Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-10-27
2004-02-10
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
Reexamination Certificate
active
06689584
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel transcriptional regulatory factor comprising bromodomains and the encoding gene.
BACKGROUND OF THE INVENTION
The bromodomain is a characteristic amino-acid motif seen in transcriptional regulatory factors and is believed to be involved in the interactions with other transcriptional regulatory factors. Proteins comprising the bromodomain, normally have one or two (Tamkun et al. (1992) Nuc. Acids Res. 20:2603; Haynes et al. (1992) Nuc. Acids Res. 20: 2603), but as many as five (Nicolas et al. (1996) Gene 175(12):233-240) bromodomain motifs. This motif has been identified in a wide range of animals, for example, in the homeotic gene (Digan et al. (1986) Dev. Biol. 114:161-169; Tamkun et al. (1992) Cell 68: 561-572) of the fruit fly (Drosophila), in the transcriptional regulatory genes of yeasts (Winston et al. (1987) Genetics 115:649-656; Laurent et al. (1991) Proc. Nat. Acad. Sci. USA 88:2687-2691) and in mammals (Denis et al. (1996) Genes and Devel. 10:261-271; Yang et al. (1996) Nature 382:319-324). According to a recent report (Jeanmougin et al. (1997) Trends Biochem. Sci. 22:151-153), 37 bromodomain genes, including 13 human genes are recorded in the database. In addition to the bromodomain motif of amino acid residues 59-63, the sequences adjacent to the motif are also structurally conserved, and furthermore, 4 &agr;-helixes (Z, A, B, and C) are reported to be coded within the long 110 amino acids.
When these bromodomain-comprising transcriptional regulatory factors are compared, they all regulate signal-dependent transcription in actively proliferating cells (Tamkun et al. (1992) Cell 68:561-572; Haynes et al. (1992) Nuc. Acids Res. 20:2603). This characteristic implies that oncogenesis may occur when a gene encoding a bromodomain-containing protein undergoes abnormal regulation. In reality, six bromodomain genes have been experimentally proven to associate with oncogenesis. Three of these genes HRX/ALL-1 (Tkachuk et al. (1992) Cell 71:691-700\; Gu et al. (1992) Cell 71:701-708); TIF1 (Miki et al. (1991) Proc. Nat. Acad. Sci. USA 88:5167-5171; Le Douarin et al. (1995) EMBO J. 14:2020-2033) and CBP (Borrow et al. (1996) Nature Genet. 14:33-41) are linked with the gene cleavage points in leukemia. All three of these proteins contain the C4HC3 (also called PHD/LAP/TRX) zinc-finger (Aasland et al. (1995) Trends Biochem. Sci. 20:56-59; Koken et al. (1995) CR Acad. Sci. III, 318:733-739; Saha et al. (1995) Proc. Nat. Acad. Sci. USA 92:9737-9741). Also, there are findings that CBP/P300 interact with p53 (Gu et al. (1997) Nature 387:819-823; Lill et al.(1997) Nature 387:823-827) and other various transcriptional factors, suggesting that CBP and the homologous gene P300 play a key-role in cancer.
The other three genes have been suggested to be linked with cancer in various ways. BRG1 interacts with retinoblastoma protein RB (Dunaief et al. (1994) Cell 79:119-130), inducing formation of flat, growth-arrested cells, and thereby showing a tumor-suppressive activity. RING3 has a homology with the fruit fly (Drosophila) growth control protein fsh (Haynes et al. (1989) Dev. Biol. 134:246-257) and is a serine-threonine kinase having endonuclear autophosphorylation activity. This activity has been reported to be linked to the growth phase of chronic and acute lymphocytic leukemia (Denis et al. (1996) Genes and Devel. 10:261-271). As for P/CAF, it has been reported to inhibit the interaction between E1A and p300/CBP (Yang et al. (1996) Nature 382:319-324). When P/CAF is exogenously expressed on HeLa cells, the cell cycle is inhibited. This is believed to be due to the disruption of the transcriptional regulation of E1A by the binding of P/CAF to p300/CBP. Similar to p300/CBP (Bannister and Kouzarides (1996) Nature 384:641-643), P/CAF has been reported to contain histone acetyl-transferase activity (Yang et al. (1996) Nature 382:319-324).
