Nascent-Seq Reveals Novel Features of Mouse Circadian Transcriptional Regulation [Nascent-Seq]

Over the past decade, genome-wide assays have underscored the broad sweep of circadian gene expression. A substantial fraction of the transcriptome undergoes oscillations in many organisms and tissues, which governs the many biochemical, physiological and behavioral functions under circadian control. Based predominantly on the transcription feedback loops important for core circadian timekeeping, it is commonly assumed that this widespread mRNA cycling reflects circadian transcriptional cycling. To address this issue, we directly measured dynamic changes in mouse liver transcription using Nascent-Seq. Many genes are rhythmically transcribed over the 24h day, which include precursors of several non-coding RNAs as well as the expected set of core clock genes. Surprisingly however, nascent RNA rhythms overlap poorly with mRNA abundance rhythms assayed by RNA-seq. This is because most mouse liver genes with rhythmic mRNA expression manifest poor transcriptional rhythms, indicating a prominent role of post-transcriptional regulation in setting mRNA cycling amplitude. To gain further insight into circadian transcriptional regulation, we also characterized the rhythmic transcription of liver genes targeted by the transcription factors CLOCK and BMAL1; they directly target other core clock genes and sit at the top of the molecular circadian clock hierarchy in mammals. CLK:BMAL1 rhythmically bind at the same discrete phase of the circadian cycle to all target genes, which not surprisingly have a much higher percentage of rhythmic transcription than the genome as a whole. However, there is a surprisingly heterogeneous set of cycling transcription phases of direct target genes, which even include core clock genes. This indicates a disconnect between rhythmic DNA binding and the peak of transcription, which is likely due to other transcription factors that collaborate with CLK:BMAL1. In summary, the application of Nascent-Seq to a mammalian tissue provides surprising insights into the rhythmic control of gene expression and should have broad applications beyond the analysis of circadian rhythms. Mouse liver nascent RNA profile over 6 time points of the 24h light:dark cycle, in duplicate, sequenced using Ilumina GAII (Nascent-Seq); Mouse liver mRNA profile over 6 time points of the 24h light:dark cycle, in duplicate, sequenced using Ilumina HiSeq2000 (RNA-Seq); CLK and BMAL1 DNA binding profile in the mouse liver at ZT8, sequenced along an Input sample using GAII (ChIP-Seq); Mouse liver strand-specific nascent RNA profile over 6 time points of the 24h light:dark cycle, in duplicate, sequenced using Ilumina HiSeq2000 (Strand-specific Nascent-Seq); Supplementary file NascentSeq_Mouse_Liver_NormalizedGeneSignal.txt represents Nascent RNA abundance (reads per base pair) for each sample.近十年来，全基因组水平检测研究已证实，节律性基因表达的覆盖范围极为广泛。多种生物体及组织的转录组中，有相当一部分呈现节律性波动，这些波动调控着受昼夜节律控制的各类生化、生理及行为功能。基于核心昼夜节律计时所依赖的转录反馈环路，学界普遍认为这种广泛存在的mRNA节律波动实则反映了昼夜节律性的转录循环。为解答这一问题，本研究通过新生转录组测序（Nascent-Seq）直接检测小鼠肝脏转录的动态变化。诸多基因在24小时周期内呈现节律性转录，其中包含多种非编码RNA的前体，以及预期的核心时钟基因集合。然而令人意外的是，新生RNA的节律波动与通过RNA测序（RNA-seq）检测得到的mRNA丰度节律重叠度极低。究其原因，大多数呈现mRNA表达节律的小鼠肝脏基因，其自身的转录节律并不显著，这表明转录后调控在调控mRNA循环振幅中发挥着关键作用。为进一步解析昼夜节律性转录调控机制，本研究还对转录因子CLOCK与BMAL1所靶向的肝脏基因的节律性转录进行了表征；二者可直接靶向其他核心时钟基因，且位于哺乳动物分子昼夜节律时钟层级的顶端。CLK:BMAL1复合物会在昼夜周期的同一特定时相节律性结合所有靶基因，不出所料，这些靶基因的节律性转录比例远高于全基因组平均水平。但令人意外的是，直接靶基因的节律性转录时相存在显著异质性，其中甚至包含核心时钟基因。这表明节律性DNA结合与转录峰值之间存在脱节，这一现象大概率由与CLK:BMAL1协同作用的其他转录因子所导致。综上，将新生转录组测序（Nascent-Seq）应用于哺乳动物组织的研究，为基因表达的节律调控提供了突破性的新见解，且其应用范围将远超昼夜节律分析领域。小鼠肝脏新生RNA谱数据：基于24小时光暗周期的6个时间点，设置生物学重复，采用Illumina GAII测序平台完成测序（新生转录组测序，Nascent-Seq）；小鼠肝脏mRNA谱数据：基于24小时光暗周期的6个时间点，设置生物学重复，采用Illumina HiSeq2000测序平台完成测序（RNA测序，RNA-seq）；小鼠肝脏CLK与BMAL1的DNA结合谱数据：于ZT8时相采集样本，同步设置Input对照样本，采用Illumina GAII测序平台完成测序（染色质免疫沉淀测序，ChIP-Seq）；小鼠肝脏链特异性新生RNA谱数据：基于24小时光暗周期的6个时间点，设置生物学重复，采用Illumina HiSeq2000测序平台完成测序（链特异性新生转录组测序，Strand-specific Nascent-Seq）；补充文件NascentSeq_Mouse_Liver_NormalizedGeneSignal.txt包含各样本的新生RNA丰度数据（每碱基对的读段数）。