Real-time monitoring of gene expression reveals transcription kinetics of mammalian genes.

In mammals, many aspects of behavior and physiology, and in particular cellular metabolism, are coordinated by the circadian timing system. Molecular clocks are thought to rely on negative feedback loops in clock gene expression that engender oscillations in the accumulation of transcriptional regulatory proteins, such as the orphan receptor REV-ERBα. Circadian transcription factors then drive daily rhythms in the expression of clock-controlled output genes, for example genes encoding enzymes and regulators of cellular metabolism. To gain insight into clock output functions of REV-ERBα, we carried out genome-wide transcriptome profiling experiments with liver RNA from wild-type mice, Rev-erbα knock-out mice, or REV-ERBα overexpressing mice. On the basis of these genetic loss- and gain-of-function experiments, we concluded that REV-ERBα participates in the circadian modulation of sterol regulatory element-binding protein (SREBP) activity, and thereby in the daily expression of SREBP target genes involved in cholesterol and lipid metabolism. This control is exerted via the cyclic transcription of Insig2, encoding a trans-membrane protein that sequesters SREBP proteins to the endoplasmic reticulum membranes and thereby interferes with the proteolytic activation of SREBPs in Golgi membranes. REV-ERBα also participates in the cyclic expression of cholesterol-7α-hydroxylase (CYP7A1), the rate-limiting enzyme in converting cholesterol to bile acids. Our findings suggest that this control acts via the stimulation of LXR nuclear receptors by cyclically produced oxysterols. In conclusion, our study suggests that rhythmic cholesterol and bile acid metabolism is not just driven by alternating feeding–fasting cycles, but also by REV-ERBα, a component of the circadian clockwork circuitry. The mammalian circadian timing system has a hierarchical architecture: a central pacemaker in the brain's suprachiasmatic nucleus (SCN) synchronizes subsidiary oscillators present in most peripheral cell types. In both SCN neurons and peripheral cells, circadian oscillators are thought to rely on two negative feedback loops. A major feedback loop involves the two cryptochromes CRY1 and CRY2 and the two period proteins PER1 and PER2, which serve as transcriptional repressors for their own genes. An accessory feedback loop couples the expression and activity of the transcriptional activators CLOCK and BMAL1 to the expression of cryptochrome and period proteins. The orphan nuclear receptor REV-ERBα is a key player in this accessory feedback loop, in that it periodically represses Bmal1 transcription. In liver, molecular clocks mediate the temporal gating of metabolic processes. Here we demonstrate that hepatocyte clocks participate in the control of cholesterol and bile acid homeostasis. According to this scenario, REV-ERBα shapes the circadian expression pattern of insulin-induced gene 2 (INSIG2), a resident protein of the endoplasmic reticulum that interferes with the proteolytic activation of sterol response element binding proteins (SREBPs). In turn SREBPs govern the rhythmic expression of enzymes with key functions in sterol and fatty acid synthesis. The circadian production of sterols (in particular oxysterols) may engender the cyclic activation of LXR nuclear receptors, which serve as critical activators of Cyp7a1 transcription. CYP7A1, also known as cholesterol 7α-hydroxylase, catalyzes the rate-limiting step in bile acid synthesis.

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBα as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparβ/δ and the peroxisome proliferator-activated receptor α (PPARα) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.

In mammals, including humans, nearly all physiological processes are subject to daily oscillations that are governed by a circadian timing system with a complex hierarchical structure. The central pacemaker, residing in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus, is synchronized daily by photic cues transmitted from the retina to SCN neurons via the retinohypothalamic tract. In turn, the SCN must establish phase coherence between self-sustained and cell-autonomous oscillators present in most peripheral cell types. The synchronization signals (Zeitgebers) can be controlled more or less directly by the SCN. In mice and rats, feeding–fasting rhythms, which are driven by the SCN through rest–activity cycles, are the most potent Zeitgebers for the circadian oscillators of peripheral organs. Signaling through the glucocorticoid receptor and the serum response factor also participate in the phase entrainment of peripheral clocks, and these two pathways are controlled by the SCN independently of feeding–fasting rhythms. Body temperature rhythms, governed by the SCN directly and indirectly through rest–activity cycles, are perhaps the most surprising cues for peripheral oscillators. Although the molecular makeup of circadian oscillators is nearly identical in all cells, these oscillators are used for different purposes in the SCN and in peripheral organs.

Transcription Around the Clock: The biological clock that controls daily rhythms in mammalian physiology and behavior is thought to be regulated in large part by transcriptional events (see the Perspective by Doherty and Kay ). Koike et al. (p. 349 ; published online 30 August) produced a comprehensive analysis of these transcriptional events across the entire mouse liver genome over a 24-hour period. Only ∼22% of cycling messenger RNA transcripts were driven by de novo transcription, suggesting that posttranscriptional events also play an important regulatory role in the mammalian clock. Biological timing in organisms can also respond to rhythmic cues from the environment. Morf et al. (p. 379 , published online 23 August) explored how one such cue, cycles in ambient temperature, influence circadian timing in mammalian cells. Cold-inducible RNA–binding protein (CIRP) accumulates when body temperature is low. A systematic search for binding partners of CIRP identified RNA encoding core components of the circadian clock. Loss of CIRP decreased the amplitude of circadian gene expression and cells lacking CIRP adapted more quickly to temperature cycles.

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver. In mammalian organs such as the liver, many metabolic and physiological processes occur preferentially at specific times during the 24-hour daily cycle. The timing of these rhythmic functions depends on a complex interplay between the endogenous circadian clock and environmental timing cues relayed through the master circadian clock in the suprachiasmatic nucleus, or via feeding rhythms. These rhythms can be implemented on several regulatory levels, and here we aimed at a better understanding of the transcriptional and epigenetic changes that regulate diurnal rhythms. We performed genome-wide analysis of the locations of RNA polymerase II (Pol II) and the epigenetic histone modifications H3K4me3 and H3K36me3 at specific times of day, relating these data to mRNA expression levels. Our analyses show that Pol II transcriptional rhythms are biphasic in mouse liver, having predominant peak activities in the morning and evening. Moreover, dynamic changes in histone marks lag transcription rhythms genome-wide, indicating that the epigenetic landscape can be remodeled during the 24-hour cycle. Finally, a quantitative analysis of temporal Pol II and mRNA accumulation profiles indicates that posttranscriptional regulation significantly contributes to the amplitude and phase of mRNA accumulation profiles. While many studies have analyzed how transcription and chromatin states are modified during irreversible cell differentiation processes, our work highlights how these states can evolve reversibly in a system exhibiting periodicity in time.