KR20140113135A - Screening Methods of a Substance for Preventing or Treating a Tumor Disorder Using a TFG-TEC Fusion Construct and Uses Thereof - Google Patents

Abstract

The present invention provides a method for screening a substance for preventing or treating tumor disorders using novel transcriptional activation and transactivation of the TFG-TEC fusion protein, a pharmaceutical composition thereof, and an expression vector and a composition for promoting transcriptional activity using TFG (NTD). The TFG-TEC fusion protein of the present invention has identical sequence specificity to the TEC, and more excellent ability to promote transcriptional activity. In addition, the TFG (NTD) in the TFG-TEC fusion protein of the present invention functions as a transactivation domain, and the PB1 domain and SPYGQ-rich regions of the TFG (NTD) has partial transactivation. As a result, the TFG-TEC fusion protein of the present invention promotes the expression of downstream genes (e.g., Skp2, L-Myc, SOCS2, or STAT-3), which has been known to be regulated by TEC. Thus, the method for screening a substance for inhibiting the expression of the TFG-TEC fusion protein or inhibit the activity of the TFG-TEC fusion protein and the pharmaceutical composition using the same can be useful to prevent or treat tumor disorders, diseases, or conditions.

Description

TECHNICAL FIELD [0001] The present invention relates to a screening method of a substance for preventing or treating tumor diseases using a TFG-TEC fusion construct,

The present invention relates to novel transfer and performance capabilities of TFG-TEC fusion proteins and their use.

Chromosome translocation is one of the main mechanisms by which a piece of chromosomes breaks apart and migrates to another chromosome, creating oncogenes or inducing human cancers (1). Extraskeletal myxoid chondrosarcomas (EMCs) in humans are soft tissue tumors that are characterized by specific chromosomal abnormalities, histologically of unclear origins, but mainly in musculature, It occurs in the muscle tissue of the thigh and knee (2-4). Most human EMC patients involve the characteristic chromosomal translocations t (9; 22) (q22; q12) or t (9; 17) (q22; q11.2), which translocated in extraskeletal Chondrosarcomas gene and EWS (Ewings sarcoma) gene located at 22q12 or hTAF _II 68 (human TATA-binding protein-associated factor II 68) gene located at 17q12.2 (5, 6). Recently, it has been found that some EMC patients are accompanied by a chromosomal translocation of t (3; 9) (q11-q12; q22), including the TFG (TRK fused gene) gene in the 3q11-q12 position and the TEC gene in the 9q22 position (7).

Originally, the TFG gene was identified as a fusion partner of the NTRK1 gene in human papillary thyroid carcinoma (8) and is also involved in other chromosomal translocation with ALK gene in patients with anaplastic large-cell lymphomas (9). The TFG gene encodes a cytoplasmic protein that is always located in the q-arm of human chromosome 3 (8). The total length of the TFG cDNA was 1,677 bp, with the potential functional domains such as Phox and Bem1p (PB1), one CC (coiled coil) domain and the rich region of SPYGQ (serine, proline, tyrosine, glycine, glutamine) (10). ≪ / RTI > Interestingly, TFG proteins interact with NF-κB key regulators and TRAF family member-related NF-κB active proteins, which suggests that TFG proteins play a key role in NF-κB regulation (11). However, TFG protein function in normal cells is currently unknown.

The TEC gene encodes a novel orphan nuclear receptor belonging to the steroid / thyroid hormone receptor gene superfamily (2, 4), which is a human homologue of the rat NOR-1 receptor gene (12). The TEC gene is also called CHN (2) and MINOR (13) and is located at 9q22 (14). The TEC gene was initially identified as a fusion partner of the EWS gene in human EMC patients (4). The TEC gene is present over about 40 kb and has eight exons, the exon 1 and exon 2 corresponding to the 5'-UTS (untranslated sequence) of mature TEC mRNA (14). Although the biological function of the TEC gene is unclear, continuous expression of the TEC gene results in significant cell death in thymocytes (15), which may be involved in cell proliferation through regulation of downstream target genes .

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have developed a pharmaceutical composition for the prevention or treatment of a tumor disease (for example, extraskeletal myxoid chondrosarcomas (EMCs)) caused by chromosome abnormality (for example, chromosomal translocation or chromosomal rearrangement) I tried my best to study. As a result, the inventors of the present invention found that a TFG-TEC fusion protein is a DNA-binding nuclear protein having sequence specificity such that it can not be distinguished from the DNA binding sequence of a parental TEC protein, and TFG (NTD ) Functions as a transactivation domain that leads to even greater transcriptional activation potentials than the parent TEC protein.

Accordingly, it is an object of the present invention to provide a method for screening a substance for preventing or treating a tumor disease.

It is another object of the present invention to provide an expression vector for promoting transcriptional activity.

Another object of the present invention is to provide a cell transformed with the above-mentioned expression vector.

It is still another object of the present invention to provide a composition for promoting transcriptional activity.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating tumor diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a substance for the prophylaxis or treatment of a tumor disease comprising the steps of: (a) culturing a cell comprising a TFG-encoding nucleotide sequence and a TEC-encoding nucleotide sequence Treating the test substance with the test substance; And (b) analyzing the expression of the TFG-TEC fusion gene or the activity of the TFG-TEC fusion protein in the cell, wherein the test substance expresses the TFG-TEC fusion gene or the transcription activity of the TFG-TEC fusion complex Is judged to be a therapeutic agent for a tumor disease.

According to another aspect of the present invention, the present invention provides a method of screening a substance for the prophylaxis or treatment of a tumor disease comprising the steps of: (a) (i) contacting a TFG-TEC fusion protein operably linked to a promoter, A vector comprising an encoding nucleotide sequence; And (ii) a reporter gene operably linked to a promoter that is positively regulated by the TFG-TEC fusion protein. And (b) measuring the expression level of the reporter gene in the transformed host cell of step (a). If the test substance inhibits the transcription activity by the TFG-TEC fusion protein, .

The present inventors have made extensive efforts to develop a pharmaceutical composition for the prevention or treatment of a tumor disease (for example, exoskeletal mucosal chondrosarcoma) caused by chromosomal anomalies (for example, chromosomal translocation or chromosomal rearrangement). As a result, the inventors of the present invention found that a TFG-TEC fusion protein is a DNA-binding nuclear protein having sequence specificity that is indistinguishable from the DNA binding sequence of the parent TEC protein, and that the TFG (NTD) Lt; RTI ID = 0.0 > TEC < / RTI > protein.

Chromosomal translocations cause cancer through activation of existing genes or creation of novel fusion proteins. That is, large-scale genetic abnormalities, including balanced rearrangement (e.g. translocation or inversion) and dielectric imbalance (deletion, redundancy or amplification) are common in cancer and play a pivotal role in tumorigenesis.

The genes that are affected by chromosomal translocation are roughly divided into two groups: (a) proto-oncogenes (Enforced gene product expression), which are forced to express more strongly on the transferred chromosome; And (b) a gene fusion expression in which a breakpoint exists in the intron of affected genes existing on two chromosomes and is formed by chromosomal translocation. In particular, the latter case is more common in chromosomal translocations. For example, the MLL-MLLT3 fusion gene and the FUS-CHOP fusion gene are restricted to the myeloid cells of acute myeloid leukemia and adipocytes of liposarcoma, respectively, depending on the cell type .

Typically, balanced translocations and gene fusions have been frequently observed in blood and mesenchymal tumors including sarcoma, leukemia and lymphoma (Rabbits, TH, Nature 372 (6502): 143-149 (1994) Tumor was less observed. In recent years, tumorigenic fusions have been shown to be associated with prostate (Tomlins, SA, et al . , Science 310 (5748): 644-648 (2005)), thyroid (Bongarzone, I., et al . , Cancer Res. 54 (11): 2979-2985 (1994); Kroll, TG, et al . , Science 289 (5483): 1357-1360 (2000)), and lungs (Soda, M., et al . , Nature 448 (7153): 561-566 (2007)), which shows that the tumorigenic fusants occur very frequently in carcinomas. For example, recurrent chromosomal translocation is characteristically found in many leukemias, lymphomas, and sarcomas (e.g., mesenchymal tumors). Chromosomal translocations in leukemias and lymphomas result from the destruction of normal processes of immunoglobulin or T-cell-receptor gene rearrangement, resulting in inter-chromosomal rearrangement rather than intra-chromosomal rearrangement Causing rearrangement. Chromosomal translocations occur at high frequency in non-lymphoid constituent cell types, particularly sarcomas, and some chromosomal translocations have been reported in epithelial tumors.

The present invention relates to a TFG-TEC fusion protein expressed through chromosomal translocation of t (3; 9) (q11-q12; q22) containing a TRK fused gene gene in a 3q11-q12 position and a TEC gene in a 9q22 position It is based on the fact that it is frequently found in human EMC patients.

According to some embodiments of the present invention, the TFG-TEC fusion protein described above consists of ¹ TFG (NTD) ^273- Asp- ¹ TEC ⁶²⁶ by chromosomal translocation between the 6th intron of TFG and the 2nd intron of TEC . The above-mentioned Asp (D) amino acid residue is an amino acid residue formed by a G nucleotide derived from the translocation site of TFG through chromosome translocation and A and T nucleotides derived from the translocation site of TEC.

The present inventors have found that the TFG-TEC protein is a nuclear protein bound to DNA with sequence specificity that is indistinguishable from the sequence specificity of the parent TEC protein and that TFG (NTD) in the fusion protein functions as a strong tansactivator Based on this, the present invention was applied to the development of a substance for preventing or treating tumor diseases using the TFG (NTD).

According to some embodiments of the present invention, the TFG (NTD) in the TFG-TEC fusion protein of the present invention functions as a transactivation domain and the PB1 domain and the SPYGQ-rich region of the TFG (NTD) rich regions have partial transactivation.

According to the present invention, the test substance is first contacted with cells containing a TFG-encoding nucleotide sequence and a TEC-encoding nucleotide sequence. The cells containing the nucleotide sequence of the present invention are not particularly limited, and specifically include 293T cells. The term "test substance" used in referring to the screening method of the present invention means an unknown substance used in screening to check whether expression of the TFG-TEC fusion gene or the activity of the fusion protein is affected. Such test materials include, but are not limited to, chemicals, antisense oligonucleotides, small interference RNA (siRNA), shRNA (small hairpin RNA or short hairpin RNA), miRNA (microRNA), peptides and natural extracts.

When the test substance to be analyzed by the screening method of the present invention is a chemical, it may be a single compound or a mixture of compounds (e.g., a cell or a tissue culture). The test substance can be obtained from a library of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the desired synthetic library method, "1-bead 1-compound" library method, and by synthetic library methods using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt, et al . , Proc . Natl . Acad . Sci . USA 90, 6909, 1993; Erb, et al . , Proc . Natl . Acad. Sci . USA 91, 11422, 1994; Zuckermann, et al . , J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell, et al . , Angew . Chem . Int . Ed . Engl . 33,2059,1994; Carell, et al. , Angew . Chem . Int . Ed . Engl . 33, 2061; Gallop, et al . , J. Med . Chem . 37, 1233, 1994, and the like.

The term "antisense oligonucleotide " in the present invention means DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein . As used herein, the term "complementary" means that the antisense oligonucleotide is sufficiently complementary to a target (e.g., a TFG-TEC fusion gene) under certain hybridization or annealing conditions, preferably physiological conditions Means one or more mismatch nucleotide sequences and is meant to encompass both substantially complementary and perfectly complementary sequences, . The length of the antisense oligonucleotide is 6 to 100 bases, more specifically 8 to 60 bases, most specifically 10 to 40 bases.

The term "siRNA" in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914). Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method. siRNA was first found in plants, insects, fruit flies and parasites, but recently it has been applied to mammalian cell research by developing si / siRNA (Degot S, et al . , 2002; Degot S, et al . , 2004; Ballut L, et al . , 2005).

SiRNA molecules that may be used in the present invention include siRNA molecules that are complementary to a sense strand (e.g., the corresponding sequence of a TFG-TEC fusion gene mRNA sequence) and an antisense strand (e.g., a TFG-TEC fusion gene mRNA sequence Sequence) may be located on opposite sides to form a double-stranded structure. In addition, siRNA molecules that may be used in the present invention may have a single stranded structure with self-complementary sense and antisense strands.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. Specifically, the total length is 10 to 100 bases, more specifically 15 to 80 bases, and even more specifically 20 to 70 bases.

The term "shRNA (small hairpin RNA or short hairpin RNA) " in the present invention refers to a sequence of RNA that produces a robust hairpin turn, which can be used to silence gene expression through RNA interference. shRNA uses a vector for introducing cells and mainly uses a U6 promoter capable of expressing shRNA. These vectors are always transferred to daughter cells, allowing genetic silencing to be inherited. The shRNA hairpin structure is degraded into an intracellular machinery siRNA and bound to an RNA-induced silencing complex. The above-described complex binds to and degrades mRNA matched to the siRNA bound thereto. shRNAs are transcribed by RNA polymerase III, and production of shRNAs in mammalian cells may cause interferon responses as cells recognize shRNA as a viral attack and find defensive means. In addition, shRNAs can also be used in plants and other systems, and the U6 promoter is not necessary. In the case of plants, the CaMV (cauliflower mosaic virus) 35S promoter, which is a conventional promoter having a very strong continuous expression ability, can be used.

