to a method of detecting biologically active substances using GFPs ; Heim & Tsien (1996) Current Biology 6, 178-182 relates to engineered GFPs with improved brightness, longer wavelengths and fluorescence resonance energy transfer (FRET); Poppenborg et al (1997) J. Biotechnol. 58,79-88 relates to GFPs as a reporter for bioprocess monitoring; Park & Raines (1997) Protein Science 6,2344-2349 relates to a GFP as a signal for protein-protein interactions; Niswender et al (1995) J.

Microscopy 180,109-116 relates to quantitative imaging of GFP in cultured cells; Chalfie et al (1994) Science 263, 802-805 relates to GFP as a marker for gene expression; Hampton et al (1996) Proc. Natl. Acad. Sci. USA 93, 828-833 relates to the in vivo examination of membrane protein localisation and degradation with GFP; Heim et al (1995) Nature 373,663-664 relates to mutant GFPs with altered fluorescent properties; Mosser et al (1997)

BioTechniques 22,150-161 relates to the use of a dicistronic expression cassette encoding GFP for the screening and selection of cells expressing inducible gene products; Suarez et al (1997) Gene 196,69-74 relates to GFP-based reporter systems for genetic analysis of bacteria; Niedenthal et al (1996) Yeast 12,773-778 relates to GFP as a marker for gene expression and subcellular localisation in budding yeast; Mahajan et al (1998) Nature Biotech. 16,547-552 relates to the probing of Bel-2 and Bax interactions in mitochondria using GFPs and FRET; and Prescott et al (1997) FEBS Lett 411,97-101 relates to the use of GFP as a marker for assembled mitochondrial ATP synthase in yeast. GFPs and their uses have been reviewed in Pozzan et al (1997) Nature 388, 8340-835, Misteli & Spector (1997) Nature Biotechnology 15,961-964 ; and Cubitt et al (1995) Trends Biochem. Sci. 20, 448-455.

GFPs may be particularly useful in that more than one reporter gene may be introduced into the cell (as discussed further below), each expressing a GFP with a different wavelength of excitation and/or emission, so that the output of each reporter gene may be distinguished.

As a further alternative the reporter gene may encode chloramphenicol acetyltransferase (CAT), as well known to those skilled in the art. CAT catalyses the transfer of the acetyl group from acetyl-CoA to choramphenicol. CAT may be quantified immunologically, for example using an ELISA assay, as well known to those skilled in the art.

Alternatively, it may be quantified by measuring enzymic activity, for example by following the appearance of acetylated chloramphenicol (for example using radioactively labelled choramphenicol (for example [14C] choramphenicol) and physical separation means (for example thin layer chromatography or organic extraction) ). The reporter gene may alternatively encode secreted alkaline phosphatase (SEAP), which can be

tested using medium from infected cells (Cullen & Malim (1992) Meth.

Enzymol. 216: 362-368; Kain (1996) Use of secreted alkaline phosphatase as a reporter of gene expression in mammalian cells. Methods in Molecular Biology vol 63 Humana Press Totowa NJ). The reporter gene may alternatively encode a tagged protein, for instance with a 6-His tag that my be detected using non-immunological methods.

Methods and apparatus for detecting and/or quantifying reporter gene products or their enzymic products will be well known to those skilled in the art, and may include measurements using a spectrophotometer, fluorometer or luminometer. Other suitable methods, systems or instruments (depending on the reporter gene, as will be clear to the skilled person) include Light-Cycler (D, Taqman (g), histology, microscopy, radiography or LCM.

The term"viral vector"will be well known to those skilled in the art. It is particularly preferred that the viral vector is an adenoviral vector, though it may alternatively be a lentiviral vector. It encompasses any viral vector, for example adenoviral vector or lentiviral vector that is suitable for introducing recombinant nucleic acid into a eukaryotic cell, preferably a human cell.

HSV, AAV, vaccina and parvovirus vectors may also be suitable.

Preferred lentiviral vectors include those described in Verma & Somia (1997) Nature 389,239-242. Preferred vectors include lentivirus vectors and adenoviral vectors, for example vectors similar to those described in Foxwell et al (2000) Atzn Rheum Dis 59 Suppl 1, I54-59 or Bondeson et al (2000) JRheumatol 27 (9), 2078-2089.

The adenoviral vector (for example) may be any serotype of adenovirus but is preferably one that is capable of infecting or otherwise introducing

recombinant nucleic acid into a human cell. Preferably it is of serotype 5 (see, for example, Shenk (1996) Fields Virology Fields et al Eds (Lippincott, Philadelphia), pp 2111-2148; Horwitz (1996) Fields Virology Fields et al Eds (Lippincott, Philadelphia), pp 22149-2171. It may comprise a complete adenoviral virion, consisting of a core of nucleic acid and a protein capsid. Alternatively, it may comprise a naked adenoviral genome or a protein capsid with a minimal adenoviral genome comprising the non- adenoviral nucleic acid and adenoviral packaging signal but with most of the adenoviral genome deleted; this may be termed a"gutless"virus.

The viral vector, for example adenovirus vector to which the primary cancer cell or cells is exposed is preferably replication-deficient. Otherwise, it is likely to replicate and kill the cell.

It is particularly preferred that the adenoviral vector is an adenoviral vector as described in He et al (1998) PNAS 95,2509-2514. Characteristics of adenoviruses and adenoviral vectors are described in this reference and in references cited therein. The recombinant adenoviral vector to which the primary cancer cell or cells is exposed may be prepared using a method based on that described in He et al (1998), as described in PCT/GB03/00878 and Kothari et al (2003) Br J Cancer 88 (7): 1071-6. This method may allow incorporation of up to about 10kb of reporter gene sequence; multiple reporter genes may be incorporated into one adenoviral vector construct. This method makes use of recombination between the adenoviral genome (which may be modified; for example to render it replication-deficient) and nucleic acid comprising the reporter gene sequence (for example comprising an lacZ coding region under the control of an ERE), for example a plasmid comprising the reporter gene.

