Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter

R. Kikuchi, M. Murakami, S. Sobue, T. Iwasaki, K. Hagiwara, A. Takagi, T. Kojima, H. Asano, M. Suzuki, Y. Banno, Y. Nozawa, T. Murate

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Abstract

It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5′ promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

Original languageEnglish
Pages (from-to)1802-1810
Number of pages9
JournalOncogene
Volume26
Issue number12
DOIs
Publication statusPublished - 15-03-2007

Fingerprint

Ewing's Sarcoma
Gene Expression
Proteins
Phospholipase D
Cell Line
Small Interfering RNA
ETS Motif
Erythroblasts
1-Butanol
Transcription Initiation Site
Gene Expression Regulation
Electrophoretic Mobility Shift Assay
Growth
Electrophoresis
Transcription Factors
Down-Regulation
Cell Proliferation
Messenger RNA
DNA

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Cancer Research

Cite this

Kikuchi, R. ; Murakami, M. ; Sobue, S. ; Iwasaki, T. ; Hagiwara, K. ; Takagi, A. ; Kojima, T. ; Asano, H. ; Suzuki, M. ; Banno, Y. ; Nozawa, Y. ; Murate, T. / Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter. In: Oncogene. 2007 ; Vol. 26, No. 12. pp. 1802-1810.
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title = "Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter",
abstract = "It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5′ promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.",
author = "R. Kikuchi and M. Murakami and S. Sobue and T. Iwasaki and K. Hagiwara and A. Takagi and T. Kojima and H. Asano and M. Suzuki and Y. Banno and Y. Nozawa and T. Murate",
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Kikuchi, R, Murakami, M, Sobue, S, Iwasaki, T, Hagiwara, K, Takagi, A, Kojima, T, Asano, H, Suzuki, M, Banno, Y, Nozawa, Y & Murate, T 2007, 'Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter', Oncogene, vol. 26, no. 12, pp. 1802-1810. https://doi.org/10.1038/sj.onc.1209973

Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter. / Kikuchi, R.; Murakami, M.; Sobue, S.; Iwasaki, T.; Hagiwara, K.; Takagi, A.; Kojima, T.; Asano, H.; Suzuki, M.; Banno, Y.; Nozawa, Y.; Murate, T.

In: Oncogene, Vol. 26, No. 12, 15.03.2007, p. 1802-1810.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5′ promoter

AU - Kikuchi, R.

AU - Murakami, M.

AU - Sobue, S.

AU - Iwasaki, T.

AU - Hagiwara, K.

AU - Takagi, A.

AU - Kojima, T.

AU - Asano, H.

AU - Suzuki, M.

AU - Banno, Y.

AU - Nozawa, Y.

AU - Murate, T.

PY - 2007/3/15

Y1 - 2007/3/15

N2 - It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5′ promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

AB - It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5′ promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

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