RNA Polymerase II Transcriptional Pausing and Release Regulates Circadian Transcription

Circadian rhythms regulate the harmonized gene expression, metabolism and physiology through transcriptional controls. It is widely accepted that the recruitment of circadian transcriptional factors has a critical role in rhythmic gene expressions. However, the roles of RNA polymerase II pausing and release (RNAP II P/R) on circadian rhythm remain largely unknown. From the massive and precise sequencing experiments, we found the static occupancies of RNAP II complex on the initiation sites and rhythmic elongation controls on Alas1 and Srebf1 genes. The subsequent genome-wide analysis retrieved 54 circadian RNAP II P/R rhythmic genes (91 transcripts) that are closely related to nutritional signals. Those genes kept unique rhythms in clock mutant mice and showed the dynamic responses under the reversed feeding regime. Here, we report a novel circadian transcription mechanism, RNAP II pausing & rhythmic release. Overall design: The 6 weeks old male mice were housed in the two light chambers with a 2hr shifted light:dark (LD)=12hrs:12hrs condition and fed ad libitum. After 4 weeks of the entrainment, a light/dark chamber was released to constant darkness (DD) condition 24hrs earlier than the other. The liver samples (N=3) were collected after 36 hours of DD condition beginning with the first point (CT00) and subsequent every 4hrs collections for each light chamber till CT44. The harvested liver samples were analyzed by ChIP-seq, PRO-seq and mRNA-seq.

昼夜节律（Circadian Rhythm）通过转录调控实现基因表达、代谢与生理活动的协调统一。目前学界普遍认为，昼夜节律转录因子的招募对节律性基因表达具有关键调控作用。然而，RNA聚合酶II暂停与释放（RNAP II P/R）在昼夜节律中的功能仍未得到广泛阐明。

借助大规模高精度测序实验，我们发现RNA聚合酶II复合物在基因起始位点的静态占据现象，以及Alas1与Srebf1基因上的节律性延伸调控机制。后续的全基因组分析筛选出54个与营养信号密切相关的节律性RNAP II暂停释放基因（共涉及91个转录本）。这些基因在Clock突变小鼠中呈现独特的节律模式，并在进食时间逆转的条件下表现出动态应答反应。

本研究报道了一种全新的昼夜节律转录机制：RNA聚合酶II暂停与节律性释放。实验设计概述：将6周龄雄性小鼠饲养于两个光照箱中，采用12小时光照:12小时黑暗（LD）的光周期方案，且两组光周期存在2小时偏移，小鼠自由进食。经过4周的节律同步化后，其中一个光照箱的小鼠提前24小时转入持续黑暗（DD）环境，另一组则维持原光周期。在持续黑暗（DD）条件下培养36小时后开始采集肝脏样本，首个采样点为CT00，随后每个光照箱均每隔4小时采样一次，直至CT44，每组采集样本量为3只。采集得到的肝脏样本通过ChIP-seq、PRO-seq及mRNA-seq进行测序分析。