EP0941243A1 - Polypeptides comprising gax protein domains, involved in repressing transcription and/or interacting with other proteins, corresponding nucleic acids and their use - Google Patents

Abstract

The invention concerns polynucleotides comprising GAX domains, involved in the biological activity of GAX. In particular it may concern domains involved in the interaction of GAX with other molecules or domains responsible for a biological activity. The invention also concerns the chimerical molecules comprising a functional domain of GAX. It further concerns the use of GAX for repressing the expression of genes, and the use of compounds inhibiting the interaction of GAX with certain cell partners for modulating the activity of GAX, It also concerns a method for screening and/or identifying cell partners of GAX.

Description

 POLYPEPTIDES COMPRISING DOMAINS OF PROTEIN GAX, INVOLVED

IN TRANSCRIPTION REPRESSION AND / OR INTERACTING WITH OTHERS

PROTEINS, CORRESPONDING NUCLEIC ACIDS AND USES THEREOF.

The present invention relates to the therapeutic field. It relates more particularly to new therapeutic molecules and their use for the treatment of cardiovascular pathologies.

Post-angioplasty restenosis is a localized hyperproliferative disorder that develops as a result of non-surgical intervention in the atherosclerotic plaque. Thus, the treatment of an atherosclerotic lesion by angioplasty very frequently (up to 50% of cases in some studies) results in restenosis following mechanical injury to the arterial wall.

The proliferation of smooth muscle cells (CML) in the vascular wall is a key event in the development of atherosclerosis, in restenosis after angioplasty as well as in the failure of coronary bypass surgery. SMCs are normally quiescent in the vascular wall but, following a lesion destroying the vascular endothelium, they are found in contact with blood growth factors. Unlike cardiomyocites and skeletal muscle cells, SMCs can, in response to various growth factors, differentiate and reset their proliferative cycle again. These changes are phéiiot ic CML iésukeπi profound changes in ^{'the expression} of many genes. For example, the expression of early genes, involved in cell proliferation such as c-fos or c-myc, is remarkably increased while the expression of other structural genes such as alpha-actin muscle specific smooth as well as certain myosin isoforms undergoes a down regulation. Although it is clearly established that the terminal differentiation of skeletal muscle cells is regulated by so-called myogenic transcription factors such as Myo-D, very few factors involved in the reversible differentiation of CML are known to date. Recent studies have revealed the existence of transcriptional factors possessing a homeo-box in different tissues of the cardiovascular system. Such factors, thanks to their homeo-box, recognize specific DNA sequences in the promoter regions of their target genes. and thus intervene in the regulation of differentiation, proliferation or cell migration. One of these gax (Growth-Arrest-Specific Homeobox) factors is expressed in various cardiovascular tissues including the SMCs of the vascular wall. The gax gene was initially identified from an aorta cDNA library. rat. It codes for a protein of 303 amino acids. His streak was characterized and his cDNA clone (Gorski et al., Mol.Cell.Biol. 1993, 6, 3722-3733). The human gax gene has also been cloned and sequenced (David F. Le Page et al., Genomics 1994,24,535-540). It codes for a protein of 302 amino acids. The gax gene has certain properties similar to the gas and Gadd genes since it also seems to control the G0 / G1 transition of the cell cycle. Thus the levels of gax mRNA are reduced in rat LVMC by a factor of 10 after two hours of exposure to PDGF (Gorski et al., Mol.Cell.Biol. 1993, 6, 3722-3733). The expression of the gax gene is therefore repressed during the mitogenic response of CMLV. Another characteristic of the gax gene lies in its specificity of expression. Indeed, in the adult rat, the gax gene is mainly expressed in the cardiovascular system (aorta, heart). The presence of gax mRNA has not been demonstrated by Northern Blot in the liver, brain, stomach and skeletal muscle. Furthermore, the gax gene belongs to the family of homeotic genes. These genes code for transcriptional factors which contain consensus (or homeodomain) sequences recognizing specific regions of DNA (or homeoboxes) (review: Gehring et al. Cell, 78: 211-223, 1994). The homeodomain of the rat gax protein is between amino acids 185 and 245. Interestingly, the homeotic genes identified to date are involved in the control of cell differentiation / growth during embryogenesis, which reinforces the therapeutic potential of the gax gene (review: Lawrence and Morata Cell 78: 181-189, 1994; Krumlauf, Cell 78: 191-201, 1994).

One of the characteristics of GAX is that it undergoes down regulation as soon as CML proliferate, whether in vitro in response to growth factors or in vivo following damage to the endothelium of the vascular wall. This repression is reversible because when the SMCs are made quiescent by deprivation in serum, in vitro, the expression of GAX resumes. Work in our laboratory has recently shown that overexpression of GAX in SMCs by an adenoviral vector blocks their proliferation FR 95/04234 (ST95022).

Post-angioplasty restenosis following mechanical injury is the most frequent failure factor (50% of cases in some studies). The proliferation of CML being a key element of this phenomenon, knowing its regulatory role for the proliferation of GAX, the latter appears as a therapeutic gene of choice for the preventive treatment of the vascular wall after angioplasty FR 95/04234 (ST95022).

However, the mechanism of action of GAX remains, however, very little documented. It remains to identify target DNA sequences of the homeobox of GAX. Furthermore, a study of the function of the different GAX domains has not been carried out to date. The identification of GAX functional partners finally constitutes an important facet which will make it possible to define the molecular cascade on which GAX depends or on which it is responsible. source The present invention has precisely the advantage of providing information on these points

During this work, different analytical approaches were adopted to identify molecules interacting with GAX, to determine the domain of GAX necessary for this interaction and to study the importance of the different domains in the function of GAX To do this, the system double hybrid was used to characterize proteins interacting with GAX from a human lung bank

This approach has made it possible to identify different molecules, one of which, the Ki antigen, is particularly interesting. The Ki antigen is a protein of around 32 KDa. It was identified for the first time as a nuclear autoantigen recognized by sera from lupus erythematosus patients (Nikaido et al 1989, Clin. Exp. Immunol 1990, 79, 209-214) Recent work has shown that Ki is overexpressed in proliferating cells or transformed by an oncogene (Nikaido et al 1989, Exp . Cell Res 1989, 182,284-289) Experiments in coimmunoprecipitation (Takeushi et al abstract 1995 Juntendo University, Tokyo, Japan) have shown that Ki exists in the form of a complex with PCNA a proliferation marker (Almendral et al 1987, Proc Natl Acad Sci USA, 84, 1575-1579)

The function of GAX has also been studied in transient mammalian cell transfection experiments using GAX fused to a heterologous DNA binding domain and a reporter gene system making it possible to provide information on the transcriptional activity of GAX after deletion. of different regions of the molecule II has thus been shown that GAX acts essentially as a transcription repressor This repression activity is linked more particularly to the first 32 amino acids of GAX In addition, this repressor domain is localized in the region (1- 222) of GAX required for interaction with Ki in yeast The potential applications of the repressiveness of GAX and its interaction with Ki will be developed below A first aspect of the invention therefore relates to fragments of the GAX protein endowed with biological properties

Another aspect of the invention relates to areas of GAX involved in the biological activity of GAX. These may include areas involved in the interaction of GAX with other molecules or areas responsible for biological activity. The invention also relates to chimeric molecules comprising a functional domain of GAX. It also relates to the use of GAX to suppress gene expression, as well as the use of compounds inhibiting the interaction of GAX with certain cellular partners to modulate the activity of GAX. It also relates to a method for screening and / or identifying GAX cellular partners

As indicated above, the gax gene has particularly advantageous properties for gene therapy applications of hyperproliferative disorders, in particular restenosis or other pathologies associated with a proliferation of CML II was demonstrated in application FR95 / 04234 ( ST95022) that the transfer of the gax gene in vivo makes it possible to considerably reduce the proliferation of CMLs, and thus, to inhibit the reduction in luminal diameter II was also shown in application FR 95/12871 (ST95057) that the gax gene, expressed in tumor cells, allowed to oppose the cell transformation process These different elements demonstrate the potential of the gax gene for therapeutic approaches

The present application now describes the identification of functional domains of GAX, the construction of GAX derivatives exhibiting a biological activity, the identification of partners of the GAX protein and the development of methods allowing the search for other partners and the 'identification of compounds capable of interacting on the activity of these partners More specifically, the applicant has now shown that the GAX protein is endowed with transcription repressor activity. It also showed that this activity was carried by certain regions of the GAX protein, in particular the N-terminal region. The results presented in the examples further show that the functional domains of GAX are also involved in the interaction of GAX with a cellular protein, Ki II is known in the literature Ki interacts with PCNA (Takeushi et al abstract 1995 Juntendo University, Tokyo, Japan) and that this is a cofactor essential for DNA polymerase for DNA replication (Fukuda et al. 1995, J. Biol. Chem. 270, 22527-22534). It has been demonstrated in the examples that Gax interacts with PCNA, thus it is conceivable that GAX can also be involved in replicative complexes in the cell.

A first object of the invention relates more particularly to a polypeptide characterized in that it is all or part of a fragment of the GAX protein having an activity of repressor of transcription and / or positively or negatively affecting replication DNA.

More preferably, the polypeptide according to the invention comprises at least residues 1 to 32 of the human GAX protein.

According to another embodiment, the polypeptide according to the invention comprises at least residues 104 to 230 of the human GAX protein.

As specific examples of polypeptides according to the invention, mention may be made of polypeptides comprising fragments 1-32, 33-302 and 1-104 of the human GAX protein. The examples presented below show that such polypeptides are capable of inhibiting the transcription of genes and therefore retain the transcriptional repressor properties of GAX.

More preferably, this polypeptide comprises, in addition to a fragment of the GAX protein in accordance with the present invention, a fragment of different origin. By fragment of different origin is meant a polypeptide fragment not derived from the GAX protein. It can be a synthetic, artificial fragment, a protein fragment, etc. This fragment of different origin can have different functions.

It can for example constitute a marker making it possible to detect the polypeptide and optionally to trace it in vivo. Such a marker can for example be an epitope recognized by a monoclonal antibody. In this regard, various labeling sequences, called Tag sequences, have been described in the literature and are usually used. We can mention the tag-myc sequence.

This fragment can also be a fragment having the property of stabilizing the polypeptide. For this purpose, it may be all or part of a protein having a high plasma half-life. Finally, it can also be a targeting element, allowing the polypeptide to reach specific cellular compartments more quickly and / or more specifically. Advantageously, it is a nuclear localization signal (NLS), the polypeptide mainly exercising its activity in the nucleus of cells Different NLS signals have been described in the literature, for example that of the T antigen of the SV40 virus, that of p53, etc.