The adenoviral vector may comprise a non-hormone dependent reporter gene (for example capable of expressing a GFP), for example a reporter

gene under the control of a constitutive or unregulated promoter such as the CMV promoter. This may be useful in monitoring the level of cellular infection achieved. However, the presence of such a reporter gene may lead to a higher background level of expression of the hormone-regulated reporter gene (and/or lower induced level of expression of the hormone- regulated reporter gene) and may therefore not always be desirable.

Other methods of delivery include adenoviruses carrying external DNA via an antibody-polylysine bridge (see Curiel Prog Med. Virol. 40, 1-18) and transferrin-polycation conjugates as carriers (Wagner et al (1990) Proc.

Natl. Acad. Sci. USA 87,3410-3414). In the first of these methods a polycation-antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody. The polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone. The adenovirus, because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine.

The DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.

High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89,6094-6098 may also be used. This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example

transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.

Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2,660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein. Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells, such as those described in Bischoff et al (1996) Science 274,373-376 are also useful for delivering the genetic construct of the invention to a cell.

The recombinant viral, for example adenoviral, vector may comprise more than one transcription factor-controlled reporter gene. For example, breast cancer is characterised by increased proliferation and decreased apoptosis as well as increased invasiveness of luminal epithelial cells. The recombinant viral vector may comprise reporter genes suitable for providing information on each of these properties, in order to assist clinicians with devising suitable strategies to combat these adverse properties. Alternatively, the primary cancer cell or cells may be exposed to more than one recombinant viral vector, for example one comprising a hormone-dependent reporter gene and the second or further vectors comprising the further reporter genes relating to invasion, apoptosis or proliferation.

Analysis may be performed on a single cell, but it is preferred that several cells are analysed (either individually or as a group) and the results pooled.

This may mean that the results obtained are more representative of the cancer or tumour than may be results obtained from a single cell.

Methods by which single cells may be analysed include methods in which the technique of Laser Capture Microdissection (LCM) is used. This technique may be used to collect single cells or homogeneous cell populations for molecular analysis and is described in, for example, Jin et al (1999) Lab Invest 79 (4), 511-512; Simone et al (1998) Trends Genet 14 (7), 272-276; Luo et al (1999) Nature Med 5 (1), 117-122; Arcuturs Updates, for example June 1999 and February 1999; US 5,859, 699 (all incorporated herein by reference). The cells of interest are visualised, for example by immunohistochemical methods, and transferred to a polymer film that is activated by laser pulses. Microscopes useful in performing LCM are manufactured by Arcturus Engineering, Inc. , 1220 Terra Bella Avenue, Mountain View, CA 94042, USA.

LCM may be used with other isolation/detection methods. For example, LCM may be used following an isolation/detection method which enriches the sample for the target cell type. LCM may be particularly useful in the analysis of primary cancer cells obtained from blood.

Because cells may not be alive following LCM, it may be most appropriate to use LCM in the analysis of cells following introduction of the reporter gene into the cells rather than prior to introduction of the reporter gene into the cells.

It will be appreciated that it may be desirable to expose the recombinant viral vector, for example adenovirus, to cell line cells, for example cancerous cell line cells, for example in order to determine suitable conditions for cell infection and reporter gene assays.

A twenty-third aspect of the invention provides a recombinant viral, preferably adenoviral, vector comprising a reporter gene which comprises a

hormone-dependent promoter for use in a method for identifying a compound which modulates or mimics the interaction between ERZFP and a transcription factor, for example a nuclear receptor DNA binding protein.

The method comprises: (a) providing the transcription factor, for example a nuclear receptor DNA binding protein or fragment thereof comprising the API domain or fragment thereof and, optionally, a compound as defined in the eighth aspect of the invention; (b) exposing a test compound to the transcription factor, for example a nuclear receptor DNA binding protein or fragment and, optionally, the compound as defined in the eighth aspect of the invention; (c) determining whether the test compound modulates or mimics the interaction between ERZFP and a transcription factor, for example a nuclear receptor DNA binding protein by measuring the activity of the reporter gene as a means of assessing the transcription factor activity of the transcription factor, for example a nuclear receptor DNA binding protein or fragment.

Expression of the hormone-dependent reporter gene in response to various agents may be judged by reference to expression of a control gene, or may be judged by reference to expression in the absence of the hormone (for example estrogen), for example as described in PCT/GB03/00878 and Kothari et al (2003) Br J Cancer 88 (7): 1071-6. A change of expression level by a factor of at least 2, more preferably 3,4, 5,6, 8 or 10 may be considered a significant activation or inhibition, as appropriate.

The recombinant viral, preferably adenoviral, vector of the invention (or vector for preparing such a viral, preferably adenoviral, vector) may comprise further reporter genes, as discussed above. However, in a preferred embodiment the adenoviral vector does not comprise a reporter gene under the control of a constitutive promoter, for example a GFP-

expressing reporter gene. It is preferred that the reporter gene comprising the hormone-dependent promoter exhibits hormone-dependent transcription (ie an increase in transcription (preferably of at least 2-fold, still more preferably at least 3,4 or 5-fold)), for example relative to a marker plasmid (for example GTK-CAT plasmid) in MCF7 cells transfected using the calcium phosphate method, as described in PCT/GB03/00878 and Kothari et al (2003) Br J Cancer 88 (7): 1071-6.

A twenty-fourth aspect of the invention is a fragment of ERZFP which binds to a transcription factor or the AF1 domain of a transcription factor, for example a nuclear receptor DNA binding protein, for example the estrogen receptor, for example ERa. Such a fragment may be identified by methods for assessing polypeptide interactions, as well known to those skilled in the art and as described herein.

