Project description: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.

Project description:Winter survival and maintenance strategy is crucial in temperate woody plants. Here, we demonstrate novel aspects of the transcriptional regulations adopted by perennial tree species in winter/dormancy, employing a biochemical and whole transcriptome analysis. As expected, genes related to cold hardiness and defense are over-represented. Interestingly, carbohydrate biosynthesis and transport-related genes were very actively expressed in winter/dormancy. Further biochemical analyses verified the dormancy/winter transcription phenotype. Furthermore, dormancy/winter preferential expression of genes involved in the cell wall biosynthesis/modification, circadian rhythm, the indirect transcriptional regulation (RNA metabolism), and chromatin modification/remodeling were identified. Taken together, regulation of gene expression in the winter survival and maintenance may include not only controlled by promoter binding transcription factors but may also be regulated at the post-transcriptional and chromatin levels. In the first step towards the understanding of molecular mechanisms underlying the winter survival and maintenance of perennial trees, we examined the characteristics of transcription phenotype of the dormancy/winter stem compared to the active growth/summer stem by using a whole transcriptome analysis (GeneChip Poplar Genome Array; Affymetrix). Poplar Genome Array has a total of 61,251 probe sets, representing 57,423 poplar gene models, and allows us to interrogate a total of 41,558 unique gene models because of the probe set redundancy. Slight redundancy of the probe sets within a single chip gave us a unique opportunity to have internal comparisons of the particular genes. Very high reproducibility (R2 > 0.97) between the replicated samples was found. Further confirmation of the GeneChip data was made by semi-quantitative RT-PCR analysis using several genes showing the summer or winter biased expression. Based on these results, further analysis of winter stem transcriptome against summer stem was performed.

Dataset Information

RNA-seq circadian winter 2020

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