The fragment of different origin can also confer on the polypeptide an additional biological function or promote the activity of the repressor domain. As a very particularly advantageous example, mention may be made of a protein domain capable of specifically binding DNA. This type chimeric molecule thus makes it possible to bind DNA into particular regions and to inhibit the transcription of genes located at the level of these regions. As specific examples, mention may be made of the DNA binding domain of the GAL4 protein of yeast Such constructs are described in the examples. They can be used to regulate the expression of genes in yeast or of genes placed under the control of an expression signal comprising the binding site of the GAL4 protein. Other fields DNA binding proteins can be for example Lex A, the DNA binding domain of a nuclear receptor such as the estrogen receptor es or any other member of this family, etc. It may also be a peptide allowing on the one hand the secretion of all or part of GAX, as well as its targeting towards membrane receptors allowing its internalization and thus increasing the diffusibility and the field of activity of GAX by a "Bystander" type effect. As such, it is more particularly possible to use all or part of the transferrin or fragments of the molecule of the extracellular matrix capable of recognizing integrins on the surface of CML

The polypeptides according to the invention are particularly advantageous from the therapeutic point of view and from that of applied research.

The therapeutic activity of the claimed polypeptides is linked more particularly to their ability to repress transcription or to affect DNA replication, by analogy with the GAX protein and therefore to give them a power to regulate the expression of other proteins. In this perspective, the present invention also relates to the use of the GAX protein or of a fragment as claimed for suppressing gene transcription and / or positively or negatively affecting DNA replication.

Due to this analogy of behavior with the GAX protein, these fragments can advantageously be substituted for it in its function of transcription repressor. Furthermore, taking into account the fact that they only comprise the GAX protein that all or part of its domain involved in repression of transcription, it is possible to think that they will be less sensitive to the various alterations to which the GAX protein is subject. These polypeptides as such or derivatives can therefore manifest a repressor character of the increased transcription compared to that of the natural GAX protein.

We can also consider using them to identify new partners of the GAX protein. In this regard, the present invention also relates to a method for the screening and / or identification of polypeptides interacting with the GAX protein or a GAX domain characterized in that it implements a polypeptide according to the invention. More preferentially, this polypeptide is chosen from there fragments 32 and 33 to 302 of the GAX protein.

As such, the Applicant has unexpectedly shown that a domain of the GAX protein could interact in a specific way with another cellular protein, the Ki protein. As explained above, Ki is an autoantigen that appears in autoimmune diseases such as lupus erythematosus. It is described as a nuclear molecule whose expression increases during cell proliferation as well as in fibroblasts transformed by an oncogene.

We have been able to show that, in yeast, the N-terminal part of GAX is necessary for the interaction with Ki. The recombinant protein Ki specifically recognizes GAX transferred to a nitrocellulose filter from a denaturing polyacrylamide gel.

The present invention also specifically relates to a polypeptide characterized in that it is a fragment of the GAX protein capable of interacting with the Ki protein. More preferably, it is fragment 1 to 32 or 104 to 223 of the GAX protein.

The Applicant has also very unexpectedly shown that a domain of the GAX protein can interact in a specific manner with the proliferation marker PCNA (Proliferating Cell Nuclear Antigen). This factor is essential for DNA replication and also plays a role in DNA repair phenomena. The present invention therefore relates to a polypeptide characterized in that it is a fragment of the GAX protein, capable of interacting with PCNA.

Furthermore, it has been described in the literature that Ki exists in the form of a complex with PCNA. Ki can therefore interact with both GAX and PCNA by forming an at least bipartite complex with one or other of these proteins and preferably a tripartite complex. The formation of these complexes has an important role in the progression of the cell cycle (activation or inhibition).

Thus, the present invention also relates to a polypeptide characterized in that it is a fragment of the GAX protein capable of interacting with Ki and / or PCNA and of forming an at least bipartite or at least tripartite complex with these proteins.

Another object of the present invention relates to any nucleic acid coding for a polypeptide as defined above. They may be sequences of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, hybrid sequences or synthetic or semi-synthetic sequences. This nucleic acid can be of human, animal, plant, bacterial, viral, etc. origin. It can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of banks. They can also be incorporated into vectors, such as plasmid vectors.

Advantageously, the nucleic acid according to the invention is a cDNA.

The nucleic acids of the invention can be used for the production of probes or antisense molecules making it possible, by hybridization, to detect the presence or the expression of nucleic acids coding for polypeptides carrying a repressor domain according to the invention, or of inhibiting the expression of such polypeptides. As regards probes, these preferably comprise more than 10 bases and, advantageously, from 10 to 300 bases.

The nucleic acids of the invention can be used for the expression and / or production of polypeptides in vitro, in vivo or ex vivo, in gene or cell therapy approaches.

To this end, the present invention relates to expression cassettes containing a nucleic acid as defined above, under the control of a promoter allowing its expression.

Different promoters can be used in the context of the invention. These are sequences allowing the expression of a nucleic acid in a mammalian cell.

The promoter is advantageously chosen from functional promoters in human cells. More preferably, it is a promoter allowing the expression of a nucleic acid sequence in a hyperproliferative cell (cancerous, restenosis, etc.). In this regard, different promoters can be used. It can thus be any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific way or not, inducible or not, strong or weak. Mention may in particular be made of the promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the target cell. Among the eukaryotic promoters, ubiquitous promoters can be used in particular (promoter of the ITPRT, PGK genes, alpha-actin, tubulin, DFfFR, etc.), promoters of intermediate filaments (promoter of the GFAP genes, desmin, vimentin, neurofilaments, keratin, etc), promoters of therapeutic genes (for example the promoter of the MDR, CFTR genes, Factor VIII, ApoAI, etc.), tissue-specific promoters (promoter of the pyruvate kinase gene, villin, intestinal fatty acid binding protein, alpha smooth muscle actin, etc.), promoters of specific cells of the dividing cell type such as cancer cells or of promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, glucocorticoid receptor, etc.) or so-called inducible. Likewise, they may be promoter sequences originating from the genome of a virus, such as for example the promoters of the El A and MLP genes of adenovirus, the early promoter of CMV, or the promoter of the LTR of RSV, etc. In addition, these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression.

The present invention now provides new therapeutic agents making it possible, by their ability to repress transcription, to interfere with numerous cellular dysfunctions. For this purpose, the nucleic acids or cassettes according to the invention can be injected as such at the site to treat or incubate directly with the cells to be destroyed or treat It has in fact been described that naked nucleic acids can penetrate cells without any particular vector Nevertheless, it is preferred in the context of the present invention to use an administration vector, allowing improve (i) the efficiency of cell penetration, (ii) targeting and (iii) extra- and intracellular stability

In a particularly preferred embodiment of the present invention, the nucleic acid or the cassette is incorporated into an expression vector. The vector used can be of chemical origin (liposome, nanoparticle, peptide complex, lipids or cationic polymers, etc. retrovirus, adenovirus, herpes virus, AAV, vaccinia virus, etc.) or plasmid

The use of viral vectors is based on the natural properties of transfection of viruses. It is thus possible to use, for example, adenoviruses, herpes viruses, retroviruses and associated adeno viruses. These vectors prove to be particularly effective in terms of transfection In this respect, a preferred object according to the invention resides in a defective recombinant retrovirus whose genome comprises a nucleic acid as defined above. Another particular object of the invention resides in a defective recombinant adenovirus whose genome comprises a nucleic acid as defined above

The vector according to the invention may also be a non-viral agent capable of promoting the transfer and expression of nucleic acids in eukaryotic cells. The chemical or biochemical vectors, synthetic or natural, represent an interesting alternative to natural viruses, in particular for for reasons of convenience, safety and also by the absence of a theoretical limit as regards the size of the DNA to be transfected. These synthetic vectors have two main functions, compacting the nucleic acid to be transfected and promoting its cellular fixation as well as its passage through the plasma membrane and, where appropriate, the two nuclear membranes To compensate for the polyanionic nature of nucleic acids, the non-viral vectors all have polycationic charges

The nucleic acid or vector used in the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the nucleic acid or the vector is used in an injectable form II can therefore be mixed with any pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection at the site to be treated II can be in particular sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as the case may be of sterilized water or physiological saline, allow the constitution of injectable solutes The doses of nucleic acid used can be adapted according to different parameters, and in particular according to the gene , the vector, the method of administration used, the pathology concerned or the duration of u treatment sought

The invention also relates to any pharmaceutical composition comprising at least one nucleic acid as defined above

It also relates to any pharmaceutical composition comprising at least one vector as defined above

Due to their antiproliferative properties, the pharmaceutical compositions according to the invention are very particularly suitable for the treatment of hyperproliferative disorders, such as in particular cancers and restenosis. The present invention thus provides a particularly effective method for the destruction of cells, in particular of hyperproliferative cells It is thus applicable to the destruction of tumor cells or smooth muscle cells of the vascular wall (restenosis) It is very particularly suitable for the treatment of cancers By way of example, mention may be made of colon adenocarcinomas, cancers thyroid, lung carcinoma, myeloid leukemia, colorectal cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, B lymphoma, ovarian cancer, cancer bladder, glioblastomas, hepatocarcinomas, bone cancer, skin, pancreas or again kidney and prostate cancer, esophageal cancer, larynx cancer, head and neck cancer, HPV positive anogenital cancer, EBV positive nasopharyngeal cancer, etc.

Furthermore, the present invention further extends to any use of compounds which inhibit the activity of the Ki protein and or of PCNA for inhibiting the proliferation of smooth muscle cells.

The present invention also relates to the use of compounds according to the invention for the formation of a two- or three-partite complex with the proteins Ki and / or PCNA to positively or negatively affect the progression of the cell cycle

The present invention will be more fully described with the aid of the examples and figures which follow, which should be considered as illustrative and not limiting.

LEGEND OF FIGURES

Figure 1 Northern blotting analysis of Ki protein expression from mRNAs extracted from different tissues

Figure 2 Nuclear localization of KiHA in cells transfected by the immunofluorescence method

Figures 3 In vitro interaction between the Ki and GAX proteins

A) coomassie blue

B) Far Westren Blotting

Figure 4 A) Representation of the construction of the Reporter gene expressing bacterial chloramphenicol acetyl transferase (CAT) under the control of an artificial promoter

B) Determination of the CAT enzymatic activity in cellular extracts after transfection with expression vectors incorporating respectively ER, GAL VP16 and ER-G L V 16

Figure 5 Comparison of constructs of different GAX deletion mutants versus the entire GAX protein 1-302 Figures 6. Determination of CAT enzymatic activity with different GAL-GAX chimeras

A) Without ER activator

B) in the presence of ER activator

Figure 7 ^• Interaction between GST-GAX and PCNA

Material and methods

A) MATERIAL

1) Yeast strain used

The strain YCM79 of the genus S.cerevisiae (MATa, ιιra3-52, hιs3-200, ade2-101, lys 2- 801, trpl-901, Ieu2-3, 112, canl, gal-f-542, ga / 80- 538, metI6 :: URA3-pGALl 10- LacZ, HIS3:: GAL7BLE) was used as a screening tool for the lung fusion bank by the two hybrid system

It is cultivated on the following culture media Complete YPD medium -Yeast extract (10g / l) (Difco) -Bactopeptone (2ûg / l) (Difco) -Glucose (20g / l) (Merck)

This medium was made solid by the addition of 20 g / l of agar (Difco)

Medium YNB minimum -Yeast Nitrogen Base (without amino acids) (6.7g / l) (Difco)

- Glucose (20g / l) (Merck) This medium can be made solid by adding 20g / l of agar (Difco)

To allow the growth of auxotrophic yeasts on this medium, it is necessary to add amino acids or nitrogenous bases, for which they are dependent at 50 mg / ml

2) Strains of bacteria used

The Escheriehia coli strain TG1 of genotype supE, hsdD5, thi, D (lac-proAB), F'ftra D36 pro AB lacP lacZDM15] was used as a means of amplification and isolation of the recombinant plasmids used For the production of recombinant proteins, the E coli bacteria used are BL-21 obtained from Pharmacia E coli They have the F ^" ompT hsdS _b (r _h .m _h ) gai dcm (DE3) genotype.

BL-21 is a strain of choice for the production of recombinant proteins because it has no protease and its membrane is fragile, easy to break by simple sonication E coli BL-21 has a repression system for T7 RNA polymerase This repression can be raised by IPTG which allows the control of genes placed downstream of the T7 RNA polymerase binding site The bacteria are cultured in 2 ml of LB medium (NaCl 5 g / 1, Tryptone 10 g / 1 , Yeast extract 5 g / 1) in a shaker at 37 ° C overnight 1 ml of this preculture is re-cultured in 50 ml of 2xYT medium (NaCl 5 g / 1, Tryptone 16 g / 1, Yeast extract 10 g 1) at 37 ° C until the bacteria reach an optical density (OD) of 0.4 to 600 nm (exponential growth phase) The culture of the bacteria is cooled on ice They are centrifuged at 3300 rpm for 20 mins

The E Coli bacteria, competent for the production of recombinant proteins, are prepared by the calcium chloride method. To do this, the bacteria are dissolved in 5 ml of 0.1 M CaCL2 (1/10 of the culture). They are centrifuged again at 3300 rpm for 10 min. They are redissolved in 1 ml of 0.1 M CaCl 2 containing 17.5% glycerol They are aliquoted and stored at -80 ° C.

3) Plasmids used

The plasmids used are

The vectors of the pGBT and pAS series (Clontech), shuttle plasmids which have an origin of bacterial and yeast replication allowing them to replicate at high copy number in these two microorganisms These plasmids contain a multiple cloning site located downstream of the sequence coding for the DNA binding domain of GAL4 and upstream of a eur terminal to form a fusion protein They also contain the TRP 1 gene of S. cerevisiae which makes it possible to complement the yeasts of trpl genotype in order to select them on a minimum medium containing no tryptophan These vectors carry the resistance gene with ampicillin which makes it possible to select the bacteria possessing them on a medium containing ampicillin.

. The vectors of the pGAD series, (Clontech) vectors which allow the expression in yeast of fusion proteins between the GAL4 transactivating domain and a protein of interest or encoded by the cDNA originating from a lung bank, inserted at an EcoRI Xhol site.

The vectors of the pET29 series (Novagen) of the vectors which allow the expression of recombinant proteins in coli in fusion with the tagS

The vectors of the pGEX series (Pharmacia) of the vectors which allow the expression of recombinant proteins in coli in fusion with La Glutathione S-Transferase (GST)

The vectors of the Bluescript series (Strata gene) of the vectors making it possible to carry out cloning as well as the series of pIC (J Lawrence Marsh et al Gene, 1984, 32, 481-485) and pMTL (Steve P Chambers et al, Gene, 1998, 68, 139-149) The plasmid pGEX-2T-hGAX is the plasmid used for the transformation of E Coli BL-21 This plasmid was obtained from the plasmid pGEX-2T and was supplied by the Doctor K Walsh of St Elizabeth's Medical Center in Boston The basic plasmid pGEX-2T (Pharmacia) makes it possible to produce the protein GAX fused with Glutathione-S-Transferase (GST), protein which has a strong affinity for Glutathione GST fusion -GAX will facilitate the purification of GAX by affinity on agarose beads coupled to Gluthation In addition, there is a thrombin cleavage site which subsequently makes it possible to split the chimera between GST and GAX The hGAX cDNA is inserted under control of the tac hybrid promoter and the lac operator Le represseur lad by fixing himself on the lake operator. prevents RNA polymerase from advancing This repression is relieved by a lactose analog. isopropyl-β-D- thiogalactoside (IPTG) which associates with the lad The lacI-IPTG complex can no longer be fixed on the operator lac l '-YRN pokmerase n therefore no longer has any obstacle

4) Ki protein production and purification

We cloned the gene for the Ki protein fused to a myc epitope in the plasmid pET-29 ( contained in the yeast The piasmid pET-29 produces the protein fused to the epitope called S-Tag which allows the purification of KI by affinity on agarose beads coupled to S-protein. The SmycKI chimera is under the control of the T7 promoter and 1 lac operator. The BL-21s are transformed by the plasmid pET-29-mycKI As for the GST-GAX protein, they are cultured up to an OD of 0.7 at 600 nm. Then, the expression of SmycKI is induced by 0.1 mM IPTG and by T7 RNA polymerase produced by BL-21. The bacteria are sonicated. The supernatant is then purified by the following method. The purification of SmycKI is done by affinity on agarose beads coupled to the S-protein. The method is the same as for GST-GAX except that the resin is washed in a solution containing 20 mM Tris pH 7.5, 0.15 M NaCl and 0.1% Triton X-100 Elution and dialysis are the same as for GST-GAX

B) METHODS

The methods conventionally used in molecular biology are well known to those skilled in the art and are abundantly described in the literature [Maniatis T et coli, "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory,

Cold Spring Harbor, N Y, 1982, Ausubel F M et coli (eds), "Current Protocols in

Molecular Biology ", John Wiley & Sons, New York, 1987]

1). The double-hybrid system

This system is a method of cloning by interaction in vivo in Saccharomyces cerevisiae, the principle of which is based on the modular structure of the yeast transcription factor GAL4 (7) The GAL4 transcription activator has two independent domains with different functions The binding domain DNA (GALDB for GAL DNA Binding) allows GAL4 to bind to a specific DNA sequence at the promoter region of a GAL4 gene is then sterically close to the transcriptional machinery and thanks to its transactivator domain (GALTA), it increases the frequency with which transcription is initiated on the adjacent gene, probably by interactions with RNA polymerase or associated proteins. The principle of the double-hybrid system consists in fusing GALDB and GALTA separately with two X proteins. and different Y which, when they interact, reconstitute an active transcriptional complex 2) Screening of yeasts by the PCR (Polymerase Chain Reaction) technique

The screening was done directly on the yeasts. Each yeast colony is dissolved in 10 μl of water containing 0.08% of Tween 20. Tween is a non-ionic detergent which makes it possible to increase the lysis of yeasts during the first heating step at 95 ° C. . For the subsequent steps, Tween 20 acts as a protective agent for Taq DNA polymerase. The yeast suspension is completed to a final volume of 20 μl containing the following final quantities or concentrations: 10 picomoles of primer 1, 10 picomoles of primer 2, 1 unit of Taq DNA polymerase, 75 mM of Tris pH9, 20 mM of (NH4) 2SO4, 0.01% of Tween 20, 2.5 mM of MgC12 and 125 μM of each of the 4 deoxyribonucleotides (dATP, dTTP, dGTP and dCTP). At the end of the PCR, a fraction of the mixture is analyzed on an agarose gel. It is thus possible to know, for a given pair of primers and for each yeast clone, whether it is a DNA sequence already identified.

3) Preparation of plasmid DNAs.

Large quantities of DNA are prepared using the Proméga DNA Rapid Preparation Kit.

Small amounts of DNA are prepared as follows: the bacteria containing the plasmid are cultured for at least 4 hours in 2 ml of LB medium in a shaker shaker. They are then centrifuged for 2 minutes at 14,000 rpm in Ependorf tubes, then the pellet is resuspended in 100 μl of solution I (50 mM of glucose, 25 mM of Tris HCl buffer pH8, 10 mM EDTA pH8), lysed with 200 μl of the solution II (0.2M NaOH, 1% SDS). The lysis solution is then neutralized with 150 μl of solution III (3M of potassium acetate, 1 1.5% (v / v) of glacial acetic acid). After stirring the tubes until a flaky precipitate is obtained, 150 μl of a mixture of phenol / Chloroform (50% of phenol and 50% of chloroform saturated in water) are added, and the whole is stirred for 30 seconds. The aqueous phase containing the DNA is recovered after centrifugation for 2 minutes at 14,000 rpm. The DNA is then precipitated by adding 0.5 volumes of isopropanol, then centrifuged for 5 minutes at 14,000 rpm and dried in air to finally be taken up in 20 μl. TE RNAse (10mM Tris-HCl solution and EDTA ImM with 50μg / ml RNAse).

4) Enzymatic synthesis and amplification of DNA or PCR (Polymerase Chain Reaction)

The PCR reactions are carried out in a final volume of 100 μl in the presence of the DNA matrix, dNTP (0.2 mM), PCR buffer (Tris-HCL pH 8.5 10 mM, MgCl2 ImM, KC1 5 mM, 0.01% gelatin), 0.5 μg of each of the oligonucleotides and 2.5 IU of Amplifier Taq DNA polymerase (Perkin Elmer) with or without formamide (5%) The mixture is covered with 2 drops of oil of paraffin to limit evaporation of the sample The device used is the "Crocodile II" from App gene

We used a denaturation temperature of the matrix of 90 ° C, a hybridization temperature of the oligonucleotides to the matrix 5 to 10 degrees lower than the temperature of separation of the oligonucleotides and a temperature of elongation by the enzyme to 72 ° C

The fragments obtained by PCR, used for cloning are systematically resequenced once cloned, so as to verify the absence of any mutation which appeared during amplification.