As used herein, the term "microRNA (miRNA)" refers to a material that binds to the 3-UTR of mRNA (messenger RNA) as a single strand RNA molecule of 21-25 nucleotides and controls gene expression of eukaryotes (Bartel DP , meat al . , Cell, 23: 116 (2): 281-297 (2004)). The production of miRNA is made into a stem-loop structure precursor miRNA (pre-miRNA) by Drosha (RNase III type enzyme), and it is transferred to the cytoplasm and cleaved by Dicer to make mature miRNA [Kim VN, et al . , Nat Rev Mol Cell Biol., 6 (5): 376-385 (2005)]. MiRNAs prepared as described above are involved in development, cell proliferation and death, lipid metabolism, tumor formation, etc. by controlling the expression of target proteins [Wienholds E, et al . , ≪ / RTI > Science, 309 (5732): 310-311 (2005); Nelson P, et al . , Trends Biochem Sci., 28: 534-540 (2003); Lee RC, et al . , Cell, 75: 843-854 (1993); And Esquela-Kerscher A, et al. , Nat Rev Cancer, 6: 259-269 (2006)].

As used herein, the term "peptide" refers to a linear molecule formed by peptide bonds and amino acid residues joined together. The peptides of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer . Chem . Soc . 85: 2149-54 (1963) ; Stewart, et al . , Solid Phase Peptide Synthesis , 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).

Next, the expression of the TFG-TEC fusion gene or the activity of the TFG-TEC fusion protein is measured in the cells treated with the test substance. Measurement of expression level and activity can be carried out as described below. As a result of the measurement, when the expression of the fusion gene which is the marker of the present invention or the activity of the fusion protein is inhibited, the test substance is used for the prevention or treatment of tumor diseases It can be judged as a substance.

The TFG-encoding nucleotide sequence, the TEC-encoding nucleotide sequence, the TFG-TEC fusion gene-encoding nucleotide sequence, and the TFG (NTD) -encoding nucleotide sequence used in the present invention are shown in Sequence Listing 1 (GenBank Accession No. NM_001007565.2 ), SEQ ID No. 2 (GenBank Accession No. NM_006981.3), SEQ ID No. 3 (fusion sequence of GenBank Accession Nos. NM_001007565.2 and NM_006981.3) and SEQ ID No. 4 (GenBank Accession No. NM_001007565 .2). The amino acid sequences of the proteins expressed from each nucleotide sequence are shown in SEQ ID NO: 5 (GenBank Accession No. NP_001007566.1), SEQ ID NO: 6 (GenBank Accession No. NP_008912.2 ), SEQ ID NO: 7 (fusion sequences of GenBank Accession Nos. NP_001007566.1 and NP_008912.2) and SEQ ID NO: 8 (some sequences of GenBank Accession No. NP_001007566.1).

According to some embodiments of the present invention, the expression of the fusion gene of the present invention can be detected according to a polymerase chain reaction (PCR). According to some embodiments of the present invention, the primers of the present invention are used for amplification reactions.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. The term "amplification reaction" as used herein refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including PCR (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT- Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822), multiplex PCR (McPherson and Moller, 2000 ), Ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) / 10315), self sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence priming polymerase The consensus sequence primed polymerase chain reaction (CP) -PCR; U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR; U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification NASBA; U.S. Patent Nos. 5,130,238; 5,409,818; 5,554,517; and 6,063,603) and strand displacement amplification, the teachings of which are incorporated herein by reference. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and U.S. Patent No. 09 / 854,317.

As used herein, the term "primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis at suitable temperature and pH conditions. Specifically, the primer is a deoxyribonucleotide and a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer. As used herein, the term " annealing "or" priming "means that oligodeoxynucleotides or nucleic acids are apposied to a template nucleic acid, wherein the polymerase polymerizes the nucleotides to form a nucleic acid complementary to the template nucleic acid, To form molecules.

When the method of the present invention is carried out using a primer, the gene amplification reaction is carried out to extract mRNA from an analyte (for example, 293T cell or TFG-TEC positive EMC cell) and detect it. Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from a sample (specifically, cells). The isolation of the total RNA can be carried out according to conventional methods known in the art (see Sambrook, J., et al . , Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C., et al . , Plant Mol. Biol. Rep., 9: 242 (1991); Ausubel, FM, et al . , Current Protocols in Molecular Biology, John Willey & Sons (1987); And Chomczynski, P., et al . , Anal. Biochem. 162: 156 (1987)). For example, Trizol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Because the total RNA of the present invention is isolated from eukaryotic cells, it has a poly-A tail at the end of mRNA, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptase using such sequence characteristics : PNAS USA, 85: 8998 (1988); Libert F, et al . , Science, 244: 569 (1989); And Sambrook, J., et al . , Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

The term "hybridization " in this specification means that two single-stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization can occur either in perfect match between single stranded nucleic acid sequences or in the presence of some mismatching nucleotides. The degree of complementarity required for hybridization can vary depending on hybridization reaction conditions, and can be controlled by temperature. The terms "annealing" and "hybridization" are no different and are used interchangeably herein.

A variety of DNA polymerases can be used in the amplification of the present invention, including the Klenow fragment of E. coli DNA polymerase I, the thermostable DNA polymerase, and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , Pyrococcus furiosus (Pfu), Thermus antranikianii , Thermus caldophilus , Thermus chliarophilus , Thermus flavus , Thermus igniterrae , Thermus lacteus , Thermus oshimai , Thermus ruber , Thermus rubens , Thermus scotoductus, Thermus silvanus , Thermus species Z05 , Thermus species sps 17, Thermus thermophilus , Thermotoga maritima , Thermotoga neapolitana and Thermosipho but are not limited to, DNA polymerases of A. africanus .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with such joins as Mg ^{2 +} , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

Therefore, when the method of the present invention is carried out based on an amplification reaction using cDNA, specifically, (i) a primer sequence complementary to a TFG (NTD) nucleotide sequence in a TFG-TEC fusion gene and a TEC Performing an amplification reaction using a primer sequence complementary to a nucleotide sequence; And (ii) analyzing the result of the amplification reaction.

The result of the above amplification reaction is subjected to gel electrophoresis and the resultant band is observed and analyzed to confirm the expression and existence of the TFG-TEC fusion gene.

In addition, changes in the amount of TFG-TEC fusion protein can be detected by immunodiffusion, radioimmunoassay, radioimmunoprecipitation, Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay, capture-ELISA, inhibition or competitive assay, But are not limited thereto.

Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotope is an antibody labeled (e.g., C ^14, I ^125, P ³² and S ³⁵⁾ of detecting the marker molecules of the present invention .

When the method of the present invention is carried out by an ELISA method, the present invention relates to a method for screening a solid substrate, comprising the steps of: (i) coating the surface of a solid substrate with an analyte of an unknown cell sample to be analyzed; (ii) reacting said cell lysate with an antibody to a target as a primary antibody; (iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and more particularly microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable that the substrate is selected from the group consisting of bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'- Azine-di [3-ethylbenzthiazoline sulfonate]), o - phenylenediamine (OPD) and naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.

When the method of the present invention is carried out in a Capture-ELISA fashion, certain embodiments of the present invention may include (i) contacting the antibody against a target of the invention (e. G., A TFG-TEC fusion protein) as a capturing antibody, To the surface of the substrate; (ii) reacting the capture antibody with a cell sample; (iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating label and specifically reacts with a TFG-TEC fusion protein; And (iv) measuring a signal originating from the label.

The detection antibody has a label that generates a detectable signal. The label may be a chemical (e.g. biotin), an enzyme (alkaline phosphatase, -galactosidase, horseradish peroxidase and cytochrome P ₄₅₀ ), a radioactive material (e.g. C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. The detection of such signals enables a qualitative or quantitative analysis of the target of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

On the other hand, the activity of the TFG-TEC fusion protein can be analyzed by measuring the expression of a downstream gene that is transcriptionally regulated by TEC.

According to some embodiments of the present invention, the TFG-TEC fusion protein of the present invention is a nuclear protein.

According to some embodiments of the present invention, the TFG-TEC fusion protein of the present invention binds to a consensus sequence (SEQ ID NO: 9) to facilitate transcription of downstream genes.

According to some embodiments of the present invention, downstream genes whose transcription is facilitated by the TFG-TEC fusion protein of the present invention include, but are not limited to Skp2 , L- Myc , SOCS2 and STAT- 3 .

Meanwhile, the method of the present invention may be carried out by an assay method using a reporter gene.

(A) (i) a vector comprising a TFG-TEC fusion protein-encoding nucleotide sequence operatively linked to a promoter; And (ii) a reporter gene operably linked to a promoter that is positively regulated by the TFG-TEC fusion protein.

As used herein, the term "promoter " means a DNA sequence that regulates the expression of a coding sequence or functional RNA. The TFG-TEC fusion protein-encoding nucleotide sequence in the expression vector of the present invention is operably linked to the promoter. As used herein, the term "operatively linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter sequences, signal sequences, or transcription factor binding sites) , Whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

The vectors that can be used in the present invention include plasmids such as pCMV, pGEX series, pcDNA series, pM, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, :? gt4.? B,? -charon,?? z1 and M13, etc.) or viruses (e.g. SV40 and the like).

When the expression vector of the present invention is applied to a eukaryotic cell, the promoter that can be used is one capable of regulating the transcription of the TFG-TEC fusion gene of the present invention, including a promoter derived from a mammalian virus, Derived cytomegalovirus promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk Promoter of the human IL-4 gene, promoter of the human lymphotoxin gene, promoter of the human IL-2 gene, promoter of the human IL-2 gene, promoter of the human IL- A promoter of GM-CSF gene, a promoter of S. cerevisiae TDH3 (glyceraldehyde-3-phosphate dehydrogenase 3), a S. cerevisiae GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) promoter, a yeast ( S. cerevisiae ) GAL10 promoter, yeast ( Pichia pastoris AOXl and yeast AOX2 promoters. Most specifically, it is a CMV (cytomegalo virus) promoter.

In addition, the expression vectors used in the present invention include polyanenylation sequences (e.g., ADH1 terminator, bovine growth hormone terminator and SV40-derived polyadenylation sequence).

In addition, the vector of the present invention additionally comprises a selection marker. According to a preferred embodiment of the present invention, the vector of the present invention includes an antibiotic resistance gene commonly used in the art, for example, kanamycin, hygromycin, ampicillin, streptomycin, penicillin, chloramphenicol, gentamicin, But are not limited to, resistance genes for benicillin, geneticin, neomycin and tetracycline.

Methods of delivering an expression vector of the present invention into a host cell can be performed by a variety of methods known in the art, for example, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc . Natl ... Acac Sci USA, 9 : 2110-2114 (1973)), a method (Cohen, SN et al, Proc Natl Acac Sci USA, 9:..... 2110-2114 (1973); and Hanahan, D., J. Mol . Biol . , 166: 557-580 (1983)) and electroporation (Dower, WJ et al., Nucleic Acids Res . Transduction, electroporation, lipofection, microinjection, particle bombardment, and the like, in the case of eukaryotic cells. , Yeast spheroid / cell fusion used in YAC, Agrobacterium-mediated transformation used in plant cells, and the like.

Next, the expression level of the reporter gene is measured in the transformed host cell of step (a).

If the test substance inhibits the transcriptional activity of the TFG-TEC fusion protein and inhibits the expression of the reporter gene, it is judged to be a substance for preventing or treating tumor diseases.

According to some embodiments of the present invention, the reporter gene that may be used in the present invention may be a fluorescent protein,? -Galactosidase, chloramphenicol acetyltransferase, human growth hormone, urease, and alkaline phosphatase But are not limited thereto.

According to some embodiments of the present invention, the fluorescent protein that can be used as the reporter gene of the present invention is selected from the group consisting of luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) fluorescent protein, BFP (blue fluorescent protein), and variants thereof.

The expression level of the reporter gene can be measured by various methods. For example, the expression of the luciferase gene is described in de Wet J. et al, Mol . Cell Biol . , 7: 725-737 (1987), the expression of the chloramphenicol acetyltransferase gene is described by Gorman C. et al, Mol . Cell Biol . , 2: 1044-1051 (1982), the expression of the galactosidase gene is described by Hall CV et al, J. Mol . Appl . Genet . , 2: 101-109 (1983), the expression of the human growth hormone gene is described in Selden R. et al., Mol . Cell Biol . , 6: 3173-3179 (1986), and expression of the green fluorescent protein gene can be measured according to the method disclosed in Chalfie M. et al, Science , 263: 802-805 (1994).

