NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed.

The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of PERIOD (PER) proteins. Using Drosophila, we identify a time-delay circuit based on hierarchical phosphorylation that controls the daily downswing in PER abundance. Phosphorylation by the NEMO/NLK kinase at the "per-short" domain on PER stimulates phosphorylation by DOUBLETIME (DBT/CK1δ/ɛ) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by DBT at other more distal sites on PER, including those required for recognition by the F box protein SLIMB/ß-TrCP and proteasomal degradation. Highly phosphorylated PER has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing PER susceptibility to degradation. Our findings identify NEMO as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed.

Parallel clinal variation in the mid-day siesta of Drosophila melanogaster implicates continent-specific targets of natural selection.

Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as ambient temperature rises, an adaptive response aimed at minimizing exposure to heat. Mid-day siesta levels are partly regulated by the thermosensitive splicing of a small intron (termed dmpi8) found in the 3' untranslated region (UTR) of the circadian clock gene period (per). Using the well-studied D. melanogaster latitudinal cline along the eastern coast of Australia, we show that flies from temperate populations sleep less during the day compared to those from tropical regions. We identified combinations of four single nucleotide polymorphisms (SNPs) in the 3' UTR of per that yield several different haplotypes. The two most abundant of these haplotypes exhibit a reciprocal tropical-temperate distribution in relative frequency. Intriguingly, transgenic flies with the major tropical isoform manifest increased daytime sleep and reduced dmpi8 splicing compared to those carrying the temperate variant. Our results strongly suggest that for a major portion of D. melanogaster in Australia, thermal adaptation of daily sleep behavior included spatially varying selection on ancestrally derived polymorphisms in the per 3' UTR that differentially control dmpi8 splicing efficiency. Prior work showed that African flies from high altitudes manifest reduced mid-day siesta levels, indicative of parallel latitudinal and altitudinal adaptation across continents. However, geographical variation in per 3' UTR haplotypes was not observed for African flies, providing a compelling case for inter-continental variation in factors targeted by natural selection in attaining a parallel adaptation. We propose that the ability to calibrate mid-day siesta levels to better match local temperature ranges is a key adaptation contributing to the successful colonization of D. melanogaster beyond its ancestral range in the lowlands of Sub-Saharan Africa.

A role for O-GlcNAcylation in setting circadian clock speed.

Post-translational modifications of one or more central "clock" proteins, most notably time-of-day-dependent changes in phosphorylation, are critical for setting the pace of circadian (≅24 h) clocks. In animals, PERIOD (PER) proteins are the key state variable regulating circadian clock speed and undergo daily changes in abundance and cytoplasmic-nuclear distribution that are partly driven by a complex phosphorylation program. Here, we identify O-GlcNAcylation (O-GlcNAc) as a critical post-translational modification in circadian regulation that also contributes to setting clock speed. Knockdown or overexpression of Drosophila O-GlcNAc transferase (ogt) in clock cells either shortens or lengthens circadian behavioral rhythms, respectively. The Drosophila PERIOD protein (dPER) is a direct target of OGT and undergoes daily changes in O-GlcNAcylation, a modification that is mainly observed during the first half of the night, when dPER is predominantly located in the cytoplasm. Intriguingly, the timing of when dPER translocates from the cytoplasm to the nucleus is advanced or delayed in flies, wherein ogt expression is reduced or increased, respectively. Our results suggest that O-GlcNAcylation of dPER contributes to setting the correct pace of the clock by delaying the timing of dPER nuclear entry. In addition, OGT stabilizes dPER, suggesting that O-GlcNAcylation has multiple roles in circadian timing systems.

A morning-induced, phosphorylation-gated repressor times evening gene expression: a new way for circadian clocks to use an old trick.

In this issue of Molecular Cell, Sancar et al. (2011) show that a morning-induced transcriptional repressor with a phosphorylation-gated half-life is a key cog in driving evening gene expression, adding new insights into how circadian clocks achieve phase-specific gene expression.

A master CLOCK hard at work brings rhythm to the transcriptome.

In this issue of Genes & Development, Abruzzi et al. (pp. 2374-2386) use chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) to show that physical interactions between circadian (≅24-h) clock machineries and genomes are more widespread than previously thought and provide novel insights into how clocks drive daily rhythms in global gene expression.

The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila.

Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription.

Mid-day siesta in natural populations of D. melanogaster from Africa exhibits an altitudinal cline and is regulated by splicing of a thermosensitive intron in the period clock gene.