The oligodeoxynucleotides are chemically synthesized according to the phosphoramidite method using β-cyanoethyl protective groups (Sinha 1984) After synthesis, the protective groups are removed by treatment with ammonia and two precipitations with butanol make it possible to purify and concentrate the oligodeoxynucleotides ( Sawadogo, 1991) The DNA concentration is determined by measuring the optical density at 260 nm

5) Ligatures

All ligation reactions are carried out at + 14 ° C overnight in a final volume of 10 μl in the presence of 100 to 200 ng of vector, 0 5 to 2 μg of insert, 40 IU of T4 DNA ligase enzyme ( Biolabs) and a ligation buffer (50 mM Tris-HCl pH 7.8, 10 mM MgC ^* i2, 10 mM DTT, 1 mM ATP) The negative control consists of ligation of the vector in the absence of insert The filling of the prominent 5 ′ ends is carried out if necessary before ligation with the Klenow fragment of the DNA Polymerase I of £. coli (Biolabs) according to the supplier's specifications The destruction of the protruding 3 'ends is carried out in the presence of DNA polymerase from phage T4 (Biolabs) used according to the manufacturer's recommendations

6) Transformation of bacteria

The transformation of the bacteria with a plasmid is carried out according to the following protocol. The entire volume of ligation (10 μl) is used to transform the TGl bacteria made competent by the method of Chung et al, (PNAS, 1988 86, 2172-2175).

The TGl bacteria are cultured in a liquid LB medium for a few hours in a stirring oven at 37 ° C., until an OD of 0.6 to 600 nm is obtained. The medium is then centrifuged at 6000 rpm for 10 min. The bacteria are made competent by taking up the bacterial pellet with a volume of TSB (LB medium + 100 g / 1 of PEG 4000, 5% of DMSO, 10 mM of MgCl2, 10 mM of MgSO / Q corresponding to 1/10 of the volume of the medium of the initial culture After incubation at 4 ° C for 30 to 60 minutes, 200 μl of bacteria are brought into contact with the ligation products for 15 minutes on ice After adding 200 μl of LB, the bacteria are incubated for 30 minutes at 37 ° C. then spread on LB + ampicillin medium

7) Separation and extraction of DNA

The DNA is separated according to their size by electrophoresis. To do this, different gels are used depending on the size of the fragments to be separated.

- 1% agarose gel (Gibco BRL) in TBE buffer (Tris base 90mM,

Borate 90mM, EDTA 2mM) for the separation of large DNA fragments (greater than 500 bp)

- 2% NuSieve agarose gel (FMC Bioproducts) in TBE buffer for the separation of small fragments (less than 500 bpd)

Any migration on agarose gel or on polyacrylamide gel is carried out in a TBE buffer and in the presence of a molecular weight marker (1Kb ladder, Gibco BRL) The DNA is mixed with 1/10 of the volume of the deposition blue (200g / l of Ficoll,

0.5 g l bromophenol blue, 50 mM EDTA) before being deposited on gel After migration at 100 volts and staining with ethydium bromide (concentration 0.5 μg / ml of gel), the bands are visualized under the UV lamp

The extraction of DNA from the band of an agarose gel is carried out by electroelution as follows: The piece of gel containing the DNA fragment is cut with a scalpel and placed in a dialysis rod closed by two forceps and containing 100 to 500μl of TBE. The whole is placed in an electrophoresis tank where it undergoes an electric field of 100 volts. The DNA, after having left the gel, is then purified by two extractions with phenol / chloroform followed by two extractions with chloroform, then precipitated in the presence 0.3M sodium acetate and 2.5 volumes of absolute ethanol After centrifugation (5 min at 14,000 rpm) the DNA pellet is dried and then taken up in 20 μl of water

8) Fluorescent sequencing of plasmid DNAs

The sequencing is done according to the Sanger method using 4 dideoxyribonucleotides having a different fluorescent marker The incorporation of one of these dideoxyribonucleotides produces a stop in the replication by Taq polymerase of the DNA to be sequenced This reaction will give fragments of 'DNA of different sizes, all terminated in 3' by one of the 4 dideoxyribonucleotides One μg of a plasmid and 4 picomoles of a primer are added to 9.5μl of a "premix" supplied by Applied Biosystems under the name Prism The final volume must be 20 μl to carry out a PCR for 25 cycles, decomposing into a denaturation step at 96 ° C for 30 seconds, a hybridization step at 50 ° C for 15 seconds and an elongation step at 60 ° C for 4 minutes

The DNA fragments, obtained after amplification, are purified on an exclusion column (Chromaspin-30 from Clontech), and are then dried with Speed Vac. The whole is taken up in 5 μl of a mixture formed of 24 μl of EDTA ( 50mM) and 120μl of deionized formamide After denaturation at 96 ° C for 3 minutes, 3 to 5μl are deposited on an electrophoresis gel The different DNA fragments are separated according to their size and will pass successively in front of a laser reader of the 370 DNA sequencer device (Applied Biosystems) where the different fluorescences will be detected

9) Preparation of plasmids from the lung bank (Clontech®) The lung cDNA fusion library is sold as bacteria. This library comes from the cloning, at the EcoRI-Xhol site of the plasmid pGAD424 (Materials and Methods), of cDNA corresponding to the total RNA of human lung cells.

After checking the title of the bank, 2 μl of bacteria from the lung fusion bank, put beforehand in 8 ml of LB, are spread non-confluently on a solid medium in order to keep the representativeness of this bank. We

2 thus spread 16 boxes of 770 cm containing an LB + ampicillin medium. The colonies that appear are taken up for each of the dishes with 30 ml of LB + liquid ampicillin. The suspensions obtained are then placed in an Erlenmeyer flask and incubated in a shaker at 37 ° C for 3 hours. DNA is then extracted from these strains by the Maxiprep technique. The DNA concentration will be determined at 260nm.

10) Transformation of the yeast with a plasmid.

The yeasts previously cultivated in 100 ml of liquid medium are harvested after centrifugation at 3000 rpm for 3 minutes and suspended in 1 ml of sterile water. After centrifugation at 3000 rpm for 3 minutes, the cell pellet is resuspended in 1 ml of sterile water and then centrifuged again. This operation is repeated again in order to eliminate all traces of the culture medium. The yeasts are then taken up in 1 ml of the transformation solution I (0.1A LiAc, Tris-HCl pH 7.5 lOmM, EDTA ImM). then centrifuged at 3000 rpm for 3 minutes. The cell pellet is taken up again in 1 ml of the transformation solution I. 50 μl of this yeast suspension are placed in the presence of 50 μg of salmon sperm DNA and from 1 to 5 μg of plasmid DNA. 300 μl of a transformation solution II (0.1 M LiAc, Tris-HCl pH 7.5 10 mM, EDTA ImM in 40% PEG40OO) are then added, then the whole is incubated at 28 ° C for 30 minutes. A thermal shock is then applied to the transformation mixture in a water bath at 40 ° C for 15 minutes and then the whole is centrifuged at 15,000 rpm for 1 minute in order to collect the cell pellet. This pellet is taken up in 200 μl of water and then spread over a minimum agar medium which does not contain the amino acids corresponding to the markers provided by the transforming plasmid. The yeasts are then placed in culture for 72 hours at 28 ° C.

In the particular case of the transformation of yeast by the lung cDNA library, the procedure is as follows. The yeast used contains the plasmid pGAL4DB-GAX expressing the GAX protein in the form fused to the DNA binding domain of GAL4. It is cultivated in

7 250 ml of minimum YPG medium at 28 ° C. with stirring up to a density of 10 cells / ml. The cells are harvested by centrifugation at 3000 rpm for 10 minutes and taken up in 250 ml of water. After another centrifugation, the cell pellet is taken up in 100 ml of water and again centrifuged. The pellet is then taken up in 10 ml of the transformation solution I and incubated for 1 hour at 28 ° C. with shaking. After centrifugation, the cells are again taken up in 2.5 ml of the transformation solution I, 100 μl of the lung cDNA bank and 20 ml of the transformation solution II, then incubated for 1 hour at 28 ° C with stirring. A thermal shock is carried out on this transformation mixture at 42 ° C for 20 minutes. Centrifugation (3000 rpm for 5 minutes) is repeated 3 times in succession, each time taking up the pellet with 10 ml of sterile water. The third time the pellet is taken up with 2.5 ml of PB S. Thus the toxic PEG for the cells has been eliminated. 2.4 ml of this suspension are used to inoculate 250 ml of minimum medium containing the amino acids His, Lys, Met and the bases Ura and Ade and cultured overnight in a shaker at 28 ° C. The remaining 100 μl of this suspension is used to verify the efficiency of the transformation; for this dilutions of 10 -2, 10 -3 and 10 -4 of this suspcffsion were made. The overnight culture is centrifuged (30 ° -pm for 5 min) and washed with sterile water twice in succession. The residue is then taken up in 2.5 ml of water. 2.4 ml, the volume of which is brought to 10 ml in sterile water, are used to inoculate 10 boxes of 435 cm 2 containing a medium

YNB + Lys + Met + His + Ade, and incubated for 3 days. The remaining 100 μl are used to carry out the same operations as when determining the transformation rate, in order to determine the rate of amplification of the number of colonies during a culture night.

11) Preparation of yeast DNA (genomic and plasmid)

The value of an average loop of a yeast clone is put in 200 μl of a TELT solution (Triton XI 00 2%, SDS 1%, NaCl lOOmM, Tris pH8 lOmM, EDTA ImM), in the presence of 3g of beads of glass 450 μm in diameter and 200 μl of phenol / chloroform. This mixture is vorified for 15 minutes, then centrifuged for 2 minutes at 14,000 rpm. The supernatant is collected without removing the protein cake and the DNA contained in this phase is precipitated with 2.5 volumes of absolute ethanol. After centrifugation for 2 minutes at 14,000 rpm, the DNA pellet is dried and taken up in 20 μl of TE-RNAse This DNA solution, which corresponds to a mixture of genomic and plasmid DNA, is used directly to transform bacteria. Only plasmid DNA is capable of replicating in bacteria and can be analyzed by the miniprep technique

12) β-galactosidase activity test

A nitrocellulose sheet is previously deposited on the Petri dish containing the individualized yeast clones. Thanks to the adsorption phenomenon, a faithful image of the location of the clones is obtained. This sheet is then immersed in liquid nitrogen for 30 seconds in order to burst the yeasts and thus release the β-galactosidase activity After thawing, the nitrocellulose sheet is deposited, colonies upwards, in another petri dish containing Whatman paper previously soaked in 1.5 ml of PB S solution ( Na2HPO4 60mM, NaH2PÛ4 40mM, KC1 10MM, MgSθ4 ImM, pH7) and from 10 to 30μl of X-Gal (5-bromo-4-chloro-3-indoyl-bD- galactoside) at 50mg / ml in N, N- dimethylformamide The box is then placed in an oven at 37 ° C with the lid closed to prevent drying. The time of appearance of the blue color can be very variable, from a few minutes to several hours. This test must always be done in advance. sence of a positive control whose interaction is known and quickly turns blue