According to some embodiments of the present invention, the tumor disease of the present invention is selected from the group consisting of extraskeletal myxoid chondrosarcoma, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, colorectal adenocarcinoma, Cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of endotheliosarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, lubulocyte, mesothelioma, Ewing's tumor, leiomyosarcoma, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis of cancer of the pancreas, Including in tumors, pineal species, vascular neuroblastoma, acoustic neuroma species, once dendritic cells glioma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma and leukemia, but not limited to this.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising (a) a promoter; (b) a TFG (NTD) -encoding nucleotide sequence of Sequence Listing 4 of the sequence operably linked to the promoter; (c) a transcription activator-encoding nucleotide sequence attached to the N-terminus or C-terminus of the TFG (NTD) -encoding nucleotide sequence; And (d) a terminator. ≪ / RTI >

According to another aspect of the present invention, the present invention provides a cell transformed with the aforementioned expression vector.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising (a) (i) a promoter; And (ii) a transactivation moiety comprising a TFG (NTD) -encoding nucleotide sequence of Sequence Listing 4 of the Sequence Listing operatively linked to the promoter; And (b) an expression vector comprising a DNA-binding moiety comprising a transcription activator-encoding nucleotide sequence linked to the N-terminus or C-terminus of the TFG (NTD) Or a pharmaceutically acceptable salt thereof.

Since the expression of the TFG-TEC fusion gene or the activity of the TFG-TEC fusion protein, which is a constituent of the expression vector of the present invention, is the same as the screening method of the present invention described above, the excessive complexity To avoid this, the description of common matters is omitted.

According to some embodiments of the invention, the transcription activator of the present invention comprises a DNA-binding domain.

According to some embodiments of the present invention, the DNA-binding domain that can be used in the vector of the present invention is a TEC, GAL4, GCN4, HAP1, ADR1, SWI5, STE12, MCM1, YAP1, ACE1, CUP2, XYR1, PPR1, ARG81 , LAC9, QA1F, VP16 and the DNA-binding domain of mammalian nuclear receptor proteins.

According to some embodiments of the present invention, the composition of the present invention increases the expression of a gene having a promoter comprising a conserved sequence bound by a DNA-binding domain in said transcriptional activator.

According to another aspect of the present invention, there is provided an RNAi (RNA interference) molecule that specifically binds to a TFG-TEC fusion gene, or a substance that inhibits the transcription activation promotion of the TFG-TEC fusion protein as an active ingredient Or a pharmaceutically acceptable salt thereof.

The composition of the present invention uses an RNAi molecule or a substance that inhibits the transcriptional activation promotion of the TFG-TEC fusion protein as an active ingredient, and this is the same as the screening method of the present invention described above. Therefore, excessive complexity The description of common matters is omitted.

According to some embodiments of the present invention, a substance that inhibits the transcriptional activation promotion of the TFG-TEC fusion protein of the present invention inhibits binding of the TFG-TEC fusion protein to the DNA-binding sequence motif.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one that is commonly used in the present invention and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and parenteral administration includes intracranial injection, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, the manner of administration, the age, body weight, sex, pathological condition, food, administration time, route of administration, absorption rate of the active ingredient in the body, , Excretion rate, and responsiveness. ≪ / RTI > Specifically, the dosage of the pharmaceutical composition of the present invention is 0.0001 ng / kg (body weight) to 100 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a screening method and a pharmaceutical composition for screening a substance for the prevention or treatment of tumor diseases using the novel transcriptional and transcriptional activity of a TFG-TEC fusion protein, and a method for promoting transcription activity using TFG (NTD) Lt; RTI ID = 0.0 > expression vector < / RTI >

(b) The TFG-TEC fusion protein of the present invention not only has the same sequence specificity as TEC but also has better transcriptional activity promoting ability than TEC.

(c) TFG (NTD) in the TFG-TEC fusion protein of the present invention also functions as a strong transactivation domain, and the PB1 domain and SPYGQ-rich regions of the TFG (NTD) Have partial transactivation.

(d) As a result, the TFG-TEC fusion protein of the present invention promotes the expression of downstream genes known to be regulated by TEC (e.g., Skp2 , L- Myc , SOCS2 or STAT- 3 ).

(e) Therefore, a screening method for a substance that inhibits the expression of a TFG-TEC fusion gene or inhibits the activity of a TFG-TEC fusion protein and a pharmaceutical composition using the same are useful for preventing or treating a tumor disease, Can be usefully applied.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagrammatic representation of a TFG-TEC fusion protein in human EMC. Exons / introns of TFG and TEC genes are shown. The exons are indicated by boxes and the coding regions of the TFG and TEC genes are presented as black boxes and shaded boxes, respectively. Open boxes represent non-encoding regions of the genes. Numbers represent exons of the TFG and TEC genes. Also, the amino acid position is indicated schematically above or below the TFG-TEC fusion protein. The TFG-TEC fusion protein contains the first 273 amino acid residues of TFG [1-273 residues; (Aspartic acid) which is encoded through the combination of one nucleotide (G) at the end of the exon 6 of TFG and two nucleotides (A and T) present in the 5'-UTR of the TEC and TFG (N) Lt; RTI ID = 0.0 > 1-626 < / RTI > amino acid residues of TEC. Functionally important domains in the TFG-TEC chimera are: PB1, Phox and Bem1p domains; CC, a coiled-coil domain; SPYGQ-rich, Ser, Pro, Tyr, Gly, Gln-rich domains; AF1, an N-terminal transactivation domain; DBD, DNA-binding domain; LBD, ligand-binding domain; And AF2, C-terminal transactivation domain. The two nucleotide sequences present in the ATG start codon upstream of TEC are indicated in lower case and the ATG start codon of TEC is indicated in the underlined upper case in TFG-TEC.
Figure 2 shows the results showing sequence-specific DNA binding by the TFG-TEC chimera. Figure 2a shows Western blotting results for in vitro -translated TFG-TEC and TEC proteins. The rabbit reticulocyte lysate was programmed with vector mRNA (lane 1), Flag-TEC mRNA (lane 2), or Flag-TFG-TEC mRNA (lane 3). The translation reaction product (5 μl) was electrophoresed on an 8% SDS-PAGE gel and analyzed by Western blotting using an anti-Flag antibody (M2). Movement of pre-stained molecular weight markers (New England Biolabs, NEB) is indicated on the left. Figure 2B shows the results of EMSA analysis of DNA-binding properties of TFG-TEC and TEC. As described in the Experimental Materials and Experimental Methods, the EMSA is a non-programmed reticulocyte lysate (lane 1), a vector-programmed reticulocyte lysate (lane 2, 2 μl; lane 3, 6 μl; (lane 5, 2 μl; lane 6, 6 μl; and lane 7, 10 μl) programmed with Flag-TEC mRNA, or reticulocyte lysate programmed with Flag-TFG-TEC mRNA 8, 2 μl; lane 9, 6 μl; lane 10, 10 μl), and radiolabeled probes. The in vitro -translated proteins used in each EMSA are labeled at the top of the gel. Protein-DNA complexes were electrophoresed at 4 ° C in non-denatured 4% acrylamide (bisacrylamide ratio, 37: 1) in 0.5 × TBE buffer (44.5 mM Tris.HCl, 44.5 mM boric acid, 1 mM EDTA) And then separated. The positions of free probes and protein-DNA complexes are indicated. Figure 2C shows the results showing sequence-specific DNA binding by TRF-TEC. Competition experiments were carried out with an excess of unlabeled wild type TEC oligonucleotides of 5 times (lane 2 and lane 7) or 10 times (lane 3 and lane 8), or 5 times (lane 4 and lane 9) or 10 times And lane 10). ≪ / RTI > The positions of free probes and protein-DNA complexes are indicated by arrows.
Figure 3 shows the mapping of the nuclear localization signal of TFG-TEC to the intracellular location and DNA-binding domain of TFG-TEC. Figure 3a is a diagrammatic illustration of Flag-tagged TFG-TEC derivatives. The intracellular location of TFG-TEC, TFG (NTD), TEC and TFG-TEC (AAAA) constructs is indicated as N (nucleotide position) or C (cytoplasmic position). Figure 3b is an immunofluorescence microscopy showing the intracellular distribution of TFG-TEC derivatives. Mammalian expression vectors encoding Flag-tagged TFG-TEC (a), TFG (NTD) (b), TEC (c) or TFG-TEC (AAAA) (d) on 293T cells cultured on cover slips Transfected. Immunofluorescence microscopy analysis was performed using anti-Flag antibody (M2; Sigma) in cells expressing Flag-tagged TFG-TEC derivative proteins.
Figure 4 shows the Western blotting results showing the interaction between U1C and TFG (NTD). Figure 4a shows the results of different binding affinities of EWS (NTD), hTAF _II 68 (NTD) or TFG (NTD) for U1C. The GST pull-down experiments were basically carried out as described in Experimental Materials and Experimental Methods. GST-TWG (NTD) pellet (lane 3), GST-hTAF _II 68 pellet (lane 4) and GST-TFG (NTD) The aliquots of the pellet (lane 5) were analyzed on a 15% SDS-PADG gel and the bound U1C protein was detected using an anti-Express antibody (Invitrogen). The identity of the GST fusion proteins is indicated at the top of the panel. The position of the molecular weight marker is indicated on the left. The U1C protein is indicated by the arrow on the right. Three independent experiments were performed, all with similar results. Figure 4B shows the quantification results of the GST fusion proteins used in the GST full-down assay. GST fusion proteins used in the pull-down assays were separated on a 15% SDS-PAGE gel and visualized in Coomassie blue stain. Three independent experiments were performed, all with similar results.
Fig. 5 shows the result of investigating the transition behavior of TFG-TEC. Figure 5A is a diagrammatic representation of expression plasmids and reporter plasmids. Expression vectors inducing the production of TFG-TEC or TEC are shown. The positions of the first amino acid and the last amino acid are indicated below each construct. The p (B1a) 8-Luc reporter plasmid contains eight TEC-recognition sites (NBRE) upstream of the primary promoter-luciferase construct. The eight copies are labeled with small black bars, the TATA box with open boxes, and the luciferase gene with a long black bar. FIG. 5B shows the results of measurement of the transferability of TFG-TEC and TEC. 293T cells were co-transfected with an expression vector encoding the indicated amount of TEC (white bar) or TFG-TEC (black bar), p (B1a) 8-Luc reporter plasmid, and Renilla luciferase. Reporter activity was normalized to renilla luciferase activity to compensate for other transfection efficiencies. The fold induction is expressed as a relative value to the co-expression vector. Each transfection was performed in three or more independent experiments, and the mean value was plotted with standard error (SEM, vertical bars). Figure 5C shows the in vivo induction of potential TEC downstream target genes by TFG-TEC. 293T cells were transiently transfected with pcDNA3-EGFP, pcDNA3-EGFP-TEC or pcDNA3-EGFP-TFG-TEC, and then the transfected cells were separated by FACS. RT-PCR analysis of Skp2, L-Myc, SOCS2 and STAT-3 mRNA was performed on 293T cells expressing EGFP, EGFP-TEC or EGFP-TFG-TEC protein. β-actin was used for standardization. After amplification, the gels were analyzed by staining with EtBr (ethidium bromide). The pcDNA3-EGFP-TEC and pcDNA3-EGFP-TFG-TEC vectors were used to express EGFP fused with TEC and TFG-TEC, respectively. The pcDNA3-EGFP expression vector was used as a control. Transiently transfected cell lines derived from the input RNAs used in the RT reactions are shown at the top of the panel.
FIG. 6 shows the results of the transition performance of TFG (NTD). Figure 6a is a diagrammatic representation of the GAL-TFG (NTD) expression plasmids and reporter plasmids used in this study. Expression vectors that induce the production of the GAL4 DNA-binding domain (1-147 amino acid residues) and GAL4-TFG (NTD) are presented. The pG5 luc reporter plasmid contains 5 copies of the GAL4-binding sites upstream of the primary promoter-luciferase construct. Five copies are marked with small black bars, the TATA box with open boxes, and the luciferase gene with a long black bar. Abbreviation: GAL4, GAL4 DNA-binding domain; TFG (NTD), TFG N-terminal domain of TFG-TEC; PB1, Phox and Bem1p domains; CC, coil-coil domain; And SPYGQ-rich, Ser, Pro, Tyr, Gly, Gln-rich domains. FIG. 6B shows the results showing the transcription activation ability by GAL4-TFG (NTD). 293T cells were transfected with 0.1 g of pG5 luc reporter plasmid, 2 ng of Renilla luciferase and 0 μg (lane 1), 0.5 μg (lane 2), 1.0 μg (lane 3), 1.5 μg (Lane 5) GAL4-TFG (NTD) plasmid. In all experiments, the total amount of transfected DNA was adjusted to a blank vector (GAL4 DNA). Reporter activity was normalized to renilla luciferase activity to compensate for other transfection efficiencies. Luciferase activity was expressed as relative activation fold activation relative to the basal level observed in the reporter plasmid and the GAL4 DNA-binding domain alone (lane 1). Each transfection was performed in three or more independent experiments, and the mean value was plotted with standard error (SEM, vertical bars).
Figure 7 shows the functional regions of TFG (NTD). The left panel graphically illustrates the deletion constructs of the TFG (NTD). Numbers represent amino acid residues. The reporter plasmid pG5 luc was co-transfected with various GAL4-TFG (NTD) deletion mutants and 293T cells. Relative transcriptional activation values are presented on the right panel as the relative mean ± standard error (SEM) assuming the transfection efficiency of GAL4-TFG (NTD) as 100%. The above results show the mean value of three independent experiments performed in two pairs.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Materials and Methods

Restriction enzymes, calf intestinal alkaline phosphatase (CIP), Klenow fragment of DNA polymerase I and T4 DNA ligase were purchased from NEB (New England Biolabs). PfuTurbo polymerase was purchased from Stratagene and [γ- ³² P] ATP (3000 Ci / mmol) was purchased from PerkinElmer. Plasmid DNA isolation, restriction digestion, DNA agarose gel electrophoresis, DNA ligation, bacterial transformation and SDS-PAGE electrophoresis of proteins were performed according to standard methods 16). In order to confirm the presence or absence of the mutant, the subclones obtained from the PCR product were sequenced by the chain termination method using a double-stranded DNA template.

