Capturing the dynamic nascent transcriptome during acute cellular responses: the serum response. Homo sapiens

Dynamic regulation of gene expression via signal transduction pathways is of fundamental importance during many biological processes such as cell state transitioning, cell cycle progression and stress responses. In this study we used serum stimulation as a cell response paradigm to apply the nascent RNA Bru-seq technique in order to capture early dynamic changes in the nascent transcriptome. Our data provides an unprecedented view of the dynamics of genome- wide transcription during the first two hours of serum stimulation in human fibroblasts. While some genes showed sustained induction or repression, other genes showed transient or delayed responses. Surprisingly, the dynamic patterns of induction and suppression of response genes showed a high degree of similarity, suggesting that these opposite outcomes are triggered by a common set of signals. As expected, early response genes such as those encoding components of the AP-1 transcription factor and those involved in the circadian clock were immediately but transiently induced. Surprisingly, transcription of important DNA damage response genes and histone genes were rapidly repressed.We also show that RNA polymerase II accelerates as it transcribes large genes and this was independent of whether the gene was induced or not. These results provide a unique genome-wide depiction of dynamic patterns of transcription of serum response genes and demonstrate the utility of Bru-seq to comprehensively capture rapid and dynamic changes of the nascent transcriptome. Overall design: One cell line was used (normal human foreskin fibroblasts; HF1, referred to as "nf" in the sample names). HF1 cells were serum-starved for 48 h. One sample ("starved") was then labeled with bromouridine (2 mM) for 30 min. For the remaining samples, serum was added and bromouridine added immediately ("30 min serum") or 30 min, 60 min, or 90 min post serum addition ("60 min serum", "90 min serum", and "120 min serum", respectively). RNA was collected immediately after the 30 min bromouridine labeling period.

通过信号转导通路（signal transduction pathways）实现的基因表达动态调控，在细胞状态转变、细胞周期进程与应激响应等诸多生物学过程中均具有核心重要性。本研究以血清刺激作为细胞响应范式，采用新生RNA（nascent RNA）Bru-seq技术，以捕捉新生转录组（nascent transcriptome）的早期动态变化。本研究数据首次全景展现了人成纤维细胞在血清刺激后最初两小时内全基因组（genome-wide）转录的动态变化特征。部分基因呈现持续诱导或持续抑制的表达模式，而另有部分基因则表现出瞬时响应或延迟响应的特征。令人意外的是，响应基因的诱导与抑制动态模式呈现高度相似性，这表明这两类截然相反的表达结果由同一组信号通路所触发。正如预期，编码AP-1转录因子（AP-1 transcription factor）组分的早期响应基因以及参与昼夜节律时钟（circadian clock）的基因，均被快速且瞬时地诱导表达。令人意外的是，重要的DNA损伤响应基因（DNA damage response genes）与组蛋白基因（histone genes）的转录过程被快速抑制。本研究同时发现，RNA聚合酶II（RNA polymerase II）在转录大型基因时会加速行进，且这一现象与基因是否被诱导表达无关。上述结果首次从全基因组层面独特地描绘了血清响应基因的转录动态模式，并证实了Bru-seq技术可全面捕捉新生转录组的快速动态变化。实验整体设计：本研究仅使用一种细胞系——正常人类包皮成纤维细胞（HF1，样本命名中简称为"nf"）。将HF1细胞进行48小时的血清饥饿处理。随后取一份样本（命名为"starved"）用2 mM溴尿苷（bromouridine）进行30分钟的标记。剩余样本则在添加血清的同时即刻加入溴尿苷（命名为"30 min serum"），或分别在血清添加后30分钟、60分钟、90分钟加入溴尿苷，对应命名为"60 min serum"、"90 min serum"与"120 min serum"。在30分钟的溴尿苷标记结束后，即刻收集样本RNA。