13) Construction of the expression vectors and of the reporter gene a) Expression vectors

The estrogen receptor (ER) cDNA is obtained by reverse transcription (RT) using a commercial kit (first strand cDNA synthesis Kit from Pharmacia), from total RNA extracted from mouse uterus, followed PCR amplification We used a pair of specific primers which hybridize, in 5 'with the first 20 nucleotides of ER and introducing an EcoR I site just upstream of the first codon (5'- gagcgααt / cATGACCATGACCCTTCACAC SEQ ID N ° 6, the nucleotides in italics represent the EcoR I site and in capitals underlined the first codon), on the 3 ′ side the back primer begins at the stop codon of ER and also introducing an EcoR I site (5′- gagcgaattc ACTGATCGTGTTGGGGAAGC SEQ ID No. 7, the nucleotides in italics represent the EcoR 1 site and in underlined capitals the stop codon) The PCR fragment obtained was purified, digested with EcoRI and then cloned at the same site in the expression vector pCDNA 3 (Invitrogen). vector is called pCMV-ER. The different GAX deletion mutants fused to the DNA binding domain of GAL4 (pCMV-GALDB / GAX, see example 9) were also cloned into the vector pCDNA 3

b) Gene reporter

The reporter gene was constructed according to the strategy described by Martinez et al (Martinez et al 1991, o /. Cell. Bwl. 1 1,2937-2945) except that the cloning vector used is pG5CAT (Clontech, CAT = Chloramphenicol Acetyl Transferase). We have synthesized a double-stranded oligonucleotide (below) containing two specific sites (in bold) one recognized by the DNA binding domain of GAL4 (Gal-17mer, Carey et al 1989, J. Mol. Bwl . 209,423-432) and the other is a consensus sequence of the "Estrogen Responsive Element" (ERE, Beato M 1989, Cell 56,335-344) which binds the receptor to estrogens. This adapter oligonucleotide has in 5 'a protruding Xhol site and 3 'an Xbal site which allowed cloning into the Xho I and Xba I sites of pG5CAT

SEQ ID N ° 8

tcgagAGGTCATATTGACCTaagcttCGGGTCGGAGTACTGTCCTCCGACTGCcatatgt cTCCAGTATAACTGGAttcgaaGCCCAGCCTCATGACAGGAGGCTGACGgtatacagatc Xhol ERE Ga.l-17mex Xba. I

The principle of this reporter gene (pEREGICAT, Fig 4, example 1 1)) is based on the fact that ER is a transcriptional activator possessing, in the case of murine ER, a constitutive activity which increases in response to its natural ligand, the oestradiol 17-β This system was used by Matinez et al to study the synergy between ER and a second transcriptional factor, NFl, whose transcription activating domain has been fused to the DNA binding domain of the yeast protein GAL4 (GALDB) This reporter makes it possible, therefore, to account for the transcription rate due solely to one or the other of the transcription factors or their cooperation without interference due to endogenous molecules. This system therefore allows us to study the transcriptional activity of all or part of the GAX protein fused to GALDB (Fig 5,6 and examples 12, 13) A plasmid expressing the luciferase gene under the control of the CMV promoter (pCMV-Luc) was constructed. This construction was obtained after cloning of a PCR fragment (flanked in 5 'and 3' from a BamHI site) containing the CMV promoter of the vector pCDNA3 (Invitrogen), in the BglII site of the vector pGL3 -Basic (Promega ). This plasmid will serve as an internal standard in all of the transient transfection experiments that follow.

14) Experiments with transient cotransfection

All the studies were carried out in murine embryonic fibroblasts NIH-3T3 (ATCC) These cells were maintained in routine in DMEM (GIBCO) supplemented with 100 U / ml of penicillin (GIBCO), 100 μg / ml of streptomycin (GBCO ), 2 mM L-glutamine (GBCO) and 10% fetal calf serum (SVF, GIBCO) This medium will be designated by the abbreviation DMEM-GPS / SVF.

For transient transfection experiments, the NIH-3T3 are seeded at a density of 100,000 cells per well of a 24-well culture plate (Falcon), in a volume of 1 ml of DMEM-GPS / SVF 16 at 20 hours later. , after attaching and spreading the cells, the cells are washed with 0.5 ml of DMEM-GPS (without SVF) and then put back into 0.5 ml of DMEM-GPS 50 μl of a transfection mixture are then added per well. culture This mixture is obtained as summarized in Table 3 below

Table 3 * The different mixtures, numbered from 1 to 4, are designed to study respectively, the basal activity of the EREGICAT promoter, the activity of ER or GADB / GAX alone and finally the simultaneous effect of ER and GALDB / GAX. ** pCDNA3 is used to complete each mixture to 10 μg total

*** Lipofectamine at 2 μg / μl (gibco) is used at a ratio of 8/1

(lipofectamy / DNA)

The cells are brought into contact with the various transfection mixtures for 4 hours at 37 ° C. under standard culture conditions. The DMEM-GPS, with the transfection mixture, is then replaced with 1 ml of DMEM-GPS / SVF then the cells are kept in culture for 24 hours Each transfection is carried out on at least 4 wells

At the end of the 24 hour period, the cells are washed with 0.5 ml of Dulbecco's PBS (GIBCO) and then harvested in 0.5 ml of a 0.2% trypsin solution in PBS (GIBCO) Trypsin is neutralized with 10 μl of FCS This cell suspension is centrifuged for 2 min at 10,000 g, the supernatant is removed and the cells are resuspended in 150 μl of 0.25 M Tris-HCl at pH 7.8 50 μl of this suspension are used to assay luciferase activity according to the Kit "luciferase Assay System" (Promega) and using the luminometer LUMAT LB 9501 (EG&G Berthold) The cytosolic proteins of the cells in the remaining 150 μl are extracted by 5 cycles of freezing / thawing (liquid nitrogen / 37 | C) These extracts, normalized with respect to luciferase activity, are then used to measure the CAT activity according to the method previously described by Gorman et al (Gorman et al 1982, Mol. Cell. Bwl . 5, 1044-1051)

15) In vitro interaction between the Ki and GAX proteins

It is carried out by the Far Western Blotting method While Western Blotting consists of an electrophoretic separation of proteins on a denaturing gel followed by an electrotransfer of proteins on a nitrocellulose membrane and a direct or indirect immunodetection, the Far - Western Blotting is a Western Blotting to which is added a protein-protein interaction step between a target immobilized on the nitrocellulose membrane and a factor in solution a) Electrophoresis

The samples are heated for 10 minutes at 95 ° C. in a protein denaturation buffer containing 50 mM Tris pH 6.8, 2% SDS (Sodium Dodecyl Sulfate), 10%> glycerol, 300 mM b-mercaptoethanol and 0 , 1% bromophenol blue 10 ml of the samples are deposited on a Tris-Glycine 14% Acrylamide gel (Tris-Glycine Gels, Novex) Migration takes place for 1 hour at a constant voltage of 120 V in a separation buffer for proteins (35 mM SDS, 1.92 M Glycine and 85 mM Tris pH 8.3) The molecular weight of proteins will be determined by the parallel migration of a colored and transferable molecular weight standard (MultiMarkTM Multi-Colored Standard , Novex) This gel is made in duplicate to be able to color one of the two with Coomassie blue (Methanol 30%, water 60%, Acetic acid 10%, Coomassie blue 0, 1%) The color is 1 hour Discoloration takes place for several hours, changing the soil several times bleaching solution (30% methanol, 10% acetic acid, 60% water) The detection limit of this method is 50 ng of proteins. This colored gel therefore allows us to visualize all the proteins deposited. The other gel is used for the " Far- Western Blotting "

b) Semi-dry transfer onto a nitrocellulose membrane The nitrocellulose membrane (Hybond C from Amersham) and the gel are taken between 3 thicknesses of Whatman paper previously immersed in the transfer buffer (Glycine 150 mM, Tris 25 mM, Methanol 20 %, water qs 1 liter, pH 8.3) Transfer takes place for 40 minutes at 2.5 mA / cm2 of gel (MilliblotTM-Graphite Electroblotter II device, Millipore)

c) Protein-protein interaction

The membrane is saturated for 30 minutes with stirring and at room temperature in PBS containing 5% (w / v) of skimmed milk powder (Gloria) and 0.2% (v / v) of Tween 20 Then it is immersed for 1 hour with stirring and at room temperature in 5 ml of PBS containing 5% (w / v) of skimmed milk powder (Gloria), 0.2% (v / v) of Tween 20 and 75 μg of KI L ' Incubation completed, the membrane is washed 3 times for 10 minutes in PBS containing 0.2% (v / v) of Tween 20 In order to visualize the interaction between GST-GAX immobilized on the nitrocellulose membrane and mycKi, this is reveal as for GST-GAX using a monoclonal antibody mouse directed against the myc epitope (Santa Cruz) and a polyclonal rabbit antibody coupled, with peroxidase, directed against mouse IgG (Nordic Immunology)

EXAMPLES

EXAMPLE 1 Construction of a vector allowing the expression of a fusion protein between GAX and the DNA-binding domain of GAL4

The screening of a library using the double hybrid system requires that the GAX protein be fused to the DNA binding domain of the GAL4 transactivating protein. The expression of this fusion protein is carried out using the vector pGBTIO (cf. materials and methods ), in which we have introduced, in the same reading frame as the sequence corresponding to the DNA binding domain of GAL4 (GAL4DB), a fragment coding for all or part of the GAX protein A particular fragment comprises the EcoRl fragment -Sall from phGAX, which is inserted at the EcoRl-Sall site of pGBTIO to give the plasmid pCM199

The construction was sequenced which makes it possible to verify that the GAX protein is indeed in the same open reading phase as that of the fragment corresponding to GAL4DB

EXAMPLE 2 Screening of the Lung Fusion Bank.