Manufacture of constructions

The pCMV-Tag2A / TFG-TEC amplifies each of the domains (TFG-NTD and TEC) by PCR and amplifies the respective regions (TFG-NTD and TEC) using an overlap extension method as previously described Lt; / RTI > A detailed description of the construction of pCMV-Tag2A / TFG-TEC has been previously reported (18). The TFG-TEC reporter plasmid p (Bla) 8-Luc has also been previously reported (3).

(5'-GATC GGATTC AATGAACGGACAGTTGGA-3 ', BamH I position is underlined) and 3'-TFGHindIII (5'-TFGHindIII) for producing pCMV-Tag2A / TFG (NTD) GATC AAGCTT ATGGCGTCCACGGATTAC-3 '; HindIII position is indicated by an underscore) primer pair, digested with pCMV-Tag2A / TFG-TEC and digested with BamHI and HindIII , inserted into pCMV-Tag2A vector (Stratagene) Respectively. to produce the pCMV-Tag2A / TFG-TEC ( AAAA), pCMV-Tag2A / a TFG was double-cut with Acc I and EcoR I and cloned into the corresponding position of the pBluescript II KS + (Stratagene) to pKSII (AccI-EcoRI) . In order to prepare pKSII (AccI-EcoRI) (AAAA) in which the KRRR amino acid sequence is substituted with AAAA, the present inventors used a mutagenic primer set 5'-mNLS (5'-CTGCCCAGTAGAC GCGGCAGCTGCA AACCGATGTCAG-3 ' QuikChange ^TM site-designated mutation kit (Stratagene) with 3'-mNLS (5'-CTGACATCGGTT TGCAGCTGCCGC GTCATCTGGGCAG-3 '; pKSII / (AccI-EcoRI) ( AAAA) and then by cutting the vector with Acc I and EcoR I and disconnect the (AccI-EcoRI) (AAAA) fragment, and cloned in the corresponding position of the pCMV-Tag2A / TEC pCMV-Tag2A / TEC (AAAA). Then, pCMV-Tag2A / TFG-TEC (AAAA) was prepared by cleaving pCMV-Tag2A / TEC (AAAA) with Xho I to isolate the XhoI (AAAA) XhoI fragment and cloning it to the corresponding position of pCMV- Tag2A / TFG- .

pGEX (4T-1) -EWS ( NTD) in order to produce a construct, one EWS (NTD) fragment is primer 5'-EcoRIEWS1 (5'-GATC GAATTC ATGGCGTCCACGGATTAC-3 '; EcoR site is underlined) And 3'-NotIEWS265 (5'-GATC GCGGCCGC TCAACTCTGCTGCCCGTA-3 '; Not I position is underlined). By cutting the PCR products with EcoR I and Not I and cloned into the same position in the pGEX (4T-1) vector (GE Healthcare) was prepared pGEX (4T-1) -EWS ( NTD). The pGEX (4T-1) -hTAE _II 68 (NTD) construct was previously reported (19). pGEX (4T-1) -TFG ( NTD) construct to produce the agent truck, TFG (NTD) fragment is primer 5'-BamHITFG (5'-GATC GGATCC ATGAACGGACAGTTGGAT-3 '; BamH I site is underlined) and Was amplified from pCMV-Tag2A / TFG-TEC by PCR method using 3'-NotITFG274 (5'-GATC GCGGCCGC TCAATCTGAATACTGAATACC-3 '; Not I position is underlined). After cutting the PCR product with BamH I and Not I and cloned into the same position in the pGEX (4T-1) vector to prepare a pGEX (4T-1) -TFG ( NTD).

for the production of pcDNA3-EGFP, EGFP gene is a primer 5'-HindIIIEGFP (5'-GATC AAGCTT ATGGTGAGCAAGGGCGAG-3 '; Hind III site is underlined) and 3'-BamHIEGFP (5'-GATC GGATCC GCTTACTTGTGCAGCTCGTC-3'; BamH I position is indicated by an underlined letter) was amplified from pEGFP-N1 (Clontech) by the PCR method. The PCR product was digested with HindIII and BamHI and cloned into the same position of the pcDNA3 vector to prepare pcDNA3-EGFP. PCR method using; (BamH I site underlined 5-GATC GGATCC GCCTTGTACAGCTCGTC-3) for the production of pcDNA3-EGFP-TEC, EGFP ( no Stop) fragment the primers 5-HindIIIEGFP and 3-BamHIEGFP (NoStop) RTI ID = 0.0 > pEGFP-N1. ≪ / RTI > The PCR product was digested with HindIII and BamHI and cloned into the same position of the pcDNA3 vector to prepare pcDNA3-EGFP (NoStop). The TEC fragment was isolated by digesting pcDNA4 / His-MaxB / TEC with BamHI . Then, pcDNA3-EGFP-TEC was prepared by cloning at the corresponding position of pcDNA3-EGFP (NoStop). The pCMV-EGFP-TFG-TEC was prepared as follows: pCMV-Tag2A / TFG-TEC was digested with BamHI to isolate the TFG-TEC fragment and cloned at a position corresponding to pcDNA3-EGFP (NoStop) -EGFP-TFG-TEC. ≪ / RTI >

GAL4-TFG (NTD) deletion mutants were prepared in the following way: (A) In the case of GAL4-TFG (NTD), the TFG (NTD) fragment the primers 5'-BamHITFG-GAL4 (5'- GATC GGATCC GAATGAACGGACAGTTGG-3 '; BamH I site is underlined) and 3'-HindIIITFG-GAL4 (5'- GATC AAGCTT CTGAATACTGAATAC-3'; Hind III site by the PCR using the underlined) pCMV-Tag2A / TFG- TEC and then cleaved with BamHI and HindIII and cloned into the corresponding positions of the pM vector (Clonetech Laboratories) to prepare the GAL4-TFG (NTD) fragment. (B) GAL4-TFG (1-124 ) For fragment, TFG (1-124) fragment the primers 5'-BamHITFG-GAL4 and 3'-HindIIITFGCC-GAL4 (5'- GATC AAGCTT TTCCAAGCTATCCAATAAC-3 '; Hind III TAG2A / TFG-TEC by PCR using the restriction enzyme site (indicated by an underlined position), followed by cleavage with BamHI and HindIII and cloning into the corresponding position of the pM vector to obtain the GAL4-TFG (1-124) fragment . (C) GAL4-TFG (97-273 ) for short, TFG (97-273) fragment the primers 5'-BamHITFGCC-GAL4 (5'- GATC GGATCC GACTTGAATCAA-3 '; BamH I site is underlined) and After amplification from pCMV-Tag2A / TFG-TEC by PCR using 3'-HindIII TFG-GAL4, the fragment was digested with BamHI and HindIII and cloned into the corresponding position of the pM vector to obtain the GAL4-TFG (97-273) fragment . (D) GAL4-TFG (PB ) for short, TFG (PB) fragment is primer 5'-BamHITFG-GAL4 and 3'-HindIIITFGPB-GAL4 (5'- GATC AAGCTT GGGTCTTGGCTGGCCATT-3 '; Hind III site is underlined The fragment was amplified from pCMV-Tag2A / TFG-TEC by PCR using a primer (SEQ ID NO: 1) and then cloned into the corresponding position of pM vector by digestion with BamHI and HindIII to prepare the GAL4-TFG (PB) fragment. For the (E) GAL4-TFG (CC) fragment, the TFG (CC) fragment was amplified from pCMV-Tag2A / TFG-TEC by PCR using primers 5'-BamHITFGCC-GAL4 and 3'- HindIIITFGCC- The GAL4-TFG (CC) fragment was prepared by cleavage with BamHI and HindIII and cloning into the corresponding position of the pM vector. (5'-GATC GGATCC GACCACCTGGAGAACCAGGAC-3 ', BamH I position is underlined) and 3'-HindIII FG (SEQ ID NO: 2) for the (F) GAL4-TFG (SPYGQ) -GAL4 and then cloned into the corresponding positions of the pM vector to obtain the GAL4-TFG (NTD) fragment. The GAL4-TFG (NTD) fragment was amplified from pCMV-Tag2A / TFG-TEC by PCR using GAL4 and then digested with BamHI and HindIII .

In bito  Transcription and translation

Flag Transcription and translation of TFG-TEC and Flag TEC in vitro was performed using a TNT kit (Promega) using pCMV-Tag2A / TFG-TEC or pCMV-Tag2A / TEC, respectively, according to the manufacturer's instructions (Promega). The in vitro translation product was electrophoresed on 8 SDS-PAGE and then analyzed by Western blotting with anti-Flag antibody (Sigma). In vitro-translated (in The protein was quantified on a ChemiDoc ^TM XRS System (Bio-Rad).

Electrophoretic mobility shift assay (EMSA)

The sequence of the synthetic oligonucleotide probe used for EMSA analysis is described in the previous report (3). Probes (0.5 ng each) were prepared by end-labeling complementary oligonucleotides with [? - ³² P] ATP using T4 polynucleotide kinase. DNA linking reaction is in vitro translation of TFG-TEC, and 10 mM using a TEC Tris-HCl (pH 8.0) , 40 mM KCl, 6% glycerol, 1 mM DTT, 0.05 NP- 40 and 10 ng / l Of poly (dIdC) (dIdC) at 4 ° C for 30 min. After the conjugation reaction, the mixture was incubated with 4 polyacrylamide gel (acrylamide / bisacrylamide ratio, 37: 1) in 0.5X TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, 1 mM EDTA) 150 V for 2-3 hours. The gel was dried and exposed to Kodak X-Omat film using an intensifying screen at -70 ° C.

Subcellular localization

Immunocytochemical assays were performed as previously reported (20). Briefly, 293T cells were plated on glass coverslips and then transfected with each plasmid using VivaMagic Reagent (Vivagen). After 48 h, the cells were washed with PBS (phosphate-buffered saline) and fixed with a mixture of acetone and methanol (1: 1, v / v) for 10 min at -20 캜. Flag-tagged TFG-TEC or TFG-TEC mutant proteins were detected using an anti-Flag antibody (M2, Sigma) and a TRITC-conjugated secondary antibody (Sigma), and fluorescence was detected using a CoolSNAP digital camera (Olympus, IX71) equipped with a fluorescence microscope (Olympus).

Purification of the GST fusion protein, GST pull-down assay, Western blot assay,

As with the bar 21, previously it reported, glutathione S- transferase (GST) -EWS (NTD), GST-68 and _GST-II hTAF TGE (NTD) protein was expressed in E. coli (Escherichia coli, E. Coli) . After binding and washing with glutathione-Sepharose (GE Healthcare), the proteins were eluted with reduced glutathione (Sigma). Protein concentration was measured by the Bradford (Bio-Rad) method. The purity and size of the eluted proteins were confirmed by coomassie blue staining of SDS-polyacrylamide gels. The GST pull-down assay was performed as previously described (20). Western blotting analysis was performed with anti-Flag (M2) or anti-Express (Invitrogen) antibodies, and reactive bands were detected by chemiluminescence using Western Ligthning ECL kit (PerkinElmer Life Scienes).

Reporter gene analysis

Cells were subjected to transient transfection of the plasmid with the VivaMagic reagent. Luciferase assay was performed with the Dual-luciferase assay system (Promega). Renilla luciferase activity was used to normalize transfection efficiency.

Reverse-PCR

Total RNA was isolated from 293T cells transiently transfected with EGFP, EGFP-TEC, or EGFP-TFG-TEC using on-column DNase treatment and RNeasy mini kit (Qiagen) (Qiagen) and cDNA was synthesized by Superscript First-Strand Synthesis System (Invitrogen) for RT-PCR. RT-PCR reactions for Skp2 , L-Myc , SOCS2 and STAT-3 were performed in triplicate using a gene-specific primer set. The primers used were: Skp2 forward primer, 5'-CTGTCTCAGTGTTCCAAGTTGCA-3 'and Skp2 reverse primer, 5'-CAGAACACCCAGAAAGGTTAAGT-3'(22); L-Myc forward primer, 5'-CAGCTTCTGGAGGAAAACG-3 'and L-Myc reverse primer, 5'-CAGCTTCTGGAGGAAAACG-3'(23); SOCS2 forward primer, 5'-CTCGGTCAGACAGGATGGTA-3 'and SOCS2 reverse primer, 5'-ACAGAGATGCTGCAGAGATG-3'(24); STAT-3 forward primer, 5'-TTGCCAGTTGTGGTGATC-3 'and STAT-3 reverse primer, 5'-AGAACCCAGAAGGAGAAGC-3'(25); And β-actin forward primer, 5'-GCTCGTCGTCGACAACGGCTC-3 'and -actin reverse primer, 5'-CAAACATGATCTGGGTCATCTTCTC-3'.