A twenty-fifth aspect of the invention is a fragment of the AF1 domain of a transcription factor, for example a nuclear receptor DNA binding protein, for example the estrogen receptor, for example ERa, which binds to ERZFP. The AF1 domain is highly variable in length, see, for example, Chawla et al (2001) Science 294,1866-1870 for a review of this topic. An example of a AF1 domain included in this aspect of the invention is amino acids 1 to 180 of human estrogen receptor a. Such a fragment may be identified by methods for assessing polypeptide interactions, as well known to those skilled in the art and as described herein.

A twenty-sixth aspect of the invention is a transgenic animal overexpressing ERZFP or a fragment, variant, derivative or fusion thereof.

A twenty-seventh aspect of the invention is a transgenic animal underexpressing ERZFP.

Transgenic animal models may be used to assess the activity of compounds of the invention to mediate the transcription factor activity of a transcription factor, for example a nuclear receptor DNA binding protein, for example an estrogen receptor. For example, a transgenic rat in which uninduced mammary tumours develop can be used for testing anti-estrogens. A further example is a nude mouse in which breast cancer cells from cell lines are injected to give tumour growth The following documents, particularly WO 95/14377, provide details on how transgenic animals may be prepared: WO 95/14377-transgenic mouse models; particularly human CD4+, mouse CD4-/-, mouse CD8-/-, human DQw6+ (hCD4+, mCD4-/-, mCD8- /). Other models are also referred to on pages 5 to 6; for example Nishimura et al (1990) J Imnaasnol 145, 353-360 describing a mouse carrying the transgenes encoding the alpha and beta chains of the human HLA-DQw6 protein).

Sriskandan et al (2001) J Infect Dis 184,166-173 and Unnikrishnan et al (2002) J Immunol 169, 2561-2569 and references therein-C57BL/10. DQ8 (carrying genomic constructs for DQA1*0301 and DQB*0302) and FVB/N. DR1 (carrying genomic constructs for DRA1*0101 and DRB 1*01 01) Welcher et al (2002) J hifect I ? es 186, 501-510 and DaSilva et al (2002) J Infect Dis 185,1754-1760 and references therein-HLA Class II transgenic mice (HLA-DR2 (HLA-DR2ß with murine IEα), HLA-DR3 and HLA- DQ8/human CD4+) on a MHC Class II knockout (Ap°) background.

A twenty-eighth aspect of the invention is a pharmaceutical composition comprising a compound as defined the eighth aspect of the invention and a drug which lowers oestrogen levels, for example an aromatase inhibitor or LHRH agonist, and a pharmaceutially acceptable carrier. Compounds that are suitable for this aspect of the invention include tamoxifen (see Ali and Coombes, (2002) Nature Cancer Reviews 2,101-112).

A twenty-ninth aspect of the invention is a pharmaceutical composition comprising a compound as defined the eighth aspect of the invention and an epidermal growth factor receptor (EGFR) antagonist and a pharmaceutially acceptable carrier. Compounds that are suitable for this aspect of the invention include Iressa (ZD1839 ; AstraZeneca).

A thirtieth aspect of the invention is a pharmaceutical composition comprising a compound as defined the eighth aspect of the invention and an inhibitor of ErbB2 or MEK signalling and a pharmaceutially acceptable carrier.

A thirty-first aspect of the invention is a pharmaceutical composition comprising a compound as defined the eighth aspect of the invention and an antiestrogen. Compounds that are suitable for this aspect of the invention include Faslodex (ICI 182,780 ; AstraZeneca), idoxifene (see Coombes et al (1995) Cancer Res 55, 1070-1074) and raloxifene.

A thirty-second aspect of the invention is a kit of parts comprising a compound as defined in the eighth aspect of the invention or a fragment as defined in the twenty-fourth aspect of the invention, and a transcription factor, for example a nuclear receptor DNA binding protein or fragment as defined in the twenty-fourth aspect of the invention.

A thirty-third aspect of the invention provides a kit of parts comprising a recombinant adenoviral vector as defined above and an ERZFP polypeptide and the hormone on which the hormone-dependent promoter is dependent (or an analogue thereof which is able to promote transcription from the reporter ie an agonist of the hormone receptor), and optionally also an antagonist of the hormone receptor and/or a partial antagonist of the hormone receptor. Thus, in a preferred embodiment, the invention provides a recombinant adenoviral vector comprising an estrogen-dependent reporter gene, estrogen (preferably 17 (3-estradiol ; E2) and optionally Tamoxifen (preferably as 4-OHT (4-hydroxytamoxifen) ) and/or Faslodex (ICI 182, 780) or other antiestrogen. The kit may also comprise a substrate for a reporter gene, if appropriate, for example a substrate for P-galactosidase.

E2 and 4-OHT may be purchased from Sigma-Aldrich Company Ltd (a catalogue may be ordered from The Old Brickyard, New Road, Gillingham, Dorest, SP8 4BR). Faslodex may be purchased from Toclis Cookson Ltd, Northpoint, Fourth Way, Avonmouth, Bristol BS 11 8TA. B-galactosiadase is part of an ELISA assays from Roche, UK (an online catalogue can be found at www. rocheuk. com).

All patent or other documents cited herein are hereby incorporated by reference.

The invention is now described further by reference to the following non- limiting Figures and Examples.

Figure 1: Amino acid and nucleotide sequence of ERZFP (A) Protein sequence of ERZFP (B) Nucleotide sequence of ERZFP

Figure 2: hERa structure and phosphorylation sites.

A schematic representation of the human ERa polypeptide. AF-1, activation function; DBD, DNA binding domain; LBD, ligand binding domain.

Figure 3: Modified yeast two-hybrid assay A schematic representation of the yeast two-hybrid assay used to identify proteins which interact with ERa. ERE, oestrogen response element; URA3, uracil biosynthesis gene; DBD, DNA binding domain; AF, activation function; AD activation domain.

Figure 4: ERZFP structure A schematic representation of the ERZFP polypeptide.

