A novel role for the EWS portion of EWS/FLI in binding GGAA-microsatellites required for oncogenic transformation in Ewing sarcoma [RNA-Seq]

Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro, and these sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. Genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal “sweet-spot” GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this was not known. We now demonstrate the absolute necessity of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene, as well as for Ewing sarcoma proliferation and oncogenic transformation. Biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion) demonstrated a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter GGAA-microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the GGAA-microsatellite was increased to the “sweet-spot” length. In contrast, a fully-functional EWS/FLI mutant (Mut9) that retains approximately half of the EWS portion of the fusion showed low affinity for smaller GGAA-microsatellites, but instead significantly increased its affinity at “sweet-spot” microsatellite lengths. Single-gene ChIP and genome-wide ChIP-seq and RNA-seq studies extended these findings to the in vivo setting. Taken together, these data demonstrate the absolute requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unsuspected novel role for the EWS portion of the EWS/FLI fusion in binding to optimal-length GGAA-microsatellites. The treatments applied to our RNA-seq A673 samples were for EWS/FLI knockdown/rescue experiments. Briefly, A673 cells were stably infected and selected for expression of a control Luc-RNAi or the EF-2-RNAi to knock-down EWS/FLI. Following 4 days of selection, cells were infected with either an empty pMSCV-hygro vector or vector expressing full length type IV EWS/FLI, the Ä22 EWS/FLI mutant, or mut9 EWS/FLI mutant to rescue EWS/FLI (or respective mutant EWS/FLI) expression in these cells.

尤因肉瘤（Ewing sarcoma）通常表达EWS/FLI融合转录因子癌蛋白。EWS/FLI调控尤因肉瘤发生发展所需的大量基因。EWS/FLI在体内外均可结合GGAA微卫星（GGAA-microsatellite）序列，且此类序列可介导EWS/FLI对报告基因构建体的激活，提示其可作为EWS/FLI应答元件发挥功能。
基因组GGAA微卫星序列具有高度的变异性与多态性。现有研究表明，存在一段最佳“黄金区间”GGAA微卫星长度（18~26次GGAA重复），可使靶基因获得最大程度的EWS/FLI应答性，但其背后的分子机制尚不明确。
本研究首次证实，EWS/FLI结合的GGAA微卫星对NR0B1基因的调控，以及尤因肉瘤的增殖与致癌转化均具有绝对必要性。利用重组Δ22（仅包含FLI部分的EWS/FLI变体）进行的生化实验表明，在较短的GGAA微卫星序列上，每两个连续的GGAA重复单元可结合一个Δ22单体，其化学计量比为1:2。令人意外的是，当GGAA微卫星长度增加至“黄金区间”时，Δ22与GGAA微卫星的结合亲和力显著下降，最终达到无法检测的水平。与之相反，保留融合蛋白约一半EWS区域的功能完整EWS/FLI突变体（Mut9），对较短的GGAA微卫星亲和力较低，但在“黄金区间”长度的微卫星序列上的亲和力则显著提升。单基因染色质免疫沉淀（Chromatin Immunoprecipitation，ChIP）、全基因组染色质免疫沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）及RNA测序（RNA-seq）实验将上述发现拓展至体内环境。综合以上数据，本研究证实GGAA微卫星作为EWS/FLI激活型应答元件在体内发挥功能的绝对必要性，并揭示了EWS/FLI融合蛋白的EWS区域在结合最佳长度GGAA微卫星过程中此前未被发现的全新作用。
本研究针对RNA-seq所用的A673细胞样本的处理方案为EWS/FLI敲低/拯救实验。具体如下：将A673细胞稳定感染并筛选获得表达对照荧光素酶RNA干扰（Luc-RNAi）或EF-2-RNAi以敲低EWS/FLI的细胞株。经过4天的筛选后，分别用空pMSCV-潮霉素（pMSCV-hygro）载体，或表达全长IV型EWS/FLI、Δ22 EWS/FLI突变体或Mut9 EWS/FLI突变体的载体感染上述细胞，以在细胞中拯救EWS/FLI（或对应突变体EWS/FLI）的表达。