PAX3-FOXO1 directs CBP/P300 via its activation domain to build RNA Pol2 clusters

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 PAX3-FOXO1 forms a small alpha helical hook that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine, and also CBP/p300. 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. Overall design: RNA-seq and Pol2 HiChIP in RMS cells treated with HAT inhibitors and degraders. Pol2 ChIP-seq in ABE RMS cells with wt P3F or C793R mutant P3F and H3K27ac ChIP-seq in RMS cells treated with HAT degraders. Pol2 HiChIP in RMS cells with P3F-FKBP12 treated with DMSO or dTAG.

致癌基因表达的激活由远程增强子区域起始于DNA结合转录因子（DNA-binding transcription factor, TF）的组装，该过程会招募如CBP/p300这类共激活因子，以修饰局部基因组环境并促进RNA聚合酶Ⅱ（RNA-Polymerase 2, Pol2）的结合。然而，绝大多数转录因子与共激活因子之间的招募关联仍未被系统阐明。本研究针对腺泡状横纹肌肉瘤（alveolar rhabdomyosarcoma, aRMS）中的致癌融合转录因子PAX3-FOXO1（简称P3F）展开研究，发现PAX3-FOXO1激活结构域（activation domain, AD）内的单个半胱氨酸残基可形成小型α螺旋钩状结构，从而将CBP/p300招募至染色质。P3F介导的转录过程同时依赖该单个半胱氨酸残基与CBP/p300。此外，本研究发现，将P3F与其靶基因相连的Pol2环簇形成，对CBP/p300存在极强的依赖性。在CBP/p300缺失的情况下，Pol2介导的远程增强子环会发生解体，Pol2会在CpG岛处聚集，且无法脱离基因体区域。上述研究结果揭示了一条可用于干预腺泡状横纹肌肉瘤中P3F活性的新型调控轴，并阐明了P3F与CBP/p300在维持致癌转录所必需的活性Pol2簇过程中的分子关联。整体实验设计：对经组蛋白乙酰转移酶（HAT）抑制剂与降解剂处理的横纹肌肉瘤（RMS）细胞开展RNA测序（RNA-seq）与Pol2染色质免疫共沉淀-高通量染色体构象捕获（Pol2 HiChIP）实验；在携带野生型（wild type, wt）P3F或C793R突变型P3F的ABE RMS细胞中开展Pol2染色质免疫共沉淀测序（Pol2 ChIP-seq），并在经HAT降解剂处理的横纹肌肉瘤细胞中开展H3K27乙酰化染色质免疫共沉淀测序（H3K27ac ChIP-seq）；对携带P3F-FKBP12融合蛋白的横纹肌肉瘤细胞分别用二甲基亚砜（DMSO）或dTAG处理后开展Pol2染色质免疫共沉淀-高通量染色体构象捕获（Pol2 HiChIP）实验。