Insect period homologs

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, the per ortholog in the housefly, Musca domestica has been identified. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the Period and Timeless proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants (Piccin, 2000).

The Musca per transcript contains an ORF encoding the 1048-amino-acid long polypeptide. The division of per into conserved (c) and nonconserved (nc) regions was introduced upon comparison of the gene in three different species: D. melanogaster, D. pseudoobscura, and D. virilis. A fourth Drosophila per gene, cloned from D. yakuba, given the short evolutionary distance of this species from melanogaster (6-15 million years, does not show much variation, even in the so-called nonconserved regions, when compared to its closely related homolog. This overall pattern of variation is largely preserved in the housefly gene; the six conserved blocks are clearly apparent upon comparison of Musca per with any of the Drosophila homologs. As in Drosophila, c1 and c2 constitute most of the N-terminal half of the protein, while c3, c4, c5, and c6 are localized in the C-terminal half and are generally less well conserved. In Musca the similarity of c1, c2, and c3 to the Drosophila proteins is very high, between 80% and 94%, slightly lower in c6 (75%-82%), and considerably lower in c4 and c5 (57%-71%) (Piccin, 2000).

The N-terminal block c1 contains the NLS. Interestingly, a second conserved putative NLS is found in c3, but a functional analysis of this signal has not been reported. The longest conserved block is c2, representing almost half the length of the entire Musca PER protein and containing the PAS dimerization region. The definition of PAS includes residues 238-496 (in the D. melanogaster sequence), and begins a few amino acids upstream of the first 51-residue PASA degenerate repeat, and ends downstream of the PASB repeat after the PAC domain. The PAC domain includes the cytoplasmic localization domain (CLD), except for a few C-terminal residues that cannot be unaligned between the species. This broad definition of PAS encompasses all the N-terminal regions that physically interact with TIM. The sites to which the perL, perS, and per01 mutations have been mapped are included within this region and are perfectly conserved in all PER proteins (Piccin, 2000).

Secondary structure analyses have identified an HLH domain located in c2 [amino acids (aa) 450-512], at the end of the CLD. The same structural motif is also found in the D. melanogaster sequence (aa 525-571). The Musca candidate HLH lies in a different region of the protein from the one suggested in the mammalian mper1 homolog. Musca c5 also contains an opa repeat (CAG), which generates a cluster of glutamines at positions 907-917, a feature associated with transcriptional activators; despite this poly-Q stretch being localized in a conserved region, none of the other Drosophila orthologues display a similar motif. A poly-Q stretch is also found in nc1 of D. virilis. In nc2 lies the Thr-Gly repeat, and as reported in other non-Drosophilid dipterans, the Musca repeat of two Thr-Gly pairs has not undergone the dramatic expansion in size observed in the Drosophila genus. Various PEST sequences and phosphorylation sites are also found within the PER proteins. One putative site for casein kinase II phosphorylation is found in all the Dipteran sequences within the C-terminal conserved PEST motif (Piccin, 2000).

The phylogeny of the six species D. melanogaster, D. yakuba, D. pseudoobscura, D. virilis, M. domestica, and A. pernyi is well known from traditional taxonomic approaches. A. pernyi belongs to the order Lepidoptera, which was already well differentiated at the end of the Triassic era 200 mya. The group Calyptratae (to which M. domestica belongs) diverged from the group Acalyptratae (which includes the Drosophilidae) 100 mya; the time of divergence of D. melanogaster and D. virilis is estimated to be ~40 mya; the obscura group (to which D. pseudoobscura belongs) separated from the melanogaster group between 25 mya and 30 mya, and the phylogenetic distance between D. melanogaster and D. yakuba is 6-15 mya. Although there are uncertainties about the exact time of divergence, there are no ambiguities in the branching order of these species. The phylogenetic approach was used to examine whether there is any significant difference between the species tree and the Per protein tree, which could be taken as an indicator of unusual events in the evolution of Per protein sequences. Of particular interested is the PAS domain, which has been implicated in the protein-protein interactions between Per and Tim (Piccin, 2000).

