REV-ERBa and REV-ERBb function as key factors regulating Mammalian Circadian Output. REV-ERBa and REV-ERBb function as key factors regulating Mammalian Circadian Output

The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERBa and REV-ERBb are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erba/Rev-erbb double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the complete knockout of both Rev-erba and Rev-erbb does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERBa/b deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2, are significantly upregulated. This study emphasizes REV-ERBs function to form an essential link between the circadian clock and a wide variety of cellular physiological functions. Overall design: Control (WT) and Rev-erba/Rev-erbb deficient (KO) mouse ES cells on Per2::Luciferase knock-in background were differentiated for 28 days upon embryoid body formation, treated with 100 nM dexamethasone, and subjected to RNA extraction at 4-h intervals from 12 h to 56 h after dexamethasone stimulation.

生物钟（circadian clock）以24小时为周期调控行为与生理过程。核受体REV-ERBa与REV-ERBb作为转录抑制因子，参与细胞自主的昼夜节律转录-翻译反馈环路。多项研究还证实，REV-ERBs在代谢、神经与炎症功能调控中发挥关键作用，涵盖胆汁酸代谢、脂质代谢及炎症细胞因子生成等过程。鉴于REV-ERBs的多功能特性，阐明其发挥功能的分子机制具有重要学术意义。为此，我们构建了Rev-erba/Rev-erbb双敲除小鼠胚胎干细胞（ES）模型，并对生物钟及受生物钟调控的输出基因表达情况进行了分析。全面的mRNA测序（mRNA-seq）分析结果显示，同时完全敲除Rev-erba与Rev-erbb并不会消除E-box调控的核心生物钟基因的表达节律，但会显著改变大量其他节律性表达的输出基因的节律模式。值得注意的是，REV-ERBa/b缺失并不会影响PER2的昼夜表达节律，而REV-ERB的靶基因Bmal1与Npas2则显著上调。本研究揭示，REV-ERBs是连接生物钟与多种细胞生理功能的关键纽带。实验设计：以Per2::荧光素酶（Per2::Luciferase）敲入为遗传背景的野生型（wild type, WT）对照细胞与Rev-erba/Rev-erbb缺陷型（knockout, KO）小鼠ES细胞，经胚状体形成诱导分化28天后，用100 nM地塞米松处理，并在地塞米松刺激后12 h至56 h的时间段内，每隔4 h收集样本进行RNA提取。