Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.

长程增强子对致癌基因表达的激活是由 DNA 结合转录因子 (TF) 的组装启动的，导致招募 CBP/p300 等共激活子来修改局部基因组背景并促进 RNA 聚合酶 2（ Pol2) 结合。然而，大多数 TF 与共激活因子的招募关系仍未确定。在此，研究来自肺泡横纹肌肉瘤 (aRMS) 的致癌融合 TF PAX3-FOXO1 (P3F)，我们发现 P3F 激活域 (AD) 中的单个半胱氨酸对于将 CBP/p300 招募到染色质的小 α 螺旋线圈非常重要。 P3F 驱动的转录需要单个半胱氨酸和 CBP/p300。半胱氨酸突变体减少 aRMS 细胞增殖并诱导细胞分化。此外，我们发现将 P3F 与其靶基因连接的 Pol2 环的聚类对 CBP/p300 具有深刻的依赖性。在缺少 CBP/p300 的情况下，Pol2 长程增强子环崩溃，Pol2 积聚在 CpG 岛中并且无法退出基因体。这些结果揭示了 aRMS 中 P3F 治疗干扰的潜在新轴，并阐明了 P3F 和 CBP/p300 在维持致癌转录所必需的活性 Pol2 簇方面的分子关系。