Initially, a molecular phylogeny was computed on the DNA sequence coding for the PAS domain. There is no ambiguity in the estimation of the evolutionary tree because the species tree is faithfully reproduced. A phylogeny was generated based on the alignable amino acid sequence from non-PAS regions c1 and c3, which takes into account the fact that some amino acid replacements occur at higher frequencies than others, irrespective of the necessary number of nucleotide substitutions. A tree similar to the DNA tree was obtained, except that D. virilis and D. pseudoobscura have swapped positions (Piccin, 2000).

The third fragment of Per used in this analysis was the PAS region from c2 (including PAC/CLD, residues 238-496 of the D. melanogaster sequence), which represents a functional domain of PER. The PAS tree places Musca PAS closer to D. melanogaster than D. pseudoobscura and D. virilis, contradicting the species tree drawn from per DNA. A similar switching of positions of the Musca and D. pseudoobscura/virilis groups, with similarly high bootstrap values, was also observed using a protein distance matrix; therefore, the tree is reasonably robust. This unusual PAS phylogeny is reflected in the smaller number of differences between the D. melanogaster/M. domestica pairwise comparison (29 aa changes and 1 aa deletion) compared to that of D. melanogaster/D. pseudoobscura (33 aa changes) or D. melanogaster/D. virilis (44 replacements) (Piccin, 2000).

Previous research showed that age-related division of labor in honey bees is associated with changes in activity rhythms; young adult bees perform hive tasks with no daily rhythms, whereas older bees forage with strong daily rhythms. This division of labor is also associated with differences in both circadian rhythms and mRNA levels of period, a gene well known for its role in circadian rhythms. The level of period mRNA in the brain oscillates in bees of all ages, but is significantly higher at all times in foragers. Elevated period mRNA levels cannot be attributed exclusively to aging, because bees induced to forage precociously because of a change in social environment have levels similar to normal age foragers. These results extend the regulation of a 'clock gene' to a social context and suggest that there are connections at the molecular level between division of labor and chronobiology in social insects (Toma, 2000).

Period (PER) protein regulation was studied in the brain of the silkmoth Antheraea pernyi. PER expression is restricted to the cytoplasm and axons of eight neurons, with no evidence of temporal movement into the nucleus. In contrast, in Drosopila, PER is found in may more cells, and exhibits circadian movement into the nucleus. The silkmoth neurons appear to be circadian clock cells, because PER and per mRNA are colocalized and their levels oscillate in these cells. Timeless protein immunoreactivity is coexpressed in each PER-positive neuron, and PER protein and mRNA oscillations are all suppressed in these neurons by constant light. A PER antisense RNA oscillation was detected that is spatially restricted to per-expressing cells, suggesting a novel mechanism of per regulation. PER-positive neurons and their projections are strategically positioned for regulating prothoracicotropic hormone and eclosion hormone, two neurohormones under circadian control. PTTH (see see Bombyx and Manduca prothoracicotropic hormone) is present in a pair of cells adjacent to the lateral pair of per-expressing cells. Eclosion hormone-immunoreactive cells are found in the dorsal medial region of each brain hemisphere, with each pair sending ipsilateral projections through the subesophageal ganglion to the ventral nervous cord. Axonal projections of PER-expressing cells and their arborization are in the immediate vicinity of EH cells (Sauman, 1996a).

The molecular basis of the circadian control of egg hatching behavior was examined in the silkmoth Antheraea pernyi. Egg hatching is rhythmically gated, persists under constant darkness, and can be entrained by light by midembryogenesis. The time of appearance of photic entrainment by the silkmoth embryo coincides with the appearance of Period (PER) and Timeless (TIM) proteins in eight cells in embryonic brain. Although daily rhythms in PER and/or TIM immunoreactivity in embryonic brain were not detected, a robust circadian oscillation of PER immunoreactivity is present in the nuclei of midgut epithelium. PER antisense oligodeoxynucleotide treatment of pharate larvae on the day before hatching consistently abolishes the circadian gate of egg hatching behavior. per antisense treatment also causes a dramatic decrease in PER immunoreactivity in newly hatched larvae. The results provide direct evidence that PER is a necessary element of a circadian clock in the silkmoth (Sauman, 1996b).