Figure 5: Alignment of ERZFP with similar vertebrate polypeptides Sequence similarity between the human ERZFP polypeptide and related murine and fugu sequences. Murine, GenBank accession number XP-138810 ; Fugu, GenBank accession number AAC60294; Human, Genbank Accession No NM-152625 ; Porcine, Genbank Accession No BI184476.

Figure 6: ERZFP interacts with ERa in yeast Growth of yeast colonies in the absence of uracil when transformed with the indicated vectors. A description of the vectors is given in Example 1.

Transformation of zinc finger clone reproduced the uracil independent growth seen in the original screening. Interaction takes place with full length receptor in the presence of E2 and not in its absence (data not shown). ERZFP enables growth in the presence of OHT providing further evidence of its interaction with API domain.

Figure 7: ERZFP protein interacts with hERa AF1 domain and AF2 domain in the presence of oestrogen.

A description of the experimental methods and vectors used is given in Example 1.

Figure 8: ERZFP interacts with a known co-repressor protein (A) GST pull down experiment showing interaction between ERZFP and RIP and CtBP.

(B) Transcription factor activity of ERa as measured by luciferase activity.

Figure 9: Northern blot analysis of ERZFP tissue expression Expression of ERZFP in human tissues. The methodology used is given in Example 1.

Figure 10: Effect of ERZFP on the transcription factor activity of ERa.

The effect of varying levels of ERZFP on the transcription factor activity of ERa was measued using the LUC activity of a ERa responsive reporter gene. The methods used are outlined in Example 1.

Figure 11. Shown is the predicted sequence of ERZNF/ZNF366 (human), together with the predicted amino acid sequence (A). The ZNF366 gene encodes a 744 amino acid polypeptide, containing 11 C2H2-type zinc finger motifs, two putative CtBP binding motifs (shaded box) and a putative nuclear receptor L-X-X-L-L-type nuclear receptor co-activator motif (open box). The cDNA clone identified through the yeast 2-hybrid screen encodes bp 443-2755 of the predicted ZNF366 gene sequence. (B) A schematic representation of the ZNF366 open reading frame showing the positions of the 11 C2H2-type zinc fingers, the potential CtBP binding motifs and the nuclear receptor (NR) co-activator motif.

Figure 12. A) One colony from co-transformation of PLIa yeast strain with pACTII-ZNF366 clone and 1) pBridge vector, 2) pBridge-HE15 (ERa- AAF2), 3) pBridge-HE15118A (in which a serine residue at position 118 has been substituted by alanine), 4) pBridge-HE15118E, or 5) pACTII vector, was diluted in 100 pl of water, followed by plating of 5ß1 of 10-3, 10- 4, 10-5 and 10-6 dilutions on YNB plates lacking uracil, tryptophan and leucine.

B) One colony from co-transformation of PLla yeast strain with pACTII- ZNF366 clone and 1) pBridge vector, 2) pBridge-ER (x-llSE, or 3) pACTII vector, were plated on YNB medium lacking uracil, tryptophan or leucine and containing 10-9 M 17B-estradiol (E2), 10-6 M 4-hydroxytamoxifen (OHT) or with an equivalent volume of ethanol (used to solubilise E2 and OHT), was diluted in 100 gl of water, followed by plating of 51 of 10-3, 10- 4, 10-5 and 10-6 dilutions on YNB plates lacking uracil, tryptophan and leucine.

Figure 13. Schematic representation of ZNF366 and deletion mutants used in the studies outlined here. The deletion mutants were generated by in Vit7'0 site-directed mutagenesis using a kit from Stratagene UK. For the generation of C-terminal deletions, site-directed mutagenesis was used to introduce a stop codon and a XhoI site at the 3'end, thus enabling recloning of the portion of interest. For the generation of deletion mutants lacking N- terminal sequences a BamHI site was introduced by site-directed mutagenesis. The numbers refer to amino acid numbers, thus ZNF366- A252-744 lacks amino acids 252-744, as define in figure 1. All mutants were verified by automated DNA sequencing.

Figure 14. Schematic representation of human estrogen receptor a and deletion mutants used in the studies outlined here. ERa-AF2 used for

making GST fusion proteins is the mouse ERa AF2. These constructs have been described (Chen et al. (2003) J. Biol. Chem. 278: 38586-38592).

Figure 15. GST pulldowns of [35S]-labelled ZNF366 (here named ERZFP) were performed using glutathione-S-transferase (GST), GST-ERa-AF2 or GST-HE15 (ERa-AAF2). Ligands (17B-estradiol (E2), 4-hydroxytamoxifen (OHT) or ICI 182,780 (ICI) ) were added in the case of GST-AF2 to a final concentration of 1 u. M. The input lane represents 10% of the in vitro translation used for the pulldowns for each protein. In vitro transcription/translation was performed using a kit from Promega, UK. GST fusion proteins were made as described (Chen et al. (2003) J. Biol. Chem.

278: 38586-38592).

Figure 6. GST pulldowns of [35S]-labelled ERa were performed using glutathione-S-transferase (GST), or GST-ERZFP. Ligands (17B-estradiol (E2), 4-hydroxytamoxifen (OHT) or ICI 182, 780 (ICI) ) were added to a final concentration of 1 uM. As the ligands were prepared in ethanol, an equal volume of ethanol (etoh) was added to the no ligand control. The input lane represents 10% of the in vitro translation used for the pulldowns for each protein. In vitro transcription/translation was performed using a kit from Promega, UK. GST fusion proteins were made as described (Chen et al. (2003) J. Biol. Chem. 278: 38586-38592).

Figure 7. GST pulldowns of [35S]-labelled ERa were performed using glutathione-S-transferase (GST), GST-ZNF366 (lane 3), GST-ZNF366- A252-744 (lane 4), ZNF366-A453-744 (lane 5), ZNF366-A568-744 (lane 6), ZNF366-A1-238 (lane 7), ZNF366-A1-444 (lane 8), or ZNF366-A1-557 (lane 9). 100 nM 17B-estradiol (E2), 4-hydroxytamoxifen (OHT) or ICI 182,780 (ICI) ) was added, as appropriate. The input lane represents 10% of

the in vitro translation used for the pulldowns for each protein. In vitro transcription/translation was performed using a kit from Promega, UK. GST fusion proteins were made as described (Chen et al. (2003) J. Biol. Chem.

