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Mutant screening for oncogenes of Ewing’s sarcoma using yeast

  • Applied genetics and molecular biotechnology
  • Published:
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Abstract

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.

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Acknowledgments

We thank Yukie Misumi for their technical support. The authors would like to acknowledge the utilization of the LAS-1000 and a gift of pGL4.74[hRluc/TK] vector and HEK293 cells from the DNA Core Facility of the Center for Gene Research, Yamaguchi University, supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.

Conflict of interest

The authors declare that they have no competing financial interests.

Author information

Author notes

  1. Hajime Okita

    Present address: Department of Pathology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

Authors and Affiliations

  1. Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan

    Takao Kitagawa, Byron Baron, Kazuhiro Tokuda, Yufeng Wang, Junko Akada & Yasuhiro Kuramitsu

  2. Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo, 157-8535, Japan

    Hajime Okita

  3. Innovative center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan

    Mikiko Nakamura

  4. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan

    Hisashi Hoshida & Rinji Akada

  5. Center of Clinical Laboratories in Tokuyama Medical Association Hospital, Shunan, 745-0846, Japan

    Kazuyuki Nakamura

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  1. Takao Kitagawa
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Correspondence to Takao Kitagawa.

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Kitagawa, T., Okita, H., Baron, B. et al. Mutant screening for oncogenes of Ewing’s sarcoma using yeast. Appl Microbiol Biotechnol 99, 6737–6744 (2015). https://doi.org/10.1007/s00253-015-6621-2

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  • DOI: https://doi.org/10.1007/s00253-015-6621-2

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