BACKGROUND: Many diurnal animals exhibit a mid-day 'siesta', generally thought to be an adaptive response aimed at minimizing exposure to heat on warm days, suggesting that in regions with cooler climates mid-day siestas might be a less prominent feature of animal behavior. Drosophila melanogaster exhibits thermal plasticity in its mid-day siesta that is partly governed by the thermosensitive splicing of the 3'-terminal intron (termed dmpi8) from the key circadian clock gene period (per). For example, decreases in temperature lead to progressively more efficient splicing, which increasingly favors activity over sleep during the mid-day. In this study we sought to determine if the adaptation of D. melanogaster from its ancestral range in the lowlands of tropical Africa to the cooler temperatures found at high altitudes involved changes in mid-day sleep behavior and/or dmpi8 splicing efficiency. RESULTS: Using natural populations of Drosophila melanogaster from different altitudes in tropical Africa we show that flies from high elevations have a reduced mid-day siesta and less consolidated sleep. We identified a single nucleotide polymorphism (SNP) in the per 3' untranslated region that has strong effects on dmpi8 splicing and mid-day sleep levels in both low and high altitude flies. Intriguingly, high altitude flies with a particular variant of this SNP exhibit increased dmpi8 splicing efficiency compared to their low altitude counterparts, consistent with reduced mid-day siesta. Thus, a boost in dmpi8 splicing efficiency appears to have played a prominent but not universal role in how African flies adapted to the cooler temperatures at high altitude. CONCLUSIONS: Our findings point towards mid-day sleep behavior as a key evolutionary target in the thermal adaptation of animals, and provide a genetic framework for investigating daytime sleep in diurnal animals which appears to be driven by mechanisms distinct from those underlying nighttime sleep.

Phosphorylation of a central clock transcription factor is required for thermal but not photic entrainment.

Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk(out) flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.

Phosphorylation of the transcription activator CLOCK regulates progression through a ∼ 24-h feedback loop to influence the circadian period in Drosophila.

Circadian (≅ 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.

DAYWAKE implicates novel roles for circulating lipid-binding proteins as extracerebral regulators of daytime wake-sleep behavior.

Sleep during the midday, commonly referred to as siesta, is a common trait of animals that mainly sleep during the night. Work using Drosophila led to the identification of the daywake (dyw) gene, found to have anti-siesta activity. Herein, we show that the DYW protein undergoes signal peptide-dependent secretion, is present in the circulatory system, and accumulates in multiple organs, but, surprisingly, it is not detected in the brain where wake-sleep centers are located. The abundance of DYW in adult flies is regulated by age, sex, temperature, and the splicing efficiency of a nearby thermosensitive intron. We suggest that DYW regulates daytime wake-sleep balance in an indirect, extracerebral manner, via a multi-organ network that interfaces with the circulatory system.

Did a small thermosensitive intron contribute to the temperate adaptation of Drosophila melanogaster?

Drosophila melanogaster was first used for research in the early 1900's by scientists located in the northeastern corridor of the United States, gaining prominence with the establishment of the famous "fly room" by Thomas Hunt Morgan at Columbia University circa1908. Several reasons for using D. melanogaster in research are well known; easy and inexpensive to breed, short lifespan, amongst others. But why was this insect species flourishing in a temperate northeast region of the New World during the late 1800's when they originated in the tropical forests of sub-Saharan Africa millions of years ago? The purpose of this review is to provide an overview of the experimental underpinnings for a temperature sensitive mechanism that likely contributed to the rather unique ability of Drosophila melanogaster to successfully colonize temperate regions on a global scale. It also furnishes an interesting historical insight into how ancestral genetics serendipitously held the keys to the journey of D. melanogaster becoming such a popular research organism. While numerous papers have been published detailing different aspects of the work, this is the first comprehensive review. Herein, I discuss the discovery of a small thermosensitive intron in D. melanogaster (termed dmpi8) that controls midday siesta levels. Like many day-active animals, Drosophila exhibits a robust genetically based midday siesta that is protective in warm climates. Yet long bouts of daytime inactivity might be counterproductive in temperate climates, especially since daylength in these regions is shorter during the cooler months. Evidence discussed in this review strongly indicates that targeting of dmpi8 splicing efficiency by natural selection enhanced the ability of D. melanogaster to scale daytime sleep levels commensurate with a wide range of local climates. Surprisingly, dmpi8 splicing regulates midday siesta levels in trans by controlling the expression of a nearby anti-siesta gene called daywake. The "fortuitous" genetic arrangement of a thermosensitive intron in proximity to an anti-siesta gene might have contributed to the cosmopolitan nature of D. melanogaster and its historical journey in becoming a popular research organism.

