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.
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.
Kikuchi
R
R
Nagoya University Graduate School of Medicine, Nagoya University School of Health Sciences, Nagoya, Aichi, Japan.
Murakami
M
M
Sobue
S
S
Iwasaki
T
T
Hagiwara
K
K
Takagi
A
A
Kojima
T
T
Asano
H
H
Suzuki
M
M
Banno
Y
Y
Nozawa
Y
Y
Murate
T
T
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D047468:Immunoprecipitation; D008840:Microfilament Proteins / Q000378:metabolism / Q000502:physiology*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism / Q000502:physiology*; D010739:Phospholipase D / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051789:Proto-Oncogene Protein c-fli-1 / Q000378:metabolism / Q000502:physiology*; D034802:RNA-Binding Protein EWS; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators; D014162:Transfection
D017931:DNA Primers; C106031:EWS-FLI fusion protein; C098179:FLII protein, human; D008840:Microfilament Proteins; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1