Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-10-15
2003-01-07
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06504009
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel transcriptional regulator containing a bromodomain and a gene encoding it.
BACKGROUND ART
The bromodomain is a characteristic motif of amino acids found in transcriptional regulators, and is believed to be involved in interaction with other proteins, such as other transcriptional regulators. Proteins having a bromodomain usually contain one or two (Tamkun, J. W. et al., (1992) Cell, 68:561-572, Haynes, S. R. et al., (1992) Nuc. Acids Res., 20:2603), but sometimes as many as five bromodomain motifs (Nicolas, R. H. and Goodwin, G. H. (1996), Gene, 175(12):233-240). This motif is found in a wide variety of animals. For example, it is identified in yeast (Winston, F. et al., (1987), Genetics, 115:649-656; Laurent, B. C. et al., (1991), Proc. Natl. Acad. Sci. USA, 25 88:2687-2691), Drosophila (Digan, M. E. et al., (1986), Dev. Biol., 114:161-169; Tamkun, J. W. et al., (1992), Cell, 68:561-572), and mammals (Denis, G. V. and Green, M. R. (1996), Genes and Devel., 10:261-271; Yang, X. J. et al., (1996), Nature, 382:319-324).
All transcriptional regulators containing a bromodomain serve to control signal-dependent transcription in actively proliferating cells (Tamkun, J. W. et al., (1992), Cell, 68:561-572; Haynes, S. R. et al., (1992), Nuc. Acids Res., 20:2603). Due to this feature, it is suggested that cancer may develop if the gene for the protein containing a bromodomain is not normally controlled. In fact, several studies have shown that human transcriptional regulators with a bromodomain RING3, p300/CBP, and PCAF may be involved in oncogenesis.
RING3 was identified during an extensive analysis of the sequences of human class II major histocompatibility systems (Beck et al., (1992) DNA Seq. 2:203-210). The protein encoded by RING3 is homologous to D26362, a human gene (Nomura et al., (1994) DNA Res. 1:223-229) and fsh, a drosophila gene (Digan et al., (1986), Dev. Biol., 114: 161-169). All three genes encode proteins contain two copies of a bromodomain and a PEST sequences. The bromodomain is a motif consisting of 59 to 63 amino acid residues and is considered to be involved in protein-protein interactions. It is found among the transcriptional regulator proteins (Tamkun, J. W. et al., (1992) Cell, 68:561-572; Haynes, S. R. et al., (1992) Nuc. Acids Res. 20:2603). The PEST sequence is a cluster of proline (P), glutamic acid (E), serine (S) and threonine (T), which characterizes the proteins that undergo rapid proteolysis in the cell.
The protein encoded by RING3 has a molecular weight of 90 kD and has serine-threonine activity (Denis and Green, (1996) Genes and Devel. 10:261-271). Comparison of the sequences of RING3 and fsh with those of kinase domains of known serine-threonine kinases revealed that the sub-domains of the kinase motif are conserved, though in no particular order (most of them are similar to the corresponding sub-domains of a proto-oncogene c-mos). Kinase activity of RING3 is stimulated by interleukin-1 (IL-1) and forskolin, but not by a certain category of cytokines (Denis and Green, (1996) Genes and Devel. 10:261-271). A close relationship between kinase activity and growth phase in chronic and acute lymphocytic leukemia suggests the role RING3 plays in the leukemogenesis regulatory pathway (Denis and Green, (1996) Genes and Devel. 10:261-271). The analysis of the drosophila fsh gene suggested the interaction with the trithorax transcriptional regulator, a possible target for the putative phosphorylative activity of fsh (Digan et al., (1986) Dev. Biol. 114:161-169; Mozer and Dawid, (1989) Proc. Natl. Acad. Sci. USA 86:3738-3742). The triathorax gene and its homologue ALL-1 have a C4HC3 zinc finger, a motif commonly found among the genes present at leukemia breakpoints(Aasland et al., (1995) Trends Biochem. Sci. 20:56-59; Saha et al., (1995)
Proc. Natl. Acad. Sci. USA
92:9737-9741).
