The Oncogenic EWS-FLI1 Protein Binds <em>In Vivo</em> GGAA Microsatellite Sequences with Potential Transcriptional Activation Function

The fusion between EWS and ETS family members is a key oncogenic event in Ewing tumors and important EWS-FLI1 target genes have been identified. However, until now, the search for EWS-FLI1 targets has been limited to promoter regions and no genome-wide comprehensive analysis of in vivo EWS-FLI1 binding sites has been undertaken. Using a ChIP-Seq approach to investigate EWS-FLI1-bound DNA sequences in two Ewing cell lines, we show that this chimeric transcription factor preferentially binds two types of sequences including consensus ETS motifs and microsatellite sequences. Most bound sites are found outside promoter regions. Microsatellites containing more than 9 GGAA repeats are very significantly enriched in EWS-FLI1 immunoprecipitates. Moreover, in reporter gene experiments, the transcription activation is highly dependent upon the number of repeats that are included in the construct. Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation. Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis.EWS与ETS家族成员的融合是尤文肉瘤（Ewing tumors）发生过程中的关键致癌事件，目前已鉴定得到多个重要的EWS-FLI1靶基因。然而迄今为止，针对EWS-FLI1靶基因的筛选仅局限于启动子区域，尚未开展全基因组水平的体内EWS-FLI1结合位点系统性分析。本研究通过染色质免疫共沉淀测序（ChIP-Seq）技术，对两株尤文肉瘤细胞系中与EWS-FLI1结合的DNA序列进行分析，结果显示该嵌合转录因子（chimeric transcription factor）偏好结合两类序列：保守型ETS基序（ETS motifs）与微卫星序列（microsatellite sequences）。绝大多数结合位点位于启动子区域之外。包含9个以上GGAA重复单元的微卫星序列在EWS-FLI1免疫沉淀物中显著富集。此外，在报告基因实验（reporter gene experiments）中，转录激活活性高度依赖于载体中所包含的重复单元数量。值得注意的是，体内与EWS-FLI1结合的微卫星序列与EWS-FLI1介导的基因激活显著相关。综合上述研究结果，本研究揭示了微卫星元件可能在远距离转录调控及肿瘤发生过程中发挥潜在作用。