Stimulation of Oct‐4 Activity by Ewing's Sarcoma Protein

• Published in: Stem cells (Dayton, Ohio) Ohio), 2005-06, Vol.23 (6), p.738-751
• Main Authors: Lee, Jungwoon, Rhee, Byung Kirl, Bae, Gab‐Yong, Han, Yong‐Mahn, Kim, Jungho
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Animals; Bacterial two‐hybrid screening; Blotting, Northern; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Nucleus - metabolism; COS Cells; DNA, Complementary - metabolism; DNA-Binding Proteins - biosynthesis; Embryo, Mammalian - cytology; Embryonic stem cells; Ewing's sarcoma protein; Gene Expression Regulation, Neoplastic; Gene Library; Genes, Reporter; Glutathione Transferase - metabolism; Histidine - chemistry; Humans; Immunoprecipitation; Mice; NIH 3T3 Cells; Octamer Transcription Factor-3; Oct‐4; Promoter Regions, Genetic; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; Protein—protein interaction; Proto‐oncogene; RNA - metabolism; RNA-Binding Protein EWS - metabolism; RNA-Binding Protein EWS - physiology; Stem Cells - cytology; Transcription Factors - biosynthesis; Transcription, Genetic; Transcriptional Activation; Transcriptional coactivator; Two-Hybrid System Techniques
• ISSN: 1066-5099; 1549-4918
• DOI: 10.1634/stemcells.2004-0375

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.