Thus, regulatory abnormalities of transcriptional regulatory factors comprising bromodomains are envisaged to be closely associated with various diseases, particularly, cancer and other cell-proliferation-linked diseases. Hence, attention has been focused on transcriptional regulatory factors comprising bromodomains in the recent years as novel targets for the treatment of cancer and other cell-proliferation-linked diseases.
SUMMARY OF THE INVENTION
The present invention provides a novel transcriptional regulatory factor comprising bromodomains, the encoding gene, a method of production, and a screening method for a drug-candidate compound that utilizes the protein and the gene of the present invention.
In order to solve the above-mentioned problems, EST databases were BLAST searched using various nucleotide sequences encoding known bromodomain motifs. As a result, several potential bromodomain-gene-encoding ESTs were found by the search using nucleotide sequence of
Tetrahymena thermophila
HAT A1 gene. One of these ESTs, the fetal lung cDNA library-derived EST (W17142) was found to encode an unknown gene. Therefore, isolation of full-length cDNA of EST W17142 was initiated. Specifically, primers were designed based on the EST W17142 sequence, and an amplification product was obtained by the polymerase chain reaction using testicular cDNA as the template. Then, the testicular cDNA library was screened using this amplification product as the probe, and a re-screening of the library was done using the cDNA clone comprising the above-mentioned EST sequence, thereby successfully isolating a full-length cDNA corresponding to EST W17142. By structural analysis of the protein encoded by the isolated cDNA, the present Inventors found that apart from the bromodomain, said protein had several regions and domains conserved in transcriptional regulatory factors.
Also, they found that the protein encoded by the isolated cDNA interacts with hSNF2H and hSNF2L that are implicated in the series of processes related to the chromatin-mediated transcriptional regulatory mechanism, and also with the transcription co-activator NcoA-62/Skip, which interacts with the ligand-binding domains of various nuclear receptors (VDR, RAR) and the Ski viral oncoprotein.
The transcriptional regulatory factor and the encoding gene revealed by the Inventors can be utilized for the screening of compounds inhibiting the binding between said transcriptional regulatory factor and an interacting factor, and compounds which regulate the binding activity. The compounds thus isolated are expected to be applied as pharmaceuticals.
Namely, the present invention relates to a novel transcriptional regulatory factor comprising a bromodomain and the encoding gene, as well as methods of production, and a screening method for related-factors and drug-candidate compounds that utilize the protein and the gene of the present invention. Specifically, the present invention relates to:
1. a protein comprising the amino acid sequence of SEQ ID NO:1 or 10;
2. a transcriptional regulatory factor comprising a bromodomain and the amino acid sequence of SEQ ID NO:1 or 10, wherein one or more amino acids are replaced, deleted, added, and/or inserted;
3. a protein comprising the amino acid sequence of SEQ ID NO:1 or 10 wherein one or more amino acids are replaced, deleted, added, and/or inserted, and having an activity to bind to a protein selected from the group consisting of hSNF2H,hSNF2L,NCoA-62/Skip and homologues thereof;
4. a transcriptional regulatory factor comprising a bromodomain, and encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9;
5. a transcriptional regulatory factor encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9, and having an activity to bind to a protein selected from the group consisting of hSNF2H, hSNF2L,NCoA-62/Skip and homologues thereof;
6. a DNA encoding the transcriptional regulatory factor of any one of (1) to (5);
7. the DNA of (6), which contains the coding region of the nucleotide sequence of SEQ ID NO:2 or 9;
8. a vector containing the DNA of
Carlson Karen Cochrane
Chugai Seiyaku Kabushiki Kaisha
Fish & Richardson P.C.
Snedden Sheridan
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