The screening of a fusion library makes it possible to identify clones producing proteins fused to the GAL4 transactivating domain, which can interact with the GAX protein or domains thereof. This interaction makes it possible to reconstitute a transactivator which will then be capable of induce the expression of the reporter genes URA3, BLE and LacZ in the strain YCM79 To carry out this screening we have chosen a fusion library produced from cDNA originating from human lung As this library was supplied to us in the form of bacteria, the plasmid DNA of the bank was first purified

2 1) Preparation of the plasmid DNA of a fusion library The plasmid DNA of the lung cDNA bank was extracted according to the Clontech protocol (see materials and methods, § 10). During this preparation it was important to preserve the representativeness of the bank, that is to say, keep the number of independent plasmids which constitute it and which are 7 10 plasmids

In order to protect us from the loss of plasmids from the library during this preparation, the batch of plasmid DNA that we have assembled was obtained from a number of isolated bacterial colonies corresponding to a little more than three times the representativeness of the bank, i.e. 25 10 colonies

2 2) Transformation by lung bank and selection by the beta-galactosidase activity test

During screening, it is necessary to preserve the probability that each plasmid independent of the fusion bank is present in at least one yeast at the same time as the plasmid GAL4DB-GAX To preserve this probability it is important to have good transformation efficiency. yeast For this we have chosen a yeast transformation protocol giving an efficiency of 10 cells transformed by μg of DNA In addition, as the cotransformation of yeast by two different plasmids reduces this efficiency, we preferred to use yeast beforehand transformed by the plasmid pGAL4DB-GAX This yeast strain was transformed by l OOμg of plasmid DNA from the fusion bank This quantity of DNA allowed us to obtain after estimation (see materials and methods) 4.2 10 cells transformed, which corresponds to a number greater than the number of independent plasmids that constitute the bank According to this res Ultat we can think that almost all of the plasmids from the library were used to transform the yeasts. The selection of transformed cells, capable of reconstituting a functional GAL4 transactivator, was made on a YNB + Lys + Met + His + Ade A l medium. from this selection 190 clones with a Ura + and bGal + phenotype were obtained

EXAMPLE 3 Identification of the inserts of the selected plasmids: Demonstration of an interaction with Ki The plasmids are extracted from the yeast, introduced into the bacteria and then prepared as described in the materials and methods section. The sequencing was carried out from 1 oligonucleotide CTATTCGATGATGAAGATACCCC (SEQ ID No. 1) complementary to the GAL4TA region near the insertion site of the lung cDNA bank, at 52 bpd from the EcoRI site.

Comparison of the sequences with the sequences contained in the GENBank and EMBL (European Molecular Biology Lab) databases has shown that one of the plasmids thus identified contains a cDNA identical to factor Ki, identified in patients with lupus as autoanti gene. This plasmid is called pCM282 This plasmid, when it is cotransformed in yeast with pCM199, is well capable of activating the reporter genes thereof as shown in Table 1 below This Table further shows that the activation is specific, which indicates that Ki is able to interact in a specific way with GAX

Table 1

EXAMPLE 4 Construction of Vectors allowing the Expression in Yeast of a Fusion Protein Between Different Deletants of GAX and the DNA Binding Domain of GAL4

This example describes the construction of vectors coding for different variants of the Gax protein, which can be used to determine the structure / function of this protein and in particular, to highlight the active domains and the domains responsible for the interaction with the Ki protein. The deletion of the 153 C-terminal amino acids is obtained by digesting the plasmid pCM199 by Eagl-Sall, then the small fragment is eliminated, the ends are made blunt by treatment with klenow and religated The plasmid thus obtained is called pCM238 II code for a protein with residues 1-104 of GAX

Total digestion with Bgl2 and SalI of pCM199 and then the religation of the vector after treatment with klenow makes it possible to conserve the first 32 amino acids of GAX. The plasmid thus obtained is called pCM244 II code for a protein comprising residues 1-32 of GAX

Total digestion with Bgl2 and SalI of pCM199 makes it possible to isolate a fragment of approximately 270 bp and 572 bp. The first fragment is cloned in pGBTl 1 at the Bamhl-SalI site to obtain the plasmid pCM245 which allows the fusion of the 79 amino acids C-ter GAL4DB The second fragment is cloned into pGBTIO at the Bamhl site to obtain the plasmid pCM246 which allows the fusion of amino acids 33 to 222 with GAL4DB

The plasmid pCM280 is obtained by digesting pCM246 with Dra3 and pstl After treatment with T4 polymerase the plasmid is closed on itself II codes for a protein comprising residues 33-63 of GAX

The plasmid pCM199 is digested with NdeI and Pst1 The insert is cloned into the plasmid pAS 1 to give the plasmids pCM301 This plasmid allows the expression of the GAX protein deleted from these first 32 amino acids fused to GAL4DB II codes for a protein comprising the GAX residues 33-302

The structure of the fusion protein expressed by these different vectors is shown in Figure 1

EXAMPLE 5: Location of the GAX interaction zone with KL

The yeast strain yCM79 is transformed by the different vectors described in examples 1 and 4 at the same time as pCM282 or that pGAD424 The Betalgal activity is revealed as described in the material and methods The results obtained are presented in Table 2 below

Table 2

These results make it possible to reveal the region of GAX which interacts with the factor Ki (Table 2) In fact, the regions causing the activity to lose by their absence make it possible to conclude that they are necessary for the interaction but not sufficient Thus the regions 1 to 32 and 104 to 223 seem important for the interaction with Ki The fact that pCM238 does not give a positive signal in Beta-gal, suggests that there is a C-Term region 104-302 of Gax necessary for the interaction yeast EXAMPLE 6 Profile of expression of mRNA in different tissues and nuclear localization of the protein expressed transiently:

In order to identify GAX partners we have transformed our double hybrid yeast strain, already possessing the plasmid allowing the expression of the GAL-GAX fusion (1-302) as bait, by a lung cDNA library fused to GAL4 TA, available in the laboratory (example 2) We were able to obtain, before amplification, more than 41 million independent clones and only 190 clones in which the three selection genes (the e URA3 gene, the LacZ gene and the BLE gene) are expressed 9 cDNAs coding for different proteins could be identified among these 190 clones We were more particularly interested in that coding for the Ki antigen

The Ki gene seems to be ubiquitous as suggested by the analysis by northern blotting (Figure 1) of its expression from mRNA extracted from different tissues (human Multiple Tissue Northern Blots, Clontech) entertaining an mRNA of approximately 3 kb We can note the presence of a form of mRNA undergoing different maturation (1, 4 kb) in the heart and skeletal muscles

The construction of a vector allowing the expression of the Ki protein fused to the HA tag in mammalian cells was carried out as follows: the Nco l -Xho l fragment of the plasmid pCM282 is inserted into the plasmid pAS l at the level of Nco l -Xho l sites to obtain the plasmid pCM322 Then the EcoRl -Dra l fragment of the plasmid pCM322 is inserted into the expression vector pCDNA3 at the EcoRl-EcoRV sites The plasmid obtained, pCM323 allows the expression of the protein ki fused to the HA tag in mammalian cells

By indirect immunofluorescence it is shown that KiHA has a nuclear localization in cells transfected with this chimera (Figure 2)

EXAMPLE 7 Expression and purification of the Ki protein fused to the S tag and yc tag The following oligos are hybridized together, phosphoπles then ligated with pET29B previously digested with Nco 1

CATCAAGCTTATGGAGCAGAAGCTGATCTCCGAGGAGGACCTGCAGCTTC (SEQ ID N ° 2) and CATGGATCCACGTGCAGGTCCTCCTCGGAGATCAGCTTCTGCTCCATAAGC

TT (SEQ ID N ° 3)

The plasmid obtained is named pCM320 The gene coding for the protein Ki is amplified by PCR with the oligos CGCGGATCCCATGGCCTCGTTGCTG (SEQ ID No. 4) and GTAGAGCTCGAGTCAGTACAGAGTCTCTGC (SEQ ID No. 5) The fragment obtained is digested with Bamhl Xhol and then ligated , at the bamHl-Xhol sites of the plasmid pBCks to give the plasmid pCM305 The latter is then digested with Bamhl Xhol The insert thus released is ligated at the Bamhl and Xhol site of pCM320 to give the plasmid pCM321 The expression and the purification of the protein recombinant are carried out according to the supplier's instructions (Novagen) from pCM321

EXAMPLE 8 Expression and purification of the GAX protein fused to GST

Colonies of BL-21 transformed with the plasmid pGEX-2T-h-GAX are placed in preculture in 30 ml of LB with ampicillin (50 μg / ml) at 37 ° C overnight 5 ml of this preculture are returned to culture in 500 ml of LB with ampicillin at 37 ° C up to an optical density of 0.7 at 600 nm The expression of GST-GAX is induced by 0.1 mM IPTG for 2 hours at 30 ° C. The culture is centrifuged at 6000 rpm for 10 min The bacteria pellets are redissolved in 10 ml of cold PBS and then distributed in 10 eppendorf tubes The bacterial proteins are extracted by sonication for 10 minutes with 12-second cycles and 24-second breaks followed centrifugation at 15,000 rpm for 15 minutes The supernatants are combined and constitute the soluble fraction which will be used for purification The remaining pellets represent the insoluble fraction up to an optical density of 0.7 to 600 nm. The expression of GST-GAX is induced by 0.1 mM IPTG for 2 hours at 30 ° C. The culture is centrifuged at 6000 rpm for 10 min. The bacteria pellets are redissolved in 10 ml of cold PBS and then distributed in 10 eppendorf tubes. Bacterial proteins are extracted by sonication for 10 minutes with 12-second cycles and 24-second pauses followed by centrifugation at 15,000 rpm for 15 minutes. The supernatants are combined and constitute the soluble fraction which will be used for the purification. The remaining pellets represent the insoluble fraction.

The purification of GAX is done by affinity of GST on agarose beads coupled to Glutathione.

The supernatant obtained after sonication of the bacteria is incubated with the resin for one hour on a rotary shaker at room temperature. Then, the resin on which the protein is bound is centrifuged at 1000 rpm for 10 minutes. The supernatant removed, the resin is washed 3 times with 50 ml of PBS containing 1% Triton X-100 for 20 minutes on 1 rotary shaker. GST-GAX can be eluted from the resin with guanidine thiocyanate. Elution is carried out by 1.5 times the volume of resin of 2M guanidine thiocyanate, 20 mM Tris pH 7.5, 0.15 M NaCl and 0.1% Triton X-100 on a rotary shaker during 30 minutes. The eluate is dialyzed against PBS overnight to remove the guanidine thiocyanate. The protein is stored in PBS at 4 ° C. A cocktail of protease inhibitors of equal quantity (Leupeptine lmg / ml, Pepstatin lmg / ml, Aprotinin l mg / ml. Benzamidine 500 mM) is added to the 1/200 th to the protein preparation.

EXAMPLE 9 In Vitro Interaction Between the Ki and GAX Proteins

To study the interaction of GAX and Ki in vitro, we produced two chimeric recombinant proteins in Escherichia Coli. GST-GAX was purified on a resin coupled to glutathione thanks to the catalytic site of glutathione S-transferase (GST); SmycKi carrying a myc epitope and the S epitope allowing purification on a resin coupled to protein S.

The indicated amounts of bovine albumin, GST protein or GST-GAX after digestion with thrombin (site created between GST and GAX), as well as increasing amounts of GST-GAX, were separated on a denaturing polyacrylamide gel and then stained with coomassie blue (Figure 3 A).

