A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation.DOI: http://dx.doi.org/10.7554/eLife.02510.001
Many aspects of behavior and physiology, including sleep/ awake cycles and hormone levels, keep a rhythm with about a 24-h period, even under constant conditions without any external time cues (1). Circadian rhythms are generated by a self-sustaining time-measuring system called the circadian clock. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) functions as the master clock, and circadian clocks are also located in peripheral tissues such as the liver (2-5). In individual cells, clock genes and their products form transcriptional/translational feedback loops (6). The basic helix-loop-helix (bHLH)-PAS transcription factors CLOCK and BMAL1 play a role as positive factors in the loops, and the heterodimer of these proteins binds to the CACGTG E-box or related E-box-like sequences to transactivate a wide range of target genes, including Per and Cry (7-10). Translated PER and CRY proteins then bind to the CLOCK-BMAL1 complex, leading to the suppression of E-box-dependent transactivation. This negative-feedback mechanism forms a molecular clock generating circadian rhythms. In addition to the Ebox element, the D-box element and the REV-ERB/ROR-binding element (RRE) form a regulatory network of gene expression, governing coordinately circadian transcriptional oscillations (11,12). The D-box element is activated and repressed by DBP and E4BP4, respectively, while RRE is activated and repressed by RORs and REV-ERBs, respectively.During the circadian cycling of the transcriptional/translational steps, posttranslational modifications, such as phosphorylation, regulate the clock proteins, in terms of activity, stability, localization, and interaction (13). It was reported previously that CLOCK and BMAL1 are phosphorylated in a time-of-day-dependent manner (14)(15)(16)(17). CLOCK phosphorylation at its DNA-binding domain (16, 18) may be important for rhythmic inhibition of the ability of the CLOCK-BMAL1 complex to bind to the E-box element. This is consistent with the observation that the CLOCK-BMAL1 complex rhythmically dissociates from the E-box in the locus of the Dbp gene (19). Here we found in vivo binding sites of CLOCK protein in the mouse liver in a genome-wide manner by chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis. Previous ChIP-Seq studies of circadian clocks confirmed CLOCK-BMAL1 binding to canonical motifs instead of finding all potential binding motifs (20)(21)(22)(23). In this study, significant CLOCK-binding motifs were comprehensively examined by developing a bioinformatics method, MOCCS (motif centrality analysis of ChIP-Seq), which analyzes the frequency distribution of DNA sequences centered at DNA-binding sites found by ChIPSeq analyses. In parallel, all the rhythmic transcripts in the liver were identified by circadian deep-sequencing analysis of poly(A)-tailed RNA and small RNA. Based on these data, we demonstrate the functional importance of rhythmic posttranscriptional regulations, such as microRNA (miRNA)-mediated gene silencing, in dynamic circadian RNA rhythms.
MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. MiRNAs are implicated in various biological processes associated with obesity, including adipocyte differentiation and lipid metabolism. We used a neuronal-specific inhibition of miRNA maturation in adult mice to study the consequences of miRNA loss on obesity development. Camk2a-CreERT2 (Cre+) and floxed Dicer (Dicerlox/lox) mice were crossed to generate tamoxifen-inducible conditional Dicer knockouts (cKO). Vehicle- and/or tamoxifen-injected Cre+;Dicerlox/lox and Cre+;Dicer+/+ served as controls. Four cohorts were used to a) measure body composition, b) follow food intake and body weight dynamics, c) evaluate basal metabolism and effects of food deprivation, and d) assess the brain transcriptome consequences of miRNA loss. cKO mice developed severe obesity and gained 18 g extra weight over the 5 weeks following tamoxifen injection, mainly due to increased fat mass. This phenotype was highly reproducible and observed in all 38 cKO mice recorded and in none of the controls, excluding possible effects of tamoxifen or the non-induced transgene. Development of obesity was concomitant with hyperphagia, increased food efficiency, and decreased activity. Surprisingly, after reaching maximum body weight, obese cKO mice spontaneously started losing weight as rapidly as it was gained. Weight loss was accompanied by lowered O2-consumption and respiratory-exchange ratio. Brain transcriptome analyses in obese mice identified several obesity-related pathways (e.g. leptin, somatostatin, and nemo-like kinase signaling), as well as genes involved in feeding and appetite (e.g. Pmch, Neurotensin) and in metabolism (e.g. Bmp4, Bmp7, Ptger1, Cox7a1). A gene cluster with anti-correlated expression in the cerebral cortex of post-obese compared to obese mice was enriched for synaptic plasticity pathways. While other studies have identified a role for miRNAs in obesity, we here present a unique model that allows for the study of processes involved in reversing obesity. Moreover, our study identified the cortex as a brain area important for body weight homeostasis.
A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation.
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