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

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);近十年来，全基因组检测（genome-wide assays）已凸显出节律基因表达（circadian gene expression）的广泛分布。众多生物体与组织的转录组中，有相当比例的转录本呈现节律性波动，这些波动调控着诸多受节律控制的生化、生理及行为功能。现有研究大多基于核心节律计时所依赖的转录反馈环路（transcription feedback loops），普遍认为这种广泛存在的mRNA节律性波动（mRNA cycling）反映了节律性转录循环（circadian transcriptional cycling）。 为解决这一问题，我们通过新生RNA测序（Nascent-Seq）直接检测了小鼠肝脏的转录动态变化。24小时周期内，众多基因呈现节律性转录，其中包含多种非编码RNA（non-coding RNAs）的前体，以及预期的核心时钟基因（core clock genes）集合。然而令人意外的是，新生RNA节律与通过RNA测序（RNA-Seq）检测得到的mRNA丰度节律（mRNA abundance rhythms）重合度极低。这是因为大多数呈现mRNA节律性表达的小鼠肝脏基因，其转录节律性并不显著，表明转录后调控（post-transcriptional regulation）在决定mRNA波动振幅方面发挥着关键作用。 为进一步解析节律性转录调控机制，我们还对转录因子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，且处于哺乳动物分子节律时钟层级的顶端。CLK:BMAL1可在节律周期的同一离散相位与所有靶基因发生节律性结合；不出所料，这些靶基因的节律性转录比例远高于全基因组水平。但令人意外的是，直接靶基因的转录波动相位呈现出显著异质性，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，这种现象大概率是由与CLK:BMAL1协同作用的其他转录因子所导致的。 综上，将新生RNA测序（Nascent-Seq）应用于哺乳动物组织，为基因表达的节律性调控带来了突破性的意外见解，其应用潜力将远超节律性分析领域。本数据集包含如下内容： 1. 小鼠肝脏在24小时光暗周期下6个时间点的新生RNA测序（Nascent-Seq）数据，设置生物学重复，采用Illumina GAII进行测序； 2. 小鼠肝脏在24小时光暗周期下6个时间点的RNA测序（RNA-Seq）数据，设置生物学重复，采用Illumina HiSeq2000进行测序； 3. 小鼠肝脏在ZT8时刻的CLOCK与BMAL1 DNA结合谱数据，与Input样本一同采用Illumina GAII进行测序（染色质免疫共沉淀测序，ChIP-Seq）； 4. 小鼠肝脏在24小时光暗周期下6个时间点的链特异性新生RNA测序（Strand-specific Nascent-Seq）数据，设置生物学重复，采用Illumina HiSeq2000进行测序；