AbstractThe EWS‐Oct‐4 protein is a chimeric molecule in which the amino terminal domain (NTD) of the EWS becomes fused to the carboxy terminal domain (CTD) of the Oct‐4 transcription factor. It was identified in human bone and soft‐tissue tumors associated with t(6;22)(p21;q12). Using in vitro and in vivo systems, we found that the EWS‐Oct‐4 protein self‐associates. The major domains required for self‐association mapped to the EWS NTD (amino acids 70–163) and the POU DNA‐binding domain. EWS‐Oct‐4 protein also associated with EWS‐Oct‐4 (V351P), which contains a mutation in the POU DNA‐binding domain. Using electrophoretic mobility shift assays, we found that the EWS‐Oct‐4 (V351P) mutant interfered with wild‐type EWS‐Oct‐4 DNA‐binding activity. In addition, we found that EWS‐Oct‐4‐mediated transcriptional activation was inhibited by EWS‐Oct‐4 (V351P) protein in vivo. Thus, this mutation in the POU DNA‐binding domain results in a dominant negative protein. These findings suggest that the biological functions of the EWS‐Oct‐4 oncogene can be modulated by the dominant negative mutant EWS‐Oct‐4 (V351P). © 2008 Wiley‐Liss, Inc.