The self-renewal function of Oct-4 can be replaced by the EWS-Oct-4 fusion protein in embryonic stem cells.

Octamer-binding transcription factor 4 (Oct-4) is essential for maintenance and pluripotency of embryonic stem (ES) cells. Despite the structural similarities between Oct-4 and its homologs (Oct-1, Oct-2, and Oct-6), these homologs cannot serve as substitutes for Oct-4 when generating stem cell colonies. While nuclear receptor subfamily 5, group A, member 2 (Nr5a2) can temporarily serve as a substitute for Oct-4 during cellular reprogramming, it is insufficient to maintain these functions in ES cells. The EWS-Oct-4 fusion protein, which was identified in human tumors, is a viable alternative that can potentially sustain and enhance ES cell functions. This study used ZHBTc4 ES cells, which have tetracycline-regulated Oct-4 expression, to explore the capabilities of EWS-Oct-4. It employed a variety of assays, including western blotting, immunocytochemistry, RT-PCR, luciferase reporter assays, flow cytometry, and teratoma formation assays. EWS-Oct-4 preserved the self-renewal capacity of Oct-4-null ES cells, as demonstrated by their undifferentiated morphology and increased expression of pluripotency markers such as Sox2, Nanog, and SSEA-1. It also boosted cell proliferation and influenced cell cycle dynamics by downregulating p21 and upregulating Oct-4 target genes, including Rex-1 and fibroblast growth factor-4. Epithelial markers were upregulated and mesenchymal markers were downregulated, suggesting a shift toward an epithelial phenotype. Prominent teratoma formation further confirmed the functionality of EWS-Oct-4 in vivo. The integrity and specific functional domains of EWS-Oct-4 were critical for these effects. Finally, comparative transcriptomic analysis revealed that ES cells expressing EWS-Oct-4 and those expressing Oct-4 had highly similar global gene expression profiles, with distinct variations in differentially expressed genes. These findings indicate that EWS-Oct-4 can effectively replace Oct-4, which has significant implications for advancements in stem cell research and regenerative medicine.