A hierarchical phosphorylation cascade that regulates the timing of PERIOD nuclear entry reveals novel roles for proline-directed kinases and GSK-3beta/SGG in circadian clocks.

The daily timing of when PERIOD (PER) proteins translocate from the cytoplasm to the nucleus is a critical step in clock mechanisms underpinning circadian rhythms in animals. Numerous lines of evidence indicate that phosphorylation plays a prominent role in regulating various aspects of PER function and metabolism, including changes in its daily stability and subcellular distribution. In this report, we show that phosphorylation of serine 661 (Ser661) by a proline-directed kinase(s) is a key phospho-signal on the Drosophila PER protein (dPER) that regulates the timing of its nuclear accumulation. Mutations that block phosphorylation at Ser661 do not affect dPER stability but delay its nuclear entry in key pacemaker neurons, yielding longer behavioral rhythms. Intriguingly, abolishing phosphorylation at Ser661 also attenuates the extent of dPER hyperphosphorylation in vivo, suggesting the phosphorylated state of Ser661 regulates phosphorylation at other sites on dPER. Indeed, we identify Ser657 as a site that is phosphorylated by the glycogen synthase kinase GSK-3ß (SHAGGY; SGG) in a manner dependent on priming at Ser661. Although not as dramatic as mutating Ser661, mutations that abolish phosphorylation at Ser657 also lead to longer behavioral periods, suggesting that a multi-kinase hierarchical phosphorylation module regulates the timing of dPER nuclear entry. Together with evidence in mammalian systems, our findings implicate proline-directed kinases in clock mechanisms and suggest that PER proteins are key downstream targets of lithium therapy, a potent inhibitor of GSK-3ß used to treat manic depression, a disorder associated with clock malfunction in humans.

Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription.

Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dCLOCK (dCLK)-CYCLE (CYC) transcriptional activity by dPERIOD (dPER) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPER function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPER interacts with dCLK-CYC to promote repression is not known. Here, we identified a small conserved region on dPER required for binding to dCLK, termed CBD (for dCLK binding domain). In the absence of the CBD, dPER is unable to stably associate with dCLK and inhibit the transcriptional activity of dCLK-CYC in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the DOUBLETIME kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPER missing the CBD (dPER(ΔCBD)) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPER(ΔCBD) and dCLK is TIM-dependent and modulated by light, revealing a novel and unanticipated in vivo role for TIM in circadian transcription. Finally, dPER(ΔCBD) does not provoke the daily hyperphosphorylation of dCLK, indicating that direct interactions between dPER and dCLK are necessary for the dCLK phosphorylation program but are not required for other aspects of dCLK regulation.

Circadian regulation in the ability of Drosophila to combat pathogenic infections.

We sought to determine if the innate immune response is under circadian regulation and whether this impacts overall health status. To this end, we used infection of Drosophila with the human opportunistic pathogenic bacteria Pseudomonas aeruginosa as our model system [1]. We show that the survival rates of wild-type flies vary as a function of when, during the day, they are infected, peaking in the middle of the night. Although this rhythm is abolished in clock mutant flies, those with an inactive period gene are highly susceptible to infection, whereas mutants with impairment in other core clock genes exhibit enhanced survival. After an initial phase of strong suppression, the kinetics of bacterial growth correlate highly with time of day and clock mutant effects on survival. Expression profiling revealed that nighttime infection leads to a clock-regulated transient burst in the expression of a limited number of innate immunity genes. Circadian modulation of survival also was observed with another pathogen, Staphylococcus aureus. Our findings suggest that medical intervention strategies incorporating chronobiological considerations could enhance the innate immune response, boosting the efficacy of combating pathogenic infections.

A DOUBLETIME kinase binding domain on the Drosophila PERIOD protein is essential for its hyperphosphorylation, transcriptional repression, and circadian clock function.

