Genome-wide map of PAX3-FKHR binding sites in rhabdomyosarcoma. Homo sapiens

We report the genome-wide maps of PAX3-FKHR binding sites. Chromatin immunoprecipitation was performed against PAX3-FKHR positive (Rh4) and PAX3-FKHR negative (RD) rhabdomyosarcoma cells with a monoclonal antibody (pFM2) specific for the fusion region of PAX3-FKHR. We obtained 4 million sequence tags for both input and ChIP DNA that aligned to the human genome. We identified 1,463 binding sites from ChIP-seq of Rh4 cells, none of which appeared from ChIP-seq of fusion negative RD cells. The PAX3-FKHR binding sites were found to associate with 1,072 genes in RMS cells. The data shows that PAX3-FKHR binds to the same sites as PAX3, at the enhancers for MYF5, FGFR4, and the MYOD core enhancer previously shown to be regulated by PAX3. Moreover, our dataset has the precision for rapid identification and validation of novel and specific sequences required for the enhancer activity of MYOD and FGFR4. The genome wide analysis reveals that the PAX3-FKHR sites are: 1) mostly distal to transcription start sites; 2) conserved; 3) enriched for PAX3 motifs; and 4) strongly associated with genes over-expressed in PAX3-FKHR positive RMS cells and tumors. There is little evidence in our dataset for PAX3-FKHR binding at the promoters. In one instance, we show two intronic enhancer elements for MET, rather than at the previously described promoter. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common co-regulation for many target genes. The map of PAX3-FKHR binding sites provides new links for PAX3 and PAX3-FKHR functions and new targets for RMS therapy. Overall design: Examination of PAX3-FKHR binding sites in translocation-positive rhabdomyosarcoma cells via ChIP-seq with an antibody specific for the fusion protein.

本研究报道了PAX3-FKHR结合位点的全基因组图谱。本研究针对PAX3-FKHR阳性（Rh4）及PAX3-FKHR阴性（RD）横纹肌肉瘤（rhabdomyosarcoma, RMS）细胞，使用针对PAX3-FKHR融合区域的单克隆抗体（pFM2）开展染色质免疫共沉淀（ChIP）实验。我们为输入DNA及ChIP DNA均获取了400万条可比对至人类基因组的序列标签。通过对Rh4细胞的染色质免疫共沉淀测序（ChIP-seq），我们共鉴定出1463个结合位点，而融合蛋白阴性的RD细胞的ChIP-seq未检测到任何此类结合位点。研究发现，横纹肌肉瘤细胞中PAX3-FKHR的结合位点与1072个基因存在关联。数据显示，PAX3-FKHR与PAX3结合于相同的位点，包括MYF5、FGFR4的增强子以及此前被证实受PAX3调控的MYOD核心增强子。此外，本数据集具备快速鉴定并验证MYOD与FGFR4增强子活性所需的新型特异性序列的精度。全基因组分析显示，PAX3-FKHR结合位点具有以下特征：1）多数位于转录起始位点远端；2）具有进化保守性；3）富集PAX3结合基序；4）与PAX3-FKHR阳性横纹肌肉瘤细胞及肿瘤中高表达的基因显著相关。本数据集几乎未发现PAX3-FKHR结合于启动子区域的证据。在一例案例中，我们证实MET存在两个内含子增强子元件，而非此前报道的启动子区域。全基因组分析进一步揭示，这些结合位点中PAX3与E-box基序存在显著关联，提示众多靶基因存在共同的共调控机制。PAX3-FKHR结合位点图谱为PAX3及PAX3-FKHR的功能提供了新的关联线索，同时为横纹肌肉瘤治疗提供了新的靶点。
实验设计：通过使用针对融合蛋白的特异性抗体开展染色质免疫共沉淀测序（ChIP-seq），检测易位阳性横纹肌肉瘤细胞中的PAX3-FKHR结合位点。