Most studies of responses to transcriptional stimuli measure changes in cellular mRNA concentrations. By sequencing nascent RNA instead, it is possible to detect changes in transcription in minutes rather than hours and thereby distinguish primary from secondary responses to regulatory signals. Here, we describe the use of PRO-seq to characterize the immediate transcriptional response in human cells to celastrol, a compound derived from traditional Chinese medicine that has potent anti-inflammatory, tumor-inhibitory, and obesity-controlling effects. Celastrol is known to elicit a cellular stress response resembling the response to heat shock, but the transcriptional basis of this response remains unclear. Our analysis of PRO-seq data for K562 cells reveals dramatic transcriptional effects soon after celastrol treatment at a broad collection of both coding and noncoding transcription units. This transcriptional response occurred in two major waves, one within 10 min, and a second 40–60 min after treatment. Transcriptional activity was generally repressed by celastrol, but one distinct group of genes, enriched for roles in the heat shock response, displayed strong activation. Using a regression approach, we identified key transcription factors that appear to drive these transcriptional responses, including members of the E2F and RFX families. We also found sequence-based evidence that particular transcription factors drive the activation of enhancers. We observed increased polymerase pausing at both genes and enhancers, suggesting that pause release may be widely inhibited during the celastrol response. Our study demonstrates that a careful analysis of PRO-seq time-course data can disentangle key aspects of a complex transcriptional response, and it provides new insights into the activity of a powerful pharmacological agent.

对转录刺激反应的大多数研究都测量细胞mRNA浓度的变化。通过对新生RNA进行测序，可以在数分钟而不是数小时内检测到转录变化，从而区分对调节信号的主要反应和次要反应。在这里，我们描述了使用PRO-seq来表征人细胞对Celastrol的即时转录反应，celastrol是一种源自中药的化合物，具有有效的抗炎，抑制肿瘤和控制肥胖的作用。已知Celastrol会引发类似于热休克的细胞应激反应，但该反应的转录基础仍不清楚。我们对K562细胞的PRO-seq数据进行的分析显示，在Celastrol处理后不久，在广泛的编码和非编码转录单位集合中，戏剧性的转录作用。这种转录反应发生在两个主要的波中，一个在10分钟内发生，第二个在治疗后40-60分钟内发生。转录活性通常被Celastrol抑制，但是一组独特的基因在热休克反应中富集，表现出很强的激活作用。使用回归方法，我们确定了可能驱动这些转录反应的关键转录因子，包括E2F和RFX家族的成员。我们还发现了基于序列的证据，即特定的转录因子驱动增强子的激活。我们观察到在基因和增强子处聚合酶停顿增加，提示在天体反应期间可能会广泛抑制暂停释放。我们的研究表明，对PRO-seq时程数据进行仔细的分析可以使复杂的转录反应的关键方面脱颖而出，并且它为强大的药理活性提供了新的见解。