Neuronal circadian clock protein oscillations are similar in behaviourally rhythmic forager honeybees and in arrhythmic nurses

Internal clocks driving rhythms of about a day (circadian) are ubiquitous in animals, allowing them to anticipate environmental changes. Genetic or environmental disturbances to circadian clocks or the rhythms they produce are commonly associated with illness, compromised performance or reduced survival. Nevertheless, some animals including Arctic mammals, open sea fish and social insects such as honeybees are active around-the-clock with no apparent ill effects. The mechanisms allowing this remarkable natural plasticity are unknown. This study generated and validated a new and specific antibody against the clock protein Period of the honeybee Apis mellifera (amPER) and used it to characterize the circadian network in the honeybee brain. Many similarities to Drosophila melanogaster and other insects were found, suggesting common anatomical organization principles in the insect clock that have not been appreciated before. Time course analyses revealed strong daily oscillations in amPER levels in foragers, which show circadian rhythms, and also in nurses that do not, although the latter have attenuated oscillations in brain mRNA clock gene levels. The oscillations in nurses show that activity can be uncoupled from the circadian network and support the hypothesis that a ticking circadian clock is essential even in around-the-clock active animals in a constant physical environment (Fuchikawa, 2017).

Daily activity of the housefly, Musca domestica, is influenced by temperature independence of 3' UTR period gene splicing

Circadian clocks orchestrate daily activity patterns and free running periods of locomotor activity under constant conditions. While the first often depends on temperature, the latter is temperature-compensated over a physiologically relevant range. This study explored the locomotor activity of the temperate housefly, Musca domestica Under low temperatures, activity was centered round a major and broad afternoon peak, while high temperatures resulted in activity throughout the photophase with a mild mid-day depression, which was especially pronounced in males exposed to long photoperiods. While period (per) mRNA peaked earlier under low temperatures, no temperature-dependent splicing of the last per 3' end intron was identified. The expression of timeless, vrille, and Par domain protein 1 was also influenced by temperature, each in a different manner. These data indicated that comparable behavioral trends in daily activity distribution have evolved in Drosophila melanogaster and Musca domestica, yet the behaviors of these two species are orchestrated by different molecular mechanisms (Bazalova, 2017).

frequency and PAS protein incoding white collar-1 and white collar-2 are components of the Neurospora circadian oscillator

The frequency (frq) gene of Neurospora encodes central components of the transcription/translation-based negative-feedback loop comprising the core of the Neurospora circadian oscillator. Frequency is a novel protein with a predicted helix-turn-helix region but with no identifiable homologies to Drosophila Period protein (Lewis, 1997). Posttranscriptional regulation associated with Frq is surprisingly complex. Alternative use of translation initiation sites gives rise to two forms of Frq: their levels peak 4-6 hr following the peak of FRQ transcript. Each form of Frq is progressively phosphorylated over the course of the day, thus providing a number of temporally distinct FRQ products. The kinetics of these regulatory processes suggest a view of the clock wherein relatively rapid events involving translational regulation in the synthesis of Frq and negative feedback of Frq on FRQ transcript levels are followed by slower posttranslational regulation, ultimately driving the turnover of Frq and reactivation of the frq gene (Garceau, 1997).

