Stimulation of Oct-4 activity by Ewing's sarcoma protein.
The Oct-4 gene encodes a transcription factor that is expressed in embryonic stem (ES) cells and germ cells. Oct-4 is known to function as a transcriptional activator of genes involved in maintaining an undifferentiated totipotent state and possibly in preventing expression of genes activated during differentiation. In addition, it is a putative proto-oncogene and a critical player in the genesis of human testicular germ cell tumors. Although much effort has gone toward characterizing Oct-4, there is still little known about the molecular mechanisms and the proteins that regulate Oct-4 function. To identify cofactors that control Oct-4 function in vivo, we used a recently developed bacterial two-hybrid screening system and isolated a novel ES cell-derived cDNA encoding Ewing's sarcoma protein (EWS). EWS is a proto-oncogene and putative RNA-binding protein involved in human cancers. By using glutathione-S-transferase (GST) pull-down assays, we were able to confirm the interaction between Oct-4 and EWS in vitro, and moreover, coimmunoprecipitation and colocalization studies have shown that these proteins also associate in vivo. We have mapped the EWS-interacting region to the POU domain of Oct-4. In addition, three independent sites on EWS are involved in binding to Oct-4. In this study, we report that Oct-4 and EWS are coexpressed in the pluripotent mouse and human ES cells. Consistent with its ability to bind to and colocalize with Oct-4, ectopic expression of EWS enhances the transactivation ability of Oct-4. Moreover, a chimeric protein generated by fusion of EWS (1-295) to the GAL4 DNA-binding domain significantly increases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of a strong activation domain within EWS. Taken together, our results suggest that Oct-4-mediated transactivation is stimulated by EWS.
Lee
Jungwoon
J
Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul 121-742, Korea.
Rhee
Byung Kirl
BK
Bae
Gab-Yong
GY
Han
Yong-Mahn
YM
Kim
Jungho
J
D020816:Amino Acid Motifs; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004622:Embryo, Mammalian / Q000166:cytology; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D041681:NIH 3T3 Cells; D050814:Octamer Transcription Factor-3; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D012313:RNA / Q000378:metabolism; D034802:RNA-Binding Protein EWS / Q000378:metabolism / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C000715470:MAS1 protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C494174:Pou5f1 protein, mouse; D000090063:Proto-Oncogene Mas; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors; D006639:Histidine; D012313:RNA; D005982:Glutathione Transferase