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.

大多数对转录刺激反应的研究都会测量细胞 mRNA 浓度的变化。通过对新生 RNA 进行测序，可以在几分钟而不是几小时内检测转录的变化，从而区分对调节信号的初级和次级反应。在这里，我们描述了使用 PRO-seq 来表征人类细胞对雷公藤红素的即时转录反应，雷公藤红素是一种源自传统中药的化合物，具有有效的抗炎、抑制肿瘤和控制肥胖的作用。已知雷公藤红素会引发类似于热休克反应的细胞应激反应，但这种反应的转录基础仍不清楚。我们对 K562 细胞的 PRO-seq 数据的分析揭示了雷公藤红素处理后不久对大量编码和非编码转录单元的显着转录效应。这种转录反应发生在两次主要波中，一次在 10 分钟内，第二次在治疗后 40-60 分钟内。转录活性通常受到雷公藤红素的抑制，但一组在热休克反应中发挥重要作用的独特基因表现出强烈的激活作用。使用回归方法，我们确定了似乎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。我们还发现基于序列的证据表明特定转录因子驱动增强子的激活。我们观察到基因和增强子处的聚合酶暂停增加，这表明在雷公藤红醇反应期间暂停释放可能被广泛抑制。我们的研究表明，对 PRO-seq 时程数据的仔细分析可以解开复杂转录反应的关键方面，并为强大的药理活性提供新的见解。