278 : 38586-38592).

Figure 8. COS-1 cells were grown in Dulbecco's medium without phenol red (GIBCO) supplemented with 5% dextran-coated stripped serum, and 5mls of L-Glutamine-penicillin-Streptomycin solution (SIGMA) to 80- 100% confluency. At this point cells were trypsinized and counted, plating them at 5 x 105 cells per well in 24 well plates. Transient transfections were carried using lipofectamine 2000 Reagent (Invitrogen, UK) as indicated in the manufacturer's instructions. COS-1 cells were transfected with a luciferase reporter gene, pXPERE3, controlled by a B-globin minimal promoter and 3 EREs from the Chicken Vitallogenin gene (100ng), renilla control reporter gene pRLTK (100ng), wild type ERa (lOng of pSG5- HEGO), carrier DNA (500ng of BSM) and increasing amounts of pCMVSPORT (6)-ZNF366 (0, 5,10, 25,50, 75 or 100 ng). Amounts of DNA were made equal for each transfection by adding corresponding amounts of pSG5 vector (0-100ng). Estrogen (E2; 10 nM), 4- hydroxytamoxifen (OHT; 100 nM) or ICI 182, 780 (ICI; 100 nM) were added 5 hours after transfection. Cells were harvested after a further 24 hours. All raw data was made relative to the internal renilla control and induction of the reporter in the presence of E2 was adjusted to 100%. All experiments were repeated at least 3 times and results are presented as the standard error of the mean error bars. Details have previously been described (Chen et al. (2003) J. Biol. Chem. 278: 38586-38592).

Figure 19. Multiple tissue northern (MTM) blots I and II (Clontech) were hybridized with cDNA probes by standard methods, according to the manufacturers instructions. Probes were synthesized by random primer

labelling with a-32P dCTP using the Rediprime II kit (Amersham) and purified over Microspin G-50 columns (Amersham). Blots were prehybridized for 2 hours at 65 C prior to addition of denatured probe, then hybridized overnight. Blots were washed three times in 1X SSC, 0.1% SDS at room temperature, then three times in 0. 1X SSC, 0. 1% SDS at 65 C.

Blots were viewed by phosphorimager analysis (Molecular Dynamics Typhoon 8600). The ERZFP probe template was a 800bp PCR product from ZNF366, corresponding to the NH2-terminus coding sequence lacking zinc- finger motifs. The SRC1 template was a 700bp restriction fragment lacking LXXLL coding motifs. The ERa template was full-length human estrogen receptor a coding sequence. B-actin template was provided in the kit.

Example 1: Determination of the mechanisms by which phosphorylation of the oestrogen receptor alpha modulates its activity Abstract Oestrogen regulates growth of a large proportion of breast cancers. Its action is mediated by two transcription factors in the nuclear receptor superfamily, oestrogen receptor a and ß (ERa and ER p). Current understanding indicates that oestrogen action in breast cancer is principally mediated through the action of ERa. ERa is overexpressed in a large proportion of breast cancers. Adjuvant therapy for ERa-positive breast cancers is therefore based on inhibiting the activity of ERa using inhibitors of oestrogen synthesis or oestrogen antagonists such as armidex and tamoxifen respectively. However, long term treatment leads to drug

resistance even though tumours remain ERa positive indicating a continued role for ERa in breast cancer cell proliferation.

In our lab we are investigating the mechanisms by which oestrogen receptors activate gene expression, and in particular the role of phosphorylation in regulating oestrogen receptor action. Phosphorylation of ERa at various residues exerts important effects on its function.

Phosphorylation of serine 118 in transcription activation function AF-1 by cdk7 and MAPK for instance result in ligand-independent transcriptional activation by ERa. It has therefore been proposed that increased ERa phosphorylation could result in its constitutive activation, thereby overcoming drug inhibition in breast cancer treatment.

At present the mechanisms through which AF-1 participates in transcriptional activation and in particular how phosphorylation stimulates AF-1 activity remain to be determined. One possibility is that phosphorylation results in the preferential recruitment of"co-activators"to AF-1. In order to identify interacting proteins that might preferentially interact with ERa phosphorylated at S118, a yeast two hybrid system was set up, using a mutant in which S118 has been replaced with glutamic acid as bait. We have identified a novel Cys2His2 zinc finger protein, that is highly conserved from the mouse to fugu. Interaction with ERa was confirmed in vitro by means of GST pull down experiments and demonstrated that this protein interacts with the AF-1 domain of the receptor in a ligand independent manner.

Background information Structural features of ERa The principal functional domains of ERa are a DNA binding domain composed of two zinc fingers, a ligand binding domain and two transactivation domains. Transcription activation is mediated by transcription activation function AF-1 and AF-2. A schematic diagram of ERa is shown in Figure 2.

ERa is extensively modified post-translationally (phosphorylation, acetylation, glycosylation). Phosphorylation of human ERa at its AF1 domain has been demonstrated on serine 104 and/or serine 106, serines 118 and 167. Phosphorylation on serine 104 and/or serine 106 has been attributed to Cdk2. Serine 167 is phosphorylated by pp9OrSkl and AKT.

Serine 118 is phosphorylated by MAPK in a ligand independent manner and by Cdk7 in the presence of E2. ERa phosphorylation is also stimulated by activation of a number of growth factor receptor signalling cascades. For example : EGF 4 MAPK 4 S I 18 phosphorylation in AF1 This can cause a ligand-independent and/or syngergistic increase in transcriptional activation. Therefore, phosphorylation causes a modulation of ERa activity.