Two forms of Frq protein, a central component of the Neurospora circadian clock, arise through alternative in-frame initiation of translation. Either form alone suffices for a functional clock at some temperatures, but both are always necessary for robust rhythmicity. Temperature regulates the ratio of Frq forms by favoring different initiation codons at different temperatures; when either initiation codon is eliminated, the temperature range permissive for rhythmicity is demonstrably reduced. This temperature-influenced choice of translation-initiation site represents a novel adaptive mechanism that extends the physiological temperature range over which clocks function. A temperature-dependent threshold level of Frq is required to establish the feedback loop comprising the oscillator. These data may explain how temperature limits permissive for rhythmicity are established, thus providing a molecular understanding for a basic characteristic of circadian clocks (Liu, 1997).

The frequency (frq) gene in Neurospora encodes central components of a circadian oscillator, a negative feedback loop involving frq mRNA and two forms of FRQ protein. FRQ is a nuclear protein; nuclear localization is essential for its function. Deletion of the nuclear localization signal (NLS) renders FRQ unable to enter the nucleus and abolishes overt circadian rhythmicity, while reinsertion of the NLS at a novel site near the N-terminus of FRQ restores its function. Each form of FRQ enters the nucleus soon after its synthesis in the early subjective day; there is no evidence for regulated sequestration in the cytoplasm prior to nuclear entry. The kinetics of the nuclear entry are consistent with previous data showing rapid depression of frq transcript levels, following the synthesis of FRQ, and suggest that early in each circadian cycle, when FRQ is synthesized, it enters the nucleus and depresses the level of its own transcript (Luo, 1998).

Circadian rhythmicity is universally associated with the ability to perceive light, and the oscillators ('clocks') giving rise to these rhythms, which are feedback loops based on transcription and translation, are reset by light. Although such loops must contain elements of positive and negative regulation, the clock genes analyzed to date -- frq in Neurospora and per and tim in Drosophila -- are associated only with negative feedback and their biochemical functions are largely inferred. The white collar-1 and white collar-2 genes, both global regulators of photoresponses in Neurospora, encode DNA binding proteins that contain PAS domains and are believed to act as transcriptional activators. Data shown in this study suggest that wc-1 is a clock-associated gene and wc-2 is a clock component; both play essential roles in the assembly or operation of the Neurospora circadian oscillator. Thus DNA binding and transcriptional activation can now be associated with a clock gene that may provide a positive element in the feedback loop. In addition, similarities between the PAS-domain regions of molecules involved in light perception and circadian rhythmicity in several organisms suggest an evolutionary link between ancient photoreceptor proteins and more modern proteins required for circadian oscillation (Crosthwaite, 1997).

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock-associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so that identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase-response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system (Heintzen, 2001).

VVD, a novel member of the PAS protein superfamily, identifies an autoregulatory negative feedback loop that closes outside of the core oscillatory system but impacts all aspects of circadian timing. Expression of vvd is clock influenced, and VVD feeds back to regulate the expression of light-regulated genes including wc-1 and frq. VVD's only distinct functional motif is the PAS domain; thus, an attractive model posits an interaction and transient downregulation of the activity of the WCC. Indeed, VVD influences wc-1 transcript and the phosphorylation and abundance of WC-1 protein, as well as frq transcript levels. Since WCC is required for light induced transcription of most Neurospora genes and is also required for the activation of frq and expression of circadian rhythms in the dark, the loss of VVD has far-reaching effects on the perception of light and on the entire circadian system, ranging from input as seen in the phase-response curve, to output as manifested in the phasing and expression levels of clock-controlled genes and the overt rhythm. Clock regulation of the immediate and transient repressor VVD could contribute to circadian entrainment by influencing phase following dark to light transitions, and VVD contributes to the gating response whereby the clock regulates its own input. The PAS protein VVD provides an example of a molecular feedback of clock-controlled output onto light input pathways, thus providing a principle that illuminates light adaptation and gating of input (Heintzen, 2001).