Experiment result

The TFG-TEC fusion protein binds to the TEC consensus sequence

Human EMC patients share recurrent translocations that fuse the 22q12 EWS gene or the 17q11.2 hTAF _II 68 gene with the TEC nuclear receptor gene at 9q22. Recent reports suggest that one of the chromosomal translocation events in human EMC leads to in-frame fusion of TEC and TFG in chromosome 3q11-q12, resulting in chromosome translocation of t (3; 9) (q11-q12; q22) (Table 1). The breakpoint of the TFG gene is present in intron 6 and the translocation point of the TEC is the two nucleotides (A and T) located upstream of the ATG start codon. The TFG protein is a cytoplasmic protein containing the PB1 and CC domains located at the N-terminus and the SPYGQ-rich transcriptional activator-like domains located at the N- and C-termini (8, 10). The expected chimeric protein is a protein in which the N-terminal PB1, CC and SPYGQ-rich domains of TFG are fused with the full-length TEC (Fig. 1).

The DNA-binding properties of the TFG-TEC protein were analyzed by EMSA using a synthetic oligonucleotide probe and an in vitro transcribed / translated TFG-TEC protein. The DNA-binding domain (DBD) of TEC is a conserved DBD that binds to the NBI (NGFI-B Response Element) sequence motif (5'-AAAAGGTCA-3 ') (3). Although there is a significant structural variation between the TFG-TEC and TEC proteins, the DBD of TEC is normal in both proteins. In order to determine whether the TFG-TEC protein binds to the physiological targets of the TEC, EMSA using the NBRE sequence motif in the binding reaction was performed. Flag- tagged full-length protein TFG-TEC TEC and the in vitro synthesized RNA produced by in vitro transcription of the protein to the cell-was used to program a rabbit reticulocyte lysate member, so that the in vitro translation a Flag- tagged TFG-TEC protein and Flag-tagged TEC protein products were quantitated by SDS-PAGE and Western blotting with anti-Flag antibody (M2) (Fig. 2a). Equimolar amounts of in-vitro -translated TFG-TEC and TEC proteins were added to each assay. Quantification of in vitro -translated proteins was performed using the ChemiDoc ^TM XRS System (Bio-Rad). EMSA was performed using the same concentration of probe and increasing amount of in vitro -translated protein. Protein-DNA complexes are formed in both the TFG-TEC protein (Figure 2b, lane 8-10) and the TEC protein (Figure 2b, lanes 5-7), whereas the unprogrammed reticulocyte lysate has very low levels of binding (Fig. 2B, lane 2-4). In that the complex is destroyed only by unlabeled oligonucleotides containing NBRE sequence motifs added at 5-fold and 10-fold excess, rather than oligonucleotides containing mutated NBRE sequence motifs not recognized by TEC DBD, The interaction was specific (Figure 2c). The present inventors from the above experimental results could be concluded that DNA binds to a similar specificity showing TFG-TEC TEC protein and protein-DNA- binding activity similar in vitro.

DBD  of mine KRRR  The sequence is TFG - TEC  Protein to nucleus Target

Since TFG is a cytoplasmic protein (8) and TFG-TEC protein contains the NTD (N-terminal domain) of TFG fused to full-length TEC, we intracellularly localize the TFG- Respectively. Non-direct immunofluorescence analysis was performed using 293T cells, since TFG-TEC-positive human EMC cell lines or derivatives thereof are not currently available. 293T cells were transfected with a co-expression vector (pCMV-Tag2A; results not shown) or pCMVTag2A / TFG-TEC (Fig. 3b (a)) and then examined by fluorescence immunohistochemistry. The chimeric TFG-TEC was located in the nucleus (Fig. 3 (b)), indicating that the TFG-TEC protein is a nuclear protein. Next, the present inventors used a series of TFG-TEC deletion mutants to map a site responsible for nuclear localization of TFG-TEC (FIG. 3A). After 293T cells were transfected with an expression vector for Flag-tagged TFG-TEC truncation mutants, the positions of the Flag-tagged proteins were examined by fluorescence immunohistochemistry. Flag-tagged TFG (NTD) was located in the cytoplasm (Figure 3b (b)) while Flag-tagged TEC was located in the nucleus (Figure 3b (c)). In order to further define the nuclear localization signal present in the DBD of the TFG-TEC protein, the present inventors used the position-designated mutagenesis method to isolate the highly conserved basic amino acid sequence ⁶¹² KRRR ⁶¹⁵ from DBD Mutants were prepared. Substitution of the ⁶¹² KRRR ⁶¹⁵ sequence by alanine resulted in cytoplasmic accumulation of Flag-tagged TFG-TEC (Fig. 3b (d)). The above results show that clusters of basic amino acids in DBD function as nuclear localization signals of TFG-TEC.

TFG ( NTD ) U1C Can not be combined with

EWS (NTD) contains the interaction motif (s) for the splicing factor U1C protein. The interaction between EWS (NTD) and U1C negatively regulates EWS-fusion-mediated transactivation (26). Due to the relationship between EWS NTD and TFG NTD (7, 10), we investigated whether TFG could interact with U1C. To do this, an in vitro GST pull-down assay using bacterially expressed GST fusion EWS (NTD), hTAF _II 68 (NTD) and His ₆ -tagged full length U1C protein was performed (Figure 4). As previously reported (26), the U1C protein specifically bound to GST-EWS (NTD) (Fig. 4A, lane 3) but not to GST alone (Fig. 4A, lane 2). Interestingly, the U1C protein also interacted with another member of the TET family, hTAF _II 68 (Figure 4a, lane 4), indicating that the EWS (NTD) / U1C interaction remained significant in the hTAF _II 68 (NTD) . In contrast, TFG (NTD) did not bind U1C (Fig. 4A, lane 5), indicating that U1C can not regulate TFG-TEC activity in human EMCs. The amount of GST fusion protein used in the assays described above was fractionated on a 15% SDS-PAGE gel and visualized by Coomassie blue staining. As a result, it was confirmed that a similar amount of protein was used in the pool-down assay (Fig. 4B).

TFG - TEC The TEC see increasingly  Powerful Warrior Is an activator

The transcriptional characteristics of TFG-TEC and TEC were studied by co-transfection of reporter plasmids and expression vectors. NTDs of EWS and hTAF _II 68 appear to contribute to transcriptional activation by EWS- or hTAF _II 68-fusion proteins by providing a strong transactivation domain (27). In order to evaluate the transcriptional activation effect of NTD-TFG in TFG-TEC, the present inventors used a reporter plasmid containing eight copies of TEC binding sites (NBREs) and a TATA box upstream of the luciferase gene and respective expression vectors - transfected to compare the transcriptional activity of TFG-TEC and TEC (Fig. 5A). Also included was a control plasmid containing the cytomegalovirus-driven Renilla luciferase gene. The co-transfection of TFG-TEC induces approximately 635-fold increase in reporter expression (Fig. 5b, bars 2 and 4) in 293T cells, whereas induction of approximately 220-fold increase in expression by TEC alone 5b, bars 1 and 3). Thus, it is clear that TFG-TEC is a more potent transcriptional activator than TEC. Thereafter, the results of a reporter assay that TFG-TEC has better transactivation activity than TEC have been further investigated. In order to evaluate the in vivo transactivation potential of TFG-TEC, we transiently transfected pcDNA3-EGFP, pcDNA3-EGFP-TEC and pcDNA3-EGFP-TFG-TEC constructs to 293T cells (FIG. 5C), potential downstream genes Skp2 (28), L- Myc , SOCS2 And STAT -3 were measured to compare the transcriptional activity of TFG-TEC and TEC. As can be seen in Figure 5c, Skp2 (28), L- Myc , SOCS2 And STAT -3 were up-regulated to a higher degree in 293T cells transfected with TFG-TEC than in TEC transfected 293T cells (Fig. 5c, lane 2 and lane 3). Taken together, the above data show that TFG-TEC is a more potent transcriptional activator than TEC in both in vitro and in vivo . β-actin was used as a control.

TFG ( NTD ) ≪ / RTI > Function

According to the present invention, TFG-TEC was a more potent transcriptional activator than TEC (Figure 5). Although the TFG-TEC chimeric proteins contained normal TEC functional domains (Figure 1), protein-DNA complexes were formed in both TFG-TEC and TEC proteins (Figure 4) . This may be due to differences in the NTDs of both proteins. However, it is unclear whether TFG (NTD) can activate transcription. Thus, an expression vector containing a GAL4-TFG fusion gene was prepared to examine the transcriptional activity of NTD-TFG (FIG. 6A). A pG5 luc reporter containing five GAL4 DNA-binding sites upstream of the TATA box was used as the reporter. TFG (NTD) reactivity to pG5 luc reporter was co-transfected with 0.1 μg of pG5 luc construct and 2 ng of Renilla plasmid together with an increasing amount of GAL4-TFG (NTD) expression construct in 293T cells Respectively. The expression vector promoting the synthesis of GAL4 DBD alone (GAL4) did not show a significant effect on the level of luciferase produced by pG5 luc in transfection into 293T cells (Fig. 6b, lane 1). In contrast, pGAL4-TFG (NTD) activated luciferase production in a dose-dependent manner (up to 12-fold; Fig. 6b, lane 2-5). The above results demonstrate that TFG (NTD) contains the transactivation domain (s).

Two functional domains TFG ( NTD ) ≪ / RTI >

Transient transfection experiments with GAL4-fused TFG (NTD) functional domains were performed to characterize functional regions in TFG (NTD) essential for transactivation. The structure of the TFG (NTD) functional domains is schematically shown in FIG. The data in the right panel assumes the value obtained by GAL4-TFG (NTD) as 100% and represents the relative transcriptional activation values for this. The polypeptide [GAL4-TFG (1-123)] in which the amino acid residues of the SPYGQ-rich region (125-273) was deleted exhibited a decrease of about 75% as compared with that of GAL4-TFG (NTD). The polypeptide with deletion of the PB1 domain [GAL4-TFG (97-273)] showed about 50% less reduction in transactivation activity. The above results indicate that the domains described above are important for TFG (NTD) -mediated transactivation activity. The fusion construct with the SPYGQ-rich region, designated GAL4-TFG (SPYGQ), increased luciferase production from the pG5 luc vector by approximately 30% because the SPYGQ-rich region of TFG (NTD) . ≪ / RTI > In addition, the PB1 domain of TFG (NTD) also affected the level of luciferase (about 13%) produced from the pG5 luc vector when transfected into 293T cells. On the other hand, the construct containing the CC domain alone produced only about 6% of the polypeptide compared to the transcriptional activity of the polypeptide produced by GAL4-TFG (NTD). Thus, the above results indicate that the PB1 domain and SPYGQ-rich region of TFG (NTD) can partially activate transcription and that the integration of the entire TFG (NTD) is required for complete transfer performance.

Additional discussion

Originally, the TFG gene was used in studies to identify genes encoding protein sites similar to the N-terminal regions of EWS and TLS / FUS proteins. In the Expression Sequence Tag (EST) database using the SPYGQ-rich region as a search sequence, and homology searches (10). Although the amino acid sequence described above is commonly found in transcriptional activation domains (27), the function of the region in TFG-TEC has not been known yet. In order to investigate the biological function of the chimeric gene, the present inventors have characterized the characteristics of the TFG-TEC fusion protein. In this study, the present inventors confirmed that TFG-TEC is a nucleoprotein that binds to DNA with sequence specificity that is indistinguishable from the sequence specificity of the parental TEC protein. The fusion gene encodes a tansactivator that is stronger than TEC.

The U1C protein, which functions as a splicing factor and is important in the early stages of spliceosome formation, binds EWS-Fli-1 (26, 29). The interaction regulates EWS-Fli-1 activity by inhibiting EWS-Fli-1-mediated transcriptional activation mediated through the NTD of EWS (26). Interestingly, the U1C / EWS (NTD) interaction remains at a reliable level in hTAF _II 68 (NTD) (Fig. 4). However, TFG (NTD) does not bind to U1C (Figure 4), because the spectrum for the interaction partners of TFG (NTD) is not the same as the spectrum of the EWS (NTD) -centered protein interaction network . Thus, it was tested whether TFG-TEC could interact with c-Abl (30), v-Src (31) and ZFMl (32), known as EWS It can be a very interesting subject.

(33, 34), EWS-WT1 (35), and EWS-Oct-4 (36, 37), which are capable of transforming into NIH3T3 cells or causing increased tumor growth in nude mice Unlike other fusion products, the expected transfection activity of TFG-TEC has not yet been determined. The tumorigenic potential of the TFG-TEC gene product is consistent with the idea that TFG-TEC plays a crucial role in the formation of human EMCs. Since the DNA-binding specificity of the TFG-TEC protein is similar to that previously reported in TEC (Fig. 2) and the TFG-TEC fusion protein encodes a strong transcriptional activator (Fig. 5) -Reducing the expression of reactive genes, thereby contributing to the tumorigenesis process. However, although the TFG-TEC fusion protein appears to function as an oncogenic function through inadequate activation of TEC target genes similar to inadequate activation of other TEC fusion proteins in human EMCs, a clear identification of the key target gene (s) need.