Activation of ERa by oestrogenic ligands is associated with increase in overall receptor phosphorylation. Serine 118 is a major phosphorylation site in ERa. S 118 phosphorylation modulates transactivation by AF-1.

Results Identification of a polypeptide that interacts with era We wished to identify potential AF1 co-activator proteins that preferentially interact with hERa phosphorylated at S 118. Such proteins may mediate the transcription factor activity of ERa. Therefore, we used a ERa mutant-in which the serine at position 118 (Sl 18) of the API domain was replaced by glutamic acid such that the resulting ERa polypeptide would mimic S118 phosphorylated ERa.

The mutated API domain of the ERa polypeptide was used as the bait (HE15118E) in a modified yeast two-hybrid screen in which the prey library screened was a human placental cDNA expression library. A schematic outline of the screening strategy is shown in Figure 3.

From this screen we identified a novel Cys2His2 zinc finger protein as a potential ERa co-activator protein which we called ERZFP. A schematic diagram of the structure of ERZFP is shown in Figure 4. ERZFP was found to be similar to GenBank Accession Number NM_152625, a hypothetical Cys2His2 zinc finger protein.

By database searching we identified similar murine, porcine and fugu Cys2His2 zinc finger proteins (see Figure 5). The fugu protein had previously been identified as a zinc finger protein called ZP366 (Gilligan et al (2002) Gene 294,35-44).

We then confirmed that ERZFP reproduced the uracil independent growth seen in the yeast two-hybrid screen using both the mutated AF1 domain and the mutated full length ERa polypeptide (see Figure 6). As can be seen,

ERZFP interacts with the AF-1 domain of the receptor in a ligand independent manner and with the AF-2 domain in the presence of ligand.

From this we concluded that ERZFP is a polypeptide that can interact with ERa.

We then confirmed that ERZFP interacted with ERa i71 vitro by using a GST pull down experiment. The data from this is shown in Figure 7. This data demonstrates that the two proteins interact in a oestrogen dependent manner.

We have also shown that ERZFP interacts with known co-repressor proteins (see Figure 8). RIP140 is a nuclear factor which modulates transcriptional activation by the estrogen receptor (Cavailles et al (1995) EMBO J. 14 (15), 3741-3751; Genbank Accession No NM003489). CtBP is a transcriptional repressor Genbank Accession No U37408.

We also determined the tissue specific expression profile of ZN366 by northern blot analysis. As can been from Figure 9, the gene is widely expressed with the highest transcript levels to be found in placenta, spleen and heart.

ERZFP mediated ERa transcription factor activity.

We also assayed the effect of the protein on the transcription factor activity of Era using COS-1 cells. The results of this experiment are shown in Figure 10. From this it can be seen that ERZFP acts to reduce the E2- dependent transcription factor activity of ERa. This is dependent on the quantity of ERZFP present in the assay.

Materials and Methods

Recombinants. The mammalian expression vector pSG5 encoding human ERa (HEGO) and the deletion mutant lacking amino acids 282-595 of human ERa (HE15) have previously been described (Tora et al. , 1989), as have mutants of HEGO and HE15 in which serine 118 has been replaced with alanine or with glutamic acid (Ali et al. , 1993a). Constitutively active ERK2, generated by fusion of ERK2 with MEK1 (ERK/MEK) was a kind gift of Dr. M. Cobb and has been described (Robinson et al. , 1998). In order to delete the GAL4 DBD encoding sequences of the bridge yeast expression plasmid (Clontech, UK) an EcoRI site was introduced just 5'to the ATG, using oligonucleotides with the sequences 5'- GAAGACAGTAGCTTCAGAATTCAGGAGGCTTGCTTC-3'and 5'- GAAGCAAGCCTCCTGAATTCTGAAGCTACTGTCTTC-3', followed by digestion with EcoRI to remove sequences between the GAL4 DBD ATG and the EcoRI site in the multiple cloning site of pBridge. HEGO, HE15 and phosphorylation mutants were cloned into pBridge-AGAL at the EcoRI site. Mutants of ERa generated for this study were made using the QuickChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Oligonucleotide sequences for these mutants have been described (Ali et al. , 1993a; Chen et al. , 2000).

PGEX-HE15 and pGEX-AF2 have been described (Danielian et al. , 1992; Chen et al. , 1999; Chen et al., 2000). All constructs were verified by automated sequencing.

GST pulldowns. In vitro transcription/translations were performed using TNT rabbit reticulocyte lysates (Promega, UK) in the presence of [35S] methionine. GST proteins were induced and lysates prepared as described (Cavailles et al. , 1995). Briefly, 10ml overnight cultures of the Ecoli strain BL21 (Studier et al. , 1990), transformed with the appropriate pGEX

expression construct, were diluted 1: 10 in LB-Amp (100 ug/ml) and incubated for 1 hour at 37°C. The GST protein or the GST fusion proteins were produced after induction with 0.1 mM IPTG for 90 minutes. Cells were collected by centrifugation at 5000 rpm at 4C for 10 minutes. The pellets were resuspended in 5 ml NETN (20 mM Tris pH8, 100 mM NaCI, 1 mM EDTA, 0.5% NP40) with protease inhibitors. Cells were sonicated on ice two times for 10 seconds at a burst power of 22. Sonicated bacteria were centrifuged at 10000 rpm for 5 minutes at 4°C, the supernatant was transferred to a clean tube. GST fusion proteins were purified by affinity chromatography on glutathione-agarose beads and retained as a 50% slurry in buffer C (20 mM Hepes pH 7.6, 100 mM KC1, 1 mM EDTA, 1 mM DTT, 20% glycerol, protease inhibitors). 100 1ll of glutathione agarose beads loaded with 10 pg of GST fusion proteins were then used directly in binding assays with 10 u. l radiolabelled polypeptides from in vitro translation by addition of 890 ul of low salt buffer (50 mM Hepes pH 7.6, 250 mM NaCl, 0. 5% NP40,5 mM EDTA, 0.1% BSA, 0.5 mM DTT, 0.005% SDS, protease inhibitors). Following 1 h incubation at room temperature the beads were washed twice with low-salt binding buffer and twice with high-salt binding buffer (low salt buffer containing 1 M NaCl).

