Nascent-Seq Reveals Novel Features of Mouse Circadian Transcriptional Regulation [RNA-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 RNASeq_Mouse_Liver_NormalizedGeneSignal.txt represents mRNA abundance (reads per base pair) for each sample.近十年来，全基因组分析技术已揭示昼夜节律基因表达的广泛分布特征。在诸多生物与组织中，转录组内有相当比例的基因会发生节律性波动，此类波动调控着受昼夜节律支配的各类生化、生理及行为功能。 基于核心生物钟调控中至关重要的转录反馈环路，学界普遍认为这种广泛存在的mRNA节律性波动，实则反映了转录层面的昼夜节律性循环。 为解答这一科学问题，本研究采用新生转录组测序（Nascent-Seq）技术，直接检测了小鼠肝脏中转录的动态变化。在24小时周期内，诸多基因呈现节律性转录，其中包含数种非编码RNA（non-coding RNA）的前体，以及预期中的核心时钟基因集合。但令人意外的是，新生RNA的节律模式与通过RNA测序（RNA-seq）检测得到的mRNA丰度节律模式重合度极低。究其原因，多数呈现mRNA节律性表达的小鼠肝脏基因，其转录层面并未表现出明显的节律性，这表明转录后调控在维持mRNA波动幅度中发挥着关键作用。 为进一步探究昼夜节律的转录调控机制，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；这两类因子可直接靶向其他核心时钟基因，且处于哺乳动物分子生物钟层级的顶端。CLK:BMAL1复合物会在昼夜周期的同一特定时相与所有靶基因结合；不出所料，这些靶基因呈现节律性转录的比例远高于全基因组平均水平。但令人意外的是，直接靶基因的转录节律时相呈现出显著的异质性，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，这种现象很可能是由与CLK:BMAL1协同作用的其他转录因子所导致的。 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颠覆性认知，且其应用范围将远超昼夜节律分析领域。 本数据集包含以下测序数据： 1. 采用Illumina GAII测序平台完成测序的小鼠肝脏新生RNA测序数据（Nascent-Seq）：样本取自24小时光暗周期的6个时间点，设置生物学重复两份； 2. 采用Illumina HiSeq2000测序平台完成测序的小鼠肝脏mRNA测序数据（RNA-seq）：样本取自24小时光暗周期的6个时间点，设置生物学重复两份； 3. 采用GAII测序平台完成测序的小鼠肝脏CLK与BMAL1 DNA结合谱数据（ChIP-Seq）：样本采集于节律时间ZT8，同时设置Input对照样本； 4. 采用Illumina HiSeq2000测序平台完成测序的小鼠肝脏链特异性新生RNA测序数据（Strand-specific Nascent-Seq）：样本取自24小时光暗周期的6个时间点，设置生物学重复两份； 补充文件RNASeq_Mouse_Liver_NormalizedGeneSignal.txt包含所有样本的mRNA丰度信息，单位为每碱基对的读段数。