Charalambos P. Kyriacou

Circadian Cycling of the Mouse Liver Transcriptome, as Revealed by cDNA Microarray, Is Driven by the Suprachiasmatic Nucleus

BACKGROUND Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecular basis of circadian timing in peripheral tissue is not well understood. We used a custom-made cDNA microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions. RESULTS Using two independent tissue sampling and hybridization regimes, we show that approximately 9% of the 2122 genes studied show robust circadian cycling in the liver. These transcripts were categorized by their phase of abundance, defining clusters of day- and night-related genes, and also by the function of their products. Circadian regulation of genes was tissue specific, insofar as novel rhythmic liver genes were not necessarily rhythmic in the brain, even when expressed in the SCN. The rhythmic transcriptome in the periphery is, nevertheless, dependent on the SCN because surgical ablation of the SCN severely dampened or destroyed completely the cyclical expression of both canonical circadian genes and novel genes identified by microarray analysis. CONCLUSIONS Temporally complex, circadian programming of the transcriptome in a peripheral organ is imposed across a wide range of core cellular functions and is dependent on an interaction between intrinsic, tissue-specific factors and extrinsic regulation by the SCN central pacemaker.

Peroxiredoxins are conserved markers of circadian rhythms

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Inhibition of calcium/calmodulin-dependent protein kinase in Drosophila disrupts behavioral plasticity

One of the major mediators of calcium action in neurons is the multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase), an enzyme with the capability of directly regulating its own activity by autophosphorylation. To assess the involvement of CaM kinase in experience-dependent behavior in an intact animal, we have designed a specific peptide inhibitor of CaM kinase and made transgenic Drosophila that express it under control of an inducible promoter. These flies fail to learn normally in two behavioral plasticity paradigms: acoustic priming, a nonassociative measure of sensitization, and courtship conditioning, a measure of associative learning. The magnitude of the learning defect in the associative paradigm appears to be proportional to the level of expression of the peptide gene in the two transgenic lines and can be increased by heat shock induction of gene expression. These results suggest that CaM kinase activity is required for plastic behaviors in an intact animal.

Natural Selection Favors a Newly Derived timeless Allele in Drosophila melanogaster

Circadian and other natural clock-like endogenous rhythms may have evolved to anticipate regular temporal changes in the environment. We report that a mutation in the circadian clock gene timeless in Drosophila melanogaster has arisen and spread by natural selection relatively recently in Europe. We found that, when introduced into different genetic backgrounds, natural and artificial alleles of the timeless gene affect the incidence of diapause in response to changes in light and temperature. The natural mutant allele alters an important life history trait that may enhance the flys adaptation to seasonal conditions.

P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic drosophila melanogaster

Mutations at the period (per) locus of Drosophila melanogaster disrupt several biological rhythms. Molecular cloning of DNA sequences encompassing the per+ locus has allowed germ-line transformation experiments to be carried out. Certain subsegments of the per region, transduced into the genome of arrhythmic pero flies, restore rhythmicity in circadian locomotor behavior and the males courtship song.

Unexpected features of Drosophila circadian behavioural rhythms under natural conditions

Circadian clocks have evolved to synchronize physiology, metabolism and behaviour to the 24-h geophysical cycles of the Earth. Drosophila melanogaster’s rhythmic locomotor behaviour provides the main phenotype for the identification of higher eukaryotic clock genes. Under laboratory light–dark cycles, flies show enhanced activity before lights on and off signals, and these anticipatory responses have defined the neuronal sites of the corresponding morning (M) and evening (E) oscillators. However, the natural environment provides much richer cycling environmental stimuli than the laboratory, so we sought to examine fly locomotor rhythms in the wild. Here we show that several key laboratory-based assumptions about circadian behaviour are not supported by natural observations. These include the anticipation of light transitions, the midday ‘siesta’, the fly’s crepuscular activity, its nocturnal behaviour under moonlight, and the dominance of light stimuli over temperature. We also observe a third major locomotor component in addition to M and E, which we term ‘A’ (afternoon). Furthermore, we show that these natural rhythm phenotypes can be observed in the laboratory by using realistic temperature and light cycle simulations. Our results suggest that a comprehensive re-examination of circadian behaviour and its molecular readouts under simulated natural conditions will provide a more authentic interpretation of the adaptive significance of this important rhythmic phenotype. Such studies should also help to clarify the underlying molecular and neuroanatomical substrates of the clock under natural protocols.

Glucocorticoid signaling synchronizes the liver circadian transcriptome.

Circadian control of physiology is mediated by local, tissue‐based clocks, synchronized to each other and to solar time by signals from the suprachiasmatic nuclei (SCN), the master oscillator in the hypothalamus. These local clocks coordinate the transcription of key pathways to establish tissue‐specific daily metabolic programs. How local transcriptomes are synchronized across the organism and their relative contribution to circadian output remain unclear. In the present study we showed that glucocorticoids alone are able to synchronize expression of about 60% of the circadian transcriptome. We propose that synchronization occurs directly by the action of glucocorticoids on a diverse range of downstream targets and indirectly by regulating the core clock genes mPer1, Bmal1, mCry1, and Dbp. We have identified the pivotal liver transcription factor, HNF4α, as a mediator of circadian and glucocorticoid‐regulated transcription, showing that it is a key conduit for downstream targeting. Conclusion: We have demonstrated that by orchestrating transcriptional cascades, glucocorticoids are able to direct synchronization of a diverse range of functionally important circadian genes. (HEPATOLOGY 2007;45:1478–1488.)

Germ-Line Transformation Involving DNA from the period Locus in Drosophila melanogaster: Overlapping Genomic Fragments that Restore Circadian and Ultradian Rhythmicity to per0 and per− Mutants

P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the flys circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNAs oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.

A latitudinal cline in a Drosophila clock gene

The clock gene period determines biological rhythmicity in Drosophila melanogaster and encodes a protein characterized by an alternating series of threonine-glycine pairs. The minisatellite region encoding the threonine-glycine repeat is polymorphic in length in natural Drosophila melanogaster populations. In this paper we report the geographical analysis of this polymorphism within Europe and North Africa. A robust clinal pattern is observed along a north-south axis. We suggest the possibility that the length polymorphism could be maintained by thermal selection because the threonine-glycine region has been shown to provide thermostability to the circadian phenotype.

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY

BACKGROUND The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.