Mutation in the DNA-binding domain of the EWS-Oct-4 oncogene results in dominant negative activity that interferes with EWS-Oct-4-mediated transactivation.
The 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).
Kim
Sol
S
Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul, Korea.
Lee
Jungwoon
J
Kim
Ja Young
JY
Lim
Bobae
B
Shin
Eung-Kyun
EK
Han
Yong-Mahn
YM
Kim
Sung-Su
SS
Song
Jin-Ho
JH
Kim
Jungho
J
D001483:Base Sequence; D002892:Chromosomes, Human, Pair 22; D002896:Chromosomes, Human, Pair 6; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005799:Genes, Dominant; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000502:physiology; D009857:Oncogenes; D034802:RNA-Binding Protein EWS / Q000235:genetics* / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*
D017931:DNA Primers; D050814:Octamer Transcription Factor-3; D034802:RNA-Binding Protein EWS; D004247:DNA