Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription. Homo sapiens

Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome-depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately transcriptional dysregulation. Overall design: Examination of two transcription factors in two different cell types, as well as three histone methylation marks and FAIRE

本研究以EWS-FLI（EWS-FLI）及其亲本转录因子FLI1（FLI1）为研究对象，构建了一套独特的实验体系，用以解析嵌合转录因子（chimeric transcription factor）致癌的基因组调控机制。研究发现，尽管二者拥有完全一致的DNA结合结构域（DNA-binding domain），但在肿瘤细胞中，EWS-FLI所靶向的基因组区域与FLI1存在显著差异。但在原代内皮细胞（primary endothelial cell）中，EWS-FLI与FLI1的基因组靶向模式则高度相似。为解析这一异常靶向（mistargeting）现象的成因，我们对染色质组织（chromatin organization）状态进行了分析。EWS-FLI的靶向区域在原代内皮细胞中通常处于转录抑制状态，且以核小体富集的形式存在。而在肿瘤细胞中，EWS-FLI结合的区域则呈现核小体缺失（nucleosome-depleted）特征，并兼具增强子的染色质标记（chromatin signature of enhancers）。后续实验证实，通过嵌合融合（chimerism），EWS-FLI获得了重塑染色质结构的能力。诱导EWS-FLI的表达可导致其靶向位点发生核小体缺失；而在肿瘤细胞中沉默EWS-FLI，则可恢复该位点的核小体占据率（nucleosome occupancy）。综上，EWS-FLI嵌合蛋白获得了染色质重塑活性，进而引发异常靶向、染色质紊乱，并最终导致转录调控失衡（transcriptional dysregulation）。
实验整体设计：在两种不同细胞类型中分析两种转录因子，同时检测三种组蛋白甲基化修饰标记（histone methylation mark）与FAIRE（甲醛辅助分离调控元件，Formaldehyde-Assisted Isolation of Regulatory Elements）。