de Bruijn D R, dos Santos N R, Thijssen J, Balemans M, Debernardi S, Linder B, Young B D, Geurts van Kessel A
Department of Human Genetics, University Hospital, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Oncogene. 2001 May 31;20(25):3281-9. doi: 10.1038/sj.onc.1204419.
As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.
由于滑膜肉瘤相关的t(X;18)易位，18号染色体上的人类SYT基因与X染色体上的SSX1或SSX2基因融合。尽管初步证据表明这些基因编码的（融合）蛋白可能在转录调控中起作用，但对其确切功能知之甚少。我们以SYT为诱饵，通过酵母双杂交筛选人cDNA文库来分离相互作用蛋白。在分离出的阳性克隆中，发现有两个对应于急性白血病t(10;11)相关的AF10基因，它是MLL的融合伴侣。通过从细胞系提取物中对内源性和外源性、表位标记的SYT和AF10蛋白进行共免疫沉淀，以及在转染细胞中表位标记的SYT和AF10蛋白的共定位，证实了这些结果。随后的序列突变分析揭示了SYT蛋白的N端片段与AF10蛋白的C端片段之间存在高度特异性的相互作用。还发现SYT蛋白的N端相互作用结构域存在于几种SYT直系同源物和同源物中。AF10的C端相互作用结构域位于已知功能结构域之外。基于这些结果，提出了一个模型，其中SYT和AF10蛋白协同作为二元转录因子发挥作用。该模型对人类滑膜肉瘤和急性白血病发生的分子机制具有启示意义。