A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins from hypo- to hyperphosphorylated species, events that are highly dependent on casein kinase 1 epsilon (termed DOUBLETIME [DBT] in Drosophila melanogaster) and necessary for normal clock progression. Drosophila PER (dPER) functions in the negative limb of the clockworks by presumably binding to the transcription factor CLOCK (CLK) and inhibiting its transactivation activity. Here, we identify a small region on dPER that is conserved with mammalian PERs and contains the major in vivo DBT binding domain, termed dPDBD (for dPER DBT binding domain). This domain is required for the manifestation of molecular and behavioral rhythms in vivo. In the absence of the dPDBD, the dPER protein is present at constant high levels throughout a daily cycle, undergoes little phosphorylation, and is severely impaired in its ability to function as a transcriptional repressor. Our findings indicate that the binding of dPER to CLK is not sufficient for transcriptional inhibition, implicating a more indirect mode of action whereby dPER acts as a molecular bridge to "deliver" DBT and/or other factors that directly repress CLK-dependent gene expression.

Daywake, an Anti-siesta Gene Linked to a Splicing-Based Thermostat from an Adjoining Clock Gene.

Sleep is fundamental to animal survival but is a vulnerable state that also limits how much time can be devoted to critical wake-dependent activities [1]. Although many animals are day-active and sleep at night, they exhibit a midday nap, or "siesta," that can vary in intensity and is usually more prominent on warm days. In humans, the balance between maintaining the wake state or sleeping during the day has important health implications [2], but the mechanisms underlying this dynamic regulation are poorly understood. Using the well-established Drosophila melanogaster animal model to study sleep [3], we identify a new wake-sleep regulator that we term daywake (dyw). dyw encodes a juvenile hormone-binding protein [4] that functions in neurons as a day-specific anti-siesta gene, with little effect on sleep levels during the nighttime or in the absence of light. Remarkably, dyw expression is stimulated in trans via cold-enhanced splicing of the dmpi8 intron [5] from the reverse-oriented but slightly overlapping period (per) clock gene [6]. The functionally integrated dmpi8-dyw genetic unit operates as a "behavioral temperate acclimator" by increasingly counterbalancing siesta-promoting pathways as daily temperatures become cooler and carry reduced risks from daytime heat exposure. While daily patterns of when animals are awake and when they sleep are largely scheduled by the circadian timing system, dyw implicates a less recognized class of modulatory wake-sleep regulators that primarily function to enhance flexibility in wake-sleep preference, a behavioral plasticity that is commonly observed in animals during the midday, raising the possibility of shared mechanisms.

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.

Circadian regulation of a limited set of conserved microRNAs in Drosophila.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules that target mRNAs to control gene expression by attenuating the translational efficiency and stability of transcripts. They are found in a wide variety of organisms, from plants to insects and humans. Here, we use Drosophila to investigate the possibility that circadian clocks regulate the expression of miRNAs. RESULTS: We used a microarray platform to survey the daily levels of D. melanogaster miRNAs in adult heads of wildtype flies and the arrhythmic clock mutant cyc01. We find two miRNAs (dme-miR-263a and -263b) that exhibit robust daily changes in abundance in wildtype flies that are abolished in the cyc01 mutant. dme-miR-263a and -263b reach trough levels during the daytime, peak during the night and their levels are constitutively elevated in cyc01 flies. A similar pattern of cycling is also observed in complete darkness, further supporting circadian regulation. In addition, we identified several miRNAs that appear to be constitutively expressed but nevertheless differ in overall daily levels between control and cyc01 flies. CONCLUSION: The circadian clock regulates miRNA expression in Drosophila, although this appears to be highly restricted to a small number of miRNAs. A common mechanism likely underlies daily changes in the levels of dme-miR-263a and -263b. Our results suggest that cycling miRNAs contribute to daily changes in mRNA and/or protein levels in Drosophila. Intriguingly, the mature forms of dme-miR-263a and -263b are very similar in sequence to several miRNAs recently shown to be under circadian regulation in the mouse retina, suggesting conserved functions.

A role for CK2 in the Drosophila circadian oscillator.

The posttranslational modification of clock proteins is critical for the function of circadian oscillators. By genetic analysis of a Drosophila melanogaster circadian clock mutant known as Andante, which has abnormally long circadian periods, we show that casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the beta subunit of CK2 and is predicted to perturb CK2beta subunit dimerization. It is associated with reduced beta subunit levels, indicative of a defect in alpha:beta association and production of the tetrameric alpha2:beta2 holoenzyme. Consistent with a direct action on the clock mechanism, we show that CK2beta is localized within clock neurons and that the clock proteins Period (Per) and Timeless (Tim) accumulate to abnormally high levels in the Andante mutant. Furthermore, the nuclear translocation of Per and Tim is delayed in Andante, and this defect accounts for the long-period phenotype of the mutant. These results suggest a function for CK2-dependent phosphorylation in the molecular oscillator.