In addition to RING3, at least two other bromodomain proteins, p300/CBP and PCAF, are associated with oncogenesis. Although p300 protein and CBP protein are encoded by different genes, they are extremely closely related, and therefore, they are often called p300/CBP. Mutations in CBP are often found in familial and sporadic cancers. Mutations in CBP sometimes result in Rubinstein-Taybi syndrome, which causes patients to develop various malignant tumors (Petrij et al., (1995) Nature 376:348-51). Furthermore, CBP is fused with MOZ at t(8;6)(p11;p13) translocation (Borrow et al., (1996) Nature Genet. 14:33-41). This fusion protein possibly causes leukemogenesis by its aberrant acetyltransferase activity (Brownwell and Allis, (1996) Curr. Opin. Genet. Devel. 6:176-184). Mutation in p300 is found in sporadic colon and gastric cancers (Muraoka et al., (1996) Oncogene 12:1565-1569), and p300 has been suggested to be a gene for a tumor-suppressing factor located on chromosome 22q. Another fact that suggests the role of p300/CBP in cancer is that it interacts with the known oncogenes. For example, it is a co-activator of c-Myb (Dai, et al., (1996) Genes and Devel. 10:528-540) and c-Fos (Bannister and Kouzarides, (1996) Nature 384:641-643) transcriptional factors, to which the E1A protein of Adenovirus bind (Yang et al., (1996) Nature 382:319-324). The interaction between E1A and p300/CBP is inhibited by PCAF, a bromodomain protein.
Like p300/CBP, PCAF also has histone acetyltransferase activity. PCAF, when exogenously expressed, can reduce the proliferation associated with E1A in cultured cells (Yang et al., (1996) Nature 382:319-324). Therefore one of the first mechanisms of the activity of the E1A oncogene may be to inhibit the interaction between PCAF and p300.
Thus, it is thought that aberrant regulation of transcriptional regulators containing a bromodomain may be closely related to various diseases, for example, cancer. Transcriptional regulators containing a bromodomain have thus recently received much attention as novel targets for treating cancer.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a novel transcriptional regulator having a bromodomain and DNA encoding said transcriptional regulator. Another objective of the present invention is to provide a vector carrying said DNA, a transformant retaining said DNA, and a process for producing a recombinant protein by utilizing said transformant. A further objective of the present invention is to provide a method of screening a compound that binds to said transcriptional regulator.
To solve the problems described above, the inventors searched a database based on the sequence of RING3 transcriptional regulator with a bromodomain and found several EST sequences highly homologous to RING3. Primers were then prepared based on one of the EST sequences, and polymerase chain reaction was performed in a human testis cDNA library using the primers. As a result, a gene encoding a novel transcriptional regulator with a bromodomain was successfully isolated. By analyzing the expression of this gene, the inventors discovered that the gene is highly expressed in the testis cells. Moreover, the inventors found that a factor interacting with the transcriptional regulator, or a candidate pharmaceutical compound that regulates activity of the transcriptional regulator, can be screened by utilizing the transcriptional regulator and the gene encoding it.
Thus, the present invention relates to novel transcriptional regulators each having a bromodomain and the genes encoding them, and to a method of screening for a related factor or a candidate compound as a medicament using said proteins or genes, and more specifically relates to:
(1) a transcriptional regulator having a bromodomain, which comprises the amino acid sequence shown in SEQ ID NO:1, or said sequence wherein one or more amino acids are substituted, deleted, or added;
(2) a transcriptional regulator having a bromodomain, which is encoded by DNA hybridizing with DNA comprising the nucleotide sequence shown in SEQ ID NO:2;
(3) DNA coding for the transcriptional regulator according to (1) or (2);
(4) a vector
Carlson Karen Cochrane
Chugai Seiyaku Kabushiki Kaisha
Fish & Richardson PC
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