A role for microRNAs in the Drosophila circadian clock.

Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs-clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.

Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component.

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila.

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cry(b). The period mutant per(L) manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of per(L) flies, because a per(L); cry(b) strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.

A small conserved domain of Drosophila PERIOD is important for circadian phosphorylation, nuclear localization, and transcriptional repressor activity.

We identify in this study a 27-amino-acid motif which is conserved between the Drosophila melanogaster period protein (PER) and the three mammalian PERs. Characterization of PER lacking this motif (PER Delta) shows that it is important for phosphorylation of Drosophila PER by casein kinase I epsilon (CKI epsilon; doubletime protein or DBT) and CKII. S2 cell assays indicate that the domain also contributes significantly to PER nuclear localization as well as to PER transcriptional repressor activity. These two phenomena appear linked, since PER Delta transcriptional repressor activity in S2 cells was restored when nuclear localization was facilitated. Two less direct assays of PER Delta activity in flies can be interpreted similarly. The separate assay of nuclear import and export suggests that the domain functions in part to facilitate PER phosphorylation within the cytoplasm, which in turn promotes nuclear entry. As there is evidence that the kinases also function within the nucleus to promote transcriptional repression, we suggest that there is a subsequent collaboration between phosphorylated PER and the kinases to repress CLK-CYC activity, probably through the phosphorylation of CLK. This is then followed by additional PER phosphorylation, which occurs within the nucleus and leads to PER degradation.

The Drosophila circadian network is a seasonal timer.

Previous work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. We have manipulated separately the circadian photoentrainment pathway within E- and M-cells and show that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season.

A resetting signal between Drosophila pacemakers synchronizes morning and evening activity.

The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: we and others have recently shown that a group of adult brain clock neurons expresses the neuropeptide PDF and controls morning activity (small LN(v) cells; M-cells), whereas another group of clock neurons controls evening activity (CRY+, PDF- cells; E-cells). We have generated transgenic mosaic animals with different circadian periods in morning and evening cells. Here we show, by behavioural and molecular assays, that the six canonical groups of clock neurons are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity.

Assaying the Drosophila negative feedback loop with RNA interference in S2 cells.

Transcriptional negative feedback loops play a critical role in the molecular oscillations of circadian genes and contribute to robust behavioral rhythms. In one key Drosophila loop, CLOCK and CYCLE (CLK/CYC) positively regulate transcription of period (per). The period protein (PER) then represses this transcriptional activation, giving rise to the molecular oscillations of per RNA and protein. There is evidence that links molecular oscillations with behavioral rhythms, suggesting that PER also regulates the expression of downstream genes, ultimately resulting in proper behavior rhythmicity. Phosphorylation of PER has also been shown to be critical for rhythms. Doubletime (DBT) and casein kinase II (CKII) have been implicated in the phosphorylation of PER, which affects its stability as well as nuclear localization. We investigated the role of these kinases on PER transcriptional repression using the Drosophila S2 cell line in combination with RNA interference (RNAi) to knock down specific gene expression. This article describes the methods used to study PER repression activity in the S2 cell system as well as to exploit RNAi in this system. We also include protocols for immunocytochemistry and the application of leptomycin to differentiate direct effects on repression from indirect effects on subcellular localization. Finally, we discuss the generation of stable cell lines in the S2 cell system; these will be useful for experiments requiring homogeneous cell populations.

The doubletime and CKII kinases collaborate to potentiate Drosophila PER transcriptional repressor activity.

In all genetically studied model organisms, a negative feedback loop of gene expression contributes to the circadian rhythm mechanism. In the Drosophila system, it has been proposed that the delay between the synthesis and function of clock proteins is due to phosphorylation-regulated nuclear entry. To test this hypothesis, we assayed the relationship between PER phosphorylation, nuclear localization, and transcriptional repression activity in cultured S2 cells. The results indicate that the two putative PER kinases DBT and CKII work together to phosphorylate PER and increase repression activity. Experiments combining kinase inhibition with inhibition of PER nuclear export suggest that phosphorylation directly affects PER repression activity and that PER nuclear localization is an indirect consequence of the association of active PER with DNA or chromatin. This interpretation suggests further that the circadian regulation of PER nuclear localization in flies reflects changes in PER transcriptional activity rather than in PER nuclear import or export activity.