Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul 121-742, Korea.
Br J Cancer. 2010 Jan 19;102(2):436-46. doi: 10.1038/sj.bjc.6605516. Epub 2010 Jan 5.
Characterisation of EWS-Oct-4 translocation fusion product in bone and soft-tissue tumours revealed a chimeric gene resulting from an in-frame fusion between EWS (Ewing's sarcoma gene) exons 1-6 and Oct-4 exons 1-4. Recently, an alternative form of the fusion protein between the EWS and Oct-4 genes, named EWS-Oct-4B, was reported in two types of epithelial tumours, a hidradenoma of the skin and a mucoepidermoid carcinoma of the salivary glands. As the N-terminal and POU domains of the EWS-Oct-4 and EWS-Oct-4B proteins are not structurally identical, we decided to investigate the functional consequences of the EWS-Oct-4B fusion.
In this report, we have characterised the EWS-Oct-4B fusion protein. To investigate how the EWS-Oct-4B protein contributes to tumourigenesis in human cancers, we analysed its DNA-binding activity, subcellular localisation, transcriptional activation behaviour, and oncogenic properties.
We found that this new chimeric gene encodes a nuclear protein that binds DNA with the same sequence specificity as the parental Oct-4 protein or the fusion EWS-Oct-4 protein. We show that the nuclear localisation signal of EWS-Oct-4B is dependent on the POU DNA-binding domain, and we identified a cluster of basic amino acids, (269)RKRKR(273), in the POU domain that specifically mediates the nuclear localisation of EWS-Oct-4B. Comparison of the properties of EWS-Oct-4B and EWS-Oct-4 indicated that EWS-Oct-4B is a less-potent transcriptional activator of a reporter construct carrying the Oct-4-binding sites. Deletion analysis of the functional domains of EWS-Oct-4B revealed that the EWS N-terminal domain (NTD)(B), POU, and C-terminal domain (CTD) are necessary for its full transactivation potential. Despite its reduced activity as a transcriptional activator, EWS-Oct-4B regulated the expression of fgf-4 (fibroblast growth factor-4) and nanog, which are potent mitogens, as well as of Oct-4 downstream target genes, the promoters of which contain potential Oct-4-binding sites. Finally, ectopic expression of EWS-Oct-4B in Oct-4-null ZHBTc4 ES cells resulted in increased tumourigenic growth potential in nude mice.
These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS-Oct-4B chimeric protein, and that alternative fusion of the EWS amino terminal domain to the Oct-4 DNA-binding domain produces another transforming chimeric product in human epithelial tumours.
在骨和软组织肿瘤中对 EWS-Oct-4 易位融合产物的特征描述揭示了一种嵌合基因，它是由 EWS（尤因肉瘤基因）外显子 1-6 和 Oct-4 外显子 1-4 之间的框内融合产生的。最近，在两种上皮肿瘤，皮肤的汗腺瘤和唾液腺的黏液表皮样癌中，报告了 EWS 和 Oct-4 基因之间的另一种融合蛋白形式，称为 EWS-Oct-4B。由于 EWS-Oct-4 和 EWS-Oct-4B 蛋白的 N 端和 POU 结构域在结构上并不完全相同，因此我们决定研究 EWS-Oct-4B 融合的功能后果。
在本报告中，我们对 EWS-Oct-4B 融合蛋白进行了表征。为了研究 EWS-Oct-4B 蛋白如何促进人类癌症的肿瘤发生，我们分析了其 DNA 结合活性、亚细胞定位、转录激活行为和致癌特性。
我们发现这个新的嵌合基因编码一种核蛋白，它与亲本 Oct-4 蛋白或融合 EWS-Oct-4 蛋白具有相同的 DNA 结合序列特异性。我们表明，EWS-Oct-4B 的核定位信号依赖于 POU DNA 结合域，并且我们在 POU 结构域中鉴定出一个碱性氨基酸簇（269）RKRKR（273），该簇特异性介导 EWS-Oct-4B 的核定位。EWS-Oct-4B 和 EWS-Oct-4 的性质比较表明，EWS-Oct-4B 是携带 Oct-4 结合位点的报告构建体的转录激活较弱的激活剂。EWS-Oct-4B 的功能结构域缺失分析表明，其完整的转录激活潜能需要 EWS N 端结构域（NTD）（B）、POU 和 C 端结构域（CTD）。尽管作为转录激活剂的活性降低，但 EWS-Oct-4B 调节了成纤维细胞生长因子 4（fibroblast growth factor-4）和 Nanog 的表达，它们是有效的有丝分裂原，以及 Oct-4 下游靶基因的表达，其启动子含有潜在的 Oct-4 结合位点。最后，在 Oct-4 缺失的 ZHBTc4 ES 细胞中异位表达 EWS-Oct-4B 导致裸鼠肿瘤生成潜力增加。
这些结果表明，t（6;22）易位的致癌作用归因于 EWS-Oct-4B 嵌合蛋白，并且 EWS 氨基末端结构域与 Oct-4 DNA 结合结构域的另一种融合在人类上皮肿瘤中产生了另一种转化嵌合产物。
