Nascent-Seq reveals novel features of mouse circadian transcriptional regulation

RESEARCH ARTICLE elife.elifesciences.org Nascent-Seq reveals novel features of mouse circadian transcriptional regulation Jerome S Menet*, Joseph Rodriguez, Katharine C Abruzzi, Michael Rosbash* Howard Hughes Medical Institute, National Center for Behavioral Genomics, and Department of Biology Brandeis University, Waltham, United States Abstract A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues. DOI: 10.7554/eLife.00011.001 Introduction *For correspondence: rosbash@ brandeis.edu (MR); menet@ brandeis.edu (JSM) Competing interests: The authors have declared that no competing interests exist Funding: See page 23 Received: 22 June 2012 Accepted: 16 September 2012 Published: 13 November 2012 Reviewing editor: Todd C Mockler, Donald Danforth Plant Science Center, United States Copyright Menet et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Most organisms from bacteria to humans possess circadian rhythms, which generate oscillations in biochemistry, physiology and behavior. The circadian system in eukaryotes is based on cell-autonomous molecular oscillators, which rely on transcriptional feedback loops. In mammals, the transcription factor BMAL1 acts as a dimer with either CLOCK (CLK) or Neuronal PAS domain protein 2 (NPAS2) to activate the transcription of many genes, including the transcriptional repressors Period (Per1, Per2 and Per3) and Cryptochrome (Cry1 and Cry2). The PERs and CRYs are expressed, post-translationally modified, feedback to inhibit their own transcription and are then rhythmically degraded to lead to a new round of BMAL1:CLK or BMAL1:NPAS2 -mediated transcription (reviewed in Ko and Takahashi, 2006; Dardente and Cermakian, 2007). This temporal regulation of clock gene transcription cycles with a period of about 24 hr and probably underlies much of circadian biology. Over the past decade, clock gene transcriptional regulation has been described in many species and tissues, where it drives the rhythmic expression of a large fraction of the mRNA population (up to 10–15% of all mRNAs in a single mammalian tissue; Lowrey and Takahashi, 2004; Vollmers et al., 2009). Rhythmic mRNA expression has mostly been characterized by analyzing temporal changes of steady-state mRNA levels, using techniques such as microarrays (e.g., McDonald and Rosbash, 2001; Panda et al., 2002; Storch et al., 2002) and more recently high-throughput sequencing (Hughes et al., 2012). It is generally assumed that these rhythms in mRNA expression directly result from temporal changes in transcription. There are, however, a few reports indicating that post-transcriptional regulation contributes to rhythmic mRNA expression of several genes, including core clock genes (reviewed in Kojima et al., 2011; Staiger and Green, 2011; Staiger and Koster, 2011; Zhang et al., 2011), but this has never been studied in detail at the genome-wide level. Circadian post-transcriptional regulation may impact rhythmic mRNA expression at many different levels, such as mRNA splicing, stability and translation. For example, post-transcriptional events rhythmically regulate the mRNA half-life of the mammalian clock genes Per2 and Cry1 and the Drosophila clock gene per (So and Rosbash, Menet et al. eLife 2012;1:e00011. DOI: 10.7554/eLife.00011 1 of 25 Research article Genes and chromosomes | Genomics and evolutionary biology eLife digest Many biological processes oscillate with a period of roughly 24 hr, and the ability of organisms as diverse as bacteria and humans to maintain such circadian rhythms, even under conditions of continuous darkness, influences a range of phenomena, including sleep, migration and reproduction. One characteristic of circadian rhythms is that they can adjust to local time (with humans suffering from jet lag as they wait for this to happen). Experiments have shown that the circadian system in mammals relies on feedback loops that operate at the level of individual cells. These loops are controlled by two particular proteins, which comprise the transcription factor complex called BMAL1:CLK. Transcription factors cause particular sequences of bases in the DNA of cells to be transcribed into messenger RNA, thus starting the process by which target genes are expressed as proteins. In the case of BMAL1:CLK, these proteins are then modified, which inhibits any further transcription of the target genes. A reversal of these modifications is then followed by the synthesis of new proteins, which allows a new cycle of the transcription process to begin. The amounts of many messenger RNAs (mRNAs) in a cell also increases and decreases with a period of 24 hr, and it was generally assumed that this was due to the changes in the level of transcription. More recently, however, it was suggested that other processes, such as splicing and translation, might also contribute to rhythmic changes in the amount of mRNA associated with particular genes. Such post-transcriptional processes are known to have a role in other areas of cell biology, including aspects of the circadian system, but until very recently this had not been studied in detail for all genes. Now Menet et al. have directly assayed rhythmic transcription by measuring the amount of nascent mRNA being produced at a given time, six times a day, across all the genes in mouse liver cells using a high-throughput sequencing approach called Nascent-Seq. They compared this with the amount of liver mRNA expressed at six time points of the day. Although the authors found that many genes exhibit rhythmic mRNA expression in the mouse liver, about 70% of them did not show comparable transcriptional rhythms. Post-transcriptional regulation must, therefore, have a major role in the circadian system of mice and, presumably, other mammals. Menet et al. also found that the influence of CLK:BM (...truncated)