As shown in FIG. 5C, the overexpressed TFG-TEC and TEC proteins up-regulated the expression of Skp2, L-Myc, SOCS2 and STAT-3 genes. In addition, the inventors noted that the above-mentioned potential target genes are more strongly up-regulated by TFG-TEC than TEC (Fig. 5C). Although the NOR1 protein functions as a mitogen (38-40), the mechanisms involved in this protein activity are not yet known. Recently, NOR1 protein has been reported to induce the expression of the Skp2 gene responsible for the degradation of p27 (41, 42), a cyclin-dependent kinase inhibitor (28). Over-expressed TFG-TEC also up-regulated the expression of L- Myc , SOCS2 and STAT-3 . Interestingly, the expression of L- Myc , SOCS2 and STAT- 3 has also been observed in the pathogenesis of human cancers (43-46). The above findings are important because TFG-TEC can explain how it functions as an oncogenic function in the onset of human extraskeletal myxoid chondrosarcomas (EMCs).

The production of novel chimeric transcription factors with increased functionality by chromosome translocation is an unchanging interesting subject for EWS- and hTAF _II 68-TEC fusion proteins in human EMCs (27). According to a previously reported by the present inventors, hTAF _II 68-TEC of hTAF _II 68 NTD is to contribute to the functional activity by providing a novel activation domain, that these differences in the transition activation capacity between hTAF _II 68-TEC and TEC Gt; < RTI ID = 0.0 > NTDs. ≪ / RTI > Consistent with this hypothesis, TFG (NTD) of TFG-TEC was able to activate transcription with GAL4 DBD (FIG. 6). In addition, the above results were consistent in the presence of the activation domain (s) in TFG (NTD). TFG (NTD) shares sequence homology with both hTAF _II 68 and EWS (7). Thus, TFG (NTD) can stimulate transcription by acting in a manner similar to that proposed in hTAFII68 (48), ie by altering the preinitiation complex from closed conformation to open conformation have. Additional experiments are required to explore this possibility.