The frequency gene, the central component of the frq-based circadian negative feedback loop, regulates various aspects of the circadian clock in Neurospora: However, the biochemical function of its protein products, FRQ, is poorly understood. In this study, it is demonstrated that the most conserved region of FRQ forms a coiled-coil domain. FRQ interacts with itself in vivo, and the deletion of the coiled-coil region results in loss of the interaction. Point mutations, which are designed to disrupt the coiled-coil structure, weaken or completely abolish the FRQ self-association and lead to the arrhythmicity of the overt rhythm. Mutations of the FRQ coiled-coil that inhibit self-association also prevent its interaction with two other key components of the Neurospora: circadian clock, namely WC-1 and WC-2, the two PAS domain-containing transcription factors. Taken together, these data strongly suggest that the formation of the FRQ-FRQ and FRQ-WC complexes is essential for the function of the Neurospora clock (Cheng, 2001).

To understand the role of white collar-2 in the Neurospora circadian clock, alleles of wc-2 thought to encode partially functional proteins were examined. wc-2 allele ER24 contains a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation alters a second canonical property of the clock, temperature compensation: as temperature increases, period length decreases substantially. This temperature compensation defect correlates with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2 (ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. It is suggested that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator of frq, is critical for establishing period length and temperature compensation in this circadian system (Collett, 2001).

The finding of period length defects caused by a mutation in wc-2 adds to the similarities between the Neurospora clock and the clocks of mammals and fruit flies. The hypomorphic mutation in wc-2 resulting in lengthened period and eventual damping of the rhythm is a similar phenotype to that possessed by mice homozygous for the Clock mutation and Drosophila flies heterozygous for the Cyc or ClkJrk mutation. These three mutations all affect genes, like wc-2 and wc-1, that encode PAS domain-containing positive-acting transcription factors, which are understood to activate transcription of the negatively acting clock genes per and tim in flies and the mper genes in mammals. In agreement with the role of these genes as positive factors, all three mutations result in lowered mRNA levels of the relevant clock genes (Collett, 2001 and references therein).

The temperature compensation defect in wc-2 (ER24), however, is novel and unexpected; until now, defects in temperature compensation caused by mutations in single genes have only been noted in alleles of the negative elements frq, per, and tim and in the hamster tau mutant, which encodes casein kinase I epsilon hypomorphic for phosphorylation of mPER1. The reduced temperature compensation in a wc-2 (ER24)-containing strain suggests that temperature compensation probably results from an interplay between positively and negatively acting elements in the circadian cycle. The period shortening as temperature increases in ER24 is correlated with the temperature-dependent increase in FRQ levels. However, this increase in FRQ levels is also observed in the wild type, with only a mild period shortening effect observed, prompting the question of why this effect is so great in ER24 compared to the wild-type strain. A possible explanation is that, relative to FRQ levels at 25oC, the increase in FRQ with temperature in ER24 is greater than the corresponding increase in the wild type. This greater relative increase in FRQ might lead to a period-shortening effect, partially rescuing the decreased levels of FRQ found in wc-2 (ER24) and leading to a shortened period at higher temperatures. The greater increase in FRQ in ER24 suggests that there may be a mechanism regulating FRQ levels with temperature. Perhaps once FRQ exceeds a given level at a given temperature, the excess FRQ is degraded. However, FRQ levels in ER24 would be so low that this mechanism would have only a very small effect on FRQ levels in the mutant (Collett, 2001).

It is clear that wc-2 is a positively acting component of the Neurospora clock, a positive regulator of levels of frq mRNA. Determination of the mechanism of action of WC-2 on the frq promoter, be it direct or indirect (through other proteins), is critical for a future understanding of the clock in Neurospora (Collett, 2001).

Frequency (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1epsilon (Drosophila homolog: Doubletime). CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frqDeltaPEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frqDeltaPEST-1 RNA and FRQPEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components (Gör, 2002).

Phosphorylation of the Neurospora circadian clock protein Frequency (FRQ) regulates its degradation and the proper function of the clock. FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, these results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock (He, 2003).

CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. This study shows that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1aL strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbtL mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ~32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1aL strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1aL strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the ckaRIP strain, which carries the disruption of the catalytic subunit of casein kinase II. In the ckaRIP strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop (He, 2006).

