Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers.

转录控制是一个高度动态的过程，它会响应各种细胞和细胞外信号而迅速变化，因此很难使用缓慢的遗传方法来定义转录因子的功能机制。我们使用化学遗传学方法快速降解经典转录激活因子 PAX3-FOXO1，以确定其调节基因表达程序的机制。通过将快速蛋白质降解与短时间过程中的新生转录分析相结合，并将 CUT&RUN、ATAC-seq 和 eRNA 分析与深度蛋白质组分析相结合，我们在直接转录靶标的小型网络中定义了 PAX3-FOXO1 功能。 PAX3-FOXO1 降解会损害大多数受调控基因靶标的 RNA 聚合酶暂停释放和转录延伸。此外，PAX3-FOXO1 在控制该核心网络的增强子上的活性令人惊讶地具有选择性，影响超级增强子中的单个元件。该组合分析表明，持续需要 PAX3-FOXO1 来维持受调节增强子处的染色质可及性和增强子结构。