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

Abstract

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. Stem Cells 2005;23:738-751

POU5F1, encoding a key regulator of stem cell pluripotency, is fused to EWSR1 in hidradenoma of the skin and mucoepidermoid carcinoma of the salivary glands

“…In fact, POU5F1 has been identified as an EWSR1-interacting partner [34]. In all our experiments, especially when using the NANOG promoter and POU5F1 enhancer constructs, POU5F1 wt 1 activated transcription more efficiently than POU5F1 wt 2 which is in agreement with previous findings [35].…”

Gene amplification and associated loss of 5′ regulatory sequences of CoAA in human cancers

“…CoAA protein is significantly expressed in mouse embryos, embryonic stem cells and all cancer cell lines analysed, but its expression is decreased during normal differentiation (unpublished observation); it thus remains to be studied if deregulation of CoAA might be involved in the differentiation of cancer progenitor cells . Consistent with this notion, EWS has recently been reported to regulate stem cell differentiation (Lee et al, 2005). Therefore, regulation of alternative splicing might be an important issue in differentiation as well as cancer development.…”

EWSR1 regulates mitosis by dynamically influencing microtubule acetylation

“…10 For example, several studies reported the participation of EWSR1 in transcriptional regulation through its association with the general transcription factor TFIID and the tissue-specific transcription factors Oct4 and Brn3a. [11][12][13] The association of EWSR1 with YB-1, a shuttling splicing activator, demonstrates the involvement of EWSR1 in splicing through its C-terminal domain. 14 Some lines of evidence, such as the interaction of EWSR1 with Drosha, also suggest a relationship between EWSR1 and micro-RNA processing.…”