Samples were boiled for 10 min in 80 ul of Laemmli buffer and fractionated by SDS-PAGE. Gels were dried and autoradiographed.

Miniprep plasmid DNA preparation. Colonies were picked and grown in 2mls of L-Broth (171mM NaCl, 1% (w/v) tryptone, 0.5% (w/v) yeast extract) containing ampicillin (50 pg/ml). lml of the overnight culture was then centrifuged at 13000g for 1.5 minutes and the supernatant discarded.

All small-scale plasmid preparations were made by using the Promega Wizard miniprep kit (Promega, UK) following the manufacturer's instructions.

Large scale DNA preparation. Plasmid preparations were prepared using the Qiagen plasmid maxi kit (Qiagen, UK) following the manufacturer's instructions. A volume of 100-200 ml of bacterial culture with intermediate and high copy number plasmids grown overnight yielded approximately 200-900 ug of plasmid DNA, as determined by spectrophotometry at 260 nm in quartz cuvettes. The purity of the double stranded DNA was estimated by the ratio of the O. D. readings at 260 nm and 280 nm.

Restriction digestion analysis of DNA. Restriction endonucleases were purchased from Roche UK and used as recommended by the suppliers.

Generally the restriction digests were performed with 10 U of restriction enzyme per 1 llg of DNA and an incubation of 1-3 hours at 37°C. The digested DNA fragments were separated by electrophoresis in 1% (w/v) HGT agarose (Flowgen) horizontal slab gels made with Ix TBE (90 mM Tris-borate, 1 mM EDTA) containing 0.05 llg/ml ethidium bromide (Sigma). Fragment sizes were assessed by comparison to molecular weight standards of known sizes.

DNA ligations. Ligations were performed using T4 DNA ligase (Promega, UK). The standard reaction mix contained the following reagents, 1X ligation Buffer, 10 units of T4 DNA ligase, 20 ng of linearised vector of interest and a 3 fold molar excess of insert and sterile water to 10 ul. This was incubated at 15°C overnight prior to bacterial transfromation.

Bacterial transformation. For transformation of DNA, generally DH5a or XL-lblue cells were used. 100 ul of competent cells were aliquoted into pre-chilled 1.5 ml Eppendorf tube and half of the ligation reaction, usually 5 u. l was added to the cells. The cells were then incubated in ice for 30 minutes after which they were heat-shocked at 42°C for 45 seconds and then placed back on ice for 2 minutes. 1 ml of LB medium (171 mM NaCl, 1 % (w/v) tryptone, 0.5% (w/v) yeast extract) was added to each transformation and incubated at 37°C with shaking at 200 rpm for 1 hour to

allow cells to recover and start expressing the corresponding antibiotic resistance gene. Cells were centrifuged briefly, medium was removed leaving ~100 pl of LB. The cells were re-suspended in the remaining supernatant which was spread on an LB agar (171 mM NaCl, 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 15% (w/v) bacto agar) plate made up with the appropriate antibiotic and incubated overnight at 37C.

Western Blotting. 10% acrylamide gels were prepared using 30: 1 acrylamide: bis-acrylamide (National Diagnostics). Protein samples were mixed with an equal volume of 2X Laemmli buffer (50 mM Tris. HCl pH 6.8, 15% Glycerol, 2% SDS, 10 mg bromophenol blue dye, 1 mM DTT), denatured by boiling at 100C for 5 minutes and loaded on the gel. The molecular weights of the protein bands were identified by comparison with the Rainbow marker (Amersham, UK). The samples were electrophoresed at 90 volts for 2 hours using Running buffer (250mM Tris base, 1.92 M Glycine, 1% SDS). A nitrocellulose membrane (Amersham Pharmacia Biotech) and the gel were sandwhiched between Whatman paper and were pre-wetted in Electrotransfer Buffer (5 mM Tris base, 38. 4 mM Glycine).

The sandwich was then placed in the electrotransfer apparatus and was run at 90 volts for 90 minutes. The membrane was removed from the apparatus and was blocked with 3% fat free dried milk (Marvel) in PBS (138 mM NaCl, 20.4 mM Na2HP04, 2.7 mM KCL, 1.47 mM KH2PO4) for 30 minutes to decrease the non specific binding of the antibody to the membrane. The first antibody, diluted in PBS was added to the membrane and incubated for 2 hours at room temperature with gentle shaking. The membrane was washed 4x over 1 hour with PBS containing 0.05% Tween-20 and blocked with 0.3% fat free milk in PBS for 30 minutes. The membrane was incubated with the secondary antibody (Alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG) diluted in PBS for 1.5 hours at room temperature, washed 4x over 1 hour with PBS containing 0.05% Tween-20 and 2x in AP buffer (100 mM Tris base, 100 mM NaCl, 5 mM MgCl2), followed by revelation using BCIP/NTB (Promega, UK).

Yeast transformations. Yeast tramsformations were carried out using the Alkali-Cation yeast transformation kit (BIO 101 Systems) following the manufacturer's instructions. A single colony was used to inoculate 100 ml of selective media (0.675% w/v Yeast nitrogen base without amino acids, 50ml of 40% glucose solution/L, 1.8% Difco agar + appropriate amino acid drop/out supplement), which were grown with aeration at 30°C to mid log phase (2 x 10-6 to 2 x-10-7 cells/ml) over-24 hours. Cells were pelleted at 400 x g for 5 minutes and resuspended in 9 ml TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) in order to remove residual medium. Cells were centrifuged a at 400 x g for 5 minutes, resuspended in 5 ml of Lithium/Caesium Acetate solution (BIO 101 Systems) and incubated at 30°C for 30 minutes with gentle shaking. Cells were then pelleted and resuspended in 1 ml of TE. 5 111 of carrier DNA (lOmg/ml), 5 ul histamine solution (BIO 101 Systems) and 0.01-1 ug ofplasmid DNA in a total volume of 10 pl was added to 100 ul of competent yeast cells.

