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The circadian clock orchestrates gene expression rhythms. Regulation at the level of gene transcription is essential for molecular and cellular rhythms. Pol II pause release is a critical step of transcription regulation. However, whether and how Pol II pause release is regulated during daily transcription have not been characterized. In this study, we performed Pol II ChIP-seq across the day in the mouse liver and quantitatively analyzed binding signals within the transcription start site (TSS) region and the gene body. We frequently found discordant changes between Pol II near the TSS ([Pol II] TSS , paused Pol II) and that within the gene body ([Pol II] GB , transcribing Pol II) across the genome, with only [Pol II] GB  always reflecting transcription of clock and clock-controlled genes. Accordingly, Pol II traveling ratios of more than 7000 genes showed significant daily changes (&gt;1.5-fold). Therefore, there is widespread regulation of Pol II pausing in the mouse liver. Interestingly, gene transcription rhythms exhibited a bimodal phase distribution. The transcription of ~400 genes peaked near ZT0, coincident with a genome-wide increase in [Pol II] TSS  and traveling ratio (TR). The transcription of ~300 other genes peaked ~12 h later, when there was a global decrease in [Pol II] TSS  and TR. ChIP-seq against TATA-binding protein (Tbp), a preinitiation complex (PIC) component, revealed that Pol II recruitment mainly played an indirect role in transcriptional output, with transcriptional termination and pause release functioning prominently in determining the fate of initiated Pol II and its pausing status. Taken together, our results revealed a critical, albeit complex role of Pol II pausing control in regulating the temporal output of gene transcription.

Regulation of Pol II Pausing Is Involved in Daily Gene Transcription in the Mouse Liver