The eukaryotic circadian clocks are composed of auto- regulatory circadian negative feedback loops including both positive and negative elements. In Neurospora, Drosophila, and mammals, the positive elements are all heterodimeric complexes, consisting of two PER-ARNT-SIM (PAS) domain-containing transcriptional factors that bind to the cis-elements in the promoter of the negative elements to activate their transcription. In contrast, the negative elements repress their own transcription by inhibiting the activity of the positive elements through their physical interactions. It is unclear how negative elements inhibit the activity of positive elements to close the circadian negative feedback loops. Since the identification of the Drosophila doubletime (dbt) gene, which encodes for a casein kinase I (CKI) homolog, it has become clear that post-translational protein phosphorylation is essential for the function of circadian clocks. Despite the evolutionary distance, remarkable conservation of post-translational regulation exists among different eukaryotic systems from fungi to human (He, 2006).

In the filamentous fungus Neurospora crassa, the core circadian negative feedback loop consists of four essential components: WHITE COLLAR-1 (WC-1), WC-2, FREQUENCY (FRQ), and a FRQ-interacting RNA hecliase FRH. WC-1 and WC-2, two PAS domain-containing transcription factors, form a heterodimeric WC complex (D-WCC) in the dark, which binds to the Clock (C)-box in the frq promoter to activate frq transcription. Thus, WCC is the positive element in the Neurospora circadian negative feedback loop. In contrast, FFC, the complex formed by FRQ and FRH functions as the negative element. To repress the transcription of frq, FFC possibly mediates the inhibition of the WCC activity through their physical interaction. In a wild-type strain, this circadian negative feedback loop generates robust daily rhythms of frq RNA and FRQ protein. When the circadian negative feedback loop is disrupted by mutation of frq or down-regulation of frh, frq RNA levels stay at constant higher levels, resulting in arrhythmici- ties. In addition to their role in the circadian negative feedback loop in the dark, WC-1and WC-2 are also essential components for the light responses and light resetting of the clock, with WC-1 being the blue-light photoreceptor (He, 2006).

In addition to the repression of D-WCC activity in the dark, FRQ promotes the accumulation of WC-1 and WC-2, forming positive feedback loops that are inter-locked with the negative loop, a feature that is shared by animal circadian systems. In Neurospora, these positive feedback loops have been shown to be important for the robustness and function of the clock. It was recently shown that the phosphorylation of the PEST-2 region of cytoplasmic FRQ is important for the accumulation of WC-1 but not WC-2 (He, 2006).

FRQ, WC-1, and WC-2 are regulated by phosphorylation events. After its synthesis, FRQ is immediately phosphorylated and becomes progressively more phosphorylated over time before its degradation through the ubiquitin–proteasome pathway mediated by FWD-1. Thus, in the dark, FRQ is not only robustly rhythmic in quantity, but also in its phosphorylation states. CK-1a (casein kinase 1a), CKII (casein kinase II), and CAMK-1 are the three identified FRQ kinases. However, only CKII's role in mediating FRQ phosphorylation is firmly established in vivo. In vitro, CKII is one of the main kinases that phosphorylate FRQ. In strains in which either the CKII catalytic subunit (cka) or one of its regulatory subunits (ckb1) is disrupted, FRQ is both hypophosphorylated and more stable, and the clock function is either completely abolished (cka mutant) or oscillates with a severely damped amplitude (ckb1 mutant). Furthermore, in the cka mutant strain that has no CKII activity, frq mRNA levels are constantly high, which is reminiscent of the frq RNA levels in strains with a disrupted circadian negative feedback loop. These data suggest that CKII not only promotes FRQ degradation, but it is also required for the repressor function of FRQ. The mechanism by which CKII carries this latter function is not known (He, 2006).

