Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-02-10
2001-02-27
Pak, Michael (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C424S185100
Reexamination Certificate
active
06194547
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, inter alia, to the ERF gene and to the products encoded by this gene. More particularly, the present invention relates to DNA sequences encoding ERF and AERF; polypeptides encoded by such DNA sequences; ERF chimeric molecules; and methods of using ERF and ERF chimeric molecules to reduce tumorigenicity in a tumor cell.
BACKGROUND OF THE INVENTION
Transcriptional regulators in eukaryotic cells are organized into families of genes characterized by their DNA-binding domain and DNA sequence specificity (homeo domain, b-ZIP, HLH, ets domain, Rel homology region, etc.) (see, e.g., Pabo, et al.,
Annu. Rev. Biochem
., 61:1015-1023 (1992)). Although some functional redundancy exists, the specific function of each member of a given family is defined by a configuration of factors involving tissue-specificity and cell cycle regulation, response to extracellular signals, level of expression, interacting proteins, target specificity, kinetics of interaction and effect on transcription (positive or negative). However, the members of a family are usually involved in a common function (e.g., homeo genes and development, AP1 genes and mitogenic stimulation, etc.). The ets family of genes recognize a common target sequence and have been implicated in cellular proliferation and tumorigenesis (see, e.g., Seth, et al.,
Cell Growth Differ
, 3:327-334 (1992); Macleod, et al.,
TIBS
, 17:251-256 (1992); Wasylyk, et al.,
Eur. J. Biochem
., 211:8-19 (1993); and Janknecht, et al.,
Biochim. Biophys. Acta
, 1155:346-356 (1993)). Several growth-related genes contain a functional ets-binding site (EBS) in their regulatory region, including c-fos (for review see Treisman, et al.,
Curr. Opinion Genet. Dev
., 4:96-101 (1994); Karin, et al.,
Curr. Opinion Cell Biol
., 6:415-424 (1994)), Jun-B (Coffer, et al.,
Oncogene
, 9:911-921 (1994)), Rb (Savoysky, et al.,
Oncogene
, 9:1839-1846 (1994)), c-MYC (Roussel, et al.,
Oncogene
, 9:405-415 (1994)) and ETS2 (Mavrothalassitis, et al.,
Cell Growth Differ
, 2:215-224 (1991)), indicating that members of the ets family may play a key role during mitogenic stimulation by regulating the transcription of these genes. Furthermore, recent reports indicate that the activity of some ets family members can be regulated by the ras/MAPK pathway (Janknecht, et al.,
EMBO
, 12:5097-5104 (1993); Rao, et al.,
Oncogene
, 9:1855-1860 (1994); O'Neill, et al.,
Cell
, 78:137-147 (1994); Brunner, et al.,
Nature
, 370:386-389 (1994) and, thus, provide a possible mechanism for the coordinated regulation of the function of some ets genes during mitogenic stimulation.
In an efforts to analyze regulation of the ETS2 gene, its promoter was isolated and characterized (Mavrothalassitis, et al.,
Oncogene
, 5:1337-1342 (1990), Mavrothalassitis, et al.,
Proc. Natl. Sci. USA
, 87:1047-1051 (1990)), and sites where protein interaction is required for promoter function were identified (Mavrothalassitis, et al.,
Cell Growth Differ
., 2:215-224 (1991)). One of the DNA-protein interaction sites on the ETS2 promoter, designated H1, is an ets-binding site, thus suggesting a regulatory loop among the members of the ets family. However, neither ETS1 nor ETS2 are capable of regulating ETS2 transcription via this site, and they both have a low affinity for this particular sequence, indicating that other members of the family are responsible for the transcriptional regulation of the ETS2 gene.
As such, there remains a need in the art for the identification of the gene responsible for the transcriptional regulation of the ETS2 gene.