The proteins were transferred to a nitrocellulose membrane from a gel identical to that at A. The membrane was successively incubated in the presence of SmycKi, in a solution containing a mouse monoclonal antibody directed against the myc epitope (Santa Cruz) and finally in a solution for the detection of the anti-myc antibody.

Our results clearly show that Ki specifically recognizes GST-GAX and in a dose-dependent manner, while no reaction is observed with bovine albumin or GST. Note that after digestion with thrombin Ki still interacts with GAX (Figure 3B).

EXAMPLE 10 Construction of Vectors Permitting the Expression in Mammalian Cells of a Fusion Protein Between Different Deletants of GAX and the DNA Binding Domain of GAL4

10.1. Construction of plasmid vectors

The plasmids pCM199 and pCM301 are digested with HindIII. The inserts are cloned in the correct orientation into pCDNA3 (invitrogen) at the HindIII site to give the plasmids pCM291 and pCM327 respectively, allowing expression of fusions in mammalian cells.

The plasmids pCM238, pCM244, pCM301, pCM246, and pCM280 are digested with HindIII and Nael. The inserts are cloned in the correct orientation in pCDNA3 (invitrogen) at the HindIII-EcoRV site to give the plasmids pCM292, pCM326, pCM327, pCM294 and ρCM295, respectively, allowing the expression of fusions in mammalian cells.

10.2. Construction of viral vectors According to a particular embodiment, the invention resides in the construction and the use of viral vectors allowing the transfer and the expression in vivo of nucleic acids as defined above.

With regard more particularly to adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized. Among these serotypes, it is preferred to use, in the context of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO94 / 26914). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example]. Preferably, in the context of the invention, adenoviruses of human or canine or mixed origin are used.

Preferably, the defective adenoviruses of the invention comprise ITRs, a sequence allowing the packaging and a nucleic acid according to the invention. Even more preferentially, in the genome of the adenoviruses of the invention, the region E 1 at least is non-functional. The viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the gene or genes considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents. Other regions can also be modified, and in particular the region E3 (WO95 / 02697), E2 (W094 / 28938), E4 (WO94 / 28152, W094 / 12649, WO95 / 02697) and L5 (WO95 / 02697). According to a preferred embodiment, the adenovirus according to the invention comprises a deletion in the regions E1 and E4. According to another preferred embodiment, it comprises a deletion in region E1 at the level of which the region E4 and the nucleic sequence of the invention are inserted (Cf FR94 13355). In the viruses of the invention, the deletion in the E1 region preferably extends from nucleotides 455 to 3329 on the sequence of the adenovirus Ad5.

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185,573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia a nucleic sequence or a combination of nucleic sequences of the invention. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697 or in Yeh et al., J. Virol. 70 (1996) 559. Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology.

Concerning adeno-associated viruses (AAV), these are DNA viruses of relatively small size, which integrate into the genome of the cells they infect, in a stable and site-specific manner. They are capable of infecting a wide spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. Furthermore, they do not seem to be involved in pathologies in humans. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4,700 bases, and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as an origin of replication for the virus. The rest of the genome is divided into 2 essential regions carrying the packaging functions: the left part of the genome, which contains the rep gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene coding for the capsid proteins of the virus. The use of vectors derived from AAVs for gene transfer in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; US 4,797,368, US5, 139,941, EP 488,528). These applications describe various constructs derived from AAVs, in which the rep and / or cap genes are deleted and replaced by a gene of interest, and their use for transferring in vitro (onto cells in culture) or in vivo (directly into an organism ) said gene of interest. The defective recombinant AAVs according to the invention can be prepared by cotransfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing a nucleic sequence or a combination of nucleic sequences of the invention bordered by two inverted repeat regions (ITRs) of AAV, and of a plasmid carrying the packaging genes (rep and cap genes) of AAV. A usable cell line is, for example, line 293. Other production systems are described, for example, in applications WO95 / 14771; W095 / 13365; WO95 / 13392 or WO95 / 06743. The recombinant AAVs produced are then purified by conventional techniques.

Concerning herpes viruses and retroviruses, the construction of recombinant vectors has been widely described in the literature: see in particular Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EPI 78220, Bernstein et al. broom. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc. In particular, retroviruses are integrative viruses, selectively infecting dividing cells. They therefore constitute vectors of interest for cancer applications. The genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses; the gag, pol and env genes are generally deleted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest. These vectors can be produced from different types of retroviruses such as in particular MoMuLV ("murine Moloney leukemia virus"; also designated MoMLV), MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus") ; SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") or the Friend virus. To construct recombinant retroviruses according to the invention comprising a nucleic nucleic sequence or a combination of nucleic sequences according to the invention, a plasmid comprising in particular the LTRs, the packaging sequence and said nucleic sequence is constructed, then used to transfect a line so-called packaging cell, capable of providing trans retroviral functions deficient in the plasmid. Generally, the packaging lines are therefore capable of expressing the gag, pol and env genes. Such packaging lines have been described in the prior art, and in particular the line PA317 (US4,861,719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150). In addition, recombinant retroviruses may include modifications to the LTRs to suppress transcriptional activity, as well as packaging sequences. extended, comprising part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). The recombinant retroviruses produced are then purified by conventional techniques.

10.3 - Chemical vectors

The nucleic acids or the plasmid expression vectors described in the preceding examples can be administered as they are in vivo or ex vivo. It has indeed been shown that naked nucleic acids can transfect cells. However, to improve the transfer efficiency, it is preferred to use, within the framework of the invention, a transfer vector. It can be a viral vector (example 9.2.) Or a synthetic transfection agent.

Among the synthetic vectors developed, it is preferred to use, within the framework of the invention, cationic polymers of polylysine type, (LKLK) n, (LKKL) n, (PCT / FR / 00098) polyethylene imine (WO96 / 02655) and DEAE dextran or cationic or lipofectant lipids. They have the property of condensing DNA and promoting its association with the cell membrane. Among these, mention may be made of lipopolyamines (lipofectamine, transfectam, etc.), different cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.) as well as peptides of nuclear origin. In addition, the concept of targeted transfection has been developed, mediated by a receptor, which takes advantage of the principle of condensing DNA thanks to the cationic polymer while directing the binding of the complex to the membrane thanks to a chemical coupling between the cationic polymer and the ligand of a membrane receptor, present on the surface of the cell type that is to be grafted. The targeting of the transferrin, insulin receptor or the hepatocyte asialoglycoprotein receptor has thus been described. The preparation of a composition according to the invention using such a chemical vector is carried out according to any technique known to those skilled in the art, generally by simple contacting of the various components.

EXAMPLE 11: Development of a method for studying the transcriptional activity of GAX

We used a transient transfection system to study the transcriptional activity specific to GALDB-GAX fusions as well as their effect on transcriptional activators such as the estrogen receptor (ER) or the domain. activation of protein VP16 from the Herpes simplex virus fused to GALDB. In order to quantify the transcriptional activity of each or of the combination of several factors, we have constructed a target gene (Reporter) expressing the bacterial chloramphenicol acetyl transferase (CAT) under the control of an artificial promoter. This promoter contains a TATA box, originating from the E1B gene of the adenovirus Ad2 (E1B TATA), upstream from the site of initiation of the transcription of the CAT gene (FIG. 4 A). A TBP protein from the TFIID complex recognizes the TATA box and positions the preinitiation complex (TFIID + TFII A, B, E, F) which recruits RNA polymerase type II (PolII) which will initiate the transcription of the CAT gene. Upstream of this basic promoter we have inserted a GALDB binding site (17 mer) which will be recognized by the chimeras GALDB-GAX or GALDB-VP16. We also cloned an estrogen response element (ERE) upstream of the 17th sea on which ER binds to activate transcription. This reporter will allow us to study the different schematic combinations, activation or repression of transcription will result in a change in the amount of CAT in a cell. The ^" assay of CAT enzyme activity in cell extracts after transfection is used to measure the transcriptional effect.

ER and GAL4-VPÎ6 sυnLdes ^~ very strong transcriptional activators. The CAT activity is increased by more than 100 times by ER and approximately 85 times by GAL4-VP16. The coexpression of ER and GAL4-VP16 results in a CAT activity of more than 300 times that of the unactivated reporter and greater than the sum of the two activators expressed individually. This suggests that ER and GAL4-VP16, despite steric hindrance, can occupy and activate the same promoter (Figure 4B).

EXAMPLE 12 Study of the transcriptional activity of GALDB-GAX fusions and identification of a repressor domain for transcription

The GAX gene codes for a protein of 302 amino acids in humans. The primary structure of this protein has different domains whose function remains unknown. GAX belongs to a family of genes called homeoboite (HOMEOBOX). Homeobox is necessary for the recognition and binding of these proteins on specific DNA sequences present on the promoter of the target genes whose control the expression. To date, no DNA sequence recognized by GAX has been identified. GAX has a region rich in Histidine residues (HIS) in its N-terminal part whose function is unknown.

To understand the function of GAX, different regions of the human GAX gene have been deleted to produce truncated proteins in both their N-terminal and C-terminal parts, the numbers indicate the position of the first and last amino acids of each deletion mutant. compared to whole GAX 1-302 (Figure 5). Not knowing a DNA sequence recognized naturally by GAX, we have created chimeras between the different deletion mutants and a heterologous DNA binding domain (fused to their N-terminal part) originating from the yeast transcription factor GAL4 (GALDB). Expression vectors have been constructed to transiently express the different chimeras in cultured mammalian cells

The different GAL-GAX chimeras were expressed alone or in the presence of ER to study their effect respectively on the basal transcription or on the transcriptional activity of ER.

Whole GAX. in the context of the GAL-GAX 1 -302 merger, decreases CAT activity by more than 8 times compared to the non-activated reporter. This repression is slightly affected when the 32 N-terminal amino acids of GAX are absent (GAL-GAX33-302) but it is completely lifted when an additional deletion removes the C-terminal part of GAX (GAL-GAX33-222). These 32 amino acids are sufficient in the context of GAL-GAX 1-32 to decrease the CAT activity (Figure 6 A).

The coexpression of GAL-GAX 1 -302 and ER results in a CAT activity more than 25 times lower than that obtained with ER alone. This repression as in A is due to the first 32 amino acids of GAX (compare GAL-GAX1-302, GAL-GAX33-302 and GAL-GAX33-222). These 32 N-terminal amino acids (GAL-GAX 1-32) decrease the activity of ER only by 2 times (compare GAL-GAX 1-32, GAL-GAX 1 - 104) and require at least the presence of residues 33 to 222 for maximum activity (Figure 6B). The sequences making lose the activity when they are absent are sought, allowing to conclude that they are necessary for the interaction but not sufficient.

