Nick R. J. Glossop

vrille, Pdp1, and dClock Form a Second Feedback Loop in the Drosophila Circadian Clock

The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, dCLOCK/CYCLE activates period expression, and PERIOD protein then inhibits dCLOCK/CYCLE activity. dClock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related transcription factors whose expression is directly activated by dCLOCK/CYCLE. We show here that VRI and PDP1 proteins feed back and directly regulate dClock expression. Repression of dClock by VRI is separated from activation by PDP1 since VRI levels peak 3-6 hours before PDP1. Rhythmic vri transcription is required for molecular rhythms, and here we show that the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, VRI and PDP1, together with dClock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of dClock, and probably of other genes, to generate accurate circadian rhythms.

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

VRILLE Feeds Back to Control Circadian Transcription of Clock in the Drosophila Circadian Oscillator

The Drosophila circadian oscillator consists of interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLK and CYCLE (CYC) activate per and tim transcription at the same time as they repress Clk transcription, thus controlling the opposite cycling phases of these transcripts. CLK-CYC directly bind E box elements to activate transcription, but the mechanism of CLK-CYC-dependent repression is not known. Here we show that a CLK-CYC-activated gene, vrille (vri), encodes a repressor of Clk transcription, thereby identifying vri as a key negative component of the Clk feedback loop in Drosophilas circadian oscillator. The blue light photoreceptor encoding cryptochrome (cry) gene is also a target for VRI repression, suggesting a broader role for VRI in the rhythmic repression of output genes that cycle in phase with Clk.

Drosophila CLOCK Protein Is under Posttranscriptional Control and Influences Light-Induced Activity

In the Drosophila circadian clock, daily cycles in the RNA levels of dclock (dClk) are antiphase to those of period (per). We altered the timing/levels of dClk expression by generating transgenic flies whereby per circadian regulatory sequences were used to drive rhythmic transcription of dClk. The results indicate that posttranscriptional mechanisms make substantial contributions to the temporal changes in the abundance of the dCLK protein. Circadian regulation is largely unaffected in the transgenic per-dClk flies despite higher mean levels of dCLK. However, in per-dClk flies the duration of morning activity is lengthened in light-dark cycles and light pulses evoke longer lasting bouts of activity. Our findings suggest that, in addition to a role in generating circadian rhythms, dCLK modulates the direct effects of light on locomotion.

The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

Analysis of the Drosophila Clock Promoter Reveals Heterogeneity in Expression between Subgroups of Central Oscillator Cells and Identifies a Novel Enhancer Region

The CLOCK-CYCLE (CLK-CYC) heterodimer lies at the heart of the circadian oscillator mechanism in Drosophila, yet little is known about the identity of transcription factors that regulate the expression of Clk and/or cyc. Here, the authors have used a transgenic approach to isolate regions of the Clk locus that are necessary for expression in central oscillator neurons in the adult fly brain. This analysis shows that central clock cells can be subdivided into 2 distinct groups based on Clk gene regulation. Expression in the lateral neuron (LN), dorsal neuron 1 anterior (DN1a) and 2 (DN2) clusters requires cis-elements located in a 122 base-pair (bp) region (–206 to –84) of the Clk promoter. Expression in the remaining dorsal neurons, 1 posterior (DN1p) and 3 (DN3) and the lateral posterior neurons (LPN), requires regulatory elements located in the –856 to –206 region. In addition, expression in photoreceptors of the compound eye is enhanced by cis-elements located in a 3rd region of the Clk locus (–1982 to –856). This region also enhances expression in nonoscillator cells in the brain including the Kenyon cells, but expression in these neurons is suppressed by regulatory sites located further upstream of –1982. The authors’ analysis reveals clear heterogeneity in Clk gene expression in the adult brain and provides a necessary focus to isolate novel transcription factors that bind at the Clk locus to regulate expression in different oscillator neuron subgroups. These results also suggest that the DN1a/DN2 neurons may have more molecular commonality with the LNs than they do with the DN1p/DN3/LPN neurons. Finally, this analysis has generated new transgenic lines that will enable genes to be misexpressed in subgroups of central oscillator cells that have previously been resistant to discrete genetic manipulation. Hence, these lines provide important new tools to facilitate a more complete dissection of the neural network that regulates output rhythms in physiology and behavior.

Circadian timekeeping in Drosophila melanogaster and Mus musculus

The discovery of the period gene mutants in 1971 provided the first evidence that daily rhythms in the sleep-wake cycle of a multicellular organism, the fruit fly Drosophila melanogaster, had an underlying genetic basis. Subsequent research has established that the biological clock mechanism in flies and mammals is strikingly similar and functions as a bimodal switch, simultaneously turning on one set of genes and turning off another set and then reversing the process every 12 h. In this chapter, the current model of the clock mechanism in Drosophila will be presented. This relatively basic model will then be used to outline the general rules that govern how the biological clock operates in mammals.

Effects of TWIN-OF-EYELESS on Clock Gene Expression and Central-Pacemaker Neuron Development in Drosophila.

Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LNvs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LNvs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs.