This review examines the connections between circadian and metabolic rhythms. Examples from a wide variety of well-studied organisms are used to illustrate some of the genetic and molecular pathways linking circadian timekeeping to metabolism. The principles underlying biological timekeeping by intrinsic circadian clocks are discussed briefly. Genetic and molecular studies have unambiguously identified the importance of gene expression feedback circuits to the generation of overt circadian rhythms. This is illustrated particularly well by the results of genome-wide expression studies, which have uncovered hundreds of clock-controlled genes in cyanobacteria, fungi, plants, and animals. The potential connections between circadian oscillations in gene expression and circadian oscillations in metabolic activity are a major focus of this review.

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study we examine the relationship between autonomous transcript oscillations and light-driven transcript responses. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12- to 48-h period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many light-responsive transcripts are preferentially expressed in the compound eyes and the phospholipase C component of phototransduction, NORPA (no receptor potential), is required for their light-dependent regulation. Although there is evidence for the existence of multiple molecular clock circuits in cyanobacteria, protists, plants, and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod. Daily changes in sunlight dramatically affect the environment of animals like the fruit fly, a genetic model. To anticipate these changes, the fruit fly possesses a circadian molecular clock that regulates its behavioral activity and other physiology according to the time of day. The clock's mechanism is comprised of genes and their products (proteins and RNAs). One way the clock modulates physiology is by regulating other genes' RNAs. The signature of a circadian RNA is that its levels oscillate once each day. The present study uses microarrays and a novel statistical strategy to resolve how many genes are oscillating in the fly head and verify that there is only a single transcriptional clock. However, this study also finds a large number of genes that are directly regulated by the presence of light. These genes respond to light even in the absence of a functioning clock. Indeed, the light response depends on a signaling component in the retina that has an established role in vision. This represents a new mechanism by which an animal's physiology may be dynamically tuned to daily time.

Circadian clocks are aligned to the environment via synchronizing signals, or Zeitgebers, such as daily light and temperature cycles, food availability, and social behavior. In this study, we found that genome-wide expression profiles from temperature-entrained flies show a dramatic difference in the presence or absence of a thermocycle. Whereas transcript levels appear to be modified broadly by changes in temperature, there is a specific set of temperature-entrained circadian mRNA profiles that continue to oscillate in constant conditions. There are marked differences in the biological functions represented by temperature-driven or circadian regulation. The set of temperature-entrained circadian transcripts overlaps significantly with a previously defined set of transcripts oscillating in response to a photocycle. In follow-up studies, all thermocycle-entrained circadian transcript rhythms also responded to light/dark entrainment, whereas some photocycle-entrained rhythms did not respond to temperature entrainment. Transcripts encoding the clock components Period, Timeless, Clock, Vrille, PAR-domain protein 1, and Cryptochrome were all confirmed to be rhythmic after entrainment to a daily thermocycle, although the presence of a thermocycle resulted in an unexpected phase difference between period and timeless expression rhythms at the transcript but not the protein level. Generally, transcripts that exhibit circadian rhythms both in response to thermocycles and photocycles maintained the same mutual phase relationships after entrainment by temperature or light. Comparison of the collective temperature- and light-entrained circadian phases of these transcripts indicates that natural environmental light and temperature cycles cooperatively entrain the circadian clock. This interpretation is further supported by comparative analysis of the circadian phases observed for temperature-entrained and light-entrained circadian locomotor behavior. Taken together, these findings suggest that information from both light and temperature is integrated by the transcriptional clock mechanism in the adult fly head. A key adaptation to life on Earth is provided by internal daily time-keeping mechanisms that allow anticipation of the alternations between night and day. To act as reliable time-keeping mechanisms, circadian clocks have to be able to synchronize to environmental time cues, maintain ∼24-h rhythms under constant conditions, run at approximately the same pace over a range of environmental temperatures, and efficiently communicate time-of-day information to other biological systems. Clock-controlled oscillations in gene expression play an essential role in producing overt circadian rhythms. For most organisms, light/dark cycles appear to constitute the most powerful entrainment cue, but daily temperature cycles have also been demonstrated to efficiently synchronize circadian rhythms. This study uses the fruit fly Drosophila melanogaster as a model to compare the clock-dependent and clock-independent daily gene expression rhythms produced in response to light/dark cycles versus temperature cycles. A broad temperature-driven expression program was found in the heads of both wild-type and arrhythmic mutant flies, but wild-type flies also exhibited a more specific temperature-entrained circadian expression response that resembled the circadian response following light entrainment. The phase relationship between the temperature- and light-entrained circadian rhythms suggests that in nature light and temperature act cooperatively to synchronize the circadian clock.

In yeast, the G1 cyclin Cln3 promotes cell cycle entry by activating the transcription factor SBF. In mammals, there is a parallel system for cell cycle entry in which cyclin dependent kinase (CDK) activates transcription factor E2F/Dp. Here we show that Cln3 regulates SBF by at least two different pathways, one involving the repressive protein Whi5, and the second involving Stb1. The Rpd3 histone deacetylase complex is also involved. Cln3 binds to SBF at the CLN2 promoter, and removes previously bound Whi5 and histone deacetylase. Adding extra copies of the SBF binding site to the cell delays Start, possibly by titrating Cln3. Since Rpd3 is the yeast ortholog of mammalian HDAC1, there is now a virtually complete analogy between the proteins regulating cell cycle entry in yeast (SBF, Cln3, Whi5 and Stb1, Rpd3) and mammals (E2F, Cyclin D, Rb, HDAC1). The cell may titrate Cln3 molecules against the number of SBF binding sites, and this could be the underlying basis of the size-control mechanism for Start. Cells seem to divide only after they have grown “big enough.” Entry into the cell cycle, at a point called Start in budding yeast, is triggered by activation of the Cln3 cyclin-dependent kinase (CDK), which in turn activates downstream transcription. We find that the Cln3-CDK acts through a histone deacetylase, as well as through the previously discovered repressor Whi5, to activate the SBF transcription factor and trigger entry into the cell cycle. The system is strikingly similar to the one in mammalian cells, which relies on Cyclin D, CDK, the transcription factor E2F, its repressor Rb, and the histone deacetylase system. There is preliminary evidence that as the yeast cell grows in size, the increasing number of Cln3 molecules is titrated against the fixed number of Cln3-CDK-SBF binding sites in genomic DNA, and that this cell size-dependent titration could be the mechanism that makes cell cycle entry dependent on cell size.