The results presented show that GAX represses transcription of the reporter gene used. This repression is essentially due to an N-terminal peptide (1 to 32) whose optimal activity requires the presence of the region 33 to 222 of GAX and preferably the presence of the region 33-104 of Gax.

Using the different deletions of GAX fused to the DNA binding domain of GAL4, we were able to identify in yeast (by the double hybrid system, Example 5) the regions of GAX important for the interaction with Ki. This is the N-terminal part up to residue 222. This region includes, as we have shown above, the repressor domain of GAX.

EXAMPLE 13 In vitro interaction between GAX and PCNA

13.1 Cloning of the Human Proliferating Cell Nuclear Antigen (PCNA) cDNA and Production of a Recombinant Protein

The cloning of the PCNA cDNA is carried out by reverse transcription and PCR. The first reverse transcription (RT) step is carried out with the "First Strand cDNA synthesis" kit from Pharmacia. Total RNA is extracted from human smooth muscle cells in primary culture (Clonetics) according to the method described by P. Chomczynski and N. Sacchi (Anal. Biochem. 1987, 162: 156-159). 10 μg of this total RNA is taken up in 20 μl of water are heated for 10 minutes to 65 ° C., cooled on ice and then mixed with 11 μl of "Bulk First Strand reaction mix" from the RT kit (Cloned, FPLCpwre®, Murine Reverse Transcriptase, RNase / DNase-Free BSA, dATP, dCTP, dGTP, and dTTP), 1 μl of DTT and 1 μl of pD (N) primers ₆ . The RT reaction is incubated for 1 hour at 37 ° C. The reaction is stopped by heating for 5 minutes at 90 ° C. and then cooled on ice.

The second step consists in PCR amplification of the PCNA cDNA. The primers used for the PCR reaction are the following:

- CGCGαaattcTGTTCGAGGCGCGCCTGGTCCAGG- 3 F E A R L V Q G

2 5 '- GGTCσaattcTAAGATCCTTCTTCATCCTCGATC - 3' * S G E E D E I K

The two primers introduce an EcoRI site (lowercase underlined). The PCNA amino acids contained in the primers are represented by their code (capital letters) below the corresponding codons. * represents the stop codon of PCNA.

8 μl of the RT reaction, described above, are completed to a final volume of 50 μl containing the following final quantities or concentrations: 50 picomoles of primer 1, 50 picomoles of primer 2, one unit of Taq DNA polymerase (Perkin Elmer), 5 μl of MgC12 (25 mM), 2 μl of a 200 mM mixture of the 4 deoxynucleotides (dATP, dTTP, dGTP and dCTP) and 5 μl of the 10-fold concentrated PCR buffer (Perkin Elmer). The PCR amplification is carried out in Micoamp ™ tubes (Perkin Elmer) using a PTC-100 ™ thermocycler (MJ Research, Inc.). This amplification consists of a denaturation step at 95 ° C for 2 min followed by 30 cycles including a denaturation step of 15 sec at 95 ° C, a hybridization step of 30 sec at 55 ° C and an extension step 1 min at 72 ° C. These thirty cycles are followed by an additional extension of 5 min, then the PCR reactions are stored at 10 ° C.

The practical cloning of PCNA in the vector PCRII (Invitrogen) is carried out with the "Original TA Cloning® Kit" (Invitrogen). 3 μl of PCR product, 1 μl of 10X ligation buffer, 2 μl of pCRII vector (25 ng / μl), 3 μl of water and 1 μl of T4 DNA ligase are incubated at 16 ° C for 16 hours.

2 ml of this ligation reaction are used to transform competent TG1 bacteria as previously described. The bacteria are then cultivated at 37 ° C. for 16 hours on LB solid medium containing 1.5% Agar, 100 μg / ml of ampicillin in the presence of X-gal for the blue white selection of the recombinant clones (alpha complementation β-galactosidase).

The recombinant clones (white colonies) are taken up in 10 μl of water and confirmed under the same conditions as the PCR after the RT reaction described above except that of triton X-100 at a final concentration of 0.01% v / v is added to the reaction. Several positive clones are used to make DNA mini-preparations, those in which the 5 ′ part of the PCNA insert is positioned downstream of the EcoRV site of pCRII are sequenced and selected for the next cloning step.

Cloning of PCNA into the plasmid pET29 is carried out as follows: the PCNA EcoRV-Hind III fragment derived from PCRII-PCNA is inserted at the EcoRV-Hind III site of pET-29. This plasmid is then used for the production of the chimeric recombinant protein S-PCNA. The stages of transformation, production and purification of S-PCNA are identical to those used for the production of the Ki antigen fused to the myc epitope.

13.2 Study of the in vitro interaction of GAX with PCNA

This study was carried out using the same method used in Example 9 to study the interaction between the recombinant proteins GST-GAX and SmycKi. Increasing amounts of recombinant GST and the chimera GST-GAX (50, 250, 500 and 750 ng) were separated on a 14% denaturing polyacrylamide gel (Novex). The proteins are transferred from the gel to a nitrocellulose membrane, which is then incubated in a solution containing the recombinant protein S-PCNA followed by immunostaining of PCNA using an anti-PCNA monoclonal antibody (Santa Cruz).

Figure 7 shows a weak interaction between GST and PCNA only at high doses of GST (500 and 750 ng). The affinity of GST-GAX for PCNA is very much higher than that with GST. This specific interaction between GAX and PCNA suggests that the antiproliferative activity of GAX is mediated by PCNA sequestration. The fact that Ki can interact both with GAX and with PCNA is in favor of the formation of bi or tripartite complexes which could play an important role (activation or inhibition) in the progression of the cell cycle.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(î) DEPOSITOR: (A) NAME: RHONE-POULENC RORER S.A.

(B) STREET: 20, avenue Raymond ARON

(C) CITY: ANTONY

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 92165 (n) TITLE OF THE INVENTION: Polypeptides comprising domains of the GAX protein involved in repression of transcription and / or interacting with other proteins, corresponding nucleic acids and their uses.

(in) NUMBER OF SEQUENCES: 8 (IV) FORM READABLE BY COMPUTER:

(A) TYPE OF SUPPORT: Tape

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: simple

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (ni) HYPOTHETIC: NO (lll) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

CTATTCGATG ATGAAGATAC CCC 23

2) INFORMATION FOR SEQ ID NO: 2: (1) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (il) TYPE OF MOLECULE: cDNA (m) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

CATCAAGCTT ATGGAGCAGA AGCTGATCTC CGAGGAGGAC CTGCAGCTTC 50

(2) INFORMATION FOR SEQ ID NO: 3:

(î) CHARACTERISTICS OF THE SEQUENCE:

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(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(m) HYPOTHETIC: NO

(m) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

CATGGATCCA CGTGCAGGTC CTCCTCGGAG ATCAGCTTCT GCTCCATAAG CTT 53

(2) INFORMATION FOR SEQ ID NO: 4:

(l) CHARACTERISTICS OF THE SEQUENCE

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (m) HYPOTHETIC: NO (m) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

CGCGGATCCC ATGGCCTCGT TGCTG 25 (2) INFORMATION FOR SEQ ID NO: 5:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: Single

(D) CONFIGURATION: linear

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(m) HYPOTHETIC: NO

(m) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

GTAGAGCTCG AGTCAGTACA GAGTCTCTGC 30

(2) INFORMATION FOR SEQ ID NO: 6:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(m) HYPOTHETIC: NO

(m) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6.

GAGCGAATTC ATGACCATGA CCCTTCACAC 30

(2) INFORMATION FOR SEQ ID NO: 7:

(î) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GAGCGAATTC ACTGATCGTG TTGGGGAAGC 30

(2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N ° 8:

TCGAGAGGTC ATATTGACCT AAGCTTCGGG TCGGAGTACT GTCCTCCGAC TGCCATATGT

CTCCAG TATAACTGGA TTCGAAGCCC AGCCTCATGA CAGGAGGCTG ACGGTATACAGATC

Claims

1. Polypeptide characterized in that it is all or part of a fragment of the GAX protein possessing transcription repressor activity and / or positively or negatively affecting DNA replication.

2. Polypeptide according to claim 1 characterized in that it comprises at least residues 1 to 32 of the human GAX protein.

3. Polypeptide according to claim 1 or 2 characterized in that it comprises at least residues 140 to 230 of the human GAX protein.

4. Polypeptide characterized in that it comprises at least one fragment chosen from fragments 1-32, 33-302 and 1-104 of the human GAX protein.

5. Polypeptide characterized in that it is a fragment of the GAX protein capable of interacting with the Ki protein.

6. Polypeptide according to claim 5 characterized in that it is the fragment 1 to 32 or 104 to 223 of the GAX protein.

7. Polypeptide characterized in that it is a fragment of the GAX protein, capable of interacting with PCNA.

8. Polypeptide characterized in that it is a fragment of the GAX protein capable of interacting with Ki and / or PCNA and of forming an at least bipartite or at least tripartite complex with these proteins.

9. A polypeptide characterized in that it comprises, in addition to a fragment of the GAX protein having transcription repressor activity, at least one fragment of different origin.

10. Polypeptide according to claim 9 characterized in that this fragment of different origin is endowed with a biological activity, is a marker or a targeting element.

1 1. Nucleic acid encoding a polypeptide according to one of claims 1 to 9.

12. Nucleic acid according to claim 1 1 characterized in that it is a cDNA.

13. Expression cassette comprising a nucleic acid according to claim 1 1 or 12 under the control of a promoter.

14. Expression vector comprising a nucleic acid according to claim 1 1 or 12 or an expression cassette according to claim 13.

15. Vector according to claim 14 characterized in that it is a viral vector.

16. Vector according to claim 15 characterized in that it is an adenovirus.

17. Vector according to claim 15 characterized in that it is a retrovirus.

18. Use of the GAX protein or of a polypeptide according to claim 1 to 9 for suppressing the transcription of genes and / or positively or negatively affecting DNA replication.

19. Use of compounds which inhibit the activity of the Ki protein and or of PCNA to inhibit the proliferation of smooth muscle cells.

20. Use of compounds for the formation of a two- or three-partite complex with the proteins Ki and / or PCNA to positively or negatively affect the progression of the cell cycle.

21. Pharmaceutical composition, characterized in that it comprises at least one polypeptide according to one of claims 1 to 9 or a vector according to one of claims 14 to 17.

22. A method for screening and / or identifying polypeptides interacting with the GAX protein or a GAX domain, characterized in that it implements a polypeptide according to one of claims 1 to 9.

> 23. Method according to claim 22 characterized in that it is a polypeptide chosen from there fragments 32 and 33 to 302 of the GAX protein