The contents of the tube were gently mixed and incubated at room temperature for 15 minutes. In a separate tube 0.8 ml PEG and 0.2 ml TE/Cation MIXX (BIO 101 Systems) were combined and then added to each transformation and mixed by gentle pippeting. Each transformation reaction was then incubated at 30°C for 30 minutes and then heat shocked at 42C for another 10 minutes. Tubes were then allowed to cool down to 30°C, cells were pelleted in a microcentrifuge at high power for 5 seconds and the supernatant was removed. Cells were resuspended in appropriate selective media and plated selective media and incubated at 30°C for 48-72 hours, until transformant colonies appeared.

For the screen using pBridge-HE15118E bait, 20 individual transformations were carried out using lu. g of library and 100p1 of competent cells in each. For the screen using pBridge-HEG0118E, 251lg of DNA were used for every 100p1 of cells. Following transformation the cells were plated onto 245mm diameter YNB plates lacking tryptophan, leucine and uracil. The equivalent of 0. 01 Og DNA transformed yeast was plated onto plates lacking tryptophan and leucine to assay the total number of independent clones screened.

Yeast extracts. Yeast extracts were prepared according to Clontech Laboratories'protocol obtained from Clontech's Yeast Protocols Handbook. 5 ml of appropriate selective media were inoculated with a single isolated colony and placed at 30C overnight, with shaking. The overnight culture was used to inoculate 50 ml of YPG medium (2% w/v Difco Yeast extract, 1% w/v Difco Bacto Peptone, 50 ml of 40% glucose solution/L), which was incubated at 30°C with shaking until the OD600 reached 0.4-0. 6 (4-8 hours). The culture was poured into two pre chilled 50ml tubes filled halfway with ice and centrifuged at 1000 x g for 5 minutes at 4°C. The supernatant was discarded and the cells resuspended in 50 ml ice-cold H20. The pellet was recovered by centrifugation at 1000 x g for 5 minutes at 4°C and frozen by placing the tube in liquid nitrogen. Cells were then quickly thawed by resuspending in prewarmed Cracking buffer (8M urea, 5% w/v SDS, 40mM Tris-HCl pH 6.8, O. lmM, 0.4mg/ml Bromophenol blue) with 10 Ill of (3-mercaptoethanol, 70p1 of protease inhibitor solution (0.1 mg/ml Pepstatin A, 0.03 mM Leupetin, 145 mM Benzamidine and 0.37 mg/ml Aprotinin) and 50 1 of PMSF (Sigma) for every 1ml of cracking buffer. The cell suspension was transferred to a microcentrifuge tube containing 80, u1 of glass beads (Sigma) per 7.5 OD600 units of cells, heated at 70C for 10 minutes and vortexed vigorously for 1 minute. Debris and unbroken cells were pelleted by centrifugation at 14,000 rpm for 5 minutes. The supernatants were then transferred to a clean tube and loaded on a gel or stored at-70°C.

Isolation of plasmid DNA from yeast. 2 ml of selective media were inoculated with a single yeast colony and incubated at 30°C with shaking overnight. Cells were then centrifuged at high speed using a microcentrifuge, the supernatant was discarded and the cells resuspended in 100 nl of TE buffer. 10 Ill of 10 units/pl lyticase (Sigma, UK) were added to the resuspended cells and incubated at 37°C for 1-2 hours after which the cells were quickly frozen with liquid nitrogen and thawed at 37°C. Plasmid

preparations were then made by using the Promega Wizard miniprep kit following the manufacturer's instructions.

Transient transfection protocol for cos-1 cells (african green monkey kidney cells). Cells were grown in Dulbecco's medium without phenol red (GIBCO) supplemented with 5% double stripped serum, and 5mls of L- Glutamine-penicillin-Streptomycin solution (SIGMA) to 80-100% confluency. At this point cells were trypsinized and counted, plating them at 5 x 105 cells per well in 24 well plates.

Transient transfections were carried using lipofectamine 2000 Reagent (INVITROGEN) as indicated in the manufactures instructions.

COS-1 cells were transfected with a luciferase reporter gene, pXPERE3, controlled by a B-globulin minimal promoter and 3 EREs from the Chicken Vitallogenin 5'flank (100ng), renilla control reporter gene pRLTK (100ng), wild type ERa (lOng of psg5 HEGO), carrier DNA (500ng of BSM) and increasing amounts of pCMVSPORT (6) -ZNF366 (0-100ng). Amounts of DNA were made equal for each transfection by adding corresponding amounts of psg5 vector (0-100ng).

All raw data was made relative to the internal renilla control and induction of the reporter in the presence of oestrogen was adjusted to 100%. All experiments were repeated at least 3 times and results are presented as the standard error of the mean error bars.

ERZFP northern Human multiple tissue northern (MTM) blots I and II (Clontech) were hybridized with cDNA probes by standard methods, according to the manufacturers instructions. Probes were synthesized by random primer labelling with (X32p dCTP using the Rediprime II kit

(Amersham) and purified over Microspin G-50 columns (Amersham).

Blots were prehybridized for 2 hours at 65 C prior to addition of denatured probe, then hybridized overnight. Blots were washed three times in 1X SSC, 0. 1% SDS at room temperature, then three times in O. 1X SSC, 0.1% SDS at 65 C. Blots were viewed by phosphorimager analysis (Molecular Dynamics Typhoon 8600). The ERZFP probe template was a 800bp PCR product from ZF366, corresponding to the NH2-terminus coding sequence lacking zinc-finger motifs. The SRC1 template was a 700bp restriction fragment lacking LXXLL coding motifs. The ER template was full-length human oestrogen receptor a coding sequence. P-actin template was provided in the kit.

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