CK-1a is one of the two Neurospora CKI homologs and it can phosphorylate the PEST-1 and PEST-2 domains of FRQ in vitro. The deletion of the PEST-1 domain resulted in the increased stability of FRQ and a long period rhythm. More importantly, CK-1a was found to associate with FRQ, suggesting that it may phosphorylate FRQ in vivo. However, in vivo evidence for the involvement of CK-1a in the clock was not available because CK-1a is essential for cell survival in Neurospora (He, 2006).

Similar to FRQ, both WC-1 and WC-2 are phosphorylated both in the dark and in a light-dependent manner. Their phosphorylation plays important roles in regulating WCC activity. Five major in vivo WC-1 phosphorylation sites, located immediately downstream from its DNA-binding domain, have been identified. Mutation of these light-independent sites suggested that they are critical for circadian clock function and they negatively regulate the D-WCC activity. The importance of WC phosphorylation in the circadian clock was later confirmed by the surprising observation that the WC phosphorylation is FRQ dependent. In the frq-null strain, both WC-1 and WC-2 are hypophosphorylated. In a wild- type strain, WC-2 exhibits a robust circadian rhythm of its phosphorylation profile when analyzed on two-dimensional electrophoresis. Importantly, the activation of frq transcription correlates with the hypophosphorylation of the WCs. Consistent with these data, it has been shown that dephosphorylation of the Neurospora WCC significantly promotes its binding to the C-box. Together, these results suggest a model in which FFC inhibits the WCC activity by promoting the phosphorylation of WC proteins. Interestingly, PER-dependent phosphorylation of CLK has also been observed in Drosophila, suggesting a common mechanism that closes the circadian negative feedback loops (He, 2006).

The kinase(s) recruited by FRQ to phosphorylate the WC proteins has not been identified. How WCC activity is affected by the phosphorylation mediated by this kinase(s) is also not known. In this study, it is shown that like CKII, CK-1a phosphorylates FRQ in vivo to promote its degradation. More importantly, both kinases mediate the FRQ-dependent phosphorylation of WCC, which inhibits its activity to close the circadian negative feedback loop. In addition to CK-1a's role in the negative feedback loop, it is also required for the function of the circadian positive feedback loops by increasing WC levels (He, 2006).

LIN-42, a heterochronic gene of C. elegans, a PAS-domain protein that regulates molting cycles

The C. elegans heterochronic genes control the relative timing and sequence of many events during postembryonic development, including the terminal differentiation of the lateral hypodermis, which occurs during the final (fourth) molt. During the fourth molt, the lateral hypodermal seam cells terminally differentiate; they exit the cell cycle and secrete a morphologically distinct adult cuticle. Mutations in the heterochronic genes alter the timing of this event. Loss-of-function mutations in the heterochronic genes lin-14, lin-28, and lin-42 cause precocious phenotypes in which seam cell terminal differentiation is executed prematurely, whereas mutation of lin-4 or lin-29 retards this differentiation event. These latter mutants undergo additional rounds of ecdysis during which the hypodermis remains undifferentiated. These genes form a negative regulatory pathway that restricts seam cell terminal differentiation to the fourth molt. The transcription factor LIN-29 is the most direct trigger of the switch to the adult hypodermal program; the remaining genes act upstream, ensuring that lin-29 activity is correctly timed. Inactivation lin-42 causes hypodermal terminal differentiation to occur precociously, during the third molt. lin-42 mRNA levels oscillate, but with a faster rhythm than do levels of Drosophila Per; the oscillation occurs relative to the approximately 6-hour molting cycles of postembryonic development (Jeon, 1999).