In conclusion, the findings of this study demonstrate that TFG-TEC is a potential tumorigenic gene necessary for tumorigenesis in human EMCs, although additional genes may be required for co-action with TFG-TEC or for tumor progression to provide. Presumably, TFG-TEC regulates key TEC downstream target genes and contributes to oncogenesis. It would therefore be very interesting to determine whether the downstream gene is essential for tumorigenesis and whether TFG-TEC works in partnership with this gene (s) to develop human EMCs. In future experiments, the inventors will focus on identifying key endogenous targets regulated by TFG-TEC in human EMCs.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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<110> Industry-University Cooperation Foundation Sogang University <120> Screening Methods of a Substance for Preventing or Treatin          Tumor Disorder Using a TFG-TEC Fusion Construct and Uses Thereof <130> PN130091 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 1200 <212> DNA <213> Homo sapiens <400> 1 atgaacggac agttggatct aagtgggaag ctaatcgtca aagctcaact tggggaggat 60 attcggcgaa ttcctattca taatgaagat attacttatg atgaattagt gctaatgatg 120 caacgagttt tcagaggaaa acttctgagt aatgatgaag taacaataaa gtataaagat 180 gaagatggag atcttataac aatttttgat agttctgacc tttcctttgc aattcagtgc 240 agtaggatac tgaaactgac attatttgtt aatggccagc caagacccct tgaatcaagt 300 caggtgaaat atctccgtcg agaactgata gaacttcgaa ataaagtgaa tcgtttattg 360 gatagcttgg aaccacctgg agaaccagga ccttccacca atattcctga aaatgatact 420 gtggatggta gggaagaaaa gtctgcttct gattcttctg gaaaacagtc tactcaggtt 480 atggcagcaa gtatgtctgc ttttgatcct ttaaaaaacc aagatgaaat caataaaaat 540 gttatgtcag cgtttggctt aacagatgat caggtttcag ggccacccag tgctcctgca 600 gaagatcgtt caggaacacc cgacagcatt gcttcctcct cctcagcagc tcacccacca 660 ggcgttcagc cacagcagcc accatataca ggagctcaga ctcaagcagg tcagattgaa 720 ggtcagatgt accaacagta ccagcaacag gccggctatg gtgcacagca gccgcaggct 780 ccacctcagc agcctcaaca gtatggtatt cagtattcag caagctatag tcagcagact 840 ggacctcaac aacctcagca gttccaggga tatggccagc aaccaacttc ccaggcacca 900 gctcctgcct tttctggtca gcctcaacaa ctgcctgctc agccgccaca gcagtaccag 960 gcgagcaatt atcctgcaca aacttacact gcccaaactt ctcagcctac taattatact 1020 gtggctcctg cctctcaacc tggaatggct ccaagccaac ctggggccta tcaaccaaga 1080 ccaggtttta cttcacttcc tggaagtacc atgacccctc ctccaagtgg gcctaatcct 1140 tatgcgcgta accgtcctcc ctttggtcag ggctataccc aacctggacc tggttatcga 1200                                                                         1200 <210> 2 <211> 1878 <212> DNA <213> Homo sapiens <400> 2 atgccctgcg tccaagccca atatagccct tcccctccag gttccagtta tgcggcgcag 60 acatacagct cggaatacac cacggagatc atgaaccccg actacaccaa gctgaccatg 120 gaccttggca gcactgagat cacggctaca gccaccacgt ccctgcccag catcagtacc 180 ttcgtggagg gctactcgag caactacgaa ctcaagcctt cctgcgtgta ccaaatgcag 240 cggcccttga tcaaagtgga ggaggggcgg gcgcccagct accatcacca tcaccaccac 300 caccaccacc accaccacca tcaccagcag cagcatcagc agccatccat tcctccagcc 360 tccagcccgg aggacgaggt gctgcccagc acctccatgt acttcaagca gtccccaccg 420 tccaccccca ccacgccggc cttccccccg caggcggggg cgttatggga cgaggcactg 480 ccctcggcgc ccggctgcat cgcacccggc ccgctgctgg acccgccgat gaaggcggtc 540 cccacggtgg ccggcgcgcg cttcccgctc ttccacttca agccctcgcc gccgcatccc 600 cccgcgccca gcccggccgg cggccaccac ctcggctacg acccgacggc cgctgccgcg 660 ctcagcctgc cgctgggagc cgcagccgcc gcgggcagcc aggccgccgc gcttgagagc 720 cacccgtacg ggctgccgct ggccaagagg gcggccccgc tggccttccc gcctctcggc 780 ctcacgccct cccctaccgc gtccagcctg ctgggcgaga gtcccagcct gccgtcgccg 840 cccagcagga gctcgtcgtc tggcgagggc acgtgtgccg tgtgcgggga caacgccgcc 900 tgccagcact acggcgtgcg aacctgcgag ggctgcaagg gctttttcaa gagaacagtg 960 cagaaaaatg caaaatatgt ttgcctggca aataaaaact gcccagtaga caagagacgt 1020 cgaaaccgat gtcagtactg tcgatttcag aagtgtctca gtgttggaat ggtaaaagaa 1080 gttgtccgta cagatagtct gaaagggagg agaggtcgtc tgccttccaa accaaagagc 1140 ccattacaac aggaaccttc tcagccctct ccaccttctc ctccaatctg catgatgaat 1200 gcccttgtcc gagctttaac agactcaaca cccagagatc ttgattattc cagatactgt 1260 cccactgacc aggctgctgc aggcacagat gctgagcatg tgcaacaatt ctacaacctc 1320 ctgacagcct ccattgatgt atccagaagc tgggcagaaa agattccggg atttactgat 1380 ctccccaaag aagatcagac attacttatt gaatcagcct ttttggagct gtttgtcctc 1440 agactttcca tcaggtcaaa cactgctgaa gataagtttg tgttctgcaa tggacttgtc 1500 ctgcatcgac ttcagtgcct tcgtggattt ggggagtggc tcgactctat taaagacttt 1560 tccttaaatt tgcagagcct gaaccttgat atccaagcct tagcctgcct gtcagcactg 1620 agcatgatca cagaaagaca tgggttaaaa gaaccaaaga gagtcgaaga gctatgcaac 1680 aagatcacaa gcagtttaaa agaccaccag agtaagggac aggctctgga gcccaccgag 1740 tccaaggtcc tgggtgccct ggtagaactg aggaagatct gcaccctggg cctccagcgc 1800 atcttctacc tgaagctgga agacttggtg tctccacctt ccatcattga caagctcttc 1860 ctggacaccc tacctttc 1878 <210> 3 <211> 2700 <212> DNA <213> Artificial Sequence <220> <223> TFG-TEC fusion construct <400> 3 atgaacggac agttggatct aagtgggaag ctaatcatca aagctcaact tggggaggat 60 attcggcgaa ttcctattca taatgaagat attacttatg atgaattagt gctaatgatg 120 caacgagttt tcagaggaaa acttctgagt aatgatgaag taacaataaa gtataaagat 180 gaagatggag atcttataac aatttttgat agttctgacc tttcctttgc aattcagtgc 240 agtaggatac tgaaactgac attatttgtt aatggccagc caagacccct tgaatcaagt 300 caggtgaaat atctccgtcg agaactgata gaacttcgaa ataaagtgaa tcgtttattg 360 gatagcttgg aaccacctgg agaaccagga ccttccacca atattcctga aaatgatact 420 gtggatggta gggaagaaaa gtctgcttct gattcttctg gaaaacagtc tactcaggtt 480 atggcagcaa gtatgtctgc ttttgatcct ttaaaaaacc aagatgaaat caataaaaat 540 gttatgtcag cgtttggctt aacagatgat caggtttcag ggccacccag tgctcctgca 600 gaagatcgtt caggaacacc cgacagcatt gcttcctcct cctcagcagc tcacccacca 660 ggcgttcagc cacagcagcc accatataca ggagctcaga ctcaagcagg tcagattgaa 720 ggtcagatgt accaacagta ccagcaacag gccggctatg gtgcacagca gccgcaggct 780 ccacctcagc agcctcaaca gtatggtatt cagtattcag atatgccctg cgtccaagcc 840 caatatagcc cttcccctcc aggttccagt tatgcggcgc agacatacag ctcggaatac 900 accacggaga tcatgaaccc cgactacacc aagctgacca tggaccttgg cagcactgag 960 atcacggcta cagccaccac gtccctgccc agcatcagta ccttcgtgga gggctactcg 1020 agcaactacg aactcaagcc ttcctgcgtg taccaaatgc agcggccctt gatcaaagtg 1080 gaggaggggc gggcgcccag ctaccatcac catcaccacc accaccacca ccaccaccac 1140 catcaccagc agcagcatca gcagccatcc attcctccag cctccagccc ggaggacgag 1200 gtgctgccca gcacctccat gtacttcaag cagtccccac cgtccacccc caccacgccg 1260 gccttccccc cgcaggcggg ggcgttatgg gacgaggcac tgccctcggc gcccggctgc 1320 atcgcacccg gcccgctgct ggacccgccg atgaaggcgg tccccacggt ggccggcgcg 1380 cgcttcccgc tcttccactt caagccctcg ccgccgcatc cccccgcgcc cagcccggcc 1440 ggcggccacc acctcggcta cgacccgacg gccgctgccg cgctcagcct gccgctggga 1500 gccgcagccg ccgcgggcag ccaggccgcc gcgcttgaga gccacccgta cgggctgccg 1560 ctggccaaga gggcggcccc gctggccttc ccgcctctcg gcctcacgcc ctcccctacc 1620 gcgtccagcc tgctgggcga gagtcccagc ctgccgtcgc cgcccagcag gagctcgtcg 1680 tctggcgagg gcacgtgtgc cgtgtgcggg gacaacgccg cctgccagca ctacggcgtg 1740 cgaacctgcg agggctgcaa gggctttttc aagagaacag tgcagaaaaa tgcaaaatat 1800 gtttgcctgg caaataaaaa ctgcccagta gacaagagac gtcgaaaccg atgtcagtac 1860 tgtcgatttc agaagtgtct cagtgttgga atggtaaaag aagttgtccg tacagatagt 1920 ctgaaaggga ggagaggtcg tctgccttcc aaaccaaaga gcccattaca acaggaacct 1980 tctcagccct ctccaccttc tcctccaatc tgcatgatga atgcccttgt ccgagcttta 2040 acagactcaa cacccagaga tcttgattat tccagatact gtcccactga ccaggctgct 2100 gcaggcacag atgctgagca tgtgcaacaa ttctacaacc tcctgacagc ctccattgat 2160 gtatccagaa gctgggcaga aaagattccg ggatttactg atctccccaa agaagatcag 2220 acattactta ttgaatcagc ctttttggag ctgtttgtcc tcagactttc catcaggtca 2280 aacactgctg aagataagtt tgtgttctgc aatggacttg tcctgcatcg acttcagtgc 2340 cttcgtggat ttggggagtg gctcgactct attaaagact tttccttaaa tttgcagagc 2400 ctgaaccttg atatccaagc cttagcctgc ctgtcagcac tgagcatgat cacagaaaga 2460 catgggttaa aagaaccaaa gagagtcgaa gagctatgca acaagatcac aagcagttta 2520 aaagaccacc agagtaaggg acaggctctg gagcccaccg agtccaaggt cctgggtgcc 2580 ctggtagaac tgaggaagat ctgcaccctg ggcctccagc gcatcttcta cctgaagctg 2640 gaagacttgg tgtctccacc ttccatcatt gacaagctct tcctggacac cctacctttc 2700                                                                         2700 <210> 4 <211> 820 <212> DNA <213> Artificial Sequence <220> <223> TFG (NTD) construct <400> 4 atgaacggac agttggatct aagtgggaag ctaatcgtca aagctcaact tggggaggat 60 attcggcgaa ttcctattca taatgaagat attacttatg atgaattagt gctaatgatg 120 caacgagttt tcagaggaaa acttctgagt aatgatgaag taacaataaa gtataaagat 180 gaagatggag atcttataac aatttttgat agttctgacc tttcctttgc aattcagtgc 240 agtaggatac tgaaactgac attatttgtt aatggccagc caagacccct tgaatcaagt 300 caggtgaaat atctccgtcg agaactgata gaacttcgaa ataaagtgaa tcgtttattg 360 gatagcttgg aaccacctgg agaaccagga ccttccacca atattcctga aaatgatact 420 gtggatggta gggaagaaaa gtctgcttct gattcttctg gaaaacagtc tactcaggtt 480 atggcagcaa gtatgtctgc ttttgatcct ttaaaaaacc aagatgaaat caataaaaat 540 gttatgtcag cgtttggctt aacagatgat caggtttcag ggccacccag tgctcctgca 600 gaagatcgtt caggaacacc cgacagcatt gcttcctcct cctcagcagc tcacccacca 660 ggcgttcagc cacagcagcc accatataca ggagctcaga ctcaagcagg tcagattgaa 720 ggtcagatgt accaacagta ccagcaacag gccggctatg gtgcacagca gccgcaggct 780 ccacctcagc agcctcaaca gtatggtatt cagtattcag 820 <210> 5 <211> 400 <212> PRT <213> Homo sapiens <400> 5 Met Asn Gly Gln Leu Asp Leu Ser Gly Lys Leu Ile Val Lys Ala Gln   1 5 10 15 Leu Gly Glu Asp Ile Arg Arg Ile Pro Ile His Asn Glu Asp Ile Thr              20 25 30 Tyr Asp Glu Leu Val Leu Met Met Gln Arg Val Phe Arg Gly Lys Leu          35 40 45 Leu Ser Asn Asp Glu Val Thr Ile Lys Tyr Lys Asp Glu Asp Gly Asp      50 55 60 Leu Ile Thr Ile Phe Asp Ser Ser Asp Leu Ser Phe Ala Ile Gln Cys  65 70 75 80 Ser Arg Ile Leu Lys Leu Thr Leu Phe Val Asn Gly Gln Pro Arg Pro                  85 90 95 Leu Glu Ser Ser Gln Val Lys Tyr Leu Arg Arg Glu Leu Ile Glu Leu             100 105 110 Arg Asn Lys Val Asn Arg Leu Leu Asp Ser Leu Glu Pro Pro Gly Glu         115 120 125 Pro Gly Pro Ser Thr Asn Ile Pro Glu Asn Asp Thr Val Asp Gly Arg     130 135 140 Glu Glu Lys Ser Ala Ser Asp Ser Ser Gly Lys Gln Ser Thr Gln Val 145 150 155 160 Met Ala Ala Ser Met Ser Ala Phe Asp Pro Leu Lys Asn Gln Asp Glu                 165 170 175 Ile Asn Lys Asn Val Met Ser Ala Phe Gly Leu Thr Asp Asp Gln Val             180 185 190 Ser Gly Pro Ser Ala Pro Ala Glu Asp Arg Ser Gly Thr Pro Asp         195 200 205 Ser Ile Ala Ser Ser Ser Ala Ala His Pro Pro Gly Val Gln Pro     210 215 220 Gln Gln Pro Pro Tyr Thr Gly Ala Gln Thr Gln Ala Gly Gln Ile Glu 225 230 235 240 Gly Gln Met Tyr Gln Gln Tyr Gln Gln Gln Ala Gly Tyr Gly Ala Gln                 245 250 255 Gln Pro Gln Ala Pro Pro Gln Gln Pro Gln Gln Tyr Gly Ile Gln Tyr             260 265 270 Ser Ala Ser Tyr Ser Gln Gln Thr Gly Pro Gln Gln Pro Gln Gln Phe         275 280 285 Gln Gly Tyr Gly Gln Gln Pro Thr Ser Gln Ala Pro Ala Pro Ala Phe     290 295 300 Ser Gly Gln Pro Gln Gln Leu Pro Ala Gln Pro Pro Gln Gln Tyr Gln 305 310 315 320 Ala Ser Asn Tyr Pro Ala Gln Thr Tyr Thr Ala Gln Thr Ser Gln Pro                 325 330 335 Thr Asn Tyr Thr Val Ala Pro Ala Ser Gln Pro Gly Met Ala Pro Ser             340 345 350 Gln Pro Gly Ala Tyr Gln Pro Arg Pro Gly Phe Thr Ser Leu Pro Gly         355 360 365 Ser Thr Met Thr Pro Pro Pro Ser Gly Pro Asn Pro Tyr Ala Arg Asn     370 375 380 Arg Pro Pro Phe Gly Gln Gly Tyr Thr Gln Pro Gly Pro Gly Tyr Arg 385 390 395 400 <210> 6 <211> 626 <212> PRT <213> Homo sapiens <400> 6 Met Pro Cys Val Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly Ser Ser   1 5 10 15 Tyr Ala Gln Thr Tyr Ser Ser Glu Tyr Thr Thr Glu Ile Met Asn              20 25 30 Pro Asp Tyr Thr Lys Leu Thr Met Asp Leu Gly Ser Thr Glu Ile Thr          35 40 45 Ala Thr Ala Thr Thr Ser Leu Ser Ser Ser Thr Phe Val Glu Gly      50 55 60 Tyr Ser Ser Asn Tyr Glu Leu Lys Pro Ser Cys Val Tyr Gln Met Gln  65 70 75 80 Arg Pro Leu Ile Lys Val Glu Glu Gly Arg Ala Pro Ser Tyr His His                  85 90 95 His His His His His His His His His His His Gln Gln Gln His             100 105 110 Gln Gln Pro Ser Ile Pro Pro Ala Ser Ser Pro Glu Asp Glu Val Leu         115 120 125 Pro Ser Thr Ser Met Tyr Phe Lys Gln Ser Pro Pro Ser Thr Pro Thr     130 135 140 Thr Pro Ala Phe Pro Pro Gln Ala Gly Ala Leu Trp Asp Glu Ala Leu 145 150 155 160 Pro Ser Ala Pro Gly Cys Ile Ala Pro Gly Pro Leu Leu Asp Pro Pro                 165 170 175 Met Lys Ala Val Pro Thr Val Ala Gly Ala Arg Phe Pro Leu Phe His             180 185 190 Phe Lys Pro Ser Pro Pro His Pro Pro Ala Pro Ser Pro Ala Gly Gly         195 200 205 His His Leu Gly Tyr Asp Pro Thr Ala Ala Ala Leu Ser Leu Pro     210 215 220 Leu Gly Ala Ala Ala Ala Ala Glu Ser Gln Ala Ala Leu Glu Ser 225 230 235 240 His Pro Tyr Gly Leu Pro Leu Ala Lys Arg Ala Ala Pro Leu Ala Phe                 245 250 255 Pro Pro Leu Gly Leu Thr Pro Ser Pro Thr Ala Ser Ser Leu Leu Gly             260 265 270 Glu Ser Pro Ser Leu Pro Ser Pro Pro Ser Ser Ser Ser Ser Ser Gly         275 280 285 Glu Gly Thr Cys Ala Val Cys Gly Asp Asn Ala Ala Cys Gln His Tyr     290 295 300 Gly Val Arg Thr Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Thr Val 305 310 315 320 Gln Lys Asn Ala Lys Tyr Val Cys Leu Ala Asn Lys Asn Cys Pro Val                 325 330 335 Asp Lys Arg Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys             340 345 350 Leu Ser Val Gly Met Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys         355 360 365 Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Leu Gln Gln     370 375 380 Glu Pro Ser Gln Pro Ser Pro Pro Ser Pro Pro Ile Cys Met Met Asn 385 390 395 400 Ala Leu Val Arg Ala Leu Thr Asp Ser Thr Pro Arg Asp Leu Asp Tyr                 405 410 415 Ser Arg Tyr Cys Pro Thr Asp Gln Ala Ala Ala Gly Thr Asp Ala Glu             420 425 430 His Val Gln Gln Phe Tyr Asn Leu Leu Thr Ala Ser Ile Asp Val Ser         435 440 445 Arg Ser Trp Ala Glu Lys Ile Pro Gly Phe Thr Asp Leu Pro Lys Glu     450 455 460 Asp Gln Thr Leu Leu Ile Glu Ser Ala Phe Leu Glu Leu Phe Val Leu 465 470 475 480 Arg Leu Ser Ile Arg Ser Asn Thr Ala Glu Asp Lys Phe Val Phe Cys                 485 490 495 Asn Gly Leu Val Leu His Arg Leu Gln Cys Leu Arg Gly Phe Gly Glu             500 505 510 Trp Leu Asp Ser Ile Lys Asp Phe Ser Leu Asn Leu Gln Ser Leu Asn         515 520 525 Leu Asp Ile Gln Ala Leu Ala Cys Leu Ser Ala Leu Ser Met Ile Thr     530 535 540 Glu Arg His Gly Leu Lys Glu Pro Lys Arg Val Glu Glu Leu Cys Asn 545 550 555 560 Lys Ile Thr Ser Ser Leu Lys Asp His Gln Ser Lys Gly Gln Ala Leu                 565 570 575 Glu Pro Thr Glu Ser Lys Val Leu Gly Ala Leu Val Glu Leu Arg Lys             580 585 590 Ile Cys Thr Leu Gly Leu Gln Arg Ile Phe Tyr Leu Lys Leu Glu Asp         595 600 605 Leu Val Ser Pro Pro Ser Ile Ile Asp Lys Leu Phe Leu Asp Thr Leu     610 615 620 Pro Phe 625 <210> 7 <211> 900 <212> PRT <213> Artificial Sequence <220> <223> TFG-TEC fusion protein <400> 7 Met Asn Gly Gln Leu Asp Leu Ser Gly Lys Leu Ile Val Lys Ala Gln   1 5 10 15 Leu Gly Glu Asp Ile Arg Arg Ile Pro Ile His Asn Glu Asp Ile Thr              20 25 30 Tyr Asp Glu Leu Val Leu Met Met Gln Arg Val Phe Arg Gly Lys Leu          35 40 45 Leu Ser Asn Asp Glu Val Thr Ile Lys Tyr Lys Asp Glu Asp Gly Asp      50 55 60 Leu Ile Thr Ile Phe Asp Ser Ser Asp Leu Ser Phe Ala Ile Gln Cys  65 70 75 80 Ser Arg Ile Leu Lys Leu Thr Leu Phe Val Asn Gly Gln Pro Arg Pro                  85 90 95 Leu Glu Ser Ser Gln Val Lys Tyr Leu Arg Arg Glu Leu Ile Glu Leu             100 105 110 Arg Asn Lys Val Asn Arg Leu Leu Asp Ser Leu Glu Pro Pro Gly Glu         115 120 125 Pro Gly Pro Ser Thr Asn Ile Pro Glu Asn Asp Thr Val Asp Gly Arg     130 135 140 Glu Glu Lys Ser Ala Ser Asp Ser Ser Gly Lys Gln Ser Thr Gln Val 145 150 155 160 Met Ala Ala Ser Met Ser Ala Phe Asp Pro Leu Lys Asn Gln Asp Glu                 165 170 175 Ile Asn Lys Asn Val Met Ser Ala Phe Gly Leu Thr Asp Asp Gln Val             180 185 190 Ser Gly Pro Ser Ala Pro Ala Glu Asp Arg Ser Gly Thr Pro Asp         195 200 205 Ser Ile Ala Ser Ser Ser Ala Ala His Pro Pro Gly Val Gln Pro     210 215 220 Gln Gln Pro Pro Tyr Thr Gly Ala Gln Thr Gln Ala Gly Gln Ile Glu 225 230 235 240 Gly Gln Met Tyr Gln Gln Tyr Gln Gln Gln Ala Gly Tyr Gly Ala Gln                 245 250 255 Gln Pro Gln Ala Pro Pro Gln Gln Pro Gln Gln Tyr Gly Ile Gln Tyr             260 265 270 Ser Asp Met Pro Cys Val Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly         275 280 285 Ser Ser Tyr Ala Gln Thr Tyr Ser Ser Glu Tyr Thr Thr Glu Ile     290 295 300 Met Asn Pro Asp Tyr Thr Lys Leu Thr Met Asp Leu Gly Ser Thr Glu 305 310 315 320 Ile Thr Ala Thr Ala Thr Thr Ser Leu Ser Ser Ser Thr Phe Val                 325 330 335 Glu Gly Tyr Ser Ser Asn Tyr Glu Leu Lys Ser Ser Cys Val Tyr Gln             340 345 350 Met Gln Arg Pro Leu Ile Lys Val Glu Glu Gly Arg Ala Pro Ser Tyr         355 360 365 His His His His His His His His His His His His His His Gln Gln     370 375 380 Gln His Gln Gln Pro Ser Ile Pro Pro Ala Ser Ser Pro Glu Asp Glu 385 390 395 400 Val Leu Pro Ser Thr Ser Met Tyr Phe Lys Gln Ser Pro Pro Ser Thr                 405 410 415 Pro Thr Thr Pro Ala Phe Pro Pro Gln Ala Gly Ala Leu Trp Asp Glu             420 425 430 Ala Leu Pro Ser Ala Pro Gly Cys Ile Ala Pro Gly Pro Leu Leu Asp         435 440 445 Pro Pro Met Lys Ala Val Pro Thr Val Ala Gly Ala Arg Phe Pro Leu     450 455 460 Phe His Phe Lys Pro Ser Pro Pro His Pro Pro Ala Pro Ser Pro Ala 465 470 475 480 Gly Gly His His Leu Gly Tyr Asp Pro Thr Ala Ala Ala Leu Ser                 485 490 495 Leu Pro Leu Gly Ala Ala Ala Ala             500 505 510 Glu Ser His Pro Tyr Gly Leu Pro Leu Ala Lys Arg Ala Ala Pro Leu         515 520 525 Ala Phe Pro Pro Leu Gly Leu Thr Pro Ser Pro Thr Ala Ser Seru     530 535 540 Leu Gly Glu Ser Pro Ser Leu Pro Ser Pro Pro Ser Ser Ser Ser Ser 545 550 555 560 Ser Gly Glu Gly Thr Cys Ala Val Cys Gly Asp Asn Ala Ala Cys Gln                 565 570 575 His Tyr Gly Val Arg Thr Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg             580 585 590 Thr Val Gln Lys Asn Ala Lys Tyr Val Cys Leu Ala Asn Lys Asn Cys         595 600 605 Pro Val Asp Lys Arg Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln     610 615 620 Lys Cys Leu Ser Val Gly Met Val Lys Glu Val Val Arg Thr Asp Ser 625 630 635 640 Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Leu                 645 650 655 Gln Gln Glu Pro Ser Gln Pro Ser Pro Pro Ser Pro Pro Ile Cys Met             660 665 670 Met Asn Ala Leu Val Arg Ala Leu Thr Asp Ser Thr Pro Arg Asp Leu         675 680 685 Asp Tyr Ser Arg Tyr Cys Pro Thr Asp Gln Ala Ala Gly Thr Asp     690 695 700 Ala Glu His Val Gln Gln Phe Tyr Asn Leu Leu Thr Ala Ser Ile Asp 705 710 715 720 Val Ser Arg Ser Trp Ala Glu Lys Ile Pro Gly Phe Thr Asp Leu Pro                 725 730 735 Lys Glu Asp Gln Thr Leu Leu Ile Glu Ser Ala Phe Leu Glu Leu Phe             740 745 750 Val Leu Arg Leu Ser Ile Arg Ser Asn Thr Ala Glu Asp Lys Phe Val         755 760 765 Phe Cys Asn Gly Leu Val Leu His Arg Leu Gln Cys Leu Arg Gly Phe     770 775 780 Gly Glu Trp Leu Asp Ser Ile Lys Asp Phe Ser Leu Asn Leu Gln Ser 785 790 795 800 Leu Asn Leu Asp Ile Gln Ala Leu Ala Cys Leu Ser Ala Leu Ser Met                 805 810 815 Ile Thr Glu Arg His Gly Leu Lys Glu Pro Lys Arg Val Glu Glu Leu             820 825 830 Cys Asn Lys Ile Thr Ser Ser Leu Lys Asp His Gln Ser Lys Gly Gln         835 840 845 Ala Leu Glu Pro Thr Glu Ser Lys Val Leu Gly Ala Leu Val Glu Leu     850 855 860 Arg Lys Ile Cys Thr Leu Gly Leu Gln Arg Ile Phe Tyr Leu Lys Leu 865 870 875 880 Glu Asp Leu Val Ser Pro Pro Ser Ile Ile Asp Lys Leu Phe Leu Asp                 885 890 895 Thr Leu Pro Phe             900 <210> 8 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> TFG (NTD) protein <400> 8 Met Asn Gly Gln Leu Asp Leu Ser Gly Lys Leu Ile Val Lys Ala Gln   1 5 10 15 Leu Gly Glu Asp Ile Arg Arg Ile Pro Ile His Asn Glu Asp Ile Thr              20 25 30 Tyr Asp Glu Leu Val Leu Met Met Gln Arg Val Phe Arg Gly Lys Leu          35 40 45 Leu Ser Asn Asp Glu Val Thr Ile Lys Tyr Lys Asp Glu Asp Gly Asp      50 55 60 Leu Ile Thr Ile Phe Asp Ser Ser Asp Leu Ser Phe Ala Ile Gln Cys  65 70 75 80 Ser Arg Ile Leu Lys Leu Thr Leu Phe Val Asn Gly Gln Pro Arg Pro                  85 90 95 Leu Glu Ser Ser Gln Val Lys Tyr Leu Arg Arg Glu Leu Ile Glu Leu             100 105 110 Arg Asn Lys Val Asn Arg Leu Leu Asp Ser Leu Glu Pro Pro Gly Glu         115 120 125 Pro Gly Pro Ser Thr Asn Ile Pro Glu Asn Asp Thr Val Asp Gly Arg     130 135 140 Glu Glu Lys Ser Ala Ser Asp Ser Ser Gly Lys Gln Ser Thr Gln Val 145 150 155 160 Met Ala Ala Ser Met Ser Ala Phe Asp Pro Leu Lys Asn Gln Asp Glu                 165 170 175 Ile Asn Lys Asn Val Met Ser Ala Phe Gly Leu Thr Asp Asp Gln Val             180 185 190 Ser Gly Pro Ser Ala Pro Ala Glu Asp Arg Ser Gly Thr Pro Asp         195 200 205 Ser Ile Ala Ser Ser Ser Ala Ala His Pro Pro Gly Val Gln Pro     210 215 220 Gln Gln Pro Pro Tyr Thr Gly Ala Gln Thr Gln Ala Gly Gln Ile Glu 225 230 235 240 Gly Gln Met Tyr Gln Gln Tyr Gln Gln Gln Ala Gly Tyr Gly Ala Gln                 245 250 255 Gln Pro Gln Ala Pro Pro Gln Gln Pro Gln Gln Tyr Gly Ile Gln Tyr             260 265 270 Ser     <210> 9 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> TEC binding sequence <400> 9 aaaaggtca 9

