Mutant screening for oncogenes of Ewing's sarcoma using yeast.
Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.
Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan, takao-k@yamaguchi-u.ac.jp.
Takao
T
Kitagawa
Hajime
H
Okita
Byron
B
Baron
Kazuhiro
K
Tokuda
Mikiko
M
Nakamura
Yufeng
Y
Wang
Junko
J
Akada
Hisashi
H
Hoshida
Rinji
R
Akada
Yasuhiro
Y
Kuramitsu
Kazuyuki
K
Nakamura
D017500:Adenovirus E1A Proteins / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012512:Sarcoma, Ewing / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D000071230:Transcriptional Regulator ERG; D015003:Yeasts / Q000235:genetics
D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; C072312:ERG protein, human; C079853:ETV4 protein, human; C078433:FLI1 protein, human; D050505:Mutant Proteins; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG