The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse.

In mammals, the circadian oscillator generates approximately 24-h rhythms in feeding behavior, even under constant environmental conditions. Livers of mice held under constant darkness exhibit circadian rhythm in abundance in up to 15% of expressed transcripts. Therefore, oscillations in hepatic transcripts could be driven by rhythmic food intake or sustained by the hepatic circadian oscillator, or a combination of both. To address this question, we used distinct feeding and fasting paradigms on wild-type (WT) and circadian clock-deficient mice. We monitored temporal patterns of feeding and hepatic transcription. Both food availability and the temporal pattern of feeding determined the repertoire, phase, and amplitude of the circadian transcriptome in WT liver. In the absence of feeding, only a small subset of transcripts continued to express circadian patterns. Conversely, temporally restricted feeding restored rhythmic transcription of hundreds of genes in oscillatordeficient mouse liver. Our findings show that both temporal pattern of food intake and the circadian clock drive rhythmic transcription, thereby highlighting temporal regulation of hepatic transcription as an emergent property of the circadian system. CREB ͉ metabolism ͉ mouse liver ͉ circadian rhythms

Circadian rhythms help organisms adapt to predictable daily changes in their environment. Light resets the phase of the underlying oscillator to maintain the organism in sync with its surroundings. Light also affects the amplitude of overt rhythms. At a critical phase during the night, when phase shifts are maximal, light can reduce rhythm amplitude to nearly zero, whereas in the subjective day, when phase shifts are minimal, it can boost amplitude substantially. To explore the cellular basis for this reciprocal relationship between phase shift and amplitude change, we generated a photoentrainable, cell-based system in mammalian fibroblasts that shares several key features of suprachiasmatic nucleus light entrainment. Upon light stimulation, these cells exhibit calcium/cyclic AMP responsive element-binding (CREB) protein phosphorylation, leading to temporally gated acute induction of the Per2 gene, followed by phase-dependent changes in phase and/or amplitude of the PER2 circadian rhythm. At phases near the PER2 peak, photic stimulation causes little phase shift but enhanced rhythm amplitude. At phases near the PER2 nadir, on the other hand, the same stimuli cause large phase shifts but dampen rhythm amplitude. Real-time monitoring of PER2 oscillations in single cells reveals that changes in both synchrony and amplitude of individual oscillators underlie these phenomena.circadian rhythm ͉ melanopsin ͉ singularity C ircadian oscillators enable organisms to anticipate and synchronize their behavior and physiology to periodic changes in the environment. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker, coordinating tissue-autonomous oscillators to generate overt rhythms (1, 2). At the molecular level, circadian rhythms are generated by a transcription-translation feedback loop. In this circuit, the transcriptional activators CLOCK and BMAL1 drive expression of the Cryptochrome 1 (Cry1), Cry2, Period 1 (Per1) and Per2 genes, whose protein products, in turn, repress CLOCK/BMAL1 transcriptional activity (reviewed in ref.3). The phase of rhythms in mRNA and protein levels of these repressors, particularly Per2, reflects the phase of the oscillator (4).Light entrains the SCN pacemaker, which relays phase information to peripheral oscillators via humoral and synaptic mechanisms. The retinorecipient cells of the SCN receive direct synaptic input from the intrinsically photosensitive retinal ganglion cells (ipRGCs) that express melanopsin. Upon photostimulation, the ipRGCs release neurotransmitters, which act via their cognate receptors to phosphorylate the calcium/cAMPresponsive element-binding protein (CREB). In turn, transcriptionally active phospho-CREB (pCREB) binds to Per1 and Per2 promoter CRE sites and activates transcription, subsequently resetting the phase of the molecular oscillator (reviewed in refs. 5 and 6). Mice bearing a targeted mutation in the CREB phosphorylation site (7) or perturbed Per function (8, 9) exhibit attenuated phase resetting.

In both the suprachiasmatic nucleus and peripheral tissues, the circadian oscillator drives rhythmic transcription of downstream target genes. Recently, a number of studies have used DNA microarrays to systematically identify oscillating transcripts in plants, fruit flies, rats and mice. These studies have identified several dozen to many hundred rhythmically expressed genes by sampling tissues every four hours for one, two, or more days. To extend this work, we have performed DNA microarray analysis on RNA derived from the mouse pituitary sampled every hour for two days. COSOPT and Fisher's G-test were employed at a false-discovery rate less than 5% to identify more than 250 genes in the pituitary that oscillate with a 24-hour period length. We found that increasing the frequency of sampling across the circadian day dramatically increased the statistical power of both COSOPT and Fisher's G-test, resulting in considerably more high-confidence identifications of rhythmic transcripts than previously described. Finally, to extend the utility of these data sets, a web-based resource has been constructed at http://wasabi.itmat.upenn.edu/circa/mouse that is freely available to the research community.