Claims (22)

A method of screening for a substance for the prophylaxis or treatment of a tumor disease comprising the steps of:
(a) treating the test material with a cell comprising a TFG-encoding nucleotide sequence and a TEC-encoding nucleotide sequence; And
(b) analyzing the expression of the TFG-TEC fusion gene or the activity of the TFG-TEC fusion protein in the cell, wherein the test substance expresses the TFG-TEC fusion gene or the transcription activity of the TFG- Wherein the compound is judged to be a therapeutic agent for a tumor disease.
A method of screening for a substance for the prophylaxis or treatment of a tumor disease comprising the steps of:
(a) (i) a vector comprising a TFG-TEC fusion protein-encoding nucleotide sequence operably linked to a promoter; And (ii) treating the test substance with a vector comprising a reporter gene operably linked to a promoter that is positively regulated by the TFG-TEC fusion protein; And
(b) measuring the expression level of the reporter gene in the transformed host cell of step (a), wherein inhibition of the expression of the reporter gene is judged to be a therapeutic agent for a tumor disease Screening method.
The method according to claim 1 or 2, wherein the tumor disease is selected from the group consisting of extraskeletal myxoid chondrosarcoma (EMC), cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, Cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, Endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, sweat gland carcinoma, sebaceous gland carcinoma , Papillary adenocarcinoma, papillary adenocarcinoma, cystadenocarcinoma, soft tissue thyroid carcinoma, bronchial carcinoma, choriocarcinoma, testicular tumor, embryonal carcinoma, Wilm's tumor, astrocytoma, Kaposi's sarcoma, hematoblastoma, craniopharyngioma, A method for screening a substance for the prophylaxis or treatment of a tumor disease, which is characterized by being a tumor, a pineal gland, an angioblastoma, an auditory neoplasm, a pterygium glioma, a meningioma, a neuroblastoma, a retinoblastoma, a myeloma, a lymphoma or a leukemia.
The method according to claim 1, wherein the TFG-TEC fusion protein is formed by chromosomal translocation between a sixth intron of TFG and a second intron of TEC. .
3. The method according to claim 1 or 2, wherein the TFG-TEC fusion protein binds to a TEC consensus sequence.
6. The method according to claim 5, wherein the TEC consensus sequence is the sequence of SEQ ID NO: 9.
The method according to claim 1 or 2, wherein the TFG-TEC fusion protein is located at the nucleus.
3. The method according to claim 1 or 2, wherein TFG (NTD) in the TFG-TEC fusion protein functions as a transactivation domain.
9. The method of claim 8, wherein the PB1 domain and the SPYGQ-rich regions of the TFG (NTD) have partial transactivation. Way.
3. The method according to claim 1 or 2, wherein said test substance down-regulates the expression of Skp2 , L- Myc , SOCS2 and STAT-3 .
3. The method according to claim 2, wherein the reporter gene is a fluorescent protein,? -Galactosidase, chloramphenicol acetyltransferase, human growth hormone, urease or alkaline phosphatase. A method for screening a substance for preventing or treating.
(a) a promoter; (b) a TFG (NTD) -encoding nucleotide sequence of Sequence Listing 4 of the sequence operably linked to the promoter; (c) a transcription activator-encoding nucleotide sequence attached to the N-terminus or C-terminus of the TFG (NTD) -encoding nucleotide sequence; And (d) a terminator.
13. The expression vector for promoting transcriptional activity according to claim 12, wherein the transcription activator comprises a DNA-binding domain.
14. The method of claim 13, wherein the DNA-binding domain is selected from the group consisting of TEC, GAL4, GCN4, HAP1, ADR1, SWI5, STE12, MCM1, YAP1, ACE1, CUP2, XYR1, PPR1, ARG81, LAC9, QA1F, Wherein the expression vector is a DNA-binding domain of a protein.
A cell transformed with the expression vector of claim 12.
(a) (i) a promoter; And (ii) a transactivation moiety comprising a TFG (NTD) -encoding nucleotide sequence of Sequence Listing 4 of the Sequence Listing operatively linked to the promoter; And (b) an expression vector comprising a DNA-binding moiety comprising a transcription activator-encoding nucleotide sequence linked to the N-terminus or C-terminus of the TFG (NTD) Or a pharmaceutically acceptable salt thereof.
18. The composition for promoting transcriptional activity according to claim 16, wherein the composition increases the expression of a gene having a promoter comprising a conserved sequence bound by a DNA-binding domain in the transcriptional activator.
A pharmaceutical composition for preventing or treating tumor diseases, which comprises an RNAi (RNA interference) molecule that specifically binds to a TFG-TEC fusion gene, or a substance that inhibits the transcription activation promotion of said TFG-TEC fusion protein.
19. The pharmaceutical composition according to claim 18, wherein the RNAi molecule specifically binding to the TFG-TEC fusion gene is an antisense oligonucleotide, siRNA, shRNA or miRNA.
19. The pharmaceutical composition according to claim 18, wherein the TFG-TEC fusion protein up-regulates the expression of Skp2 , L- Myc , SOCS2 and STAT-3 .
19. The method according to claim 18, wherein the substance that inhibits the transcriptional activation promotion of the TFG-TEC fusion protein inhibits the binding of the TFG-TEC fusion protein to DNA-binding sequence motifs Gt;
19. The method of claim 18, wherein the tumor disease is selected from the group consisting of: EMC, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, colorectal adenocarcinoma, prostate carcinoma, colon cancer, breast cancer, , Squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, choroidal carcinoma, angiosarcoma, endothelial cell sarcoma, lymphatic sarcoma, The present invention relates to a method for the treatment and prophylaxis of vascular endothelial cell sarcoma, endometrioid carcinoma, mesenchymal tumor, mesenchymal tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, glandular carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, The present invention relates to a method of treating a tumor in a patient suffering from a cancer selected from the group consisting of a malignant tumor, a testicular tumor, a germ cell tumor, a germ cell tumor, a germ cell tumor, an astrocytoma, a Kaposi sarcoma, a hematoblastoma, a craniopharyngioma, In addition A pharmaceutical composition for the prevention and treatment of tumor diseases, characterized in that the leukemia.

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