SUMMARY OF THE INVENTION
It has now been discovered that ERF (ETS2 Repressor Factor), a novel member of the ets family of genes, is the gene responsible for the transcriptional regulation of the ETS2 gene. Moreover, it has now been discovered that ERF can regulate the transcription of other genes having ets-binding sites. As such, the ERF gene can be used in vivo and in vitro to suppress or repress transcription and to elucidate transcription process and regulation. ERF is the first member of the ets family to be identified as a transcriptional repressor in mammalian cells. ERF has no significant homology to the only other known repressor member of the ets family, i.e., the Drosophila gene Yan, or to other transcriptional repressors. In addition to being a transcrptional repressor, it has also been discovered that the ERF gene possesses tumor suppressor activity and, thus, ERF can be used to suppress ets-dependent tumorigenicity as well as tumorigenicity associated with the inappropriate expression of transcription factors.
As such, in one aspect, the present invention provides isolated nucleic acid sequences, i.e., polynucleotides, which encode a family of proteins. More particularly, the present invention provides a DNA sequence (SEQ ID NO:1) which is transcribed into an mRNA of about 2.7 kb which encodes a human ERF protein having an amino acid sequence comprising SEQ ID NO: 3. Moreover, the present invention provides an isolated DNA sequence (SEQ ID NO:4) which is transcribed into an mRNA of about 2.5 kb which encodes a human AERF protein having an amino acid sequence comprising SEQ ID NO: 4. In addition, the present invention provides an isolated DNA sequence encoding a murine ERF protein having an amino acid comprising SEQ ID NO: 7.
In another aspect, the present invention provides isolated, substantially purified ERF and AERF proteins. More particularly, the present invention provides an isolated, substantially purified human ERF protein encoded by an mRNA of about 2.7 kb and having an amino acid sequence comprising SEQ ID NO: 1. Additionally, the present invention provides an isolated, substantially purified human AERF protein encoded by an mRNA of about 2.5 kb and having an amino acid sequence comprising SEQ ID NO: 4. Moreover, the present invention provides an isolated, substantially purified murine ERF protein having an amino acid sequence comprising SEQ ID NO: 7.
In yet another aspect, the present invention provides an ERF chimeric molecule, the ERF chimeric molecule comprising an ERF repressor domain in combination with a heterologous transcription factor having a binding domain. It has been discovered that ERF contains an active repressor domain located between amino acids 472 and 530, and corresponding to SEQ ID NO: 5. The ERF repressor domain does not contain any recognizable features reported for other transcriptional repressors and has no homology to previously reported repressor genes. Moreover, it has been discovered that the ERF repressor domain can be effectively transferred to transcription factors having a binding domain (e.g., GAL4, NF&kgr;B (p50 and p65), MYC, Fli-1, EST1, etc.) to generate transcriptional repressors. As such, the present invention provides ERF chimeric molecules which are effective transcriptional repressors.
In still another embodiment, the present invention provides a method for reducing ets-dependent tumorigenicity, the method comprising: transfecting a tumor cell with an ERF gene. Moreover, the present invention provides a method for reducing ets-dependent tumorigenicity, the method comprising contacting a tumor cell with a peptide expressed by an ERF gene. In addition, the present invention provides a method for reducing cell tumorigenicity associated with the inappropriate expression of a transcription factor, the method comprising contacting a tumor cell with an ERF chimeric molecule, the ERF chimeric molecule comprising an ERF repressor domain in combination with the transcription factor having a binding domain. Using these methods, one can reduce tumorigenicity associated with, inter alia, the v-mos, c-met, tpr-met, Ha-ras and gag-myb-ets oncogenes or, alternatively, one can reduce tumorigenicity associated with, inter alia, the inappropriate expression of the GAL4, NF&kgr;B (HIV), MYC (Burkitt Lymphoma), Fli-1 (Ewing's Sarcoma) and EST1 transcription factors.
Other advantages, objects, features and embodiments of the present invention will become apparent
Athanasiou Meropi A.
Beal, Jr. Gregory J.
Blair Donald G.
Fisher Robert J.
Mavrothalassitis George J.
Pak Michael
The United States of America as represented by the Department of
Townsend and Townsend / and Crew LLP
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