Structurally, LIN-42 most closely resembles the Period family of proteins from Drosophila and other organisms -- proteins that function in another type of biological timing mechanism: the timing of circadian rhythms. The most striking region of similarity includes a protein interaction domain, the PAS domain, which has recently come to be viewed as a signature feature of circadian rhythm proteins, including the insect and mammalian PER proteins, the WHITE COLLAR proteins of Neurospora, and the CLOCK and BMAL proteins of mice and their Drosophila counterparts, Clock and Cycle. The PER PAS domain is an approximately 260-amino acid region containing two divergent hydrophobic direct repeats of about 50 amino acids, known as the PASA and PASB repeats. The region of highest similarity between LIN-42 and Per encompasses the PASB repeat, and includes the cytoplasmic localization domain (CLD) of Per. The two proteins are 30% identical and 45% similar throughout this 139-amino acid region. The percent identity between LIN-42 and other Per family members in this region is also about 30% (29% for cockroach Per; 28% for human and mouse PER1). By comparison, human PER1 (hPER1) and Drosophila Per share 39% identity in this region. The similarity between vertebrate and invertebrate Per proteins is lower in the PASA repeat. Drosophila Per and LIN-42 each share 20% identity with hPER1 over the 50-amino acid PASA repeat (Jeon, 1999 and references therein).

The observed oscillations of lin-42 mRNA levels do not correspond to absolute time from hatching; rather, they are synchronized to the molting cycles as is demonstrated by the lengthening of the period of oscillation when worms are grown at a lower temperature. These experiments indicate that lin-42 mRNA levels oscillate relative to the execution of molting cycles. C. elegans undergoes four rounds of ecdysis, at approximately 6-hour intervals (at 25°C), as they develop from the newly hatched L1 larva to the adult. The lin-42 mRNA expression pattern suggests a possible role for lin-42 in promoting or coordinating aspects of the molting cycle. However, lin-42 mutants do not exhibit obvious molting defects until the execution of the final molt. At this stage, lin-42 mutants often have difficulty shedding the L4 cuticle. This observation suggests that lin-42 functions in at least this ecdysis event, and perhaps another factor supplies this function during the earlier molts. During each molt cycle, a new worm cuticle is synthesized, composed mainly of collagens. Expression levels of six collagen genes have been found to oscillate relative to the molting cycles, raising the possibility that lin-42 cycling could function in, or be synchronized with, collagen gene regulation (Jeon, 1999).

A LIN-42::GFP construct that reflects Lin-42 expression is present in the lateral hypodermis. LIN-42::GFP also accumulates in the hyp7 syncytium, which comprises the main body hypodermis, and in head and tail hypodermal cells. LIN-42::GFP expression is first detected in late embryonic stage animals, and it remains detectable into the adult stage. The intensity of lin-42::gfp expression appears higher, in general, in animals undergoing ecdysis. LIN-42::GFP is generally enriched in nuclei relative to cytoplasm, but there is no developmental time when LIN-42::GFP is observed to be entirely nuclear or entirely cytoplasmic. Cytoplasmic LIN-42::GFP signal is enhanced in lateral seam cells during the molt periods. During the first three molts, this correlates with seam cell divisions and may be related to cell cycle stage, reflecting nuclear release followed by protein turnover and replenishment after division, because there is a period during cytokinesis where LIN-42::GFP is not observed. However, the LIN-42::GFP signal is also enhanced in the seam cell cytoplasm of L4 molt animals, when cell divisions do not occur (Jeon, 1999).

The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. This study identifies the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. A spontaneous suppressor of the normally lethal let-7(n2853) allele was mapped to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42n1089 worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, it is proposed that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis (Van Wynsberghe, 2014)

Chicken Period homologs

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. Chicken pineal genes were sought that are induced by light in a time-of-day-dependent manner. Isolated was the chick homolog of bZIP transcription factor E4bp4 (cE4bp4), which shows high similarity to vrille, one of the Drosophila clock genes. cE4bp4 is expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase is nearly opposite that of the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays show that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5'-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies show that the prolongation of the light period to early subjective night maintains the high level expression of the pineal cE4bp4, and presumably as a consequence delays the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 are followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene (Doi, 2001).

Mammalian Period homologs

Period Evolutionary homologs part 2/3 | part 3/3

period: Biological Overview | Regulation | Targets of Activity and Post-transcriptional Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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